Capsaicinoids, From the plant cultivation to the production of the human medical drug

168x238_paprika_cimnegyed_vegleges.qxd

2009.04.21.

16:32

Page 1

CAPSAICINOIDS From the Plant Cultivation to the Production of the Human Medical Drug

168x238_paprika_cimnegyed_vegleges.qxd

2009.04.21.

16:32

Page 3

CAPSAICINOIDS From the Plant Cultivation to the Production of the Human Medical Drug By Gyula Mózsik, András Dömötör, Tibor Past, Viktória Vas, Pál Perjési, Mónika Kuzma, Gyula Blazics, János Szolcsányi

AKADÉMIAI

KIADÓ

168x238_paprika_cimnegyed_vegleges.qxd

2009.04.21.

16:32

Page 4

The publication of this book was supported by the grant of the National Office for Research and Technology “Pázmány Péter Program” Ret-II, 08/2005 MEDIPOLISZ

ISBN 978 963 05 8694 8

© Gyula Mózsik, 2009

Published by Akadémiai Kiadó Member of Wolters Kluwer Group P.O. Box 245, H–1519 Budapest, Hungary www.akkrt.hu

All rights reserved. No part of this book may be reproduced by any means or transmitted or translated into machine language without the written permission of the publisher.

Printed in Hungary

paprika2.qxd

4/15/09

12:17 PM

Page 5

Contents

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 1. General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 1.1. General introduction to the interactions of foods (or food components) and drugs to be used in the prevention and treatment of different diseases in patients. . . . . . . . . . . . . . . . . . . . . . . . . . 15 1.1.1. Foods and food components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 1.1.2. Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 1.1.3. Levels of the drug–food interactions in healthy human beings and in patients with different diseases . . . . . . . . . . . . . . . . . . . . . 17 1.1.4. A short historic background of the interaction between the effects of capsaicin (capsaicinoids) and NSAIDs in animal experiments and healthy human subjects . . . . . . . . . . . . . . . . . . . . . . . . 18 1.1.5. Goals of the drug (drug-combination) production. . . . . . . . . . . . . . . . . . 20 2. Capsaicin (capsaicinoids) is (are) a famous family of the spices . . . . . . . . . . . . 21 2.1. Cultural background of spices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 2.2. History of spices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 2.2.1. Terminology and modern history of Capsicum . . . . . . . . . . . . . . . . . . . . 23 2.2.2. History of Capsicum in Hungary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 3. Botanical taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 4. Cultivation of Capsicum or paprika . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 4.1. Cultivation of Capsicums in India . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 4.2. Cultivation of Capsicums in other Asian countries . . . . . . . . . . . . . . . . . . . . . . 33 4.3. Cultivation of Capsicums in Africa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.4. Cultivation of Capsicums in America . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 4.5. Cultivation of Capsicums in Europe and Hungary . . . . . . . . . . . . . . . . . . . . . . 34 4.5.1. Cultivar types in Hungary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4.5.1.1. Sweet Capsicum cultivar . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 4.5.1.2. Paprika varieties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

5

paprika2.qxd

4/15/09

12:17 PM

Page 6

5. General chemical structure and composition of Capsicums . . . . . . . . . . . . . . . 39 5.1. Macroscopic characteristics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 5.2. Microscopic characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 5.3. General chemical composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 6. Chemical taxonomy of the functional parts of the Capsicums. . . . . . . . . . . . . . 43 6.1. Capsicum: Botanical aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 6.2. Capsicum: Chemical constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 6.2.1. Volatiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 6.2.2. Coloring pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 6.2.2.1. Methods for determination of red and total carotenoids . . . . . . 53 6.2.3. Capsaicinoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 6.2.3.1. Chemistry of capsaicinoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 6.3. Capsicum: Quality control. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 6.3.1. Capsicum fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 6.3.1.1. The European Pharmacopeia (Ph. Eur. 5.0) . . . . . . . . . . . . . . . . 61 6.3.2. Capsicum extracts – Oleoresin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 6.3.2.1. Capsicum extracts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64 6.3.2.2. Capsicum Oleoresin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 6.3.3. Quantitation of capsicum pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 6.3.3.1. The Color Matching Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 67 6.3.3.2. Spectrophotometric methods . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 6.3.3.2.1. The EOA Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 6.3.3.2.2. The American Spice Trade Association (ASTA) Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68 6.3.3.2.3. The Hungarian Standard Method . . . . . . . . . . . . . . . . 68 6.3.4. Quantitation of pungent principles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 6.3.4.1. Official methods for organoleptic determination of pungency . . . . 69 6.3.4.1.1. The Scoville Method. . . . . . . . . . . . . . . . . . . . . . . . . . 69 6.3.4.1.2. The EOA Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70 6.3.4.1.3. The British Standard Method . . . . . . . . . . . . . . . . . . . 71 6.3.4.1.4. The International Standard Organization (ISO) Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 6.3.4.1.5. The ASTA Method . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 6.3.4.2. Quantitation of capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . 71 6.3.4.2.1. Early direct methods . . . . . . . . . . . . . . . . . . . . . . . . . . 72 6.3.4.2.2. Methods based on separation of capsaicinoids. . . . . . 72 6.3.4.2.3. Newer chromatographic micromethods – Thin Layer Chromatography . . . . . . . . . . . . . . . . . . . 73 6.3.4.2.4. Newer chromatographic micromethods – Gas Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . 73 6.3.4.2.5. Newer chromatographic micromethods – High Performance Liquid Chromatography . . . . . . . . 75 6.3.4.2.6. The ASTA method for determination of capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77 6.3.4.2.7. The United States Pharmacopeia (USP) . . . . . . . . . . . 77 6.3.4.3. Correlation of pungency and capsaicinoid content . . . . . . . . . . 79 6.3.4.4. Stability of capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

6

paprika2.qxd

4/15/09

12:17 PM

Page 7

7. General physiology of retinoids and carotenoids. . . . . . . . . . . . . . . . . . . . . . . . . 81 7.1. Biochemistry of retinoids and carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 7.1.1. General physiology of retinoids and carotenoids . . . . . . . . . . . . . . . . . . 82 7.1.2. Retinoids and chemical carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . 83 7.1.3. Effect of antioxidants on colorectal epithelial cell proliferation, polyp recurrence and carcinogenesis: Clinical trials in patients . . . . . . . 84 7.2. Prevalence and importance of the checked human GI diseases. . . . . . . . . . . . . 85 7.3. Gastric cytoprotection, as a special form of defensive mechanisms to gastrointestinal (GI) mucosal injury produced by retinoids . . . . . . . . . . . . . 90 7.4. New results in the biological actions of retinoids and carotenoids in animals, healthy human subjects and patients with different gastrointestinal disorders . . . 91 7.4.1. Gastrointestinal mucosal protective effects produced by retinoids in animal experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 7.4.2. Effects of vitamin A on the gastric secretory responses and indomethacin-induced gastric microbleedings in healthy human subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 7.4.3. Ulcer healing effect of vitamin A in patients with chronic gastric ulcer (multiclinical randomized, prospective study) . . . . . . . . . . . . . . . . 93 7.4.4. Changes in serum levels of retinoids in patients with different gastrointestinal inflammatory diseases . . . . . . . . . . . . . . . . . . . . . . . . . . 93 7.4.5. Changes in serum levels of retinoids in patients with different gastrointestinal cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94 7.4.5.1. Leiden mutation in patients with different gastrointestinal tumors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 7.4.5.2. Correlation between the prevalence of Leiden mutation and decrease of serum levels of vitamin A and zeaxanthin in patients with different gastrointestinal tumors. . . . . . . . . . . . 97 8. Animal and human observations with capsaicinoids . . . . . . . . . . . . . . . . . . . . 100 8.1. Physiological and pharmacological research tool by capsaicin (Short overview) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 8.1.1. The chemistry of capsaicinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 8.1.2. Source of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 8.1.3. Selective sensory effects of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . 101 8.1.4. Mechanism of action of capsaicin on sensory receptors. . . . . . . . . . . . 102 8.1.5. Capsaicin actions in the gastrointestinal tract of animals . . . . . . . . . . . 103 8.1.6. Capsaicin-sensitive sensory nerves and gastric acid secretion . . . . . . . 105 8.1.7. Molecular-pharmacological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 8.1.8. Capsaicin actions in healthy human subjects and in patients with different gastrointestinal disorders . . . . . . . . . . . . . . . . . . . . . . . . 113 8.1.8.1. Results of the comparative molecular-pharmacological studies of capsaicin, atropine, omeprazole, famotidine, ranitidine and cimetidine on the gastric basal acid output (BAO) in human subjects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115 8.1.9. Side effects of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) in the gastrointestinal tract of patients . . . . . . . . . . . . 118

7

paprika2.qxd

4/15/09

12:17 PM

Page 8

9. Toxicological studies with capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 9.1. Animal observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 9.1.1. Acute toxicology studies of capsaicin in animal experiments . . . . . . . 120 9.1.2. Acute toxicity studies with pure trans-capsaicin derivates in dogs after intravenous administration . . . . . . . . . . . . . . . . . . . . . . . . 121 9.1.2.1. Acute effects on cardiovascular and respiratory parameters . . . 123 9.1.2.2. Plasma levels of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124 9.1.3. Results of subacute toxicology of capsaicin in dogs. . . . . . . . . . . . . . . 124 9.1.3.1. Two weeks treatment with trans-capsaicin . . . . . . . . . . . . . . . 124 9.1.3.2. Clinical chemistry and hematology . . . . . . . . . . . . . . . . . . . . . 124 9.1.3.3. Organ weights, macroscopic and microscopic observations . . . 125 9.1.3.4. Pharmacokinetic data after 14-days treatment with trans-capsaicin in dogs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 9.1.3.5. Absorption and metabolism of oral application of capsaicinoids in animal experiments. . . . . . . . . . . . . . . . . . 125 9.1.3.6. Summary and conclusions of the administration of different doses of trans-capsaicin in acute and subacute experiments in dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126 9.1.3.7. Chronic toxicity studies in animals . . . . . . . . . . . . . . . . . . . . . 128 9.1.4. Metabolism of capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128 9.1.4.1. The potential routes of metabolism of capsaicin . . . . . . . . . . . 129 9.1.4.1.1. Enzymatic oxidative metabolism of capsaicin . . . . 129 9.1.4.1.2. Non-oxidative metabolism of capsaicin . . . . . . . . . 130 9.1.4.2. Role of metabolic activation in capsaicin-induced toxicity . . . . 131 9.1.5. Effects of capsaicin on xenobiotic metabolism and chemically induced mutagenesis and carcinogenesis . . . . . . . . . . . . . . . . . . . . . . . 133 9.1.6. Hepatoprotection of capsaicin in rats . . . . . . . . . . . . . . . . . . . . . . . . . . 134 9.1.7. Genotoxicity studies with capsaicin or trans-capsaicin . . . . . . . . . . . . 135 9.1.7.1. Ames assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 9.1.7.2. Mouse lymphoma cell mutation assay . . . . . . . . . . . . . . . . . . 137 9.1.7.3. Mouse in vivo micronucleus assay . . . . . . . . . . . . . . . . . . . . . 137 9.1.7.4. Chromosomal aberration in human peripheral blood lymphocytes (HPBL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138 9.1.7.5. Brief summary of the main results of observations with capsaicin in animals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140 9.2. Human observations with capsaicin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 9.2.1. Observations with capsaicin in healthy human subjects . . . . . . . . . . . . 141 9.2.1.1. Dose-response curves of capsaicin in the human stomach acute observation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 141 9.2.1.2. Changes in laboratory parameters and complaints of healthy human subjects during the study with capsaicin . . 142 9.2.2. Subchronic observations with capsaicin in healthy human subjects . . . . 142 9.2.2.1. Two weeks treatment with capsaicin . . . . . . . . . . . . . . . . . . . . 142 9.2.2.2. Biochemical measurements and complaints in healthy human subjects during two weeks capsaicin treatment . . . . . . 142 9.2.3. Human chronic observations with capsaicinoids . . . . . . . . . . . . . . . . . 143 9.2.4. Preventive effects of capsaicin against the selective and non-selective inhibitory actions produced by nonsteroidal anti-inflammatory drugs on COX-1 and COX-2 enzymes . . . . . . . . . . 143 8

paprika2.qxd

4/15/09

12:17 PM

Page 9

9.2.5. Summary of the observations with capsaicin alone or in combination with selective and non-selective inhibition of COX-1 and COX-2 enzymes by nonsteroidal anti-inflammatory compounds (drugs) in animal experiments and in human observations . . . . . . . . . . . . . . . . 145 10. Nature and characteristics of the innovative drug research. . . . . . . . . . . . . . 147 10.1. Characterization of the innovative drug research . . . . . . . . . . . . . . . . . . . . 147 11. Pharmaceutical industrial research and development . . . . . . . . . . . . . . . . . . 157 11.1. Product design and development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 11.1.1. General goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158 11.2. Chemical-pharmaceutical aspects of the product development . . . . . . . . . 158 11.2.1 Pharmaceutical form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159 11.2.2 Composition and quality of starting materials. . . . . . . . . . . . . . . . . 159 11.2.2.1. Active Pharmaceutical Ingredient (API) of the product . . . . 160 11.2.2.2. Excipients’ quality of PH EUR requirements. . . . . . . . . . 161 11.2.2.3. Coating powder mixture quality . . . . . . . . . . . . . . . . . . . . 161 11.2.3. Packaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 11.3. Formulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 11.3.1. Formulation steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 11.3.2. FP Specification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 11.3.3. Samples for clinical trial . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 11.3.3.1. Preparation of samples for clinical trial . . . . . . . . . . . . . . 163 11.3.3.2. Exposing, stability and packaging of planned final product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 11.4. Plant batch manufacturing and process validation . . . . . . . . . . . . . . . . . . . 163 11.5. Summary of the Chemical-Pharmaceutical development . . . . . . . . . . . . . 164 12. Clinical pharmacological studies with capsaicinoids alone and with combination of capsaicinoids with nonsteroidal anti-inflammatory drugs. . . . 165 12.1. Main aims of clinical pharmacology and its relation to the evidence-based medicine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 12.2. Our special scientific problems in the human clinical pharmacology of capsaicinoids alone and capsaicinoids with together application of aspirin, diclofenac and Naproxen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 12.3. Principal schedules for the human Phase I–II studies with capsaicinoids alone and together with aspririn, diclofenac and Naproxen . . . . . . . . . . . . 168 12.3.1. Preparation of protocols for the human clinical pharmacological studies (including Phase I to IV). . . . . . . . . . . . . . . . . . . . . . . . . . . 168 12.3.1.1. Medical points of the preparation of the study protocols. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 12.3.2. Control of the protocols by the National or Regional Clinical Pharmacological and Ethical Commitees.. . . . . . . . . . . . . . . . . . . . 169 12.3.3. Pharmacokinetic and pharmacodynamic effects of capsaicinoids only. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 12.3.3.1. Human Phase I clinical pharmacological study. . . . . . . . 169 12.3.3.2. Human clinical pharmacological Phase I study with capsaicinoids plus nonsteroidal anti-inflammatory drugs in healthy human subjects . . . . . . . . . . . . . . . . . . . 170

9

paprika2.qxd

4/15/09

12:17 PM

Page 10

12.3.3.2.1. Human clinical pharmacological phase I study with capsaicinoids plus aspirin . . . . . 170 12.3.3.2.2. Human clinical pharmacological Phase I study with capsaicinoids plus diclofenac. . . 170 12.3.3.2.3. Human clinical pharmacological Phase I study with capsaicinoids plus Naproxen . . . 171 12.3.3.3. Human clinical pharmacological Phase II studies in patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 12.3.3.3.1. Human clinical pharmacological Phase II study in patients with thromboembolic diseases (myocardial infarction, stroke, thromboembolic events). . . . . . . . . . . . . . . . . 171 12.3.3.3.2. Human clinical pharmacological Phase II studies in patients with different degenerative locomotor diseases . . . . . . . . . . 171 Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175 Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 1. European Commission. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 1.1 Opinion of the scientific committee on food on capsaicin . . . . . . . . . . . . . . . . 207 1.2. European Herbal Practitioners Association . . . . . . . . . . . . . . . . . . . . . . . . . . 213 1.3. Commission Decision of 18 May 2005 amending Decision 1999/217/EC as regards the register of flavouring substances used in or on foodstuffs (notified under document number C(2005) 1437) (Text with EEA relevance) (2005/389/EC) . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 2. Medical products for human use: Common Technical Document (CTD) . . . 219 2.1. Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 2.2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 2.3. Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220 2.4. Presentation of European Marketing Authorisation Applications . . . . . . . . . 221 2.5. Presentation of Applications in the Mutual Recognition Procedure or Decentralised Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221 2.6. Presentation of Follow-up Measures, Specific Obligations and PSURs . . . . 222 2.7. Reformatting of dossiers of already authorised products . . . . . . . . . . . . . . . . 222 2.8. Presentation of the application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 2.9. Administrative, regional or national information is provided in different Modules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 2.10. Preparing and Organizing the CTD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224 2.11. Pagination and Segregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225 2.12. Information about national administrative requirements . . . . . . . . . . . . . . . 226 2.13. Special guidance for different kinds of applications . . . . . . . . . . . . . . . . . . 226 2.14. Special guidance on herbal medicinal products . . . . . . . . . . . . . . . . . . . . . . 229 2.15. Variation of an ASMF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 2.16. European Certificate of Suitability of monographs of the European Pharmacopoeia (CEP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 2.17. European Community Guidelines on Quality, Safety and Efficacy . . . . . . . 231 About the authors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239

paprika2.qxd

4/15/09

12:17 PM

Page 11

Foreword

The potential role(s) of the scientific research works is (are) extremely well accepted in our days, when the Establishment of European Union celebrated its 50th years anniversary (2007). Consequently the countries of the European Union have chosen a new and common pathway for the economical, social and scientific development in Europe for the forthcoming time period (hopefully for all of the countries and for many centuries). Seven countries (France, West Germany, Italy, Spain, The Netherlands, Luxemburg, Belgium) established the European Economical Committee in Rome, Italy in 1957, when Europe demonstrated clearly the signs of survival over the Second World War to other parts of the World. Later on, this European Economical Committee has incorporated other countries including the countries liberated from the Soviet power. The final aim of these European political and economical changes is to establish an eminent, economically, politically and scientifically stable European Union. Hungary was accepted and entered the European Union by January 1, 2004. Thereafter many assets of our life have changed significantly (including economy, policy, social life, science). We have to try to find the most prominent lines still going on from the life in Hungary, which help us to reach the streams of the European Union. The pharmacological research and industry in Hungary were internationally well accepted by the different countries of Europe, North America (USA, Canada), Asia (Japan, China, India, and Pakistan). The essential and key role of so-called “innovative pharmacological research” has been especially accepted by the Hungarian Research Departments (including the Hungarian Academy of Sciences) and by the Hungarian Government. The different Regional University Science Centers (Debrecen, Budapest, Szeged and Pécs) have been established in 2004 by the Hungarian Government. Our (Pécs) University has received a grant for carrying out the innovative pharmacological research in the time period of 2005–2008 (National Office for Research and Technology “Pázmány Péter Program, RET-II, 08/2005”). The members (altogether 21) of the research are working in the University of Pécs, First Department of Medicine and Pharmaceutical Chemical Institute, Medical Faculty, University of Pécs, Hungary and PannonPharma Pharmaceutical Ltd. (Pécsvárad, Hungary). These researchers never worked together in a common project. So we found ourselves before an extremely new challenge in our common innovative pharmacologi11

paprika2.qxd

4/15/09

12:17 PM

Page 12

cal research, considering the very short time period (from 2005 to 2008) of our grant and our common success to produce new drug combinations of orally applicable capsaicin (capsaicinoids*) alone, capsaicinoids plus nonsteroidal anti-inflammatory compounds (aspirin, diclofenac, Naproxen) for patients suffering from myocardial infarction, pain, degenerative joint diseases, topically applied itraconazole (as one of the antifungal drugs) and etacrinic acid alone or in some combination to be used as an eyedrop preparation for patients suffering from glaucoma. The scale of this innovative pharmacological research is very wide and multidisciplinary. Furthermore, we had a very short time to realize the development of these scientific and industrial aims. We had several different scientific, industrial, marketing problems in the realization of our innovative pharmacological research. We met firstly with the problems of the use of capsaicin as chemical compound used as drug material. Capsaicin can be obtained from the plants, and this compound does not represent a uniform chemical entity (chemically capsaicin contains at least 5 to 6 chemical components (which are similar chemically and act in the same manner). This compound was never used as an orally applicable drug. Furthermore, we had very limited knowledge on the chemical compounds used in the cultivation of plants for extraction of capsaicin (capsaicinoids). We have learned a great deal of scientific matters from the lawyers, authorities, medical, industrial and patent work experts. The main point was focused, in these problems, to obtain the preparate of capsaicin (capsaicinoids) from the different plants. The environmental factors (application of different chemicals) played an essential role in the cultivation of capsaicin-containing plants. Briefly, the capsaicin (capsaicinoids) is (are) of plant origin (but not chemically produced) chemical compound (compounds). Consequently, the acceptance of a chemical compound of plant origin cannot be separated from the applied chemicals to produce larger quantities of the chemical compound (compounds), which may be environmentally harmful. Furthermore, we planned to produce capsaicin-containing drug combinations for oral application. During our innovative pharmacological research, we met a lot of different (medical, industrial, technological, botanical, agriculture, chemical, pharmacological, national and international juridical and patent) problems. All of us worked hard. Our aim was to give a short (but complete) summary of our common works in the years between 2005 and 2008. We hope very much that the results of our scientific endeavour can help the works of those participating in innovative research. Pécs, Hungary, September 2008

The Authors

* Important note: The term of capsaicin is used in the text, however, capsaicinoids of natural origin are used during different studies. Sometimes the term capsaicinoids is used to emphasize their plant origin in industrial research; suitable methods are used in the human clinical pharmacology (phase I–II).

12

paprika2.qxd

4/15/09

12:17 PM

Page 13

Preface

Generally, books and monographs are published to summarize the actually most considerable results of the relevant research topics. We have also published a series of different monographs, important books and congress proceedings up to now. Our studies with capsaicin (capsaicinoids) have been started from the 1980s in animal experiments and from 1997 in healthy human subjects and patients with different gastrointestinal (GI) disorders. The main point of the capsaicin research can be summarized by mentioning that capsaicin modifies significantly the function of the “capsaicin-sensitive afferent nerves” (the effects depending on the doses of applied capsaicin). The application of capsaicin is able to produce gastrointestinal mucosal defense. The gastrointestinal mucosal damage can be produced by different noxious agents, including nonsteroidal antiinflammatory drugs, NSAIDs in animal experiments and humans. The gastrointestinal mucosal defense induced by small doses of capsaicin (against Indomethacin, ethanol) has been proven in healthy human subjects, under conditions of Helsinki Declaration and respecting the rules of the Good Clinical Practice (GCP). The nonsteroidal antiinflammatory drugs (NSAIDs) are widely used in the treatment of patients with acute coronary syndrome (ACS), myocardial infarction, thrombophilia, stroke (aspirin) and with different degenerative diseases of the locomotor system [selective cyclooxygenase (COX) I inhibitors and non-selective COX-1 and COX-2 inhibitors]. These drugs produced different gastrointestinal diseases (mucosal damage, ulceration, bleedings, and gastropathia). The results of animal experiments and human observations have proved that capsaicin (given in small doses) is able to prevent the NSAIDs-induced GI complaints (mucosal damage, ulceration, bleedings, etc.). Different drugs (anticholinergic and antigastrinic drugs, histamine 2-receptor antagonists, proton-pump inhibitors, and scavengers) are widely used in the everyday medical practice to prevent the NSAIDs-induced GI side effects. The stimulation of capsaicin-sensitive nerves, by the application of capsaicin in small doses, represents a new pharmacological approach to prevent NSAIDs induced GI mucosal damage in healthy human subjects and in patients with different GI disorders. The Hungarian National Office for Research and Technology has offered us a grant for doing “innovative drug research in humans”. We submitted our application to produce a new drug (or drug combinations) to be applied in the patients treated with NSAIDs. 13

paprika2.qxd

4/15/09

12:17 PM

Page 14

The participants of this multidisciplinary group – receiving the grant (National Office for Research and Technology “Pázmány Péter Program RET-II, 08/2005”) – came from the Department of the Pharmaceutical Chemistry, First Department of Medicine, Department of Ophthalmology, Medical Faculty, Pécs University, Hungary and from the PannonPharma Pharmaceutical Ltd, Pécsvárad, Hungary (including 21 researchers altogether, among them internist, ophthalmologist, chemists, chemical engineers, biologist, bioenergineers, pharmacologist, horticultural engineer). We have met considerably different problems of medical, chemical, technological, industrial research, national and international laws, and marketing during our research work. We all together started with the “innovative drug research” and we had to learn many new things (results) from the different medical, chemical, pharmaceutical, industrial research during a very short time in 2005, in the pathways of mutually induced group education (including the research, legal, ethical, drug industrial fields; the necessity of the contact to the National Institute of Pharmacy (Országos Gyógyszerészeti Intézet, OGYI); to evaluate successfully the problems given by the National Pharmaceutical Institute in the field of our “innovative drug research”; marketing fields, etc. In our days, the role of “innovative drug research” has been widely accepted not only in Hungary but all over the World. Consequently we had a very short time to find the successful possibilities in the pathways of this research field. We had to learn the possibilities and basic requirements for the international registration of three patents of the produced new drug (or drug combinations) in the year of 2007: 1. Production of a new drug (or drug combinations): small doses of capsaicin (as orally given drug) applied alone or in combination with aspirin, diclofenac and Naproxen; 2. Production of a topically applicable antifungal drug in form of solution and in nail polish; 3. Production of a new drug combination of etachrinic acid with other chemical compounds to be applied in the patients with glaucoma. This book indicates and details the cultural history of spices (capsaicin) in the World; species, taxonomy, chemistry, medical research (in animal experiments and human observations), pharmacokinetic and pharmacodynamic determinations of drug (or drug combinations), preparation of suitable clinical pharmacological protocols, which are used in human studies (phase I–II); the actuality of the related and valid national (Hungarian) and international (European Union, United States, Japan, India, China) laws which give the principal bases of new patents and marketing. This book represents the very effective application of human power of the researchers to the enormous spiritual and physical challenges of our country, the European Union and other parts of the World in the field of introduction of a new drug (or of drug combinations). The research in this field is an extremely hard and interdisciplinary work. We believe that the experiments of our “innovative drug research” can help others, who are participating in these types of the innovative and multidisciplinary research. Pécs, Hungary, September 2008

The Authors

1. General introduction 1.1. General introduction to the interactions of foods (or food components) and drugs to be used in the prevention and treatment of different diseases in patients 1.1.1. Foods and food components The prevention and treatment of the different human diseases is the final form of our medical activities to keep the health conditions in the human beings. Nutrition and pharmacological treatment are also incorporated in this field. Nutrition alone is very important (including the consume of different macro- and micronutrients, trace elements, etc. in terms of their quantities and preparations) representing the basis of sustainance of our life, by supplying the necessary energetical background. The production of foods, establishment of the new foods, their biological and nutritional qualifications, cultivation of plants to be used for food production, industrial background cover a very wide range of natural sciences (physiology, pharmacology, nutrition, agriculture, food industry, medicine) - in terms of prevention and treatment - and of the different social sciences, marketing, etc. (Mozsik, Figler, 2005). The correct evaluation of these questions represents very urgent and important scientific aspects in our personal life, as well as in the World. The spices (chemical compounds of plant origin) have been used to modify the use of the different foods since 9500 years ago in the human culture. These spices are different chemically; they produce different changes in the process of nutritional habits, and on the other hand, are able to produce different physiological processes in healthy human beings and in patients with different diseases. Capsaicin (functionally capsaicinoids) is (are) one famous chemical compound among the spices. The historical and scientific history of capsaicin (capsaicinoids) is especially interesting: 1. capsaicin (capsaicinoids) does (do) not represent an energy supply for the man; 2. capsaicin (capsaicinoids) is (are) able to modify the carbohydrate metabolism (Domotor et al., 2006); 3. the spreading of capsaicin (capsaicinoids) has changed significantly over the World during the last 9500 years (Govindajaran, 1985, 1986a-f; Govindajaran et al., 1977, 1987; Govindajaran, Sathyanarayana 1991); 4. the cultivation of plants containing capsaicin changed significantly from time to time, especially in the last two centuries (Govindajaran, 1985, 1986a-f; Govindajaran et al., 1977, 1987; Govindajaran, Sathyanarayana 1991); 5. the discovery of a very wide scale of capsaicin-induced physiological regulatory mechanisms (Roosterman et al., 2006);

6. the discovery of the specific stimulatory effect of capsaicin on the afferent sensory nerves ("capsaicin sensitive afferent nerves") (Jancso-Gabor, 1959; Jancso et al., 1968, 1985; 1987; Jancso-Gabor, Szolcsanyi 1972; Szolcsanyi, 1977, 1982, 1983a,b, 1984a,b, 1985, 1990a,b, 1993, 1996, 2004; Buck et al., 1982; Monseerenusorn et al., 1982; Fitzgerald, 1983; Chahl et al., 1984; Hori, 1984; Russel, Burchiel, 1984; Holzer, Sametz, 1986; Buck, Burks, 1986; Maggi, Meli, 1988; Holzer, 1988, 1990, 1991a,b, 1992a,b, 1998; Maggi et al., 1989; Holzer, Lippe, 1988; Holzer et al., 1989; Maggi 1995; Jancso-Gabor et al., 1997); 7. The application of capsaicin produces different physiological (pharmacological) regulatory steps (Szolcsanyi, 1990a,b, 1993, 1996, 2004); 8. The application of capsaicin offered a new research tool to approach the different biological regulatory mechanisms in animals and human beings (Mozsik et al., 2007b) and in patients with different diseases (Domotor et al., 2005, 2006, 2007).

1.1.2. Drugs The research and production of different drugs are the keystones both in the drug industry and in the human medical treatment. The progress of medical treatments was based on two main research lines: 1. The evaluation of the results of the "classical and historical" medical practice dominantly started from Asia (from the Chinese medicine). The application of these medicines was based on the efficacy of the medical treatment carried out; 2. The planning of chemically, pharmacologically, biologically active compound(s) for production of the new drugs to be used in the treatment of patients with different diseases (that has been dominating in the last decades). Our attention is focused on the nonsteroidal anti-inflammatory drugs (NSAIDs). The most famous compound of this drug family was produced by the Pharmaceutical Company of Bayer (Germany) in the 1880s (Aspirin). This compound is very widely used in the everyday medical treatment (as analgetic, antipyretic agent, and platelet aggregation inhibitor), however, its most important indication is in the prevention of myocardial infarction, stroke, thromboembolic events and prevention of different colorectal malignancies. The main actions of NSAIDs are the following: 1. inhibitory action on the platelet aggregation; 2. production of gastric mucosal damage chemically (in the pathway of inhibition of dissociation of weak acid in the presence of a strong acid); 3. inhibition of the bicarbonate secretion produced by the pancreas; 4. inhibition of the prostaglandin (PG) synthesis and 5. modification of the blood flow in the gastrointestinal tract by the inhibition of the PG synthesis. The number of patients suffering from myocardial infarction or from different degenerative locomotor diseases is very large. The application of aspirin is generally and internationally accepted in the prevention of the myocardial reinfarction (given in doses of 100 mg/day or in large doses of 300 mg/day in so-called Aspirin 16

insensitive patients). The administration of aspirin means a long-lasting treatment in all patients with cardiological and thromboembolic events. Patients suffering from different degenerative diseases of the locomotor system are treated with one of the compounds of NSAIDs. The actions and pharmacokinetic behaviors of the NSAIDs can be modified by the application of different foods (fats, aperitive compounds, aldehyde and keto-sugars, dietary fibres, etc.).

1.1.3. Levels of the drug-food interactions in healthy human beings and in patients with different diseases There is a specific field of the human nutrition (including that of both healthy subjects and patients) considering that we use generally several macronutrients (proteins, carbohydrates, fats, etc.) together. There is no question that the absorption, metabolism, excretion and utilization of the macronutrients taken alone differ significantly from those when they are consumed together. Furthermore, the complementation of macronutrients with each other is a basic nutritional question (e.g. complementation of cereals with beans). Clinicians were taught that these problems (related to the application form of macronutrients) significantly differ in healthy subjects and in patients with different diseases. The different diseases are able to modify the digestion (all of the physiological processes which are able to prepare the foods in a suitable form for absorption), absorption and post-absorptive phases (transfer of the drugs from the small intestine to the target organ, excretion by the different pathways, utilization, including their anabolism and catabolism). The analysis of these medical questions requires a thorough medical knowledge. The detailed analysis of digestion, absorption and utilization of the different foods covers the main topics of the medical sciences (such as gastroenterology, nutrition, medical pathophysiology, organspecific topics of medicine as hepatology, nephrology, hematology, as well as different other subjects). Different drugs are used in the medical practice to modify the different physiological and pathological processes in healthy human subjects and in patients. Drug application to healthy subjects causes the transitory modification of the different functions in healthy human subjects (pain killers, coffee, laxative agents, many other drugs). Drug application in the patients with different diseases originally aims at decreasing the complaints of the patients. We have to know that these drugs modify the consumed foods (their absorption, digestion, utilization, transportation in the human body) (Table 1). A few examples will be given to explain these questions: 1. The prokinetic drugs decrease the transit time of the different foods in the GI tract of healthy humans, just like diarrhea (as disease). Consequently the time for the absorption of different foods is short; 2. Vitamin B is absorbed together with its intrinsic factor from the distal part of the small intestine. If the patients have no intrinsic factor, then the B absorption becomes insufficient, however, in patients with ileitis terminalis B absorption l 2

) 2

1 2

17

T a b l e I . T h e scientific a p p r o a c h of t h e m a i n q u e s t i o n s o f c l i n i c a l nutrition a n d dietetics a p p e a r s as f o l l o w s ( M o z s i k , Figler, 2 0 0 5 ) * 1.

2.

F o o d a n d f o o d i n t e r a c t i o n s at t h e l e v e l s of 1.1.

pre-absorptive p h a s e (digestion),

1.2.

absorptive phase,

1.3.

post-absorptive p h a s e .

T h e d r u g a n d f o o d a c t i o n s w i l l m a n i f e s t at t h e l e v e l s of 2.1.

pre-absorptive l e v e l (digestion),

2.2.

absorptive level,

2.3.

post-absorptive l e v e l .

* Mozsik, Gy., Figler, M. (2005): Metabolic Ward in Human Clinical Nutrition and Dietetics. Research Signpost, Kerala (with permission)

becomes insufficient (partly due to the extremely decreased transit time) without any absence of vitamin B and its intrinsic factor; 3. Most of the macronutrients (carbohydrates, proteins) are absorbed by an active transport process from the small intestine. Strophantin g (and other digitalis compounds) specifically inhibits the active transport processes in the human gastrointestinal tract. In non-tropical sprue (gluten sensitive enteropathy) the patients have no brush border in the small intestine and consequently the mentioned food components will not be absorbed from the GI tract (without or with application of any kind of digitalis compounds); 4. Finally, the application of different drugs (nonsteroidal anti-inflammatory compounds) can modify the transport of food by the modification of the linkage of nutritional compounds and hormones to albumin, the excretion (e.g. diuretics) and the function of the target organ (positive and negative enzyme inductors). Moreover, in patients with hypalbuminemia (when the drugs are linked in smaller quantities to albumin) we can produce easily drug intoxication in the patients during different drug treatments. These examples from the everyday medical practice clearly indicate the extreme complexity of the correlation between nutrition and drug therapy in healthy human subjects and in patients with different diseases. 1 2

1.1.4. A short historic background of the interaction between the effects of capsaicin (capsaicinoids) and NSAIDs in animal experiments and healthy human subjects The mechanism of action produced by capsaicin (capsaicinoids) is very wide both in animal experiments and human beings (including the studies with isolated and cultivated cells, in vitro and in vivo studies, and whole living individuals) (Roosterman et al., 2006). 18

Our attention is focused on the mechanisms of action of NSAIDs and capsaicin both in animals and human beings. There is a vast literature dealing with the gastrointestinal mucosal damage produced by NSAIDs. The mechanisms of the NSAIDs-induced gastrointestinal mucosal damage can be detected at the levels of: 1. physico-chemical laws (Davenport, 1970, 1973; Mozsik et al., 1981); 2. prostaglandin synthesis (many authors worked in this field); 3. cellular energy systems (Mozsik et al., 1981; Mozsik, 2006); 4. oxygen free radicals (Mozsik, 2006). Szolcsanyi and Bartho (1981) were the first who proved that capsaicin prevents the gastric mucosal damage, when it is given in a small dose. In contrast, the gastric mucosal damage is enhanced by the application of higher doses of capsaicin. These results clearly demonstrated that the effect of capsaicin on the gastrointestinal tract depends on its doses applied in the experiments. After this original observation many researchers demonstrated the gastrointestinal preventive effect of capsaicin in animal experiments (Holzer, 1988, 1990, 1991a,b, 1992a,b, 1998; Holzer etal., 1989). A very systematic research work has been carried out by our team since 1980. Its main results and critical overview were summarized in a book (Mozsik et al., 1997a). The final conclusion of this book was that the application of capsaicin in the different animal experiments offers a new research tool for understanding the different mechanisms existing in the gastrointestinal tract. Because one of us (Gy.M.) is internist, nutritional expert and clinical pharmacologist, our attention turned toward the human physiological, pharmacological aspects of gastroenterology, the gastrointestinal tract and the mechanisms of actions of capsaicin related to other fields of medicine. The human clinical nutritional studies with capsaicin have been carried out from 1997, according to the Good Clinical Practice (GCP), respecting the Helsinski Declaration, and by the permission of the Regional Ethical Committee of Pecs University, Hungary. One of the main results of these studies was: Capsaicin (capsaicinoids) prevents (prevent) the Indomethacin (which is a nonselective cyclooxygenase enzyme inhibitor)-induced gastrointestinal side effect (microbleeding), however, it prevents the topically applied ethanol-induced decrease of gastric transmucosal potential difference (GTPD), and enhances the gastric transmucosal potential difference (GTPD) in healthy human subjects in association with gastric inhibitory actions on the gastric basal acid output (BAO) (Mozsik et al., 1999, 2004, 2005a, 2007b). These results offered a new possibility to plan and to create a new drug (or drug combinations), in which the gastrointestinal mucosal damaging NSAIDs are combined with the gastroprotective capsaicin in a suitable concentration and formulation.

19

1.1.5. Goals of the drug (drug-combination) production The NSAIDs are widely used in the everyday medical practice (prevention of thromboembolic episodes, myocardial reinfarction, stroke and colorectal cancers). These compounds act at the levels of enzymes of COX-1 and COX-2 by the modification of functions of these enzymes. The functions of COX-1 and COX-2 are involved in many therapeutic steps, new pathological events in the development of different diseases (myocardial infarct, damage in the gastrointestinal tract, development of different gastrointestinal malignancies, drugs-induced gastrointestinal injuries, etc.). The further (present) drug production research was based on our earlier obtained research works in the everyday clinical (cardiological, gastrointestinal, oncological) practice. The production of a new drug (or new drug combination) is a very hard and complicated work. This research is extremely well regulated internationally, covering the chemistry, animal experiments, stability studies, genotoxicology, metabolism, pharmacokinetic observations in animals and in human beings. The authors of this book participated in the establishments of clinical pharmacology (in the 1960s), clinical nutrition (in 1977) in the production of many drugs. These investigators work in the basic pharmaceutical research as well as in clinical nutrition, clinical pharmacology and medicine. This research represented a new challenge for all of us. Because this so-called innovative drug research does not represent a "typical research" pathway to us (independently all of us participated actively in the classical medical and chemical research), we decided that we try to give the necessary information of this "innovative drug research" for those, who want to participate in this process. In the course of this process we have gained knowledge considering national and international new scientific information, organization of the "innovative research team" to estimate our national and international possibilities including our limits, preparation of patents, preparation of the preclinical dossier, organization of human phase I—II trials, medical problems, marketing, etc.).

20

2. Capsaicin (capsaicinoids) is (are) a famous family of the spices

2 . 1 . Cultural background of spices Since the time man started gathering food, the addition of small amounts of some plant materials may have been used for the powerful impact they had on the appearance or eating quality. Initially, these additions were valued for masking the off-flavor of stored, decomposing foods; later on, in medieval Europe, they were imported as rural spices for their capacity to slow down deterioration during storage. Gradually, when definitive ethnic cuisines developed, the individual spices came to be valued in the modern sense for their real contribution, which is to flavor generally insipid meat and cereal foods and to make the food more acceptable and preferred. Spices have become indispensable in the modern kitchens of individual homes, institutions, and the food manufacturing industries. They provide individually to the dishes and distinguishing gourmet foods. In the countries of their origin and growth, the Hindustan, the Spice Island, and China, the highly systemized use of spices and other aromatic plants in foods, for increased acceptance and physiological effects of the extracts, has been known through ancient Vedic texts and the Ayurvedic text of Susrata and Caraca. For several centuries Before Christ (B.C.) spices, along with silk, gold, and precious stones, were principal articles of trade between the East and West. As early as 1600 B.C., the Chinese brought spices along the Chinese coast and overland along Tibet to Kashgar. The Arabs then took the spices west to Baghdad, Alexandria, and Persian Gulf ports. These were hazardous journeys, and great mystery surrounded the origin, collection and transport such that the values of the spices were very great. Later, the spice route shifted to sea routes (and included products besides those of Gulf ports) and then to the merchants in Alexandria and Baghdad. Knowledge of spices and their uses reached Egypt and spread to the Mediterranean region. Hippocrates, the father of modern medicine, and Theophrastus, the Greek philosopher and botanist, wrote treatises on medicinal plants that included many spices. During the first few centuries A.D., many cities (Alexandria, Constantinople, Basra and Venice) underwent violent changes as they became focal points of the spice trade. The values of trade in pepper and a few other spices were so great to the economy of the kingdoms in Europe that kings sent costly expeditions to spice-growing countries. In the late 15th century, Marco Polo of Venice, Vasco da Gama and Cabral of Portugal, and later Columbus and Cortes from Spain, undertook hazardous voyages 21

to establish sea routes to trading ports in the spice-producing countries of Asia. The trading became so intense that there were wars between Portugal, The Netherlands and England, while the local populations in Asia were forcibly enslaved and crippling taxes were levied. The entry of U.S.A. into the world of spices broke the monopoly of the European nations and the important trading centers shifted to Singapore and Calcutta in the East, and New York, London and Hamburg in West. The colonization of distant lands and the internationalization of the spice trade also had desirable changes, world trade in spices was increased and spices were introduced from one region to another. Black pepper, cardamom, ginger and turmeric from Asia were introduced into South America and Africa, and Capsicum (red pepper), vanilla and other spices were introduced from the New World of Central and South America to the Old World of Europe, Asia and Africa.

2.2. History of spices Among the spices, capsicums are the most colorful in appearance, and are important in terms of their history, antiquity and influence on many cuisines of the world. During his first expedition, Columbus (1492 to 1493) observed that the natives of the New World used a colorful red fruit calls aji or axi with most of their foods. This additive was found to be much stronger than the black pepper of Asia (Piper nigrum L.) in search of which he had undertaken this expedition. He took samples of this fruit back to Spain and named it "red pepper". De Cuneo, who accompanied Columbus on the second voyage to the New World in 1495, made the more definitive observations that "rose-like bushes have fruits as long as cinnamon, full of small grains, as biting as pepper; those Caribes and Indians eat that fruit like we eat apples". Chanca, the physician with the expedition, observed that the natives used this food additive both as a condiment and in medicine. Other travelers, who extensively traveled in Spanish America in the 16th and 17th centuries, described the popular use of Uchu, a colorful fiery pod of a plant, in the food of Peruvian Indians. Since early times, the internal use of a similar fruit as a cure for cramps and diarrhea by Mayan Indians of northern Guatemala has been recorded. The highly irritating smoke from the burning dried fruit is reported to have been used by the native Indians against Spanish invaders. For many years (centuries) prior to the Spanish occupation, the native Indians had been cultivating this piquant food additive. There was a considerable variation in color, shapes and sizes in the different regions. The great variety of capsicums used by the native tribes was thus known by different names, as mentioned earlier. Chilli or Chili, a name now commonly used in Asia and Africa, is said to have come from the Nahuatl dialect of Mexico and Central America. A wealth of original material thus became available and is still available in these areas for taxonomic and developmental work.

22

2.2.1. Terminology and modern history of Capsicum The term "chilli" is a rather confusing terminology: "chilli", "aji", "paprika", "chile", "chile" and Capsicum are all used frequently and interchangeably for "chilli pepper" under the genus of Capsicum, which belongs to a dicotyledonous group of flowering plants. A particular species of Capsicum is called "chili pepper" in parts of Mexico, southwestern United States and parts of Central America. To make matters still more confusing, a "sweet bell pepper" is often referred to as "Capsicum pepper", whereas to refer to the term "chilli", "hot pepper" is used. The term "bell pepper" is used to refer to a non-pungent, chunky, sweet chilli type, whereas "chilli pepper" generally refers to a pungent chilli variety. Red peppers have been familiar to all Spanish South Americans by the Arawakan name "aji" and by the Nahuatlan name "chilli" in Mexico and Central America. The genus Capsicum, which is commonly known as "red chile", "chilli pepper", "hot red pepper", "tabasco", "paprika" and "cayenne", belongs to the family Solanaceae (Night shade family) that includes tomato, potato, petunias and tobacco (Hawkes et al., 1979; Macrae, 1993). According to some references, the popular name "chili" or "chilli" originates from the hot pepper specially cultivated in the South American country, Chile. However, the name "chilli" seems to have nothing to do with the country name, on the contrary, it is believed to have originated from a district of Central America (De, 1992, 1993, 1994, 2000). One of the very first sources of life among the Inca, the "Commentaries Reales" by Garcilaso de la Vega, "El Inca", in 1609, mentions the common or even daily use of "chillies". According to de la Vega, there are three different kinds of chilli, two of which can be identified as "aji" and "rocoto", while the third is only insufficiently described. Even though the chilli is referred to by different names within the same country, and even in different states or provinces, the botanical name of chilli is the Latin name Capsicum. The word comes from a Greek based derivate of Latin "Kapto" meaning "to bite". The word "chili" is a variation of "cil" derivated from the Nahuatl (Aztec) dialect, which referred to plants now known as Capsicum, whereas "aji" is a variation of "axi" from the Arawak dialect of the Caribbean. This brings us up to the point of the correct way to spell "chile" (Domencini, 1983). The "e" ending in chile is the authentic Hispanic spelling of the word, whereas English linguists have changed the "e" to an "i". From the Nahuatl dialect of the Aztec language, the name "Chilrepin" has been derived. This was the name given to the earliest known word chile, the combination resulting in "Flea Chile", which is believed to allude to the sharp biting taste of the chilli pepper. Down the ages the original name has been slowly reformulated as "chile + reprintl" to "chilecping" to "chilrepin" to "chilepiquin", the latter two names being frequently interchangeable. The version used depends upon the source of information. However, Capsicum annuum var. aviculare is the modern scientific name of this earliest known variety (Macrae, 1993). However, a multilinguistic nation (like India) represents a unique case, where the same specimen Capsicum annuum L. is referred to by different names in different parts of the nation. In original Sanskrit the plant is known as "Mairichi phalam 23

and "Bruchi". However, in modern Indian languages it is known by different names, and even by more than one within the same language as given in Table 2. T a b l e 2. S u m m a r y of C a p s i c u m s in t h e l a n g u a g e s of different n a t i o n s * Language

Common name

Pharm

Fructus C a p s i c i a c e r

Arabic

Felfel, B i s b a s , F u l f u l h a l u

Amharic

Mit'mita, Berbere

Assam i

Joloka

Bulgarian

C h e r v e n piper

Bengali

Lanka, M o r i c h

Burmese

N g a yut thee, Nil thee

Chinese

Lup-Chew

Danish

Chili

Dutch

S p a a n s e peper, C a y e n n e p e p e r

English

C a y e n n e pepper, R e d pepper, C h i l l i , Chili

Estonian

Kibe paprika

Finnish

Chilipippuri

French

P o i v r e r o u g e , P i m e n t e n r a g e , P i m e n t fort, P i m e n t - a i s e a u , P o i v r e d e C a y e n n e

German

R o t e r Pfeffer, C a y e n n e - P f e f f e r , Chili-Pfeffer, B e i f t b e e r e

Gujarati

Lai m a r c h a (red), Lila m a r c h a (green)

Hebrew

Pilpel adorn

Hindi

Lai m i r c h (red), H a r i m i r c h (green)

Hungarian

C s i l i p a p r i k a , Igen eros a p r o , C a y e n n e bors, C a y e n n i b o r s , M a c s k a p o c s paprika, Aranybors, Ordogbors, Chilipaprika

Icelandic

Chilipipar, Cayennepipar

Indonesian

Lombok, Cabe, Cabai

Italian

P e p e r o n e , D i a v o l e t t o , P e p e r o n c i n o , P e p e di C a i e n n e , P e p e rosso p i c a n t e

Japanese

Togarashi

Kannada

Menashinakay

Laotian

M a k p h e t kunsi

Malay

Lada merah

Malayalam

Mulagu

Marathi

Lai m i r c h y a (red), H i r v y a m i r c h y a (green)

Oriya

Lankamaricha

Pashto

Murgh

Portuguese

P i m e n t a o , Piri-piri, P i m e n t a d e c a i e n a

Punjabi

Lal-mircha

Russian

Perets krasni

Sanskrit

Marichiphala, Ujjvala

Singhalese

Rathu miris, Gasmiris

Spanish

C h i l e , G u i n d i l l a , C a y e n a inglesa, P i m i e n t a d e C a y e n a , P i m i e n t a p i c a n t e , Ajf

24

Table 2. Cont'd Language

Common name

Swahili

Pilipili h o h o

Swedish

Chilipeppar

Tagalog

S i l i n g l a b y o , Sili

Tamil

Mulagu

Telegu

Mirapakya

Thai

Pisi h u i , Prik k h e e , Prik

Tibetan

S i p e n m a r p o , Si p a n d m a r p o

Turkish

A c i kirmizi biber

Urdu

Lalmarach

Vietnamese

Ot

* After the paper of Basu, S. K., De, A. K. (2003): Capsicum: Historical and Botanical Perspectives. Taylor and Francis Ltd., London (with permission)

The chilli pepper is one of the very old domesticated plants of Middle America. In the Valle de Tehuacan (Puebla) which is one of the best documented examples of an early settlement in Mesoamerica, archeological evidence for the consumption of chilli peppers dates back to the seventh millennium B.C., long before the cultivation of maize and beans. The early findings of peppers - in coprolites and charred remains were probably harvested in the wild. However, domesticated chillies similar to their modern varieties in both size and shape can be found from the fifth millennium B.C. onwards. In pre-Columbian Mexico, chilli was one of the preferred tributes which dependent city-states had to deliver to their hegemonial powers. The paying of tribute also in the form of chilli peppers was later continued by the new Castilian rules. Capsicum has been known since the beginning of civilization in the Western Hemisphere. It has been a part of the human diet since about 7500 B.C. (MacNeish, 1964). It was the ancient ancestors of the native people, who took the wild chilli piquin and selected the values types known today. Heiser (1967) stated that between 5200 and 3400 B.C., the native Americans were growing chilli plants. This places chilli among the oldest cultivated crops of the Americas. Seeds found in early dwellings indicate that the natives were enjoying the peppers in 7000 B.C., along with potatoes in the Andes. In Mexico dry pepper fruits and seeds were received from 9500-year-old burials in Tamaulipas and Tehuacan. Domestication might have taken place 10,000 to 12,000 years ago. Christopher Columbus is believed to be the first European to discover chilli during one of his legendary travels to America around 1493. He was looking for an alternative source of black pepper which at that time was the favorite spice of Europe. What he discovered was a small fiery pod that for centuries provided seasoning for native Americans, the hot chilli pepper. It has to be emphasized that chilli or capsicum is not related to the Piper genus, which contains Piper nigrum L. of the family Piperaceae, the source of the black and white pepper. Within a century after its discovery hot chilli pepper attained a worldwide distribution. According to some 25

other sources, the American origin of Capsicum was first reported in 1494 by Chanca, a physician who accompanied Columbus in his second voyage to the West Indies (Macrae, 1993). Chilli peppers grow as a perennial shrub in suitable climatic conditions. The genus usually represents glabrous, perennial, woody subshrubs, some tending to be vines, rarely herbs, which are native to Central and South America, live for a decade or more in the tropical conditions of their natural habitat, but are mostly cultivated as annuals elsewhere. Chilli is native to the Western Hemisphere and probably evolved from an ancestor from the area of Bolivia and Peru. The first chillies consumed were probably collected from wild plants. Prehistoric Americans took the wild chilli "Piquin" and selected it for the various pod-types known today. However, domesticated chilli was apparently not grown prehistorically in New Mexico (Macrae, 1993). In fact, it is not known exactly when chillies were introduced into New Mexico. Chillies may have been used by the indigenous peoples as a medicine, a practice common among the Mayans. By the time the Spanish arrived in Mexico, Aztec plant breeders had already dozens of varieties. Undoubtedly, these chillies were the precursors to the large number of varieties found in Mexico today. Whether chillies were traded and used in New Mexican pueblos is still not clear (Macrae, 1993). Capsicum species have been thought to be of Central American origin, but one species has been reported to be introduced in Europe in the fifteenth century. By the middle of the seventeenth century, Capsicum was cultivated throughout Southern and Middle Europe as a spice and/or medicinal drug. One species was introduced into Japan and about five species were introduced into India, of which Capsicum annuum L. and C. frutescens L. were cultivated on a large scale (The Wealth of India, 1992). In commerce the description given applies to various African commercial varieties and these and the Japanese variety are sold in the United Kingdom as chillies, while the larger but less pungent Bombay and Natal fruits are sold as Capsicums. Very large Capsicum fruits that resemble tomatoes in texture and are practically nonpungent are widely grown in Southern Europe as vegetables (Evans, 1996). Records of the prehistoric Capsicum species around burial sites in Peru indicate that the original home of the chillies may be tropical South America. These seem to have been diffused from there to Mexico, or an independent origin in the latter country, where a great diversity of the genus is found. While Capsicum annuum has not been recorded in the wild state and Capsicum frutescens doubtfully so, they have now naturalized in the tropics of many countries and are easily disseminated by birds (The Wealth of India, 1992). The plant was introduced into Spain by Columbus, from where it spread widely. Subsequently, the prolonged viability, easy germination, and easy transportation assisted its spread all across the globe. The original distributions of this species appear to have been extending from the South of Mexico into Columbia (The Wealth of India, 1992). "Ginnie Pepper" was well known in England in 1597 and was grown by Gerarde (Evans, 1996). The Portuguese introduced chilli into India. Chilli is used as a condiment in large quantities in India, Africa and tropical America, where the fruit develops greater pungency than in colder regions. It has now, however, become a popular condiment 26

all over the World. The long, thin fruits constitute the source of dry chillies used for commerce. The wide popularity of chilli and its extensive cultivation are due to its being a short duration crop and its ease of cultivation under a wide range of climatic and adaptive conditions, particularly in comparison with black pepper {Pepper nigrum L.). The cultivation and utility of both Capsicum spp. are similar, except for local peculiarities (The Wealth of India, 1992). Capsicums are mentioned in a classic text of the Tibetan medical tradition, the "Blue Beryll": "Capsicum (tsi-tra-ka) increases digestive warmth of the stomach, and is the supreme medication for the alleviation of edema, hemorrhoids, animalcules, leprosy and wind". Another passage tells us "bad-kan-nad-sel tsje-'phel tsi-tra-ka mar sbrang sbyar - to alleviate diseases of phlegm and prolong the lifespan: [use] Capsicum mixed with butter and honey". Interestingly, the spice translated as cayenne pepper has another name, Yer-ma, and is generally used to treat wind disorders. In pre-Hispanic medicine, chilli peppers were used to treat a host of conditions, often in combination with other plant and mineral substances. For example, chilli was used to treat diseases of the gastrointestinal tract (infections, diarrhoea), in addition to tooth pain, cough and lack of appetite. It was popular as an aphrodisiac, as well. For that reason, the Spanish Padre Acosta, traveling through New Spain in the sixteenth century warned that high consumption of the chilli peppers would be detrimental to the "soul's health" because it "promoted sensuality" (Acosta, 1985). Chilli peppers had other uses as draconic punishment in children's education, and even in warfare - the enemy was driven out of his fortification by the employing the acrid smoke of smoldering chillies. This tactic was employed in pre-Columbian times, but also during the Mexican revolution at the beginning of the twentieth century.

2.2.2. History of Capsicum in Hungary Spain cultivated the pimientos, valued for the brilliant red color, characteristic aroma, and mild pungency. These are more and more being produced as an industry with close state control, integrating the steps of cultivation, processing, quality control and marketing. In Europe, Hungary and the Balkan countries have been producing the carefully selected cultivars of paprika since the 18th century and possibly still earlier. Hungary and the Balkan countries have been the traditional producers of highly valued burgundy red, elongated conical, fleshy smooth erect fruit of paprika for its color and flower. The main areas of cultivation in Hungary are Szeged and Kalocsa alongside the Danube (Govindarajan, 1985). The first written records of the appearance of paprika in Hungary are from the middle of the sixteenth century. Paprika was grown as a rarity in the garden of Margit Szechy, the step-mother of the distinguished general Miklos Zrinyi. It can be assumed from the information of the next two to three centuries that Capsicum was cultivated only to be used as a condiment. The cultivation of the sweet Capsicum known today began at the end of nineteenth century. Bulgarians were the first growers who cultivated Capsicum in the southern part of the country (Szentes and surroundings) (Somogyi et al., 2003). 27

At the beginning of the twentieth century, production of sweet Capsicum spread to other parts of Hungary. It is interesting to note that the sweet Capsicum growing regions are not the same as the traditional paprika producing areas. Important sweet Capsicum areas were in the middle of the country (Boldog, Nagykoros, Cegled), and in the south-southeastern part (Gyula, Baja, Bogyiszlo). But today this typical vegetable is cultivated all over the country. The production of condiment paprika in Hungary is highly labor-intensive. It became a significant crop at the end of the nineteenth century, although reports of its cultivation exist from the sixteenth century in Szeged, where the crop was mentioned as one of the crops grown. It was introduced from Turkey by monks, who excelled in healing, and was used as an effective medicine against malaria. Looking at the history of Hungarian paprika production we can distinguish several classical periods: - Until the middle of the nineteenth century feudalistic family self-sufficiency and the beginning of the production for the market; - Until the beginning of the First World War paprika production is market-oriented based on free competition; - From the end of the First World War until the end of Second World War a stateregulated production order was characteristic; - Paprika milling was under state monopoly for the period of 1940-1990; - In the last decade of the twentieth century again free market: production and processing are based on competition. Spice made of paprika is known as "Hungaricum" worldwide, and is an essential element of the Hungarian cuisine. Until the turn of the nineteenth century it was known in public life mainly as medicine. Paprika (Capsicum annuum L.) originated from South America and came to Europe - probably first to Spain in 1493, after the discovery of the American continent (Pickersgill, 1986, 1989). From there it came to Hungary across the Balkan through Turkish growers. The first paprika plants were planted at the end of the 1500s. At first paprika was considered as an ornamental plant and was grown for culinary usage at the beginning of the seventeenth century. The plant later enjoyed tremendous popularity around the time of Napoleon (Somos, 1981). The book containing its first detailed description was written by Csapo (1775). According to this book it is grown in vegetable gardens and the long red fruit is dried and crushed to a powder. Veszelszky (1798) mentioned around that time that the farmers of Fot, Palota and Dunakeszi grew paprika. The first cultivation trials were conducted at the botanical garden of the University of Pest in 1788. Since that time different Capsicum varieties were found in the "Index seminum" of the botanical garden (Augustin, 1907). In letters that Count Hoffmansegg sent his wife about his journey in Hungary, he mentioned "here I really liked a Hungarian dish, meat with paprika. I must be very healthy" (Balint, 1962). Augustin, a German traveler did not talk so nicely about the Hungarian paprika in his book, "Die Ungarn wie sie sind" (1831). He called paprika "Diablische Paprika Briihe". He wrote that for people who are not used to it, the effect on the palate is like embers or even worse (see for details Somogyi et al., 2003). 28

3. Botanical taxonomy

Capsicum is a genus of the family Solanaceae and is closely related to another genus, Solanum, which covers many economically important plants such as the potato {Solarium tuberosum L.) eggplant {S. melongene L.) tomato (Lycopersieon esculentum Mill.), and tobacco (Nicotiana tobacum L.). Some 20 to 30 species of Capsicum are reported to have their origin in the New World covering the tropical area of Northern South America, Central America, Mexico, and the islands in that region. Many of these occur in the wild as natural growth in areas of undisturbed vegetation, though their fruits were collected and distributed throughout the marketplace. Exploration and plant collection throughout the New World had given us a general but false impression of specification in the genus. Humans selected several taxa and in moving them toward domestication selected the same morphological shapes, size and colors in at least three distinct species. The early explorations in Latin America were designed to sample the flora of a particular region. Thus, any collection of Capsicum was a matter of chance and usually yielded a very limited sample of pepper from that area. Only with the advent of collecting trips designed to investigate a particular taxon did the range of variation with species begin to be understood. One needs only to borrow specimens from the international network of herbaria to appreciate what a limited sample exists for most taxa, particularly for collections made prior to 1950. The domesticated Capsicum pubescent, e.g., that is wide-spread in the mid-elevation Andes from Columbia to Bolivia, is barely represented in the herbaticum collection of the world. The taxonomy of Capsicum represents a very complex approach to different species. Many classifications were published earlier (see Govindarajan, 1985; Basu, De, 2003). In 1957 Smith and Heiser recognized five species only, which gives the "Big Five" species of Capsicum. Further recent works by Eshbaugh, Pickersgill and Hunziker identified 22 other wild species (Macrae, 1993). The wild gene pool, tightly linked to the domesticated, is designated Capsicum baccatum var. baccatum and is most common in Bolivia, Brazil, Chile and Argentina. Its flowers are solitary at each node. The pedicels are erected or declining at anthesis. Corollas are white or greenish-white, with diffuse yellow spots at the base of corolla lobes or either side of middle-vein (the flower is white with yellowish spots, anthers are white but turn brownish-yellow with age): the corolla lobes usually are 29

slightly revolving. The calyx of mature fruit is without annular constriction at its junction with the pedicel (though sometimes irregularly wrinkled), veins prolonged into prominent teeth. The fruit flesh is firm. Seeds are straw-colored. Chromosome number is 2n=24, with one pair of acrocentric chromosomes (Escabeche, Peru). The following five major species are morphologically definable: Capsicum annuum var. annuum L., Capsicum frutescences L., Capsicum chinense Jacq, Capsicum baccatum var. pendulum wild, Capsicum pubescent Riuz and Pav (Table 3) T a b l e 3 . T h e m o r p h o l o g i c a l i d e n t i f i c a t i o n of t h e f i v e m a j o r s p e c i e s * Species

Flower color

Number flw/node

Seed color

Calyx constriction

C.

annuum

white

1

tan

absent

C.

frutescens

greenish

2-5

tan

absent

C.

Chinese

white/greenish

2-5

tan

present

white with

1-2

tan

absent

1-2

black

absent

C.

baccatum

y e l l o w spot C.

pubescens

purple

* After the paper Basu S.K., De, A.K. (2003): Capsicum: Historical and Botanical Perspectives Taylor and Francis Ltd., London (with permission)

The haploid chromosomal count of the cultivated and wild species is 12. There are wide variations among and within the species, whether wild or cultivated, as no karyotype is characteristic for any single species and certain characteristics are observed among the majority of the members. Natural polyploidy is reported in the case of Capsicum, although a spontaneous tetraploidy has also been reported in an intravarietal cross. Induced polyploidy with colchicine has also been reported where the induced polyploid exhibits a high vitamin C content profile. Diploids showing mitotic abnormalities and irregulaties have also been reported (The Wealth of India, 1992). Generally, there appears to be a well-developed sterility barrier between cultivated species. It is impossible to cross Capsicum pubescens with other species. Several crosses between Capsicum annuum, Capsicum frutescens and Capsicum baccatum var. pendulum have produced a few Fl hybrids, but are mostly highly sterile. In the case of favorable crosses, the success or failure depends on the direction of the cross. Reciprocal differences were observed in the case of fertility. The results of hybridization also differed according to the parental cultivars. Viable seeds have easily been produced from Capsicum annuum X Capsicum chinense and Capsicum frutescens X Capsicum pendulum. The crosses Capsicum annuum X Capsicum frutescens and Capsicum frutescens X Capsicum chinense have yielded a few viable F l , F2 and bud cross seeds (The Wealth of India, 1992) Pronounced differences have been found in the capsaicinoid composition of the individual species, and the chemotaxonomic basis for identification is in line with the earlier classification based on floral morphology and can therefore be used as an 30

additional method. The key for the identification of the individual Capsicum species is given based on the limits of the total and the three individual capsaicinoids and their properties in Table 4. T a b l e 4 . A c h e m o t a x o n o m i c k e y t o t h e i d e n t i f i c a t i o n of c u l t i v a t e d C a p s i c u m s * N D H C fraction o v e r 9 . 5 % 1.1 C fraction o v e r 5 6 %

Capsicum

baccatum

1.2 C fraction u n d e r 5 6 %

Capsicum

annuum

var.

var.

annuum

pendulum

2.1 C f r a c t i o n , 4 2 t o 5 7 %

Capsicum

annuum

var.

annuum

2.2 C f r a c t i o n u n d e r 4 2 %

Capsicum

baccatum

var.

pendulum

Capsicum

baccatum

var.

pendulum

Capsicum

frutescens

-

Capsicum

chinense

2 N D H C C Fraction 9 . 5 %

2.3 C f r a c t i o n , 5 7 t o 7 3 % D H C fraction 2 6 t o 3 4 % Total c a p s a i c i n o i d s , u n d e r 0 . 3 5 % 2.4 C fraction o v e r 6 3 % D H C Fraction u n d e r 3 2 % Total c a p s a i c i n o i d s , o v e r 0 . 3 5 %

complex

* After Jurenitsch J . , Kubelka, W., Jentzsch, K. (1979c): Identification of Cultivated Taxa of Capsicum Taxonomy, Anatomy and Composition of Pungent Principles. Plant. Med. 35: 174-180 (with permission) Abbreviations: C-Capsaicin; NDHC-Nordihydrocapsaicin; DHC-Dihydrocapsaicin

Thus, on the basis of the above studies, one may come to the conclusion that the genus Capsicum represents a very wide and divergent taxonomic group consisting of both wild and cultivated species. Some workers consider Capsicum to consist of three principal species, C. annuum, C. frutescens and C. chinense; meanwhile others have divided the genus into a divergent spectrum of species. Probably, molecular biology and molecular genetics, phytochemistry and cell biology can contribute to a deeper recognition of the taxonomy status of the genus Capsicum.

31

4. Cultivation of Capsicum or paprika

Capsicums, indigenous to South and Central America, Mexico and West India, continue to be cultivated there and have been introduced and widely cultivated through temperate, subtropical Europe, the southern of United States of America, tropical Africa, India, East Asia and China. The cultivation - through the last 400 years in the different soils, cultivation conditions, natural hybridization and selections - has given us a variety of Capsicums. Valued solely for their color and secondarily for aroma and mild pungency are the Spanish and Hungarian paprikas. For color, medium to high pungency and aroma are the chillies grown in India, Southeast Asia, China and America, and essentially for pungency the chillies in tropical Africa. Typical practices of the cultivation of Capsicums are written briefly below.

4 . 1 . Cultivation of Capsicums in India Chillies have been cultivated in India for over 200 years and have spread to most of countries, from sea level to 1600 m above sea level with annual rainfall of 600 to 1250 mm. Throughout India, 14 states cultivate more than 10,000 ha each, and 3 of them over 150,000 ha. The major chilli-producing states are Andhra Pradesh, Maharasthra, Orissa, Karnataka and Tamil Nadu. Chilli is a warm season crop, but low humidity with high temperature results in the shedding of buds, flowers and young fruits. Very low temperatures also result in poor growth. Chilli is started in three seasons, in different parts of the country. The main crop on the southern plains in India - which produces 70% of the crop (total of nearly 500,000 tons annually - is started in May to June, transplanted in 5 to 6 weeks, and harvested from October. In the Punjab area the cultivation of crop starts at the end of the cold season (in March to April), to avoid damage from frost, and harvested in August. In the Gangetic plains the crops are started in September and harvested in February. Chilli is cultivated in many areas in the north as a rain-fed crop, but in the south it is increasingly cultivated as an irrigated crop, especially in Andhra Pradesh and Tamil Nadu. The crop is grown in many types of soils but well drained loamy soils rich in organic matter are considered the most suitable. Clayey loams which can retain moisture are those in which chilli is cultivated as a rain-fed crop. 32

A variety of chillies, combining different sizes and degrees of pungency, are grown in India, each area having their characteristic cultivar of Capsicum annuum var. annuum. Generally, the medium to long and medium to high pungency red varieties are grown in the plains and the long, deep red, thick-fleshed, low pungency types are grown at higher elevations. Some amounts of vegetable capsicums and red short blunt conical types are also grown. Minor amounts of the highly pungent, small conical variety of Capsicum frutescens are grown or collected from semi-wild growth. A typical cultivation in Tamil Nadu, India is as follows; seedlings are started and raised in carefully prepared beds with natural shade and near a water source.

4.2. Cultivation of Capsicums in other Asian countries In the Indian subcontinent, other notable producers are Pakistan and Bangladesh, where the cultivation practices are similar to that in India, in amounts of internal consumption, with only a small percentage of export of a short conical variety ("dandicut cherry"). The other consumers of a number of Capsicum annuum, with moderate to high pungency and good color, are Thailand, Indonesia and Malaysia. In Southern Asia, South Korea is a substantial producer of highly colored and rather high pungency chilli varieties, but with a very high consumption, it is not an exporter. Since the 1950s, China has emerged as a major producer. A Federal Agricultural Organization (FAO) estimate of the annual production of fresh chillies is put at 1 million ton. China has become a top exporter, equalling, and even exceeding India.

4.3. Cultivation of Capsicums in Africa In the African continent, the crop was introduced early by the European conquests, but largely remained as semi-wild plants and exists as an organized cultivation in small holdings in only the North and West African countries. Both medium-sized mild and high pungency small chillies have been produced. The mild form is preferred for local consumption, the export being dominated by the crop collected from the wild growth in East Africa, the cultivated Zanzibar type from Sierra Leone, and the mild to medium type from Kenya and Nigeria. Ethiopia is notable for the largescale cultivation of a variety of Capsicums, the mild to medium pungency red chillies and more recently, paprika for oleoresin production.

4.4. Cultivation of Capsicums in America In the Americas, the source of Capsicum, the Central and South American countries continue to produce a great variety of Capsicums by collection of wild growths and by rudimentary cultivation of specific local varieties mostly for internal consumption. The wild Capsicums are reportedly used fresh, and medium pungency types are dried product. The highly pungent small varieties from all the five species cultivated are used essentially for therapeutic use. Mexico alone in this group appears to pro33

duce mild and high pungency chillies in amounts to have a substantial surplus for export to the United States. It has been difficult to obtain information on organized cultivation from these countries. In the United States, Capsicums have been cultivated since the 1920s in California, South Carolina, Louisiana and New Mexico. Considerable developmental work on the selection of suitable cultivars adapted to this region optimal cultivation practices. Different cultivars which combine the qualities of color and pungency in the required proportions are grown and account for nearly half of the total requirement of chillies and paprika, both of which had been earlier wholly imported. Moderately pungent ("Louisiana sport", "Carolinas" or "Anaheim" in California), the high pungency ("Bahanian" in South Carolina and "Tabasco" in Louisiana), the green bell Capsicum ("California Wonder" and "Florida Giant" for the fresh market in Florida and New Jersey), paprika ("Ruby King" in California) and pimientos ("Perfection") in Georgia exist as flourishing crop, raised through modern cultivation and processing methods. Large-scale cultivation and processing practiced in the United States are based on the basic scientific information collected on the growth of capsicum plant, and the formation of functionally important components for optimized economic production. Considered as a warm season crop, Capsicums are cultivated in spring, summer and autumn in the upper south of the United States and even in the winter in the extreme south. Boswell recorded that the optimum temperature for growth is around 24°C, and night temperatures lower than 15°C or day temperature higher than 32°C are detrimental to growth and fruit set. Additionally, low humidity causes abscission of flowers, buds and small fruits. At least 3 months of warm weather are necessary for the quick maturing sweet vegetable Capsicums and 4 to 5 months for most other cultivars. The small-fruited forms, those of Capsicum frutescent, are much more tolerant of higher temperatures and also are late maturing, requiring a longer warm season. The Capsicums can be grown on well-drained soils, but a fertile loamy soil rich in lime is considered most suitable. They are generally grown in rain-fed areas having 600 to 1250 mm of rain annually and redden better under controlled irrigation. Heavy rain and water logging lead to poor fruit set or fruit rot.

4.5. Cultivation of Capsicums in Europe and Hungary In Europe, since the 18th century, Hungary and the Balkan countries have been producing the carefully selected cultivars of paprika and possibly earlier still, Spain has cultivated the pimientos, valued for their brilliant red color, characteristic aroma, and mild pungency. These are more and more being produced as an industry with close control, integrating the steps of cultivation, processing, quality control and marketing. Sweet Capsicum can be grown in any region of Hungary with the exception of the western border of the country where the precipitation is higher and the temperature is lower than average. Only 8 to 10% of the country's soil and climate conditions are suitable for growing sweet Capsicum. Nevertheless, as traditional growing regions evolved, immigrant Bulgarian market gardeners settled at the southern part 34

of the country and started vegetable production. However, in a significant part of the region we find higher, sandy loam. These soils have inadequate water management with less humus, the cultivation is easier and the soil warms up faster, at the same time the quality of the condiment paprika is lower than that obtained from the heavier soils. These lighter soils are suitable for intensive sweet Capsicum production under controlled conditions. In the region of Kalocsa the soil is mostly heavy, with relatively low humus content on the former flood plains of the Danube River.

4.5.1. Cultivar types in Hungary 4.5.1.1. Sweet Capsicum cultivar The sweet Capsicum cultivars in Hungary have changed a lot during the last centuries, especially during the last 50 to 60 years. In the previous centuries the seed was handed down from father to son and the growers passed the seed on to each other. The seeds of the best and earliest fruits were always kept for further sowing. It can be assumed that on many occasions spontaneous pollination or mutation created new types or varieties. Breeding techniques applying genetics go back only to the last 50 to 60 years in Hungary. The first sweet cultivar which received State registration was "Cecei sweet 3" bred by Angeli (see Somogyi et al., 2003). He selected this from a pungent white type and created a non-pungent cultivar. With his work, an active and efficient Capsicum improvement started and consequently the Hungarian sweet Capsicum types and an assortment of cultivars have been continuously increased and spread all over the World. The production of sweet Capsicum hybrid seed cultivars was initiated in Hungary in the middle of the 1960s (Moor, 1969). Cultivars were made by the breeding work of the state-owned Vegetable Crop Research Institute and its processors, and the Horticulture Departments of Universities until the middle of 1980s. Since then, several private breeders' cultivars received Plant Variety Protection. All the cultivars grown belong to the species of Capsicum annuum L. The sweet Capsicum cultivars (varieties) are categorized into different groups by the National Institute for Agricultural Quality Control (Table 5). Approximately 100 cultivars were registered in the National List of Varieties in 2000. One of the most important trains of sweet Capsicum cultivation is the sensitivity to the lack of light (Zatyko, 1979). This characteristic is important for determining whether the cultivar can be force-grown or not. This means establishing whether the cultivar may be grown only in the field or whether its production is economical under green-house conditions out-of-season: 1. Ciklon Fl - indeterminate, white fruit ripening to red, sweet, conical, upright fruit, for all production systems. Fruits are 12 to 15 cm long and 5 to 6 cm in diameter. Yield is 8 to 15 kg/m depending on the production technology. It contains Tm2 resistance. Under local night conditions, sowing is done at the end of September and the first 2 cm long fruits appear after a vegetation period of 125 to 130 days. This can decrease by 50% under more intense light conditions; 2

35

T a b l e 5. C a t e g o r i e s of t h e H u n g a r i a n s w e e t C a p s i c u m c a t e g o r i z e d b y t h e H u n g a r i a n N a t i o n a l Institute for A g r i c u l t u r a l Q u a l i t y C o n t r o l * W h i t e fruit, i n d e t e r m i n a t e

p u n g e n t or w i t h o u t p u n g e n c y

W h i t e fruit, d e t e r m i n a t e

without pungency

Pale g r e e n fruit, i n d e t e r m i n a t e

without pungency

Hornshaped, indeterminate

pungent, without pungency

P o i n t e d , hot, i n d e t e r m i n a t e

without pungency

P o i n t e d , hot, i n d e t e r m i n a t e

without pungency

Tomato-shaped

without pungency

California W o n d e r type

without pungency

Others

pungent, without pungency

* After Somogyi, N., Moor, A., Pek, M. (2003): The preservation and production of Capsicum in Hungary. In: De, A. K. (ed.) Capsicum. The genus Capsicum. Taylor&Francis Ltd, New York. pp. 144-162 (with permission)

2. Taltos - white fruit turning to red, indeterminate, sweet, conical, blunt, pendulous fruits, grown in the field. Fruits are 10 to 15 cm long and 5 to 6 cm in diameter. Potential yield is 30 to 35 tons/ha; 3. "Pungent apple" - white fruit turning to red, indeterminate, pungent, apple-shaped upright fruit, grown in the field. It is mainly used by the canning industry (to pickle). Fruits are 6 to 7 cm, in diameter and 4 to 5 cm long. Potential yield is 18 to 25 tons/ha; 4. Feherozon - white fruit turning red, determinate, without pungency, upright fruits, grown in the field and also under green-house conditions. Fruits are 12 to 15 cm long and 5 to 6 cm in diameter. Yield in green-house conditions is 25 to 35 tons/ha; 5. Rapires Fl - pale green turning red, indeterminate, pungent, long, conical, pendulous fruits. It can be grown in any type of controlled facilities. Fruits are 15 to 20 cm long and 3 to 4 cm in diameter. It contains Tm2 resistance. Yield depends on the technology, 7 to 8 kg/m ; 6. Tomato-shaped - green-dark green ripening to red, indeterminate, sweet, flat, round, seamed, pendulous fruits. Fruits are 8 to 12 cm in diameter and 3 to 5 cm long. The potential yield of fully ripe fruits is 18 to 20 tons/ha. 2

4.5.1.2. Paprika varieties The Hungarian condiment paprika's cultivation period is short, there are only 5 to 5 and half months available for the vegetation period. In spite of the short vegetation period, the quality of the harvested crop is excellent in most years. The high pigment and the high dry matter contents guarantee a very good base material for milling. The Hungarian varieties' yield can be up to 50% more, with improved attributes, by cultivating them in areas where the vegetation period is longer. That is based on the Hungarian-Spanish (Somogyi et al., 2003), the Hungarian-Portuguese and the Hungarian-Austrian (Derera, 2000) cooperative experiments. The full genetic potential 36

of the Hungarian cultivars is limited by the climatic conditions. Between 1993 and 2000 condiment paprika was grown on an area between 3,000 and 6,500 ha and between 26,000 and 65,000 tons of raw paprika were produced. Consequently, the quantity of the milled product varied between 3,000 and 9,400 tons. All of the condiment paprika cultivars grown in Hungary were bred in Hungary. They belong to Capsicum annuum L. covar longum by botanical classification. There are two exceptions (cv. Kalocsai A. cherry type, cv. Kalocsai M. cherry type), regarding the growth habit. They are continuous, semi-determinate and determinate. There are two types of orientation of the fruits: erect and pendulous. The categories are indicated below and the pungency is given in brackets: 1. Varieties of continuous growth habit, pendulous fruit: Szegedi 20 Szegedi 80, Szegedi 57 13, Remeny, Kalmin, Szegedi 178 (pungent), Szegedi 179 (pungent), Szegedi F-03 (pungent), Kalocsai 50, Kalocsai 90, Kalocsai V-2 (pungent), Kalocsai 15 (pungent), Csardas, Folklor; 2. Varieties of continuous growth habit, erect, Kalocsai 37 to 231; 3. Varieties of semi-determinate growth habit, pendulous fruits: Kalocsai 601, Kalocsai 702, Zuhatag; 4. Varieties of semi-determinate growth, erect fruit: Kalocsai 622, Rubin; 5. Varieties of determinate growth habit, erect fruits: Kalocsai D 601, Kalocsai D 621 (pungent); 6. The cherry-type paprika is classified as paprika, but it differs from the rest. It is Capsicum annuum covar. cerasiforme and not longum. It is pungent, its importance is found in gastronomy. If green fruit is harvested, it can be pickled or made into salad. When the ripe fruits are harvested they can be used for hot sauces or dried spice (not milled) flakes. The two cherry cultivars that differ in fruit size and growth habit are Kalocsai M and Kalocsai A. A detailed description of the most typical cultivars of each category is given below: 1. Cultivar of continuous growth habit and erect: Kalocsai 37 to 231, a sweet variety. The bush is 45 to 55 cm high, its fruits are scattered and 10 to 14 cm long, slightly bent. They are of flaming red color or dark red after post-ripening treatment. Its main value lies in its good pigment (8 to 9 g/kg) and solids content. It is a middleearly maturing variety. Its yield potential is 25-16 tons/ ha. It can be transplanted or directly seeded. It has a good tolerance to diseases; 2. Cultivar of continuous growth habit and pendulous fruit: Szegedi 80, a sweet cultivar. The fruits are 12 to 14 cm long, dark red when ripe, pigment content is 8 to 10 g/kg after post-ripening treatment. The solids content at picking is 20%. Its yield potential under intensive conditions is 20 to 25 tons/ha. It has a reasonable tolerance to diseases and can be transplanted or directly seeded. Due to an early ripeness a reasonable yield can be relied upon before the first frosts. The Szegedi F-03 is the same type but with pungency; 3. Cultivar of semi-determinate growth habit, with pendulous fruits: Kalocsai 801, a sweet variety. It has loose spreading foliage. The plant is about 40 to 45 cm long, the fruits weigh 22 to 28 g. They are straight, gradually tapering toward a pointed tip, and their color is dark red when ripe. Their pigment content at picking is 6.0 to 7.0 g/kg, going up to 8.0 to 9.0 g/kg after post-harvesting. The dry matter con37

tent is 18% when ripe. It is an early, intensive variety bringing a high yield as an exchange for watering and good nutriment supply. Its potential harvest is 20 to 22 tons/ha and it has a high tolerance to viral diseases; 4. Cultivar of semi-determinate growth habit with erect fruits: Kalocsai M 622, a sweet cultivar. The bush is 35 to 45 cm high with sparse foliage, and has a rigid stem with short internodes. Leaves are leathery and thick, so it has a good field resistance to fungal infections. The fruit is 10 to 15 cm long, gradually tapering towards a pointed tip, and is dark red when ripe. The pigment content at picking is 6.0 to 8.0 g/kg, increasing to 9.0 to 12.0 g/kg after post-harvest ripening. If transplanted, the entire crop can be harvested at the same time due to its short growing season and early, uniform ripening. It is primarily directly seeded. It is the most widely spread cultivar in the Kalocsa region. It requires intensive agronomic conditions, but the compensation is a high yield. Its yield potential is 20 to 25 tons/ha. It also has a high tolerance to diseases, which is the basis of secure production; 5. Cultivar of determinate growth habit and erect fruits: Kalocsai D 601, a sweet variety with erect fruits appearing in bunches on the stem. The bush is 30 to 35 cm high and the fruit bunches rise above the foliage and ripen uniformly. Fruits are 10 to 12 cm long, slightly bent, pointed and ripen to a deep red color. It contains 6.0 to 7.0 g/kg pigment at picking. The dry matter content is above average. It has a short growing season and early, uniform ripening. It is primarily recommended for direct seeding. On a large scale its can be harvested by machinery in one operation. The yield potential is 15 to 16 tons/ha; 6. Cherry-type paprika: Kalocsai M cherry type. It is the cultivar of pendulous fruits and loose foliage. The bush is 40-60 cm high and of continuous growth. The fruit is 3 to 3.5 cm in diameter, slightly flat and globe-shaped with a closed style point. Its surface is smooth and its pigment content is 7 to 9 g/kg. The dry matter content is 20 to 22% when ripe and the capsaicin content at picking is 120 to 140 g/100 g. It has a good tolerance against viral infections. It has a middle-early, continuous ripening and a good yield. It is primarily recommended for transplanting and requires intensive growing conditions. It needs a soil rich in organic matter that can be easily warmed up.

38

5. General chemical structure and composition of Capsicums

5.1. Macroscopic characteristics The dried fruits of the chilli variety are of varying sizes: 6 to 300 mm in length and 5 to 140 mm in width near the calyx. They present a variety of shapes, including the long thin conical pointed tips, short thin conical blunt tips, short broad conical to rounded tips, long broad with blunt tips and small round to oval in a few cultivars. They are laterally compressed except in the small seed-filled varieties and some paprikas. The vegetable Capsicum varieties are generally big, broad, blunt and blocky with slightly sloping sides, while the paprikas are of medium length, broad, blunt and round (Fig. I). The calyx and peduncle are usually attached, except in a few small-sized fruits and in longer varieties bred for easy destalking during harvest.

Fig.

7. C a p s i c u m fruits v a r y i n g in s h a p e s a n d s i z e . 1 . tiny g l o b u l a r ; 2 . C h i l l i p i q u i n ( M e x i c o ) ; 3. M u n d u ; 4 . S i n d h o o r ; 5. L o n g R e d C a y e n n e ( U S A ) ; 6. M e x i c a n A n c h o ; 7. H u n g a r i a n p a p r i k a ; 8. B e l l C a p s i c u m (all of t h e s p e c i e s Capsicum

annuum

var. annuum,

L);

9. t a b a s c o ( U S A ) ; 1 0 . bird's e y e ( A f r i c a n ) , ( 9 - 1 0 ) b o t h of t h e s p e c i e s Capsicum L; 1 1 . o b l o n g ; 1 2 . m e d i u m b r o a d ; 1 3 . l o n g p o i n t e d fruits of Capsicum pendulum

baccatum

frutescens var.

( W i l d ) (after G o v i n d a r a j a n , V . S . , 1 9 8 5 : C a p s i c u m - P r o d u c t i o n , t e c h n o l o g y ,

chemistry, a n d q u a l i t y - Part I: History, b o t a n y , c u l t i v a t i o n , a n d p r i m a r y p r o c e s s i n g . C R C , C r i t i c a l Rev. F o o d S c i . Nutr. 2 2 : 1 0 9 - 1 7 6 ) ( w i t h p e r m i s s i o n )

39

The pericarp is lustrous, smooth and its color generally ranges from deep red to brownish red or orange. A few cultivars with the ripe mature color such as deep purple or white are known as also partially bleached orange color due to poor dyeing. Paprika and pimientos are generally of intense burgundy red, while vegetable Capsicums are known with both green and deep red colors at fully maturity. The fruit has two to four loci marked by longitudinal ridges with a central placenta extending from the base under the calyx to the apex end, except in vegetable Capsicums where the placenta is confined to the base (Fig. 2). Seeds are numerous, attached to the central placenta, are yellow (black in one species), smooth, 2.1 to 5 mm, discoid with thickened edge, and prominent pointed micropyle.

Fig. 2. Cross-sections of C a p s i c u m a n d c h i l l i fruits. P e : p e r i c a r p ; D : d i s s i p i g m e n t s ; S: s e e d s ; P I : p l a c e n t a (after C o v i n d a r a j a n , V . S . , 1 9 8 5 : C a p s i c u m - P r o d u c t i o n , t e c h n o l o g y , c h e m i s t r y , a n d q u a l i t y - Part I: History, botany, c u l t i v a t i o n , a n d p r i m a r y p r o c e s s i n g . C R C , C r i t i c a l Rev. F o o d S c i . Nutr. 2 2 : 1 0 9 - 1 7 6 ) ( w i t h p e r m i s s i o n )

5.2. Microscopic characteristics The microscopic characteristics of Capsicums are seen on the cross-section. A cross-section of the fruit shows the pericarp components, the cuticular layer of cells, the epidermis of the single layer of regular rectangular cells, the hypodermis of tangentially oval cells with thick walls in one or more layers, the mesocarp of parenchyma cells, having oblong, tangentially compressed and greatly varying sizes and frequently containing droplets of the red oil, occasionally crystals of calcium oxalate. In the inner regions are the vascular bundles and giant cells causing blisters on the inner surface of the pericarp, a characteristic of all Capsicum fruits, and the endocarp of rectangular cells with thick pitted walls. The diagnostic characteristics of ground Capsicum include all tissue components described above and fragments of calyx and pedicel, all mixed up. Jackson and Snowdon (1974) described the following microscopic diagnostic characteristics of Capsicum annuum L.: 40

1. numerous pale yellow fragments of the epicarp as single layer of polygonal cells with beaded walls, some showing strongly striated cuticle; 2. the abundant parenchyma of the mesocarp, thin-walled, containing orange to red oil globules and occasionally microspheroidal crystals of calcium oxalate; 3. the sclerenchymatous endocarp as groups of polygonal cells with thickened sinuous walls and having distinct pits; 4. fragments of the epidermis of the testa composed of a layer of very large lignified and markedly varying cells, unevenly thickened on the inner walls appearing as balloon-like swellings; 5. endosperm, parenchymatous cells with drops of fixed oil and aleuronic grains; 6. fragments of calyx, the inner epidermis cells with numerous trichomes with stalk and multicellular head; 7. fragments of pedicel tissues, elongated or small polygonal cells with stomata, glandular trichomes, cellulosic parenchyma, fibers in isolated groups and parenchymatous pith with large central cavity; 8. seed structures, where present, are sinuous with thin outer and thickened, pitted inner walls.

5.3. General chemical composition Carbohydrate, protein, fat and fibre are the major components which rapidly increase from the green stage to the ripe stage. Fructose, glucose, galactose and sucrose were identified by gas chromatography and enzymatical methods. Fructose is the major sugar together with glucose amounting to about 70% of total sugars. Free sugar content in seeds was reported to be higher than in the pericarp or placenta. The most fat in Capsicums is in the seed. Values for total fat vary from 9 to 16% depending on the seed content of the variety or processed sample. The high seed fat content is largely found in cultivated species. The fibre content varies greatly between the sources and reflects the amount of pedicel included in the sample. Three functionally important characteristics in the use of chillies as spice: - red color and capsaicinoids, - the pungency stimulants, - characteristic sweet aroma of paprikas and vegetable Capsicums or the pungent aroma of chillies. Other important components of Capsicums are: citric acid is a major acid component, the others being succinic, fumaric, malic and quinic acids (Table 6). Of nutritional importance is the vitamin C content of paprika, the vegetable Capsicums and chillies. Vitamin C was firstly isolated from paprika by Szent-Gyorgyi for which he was awarded the Nobel Prize in 1937. Pound for pound paprika is higher in vitamin C content than citrus fruit. Contents of vitamin C are up to 340 mg/100 g in some varieties of paprika. The content of vitamin C in dried Capsicums is only of the order of 30 to 60 mg/100 g, which indicates a large percent of loss of vitamin C in processing these Capsicums.

T a b l e 6. C h e m i c a l c o m p o s i t i o n of C a p s i c u m s * Compounds

g/100 g fresh f r u i t ( m e a n s ± S E M )

Water

91 ±0.6

Glucose

0.85 +0.1

Fructose

0.75±0.1

Sucrose

N.D.

Starch

0.81 ±0.2

Fibre

2.2±0.3

Pectin

0.73±0.1 mg/100 g fresh fruit (means ± S E M )

Citric acid

28+12

Fumaric acid

1.1 ±0.4

Malic acid

208±18

Oxalic acid

140±24

Quininic acid

183±12

Vitamin C

24±12

Chlorophyll a

7.9±2

Chrorophyll b

3.4±0.6

All-trans-lutein

1.4±0.3

All-trans-ls-carotene

0.92±0.4

* After Lopez-Hernandez, J . , Oruna-Concha, M. J . , SimaLLozano,)., Vazquez-Bianco, M. E., Gonzales-Castro, M. J . (1996b): Chemical composition of Padron peppers (Capsicum annuum L.) grown in Galicia (N.W. Spain). Food Chemistry 57: 557-559 (with permission) Abbreviation: N.D. not detected

The vitamins A and B complexes also have been shown in Capsicums. Values reported of vitamin A are as high as 3315 international units in paprika and 3530 to 6165 IU in chilli varieties. The vitamin E content in fresh and dried paprika depends on the ripening stage and genetic factors. The presence of 3 to 10 mg/100 g in fresh ripe Capsicums could be an important source of vitamin E in the human diet. The loss in drying is about 5%. Oleoresin paprika had earlier been shown to have as much as 10 mg/g of a-tocopherol. A total of 14 amino acids in green Capsicum and 16 in paprika, from which asparagine and proline are dominant, were identified earlier. Recent analysis of sweet and hot varieties showed no differences in amino acids, but the composition of the seed was different from that of the pericarp. Chillies and paprika are evaluated for uniformity of shape, size, color and pungency combination of the trade types. The sweet or mildly pungent paprika is valued for its bright color and to a minor extent fot its aroma, while the chillies for their pungency and color. The paprika group contains less than 0.1% capsaicinoids, the pungency stimulants are 0 to 1.4%. In addition to water which makes up more than 90% of Capsicum fruit, fibre, pectin, glucose, starch and fructose represent the main components (Table 6).

6. Chemical taxonomy of the functional parts of the Capsicums 6.1 Capsicum: Botanical aspects Capsicum is a versatile plant used as vegetable, a pungent food additive, a colorant and a pharmaceutical. The genus Capsicum, which is commonly known as chili, "red chili", "tabasco", "paprika", "cayenne", etc., is a member of the family Solanaceae, and closely related to eggplant, potato, petunia, tomato and tobacco. Various authors ascribe 25 species to the genus, with new species to be discovered and named as the exploration of the South American tropics expands. After much work by taxonomists concerning the classification of the presently domesticated species, they have been considered to belong to one of five species, namely Capsicum annuum, Capsicum frutescens, Capsicum baccatum, Capsicum chinense and Capsicum pubescens (Bosland, 1994). The Capsicum plants grow as perennial shrubs in suitable climatic conditions. Capsicum fruits are considered to be vegetables, but botanically speaking, they are berries. Capsicum types are usually classified by fruit characteristics, i.e., pungency, color, fruit shape, as well as by their use. Capsicum species are commonly divided into two groups, pungent and non-pungent, also called hot and sweet. The history of chile peppers is one of the enthusiastic acceptances wherever they were taken. Chile is historically associated with the voyage of Columbus (Heiser, 1976). Columbus is given credit for introducing chile to Europe, and subsequently to Africa and to Asia. On his first voyage, he encountered a plant whose fruit mimicked the pungency of the black pepper (Piper nigrum L.). Columbus called it red pepper because the pods were red. The plant was not the black pepper, but a heretofore unknown plant that was later classified as Capsicum. Bolivia and Peru are the most probable ancestral home of the chile pepper, but in the world at large Mexico gets the credit for this plant, having cultivated the pepper types that entered the world's cuisines after Columbus's discovery. Capsicum species are used fresh or dried, whole or ground, and alone or in combination with other flavoring agents. Most of the Capsicum cultivars commercially cultivated in Europe and Amerika belong to the species of Capsicum annuum. However, there are a few principal types which belong to other Capsicum species. The most common types are "Bell", "Cayenne", "Cherry", "Jalapeno", "Mirasol", "Paprika", "Pasilla", "Pimento", "Serrano", "Squash" and "Wax" (all Capsicum annuum) as well as "Habanero" (Capsicum chinense), "Long Green" (Capsicum baccatum), "Manzano" (Capsicum pubescens) and "Tabasco" (Capsicum frutescens). 43

In many European languages, the name of this spice is somehow derived from the name of pepper, owing to the many confusions of pepper with other spices. A rather common designation of paprika is "sweet pepper": Spanish "pimiento dulce", French "piment doux", and Arabic "fulful halu". It should be noted that in most of these languages, the word for "pepper" may also mean "chile", so it would perhaps be more accurate to translate these names by "sweet chile" or "sweet chile pepper". Yet other tongues have names for paprika that mean "red pepper" e.g., Turkish "kirmizi biber", Bulgarian "cherven piper" and Russian "perets krasni". These may lead to confusion as in a plethora of other languages, similar names are reserved for chiles. Other English names are bell pepper and pod pepper because of the shape. Most confusingly, the English plural peppers always seem to imply some sort of paprika (also vegetable bell peppers or hot chile peppers), and never true black pepper! For reasons of clarity, the term "pepper" for Capsicum species will be avoided later in this chapter. Instead, the word "paprika" throughout for mild or medium-hot varieties and "chile" for hot varieties will be used. The word "paprika" was borrowed from Hungarian (paprika), it entered a great number of languages, in many cases probably via German. In the end, also "paprika" is derived from a name of black pepper, in this case Serbian papar. In most languages, "paprika" denotes the dried spice only, though in some (e.g., German) it is commonly used for the vegetable bell pepper. The form "paprika" is valid in countless European languages, while examples with slightly deviating spelling include Italian "paprica", Polish "papryka" and Bulgarian "piperka".

6.2. Capsicum: Chemical constituents Capsicum fruits contain coloring pigments, pungent principles, resin, protein, cellulose, pentosans, mineral elements and a small amount of volatile oil, while seeds contain fixed (non-volatile) oil. Besides these organic constituents Capsicum fruits also contain inorganic constituents, mostly potassium and sodium, calcium, phosphorus, iron, copper and manganese (Thresh, 1846; Brawer, Schoen, 1962; Brash et al., 1988; Pruthi, 2003). The pungent principles capsaicin and its structurally closely related homologes (so-called capsaicinoids) and analogues, are contained only in small amounts, as low as 0.001 to 0.005% in "mild" and 0.1% in "hot" cultivars. Apart from capsaicin, the taste of paprika is mostly due to the fixed oil which is comprised mainly of triglycerides of which linoleic, linolic, stearinic and other unsaturated fatty acids predominate. The fixed oil content of the Capsicum seeds also play an important role in the visual sensing of the paprika powder since it can dissolve and homogeneously distribute the colored substances during grinding of the dried fruits. The characteristic aroma and flavor of the fresh fruit are imparted by the volatile oil containing a range of alkylmethoxypyrazines (e.g., 2-methoxy-3-isobutyl-pyrazine, the "earthy" flavor) and a structurally diverse group (alcohols, aldehydes, ketones, carboxylic acids, and esters of carboxylic acids) of oxygenated hydrocarbons. Furthermore, the fresh ripe paprika contains sizable amounts (0.1 %) of vitamin C (ascorbic acid). It was the Hungarian biochemist Albert Szent-Gyorgyi who discov44

ered that the Hungarian paprika is a rich source of vitamin C. Later (1937) he won the Nobel Prize "for his discoveries in connection with the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid" (Encyclopedia Britannica). During the post-ripening period and processing a substantial amount of vitamin C undergoes degradation. Paprika powder contains only a little amount of it. Paprikas derive their color in the ripe state mainly from carotenoid pigments, which range from bright red (capsanthine, capsorubine) to yellow (cucurbitene); the total carotenoid content in dried paprika is 0.1 to 0.5%. A small number of cultivars do not produce significant amounts of carotenoids; when chlorophyll levels decrease in the last stages of ripening, these chiles develop a pale hue often referred to as "white". Due to small amounts of chlorophyll and/or yellow carotenoids, the "white" is, however, more precisely described as a pale greenish-yellow. Some varieties of paprika contain pigments of anthocyanin type and develop dark purple, aubergine-colored or almost black pods; in the last stage of ripening, however, the anthocyanins get decomposed, and the unusual darkness thus gives way to normal orange or red colors. The same anthocyanins cause the dark spots which are sometimes seen on unripe fruits or particularly the stems of paprika plants and which almost all paprika varieties can develop. In other Capsicum species, anthocyanin production is a rare phenomenon.

6.2.1. Volatiles The characteristic flavor and aroma of the fresh fruits is due to their volatile oil content. The fruits of Capsicum species have a relatively low volatile oil content, ranging from about 0.1 % to 2.6% in paprika. The total volatiles are generally isolated by steam distillation. In the case of heat-sensitive compounds present, vacuum distillation-continuous solvent extraction can be used. The pure volatile oil and concentrated extracts were analyzed by GC-MS methods. Most compounds of odor significance have been tentatively identified by their mass spectra, and the identification was confirmed by checking the retention time and mass spectra of authentic reference compounds. When Buttery et al. (1969a,b) identified 3-isobutyl-2-methoxypyrazine (1) (Fig. 3) as a characteristic aroma compound, the alkyl-methoxypyrazines aroused great interest among flavor chemists. The alkyl-methoxypyrazines have been shown to be widely distributed in vegetables and with a greenish sweet smell that possibly plays a significant role in the aroma of salad vegetables (Murray, Whitfield, 1975). In the volatiles of the green bell fruits (C. annuum var. grossum), different Capsicum fruits with strong odor, the major character-determining component 3-isobutyl-2-methoxypyrazine (1) was easily identified by GC-MS technique and sensory analysis. Other odor impact compounds identified of the bell capsicum volatiles were hexanal (2), which has a mild nutty odor, cis-3-hexen-l-ol (3) with a green leaf odor, the C(9) unsaturated ketones (non-l-en-4-one, non-trans-2-en-4-one, non-2trans-5-trans-dien-4-one), nona- and decadienals (non-2-trans-6-cis-dienal, non-2trans-4-trans-dienal), and a few terpenic compounds (e.g., limonene, trans-betaocimene, linalool (4) (Buttery, et al., (1969a) (Fig. 3). 45

CH,-CH

./CH X

N-

^OCH

,o

3

CH,

C H ,— C H — O 1 1 — CI I C I

I

-c:

3

1

CH — C H 3

2

x

/ H

H

3C

,CH

/ —

2

\

OH I

H,C

C H — C H — OH 2

/

H

C=CH—CH —CH —CH-CH=CH 2

-CH — C H = C H - C - C H 2

2

CH,

°

3

2

°,x 3

CH —CH 3

7

.CH

2

CH —CH —O /<~"2

2\

2

(' =

CH,

.C-CH -CH

/

2

3

CH,

(' \

H

8

S

6

9

Fig. 3. C h e m i c a l structure of t h e m a i n o d o r s i g n i f i c a n t c o m p o u n d s of fresh Capsicum

fruits

(see t h e text for d e t a i l e d e x p l a n a t i o n )

The pyrazines were also identified in other chile varieties, but in much smaller amounts (Murray, Whitfield, 1975; Hoffman et al., 1978). Some common fruit esters, predominantly C(6)-C(5) and C(6)-C(6) esters and related alcohols were identified in the volatiles of ripe fruits of the variety of Tabasco (Haymon, Aurand, 1971). A more elaborate search for aroma-significant compounds in the pungency-stimulating varieties of Capsicum fruits has been reported (Keller et al., 1981). Volatiles were obtained by steam-distillation from oleoresin of the highly pungent African chilies (C. frutescens), the partially concentrated hexane extract of Miscella (C. annuum variety) and the popular fresh mature green Jalapeno - a medium pungent variety of Capsicum annuum. The 102 compounds identified in the three volatiles belonged to the structural groups as follows: alcohols (e.g., hexanol, 2-methylbutanol, 4-methylpentan-l-ol, cis-3-hexen-l-ol (3)), carbonyls (e.g., 4-methyl-3-penten-2-one, 2-hexanone, P-ionone (5), 5-methyl-2-furfural), carboxylic acids (e.g., 2-methylbutyric acid, 4-methylpentanoic acid, hexanoic acid, octanoic acid), esters (methyl pentanoate, methyl hexanoate, methyl heptanoate, methyl octanoate, methyl 46

tetradecanoate), pyrazines (e.g., 2,3-dimethylpyrazine, 2,3,5-trimethylpyrazine, tetramethylpyrazine, 3-isobutyl-2-methoxypyrazine), terpene hydrocarbones (e.g., limonene, myrcene, (3-pinene, a-phelladrene, caryophyllene), and a group of miscellaneous compounds (e.g., p-xylene, 2-pentylfuran, eugenol, pentadecane, hexadecane, heptadecane). Based on GC-MS and sensory analysis of volatiles of three cultivars of C. annuum - Jalapeno, Fresno, and Anaheim - nine odor significant compounds were identified that had been found in one or the other of the earlier studies: cis-3-hexenl-ol (3), cis-3-hexenyl-isopentanoate (6) (associated with green and green-fruity odors); 3-sec-butyl- (7) and 3-isobutyl-2-methoxypyrazine (1) (with green vegetable and green bell capsicum odors); (3-ionone (5) (only in Jalapeno), linalool (6) (both with floral character); and the aromatic compounds benzaldehyde (8) and methyl salicylate (9) (with sweetish, penetrating odors) (Chitwood et al., 1983) (Fig. 3).

6.2.2. Coloring pigments The coloring pigments of red peppers are comprised of carotenoids. Carotenoids are widely distributed groups of natural pigments, responsible for the yellow, orange, and red colors of fruits, roots, flowers, fish, invertebrates, and birds. Only bacteria, algae, fungi, and green plants can synthesize carotenoids, but humans incorporate them from the diet. Especially bacterial carotenoids are most diverse. Carotenoid extracts and fruits rich in carotenoids are now being used in the food industry to color foods, thus such foods also represent carotenoid sources of the human diet. The nutritional importance of carotenoids is mostly associated with the provitamin A activity of (3-carotene and others. Besides its well-established provitamin A activity, research is under way to study the relationship between [3-carotene intake and occurrence of atherosclerosis, cardiovascular diseases, in particular degree of LDL oxidation (van Poppel, Goldbohm 1995; Hinds et al., 1997; Rodriguez-Amaya, 1997; Woodall et al., 1997; Mailard et al., 1998; Manirakiza et al., 1999, 2003). The basic carotenoid structure is a symmetrical, linear, 40-carbon tetraterpene built from eight carbon isoprenoid units joined in such a way that the order is reversed at the center. Fig. 4 shows the structure of (3-carotene (10), one of the most typical carotenoid components of capsicum fruits. 4'

4 Fig. 4. C h e m i c a l structure of 6-carotene (10) (see t h e text for further e x p l a n a t i o n )

47

Carotenoids were for a long time assumed being synthesized by the mevalonate pathway for isoprenoid biosynthesis. This view was prevalent up until the mid nineties when it was discovered that the carotenoid precursor isopentenyl-pyrophosphate (IPP) was synthesized by two independent metabolic pathways in plants (Lichtenthaler et al., 1997). The first pathway occurs in the cytoplasmic compartment from mevalonic acid and gives rise to compounds such as sterols and cytokinins. In fungi, carotenoids are derived via the mevalonate biosynthetic pathway. The second pathway, effective in bacteria and plastids of plants, is responsible for biosynthesis of gibberellins, carotenoids, abscisic acid, and also contributes to the biosynthesis of tocopherols as well as chlorophyll A and B. This plastidial pathway of isoprenoid synthesis is named after its first metabolite 1-deoxyxylulose 5-phosphate (DOXP) and has pyruvate and glycerinaldehyde-3-phosphate as precursors (Fig. 5). The overall pathway of carotenoid biosynthesis can be considered as a series of stages, as illustrated in Fig. 3. Further details can be obtained from a number of review articles (Davies, 1980; Spurgeon, Porter, 1983; Britton, 1991). A proper starting point of the carotenoid biosynthesis is the conversion of geranylgeranyl pyrophosphate (GGPP) (11) to phytoene (12) by the enzyme complex phytoene synthase (PSY) (Fig. 5). This enzyme takes two units of the C-20 compound GGPP and condensates them in a "head-to-head" manner to yield the C-40 compound phytoene. Phytoene is not a true pigment since it is unable to absorb light in the visible region. It undergoes four consecutive desaturation steps. In higher plants these reactions are catalyzed by two related desaturases. The first two steps are performed by the phytoene desaturase (PDS) and the latter two by zeta-carotene desaturase (ZDS) (Linden et al., 1991). These reactions lead to formation of the pigment lycopene (13), the main pigment of the red tomato. Lycopene produced by these two desaturase reactions is in cw-form, also called pro-lycopene (Bartley et al., 1999). There is one more enzymatic step to produce the all-trans-lycopene, that is considered to be the main substrate for the subsequent reactions. Lycopene is the major carotenoid in tomato. /4/7-frarcs-lycopene can be modified in various manners, such as hydrogenation, dehydrogenation, cyclization, double bond migration, chain shortening or extension, rearrangement, isomerization, introduction of oxygen functions or combination of these processes, resulting in a great diversity of structures. The number of naturallyoccurring carotenoids whose structures have been elucidated now numbers more than 600. The number of carotenoids so far encountered in foods is much lower, nevertheless, the carotenoid composition of a given food can be quite complex (Sandmann, 2001). Hydrocarbon carotenoids are collectively termed carotens, and those containing oxygen are called xanthophylls. The most common oxygen functions are hydroxy (OH) and epoxy groups. Hydroxy groups are common, particularly in position 3 or 4. Many carotenoids possess an epoxy group, usually in positions 5,6 or 5,8 (the 5,8epoxides are often referred to as furanoids or furanoid oxides), though some 1,2epoxides, 3,6-epoxides and 4,5-epoxides also have been reported. Carotenoids containing aldehyde, keto, carboxy, carbomethoxy, and methoxy groups, acetates and lactones, are also encountered. Carotenoids, whether carotens or oxocarotenoids (xanthophylls), may be acyclic, monocyclic, or bicyclic. Cyclization occurs at one 48

Phytoene (12) PDS ZDS

OH

HO

OH HO

HO

Violaxanthin (18)

Fig. 5. S i m p l i f i e d b i o s y n t h e t i c r o u t e of c a r o t e n o i d s (see text, for further d e t a i l e d e x p l a n a t i o n )

or both ends of the molecule, forming one or two six-membered rings (Pfander, 1987). The next two steps of the simplified carotenoid biosynthetic route (Fig. 5) result in bifurcation of the pathway, where one path is leading to the synthesis of P-caro49

tene (10) by two consecutive cyclization reactions catalysed by the enzyme P-carotene cyclase (LCY-B) (Hugueney et al., 1995), and the other branch leads to a-carotene (14), where two different cyclases are necessary. One of them is LCY-B, and the other enzyme is e-carotene cyclase (LCY-E) (Cunningham et al., 1996). a- and p-carotenes only differ in a position of a double bond in one of the end-rings, and since not all bonds are part of the conjugated system in a-carotene, this leads to a small but obvious difference in the characteristic carotenoid spectra of the two compounds. Oxygenated carotenoids are formed by hydroxylation of the precursor hydrocarbon carotenoids. For example, two consecutive hydroxylation steps convert P-carotene first to cryptoxanthin and then to zeaxanthin (15) by the action of P-carotene hydroxylase (BCH). a-Carotene is also twice hydroxylated to yield lutein (16), but by two different enzymatic reactions. The beta-ring is hydroxylated by BCH, and the epsilon-ring by £-carotene hydroxylase (ECH). The hydroxylated beta-rings of zeaxanthine are epoxidated in two steps to yield first antheraxanthin (17) and then violaxanthin (18). In each step one atom of molecular oxygen is incorporated into the substrate molecule while the other is reduced to water. During light stress violaxanthin can be converted back to antheraxanthin and zeaxanthin by the enzyme violaxanthin-deepoxidase. This flux between these three compounds, governed by light intensity, constitutes the xanthophyll cycle. The last of the ubiquitous carotenoids in higher plants is neoxanthin (19), produced from violaxanthin by the enzyme neoxanthin synthase (NXS). Neoxanthin with an allenic bond is widespread and is present in significant amounts in the leaves and green stages of fruits of higher plants (Fig. 5). Characterization of the key enzymes of the carotenoid biosynthetic pathways is summarized in a recent review along with their biotechnological applications which resulted in development of heterologous carotenoid producing bacterial and fungal systems (Sandmann, 2001). Carotenoids can be extracted from natural sources by lipid solvents. With fresh material, ethanol or acetone act both as dehydrating agent and extracting solvents. When lipids and esterified xantophylls are present (hydroxylated carotenoids generally occur as esters of fatty acids), the extracts are saponified and the free carotenoids extracted for analysis. Most carotenoids are unstable in oxygen atmosphere and light thus careful extractions and separations are generally carried out under inert atmosphere, subdued light and low temperature (Rodriguez-Amaya, 1997; Deli, Molnar, 2002). High performance liquid chromatography (HPLC) is the most powerful chromatographic technique to separate and - coupled with mass spectrometry (MS) identify carotenoids. Recent reviews on the field provide up-to-date summary of carotenoid analysis (Rodriguez-Amaya, 1997; Wall, Bosland, 1998; Deli, Molnar, 2002; Felt et al., 2005). The composition of carotenoid pigments produced by paprika have been investigated in detail. Some twenty carotenoids have been isolated so far with capsanthin and capsorubin representing the most abundant (Deli, Molnar, 2002). The ripening process is marked by the disappearance of chlorophyll and a rapid rise in the colored carotenoids (Rahman, Buckle, 1980; Rodriguez-Amaya, 1997; Hornero-Mendez et al., 2000; Gnayfeed et al., 2001; Deli, Molnar, 2002). The comprehensive analytic data by Rahman and Buckle (1980) against the background of the biosynthetic rela50

tionship among common carotenoids summarized above shows similar formation and biosynthetic relationship in Capsicum fruits at the green stages and the massive synthesis at the short ripening stage, except for the large conversion to the new type of cyclopentanol ketocarotenoids. The data presented in Table 7 are given as mg/100 g fruit to better follow the synthesis and metabolism of the components and possible relationships. The sequence of biosynthesis of formation of P-carotene, its conversion to zeaxanthin and further formation of its epoxides, the operation of the dynamic xanthoT a b l e 7. C a r o t e n o i d c o m p o s i t i o n of f i v e v a r i e t i e s of C a p s i c u m a n n u u m at four maturity l e v e l s *

Total c a r o t e n o i d s

Growth

Long red

Pacific

stage

cayenne

bell

B-Carotene (10)

oe-Carotene (14)

Z e a x a n t h i n (15)

Lutein (16)

Neoxanthin

(19)

6.2

7.0

5.3

2.9

8.1

8.2

3.1

9.8

M-3

173.0

75.6

65.3

63.1

40.9

M-4

728.0

202.0

196.9

223.4

96.4

A b s e n t f r o m b o t h stages in all v a r i e t i e s 0.0

0.4

M-3

0.0

0.0

0.0

M-4

0.0

0.0

0.1

0.4

1.0

M-1

8.0

2.0

0.8

0.4

2.0

M-2

12.0

2.0

2.0

0.4

3.0

M-3

40.0

3.0

7.0

3.0

9.0

M-4

08.0

16.0

28.0

36.0

16.0

M-1

1.0

0.0

0.1

0.0

0.0

M-2

3.0

0.0

0.2

0.0

0.0

M-3

5.0

0.4

0.4

0.0

0.4

M-4

9.0

1.0

1.0

0.0

2.0

M-1

tr.

tr.

0.1

0.1

0.2

M-2

0.4

0.1

0.2

0.2

0.4

M-3

24.0

2.0

4.0

5.0

3.0

18.0

16.0

16.0

5.0

82.0

A b s e n t f r o m b o t h stages in all v a r i e t i e s

M - 1 , M-2 3.0

0.6

0.4

0.8

0.4

21.0

7.0

3.0

3.0

5.0

M-1

5.0

2.0

1.0

0.4

0.6

M-2

11.0

2.0

1.0

0.4

1.0

M-3

16.0

3.0

6.0

6.0

7.0

M-4

41.0

15.0

17.0

19.0

10.0

M-1

8.0

2.0

2.0

0.5

3.0

M-2

12.0

2.0

2.0

0.6

4.0

M-3

0.6

0.2

0.4

0.1

9.0

M-4

0.0

0.0

0.0

0.0

9.0

M-1

2.0

1.0

0.2

0.2

0.4

M-2

4.0

2.0

0.4

0.2

0.8

M-3

4.0

1.0

0.8

0.8

0.7

M-4 (18)

wonder

42.4

M-3 Violaxanthin

Golden

gold

24.0

M-4 Antheraxanthin (17)

College

M-2

M-1

M - 1 , M-2

P h y t o e n e (12)

Ram horn

51

T a b l e 7. C o n t ' d

Capsanthin

(20)

Growth

Long red

stage

Pacific

cayenne

Ram

bell

horn

M - 1 , M-2 M-3 M-4

Capsorubin

(21)

College

Golden

gold

wonder

A b s e n t f r o m b o t h stages in all varieties 43.0

29.0

224.0

65.0

M - 1 , M-2

18.0

21.0

52.0

0.0

59.0

0.0

A b s e n t f r o m b o t h stages in all varieties

M-3

10.0

M-4

12.0

7.0

68.0

4.0

27.0

15.0

0.0

19.0

0.0

* Rahman, F.M.M., Buckle, K.A. (1980): Pigment changes in capsicum cultivars during maturation and ripening, J . FoodTechnol., 15, 241 (with permission) Notes: (1) M-1: immaturate green; M-2: mature green; M-3: half ripe; M-4: fully colored ripe stages (2) All values as mg per 100 g fruit (3) Numbers in parenthesis after individual carotenoids refer to their numbers in Figs 3-5.

phyll cycle discussed earlier could be visualized to operate in Capsicum fruits. (3-Carotene appears to be continuously formed and metabolized as it forms a substantial percentage at all stages of maturity and ripening. a-Carotene was found in four of the five varieties and increasing with ripening, though in very small absolute amounts. Lutein and neoxanthin, dominant in the green stage, decrease rapidly at the halfripened stage and are either absent or dropped to very low levels at the ripening stage. The yellow fruit variety, Golden wonder, was the only sample in which lutein increased in actual amounts. Several specific pathways for carotenoid biosynthesis have evolved in eukaryotes by novel enzymatic activities. In the Capsicum genus capsanthine-capsorubin synthase (CCS) is able to convert the epoxy-carotenoids antheraxanthin (17) and violaxanthin (18) into the red ketocarotenoids capsanthin (20) and capsorubin (21), respectively (Fig. 4) (Bouvier et al., 2003). The cyclopentanol structure characteristic of the red ketocarotenoids is conceived to be formed from a 3-hydroxy-5,6-epoxy end-group [see antheraxanthin (17) and violaxanthin (18) in Fig. 5] by a pinacolic rearrangement. Since CCS also exhibits lycopene cyclase activity, it is likely to be related to similarities in the chemical mechanims leading to the formation of betarings in P-carotene (Fig. 3) and kappa-ring in capsanthin (20) and capsorubin (21) (Fig. 6). In both mechanisms a carbenium ion at C(5) is formed, and both reactions are probably initiated by proton attack on either a carbon-carbon double bond or the oxyrane oxygen atom (Deli, Osz, 2004). The first direct evidence of the conversion of zeaxanthin epoxides into the cyclopentanyl ketocarotenoids was obtained by Camara et al. A chloroplast-enriched fraction of the semiripened fruit of Capsicum annuum effected direct conversion of violaxanthin (18) into capsorubin (21) (Camara, 1980), antheraxanthin (17) essentially into capsanthin (20) and to a small extent to capsorubin (21) (Camara, Monegar, 1981). In all experiments the control with boiled chromoplast preparation yielded no transformation of the labeled starting materials to the expected ketocarotenoids demonstrating the enzymic nature of the conversions. 52

HO

Zeaxanthin (15)

Capsanthin-5,6-epoxide (21)

Fig. 6. S i m p l i f i e d b i o s y n t h e t i c route of Capsicum

c a r o t e n o i d s (see text for further d e t a i l e d

explanation)

6.2.2.1. Methods for determination of red and total carotenoids The methods described below (6.3.3) for determining total pigments of Capsicums are based on measuring the absorbance at the isoabsorption wavelength of the different pigments or at the absorbtion maxima of the dominant pigment. The measure53

merits give only approximate values sufficient for comparative evaluation, and the measured absorbencies do not give information on the true pigment concentrations since the absorption maxima and the molar absorption differ between the red and the yellow components (Baranyai et al., 1982). The determination of the red and the yellow components is desirable for a better understanding of the variations in color that the eye discerns. Baranyai and Szabolcs described a simple spectrophotometric method based on the observation that on reduction of the paprika extracts with sodium borohydride the red ketocarotenoids yielded an equal amount of yellow components, causing an increase in the absorption at the maxima of the yellow components (Baranyai, Szabolcs, 1976). This method gave values 20 to 25% higher than the Benedek number (Benedek, 1958, 1959) usually used in Europe for total pigments since in the new method the contribution of all components is measured, even that of the cis-com¬ pounds naturally present.

6.2.3. Capsaicinoids Seven capsaicinoids and their main chemical structures were identified in Capsicum species (Table 8). T a b l e 8. S e v e n c a p s a i c i n o i d s a n d their m a i n c h e m i c a l structures i d e n t i f i e d in Capsicum N a m e s of capsaicinoids

C h e m i c a l structures (CH ) CH-CH=CH(CH ) -CO-R

Capsaicin

(CH ) CH(CH ) -CO-R

Dihyrocapsaicin

3

3

2

2

2

2

4

6

(CH ) CH(CH ) -CO-R

Nordihydrocapsaicin

(CH ) CH(CH ) -CO-R

Homodihydrocapsaicin

3

3

2

2

2

2

9

9

(CH ) CHCH=CH(CH ) -CO-R

Homocapsaicin

CH (CH ) -CO-R

Nonanoid acid vanillylamide

CH (CH ) -CO-R

Decanoid acid vanillylamide

3

3

3

2

2

2

2

species*

5

7

8

* After Anu A., Peter K. V. (2000): The Chemistry of Paprika. Indian Spices 37:15-18 (with permission) The first five compounds from the seven represent the capsaicin homologues, meanwhile the last two represent the capsaicin analogues

6.2.3.1. Chemistry of capsaicinoids Capsicum fruits have been valued for over thousands of years for the piquant taste they added to the flavorless foods, as well as for the therapeutic effects as a stimulant and counterirritant. These effects have been related to the components stimulating pungency. The chemical study of the active constituents that are responsible for pungency of the Capsicum fruits have been, however, examined only since the beginning of the 19th century, with the interest in the study of alkaloids in natural products. Bucholz in 1816 reported to have found that the pungency-stimulating compounds 54

of capsicum are extractable by maceration with organic solvents. Thresh in 1846 reported the crystallization of the active component and named it capsaicin. Micko improved the isolation method of capsaicin and showed the presence of both hydroxyl and methoxy groups and postulated the structural similarity to vanillin (Micko, 1898). Two decades later, Nelson made the significant contribution to the structure of capsaicin by identifying the compounds formed by hydrolysis (Nelson, 1919). Nelson and Dawson (1923) further determined the position of the unsaturation of the acid moiety at the C(6)-C(7) position. This established the structure as 8-methyl-6-nonenoyl-vanillylamide ((E)-N-(4-hydroxy-3-methoxybenzyl)-8-methyl-6-nonenamide) (24) (Fig. 7).

Fig. 7. C h e m i c a l structure of c a p s a i c i n ( 2 4 ) (see t h e text, for further d e t a i l e d e x p l a n a t i o n )

The degree of pungency (heat or bite) is determined by the amount of compounds called capsaicinoids in the fruit. The capsaicinoids are a family of natural products isolated from the dried fruits of chilli peppers. These substances are the principles that produce the characteristic sensations associated with ingestion of spicy cuisine as well as the agents responsible for causing severe irritation, inflammation, erythema, and transient hypo- and hyperalgesia at sites exposed to capsaicinoids. Capsaicinoids are particularly irritating to the eyes, skin, nose, tongue and respiratory tract. The nature of the causal components in the spice has been established as a mixture of acid amides of vanillylamine and C8 to C13 fatty acids, which are known generally as capsaicinoids. The major capsaicinoids in red peppers are capsaicin, (E)-N-(4-hydroxy-3-methoxybenzyl)-8-methyl-6-nonenamide (24), and dihydrocapsaicin (28), the latter being a 6,7-dihydro analogue of capsaicin. Nordihydrocapsaicin (27) is recognized as the third major capsaicinoid, and the structure is a mono-nor homologue of the acyl residue of dihydrocapsaicin, that is, N-(4-hydroxy3-methoxybenzyl)-7-methyloctanamide (Fig. 8). In commercial capsicums, capsaicin generally comprises 33 to 59%, dihydrocapsaicin accounts for 30 to 5 1 % , nordihydrocapsaicin for 7 to 15% and the remainder is less than 5% of the capsaicinoids (Reineccius, 1994). The structures of capsaicin as well as its homologues and analogues are given in Fig. 6. The seven homologous branched-chain alkyl vanillylamides are capsaicin (24), homocapsaicin I (25), homocapsaicin II (26), nordihydrocapsaicin (27), dihydrocapsaicin (28), homodihydrocapsaicin I (29) and homodihydrocapsaicin II (30) (Hoffman et al., 1983; Redly et al., 2001a,b; Karnka et al., 2002). In addition, three straight-chain analogs, octanoyl vanillylamide (31), nonoyl vanillylamide (nonivamide) (32) and decyl vanillylamide (33), have also been shown to occur in Capsicum fruits (Govindarajan, 1986f). Trace amounts of further capsaicin homologues and analogues have also been characterized (Suzuki et al., 1957; 55

Suzuki, Iwai, 1984; Govindarajan, 1986a; Collins, Bosland, 1994; Maillard et al., 1998; Bosland, Votava, 2000; Thompson et al., 2005; Schweiggert et al., 2006). Capsaicin and its natural homologues are always found in the trans (E) form because in cis (Z), the -CH(CH ) and the longer chain on the other side of the A6,7 carboncarbon double bond will be close together causing them to repel each other. This steric hindrance does not exist in the trans isomer. This additional strain imposed causes the cis isomer to be of a less stable arrangement than the trans isomer. 3

2

o C H , C K ^ r \

ll

^ C H j - N H - C - R

Name

Structure of the „ R " chain H

CH

I

C a p s a i c i n (24)

3

I

— CH —CH —CH —CH —C = C-CH-CH 2

2

2

2

3

I

H

H

CH

I

I H o m o c a p s a i c i n I (25)

3

—CH —CH —CH —CH —C = C-CH —CH—CH 2

D i h y d r o c a p s a i c i n (28)

—

H

C

H o m o d i h y d r o c a p s a i c i n I (29)

— C H

H o m o c a p s a i c i n II (26)

— C H

2

2

_

2

C

H

2

_

2

C

H

2

_

C

2

H

2

_H

H C

H

2

I

2

2

_CH C

H

3

2

_

C

_

H

I

C

H

CH

3

3

3

— CH —CH — CH — CHj-CH —CH —CH-CH 2

2

2

2

2

— C H — C H — C H — C = C— C H — C H — C H CH 2

2

2

2

3

3

3

H

H o m o d i h y d r o c a p s a i c i n 11(30)

(27)

I

CH — C H — C H — C H — C H — CH — C H — C H — CH — CH 2

2

2

2

2

3

2

2

_qh _ _a-|

3

Ncotradni h i ni d e (31) O oyydl rvoacnaipl lsyal iacm

— C H —_ C H _— CHH — _ C H — C H 2 —CCH H — C H 3 —

N o n o y l v a n i l l y l a m i d e (32)

—CH —CH —CH —CH — CH —CH —CH —CH

D e c y l v a n i l l y l a m i d e (33)

— CH — CH —CH — CH — CH — CH — CH — CH — CH

C

H

2

2

2

C

H

C

2

2

2

2

2

2

2

2

C

H

2

2

2

2

2

2

2

2

2

3

2

2

Fig. 8. C h e m i c a l structures of c a p s a i c i n h o m o l o g s a n d a n a l o g s (see t h e text for further detailed explanation)

56

2

3

3

The capsaicin biosynthetic pathway has two distinct branches, one of which utilizes L-phenylalanine (34) as the precursor of aromatic residue of capsaicinoids, presumably via rrafls-cinnamic acid (35) and its hydroxylated derivatives trans-caffeic acid (36) and rrans-ferulic acid (37) following the well-established pathways in other plants (Ishikawa, 2003). Vanillylamine (38) as precursor showed a high level of incorporation into the capsaicinoids and possibly is the immediate progenitor of natural capsaicinoids (Fig. 9). The enzymes involved in the formation of the precursors, phenolics, and fatty acids, are similar to those studied for long in other biological systems. The second branch forms the branched-chained fatty acids by elongation of deaminated valine (Fig. 9). The capsaicinoids synthetase, however, has been found to have narrow specificity in accepting only the /so-C(9:0) to C(11:0) fatty acids and in the fruit system forming predominantly the vanillylamides of evennumber branched fatty acids, capsaicin (24) and dihydrocapsaicin (28), in all the cultivated varieties of the Capsicum species (Ravishankar et al., 2003). In the isolated systems, however, the synthetase favors the formation of nordihydrocapsaicin (27) and capsaicin (24), while at the light-induced activation of the synthetase in Capsicum annuum cv. grossum, there is higher formation of nordihydrocapsaicin (27) and dihydrocapsaicin (28) (Govindarajan, 1986 a,b). Synthesis of capsaicinoids by means of recent development of biotechnological methods has been reviewed in deatails (Ravishankar et al., 2003). ,4

Kopp and Jurenitsch (1982) have found by administration of C-labeled capsaicin into the mesocarp of Capsicum fruits, that there was no interconversion of capsaicin (24) and dihydrocapsaicin (28), confirming earlier observations (Neumann, 1966) on the stability of formed capsaicinoids. The conversion of the saturated acid into the unsaturated acid should therefore take place prior to the condensation with vanillylamine to form the different capsaicin homologs. The data confirmed the earlier suggestion that capsaicinoid composition is regulated besides the substrate specificity (Fujiwake et al., 1980) more by available precursor amino acids and conditions controlling the formation of the limited branched acyl acids accepted by the enzyme capsaicinoids synthetase. The accumulated data from analysis of fruits from different species varieties and cultivars (Jurenitsch et al., 1978, 1979a,b; Jurenitsch, Leinmueller, 1980) have shown considerable variation in the total content and composition of the capsaicinoids and analogs. Total separation of major and minor capsaicinoids and related compounds have been published by Jurenitsch and Leinmueller (1980) through preliminary thin-layer chromatography clean-up, separation of capsaicinoids and related compounds, saponification, methylation and separation of the alkyl acid methyl esters by gas chromatography (Table 9). Analysis of five cultivated species of Capsicum showed capsaicin (24) and dihydrocapsaicin (28) to be the dominant constituents together forming about 80% of the total, while nordihydrocapsaicin and homodihydrocapsaicin, found in all samples, varied widely - from 1 to 12%. Other higher homologs and straight chain saturated analogs when present are at low levels. It is also worth mentioning that in spite of the large variations in the total capsaicinoid content within and between the Capsicum species, the ratio of the principal components, capsaicin and dihydrocapsaicin, has been found to vary narrowly -

57

<^

\ —

CH —CH— COOH 2

L-Phenylalanine (30)

NH, Phenylalaninea m m o n i a lyase

\ — C H = C H - C O O H

trans-Cynnamic a c i d (31)

^ ^ C H = CH-COOH

frans-p-Coumaric acid (32)

frans-Cinnamate4-monooxygenase

HO—ft

frans-p-Coumarate3-monooxygenase HO

—K

frans-Caffeic acid (33)

7—

Caffeic a c i d O-methyltransferase CH,0

—i

\—CH=CH-COOH

frans-Ferulic acid (34)

CH 0. 3

V a n i l l y l a m i n e (35)

HO

C(10:0)-, C(10:1)-/'soacids

Capsaicinoids synthetase

C(9:0)-, C(11:0)-/'soacids

—h—CH —NH—C—R 2

O II

<-

L-valine L-Leucine

Capsaicinoids

Fig. 9. S i m p l i f i e d p a t h w a y of b i o s y n t h e s i s of c a p s a i c i n o i d s (see t h e text for further d e t a i l e d explanation)

58

Table 9. C a p s a i c i n o i d c o m p o s i t i o n in fruits of f i v e c u l t i v a t e d s p e c i e s of C a p s i c u m * P e r c e n t a g e s of c o m p o n e n t c a r o t e n o i d s Species

Sample

OV

NDC

NV

C

DC

DV

No. C.

annuum var.annuum

C.

annuum var. p e n d u l u m

HC

HDC

Ratio C:CD

1

0.63

13.96

0.67

49.66

34.52

0.55

_

_

0.09

8.35

1.80

44.63

36.02

4.17

-

1:0.7

2

4.94

1:0.8

3

_

_

_

2:2.1

1.45

1:1.15

4

-

3.45

1.29

64.86

29.47

0.93

3.75

1.24

37.35

54.64

1.57

-

C.

frutescens

5

-

0.55

0.35

66.97

28.73

0.66

2.32

0.42

2.3:1

C.

chinense

6

0.54

6.04

1.06

61.66

24.75

1.44

2.73

1.79

2.5:1

C.

pubescens

7

_

3.47

1.23

36.57

45.97

0.68

12.08

1:1.3

8

0.42

8.42

1.78

29.78

46.62

8.61

4.38

1:1.6

_

-

* Jurenitsch, J . , Leinmueller, R. (1980): Quantification of nonylic acid vanillylamide and other capsaicinoids in the pungent principle of Capsicum fruits and preparations by gas4iquid chromatography on glass capillary columns (in German), J . Chromatogr., 189, 389 (with permission) Note: The capsaicinoids and analogs are given in the order of increasing gas chromatography retention of methyl esters of the respective acids obtained by saponification. OV - octanoyl vanillylamide; NDC - nordihydrocapsaicin; NV - nonoyl vanillylamide; C - capsaicin; DC - dihydrocapsaicin; DV - decyl vanillylamide; HC - homocapsaicin; DHC - homodihydrocapsaicin

about 1:1 for Capsicum annuum to 2:1 for Capsicum frutescens and Capsicum chinense (Jurenitsch, Kampelmuehler, 1980; Jurenitsch, 1981; Jurenitsch, Leinmueller, 1980). High performance liquid chromatograpy (HPLC) analysis of capsaicinoids offers a more simplified but efficient separation of capsaicinoids. The preliminary sample clean-up is generally not necessary and the separation is done on direct extracts and mostly at room temperatures, avoiding thermal degradation and loss of materials of interest. Results of reversed phase HPLC analysis of samples of natural capsaicinoid mixtures (oleoresins) from different sources and two cayenne extracts are given in Table 10 (Johnson et al., 1979). As data show, in all samples capsaicin and dihydrocapsaicin are the main constituents, and the ratio of capsaicin to dihydrocapsaicin varies from 1:0.5 to 1:1. Kozukue et al. (2005) applied reversed phase HPLC-MS analysis of eight capsaicinoids in peppers and pepper-containing foods. Quantitation was based on the UV response at 280 nm. The results also demonstrated that in all samples capsaicin and dihydrocapsaicin are the main capsaicinoids, the ratio of them, however, varied in a much wider range. All capsaicinoid analogs posses a 3-hydroxy-4-methoxy-benzylamide (vanilloid) pharmacophore, but differ from capsaicin in their hydrophobic alkyl side chain. Differences in the side chain moiety include saturation of the carbon-carbon double bond, deletion of a methyl group, and changes in the length of the hydrocarbon chain

59

T a b l e 1 0 . C a p s a i c i n o i d s c o m p o s i t i o n of C a p s i c u m extracts a n d o l e o r e s i n s * Weight

Sample

C a p s a i c i n o i d s (purified mixture)

(%)

Total

C

NDC

HC

DC

HDC

C:DC

93.20

40.59

9.39

1.37

39.35

2.50

1:0.52

Oleoresins Mannheimer

4.00

2.07

0.32

0.05

1.47

0.09

1:0.70

Stange

2.94

1.44

Kalamazoo

0.39

0.03

0.95

0.13

1:0.66

1.52

0.95

0.08

N o r d a EP-642

1.85

1.09

0.07

Norda

1.40

1.40

-

Cayenne 510

0.24

0.13

0.01

Cayenne 970

0.92

0.51

0.07

Extracts

-

-

0.01

0.49 0.69

-

0.10 0.32

-

1:0.63

-

1:0.63

0.01

1:0.52 1:0

1:0.63

* Johnson, EX., Majors, R.E., Werum, L., Reiche, P. (1979): The determination of naturally occurring capsaicins by HPLC, in Liquid Chrom. Anal. Food Beverages, Vol. 1, Charalambous, C, Ed., Academic Press, New York, pp. 1 7-29 (with permission) Note: Capsaicinoids in order of their HPLC elution

(Fig. 8). Previous structure-activity studies using models for the study of acute pain and altered pain sensitivity in mice have demonstrated a strict structural requirement for both the vanilloid ring pharmacophore and a hydrophobic alkyl chain that may be saturated or unsaturated, branched or unbranched, and consist of 8 to 12 carbon atoms for optimal binding and activation of the capsaicin receptor, TRPV1 (Walpole etal., 1993a,b,c). With regard to the pungency of capsaicinoids and their related (synthetic) compounds, it can be stated that an aromatic ring with a phenolic hydroxyl group and a methoxy in ortho position to each other is a basic prerequisite. An aliphatic side chain is also necessary the length and structure of which are also important. The pungency is greatly enhanced by an acid amide group, in this instance vanillylamide, as found in capsaicinoids (Kulka, 1967; Govindarajan, Satyanarayana, 1991). Any change in one or more of these structural features decreases or even abolishes the pungency stimulation. Careful sensory testing by threshold tests has shown that the homologs of capsaicin and dihydrocapsaicin and the straight-chain analogs stimulate only 25 to 55% of the pungency stimulated by the two dominant capsaicinoids (Table 4). Pungency is an important sensory quality of Capsicum fruits and their processed forms. As it was shown above, the stimuli in these spices are specific compounds with certain structural features. Pungency is now being clearly understood as a stimulus response phenomenon. Its evaluation involves both qualitative and quantitative aspects. In the absence of reliable analytical technique for estimation of capsaicinoids in the earlier times, spice processing companies and pharmacopeia used sensory testing as a rapid limit value method. The basic principle of pungency evaluation using the organoleptic method was established by Scoville (1912).

60

6.3. Capsicum: Quality control Capsicum is now one of the two most widely used spices. The wide popularity of the spice capsicum is attributable to its wide range of shapes and sizes and such sensory attributes as color, pungency, and distinctive aroma that make generally insipid bulk nutritive flash and cereal foods more appetizing. The quality of food should fulfill the consumers' expectations - not necessarily the maximum of each attribute, but the optimal level and combination appropriate for each food. Thus a range of quality attributes is required to make different foods of optimal quality. Quality control which can be exercised through measurement of the physical and chemical properties of the component stimuli needs to be validated by a relationship with sensorially perceived responses individually and in combination. It is obvious that the accuracy and reproducibility of any instrumental method meaningful for food quality measurement is that which correlates with the sensorially perceivable differences (Kramer, 1966). Besides the sensory attributes, capsicum, like other products used as foods and food additives, should also have certain functional properties for its optimal use in the industrial sectors, which also have to be considered. The standards of the importing countries are based on the requirements of the food processing industries and include additional emphasis on cleanliness, which progressively cover, in addition to insects and rodent parts and extrata, limits of chemical and microbiological contaminations, and absence of health-hazard organisms. These specifications assure genuineness, purity, and cleanliness, but they do not give information on the sensory attributes which the consumers require. In the case of some processed products, e.g., ground paprika and oleoresin, specifications for total color and capsaicinoids content are found in standards and manufacturer literature, these being the main selling factors in this increasing competitive market (Govindarajan, 1986c). Herbs and fruits that are used as spice, active pharmaceutical ingredients of drugs, or constituents of food additives should also fulfill even more special requirements described by the Pharmacopeias and/or other international organizations like EC, FAO, WHO, ISO, ASTA, etc., and national bodies, which guide the industry concerned in the respective activities such as manufacturing or trade. The standards are the results of continuous efforts in standardization. For lack of space, it is not possible to cover all such standards. Only those have been listed that are closely related to the quality of Capsicum and Ca/xvj'cMm-originated products that can be used for the pharmaceutical industry.

6.3.1. Capsicum fruits 6.3.1.1. The European Pharmacopeia (Ph. Eur. 5.0) The 2006 edition of European Pharmacopeia Edition 5.0 lists Capsicum fruit (Capsici fructus) and describes its Definition, Identification, Nonivamide Test, and Assay as follows.

61

CAPSICUM Capsici fructus (Ph. Eur. 5.0) DEFINITION Dried ripe fruits of Capsicum annuum L. var. minimum (Miller) Heise and smallfruited varieties of Capsicum frutescens L. Content: minimum 0.4 per cent of total capsaicinoids expressed as capsaicin ( C H N 0 ; Mw.: 305.4) (dried drug). 18

27

3

IDENTIFICATION A) The fruit is yellowish-orange to reddish-brown, oblong conical with an obtuse apex, about 1 cm to 3 cm long and up to 1 cm in diameter at the widest part, occasionally attached to a 5-toothed inferior calyx and a straight pedicel. Pericarp somewhat shrivelled, glabrous, enclosing about 10 to 20 flat, reniform seeds 3 mm to 4 mm long, either loose or attached to a reddish dissepiment. B) Reduce to a powder. The powder is orange. Examine under a microscope using chloral hydrate solution R. The powder shows the following diagnostic characters: fragments of the pericarp having an outer epicarp with cells often arranged in rows of 5 to 7, cuticle uniformly striated: parenchymatous cells frequently containing droplets of red oil, occasionally containing microsphenoidal crystals of calcium oxalate; endocarp with characteristic island groups of sclerenchymatous cells, the groups being separated by thin-walled parenchymatous cells. Fragments of the seeds having an episperm composed of large, greenish-yellow, sinuouswalled sclereids with thin outer walls and strongly and unevenly thickened radial and inner walls which are conspicuously pitted; endosperm parenchymatous cells with drops of fixed oil and aleurone grains 3 pm to 6 pm in diameter. Occasional fragments from the calyx having an outer epidermis with anisocytic stomata, inner epidermis with many trichomes but no stomata; trichomes glandular, with uniseriate stalks and multicellular heads; mesophyll with many idioblasts containing microsphenoidal crystals of calcium oxalate. C) Thin-layer chromatography. Test solution. To 0.50 g of the powdered drug (500) add 5.0 ml of ether R, shake for 5 min and filter. Reference solution. Dissolve 2 mg of capsaicin R and 2 mg of dihydrocapsaicin R in 5.0 ml of ether R. Plate: TLC octadecylyl I silica gel plate R. Mobile phase: water R, methanol R (20:80 V/V). Application: 20 pi, as bands. Development: over a path of 12 cm. Drying: in air. Detection: spray with a 5 g/1 solution of dichloroquinone chlorimide R in methanol R. Expose the plate to ammonia vapour until blue zones appear. Examine in daylight.

62

Results: see the sequence of the zones present in the chromatograms obtained with the reference solution and the test solution. Furthermore, other zones may be present in the chromatogram obtained with the test solution (Fig. 10). Top of the plate

Capsaicin: a blue zone

A blue zone (capsaicin)

Dihydrocapsaicin: a blue zone

A blue zone (dihydrocapsaicin)

Reference Solution

Test s o l u t i o n

Fig. 10. C h a r a c t e r i z a t i o n of t h e t o p of t h e p l a t e

TESTS Nonivamide. Liquid chromatography. Test solution. To 2.5 g of the powdered drug add 100 ml of methanol R. Allow to macerate for 30 min. Place in an ultrasonic bath for 15 min. Filter into a 100 ml volumetric flask, rinse the flask and filter with methanol R. Dilute to 100.0 ml with methanol R. Reference solution. Dissolve 20.0 mg of capsaicin R and 4.0 mg of nonivamide R in 100.0 ml of methanol R. Column: - size: 1 = 0.25 m, 0 = 4.6 mm, - stationary phase phenylsilyl silica gel for chromatography R (5 pm), - temperature: 30°C. Mobile phase: mixture of 40 volumes of acetonitril R and 60 volumes of a 1 g/1 solution of phosphoric acid R. Flow rate: 1.0 ml/min. Detection: spectrophotometer at 225 nm. Injection: 10 pi. System suitability: reference solution: - resolution: minimum 3.0 between the peaks due to capsaicin and nonivamide. Limit: calculate the percentage content of nonivamide - nonivamide: maximum 5.0 per cent of the total capsaicinoid content. ASSAY Liquid chromatography as described in the test for nonivamide. Calculate the percentage content of capsaicinoids (Fig. 11).

63

max

1 . nordihydrocapsaicin

70

3

60 50

2. nonivamide 3. capsaicin

40 30 20

4. dihydrocapsaicin

4

2

IA

10 0 0 Fig.

10

15

20

25

35

mm

1 7. C h r o m a t o g r a m of n o n i v a m i d e a n d t h e assay of c a p s i c u m ( P h . Eur. 5.0, p p 1175)

6.3.2. Capsicum extracts - Oleoresin 6.3.2.1. Capsicum extracts Presently, virtually every commercial spice extraction is carried out by one of two methods. One method, solvent extraction, involves treating a ground dry spice with an organic solvent such as hexane, acetone, methanol, ethanol or methylene chloride. Pursuant to this method, the spice extract is recovered by removal of the solvent, usually by distillation with heat under vacuum. The spice extract recovered in this way is known as an "Oleoresin" (Eisvale, 1981; Pruthi, 2003). In the case of oleoresin from Capsicum, the oleoresin is further treated with polar solvent, methanol, in order to separate the pungent component Oleoresin Capsicum from the color component Oleoresin paprika (see Table 11). Oleoresins are used almost exclusively by the food and pharmaceutical industries as a substitute of ground spices and spice tinctures. The composition of an oleoresin is affected by the choice of the organic solvent used in the extraction, but typically will include phospholipids, oils, waxes, sterols, resins, and a range of non-volatile and volatile compounds which make up much of the aroma and flavor of the original spice. In its use as food additive, the best oleoresin of Capsicum is that which contains the color and flavor components and that which truly recreates, when appropriately diluted in food formulations, the sensory qualities of fresh materials (Govindarajan, 1986c). The other commercial method of spice extraction is the aqueous distillation of the whole or ground, fresh or dried spice using either boiling water or steam. This method recovers only the steam volatile components of the spice; i.e. the "essential oil" which is high in aroma and flavor compounds (Simon, 1990). Many variations of these two methods are possible. The essential oil may be prepared by distillation from the original spice, or by distillation from a previously prepared solvent extracted oleoresin. These traditional processes have a number of disadvantages. Most organic solvents are toxic, and government food regulations dictate that their residues must be 64

reduced in the oleoresin to very small concentrations, generally in the range of 25 to 30 ppm or less (Pruthi, 2003). The distillation processes used to remove the solvents, or to recover essential oils, lower the content of the very light volatiles which contribute to aroma and flavor. Of more importance is the growing consumer demand for food ingredients which are completely natural and free of contact with synthetic chemicals. Extraction of spices with supercritical fluid carbon dioxide has been proposed as a means of eliminating the use of organic solvents and providing the prospect of simultaneous fractionation of the extract. The use of supercritical fluids (SCF) for extraction purposes was introduced in the late nineteenth century. A supercritical fluid is a substance at temperatures and pressures beyond its critical point at which the liquid phase of the substance will not exist. At these temperatures and pressures, the supercritical fluid has properties between gas- and liquid-phase characteristics. These properties make supercritical fluids efficient extraction solvents with high mass transfer characteristics (McHugh, Krukonis, 1986; Krukonis, 1988). Consequently, supercritical fluids are often used to selectively extract or separate specific compounds from a mixture by varying fluid density through changes in pressure and temperature. In food technology, the use of supercritical fluids is essentially limited to supercritical carbon dioxide (SCF-C0 ) extraction since carbon dioxide has the advantages of being inexpensive and nontoxic and because its critical point is easily reached. Oleoresins have several advantages over ground spices, e.g. elimination of microbial contamination, uniformity of color and flavor strength, and optimal utilization. The Essential Oil Association of America has detailed specifications for three types of Capsicum oleoresins (Table 11). Oleoresin paprika is mainly used as food coloring in meats, dairy products, soups, sauces and snacks. Oleoresin red pepper is used for both coloring and pungency, mainly in canned meats, sausages, in some snacks and in a dispersed form in some drinks such as gingerale. Oleoresin Capsicum is the most pungent and is used for its counter-irritant properties in plasters and some pharmaceutical preparations. 2

T a b l e 1 1 . N o m e n c l a t u r e of o l e o r e s i n s of C a p s i c u m s (Essential O i l A s s o c i a t i o n , 1 9 7 5 ) T y p e of o l e o r e s i n

Botanical

Capsicums

source

Oleoresin

Capsicum

( E O A N o . 244)

C.

frutescens

or C.

annuum

Preparation

Solvent

Color

Color

Scoville

value

description

Heat Units

4,000 max

C l e a r r e d , light

480,000 min

extraction

amber, or dark red

O l e o r e s i n red

pepper

C.

( E O A N o . 245)

var.

Oleoresin

C.

paprika

( E O A N o . 239)

annuum longum annuum

Solvent

20,000 max

D e e p red

Solvent

40,000-

D e e p red

extraction

100,000

240,000 min

extraction N i l or negligible

-

65

6.3.2.2. Capsicum Oleoresin The 2006 edition of the United States Pharmacopeia-National Formulary (USP30 NF25) lists Capsicum Oleoresin and describes it as follows (The USP30-NF25 Page 1611). Capsicum Oleoresin (USP30-NF25) Definition - Capsicum Oleoresin is an alcoholic extract of the dried ripe fruits of Capsicum annuum var. minimum and small fruited varieties of C. frutescens (Solanaceae). It contains not less than 8.0 percent of total capsicums [capsaicin ( C H N 0 ) , dihydrocapsaicin (C H 9N0 ), and nordihydrocapsaicin ( C H N 0 ) ] . Caution: Capsicum Oleoresin is a powerful irritant, and even in minute quantities produces an intense burning sensation when it comes in contact with the eyes and tender parts of the skin. Care should be taken to protect the eyes and to prevent contact of the skin with Capsicum Oleoresin. 18

29

3

18

2

3

]7

27

3

Identification - To about 0.5 g of it in a beaker add 5 ml of water and 10 ml of a mixture of water, 0.2 M potassium chloride, and 0.2 N hydrochloric acid, and mix. Add 5.0 ml of 0.5 M sodium nitrite and 5.0 ml of 0.02 M sodium tungstate, and mix. Heat at 55°C to 60°C for 15 minutes, allow to cool, and filter. To the fdtrate add 10 ml of 1 N sodium hydroxide: a bright yellow color is produced (presence of capsaicin). Assay Mobile phase - Prepare a mixture of methanol and 2% acetic acid (56:44), fdter through a 0.5 pm or finer porosity filter, and degas. Standard preparation - Prepare a solution of USP Capsaicin RS in methanol having a known concentration of about 0.5 mg per ml. Filter a portion of this solution through a 0.2 pm porosity filter, and use the filtrate as the Standard preparation. Assay preparation - Transfer about 1000 mg of Capsicum Oleoresin, accurately weighed, to a 100-ml volumetric flask, dissolve in and dilute with methanol to volume, and mix. Filter a portion of this solution through a 0.2 pm porosity filter, and use the filtrate as the Assay preparation. Chromatographic system - The liquid chromatograph is equipped with a 280 nm detector and a 3.9 mm x 30 cm column that contains packing LI. The flow rate is about 2 ml per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the tailing factor is not more than 2.0; and the relative standard deviation for replicate injections is not more than 2.0%. Chromatograph the Assay preparation, and record the peak responses as directed for Procedure: the relative retention times are about 0.9 for nordihydrocapsaicin, 1.0 for capsaicin, and 1.6 for dihydrocapsaicin; and the resolution, R, between the nordihydrocapsaicin peak and the capsaicin peak is not less than 1.2. Procedure: Separetely inject equal volumes (about 10 pi) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms,

66

and measure the responses for the three major peaks. Calculate the percentage of total capsaicins in the portion of Capsicum Oleoresin taken by the formula: (CP/WXru/rs) in which C is the concentration, in mg per ml, of USP Capsaicin RS in the Standard preparation; P is the designated purity, in percentage, of USP Capsaicin RS, W is the weight, in mg, of Capsicum Oleoresin taken to prepare the Assay preparation; r is the sum of the peak responses for nordihydrocapsaicin, capsaicin, and dihydrocapsaicin obtained from the Assay preparation; and r is the peak response obtained from the Standard preparation.

v

s

6.3.3. Quantitation of capsicum pigments Appearance and color, the first of the perceived attributes, directly provide a basis for a decisison of appropriateness. Size, shape, and maximum percentage of defects are easily measured specifications given in standards. The color of Capsicum fruits is basically determined by the nature and distribution of the above-described carotenoids which can be hidden or modified by other pigments such as chlorophylls and anthocyanides. The major coloring pigments in paprika are capsanthin and capsorubin, comprising the majority of the total carotenoids. Other pigments are p-carotene, zeaxanthin, violaxanthin, neoxanthin and lutein (Anu, Peter, 2000). It is also worth repeatedly mentioning that the relative amounts of the colored pigments are changing during the ripening period according to rather well investigated biochemical pathways (Rahman, Buckle, 1980; Rodriguez-Amaya, 1997; Hornero-Mendez et al., 2000; Gnayfeed et al., 2001; Deli, Molnar, 2002). It is also worth mentioning that carotenoid research in the field of plant and food chemistry is a very extensive area. The interested readers can consult recent reviews to learn the analytical methods that are currently used to analyse plant and food samples for their carotenoid contents (Rodriguez-Amaya, 1997; Wall, Bosland, 1998; Deli, Molnar, 2002; Felt et al., 2005). Some of the methods to measure coloring parameters of paprika and oleoresins currently accepted as official are summarized below.

6.3.3.1. The Color Matching Method This early method for total pigments expressed as Nesslerimeter color value used in the industry was standardized and adopted by the Essential Oils Association of America (EOA) for oleoresin capsicum [(Essential Oil Association (1975): Specification of oleoresin paprika - EOA No. 239, Oleoresin Capsicum - EOA No. 244, Oleoresin red pepper - EOA No. 245, Essential Oil Association, New York)]. The method is based on matching the color of the properly diluted oleoresin acetone solution with that of potassium dichromate ( K C r 0 ) and cobaltous chloride (CoC x 6H 0) containing reference solutions. The color values are usually in the range of 40,000 to 100,000 for Oleoresin paprika (see Table 4). 67 2

]2

2

2

7

6 . 3 . 3 . 2 . Spectrophotometric methods The alternative method uses the spectrophotometer to measure the total carotenoid pigments. 6.3.3.2.1. The EOA Method [Essential Oil Association (1975): Specification of oleoresin paprika - EOA No. 239, Oleoresin Capsicum - EOA No. 244, Oleoresin red pepper - EOA No. 245, Essential Oil Association, New York], The absorbance of a 0.01% acetone solution of oleoresin is measured at 458 nm. The absorbance value is multiplied by 61,000 (an empirical factor worked out to relate the data from the color matching method) gives the total pigment as the Nesslerimeter color value. 6.3.3.2.2. The American Spice Trade Association (ASTA) Method

By the above spectrophotometric method, results from different laboratories were not directly comparable due to differences in the spectrophotometers. In the new ASTA 20.1 Method (ASTA, 1968) a reference solution of inorganic salts (potassium dichromate and cobaltous ammonium sulfate in 1.8 M sulfuric acid solution) absorbing in the same region as the carotenoids is used to calculate an instrument factor which makes interlaboratory comparison possible. In the ASTA Method 20.1 for extractable color (pigments) in Capsicums absorbance of acetone extract of ground paprika and other capsicums is measured at 460 nm. The color value (in ASTA) is calculated using the determined instrument correction factor. A direct correlation between the earlier ASTA Method 19, measuring absorbance at 450 nm, and the new Method 20.1 cannot be established, an empirical factor (16.4) in the formula gives values nearly equal to those obtained by the earlier Method No. 19. 6.3.3.2.3. The Hungarian Standard Method

In Hungary, where specified grades of ground paprika are produced (kiilonleges, csemege, edesnemes, rozsa), the total pigments are determined by similar absorbtion measurements. Earlier, the total pigment concentration in ground capsicums or in oleoresins was calculated by using the extinction coefficient in benzene of the major pigment capsanthin ( £ 4 7 7 n m = 1826). The results were expressed in grams of capsanthin per kilogram of dry matter (Hungarian Standard. Examination of Ground Paprika Spice. Determination of Pigment Content, MSZ 9681/5-76.). At present, the measurements are performed using acetone extracts similar to the ASTA Method 20.1 (MSZ 9681-5:2002) 1%

68

6.3.4. Quantitation of pungent principles For the major portion of Capsicum species produced and traded, pungency is the important quality attribute. The nature of the causal components in the spice has been established as a mixture of seven homologous branched-chain alkyl vanillylamides, named capsaicinoids. Small amounts of three straight-chain analogues have also been shown to occur. The chemistry of these compounds has been reviewed (Suzuki, Iwai, 1984). The structures are given in Fig. 6. The average composition of these related vanillylamides in the widely traded chillis (Capsicum annuum var. annuum) varieties is capsaicin 33 to 59%; dihydrocapsaicin, 30 to 51%; nordihydrocapsaicin, 7 to 15%; and others, in the range of 0 to 5% each. Fruits of the species Capsicum frutescens, stimulating high pungency and mostly used in the pharmaceutical industry, have higher capsaicin (63 to 77%) and dihydrocapsaicin (20 to 32%) contents, with other homologues and analogues making up around 10% (Jurenitsch et al., 1978). The total capsaicinoids varied greatly (0.001 to 0.01% in paprika and 0.1 to ( 1 % in chillis), but the proportion of capsaicin and dihydrocapsain ranged from 77 to 90% in the fruits of the species C. annuum and from 89 to 98% in those from species C. frutescens (Govindarajan et al., 1987). As it was shown above (Table 4), the pungency stimulated by the different alkyl acyl vanillylamides varied greatly, all much lower compared to capsaicin and dihydrocapsaicin, which were equal (Govindarajan et al., 1987). Thus, the estimation of total capsaicinoids, reproducibly and accurately correlating with the determined pungency, should be sufficient for quality control. Where the minor capsaicin-related vanillylamides make up a larger portion (above 20%), however, their individual estimation could become necessary because they stimulate much lower pungency. It has been also known for a long time that synthetic nonoyl vanillylamide (pelargonyl vanillylamide) has considerable pungency and heat (Kuika, 1967), and has been found in varying amounts in commercial oleoresins. Therefore, it was necessary to determine the upper limits of the straight-chain analogs to determine adulteration. 6 . 3 . 4 . 1 . Official methods for organoleptic determination of pungency 6.3.4.1.1. The Scoville Method

A number of methods have been reported from time to time since 1912 for assaying the pungency or capsaicin content of Capsicum fruits and/or the processed fruits (Pruthi, 2003). The basic principle of pungency evaluation using an organoleptic method was established in 1912 by W. L. Scoville (Scoville, 1912). The method is based on sensory evaluation determining the amount of sugar to neutralize the heat from the pepper. A solution of the pepper extract variably diluted with sugar solution is tested in increasing concentration. The highest dilution at which pungency is just detected is taken as a measure of the heat value. The dilution value, in milliliters per gram, has since then been called Scoville Heat Units (SHU). The SHU for pure capsaicin is reported as 16-17 x 106 (Table 12). The Scoville Heat Units of various chilli pepper varieties are shown in Table 13. The greatest weakness of the Scoville organoleptic test is its imprecision, because it relies on human subjectivity. 69

T a b l e 1 2 . P u n g e n c y t h r e s h o l d in S c o v i l l e v a l u e s of c a p s a i c i n h o m o l o g s a n d a n a l o g s * Capsaicin homolog/analog

T o d d et a l .

Jurenitsch

(1977)

(1981)

Capsaicin

16.1 ±0.6

17.0

Dihydrocapsaicin

16.1 ±0.6

10.8

Nordihydrocapsaicin

9.3+0.4

9.6

Homocapsaicin

6.9±0.5

Homodihydrocapsaicin

8.1 ±0.7

8.3

Vanillyl pelargonamide

9.2 ±0.4

8.8

Vanillyl capramide

4.5

7.4

Vanyllyl undecanamide

3.5

-

-

* Govindarajan, VS., Rajalakshmi, D., Chand, N. (1987): Capsicum - Production, technology, chemistry and quality. Part IV. Evaluation of quality. In: Furia, TE (ed.) CRC Crit. Rev. Food Sci. Nutr., CRC Press, Boca Raton, 25: 266 (with permission) Pungency is expressed in Scoville Heat Units (millions (106) ml/mg)

T a b l e 1 3 . P u n g e n c y of v a r i o u s C a p s i c u m v a r i e t i e s * Type Habanero

H e a t rating (in Scoville H e a t Units) 200.000-300.000

Tabasco

30.000-50.000

Cayenne

35.000

Serrano

7.000-25.000

Jalapeno

3.500-4.500

Anaheim

1.000-1.400

Bell & Pimento

0

* Ravishankar, G.A, Suresh, B, Giridhar, P, Ramachandra Rao, S, Sudhakar Johnson, T. (2003): Biotechnological studies on Capsicum for metabolite production and plant improvement. In: De, A.K. (ed): Capsicum The genus of Capsicum, Taylor and Francis, London, New York, Chapter 6, pp. 99-128 (with permission) Note: 0-5.000: mild; 5.000-20.000: medium; 20.000-70.000: hot; 70.000-300.000: extremely hot

6.3.4.1.2. The EOA Method This method [(Essential Oil Association (1975): Specification of oleoresin paprika EOA No. 239, Oleoresin Capsicum E A No. 244, Oleoresin red pepper EOA No. 245, Essential Oil Association, New York)] is the codification of the procedure that was in use by the spice processing industry to check the constancy of pungency of a usual trade variety and source. The method, based on the approach of the original Scoville method, specified for oleoresins of capsicum as follows. A standard solution for testing is made by diluting a stock alcoholic solution of the oleoresin and it is tested by five trained panelists. If three of the five on a panel agree on just perceptible pungency at the given dilution (which is equivalent to 70

240,000 ml/g), this value is called the SHU of pungency of the sample. If the pungency response is strong, this first diluted standard is further diluted and the panel testing is repeated to find the dilution at which three of the five judge the pungency just perceptible. The Scoville test run shows a rather high correlation to total capsaicinoids content (1,500,000 Scoville units = 1% capsaicin). 6.3.4.1.3. The British Standard Method The British Standard Institution adapted the above industry procedure except that dilutions were rationalized in more convenient volumes [(British Standards Institution (1979): Methods for testing spices and condiments: Determination of Scoville index of chillies, BS 4548 (Part 7), BSI, London)]. 6.3.4.1.4. The International Standard Organization (ISO) Method

The British Standard Method adopted and improved by the International Standards Organization (ISO) requires the testing of a series of dilutions around the anticipated value by individuals experienced in recognizing pungency [(International Standards Organization (1981, 1997): Spices and condiments - chillies: Determination of Scoville index, ISO 3513:1977E, ISO, Geneva)]. Testing of dilutions should be done from the weakest to the strongest until a level at which three of five panelists agree on recognition of pungency. There is no published report on the extensive use and efficiency of these latter two methods, except a few which reported comparison of experimentally determined SHU values with that of calculated based on capsaicinoid content of oleoresin capsicum samples (Suzuki et al., 1957). 6.3.4.1.5. The ASTA Method

The ASTA adopted as early as 1968 Method No. 21.0 as an official method for pungency evaluation which took care of many variables that were to be controlled for good reproducibility [(ASTA (1968): Method 21.0, Pungency of capsicum spices and oleoresins (Scoville heat test), In: Official Analytical Methods, ASTA, Englewood Cliffs, N.J.)]. The procedure followed in the industry and EOA was thoroughly revised by designing it as a general method applicable to samples of a large range of capsaicinoid content by careful steps for determining the recognition threshold using an ascending concentration series.

6.3.4.2. Quantitation of capsaicinoids In addition to pungency, as a bulk characteristic of total capsaicinoids, estimation of the individual level of each capsaicinoid is also an important quality attribute of Capsicum fruits. There are over a hundred papers published on the estimation of total capsaicinoids in Capsicum fruits, the oleoresins and products containing their extracts. The methods can be grouped into four sets as follows:

71

6.3.4.2.1. Early direct methods As early as 1931, von Fodor used vanadium oxytrichloride or ammonium vanadate and hydrochloric acid to react with the phenolic hydroxyl group of capsaicinoids and measured the blue color formed. The accuracy of determinations based on color formation reactions of the phenolic moiety could be conveniently used when the color produced with the chromogenic reagent had absorption maxima far removed from the absorption range (300 to 550 nm) of the red and yellow carotenoids of Capsicum fruits. Thus the blue colors formed when the phenolic moiety of the capsaicinoids reacted with reagents such as vanadium oxychloride (Palacio, 1977) and phosphomolybdic or phosphotungstic reagents (Jentzsch et al., 1969) had their absorption maxima around 725 nm. On the other hand, the chromophore produced by the more specific 2,6-dichloro-p-benzoquinone-4-chlorimide, (Gibb's phenol reagent) depending on the reaction conditions, absorbed maximally at 590 or 615 nm (Jentzsch et al., 1969; Rajpoot, Govindarajan, 1981). The extinction coefficients for the blue colors using different reagents varied greatly and affected sensitivity. There are also reports using potassium ferricyanide plus ferric chloride (Spanyar, Blazovich, 1969), sodium nitrite molybdate reagent (Bajaj, 1980) and FolinCiocalteu reagent (Kosuge, Inagaki, 1959) as chromogenic reagents for determination of total capsaicinoids. It is worth mentioning that the color reactions could also be applied for visualizaton of thin layer chromatography (TLC) spots of separated capsaicinoids.. 6.3.4.2.2. Methods based on separation of capsaicinoids The specificity and accuracy of determination of capsaicinoids were improved by a preliminary separation of interfering pigments and other substances. In the early methods separation of capsaicinoids from the pigments was accomplished by solvent partition. Several combinations of partition system may be found in the literature (Spanyar et al., 1957; Benedek, 1959, Chem. Abstr. 1958, 1963a,b; Tirimanna, 1972), none of the methods, however, was validated by pungency tests, without which the accuracy of the determination cannot be ascertained. There have been several methods developed for the preliminary separation of capsaicinoids from the Capsicum pigments using short column clean-up methods. The purified capsaicinoids were quantitated by colorimetry after reacting with chromogenic reagents (Hollo et al., 1957, Chem. Abstr. 1958, 1963a,b; Bajaj, 1980) or directly at the absorption maxima (282 nm) of pure capsaicin (Suzuki et al., 1957; Brawer, Schoen, 1962; Chem. Abstr., 1963a,b). Suzuki et al. (1957) determined pungency values for a number of chilli and oleoresin samples by threshold testing and validated the capsaicinoids values determined by the proposed method. After several reviews, the Joint Committee (Pharmaceutical Society/Society of Analytical Chemistry) recommended the diethyl ether-alkali partition method for separation and the spectrophotometric difference method or the method using Gibb's reagent for measurement [Joint Committee (PS/SAC), 1964]. The method has been adopted by both the British Standards (BS) and International Standards Institutions (ISO) for estimation of capsaicinoids (International Standards Organization, 1981).

72

6.3.4.2.3. Newer chromatographic micromethods Thin Layer Chromatography As newer separation methods emerged (e.g. paper chromatography and TLC) which gave rapid and more efficient separation, they were quickly used in the determination of total and later individual capsaicinoids. The main body of papers in the 1960s practically relegated the earlier solvent partition and column methods to the past and published reliable, rapid micromethods. Most of the early thin layer chromatography (TLC) methods used silica gel plates with a wide range of variations in the developing solvents. The versatility of TLC method for the separation of a complex mixture of compounds could be further improved, however, by using reversed-phase plates and polar developing solvents containing silver nitrate (Todd et al., 1975, 1977). Methods of visualization applied UV light or chromogenic reagents. The estimation method also varied: visual comparison of size and intensity of spots, direct densitometry on the plate, collection of the marked spot into a tube, development of color with a chromogenic reagent and absorption measurements. Quantitation was by reference to a standard curve using pure capsaicinoids treated under the same conditions. A comprehensive listing of the methods can be found in some reviews on capsaicinoids (Suzuki, Iwai, 1984; Govindarajan et al., 1987). 6.3.4.2.4. Newer chromatographic micromethods - Gas Chromatography Gas chromatography (GC) was early used to detect adulteration of capsaicinoids with synthetic vanillylamides and individual components in crystalline capsaicinoids through the analysis of methyl esters of fatty acids derived from them (see Table 2). As early as 1967, Morrison demonstrated that capsaicinoids can be analyzed by gas chromatography without derivatization (Morrison, 1967). In order to improve peak symmetry, prevent degradation of column and improve reproducibility of measurements, however, most of the GC methods need a derivatization step to increase volatility of the capsaicinoids, and, furthermore, an efficient clean-up step is necessary (Todd et al., 1977; Iwai et al., 1979; Krajewska, Powers, 1987; Manirakiza et al., 1999, 2003) Two types of derivatization procedures have been reported: trimethylsilylation of capsaicinoids (Lee et al., 1976; Todd et al., 1977; Iwai et al., 1979; Fung et al., 1982) and hydrolysis of capsaicinoids to yield fatty acids and subsequent esterification (Jurenitsch et al., 1978; Jurenitsch, Leinmueller, 1980). To overcome the problem of tailing peaks and to avoid the use of derivatization step, Thomas et al. (1998) and Hawer et al. (1994) have recognized the use of polar capillary column for interaction with polar functional group of the molecules. In an earlier work Di Cecco (1976) also used a stable polar analytical column (CarbowaxTeflon) to analyse column purified capsaicinoids from ground capsicum. Furthermore, the use of a thermoionic selective detector (TSD) instead of flame ionization detection allowed the elimination of sample clean-up (Thomas et al., 1998). Although the direct analysis of capsaicinoids could be disandvantageous, direct GC-MS analysis of commercially available natural capsainoids has been proved to be a proper method to separate the main capsaicinoids and quantitate capsaicin and dihydrocapsaicin in Capsicum extracts as well (Kuzma et al., 2006). A typical gas chromatogram of a commercially available natural capsaicin is shown on Fig. 12. 73

09:34:47

capsaicin

28-Feb-2006

S c a n EI+

v4

17.48

140 -

TIC 1.44e6

-17.68

16.82, 6.00

8.00

v4 681 (17.477)

10.00

12.00

14.00

16.00

20.00

22.00

138

1 5 2

168 178 195 0 7 0 9 221 2

100

120

140

24.00

scan el+ 5.35e5

137

100

Fig.

18.00

160

180

200

2

220

248 262

240

260

2

81

280

3 0 5

i 307 325 341 355 357378

387

'•r r T"'T""i i f" i • i' ( 320 340 360 380 400

300

i

:

12a: G a s c h r o m a t o g r a p h i c ( G C - M S / T I C ) a n a l y s i s o f a c o m m e r c i a l l y a v a i l a b l e c a p s a i c i n

preparation. Retention times: 16.8 m i n : nordihydrocapsaicin; 17.5 m i n : c a p s a i c i n ; 17.7 m i n : d i h y d r o c a p s a i c i n

12b: G C - M S ( E l , S c a n m o d e ) of c a p s a i c i n ( K u z m a , M o l n a r , Perjesi, 2 0 0 6 : D e v e l o p m e n t

a n d a p p l i c a t i o n of a gas c h r o m a t o g r a p h i c m e t h o d for d e t e r m i n a t i o n of c a p s a i c i n o i d s , I n : A b s t r a c t s of Papers, S y m p o s i u m of D r u g R e s e a r c h C o m m i t t e of H u n g a r i a n

P h a r m a c e u t i c a l S o c i e t y , N o v e m b e r 2 4 - 2 5 , D e b r e c e n , H u n g a r y , p. 51)

74

Lee et al. (1976) used selective ion monitoring to identify and quantify individual capsaicinoids at the nanogram level in partially or fully separated and even mixed peaks from GC of trimethylsilylated capsaicinoids. Aliquots of fruit extracts were subjected to TLC or reversed phase (RP) HPLC to separate the capsaicinoids from other components of the extracts. Iwai et al. (1979) developed a similar method for determining all homologs in total extracts of capsicum fruits. By this analysis, similar to other GC-MS methods (Fung et al., 1982; Reilly et al., 2001) the straight-chain analogs (octanoyl, nonoyl and decyl vanyllilamides) - identified in minor amounts in other analyzes (Jurenitsch, et al., 1978; Jurenitsch, Leinmueller, 1980) - were not found in any of the samples. The reason for this was that the analysis was based on selected m/e values but not on monitoring the mass of octonoyl vanillylamide, and the method could not differentiate between nonoyl vanillylamide and nordihydrocapsaicin eluting in the same area. 6.3.4.2.5. Newer chromatographic micromethods - High Performance Liquid Chromatography This chromatographic technique has superior and rapid separation capabilities arising from the use of very fine and highly uniform particles, newer solid phases, and high pressure to move the eluting solvent and fractions. With all its advantages, high performance liquid chromatographic analysis is being increasingly used for routine analyses in both industrial and research laboratories. HPLC has superior separation capabilities for closely related compounds typically occurring in the case of extract of natural sources. Combined with additional operational parameters, e.g. reversedphase systems, silver-ion complexing of olefinic compounds, optical as well as mass selective detectors, the separation efficiency, sensitivity, and quantification at submicrogram levels of capsaicinoids have been demonstrated in the recent years. HPLC analysis has made possible the accurate determination of the homologs and analogs of capsaicin and, combined with mass spectral analysis, has led to identification of structural isomers of some minor components (Govindarajan, 1986b; Reilly et al., 2001; Schweiggert et al., 2006) and has made possible the determination of nanogram levels of the individual capsaicinoids as is required in biosynthetic and metabolic studies (Kozukue et al., 2005). Several HPLC methods have been published for the determination of capsaicin homologs and analogs. Since there is no space to summarize all the methods published so far, the attention of interested readers is drawn to recent reviews to get a comprehensive knowledge on the field (e.g. Govindarajan et al., 1987; Wall, Bosland, 1998; Manirakiza et al., 2003; Pruthi, 2003). Here only selective papers are summarized with data obtained. Lee et al (1976) and Iwai et al. (1979) early used HPLC for the separation of capsaicinoids in one or two fractions from total extracts for the subsequent analysis by mass spectrometry. Sticher et al. (1978) reported separation of four homologs of capsaicin in purified capsaicinoids using a reversed-phase system. Jurenitsch et al. (1979) accomplished the separation of the capsaicin homologs and analogs directly from ground fruit extracts on a reversed-phase system. Detection and quantitation were done by absorbance at 280 nm. Four samples of Capsicum fruits were analyzed 75

by this HPLC method and also by the TMS-GC method earlier used by the group (Jurenitsch et al., 1978) for comparison. There was a fairly close agreement between the methods for total capsaicinoids. Karnka et al. (2002) have reported an optimized HPLC based method for sample preparation, separation, detection and identification of the major capsaicinoid compounds in various capsicum samples. Nonoyl vanyllamide content has assumed importance since more than 3 to 4% of this analog in a natural sample is considered adulteration unless declared. The method developed by Jurenitsch et al. (1979) was modified with the inclusion of silver nitrate in the mobile phase to selectively shorten the retention time of capsaicin, thus separating it from the coeluting nonoyl vanillylamide (Jurenitsch, Kampelmuehler, 1980). Constant and coworkers (1995) also used complexation chromatography (AgN0 ) to separate norcapsaicin, zucapsaicin (civamide), capsaicin, nordihydrocapsaicin, nonivamide, homocapsaicin, dihydrocapsaicin and homodihydrocapsaicin-I. Isocratic reversed phase HPLC analysis performed in the author's laboratory allowed separation of five main capsaicinoids of a commercially available capsaicin preparation (see Fig. 13). The validated analytical method has been successfully applied to quantitate capsaicin and dihydrocapsaicin in commercially available ground paprikas (Boros et al., 2007, unpublished results). 3

mAU

D A D 1 E, Sig=281,16 Ref= 360,100 (CAPS\CAPS0001.D)

A 0 Fig.

~~5

l

~~

10

15

1 '

'

' 200

min

13. H P L C a n a l y s i s of a c o m m e r c i a l l y a v a i l a b l e c a p s a i c i n p r e p a r a t i o n . R e t e n t i o n t i m e s : 9.5 m i n : n o r d i h y d r o c a p s a i c i n ; 10.5 m i n : c a p s a i c i n ; 1 6 . 0 m i n : d i h y d r o c a p s a i c i n ; 17.5 m i n : h o m o c a p s a i c i n ( B o r o s , K u z m a , Perjesi, u n p u b l i s h e d results)

The rapid and efficient separation by HPLC directly of extracts of fruits for capsaicinoid components combined with the sensitive detection and quantitation by selective ion mass spectrometry gives an outstanding method for analysis of all the capsaicin homologs and analogs identified. The use of HPLC-MS (Reilly et al., 2001a,b) has been reported to differentiate nonivamide and capsaicin by mass-tocharge (m/e) ratio. The same authors have reported the use of LC-MS-MS with electrospray ionization source operating at selective ion monitoring mode (Reilly et al., 76

2001a,b). The quantification of capsaicinoids using LC-MS-MS was more sensitive (in the ng/ml range) and exhibited greater accuracy, even at low analyte concentrations. HPLC coupled with atmospheric pressure chemical ionization mass spectrometry has been reported to be a method of choice for separation and identification of the three groups of capsaicinoids: capsaicins possessing a methyl branched acyl residue with a carbon-carbon double bond, dihydrocapsaicins analogous to the previous class, but being saturated compounds, and capsaicin analogues (N-vanillyl-nacylamides) composed of saturated, unbranched alkyl chains (Fig. 8) (Schweiggert et al., 2006). In summary, HPLC analysis for total capsaicinoids or individual capsaicinoids is certainly rapid, reproducible, sensitive, and convenient for analysis of capsaicinods in various capsaicinoid containing matrices. 6.3.4.2.6. The ASTA method for determination of capsaicinoids In the 1980s it became clear that a more accurate and reproducible method of determining "heat" in peppers and pepper products was necessary. Under the auspices of the American Spice Trade Association (ASTA), a new High Pressure Liquid Chromatography (HPLC) Method was adopted as ASTA Method 21.1. The HPLC measurement of capsaicin has evolved over the years as new and better instrumentation has allowed the greater accuracy of analysis. In 1996 AO AC issued Method 995.03: Capsaicinoids in Capsicums and their Extractives. In 1998, in a collaborative effort with AOAC, ASTA issued a revised method of analysis, ASTA Method 21.3 (HPLC Method). Using the revised method, the accepted pungency of pure capsaicin was re-stated from 15,000,000 to 16,000,000 SHU. AOAC revised its method to coincide with ASTA Method 21.3, in 1999, and in 2003 AOAC revised the method once more. 6.3.4.2.7. The United States Pharmacopeia (USP) The 2006 edition of the USP30-NF25 lists Capsaicin and describes its Definition, Identification, Melting range, and Content of capsaicin, dihydrocapsaicin, and other capsaicinoids as follows (The USP30-NF25 Page 1609). Capsaicin (USP30-NF25) Chemical name: 6-Nonenamide, (E)-N-[(4-Hydroxy-3-methoxy-phenyl)methyl]8-methyl Formula: C H 7 N 0 Molecular weight: 305.41 (E)-8-MethyI-N vanillyl-6-nonenamide CAS-number: 404-86-4 Capsaicin contains not less than 90.0 percent and not more than 110.0 percent of the labeled percentage of total capsaicinoids. The content of capsaicin ( C H N 0 3 ) is not less than 55 percent, and the sum of the contents of capsaicin and dihydrocapsaicin ( C H N 0 ) is not less than 75 percent, and the content of other capsaicinoids is not more than 15 percent, all calculated on the dried basis. 1 8

2

3

1 8

1 8

2 9

2 7

3

77

Packaging and storage - Preserve in tight containers, protected from light, and store in a cool place. Caution - Handle Capsaicin with care. Prevent inhalation of particles of it and prevent its contact with any part of the body. Solubility - It does not dissolve in water. It well dissolves in alcohols (methanol, ethanol 96%), ethylacetate and acetonitrile. Identification - Prepare a test solution of Capsaicin in methanol containing 1 mg per ml. Prepare a Standard solution of USP Capsaicin RS in methanol containing 1 mg per ml. Separately apply 10-pl portions of the test solution and the Standard solution to a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel mixture. Develop the chromatograms in a solvent system consisting of a mixture of ether and methanol (19:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the chamber, and allow it to air-dry. Spray the plate with a 0.5% solution of 2,6-dibromoquinonechlorimide in methanol, allow to stand in a chamber containing ammonia fumes, and examine the chromatograms: the blue color and the Rvalue of the principal spot obtained from the test solution correspond to those properties of the principal spot obtained from the Standard solution. Melting range: between 57°C and 66°C, but the range between beginning and end of melting does not exceed 5°C. Content of capsaicin, dihydrocapsaicin, and other capsaicinoids Mobile phase - Prepare a mixture of diluted phosphoric acid (1 in 1000) and acetonitrile (600:400). Filter through a fdter having a porosity of 0.5 pm or finer, and degas. Make adjustments if necessary (see System Suitability under Chromatography). Standard capsaicin solution - Dissolve an accurately weighed quantity of USP Capsaicin RS quantitatively in methanol to obtain a solution having a known concentration of about 0.1 mg per ml. Standard dihydrocapsaicin solution - Dissolve an accurately weighed quantity of USP Dihydrocapsaicin RS quantitatively in methanol to obtain a solution having a known concentration of about 0.025 mg per ml. Test solution - Transfer about 25 mg of Capsaicin, accurately weighed, to a 250 mL volumetric flask, dilute with methanol to volume, and mix. Chromatographic system - The liquid chromatograph is equipped with a 281 nm detector and a 4.6-mm x 25-cm column that contains 5 pm packing LI 1 and is maintained at a constant temperature of about 30°C. Adjust the flow rate to obtain a retention time of about 20 minutes for the main capsaicin peak. Chromatograph the Standard capsaicin solution, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2%.

78

Procedure - Separately inject equal volumes (about 20 pi) of the Standard capsaicin solution, the Standard dihydrocapsaicin solution, and the Test solution into the chromatograph, record the chromatogram for a period of time that is twice that of the retention time of capsaicin, and measure the areas of the responses for all of the peaks. Calculate the percentage of capsaicin ( C H 7 N 0 ) in the portion of Capsaicin taken by the formula: l g

2

3

25,^(C/WHry/rs), in which C is the concentration, in mg per mL, of USP Capsaicin RS in the Standard capsaicin solution, W is the weight, in mg, of Capsaicin taken to prepare the Test solution, and r and r are the capsaicin peak responses obtained from the Test solution and the Standard capsaicin solution, respectively. Not less than 55% is found. Calculate the percentage of dihydrocapsaicin (CigH^NC^) in the portion of Capsaicin taken by the formula: v

s

25,000(C/W)(

r /r ), v

s

in which C is the concentration, in mg per ml, of USP Dihydrocapsaicin RS in the Standard capsaicin solution, W is the weight, in mg, of Capsaicin taken to prepare the Test solution, and r and r are the dihydrocapsaicin peak responses obtained from the Test solution and the Standard dihydrocapsaicin solution, respectively. The sum of the percentage of capsaicin found and of the percentage of dihydrocapsaicin found is not less than 75%. Using the chromatograms obtained from the Standard capsaicin solution and the Test solution, calculate the percentage of other capsaicinoids in the portion of Capsaicin taken by the formula: v

s

25,000(C/W)(rT/r ), s

in which C is the concentration, in mg per ml, of USP Capsaicin RS in the Standard capsaicin solution, W is the weight, in mg, of Capsaicin taken to prepare the Test solution, r is the sum of the peak responses of the capsaicinoids other than capsaicin and dihydrocapsaicin in the chromatogram obtained from the Test solution, and r is the capsaicin peak response obtained from the Standard capsaicin solution. Not more than 15% of other capsaicinoids is found. T

s

6.3.4.3. Correlation of pungency and capsaicinoid content Among the methods published for estimation of total capsaicinoids or individual capsaicinoids only a few have been validated by correlating the results to pungency. Suzuki et al. (1957) compared the pungency of a number of capsicum oleoresin samples determined by the organoleptic method to that of their total capsaicinoids content determined by UV absorption. They found a high correlation between the percent of total capsaicinoids and the estimated Scoville values. Hartman (1970) correlated SHU to the proportion of capsaicinoid content to GLC. Todd et al. (1977) also compared the pungency in Scoville units calculated from the GC-determined percentages of 79

individual capsaicinoids of the samples and the values determined by the modified ASTA method and found a high correlation. Similarly, high correlation was obtained between the Scoville values and the total capsaicinoids estimated by HPLC method (Woodbury, 1980). Govindarajan et al (1986d) proposed a standardized procedure for the evaluation of the pungency of SHU by which a linear regression was obtained between SHU and the capsaicin content of samples. Recently, Korel et al. (2002) used the electronic nose (EN) technology (Strike et al., 1999) to discriminate ground red pepper samples by headspace volatiles. The authors report linear correlation between capsaicin, dihydrocapsaicin, and total capsaicinoids and Scoville scores of the samples. Based on these literature data it can be concluded that estimation of capsaicinoids by any reproducible and accurate analytical method truly reflects the pungency of capsicum fruits or processed products (Govindarajan, 1986e). 6.3.4.4. Stability of capsaicinoids Information on stability of isolated capsaicin, dihydrocapsaicin as well as of other capsaicinoids is rather limited. The Material Safety Data Sheet (MSDS) of natural capsaicin (Formula: C H 7 N 0 , Molecular weight: 305.41, Chemical name: (E)-8Methyl-N-vanillyl-6-nonenamide, CAS number: 404-86-4) recommends the preparation between 2 to 8°C (Sigma-Aldrich MSDS). The USP30-NF25 prescribes the capsaicin preparatum to be preserved in tight containers, protected from light, and stored in a cool place. According to the MSDS of synthetic dihydrocapsaicin (Formula: C H 2 9 N 0 , Molecular weight: 307.48, Chemical name: 8-Methyl-N-vanillylnonanamide, CAS number: 19408-84-5), the preparation should be stored at -20°C (Caymanchem MSD). Both preparations are reported to be stable for at least 26 days at-5°C (NMAM, Method 5041, 1996). Schweiggert et al. (2006) investigated the stability of chili powder. They found that the capsaicin, dihydrocapsaicin and nordihydrocapsaicin content of the samples stored at ambient temperature over 6 months dropped by 7 to 12%. Based on their experience it was recommended that paprika (pepper) samples should be heat-treated before processing, in order to reduce the number of microorganisms producing enzymes with peroxidase activity of the fruits. Kopec et al. (2002) tested the stability of stored ethanol solution of capsaicin ranging in concentration from 0.5 to 128 pM. It was found that the solutions of 4 uM or higher concentrations are stable for a year if stored at 4°C protected from light. The result has been reinforced by the authors' investigations studying stability of capsaicin natural solutions (c = 3.1 mg/ml) stored for a month at 4°C protected from light (Boros et al., unpublished results). While investigating the stability of a capsaicin containing ointment Jaiarj et al. (2000) found the preparation to show higher stability stored at 4°C other than at ambient temperature. In conclusion, the presence of the phenolic hydroxyl group and the carbon-carbon double bond makes the capsaicinoids sensitive to oxidation. Accordingly, capsaicinoids should be protected from exposure to light, heat, moisture, and oxidizing agents, which initiate and/or catalyse the decomposition processes. 1 8

1 8

80

3

2

3

7. General physiology of retinoids and carotenoids

All of the main components can be found in the plants in association to the color materials of the plants and the spices. The chemical structures of these compounds have been detailed in Chapter 6. The Capsicums as typical characteristics of the species were prepared from "chilli" (synonym of chili or paprika) from the whole plants (e.g. paprika or chilli). These plants contain retinoids and carotenoids beside capsicum. The common biological results of the consumption of these plants appear as the common results of retinoids, carotenoids and Capsicums. However, it has to be emphasized that considering their biological effects, the three components show some similarities and differences. In the forthcoming pages we will give a short summary of the biological effects of the color chemical compounds (retinoids and carotenoids).

7.1. Biochemistry of retinoids and carotenoids Retinoids are a class of compounds consisting of four isoprenoid units bound in a head-to-tail manner. All retinoids may be formally derived from a monocyclic parent compound containing five carbon-carbon double bonds and a functional group at the terminus of the acyclic portion (IUPAC-IUB, 1982). Vitamin A is a fat-soluble substance found in animal foods and dairy products. Vitamin A is available as preformed vitamin A, contained in liver, cod liver oil, butter, eggs, or as provitamin A carotenoids, as found in dark green, red and yellow vegetables (Blaner, Oslon, 1994). These naturally occurring retinoids exist in the all-trans, 13-cis or 11-cis geometric configurations, with the great preponderance of the body's retinoids being present in the all-trans configuration (Blaner, 1993). The alcohol all-trans retinol is the parent compound for all retinoids. Retinol is precursor for the synthesis of the biologically active retinaldehyde and retinoic acid forms. Additionally, retinol is the precursor for the synthesis of retinyl esters (Blaner, 1993). Within target tissues retinol is taken up by the cells from the circulation and can either be oxidized to retinaldehyde, retinoic acid or esterified for storage (Goodman, 1984). Retinaldehyde is not known to play another essential physiological role outside the vision, aside from serving as an intermediate in the enzymatic oxidation of retinol to retinoic acid (Saari, 1991). Retinoic acid is the biologically active retinoid form which is needed for mediating and maintaining cellular differentiation (Ross, Tenuis, 1993). 81

7.1.1. General physiology of retinoids and carotenoids All retinoids in the body originate in the diet either as provitamin A carotenoids or as preformed vitamin A (Goodman, 1984). The dietary carotenoids and preformed retinoids undergo a series of metabolic conversions, extracellularly in the lumen of the intestine and intracellularly in the intestinal mucosa, which result in the preponderance of the absorbed dietary retinoid being converted to retinol (Semba, 1997). Provitamin A carotenoids, such as P-carotene, may be converted to retinaldehyde through the cleavage by carotenoids-15, 15 dioxygenase or by an excentric cleavage pathway. Approximately 50 of over 600 carotenoids found in nature may be converted to vitamin A (Semba, 1997). The bioavailability of provitamin A carotenoids is much less than that of performed vitamin A because of a variety of factors, including differences in efficacy of absorption and biochemical conversion (Semba, 1997). Although hundreds of carotenoids have been identified, only few have been found to exist in appreciable concentrations in the human serum: lutein, zeaxanthin, a- and P-carotene, a- and P-cryptoxanthin (Bieri et al., 1985). Retinol is esterified in the intestinal mucosa, packaged as retinyl ester into chylomicra, and carried to the liver via the lymphatic circulation (Blaner, 1993). A very small portion of the dietary retinoid is converted to retinoic acid (or comes in the diet as such) and enters the circulation through the portal system bound to serum albumin (Smith et al., 1973). Approximately 90% of vitamin A in the body are stored in the liver as retinyl esters (Hendriks et al., 1985; Yamada et al., 1987; Hussian et al., 1989). The liver has the capacity to store enough vitamin A to last for several months, with a longer storage capacity among adults than children (West, Sommer, 1984). Retinol is released from the liver in combination with plasma retinol-binding protein (RBP) and transthyretin (TTR) (Noy, Blaner, 1991; Soparno, Blaner, 1994). Retinol is poorly soluble in water and is carried in the blood sequestered inside the carrier proteins (Sziits, Harosi, 1991). Retinol seems to enter cells via specific receptors, although it is unclear whether all cells contain these receptors (Soparno, Blaner, 1994; Zhang et al., 1996). Vitamin A exerts its effects through retinoid receptors, which are found in the nucleus of cells. These receptors resemble steroid and thyreoid hormone receptors and support the idea that vitamin A acts much like a hormone (Ross, Ternus, 1993). Retinol is converted into its active metabolite, all-trans-retinoic acid (ATRA) in the cells. Retinoic acid can control genes through specific receptors that belong to the superfamily of thyreoid and steroid receptors (Corpet et al., 1990; Capristo et al., 1998). Retinoic acid receptors act as transcriptional activators for specific target genes. The retinoic acid receptor (RAR) is expressed as several isoforms referred to as RAR a, P and y for which all-trans-retinoic acid acts as a ligand, and retinoid X receptors (RXR) referred to as RXR a, P and y for which 9-cis retinoic acid acts as a ligand (Kliewer et al., 1992). 9-Cis retinoic acid seems to be functionally distinct from ATRA, and interconversion may exist between the two isomers. Each RAR and RXR has a specific DNA-binding domain, a retinoic acid response element (RARE) by which these nuclear receptors may affect retinoic acid transcriptional activity. RAR and RXR receptors form heterodimers which bind to DNA and control gene 82

activity. In addition, RXR receptors also can form heterodimers with vitamin D and thyreoid hormone receptors (Kliewer et al., 1992). Numerous studies have focused recently on the role of retinoid receptors in the process of carcinogenesis (Lee, Kumar, 1980; Hunter et al., 1993; Matsushima et al., 1996; Zhang et al., 1996; Benbrook et al., 1997; Lee et al., 1997; Sherman, Partidge, 1997; Waliszewski et al., 1997). Retinoids and carotenoids have numerous biological functions such as regulating the growth, morphogenesis and differentiation of cells, they have a variety of effects on the cell membrane, they can influence the activity of different enzymes, furthermore, exert also immunomodulatory effects. Carotenoids are non-enzymatic antioxidants, therefore they are able to prevent genetic changes by preventing DNA damage caused by free radicals. They modulate membrane functions and stabilize initiated cells in the promotional phase of carcinogenesis (Semba, 1997). The role of retinoids in GI mucosal prevention has been intensively investigated in our Department in the last two decades (Rumi et al., 2001). Vitamin A and (3carotene were shown to prevent the experimentally induced gastric mucosal lesions in animals (Javor et al., 1983), and these compounds were found to be effective in the treatment of patients with gastric peptic ulcer (Patty et al., 1982). Carotenoids have no inhibitory effects on gastric acid secretion either in animals (Javor et al., 1983) or in humans (Hunyady et al., 1991), however, P-carotene was able to prevent the gastric mucosal damage in different experimental models such as ethanol (96%)-, hydrochloric acid (0.6M)- or indomethacin-induced mucosal damage (Patty et al., 1982; Javor et al., 1983; Hunyady et al., 1991). Furthermore, the ulcer-healing effect of vitamin A was proven in randomized, multicenter clinical studies (Patty et al., 1982). The gastroprotection induced by carotenoids does not depend on the presence of vitamin A activity, P-ionone ring, number of unsaturated links or chemical structure of terminal part of molecules (Javor et al., 1983; Mozsik, Javor, 1991). Moreover, the analysis of gastric mucosal antioxidant mechanisms and free radical generation during P-carotene-induced gastric mucosal protection suggested that the scavenger character of P-carotene might partially explain its protective effect (Vincze et al., 1991). The p-carotene-induced gastric gastroprotection was completely abolished by acute bilateral surgical vagotomy (Vincze et al., 1997). Despite these investigations, the exact mechanism of action is not fully understood yet. Although carotenoids, such as p-carotene and vitamin A are popularly often regarded to be equivalent, there are large differences in the biological functions of these two nutrients, especially regarding their antioxidant properties, since vitamin A is a less potent antioxidant than P-carotene (Semba, 1997).

7.1.2. Retinoids and chemical carcinogenesis A suboptimal diet might be related to approximately 30-60% of all cancer cases (Peto, Doll, 1981) the major part of which is possibly preventable by adequate dietary modifications (Doll, 1992). Chemopreventive potentials are conceivable for antioxidant micronutritients (Block, et al., 1992; Block 1991, 1992, 1996). Particularly intriguing are those with polyene structure, i.e. vitamin A-type retinoids and carotenoids such as P-carotene (Bertram et al., 1991; Willett, 1991; Ziegler, 1991). 83

Several animal and in vitro studies have now been reported related to the effects of antioxidants on cell proliferation, including GI epithelial cell proliferation. It was observed 70 years ago that the deficiency of vitamin A in animals provokes abnormal keratinizations, precancerous squamous metaplasia and cancers of various epithelia (Wolbach, Howe, 1928). The effectiveness of retinoids as inhibitors of chemical carcinogenesis of the epithelia of the digestive tract has been somewhat contradictious, although positive results have been obtained in studies of stomach, esophageal, liver and pancreatic carcinogenesis. The oral administration of retinyl ester prevented the occurrence of papillomas and carcinomas in hamsters receiving polycyclic hydrocarbons (Moon, 1993). Nitrosamine-induced esophageal carcinogenesis can also markedly be inhibited by administering retinyl ester (Moon, McCromick 1982). Subsequent studies have shown that synthetic retinoids such as 13-cis-retinoic acid also exert a protective effect against the induction of esophageal tumors with nitroso compounds, although etretinate was ineffective in inhibiting esophageal carcinogenesis (Moon, 1993). Only few and contradictious data can be found concerning the effect of retinoids on chemically induced colon carcinogenesis. An antioxidant mixture containing |3-carotene and a-tocopherol reduced cell proliferation in the colon and rectum of mice (Lok et al., 1992). Organic and inorganic selenium with P-carotene reduced colonic epithelial cell proliferation while concomitantly reducing the incidence and multiplicity of colon adenocarcinomas in rats given chemical carcinogens (Nayini et al., 1991). Colorectal epithelial cell proliferation was reduced in rats given chemical carcinogens when given a diet low in fat and protein and high in vitamin E, selenium, vitamin A and fibre (Goettler et al., 1987). Synthetic retinoids are able to prevent azoxymethane-induced intestinal carcinogenesis in rats (Kawaromi et al., 1995), although natural and synthetic retinoids have little effect on colon carcinogenesis induced by aflatoxin and dimethylhydrazine (Moon, 1993). The few studies on liver carcinogenesis indicated that 13-cis-retinoic acid was highly effective in reducing the incidence of liver tumors induced by 3methyl-4-dimethylaminoazobenzene (Daoud, Griffin, 1980; Moon, 1993). Similar findings have been reported for spontaneous hepatomas in mice given various doses of retinyl ester (Moon, 1993). Several synthetic retinoids inhibited the development of azaserine-induced pancreatic tumors in rats when administered during the promotional phase of carcinogenesis (Longnecker et al., 1982). In colorectal carcinoma cell lines ascorbic acid enhanced the antiproliferative effect of vitamin A (Kandaaswani et al., 1983), emphasizing their interdependence.

7.1.3. Effect of antioxidants on colorectal epithelial cell proliferation, polyp recurrence and carcinogenesis: Clinical trials in patients The effect of carotenoids supplementation on precancerous lesions and cancer incidence has been investigated in numerous clinical trials in the last two decades. Smaller trials in humans suggest that antioxidants can reduce colorectal epithelial cell proliferation. Vitamin A, combined with ascorbic acid, a-tocopherol, further84

more selenium caused a significant reduction in the labeling index (LI) of the upper colonic crypts and the decrease in polyp recurrence in colorectal adenoma patients (De Cosse et al., 1975, 1989; Bussey et al., 1982; Mckeown-Eyssen et al., 1988; Cahill et al., 1991, 1993; Paganelli et al., 1992; Roncucci et al., 1993). Polyp formation could not be inhibited in larger full-scale trials (De Cosse et al., 1989; Vogelstein, Knizler, 1993; Greenberg et al., 1994). Prospective studies, including the particularly conclusive Basle study and the Finnish cohort study, demonstrated that lower levels of retinol and carotene at entry were associated with a significantly increased relative risk for bronchial carcinoma and all other cancers (Salonen et al., 1985; Stahelin et al., 1991). Few studies indicated a positive correlation between carotene intake and carcinogenesis. In the P-Carotene and Retinol Efficacy Trial (CARET) (Hennekens et al., 1994a,b; Omenn et al., 1996) P-carotene (30mg) and vitamin A (25000 IU) were supplemented for patients with high risk for lung cancer (cigarette smoking history or asbestos exposure), the trial was finished because of the results of the AlphaTocopherol, Beta-carotene Cancer Prevention Study (ATBC) (Hennekens et al., 1994a,b). There was an increase in cardiovascular disease and lung cancer mortality among those assigned the supplementation combination. In the Physicians Health Study (PHS) (Steering Committee of the Physician's Health Study Research Group. Final Report on the Aspirin Component of the ongoing Physician's Health Study, 1989) US male physicians were randomized to alternate daily 50 mg P-carotene and 325 mg aspirin, both active treatment, or both placebo. There was no significant effect of P-carotene on total cancer incidence. Numerous problems (Block, 1991, 1992, 1996; Tan, Chu, 1991; Ascherio et al., 1992; Block et al., 1992; Hunter et al. 1993) have been pointed out with these three studies.

7.2. Prevalence and importance of the checked human G I diseases Upper gastrointestinal (GI) ulceration from drugs and inflammatory bowel diseases (IBD) as well as GI malignancies are relatively common GI pathologies which have an appreciable GI mortality and morbidity. Furthermore, the role of environmental, genetic and nutritional factors in the etiology of various cancers and other GI disorders emphasizes the importance of research in these GI conditions (Rainsford, 1996, 2006). It has long been recognized that the relatively high incidence of GI ulceration arises from a variety of causes, among them stressful conditions, ulcerogenic drugs, infections, smoking and high intake of alcohol (Mozsik et al., 1997b,c). While the incidence of peptic ulcer disease continues to be high, the international trend is, overall, towards a moderate decline (despite some fluctuations in reported statistics in some countries) except in individuals above 60 years of age in whom the incidence is increasing. Silent or asymptomatic peptic ulcer disease which is evident in about 85

10% of patients is of particular importance because it often has a fatal outcome (Rainsford, 1996). About 10% of the total Hungarian population suffer from peptic ulcer disease during their life span. It is true that the peak of disease incidence in patients (including males and females) appears after 50 years of age, furthermore the costs of therapy are extremely high, though we still do not know exactly how much (Mozsik et al., 1997a,b,c; Mozsik, 2006). The non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the therapy of inflammatory and pain processes in patients, however, these compounds cause often gastrointestinal mucosal damage (H back diffusion, hemorrhage, bleeding) and different GI complaints (dullness, epigastric pain, vomiting) in a significant group of treated patients (Mozsik et al., 2006). The extent of NSAID consumption has increased significantly in the Hungarian population suffering from different joint diseases. The number of patients who consumed NSAIDs treating their joint diseases is about 2 to 3 millions (20 to 30% of the nation). It is unambiguous that the number of patients suffering from NSAIDs-induced gastrointestinal mucosal damage has increased in the last decade (Mozsik, 2006). This fact is enough for doing research in this field. GI mucosal lesions induced by NSAIDs are considered to involve multiple pathogenic elements, such as deficiency of prostaglandins (PGs), gastric hypermotility, disturbances in microcirculation, oxygen free radicals, neutrophil activation and direct damaging effect on the gastric mucosa (weak vs. strong acid-induced physicochemical properties, like salicylic acid) (Takeuchi et al., 1994, 1996; Mozsik et al., 2001a,b). However, these components closely interact with each other and it would be difficult to determine which pathogenic element is of prime importance in the ulcerogenic response to NSAIDs. Indeed neutrophil activation is caused by alteration of arachidonic acid metabolism, such as PG deficiency; gastric hypermotility leads to microcirculatory disturbances, resulting the enhancement of neutrophil adherence to the vascular endothelium; and the production of oxygen radicals is brought about by neutrophil-endothelium cell interaction as well as by hemodynamic alterations due to gastric hypermotility (Takeuchi et al., 1996). Nowadays, the extent and ratio of cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2) inhibition are dominantly in the international interest (Mozsik et al., 1997a,b; Karadi, Mozsik, 2000). The extents of actions of NSAIDs on COX-1 and COX-2 inhibitions can be separated from each other experimentally, but we do not have enough concrete data on the NSAIDs-induced decrease of gastric mucosal blood flow (GMBF) in patients, although it is sure that its extent cannot reach such a level to be able to produce a real tissue hypoxia in the gastric mucosa (Mozsik et al., 2000; Mozsik, 2006). Studies performed in animals can help us to understand the mechanisms of GI mucosal damage and help in prevention. The presence of tissue hypoxia has been suggested as one of the main etiological factors in the development of gastric mucosal injury (in acute ulcer phase and in other injuries in later phase) based on the basic physiological blood flow observations. (Menguy, Master, 1974a,b,c; Menguy et al., 1974). +

86

A broad biochemical-pharmacological approach of ulcer diseases has been applied in our Department in the last three decades (Mozsik, Vizi, 1976; Mozsik, Javor, 1988; Mozsik et al., 1990; 1992; Mozsik, Pfeiffer, 1992) and this shift could also be observed in international research. A special experimental approach to this problem has been established by the simultaneous measurements of the biochemical compounds of membrane-bound ATP-dependent energy systems such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and cyclic adenosine monophosphate (cAMP) which, together with the lactate level, give an excellent biochemical "cross-section" of the target (stomach) organ (Mozsik, Vizi, 1976; Mozsik, Javor, 1988; Mozsik et al., 1990, 1992; Mozsik, Pfeiffer, 1992). The energy is stored in form of ATP in the gastric mucosa and the measurement of its actual tissue level informs us about the dynamic equilibrium between the ATP resynthesis and breakdown. The gastric mucosal ATP can be splitted in direction of ADP (by membrane ATPase) or cyclic AMP (by adenylate cyclase) liberating a free energy source for the tissue functions, while ADP and cAMP transform to AMP supplying further energy liberation. AMP is a common energy-storing adenosine compound originating from both pathways of ATP-ADP and ATP-cAMP breakdowns (transformations). The liberated energy is used for the regulation of the different functions of the cells (Mozsik, Vizi, 1976; Mozsik, Javor, 1988; Mozsik et al., 1990, 1992; Mozsik, Pfeiffer, 1992). If the energy of different cellular functions originates from the gastric mucosal ATP breakdown, the results of activity of plasma membrane enzymes (membrane ATPase, adenylate cyclase) can be observed in the changes of mucosal ADP, cAMP and AMP. Furthermore, different biochemical parameters can be calculated from the values of ATP, ADP and AMP: a) adenylate pool (ATP+ADP+AMP), b) ratio of ATP/ADP and c) "energy charge" [(ATP+0.5 ADP) / (ATP+ADP+AMP)] (Atkinson, 1968). The value of energy charge is theoretically 1, when all of adenosine compounds are in phosphorylated form, and 0, when these compounds are in dephosphorylated form (Atkinson, 1968). After the basic observations of Menguy et al. (1974) and Menguy and Master (1974a,b,c), experimental and clinical researchers accepted these results as a strong correlation existing between the decrease of gastric mucosal blood flow and the decrease of gastric mucosal ATP level was considered to be a consequence of tissue hypoxia in the gastric mucosa (without the presence of any scientifically proved arguments) (Mozsik et al., 2000). The biochemical measurement of the decrease of gastric mucosal ATP is not enough proof for the existence of tissue hypoxia alone, because the simultaneous elevation of tissue lactate is also basically necessary (Mozsik et al., 2000). The biochemical measurements of adenosine compounds offer an excellent biochemical research information on the extent of tissue oxygenation, when the observations are simultaneous from the same tissue sample. Furthermore, the existence of tissue hypoxia can be characterized well by the extremely low values of tissue ATP, "energy charge" and increased levels of tissue lactate (Mozsik et al.,2000, 2001a,b). 87

It is very important to emphasize that the observations of Menguy et al. were carried out in rats with hemorrhagic shock (Menguy, Master, 1974a,b,c; Menguy et al., 1974). Earlier Davenport proved that weak acids induce gastric H back diffusion in presence of a strong acid like HC1 (Davenport, 1970, 1973). Later Mozsik et al. (1981) demonstrated the decreased breakdown of adenosine triphosphate (ATP) into adenosine diphosphate (ADP) in rats treated with sodium salicylate plus pylorus-ligation, while its value increased only in pylorus-ligated rats (Mozsik, Vizi, 1976). These and other observations proved the role of ATP-dependent energy systems in the development of gastric hyperacid secretion in this experimental model. Na-salicylate and indomethacin are both NSAIDs; however, their actions are significantly different, i.e. salicylate acts as weak acid, while indomethacin inhibits prostaglandin synthesis (Mozsik et al., 1996a,b). These previous data suggest a significant difference in the metabolism of the stomach, in time of development of the gastric mucosal damage produced by sodium salicylate and indomethacin (Davenport, 1970, 1973). However, we have only few data on drugs-induced intracellular metabolic changes in the GI mucosa, therefore our aim was to investigate this matter on the membrane-bound ATP dependent energy systems (ATP-ADP, ATP-cAMP) and to study the changes in the feedback mechanism between them (Mozsik, 1969; 2006; Mozsik etal., 1981, 1990). The incidence of IBDs is definitely on the rise in Europe and USA though few studies consider this fact partly to be the consequence of improved efficiency in their diagnosis (Monterio, Tavarela, 1995). With a total number of approximately 80 illnesses per 100,000 inhabitants they are comparatively rare. These illnesses are not only a burden for the individual but also put them under considerable psychological strain. The etiology of IBDs still remains unclear. Although hereditary factors, microorganisms harmful to the mucosa and certain nutritional (eating) habits are assumed to be causal factors, there is little hard evidence to substantiate these assumptions (Jewell, 1995; Travis 1995). The familiar association of IBDs, reinforced by the demonstration of increased incidence of both Crohn's disease and ulcerative colitis in monozygotic twins, is a quite compelling evidence for an inherited component in the etiology of both diseases (Farmer, Michener, 1986; Jewell, 1995). Genetic markers, such as HLA class II antigens and cytokine polymorphisms, have provided interesting data and may well prove additional factors in the predisposition to IBD. Of the HLA associations observed, that for the DR2 system is relatively strong. Within this system, however, there appear to be variations that may be due to ethnic origins (e.g. high frequency of DRB 1.502 alleles in Japanese and Jewish patients with ulcerative colitis but not in white non-Jewish subjects) (Jewell, 1995; Satsangi, Jewell, 1996). Crohn's disease (CD) is frequently complicated by various nutritional disturbances (Ladefoged et al., 1996; Capristo et al., 1998). Although it is important to correct these disturbances in treating CD patients, the nutritional status of CD patients has been poorly documented, especially concerning their vitamin status. Among various reports of vitamin A deficiency in different gastrointestinal and liver diseases (Jensen, Gluud, 1994; Whitington et al., 1994), relatively few studies have investigated CD (Fernandez-Banares et al., 1989; Zurakowski et al., 1997). +

88

There has been inadequate documentation of the prevalence of vitamin A deficiency in patients with CD who are a population at a risk of protein malnutrition and fat malabsorption as a result of diseased or resected small bowel. No observations can be found in the literature describing the changes of all serum carotenoids in patients with IBD or GI cancer. Colorectal adenocarcinoma is the second most common malignancy in Europe and the USA and leads to considerable mortality (Lakatos, 1994; Bousvaros, Zurakowski, 1998). The disease is rare in Asia and Africa, this difference is thought to be largely environmental rather than racial. 45% of all GI malignancies are colorectal compared with 25% stomach cancer, 15% pancreatic cancer, 11% esophageal cancer and 4% liver and bile duct cancer in West-European Countries (Jankowsky, 1994). The prevalence of GI cancers is increasing in Hungary with the exception of gastric cancer. The incidence of colorectal cancer increases with age, the average age at diagnosis is between 50 and 60 years (Lakatos, 1994). Colorectal cancer has a multifactiorial etiology; numerous environmental and genetic aspects have been investigated. It is estimated that perhaps 35% (range 10 to 70%) of all cancer mortality in the United States could be attributable to dietary factors (Willett, 1991; Potter et al., 1993). Ecologic and migration studies indicate the importance of environmental factors in colon cancer (Potter et al., 1992). Diet appears to have a particularly strong association with occurrence of this cancer, and thus it offers promise for intervention (Willett, 1991; Potter et al., 1993). The observation that higher colon cancer mortality rates occur in the countries where meat and fat consumption was higher led to the hypothesis that these food items contributed to an individual risk of developing colon cancer and was a stimulus for the current interest in dietary intake in most analytic studies of the etiology of colon cancer (Potter, 1992): 1. The oldest hypothesis asserts that fat intake increases bile acid production, ultimately increasing the exposure of the bowel mucosa to the toxic, trophic and cancer-promoting effects of bile acids. The capacity of colonic flora to transform bile acids into potential carcinogens has been found to be greater in populations with high rates of colon cancer and among meat-eating populations than in vegetarian populations (Potter, 1992); 2. A more recent hypothesis is the cooked food hypothesis. High-fat diets contain greater amounts of carcinogenic heterocyclic amines (from meat proteins) and promoters as a consequence of cooking at high temperature (cooking in fat produces higher temperature than cooking in water) (Corpet et al., 1990); 3. A third and recent hypothesis is that high consumption of meat, particularly red meat, may increase fecal concentrations of iron, which catalyzes oxidative reactions, leading to increased lipid peroxidation and oxidative DNA damage and to an increased risk of colorectal cancer (Babbs, 1990). Furthermore, the protective role of vegetable and fruit consumption, dietary fibres, calcium, vitamin D and the potential carcinogenic effect of sucrose have been proved in the last decades (Potter etal., 1993). The role of dietary components is well known in other types of GI malignancies, too. Consumption of salted, pickled and smoked foods has been associated with increased risk of gastric cancer in case-control studies (Fontham, 1997). The strong, 89

consistent inverse association between consumption of fruits and vegetables and colorectal carcinogenesis is clear. Relatively high intake of P-carotene and vitamin C is consistently associated with reduced risk of gastric cancer, the Basle study found a negative correlation between serum carotene levels and risk of gastric cancer. Similar results were found in case of esophageal cancer (Fontham, 1997).

7.3. Gastric cytoprotection, as a special form of defensive mechanisms to gastrointestinal (GI) mucosal injury produced by retinoids A special mechanism of actions of prostaglandins was described by Robert et al. (1979) on gastric mucosa in rats, namely the prostaglandins were able to prevent the gastric mucosal damage without presence of any decrease of gastric acid secretion. This phenomenon was named "cytoprotective action". In the early 1980s many researchers believed that this phenomenon is only the specific action of PGs, meanwhile later the existence of gastric cytoprotection has been proven for different agents (such as atropine, cimetidine, growth factors - for reference see Moron et al., 1983). We proved the cytoprotective effects of retinoids in 1983 (Javor et al., 1983). This phenomenon was special and surprising because the existence of gastric cytoprotection was produced by different nutritional components. That is why this phenomenon was termed as "nutritional cytoprotection". Below we give a short critical summary of our observations with retinoids obtained in different studies (animal experiments, malignant cell lines, human observations). The carotenoids are chemical components responsible for the color of plants, the number of naturally occurring carotenoids is about 600 (Mozsik et al., 1986). To study their mechanisms of actions different compounds (vitamin A, P-carotene, zeaxanthin, lutein, cryptoxanthin, capsanthol, and capsorubin) were tested. The selection of these retinoids was done on their significance of chemical properties, namely vitamin A activity, number of unsaturated double bonds and terminal (P) chemical structure: 1. Gastric cytoprotection does not depend on vitamin A activity. For example, the vitamin A activity of P-carotene (P,P-carotene) is completely eliminated by modifications such as introduction of a hydroxyl group at C-3(3'), substituting the E-end group for the a-end group. Although zeaxanthin (3,3'dihydroxy-P,P-carotene) and lutein (3,3'dihydroxy-P-carotene) are not precursors of vitamin A, they do exert a cytoprotective effect similar to that of vitamin A precursors P-carotene and P-cryptoxanthin (3-hydroxy-P,P-carotene); 2. Gastric cytoprotection cannot be related exclusively to a conjugated polyene chain present in carotenoids, because, for example, acyclic lycopene (P,P-carotene) with an undecene chromophore, and cyclic capsorubin (3,3'-dihydroxy-K,K-carotene6,6-dione) with a nonene-dione chromophore, are inactive. Furthermore, cytoprotective zeaxanthin and lutein have different chromophore systems; 3. Gastric cytoprotection by asymmetrical C40 carotenoids cannot be explained by a splitting of the molecules at the central double bond, which would result two C units with or without cytoprotective properties. According to such conversion, one 2u

90

might expect that capsanthin (3,3'-dihydroxy-(3, K-carotene-6'-one) would be cytoprotective, as its molecule can be built up from an active (1/2 zeaxanthin) and an inactive (1/2 capsorubin) C o unit. The lack of gastric cytoprotection by capsanthin argues against such a conversion. It should be noted that capsanthol, the reduction product of capsanthin, is also inactive (Javor et al., 1983) (Table 14). 2

T a b l e 1 4 . C o r r e l a t i o n s b e t w e e n t h e gastric c y t o p r o t e c t i v e efects of r e t i n o i d s , their c h e m i c a l structure a n d v i t a m i n A a c t i v i t y in rats* Retinoids

Terminal chemical structure

Vitamin A activity

Gastric mucosal prevention

Vitamin A

R=a

yes

yes

P-carotene

X=Y=a

yes

yes

fj-cryptoxanthin

X=a, Y=b

yes

yes

Zeaxanthin

X=Y=b

none

yes

Lutein

X=b, Y=c

none

yes

Capsorubin

X=Y=d

none

none

Capsanthin

X=b, Y=d

none

none

Capsanthol

X=b, Y=e

none

none

Lycopene

X=Y=f

none

none

* from Javor, T., Bata, M., Lovasz, L., Moron, F., Nagy, L., Patty, I., Szabolcs, J . , Tarnok, F., Toth, Cy., Mozsik, Cy. (1983): Gastric cytoprotective effects of vitamin A and other carotenoids. Int. J . Tiss. React. 5: 289-296 (with permission)

7.4. N e w results in the biological actions of retinoids and carotenoids in animals, healthy human subjects and patients with different gastrointestinal disorders The number of patients with gastrointestinal mucosal damage (inflammatory diseases, chemicals-, drugs-, bacterial- and virus-induced infections, precancerous states and cancers) is extremely high over the world. The role(s) of some genetic and environmental factors in the clinical manifestation of these disorders has (have) been emphasized. The results of different randomized studies are very controversial concerning these disorders. Many observations have indicated the essential role(s) of plants (as nutritional factors) in the protection (prevention) of GI mucosal damage in animal experiments and in human observations. The retinoids (as color materials of plants) were thoroughly studied in these disorders, however, their mechanisms of action remained to be unclear up to now. The different suggested mechanisms of retinoids were analyzed and summarized in animals under different experimental circumstances, in human healthy subjects and in patients with different GI diseases (inflammatory, precancerous states and cancers) (Mozsik, 2004) The potential roles of gastrointestinal mucosal protective effects of retinoids were studied in healthy human subjects, in patients with gastric 91

(GU) and duodenal (DU) ulcer (n=131), Crohn's disease (n=40), ulcerative colitis (n=30), hepatitis C (n=90), colorectal polyps (n=59), stomach- (n=21), pancreas(n=10), hepatocellular- (n=14) and colon- (n=44) cancer. The effect of vitamin A was measured on the gastric basal acid output (BAO) and pentagastrin stimulated maximal output (MAO) and indomethacin (3 x 25mg)induced gastric mucosal microbleeding in healthy human subjects. The serum levels of vitamin A, a- and P-carotene, a- and P-cryptoxanthin and lutein were measured by HPLC method in patients with different gastrointestinal chronic inflammatory precancerous states and cancer diseases.

7.4.1. Gastrointestinal mucosal protective effects produced by retinoids in animal experiments Many experiments have been carried out with retinoids in different animal models (for details see Ref. of Mozsik, 2006). It has been found in animal experiments that: 1) The retinoids prevent the drug (EtOH, HC1, sodium salicylate-, indomethacin)-induced gastric mucosal damage without any presence of inhibition of gastric acid secretion ("nutritional gastric cytoprotection"). 2) The gastric cytoprotective effects of retinoids do not depend on the a) vitamin A activity; b) number of unsaturated double bonds; c) the presence of a characteristic chemical structure of their terminal components; d) the presence of vitamin A activity; e) modification of vascular permeability. Their gastrointestinal mucosal prevention depends on 1) intact vagal nerve and 2) intact adrenals in the experimental animals; 3) gastric biochemical changes (the retinoids produce a dosedependent inhibition on the extent of ATP-transformation into ADP, meanwhile they produce an increase in the transformation of ATP into cAMP; 4) intact function of sulfhydryl groups. These gastric cytoprotective effects of retinoids differ: 1) the retinoid-induced gastric mucosal preventive effects differ from those of PGs; 2) the cAMP is an intracellular signal for the development of gastric mucosal damage produced by chemicals (EtOH, HC1, NaOH, concentrated NaCl, sodium salicylate, indomethacin) and for the prevention in gastric mucosa induced by retinoids (but not by PGs); 3) the gastric mucosal protection induced by retinoids and gastric mucosal permeability can be separated in time (Mozsik et al., 1991, 1995, 1996a,b, 1997a,b, 2001a; Mozsik, 2004, 2006).

7.4.2. Effects of vitamin A on the gastric secretory responses and indomethacin-induced gastric microbleedings in healthy human subjects The existence of gastric mucosal prevention can be proved in healthy persons (against IND treatment), in patients with gastric ulcer (GU) and duodenal ulcer (DU) without presence of any inhibitory effect on gastric acid secretion (Mozsik et al., 2007b).

Orally administered indomethacin (IND) (a nonspecific cyclooxygenase 1 and 2 enzyme inhibitor) produces gastric mucosal damage in human healthy subjects. This injury can be detected by the measurement of gastric microbleedings (Moron et al., 1984; Nagy et al., 1984). Recently, the capsaicin-induced gastric mucosal protection was observed by the direct measurement of the changes in the gastric transmucosal potential difference (GTPD) produced by intragastric administration of ethanol in human healthy subjects (Mozsik et al., 2004, 2005a).

7.4.3. Ulcer healing effect of vitamin A in patients with chronic gastric ulcer (multiclinical randomized, prospective study) A randomized, prospective study was carried out in healthy human subjects (n = 12 in each group) to test the cytoprotective effects of atropine (0.125 mg/kg i.m.), cimetidine (12.5 mg/kg i.m.) and vitamin A (100.000 IU i.m.) on the gastric basal (BAO) and maximal (MAO) acid output, and on gastric microbleeding from indomethacin (4 x 25 mg per os)-induced gastric microbleedings (Moron et al., 1984; Mozsik et al., 1986).

7.4.4. Changes in serum levels of retinoids in patients

with different gastrointestinal inflammatory diseases

The serum levels of vitamin A and zeaxanthin were decreased significantly in patients with chronic gastrointestinal inflammatory diseases (gastritis, hepatitis, ileitis terminalis, ulcerative colitis), colorectal polyposis, and different (esophageal, gastric, pancreas, hepatocellular and colorectal) malignant diseases, meanwhile the serum levels of vitamin A provitamins were unchanged and their GI mucosal preventive effects did not depend on the presence of vitamin A activity. It has been concluded that: 1) many experimental and human observations proved clearly the presence and nature of the defensive role of retinoids in the GI tract; 2) there is a correlation between the: a) scavenger properties of retinoids vs. intact vagal nerve; b) scavenging properties vs. intact adrenals; and 3) GI mucosal preventive effects of retinoids and their biochemical changes in GI mucosa; 4) the decrease of extent of ATP-ADP transformation in association with increased extent of ATPcAMP transformation vs. retinoids produced gastrointestinal mucosal prevention only partly as scavengers in chemicals-induced release of oxygen free radicals; 5) sulfhydryl groups of GI tissues take place in the development of GI mucosa (without and with application of retinoids). A lot of clinical observations were carried out in human healthy subjects and patients with different gastrointestinal disorders in connection to retionoids and carotenoids (Rumi et al., 1999, 2000, 2001; Mozsik et al., 2001a, 2005b; Mozsik, 2004, 2005, 2006).

93

According to these results, the serum level of the retinoids decreased in patients with Crohn's diesase, ulcerative colitis, hepatitis, colorectal polyps and different gastrointestinal tumors (oesophageal, stomach, pancreas, hepatocellular, and colon) (Table 15). T a b l e 1 5 . C h a n g e s o f s e r u m c a r o t e n o i d s in patients w i t h different C I d i s e a s e ( c o m p a r e d t o t h e

Oesophagus cc.

Stomach cc.

Pancreas cc.

Hepatocellular cc.

Colon cc.

u

Colorectal polyp

Vitamin A

u

11

11

1

NS

11

Ill

NS

NS

NS

NS

NS

NS

NS

1

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

1

i

Hepatitis C

Carotenoid

Ulcerative colitis

Disease

Crohn's disease

results of h e a l t h y s u b j e c t s ) *

Zeaxanthin

1 1 1 11

NS

NS

1

11

a-Cryptoxanthin

NS

NS

NS

NS

NS

NS

NS

NS

NS

P-Cryptoxanthin

NS

NS

NS

NS

NS

NS

NS

NS

NS

a-Carotene P-Carotene Lutein

NS

111 11

-1—LL"-

111= p<0.001; ll=p<0.01; l=p<0.05; NS= not significant * Rumi, Gy. jr., Par, A., Matus, Z., Rumi, Gy., Mozsik, Gy. (2001): The Defensive Effects of Retinoids in the Gastrointestinal Tract (Animal Experiments and Human Observations). Akademiai Kiado, Budapest, Hungary (with permission)

7.4.5. Changes in serum levels of retinoids in patients with different gastrointestinal cancers The retinoids are chemical compounds of color materials of plants. The literature uniformly agrees that the increased intake of plants as foods prevents the different types of GI cancers. We studied the possible role of different retinoids (vitamin A, (3and p-carotene, a- and p-cryptoxanthin, zeaxanthin, lutein) in patients with different GI cancers, and these present observations were based on our studies published earlier (Mozsik et al., 1994, 1995, 1998, 2001b; Rumi et al., 1999; 2000, 2001; Mozsik 2004). GI tumors had different locations (oesophagus, stomach, pancreas, liver, and colon). We suggest that different aetiological factors are involved in the development of these types of different GI cancers (Barrett's oesophageal metaplasia, chronic atrophic gastritis, viral infection in liver, chronic inflammatory bowel diseases). The serum levels of vitamin A and zeaxanthin were decreased significantly in all of the groups of GI cancer patients (vitamin A was not decreased in patients with pancreatic cancer). Surprisingly, the serum levels of provitamins of vitamin A were found to be normal in patients with different GI tumors. These results together indicate that probably the transformation of provitamins into vitamin A is impaired by 94

some reason(s) at the hepatic level, which suggests a key role of the liver in the tumor development of patients with different GI cancers. Similar correlations were obtained in the serum levels of retinoids in patients with hepatocellular cancers, which offers a further proof for the existence of this hypothesis. It was also interesting to evaluate the possible correlation between the terminal chemical structure, vitamin A activity and GI mucosal prevention. Our results clearly proved that there is no close correlation between the terminal chemical structure, vitamin A activity and GI mucosal protection (Javor et al., 1983; Mozsik, 1994, 1996; Mozsik et al., 2005b, 2007a) (Table 16). T a b l e 1 6 . S u m m a r y of t h e c h a n g e s of s e r u m l e v e l of retinoids in patients w i t h different gastrointestinal

cancers*

Oesophageal

Gastric

Hepatocellular

Pancreatic

Colorectal

cancer

cancer

cancer

I n situ

cancer

cancer

colon cancer

~~—-____Patients Retinoids

~~~~~— 8

21

15

10

44

9

Vitamin A

u

u

in

NS

III

ce-Carotene

NS

NS

NS

NS

NS

fl-Carotene

NS

NS

NS

NS

NS

NS

oc-Cryptoxantin

NS

NS

NS

NS

NS

NS

NS

NS

NS

NS

III 11

fi-Cryptoxantin

NS

NS

Zeaxanthin

11

I

Lutein

NS

NS

NS

NS

ill NS

NS

P values: between healthy controls vs. each group; -l=P<0.05 J4=P<0.01 111=P<0.01 * Mozsik, Cy, Rumi, Cy., Domotor, A., Figler, M., Casztonyi, B., Rapp, E., Belagyi, J . , Matus, Z., Melegh, B. (2005b): Involvement of serum retinoids and Leiden mutation in patients with esophageal, gastric, liver, pancretic, and colorectal cancers in Hungary. World J . Gastroenterol. 11: 7646-7650 (with permission)

Similar results were obtained in the animal experiments published earlier (Javor et al., 1983; Mozsik, 1994, 1996; Mozsik et al., 2001b, 2005b) (Table 17, Fig. 14). At present we have no correct information on the correlation between the serum vs. tissue levels of retinoids in patients with different GI tumors. In animal observations we proved the presence of P-carotene in the gastric mucosa in indomethacin-treated rats after acute surgical vagotomy (Mozsik et al., 1991, 2001a, 2005b, 2007a), however, no gastric mucosal protection was found, indicating the necessity of intact vagal nerve for the development of P-carotene-induced gastric cytoprotection (Mozsik et al., 2001). The mechanisms of action of GI mucosal defensive effects of retinoids are very complex. Our earlier observations indicated that the GI mucosal protection of retinoids depends on 1) intact vagal nerve, 2) intact adrenals in the experimental animals, 3) gastric mucosal biochemical changes (the retinoids produce a dose-dependent inhibition on the extent of ATP-transformation into ADP in association with a simultaneous increase in the transformation of ATP into cAMP), 4) intact function of sulfhydryl groups, and 5) scavenger properties (Mozsik et al., 1991, 1994, 1995, 1998, 2001a, 2005b, 2007a; Rumi et al., 2001).

T a b l e 1 7 . C o r r e l a t i o n s b e t w e e n t h e gastric c y t o p r o t e c t i v e effects of retinoids, their c h e m i c a l structure a n d v i t a m i n A a c t i v i t y in patients w i t h t h e different gastrointestinal disorders Terminal chemical

Retinoids

Vitamin A

Gastric mucosal

structure

activity

prevention

Vitamin A

R=a

Yes

Yes

a-Carotene

X=Y=a

Yes

Yes

(3-Cryptoxanthin

X=a, Y=b

Yes

Yes

Zeaxanthin

X=Y=b

None

Yes

Lutein

X=b, Y=c

None

Yes

Capsorubin

X=Y=d

None

None

Capsanthin

X=b, Y=d

None

None

Capsanthol

X=b, Y=e

None

None

Lycopene

X=Y=f

None

None

The results were based on the results obtained in animals Uavor, X, Bata, M , Lovasz, L., Moron, R, Nagy, L., Patty, I., Szabolcs,)., Tarnok, R, Toth, Gy., Mozsik, Gy. (1983): Gastric cytoprotective effects of vitamin A and other carotenoids. Int. J . Tiss. React. 5: 289-296] and patients with different gastrointestinal disorders [Mozsik, Gy., Rumi, Gy., Domotor, A., Figler, M., Gasztonyi, B., Papp, E., Belagyi, J . , Matus, Z., Melegh, B. (2005b): Involvement of serum retinoids and Leiden mutation in patients with esophageal, gastric, liver, pancretic, and colorectal cancers in Hungary. World J . Gastroenterol. 11: 7646-76501

Zeaxanthin, |imol/l

Healthy (57)

0.14

0.12 -|

0.10

Y = A + B*X

S t o m a c h (21)

A = 0.0107 ± 0.0037

0.08

B = 0.063 ± 0.005

• C o l o n (53)

R = 0.88 ± 0.014 P = 0.006

0.06 • Liver (15) 0.04 In situ c o l o n (9) 0.02

0.0

0.5

1.5

2.0 Vitamin A, |imol/l

Fig.

14. C o r r e l a t i o n b e t w e e n t h e s e r u m l e v e l s of z e a x a n t h i n a n d v i t a m i n A a n d patients w i t h different gastrointestinal t u m o u r s ( t h e n u m b e r of e x a m i n e d patients in parenthesis). ( M o z s i k , G y . , R u m i , G y . , D o m o t o r , A . , Figler, M . , G a s z t o n y i , B., P a p p , E., B e l a g y i , J . , M a t u s , Z . , M e l e g h , B., 2 0 0 5 b : I n v o l v e m e n t o f s e r u m retinoids a n d L e i d e n m u t a t i o n in patients w i t h e s o p h a g e a l , gastric, liver, p a n c r e t i c , a n d c o l o r e c t a l c a n c e r s in H u n g a r y . W o r l d J . Gastroenterol. 1 1 : 7646-7650) (with permission)

96

The retinoid-induced GI mucosal protections do not depend on the 1) inhibition of gastric acid secretory responses 2) vitamin A activity, 3) number of unsaturated double bonds, 4) the presence of a characteristic chemical structure of their terminal components, and 5) modification of vascular permeability (see updated review paper of Mozsik 2004, 2006; Mozsik et al., 2005b, 2007a). These results clearly proved that the beneficial effect of retinoids is much more complex than only those depending on their scavenger properties. The results of biochemical observations suggested the existence of different cAMP-dependent cellular regulatory mechanisms (including the functions of retinoid receptors, gene expressions) (Mozsik et al., 2005b, 2007a).

7.4.5.1. Leiden mutation in patients

with different gastrointestinal tumors

The presence of Leiden mutation (replacement of Arg by Glu of residue 506 in the factor V molecule, FVR, 506 Q) has been proven in thrombophilia (Bargen, Parker 1936; Dahlback et al., 1993; Bertina et al., 1994; Dahlback 1995) as well as in Crohn's disease and ulcerative colitis (Best et al., 1976; Talbot et al., 1986; Nagy et al., 2000; 2001; Papa et al., 2003), meanwhile no higher prevalence of Leiden mutation has been observed in patients with acute gastritis and hepatitis (Nagy et al., 2001). The presence of Leiden mutation (APC) is responsible for blood coagulation in thrombophilia. The involvement of vascular events was suggested in the development of different acute inflammatory processes in the GI tract (Mozsik, 2004). That was the reason why we studied the potential role of Leiden mutation in the acute and chronic gastrointestinal inflammatory processes {Helicobacter /ry/on'-induced gastritis, viral hepatitis, Crohn's disease, ulcerative colitis). The prevalence of Leiden mutation was increased in chronic inflammatory bowel diseases, but no changes were observed in gastritis and hepatitis. It was also significantly higher in the patients with oesophageal, gastric, hepatocellular, pancreatic and colorectal cancers. These results may indicate that the increased prevalence of Leiden mutation alone resistance does not take place directly in the tumor genesis of the human GI cancers. We compared the different results in the examined parameters in patients with acute and chronic gastrointestinal inflammatory diseases, and we suppose a time-sequence process between the inflammatory diseases and GI cancers in patients, suggesting a key role of retinoids in the progress of precancerous states to cancerous states (Rumi et al., 1999, 2001; Mozsik et al., 2001b, 2005b; Mozsik, 2004).

7.4.5.2. Correlation between the prevalence of Leiden mutation and decrease of serum levels of vitamin A and zeaxanthin in patients with different gastrointestinal tumors Different correlations were found between the genetic (Leiden mutation) and enviromental factors (vitamin A, zeaxanthin) in patients with different gastrointestinal tumors (Figs 15-17). 97

We found a positive and significant correlation between the following parameters: 1) prevalence of Leiden mutation vs decrease in serum vitamin A level, 2) prevalence of Leiden mutation vs. decrease of serum level of zeaxanthin, 3) decrease in serum level of zeaxanthin vs. the decrease in serum level of vitamin A (Fig. 14), 4) the increased prevalence of Leiden mutation represents a genetic factor in the different chronic precancerous states 5) interestingly the positive correlations were found between the prevalence of Leiden mutation vs. decrease in serum levels of vitamin A and zeaxanthin, which suggest the promoter roles of retinoids in the human GI tumor genesis, 6) these tumor promotor properties of retinoids do not depend on the nature of tumor histology (planocellular, adenocarcinoma and hepatocellular cancers) (Mozsik et al., 2005b) (Figs 14-17). p<0.001

Healthy 600

Oesophag. 8

Gastric

Liver

Pancreas

Colorectal

21

15

10

44

In situ c c . 9

Fig. 75. P r e v a l e n c e of L e i d e n m u t a t i o n in patients w i t h different gastrointestinal t u m o r s . T h e n u m b e r o n t h e abscissa i n d i c a t e s t h e n u m b e r of patients ( 6 0 0 b l o o d d o n o r s u s e d as c o n t r o l ) ( M o z s i k , G y . , R u m i , G y . , D o m o t o r , A . , Figler, M . , G a s z t o n y i , B., P a p p , E., B e l a g y i , J . , M a t u s , Z . , M e l e g h , B., 2 0 0 5 b : I n v o l v e m e n t of s e r u m retinoids a n d L e i d e n m u t a t i o n in patients w i t h e s o p h a g e a l , gastric, liver, p a n c r e t i c , a n d c o l o r e c t a l c a n c e r s in H u n g a r y . W o r l d J . G a s t r o e n t e r o l . 1 1 : 7 6 4 6 - 7 6 5 0 ) ( w i t h p e r m i s s i o n )

98

Vitamin A, n.mol/1

2.0

J

E Y = A + B*X

A = 3.166 ± 0 . 5 3 6

Pancreatic (10)

1.6

B = - 0 . 1 3 0 ±0.025 R = - 0.94 ± 0 . 3 7 P = 0.015

1.2 Stomach (21) 0.8

f Liver (15)

0.4

C o l o n (53) "~i—

30

25

Prevalence of Leiden, % Fig. 76. C o r r e l a t i o n b e t w e e n t h e p r e v a l e n c e of L e i d e n m u t a t i o n a n d s e r u m level of v i t a m i n A w i t h different gastrointestinal t u m o r s (the n u m b e r of e x a m i n e d patients in parenthesis) ( M o z s i k , G y . , R u m i , G y . , D o m o t o r , A . , Figler, M . , G a s z t o n y i , B., P a p p , E., B e l a g y i , J., M a t u s , Z . , M e l e g h , B., 2 0 0 5 b : I n v o l v e m e n t of s e r u m retinoids a n d L e i d e n m u t a t i o n in patients w i t h o e s o p h a g e a l , gastric, liver, p a n c r e t i c , a n d c o l o r e c t a l c a n c e r s in H u n g a r y . W o r l d J . Gastroenterol. 1 1 : 7646-7650) (with permission)

0,16

Zeaxanthin, (j,mol/l

Y =A+

B*X

A = 0.151 ± 0.034 0,14

B = -0.0043 ±0.0018

Healthy (57)

R = -0.94 ±0.28 P = 0.0101

0,12

0,10H Stomach (21) 0,08

O e s o p h a g e a l (8)

j

j

0,06

C o | o n

£ Liver^OsT

0,04

0,02 10

12

14

16

18

20

22

Fig. 17. C o r r e l a t i o n b e t w e e n t h e p r e v a l e n c e of L e i d e n m u t a t i o n a n d t h e s e r u m level of z e a x a n t h i n w i t h different gastrointestinal t u m o r s (the n u m b e r of e x a m i n e d patients in parenthesis) ( M o z s i k , G y . , R u m i , G y . , D o m o t o r , A . , Figler, M . , G a s z t o n y i , B., P a p p , E., B e l a g y i , J . , M a t u s , Z . , M e l e g h , B., 2 0 0 5 b : I n v o l v e m e n t of s e r u m retinoids a n d L e i d e n m u t a t i o n in patients w i t h e s o p h a g e a l , gastric, liver, p a n c r e t i c , a n d c o l o r e c t a l c a n c e r s in H u n g a r y . W o r l d J . Gastroenterol. 1 1 : 7646-7650) (with permission)

99

8. Animal and human observations with capsaicinoids 8 . 1 . Physiological and pharmacological research tool by capsaicin (Short overview) 8.1.1. The chemistry of capsaicinoids According to the USP30-NF25 (see Section 6.3.4.2.7) Capsaicin contains not less than 90.0% and not more than 110.0% of the labeled percentage of total capsaicinoids. The content of capsaicin (C H 7N0 ) is not less than 55%, and the sum of the contents of capsaicin and dihydrocapsaicin ( C H N 0 ) is not less than 75%, and the content of other capsaicinoids is not more than 15%, all calculated on the dried basis. 18

2

3

18

29

3

Chemical composition of Natural Capsaicin Natural Capsaicin contains a mixture of a series of structurally related compounds called capsaicinoids (see Section 6.2.3.1). They all share the common vanillin structural moiety, which is considered to be associated with their effect on the TRPV] receptors (Bevan, Szolcsanyi, 1990). The two main components of Natural Capsaicin are capsaicin and dihydrocapsaicin. There are, however, some other minor ones as well. The structures of the main components of Natural Capsaicin are shown in Fig. 7. Stability of capsaicinoids Information on the stability of isolated capsaicin, dihydrocapsaicin as well as on other capsaicinoids is rather limited (see Section 6.3.4.4). The recommended storage temperature of capsaicin and dihydrocapsaicin is 2-8°C and -20°C, respectively. According to the MDSD sheet, the Sigma-Aldrich Capsaicin natural (product number 21750, CAS number: 404-86-4) (-65% capsaicin) should be stored at 2-8°C. Literature data Kopec et al. (2002) tested the stability of (100%) ethanol solution of capsaicin of different concentrations. The 4 mM or more concentrated solutions protected from light and stored at 4°C have been proved to be stable for a period of 12 months. While investigating the stability of a capsaicin containing ointment Jaiarj et al. (2000) found the preparation to show higher stability stored at 4°C than at ambient temperature. Schweiggert et al. (2006) investigated the stability of chili powder. They found that the capsaicin, dihydrocapsaicin and nordihydrocapsaicin content of the samples decreased by 7-12%. Based on their experience it was recommended that paprika (pepper) samples should be heat-treated before processing, in order to reduce 100

the number of microorganisms producing enzymes with peroxidase activity of the fruits. The presence of phenolic hydroxyl group and that of the carbon-carbon double bond make the capsaicinoids sensitive to oxidation. Accordingly, natural capsaicin should be protected from exposure to light, heat, moisture, and oxidizing agents, which initiate and/or catalyse the decomposition processes.

8.1.2. Source of capsaicin The term Capsicum refers to the fruit of numerous species of the solanaceous genus Capsicum. The genus name Capsicum is either derived from Greek "Kapso" meaning to bite, referring to its pungency or from the Latin "Capso" or box referring to the fruit pod. Members of the genus vary widely in size, shape, flavor, and much more importantly in pungency. Red hot peppers, also called chilies, paprika, and sweet non-pungent peppers are widely consumed by humans (Maga, 1975; Rozin, 1990). Capsaicin (8-methyl-N-vanillyl-6-nonenamide) is the major pungent ingredient of hot peppers.

8.1.3. Selective sensory effects of capsaicin Apart from being used as a food additive, the compound has wide important pharmacological actions. Capsaicin is uniquely selective for mammalian small afferent neurons of dorsal root ganglia C and A8 fibers, a property which led to its use to investigate the role of these afferent fibres in a number of physiological processes (Jancso, Jancso-Gabor, 1959; Szolcsanyi, 1982; 1984a,b). In the skin the polymodal sensory receptors, chemoreceptors and warmth receptors are affected by capsaicin (Szolcsanyi, 1977, 1982, 1983a,b, 1984a,b, 1985, 1990a,b, 1993, 1996, 2004; Bevan, Szolcsanyi, 1990; Szolcsanyi et al., 1994; Holzer, 1988, 1990, 1991a,b, 1992 a,b, 1998; Holzer, Sametz, 1986; Holzer, Lippe, 1988; Holzer et al., 1989). Most of these afferents contain substance P (SP) and/or calcitonin gene-related peptide (CGRP) (Holzer, 1991a,b; Maggi, 1995). The action of capsaicin expresses itself as an initial short-lasting stimulation that can be followed by desensibilization to capsaicin itself and to other stimuli of afferent sensory neurons. Capsaicin in ng-pg/kg doses applied to the peripheral or central endings or cell bodies of sensory neurons induces transient excitation of these sensory neurons. In response to stimulation peptide mediators are released from the central and peripheral nerve endings (Szolcsanyi, 1984a,b, 1996; Maggi, 1995; Holzer, 1998, 1991a). In the periphery neuropeptide release exerts local neuroregulatory tissue responses (Szolcsanyi, 1984a,b, 1991, 1996). Neuropeptides are stored in sensory vehicles (Gulbekian et al., 1986; Merighi et al., 1988) and are released on stimulation with capsaicin in a Ca -dependent manner (Maggi et al., 1989). In this way the peripheral terminals of capsaicin sensitive nerves are not only sensory receptors for conveying impulses in the afferent direction but also effector sites from where mediators 2+

101

are released for neurotransmission (Szolcsanyi, 1984, 1996). With large doses (mg/kg) there is an initial stimulation, the duration of which is not yet defined, followed by sensory desensitization which is restricted to those types of neurons but are also activated by the drugs (Szolcsanyi, 1984a,b, 1993, 2004). Four response stages of the capsaicin-sensitive primary afferents to capsaicin have been introduced by Szolcsanyi (1984a,b, 1985) depending on the dose and duration of exposure to the drug. These are excitation, sensory blocking effect, long-term selective neurotoxic impairment and irreversible cell destruction.

8.1.4. Mechanism of action of capsaicin on sensory receptors In explaining the mechanism of these sensory effects of capsaicin it has been hypothesized that in the polymodal nociceptive primary afferent neurons there exists a capsaicin-gated cation channel which operates at the peripheral receptive terminals at the level of the cell body. Capsaicin exerts its excitatory effect by activation of this cation channel which is permeable to a wide range of cations as Na , K and Ca , but not to anions as CI" (Winter, 1987; Wood et al., 1988). The influx of Na and C a induces a membrane depolarization which, at a certain level, opens tetrotodotoxin-sensitive fast sodium channels at the regenerative region of the sensory receptor and in this way triggers nerve terminal spike and direct stimulation of transmitter release by Ca . Opening of the capsaicin-gated cation channel for shorter or longer periods of time triggers a chain of intracellular events (Bevan, Szolcsanyi, 1990). Functional blockage with reversible intracellular molecular changes (stage 2) or neurotoxic degeneration (stages 3 and 4) develops depending on the concentration and the contact time at the site of action on different parts (receptor, axon, cell body, central terminal) of the capsaicin-sensitive primary afferent neurons. Prolonged stimulation and consequently prolonged opening of this cation channel result in osmotic swelling due to intracellular NaCl accumulation together with intracellular accumulation of C a at the exposed sites of the nerve terminal which activate Ca -dependent enzymes and impair mitochondrial functions (Bevan, Szolcsanyi, 1990; Chard et al., 1995). Prolonged activation of C a is therefore the first step in sequence of events ultimately leading to cell death. In mammals, the long-lasting sensory blocking or neurotoxic effects of capsaicin on primary afferent neurons have been described following topical, periaxonal, or systemic pretreatment in adults or neonates. Autonomic efferent neural mechanisms are not affected by drug (Szolcsanyi, 1982; Maggi, Meli, 1988; Holzer, 1991a). This selective sensory blocking effect of capsaicin has been used as an experimental tool for the elimination of the capsaicin-sensitive subset of primary afferent neurons and consequently for identifying those tissue responses that are mediated by capsaicinsensitive afferent neurons (Szolcsanyi, 1996; Mozsik et al., 1997a). The later tissue responses are absent in sensory desensitized animals. +

+

2+

+

2+

2+

2+

2+

2+

102

8.1.5. Capsaicin actions in the gastrointestinal tract of animals In their experiments, Makara et al. (1965) found that intragastric capsaicin (1 mg/0.5 ml volume/rat) enhanced the development of Shay ulcer 12 hours later. After four consecutive daily doses of reserpine (1.0 mg/kg, sc.), the above dose of capsaicin given simultaneously with reserpine enhanced gastric ulceration by the latter. On the other hand, daily administration of paprika oil containing 1.0 mg/kg of capsaicin tended to accelerate rather than retard the healing process of reserpine-induced gastric ulcer in rats. This was attributed by the authors to the capsaicin-induced local hyperemia or the carotenoids and other pigments present in paprika oil. When Lee (1963) fed rabbits for 12 months on various diets (high fat, high carbohydrate or high protein) supplemented with large doses of ground hot pepper, ulcers developed in the stomach of all animals, and cirrhosis of the liver occurred in animals fed either high fat or high carbohydrate diets supplemented with capsaicin. In the study of Nopanitaya (1974) the effect of capsaicin (1.0 mg/kg) and its combination with various diets on the morphology of the duodenal mucosa of young rats was investigated for periods of 28 and 56 days. The author reported ultrastructural alterations in the mitochondria of the absorptive cells of rats fed a low protein diet and also those supplemented with capsaicin. The changes, however, were less pronounced at the 56 day than at the 2 8 day, indicating some sort of adaptation to capsaicin. In 1981, Szolcsanyi and Bartho proved that capsaicin protects against experimental gastric ulcer. Introduction of capsaicin into the stomach of pylorus-ligated (Shay)rats in small doses (5 to 50 pg) and low concentration (10 pg/ml) markedly reduced the ulcer formation in these 18 hours later. On the other hand, acute gastric ulceration induced by pylorus-ligation (Shay ulcer) or acid distension was aggravated in rats desensitized 2 weeks earlier with systematic capsaicin in high doses which selectively impairs capsaicin sensitive sensory nerves. In capsaicin desensitized rats the aggressive side of the balance remained apparently unchanged since hypersecretion of the pylorus-ligated rats did not differ from that of the controls with respect to volume, H and pepsin concentration. This suggested that it is the gastric defense mechanism which was impaired in capsaicin desensitized rats. As a result of these data a role of capsaicin-sensitive afferents in modulating gastric mucosal defenses was forwarded by the authors. In explaining the nature of this novel gastroprotective action of low-dose capsaicin, it was postulated that intragastric capsaicin exerts opposite effects on gastric ulcer formation depending on the concentration in which it is introduced into the gastric lumen. Low concentrations tend to inhibit the development of ulceration and high concentrations promote ulcer formation. Release of vasodilatation mediators from capsaicin-sensitive sensory nerve endings with the consequent enhancement of the microcirculation was proposed as the mechanism responsible for the mucosal protective effects of intragastrically administered capsaicin in low concentrations. This mechanism seems to operate under physiological conditions and provides a type of resistance or defense against ulcer formation. In capsaicin-desensitized rats, mucosal sensory receptors will be unresponsive to stimuli and consequently no th

th

+

103

release of vascular dilatator mediators will take place upon challenge with noxious agents. The result is that the gastric ulcer will be aggravated (Szolcsanyi, Bartho, 1981). These observations and the drawn conclusions were confirmed after a few years by a number of investigators. Intragastric application of capsaicin in small doses has been shown to protect the rat gastric mucosa against experimental gastric ulcerations induced by ethanol (Holzer, Lippe, 1988; Esplugues, Whittle, 1989; Esplugues et al., 1990, 1992), acidified aspirin (Holzer et al., 1989) and indomethacin (Gray et al., 1994). Furthermore, after desensitization rats exhibited more severe gastric mucosal damage than their sensory intact controls in response to chemical challenge with ethanol (Holzer, Lippe, 1988; Lippe et al., 1989; Esplugues, Whittle, 1990; Esplugues et al., 1992; Pabst et al., 1993), cysteamine (Holzer, Sametz, 1986; Gray et al., 1994), platelet activating factor (Esplugues et al., 1989; Pique et al., 1990), endothelin-1 (Whittle, Lopez-Belmonte, 1991). Capsaicin desensitization 6 days prior to cold restraint stress, however, was reported to have had a little effect on gastric ulcer. Only the number of lesions was higher in capsaicin-treated rats restrained for 3 hours (Dugani, Glavin, 1986). Acute intragastric capsaicin (40 mg/kg) followed immediately by stress resulted in significantly more frequent and more severe ulceration relative to controls or rats given a 1, 2 and 3 hours delay between capsaicin administration and application of restraint stress (Dugani, Glavin, 1985). Cysteamine-induced duodenal ulcers (Holzer, Sametz, 1986), and gastric mucosal damage evoked by 0.6 M HC1 (Takeuchi et al., 1994), were not changed after capsaicin desensitization. Studies have shown that gastric mucosal barrier disruption is accompanied by an increase of gastric mucosal blood flow (GMBF), which appears to be triggered by H rediffused through the breached mucosal defenses (Bruggeman et al., 1979; Starlinger et al., 1981). Such an increase in GMBF is thought to be a defense mechanism, whereby the increased blood flow prevents the accumulation of injurious concentration of H in the submucosa. Capsaicin-sensitive nerves which are unduly sensitive to H (Bevan, Yeats, 1990) have been shown to respond to H back-diffused through breached mucosal defenses and signal for an increase in gastric mucosal blood flow (Holzer, 1991a,b). This further strengthened the role of these nerves in maintaining mucosal integrity. Holzer (1991a,b) postulated that the acidinduced mucosal hyperemia results from local axon reflex between collaterals of afferent nerve fibres within the gastric wall. Li et al. (1992) found that gastric mucosal hyperemia in response to perfusion of the rat stomach with 0.15 M HC1 in 15 v/v ethanol could to be completely blocked by close arterial infusion of a hCGRP receptor antagonist. Lippe and Holzer (1992) reported that in rats NG-nitro-L-arginine methyl ester (L-NAME) (an inhibitor of endothelium derived nitric oxide formation) depressed the increase in GMBF produced by gastric perfusion with dilute ethanol in 0.15 M HC1. The loss of H from the lumen under these circumstances was also markedly enhanced by L-NAME. Considerable controversy still exists in the literature, however, as regards the mediators of the hyperemic response to back-diffusion of acid (Whittle, 1977; Ritchie, 1991; Gislason etal., 1995). +

+

+

+

+

104

Whether capsaicin-sensitive sensory nerves are involved in repair mechanisms of the injured mucosa has been also investigated. In anaesthetized rats, 180 min after exposure to 50 v/v ethanol, rapid repair of the injured mucosa (assessed by reduction of deep mucosal damage and partial re-epithelialization of the denuded surface) was reported to be similar in sensory denervated and sensory intact groups. This suggested that nociceptive neurons control mechanisms of defense against acute gastric mucosal injury, but they are not required for the rapid repair of the injured mucosa (Pabst et al., 1993). On the contrary, the different status of sensory neurons delayed healing of gastric ulcers provoked in rats by 0.6 M HC1. In addition, in stomachs damaged by 0.6 M HC1, capsaicin-desensitized not sensory intact animals showed no hyperemic responses to intragastric instillation of 50 mM HC1. The conclusion was that capsaicin-sensitive sensory nerves contribute to healing of gastric ulcer (lesions) by mediating the hyperemic responses associated with acid back-diffusion following injury (Takeuchi et al., 1994). Similarly, sensory denervated rats showed markedly increased area of acetic acid-induced ulceration at 1 and 2 weeks following the acetic acid injection indicating that the injury of sensory function adversely affected the healing of gastric ulcer (Tramontana et al., 1994). Capsaicin exerts protective effects on the chemical-induced mucosal injury not only in the stomach, but also in the colon. Evangelista and Meli (1989) found that systemic (one week) capsaicin neonatal pretreatment enhanced trinitrobenzene sulfonic acid-induced colic colitis in the rat. This pretreatment, however, did not affect acute colitis (24 hours) caused by trinitrobenzene sulfonic acid, ethanol or acetic acid. Reinshagen et al. (1994) employed systemic capsaicin pretreatment as a tool to investigate the role of sensory nerves in an immune-complex model of colitis in rabbits. They found that capsaicin pretreatment per se caused no histological evidence of inflammation. Meanwhile, colitis was more severe in sensory denervated than in sensory intact rabbits. The increase in ulcer index and neutrophil infiltration was more marked in the capsaicin pretreated control group at both 48 and 96 hours, respectively. The difference in neutrophil infiltration between the two groups was, however, more marked at 48 than 96 hours (Reinshagen et al., 1994). Endoh and Leung (1990) reported that topical capsaicin application protected against acetic acid-induced colitis. In the trinitrobenzene sulfonic acid-induced colitis rat model, however, partial and transient protective effect was seen by Goso et al. (1993) after topical capsaicin administration. Co-administration of 640 pM capsaicin reduced the ulcerative area from 9 1 % to 64% only when the colon was examined 1 hour later. An approximately 8-fold higher dose of capsaicin (5000 pM) yielded similar protection, while 100 pM had no protective effect. No protection by capsaicin was seen, however, when the colon was examined 24 hours after noxious challenge.

8.1.6. Capsaicin-sensitive sensory nerves and gastric acid secretion Several studies in rats have indicated the involvement of capsaicin-sensitive sensory nerves in the regulation of gastric acid secretion, however, contradictory data were reported. In most studies, the indirect approach through functional ablation of cap105

saicin-sensitive afferent nerves with systemic neonatal (Evangelista et al., 1989; Esplugues et al., 1990), adult (Alfoldi et al., 1986; 1987; Dugani, Glavin, 1986; Robert et al., 1991) or peripheral capsaicin (Raybould, Tache, 1989) treatment was used as a tool to investigate the role of capsaicin-sensitive afferent nerves in the regulation of gastric acid secretion. Adult rats treated with systemic capsaicin (60 mg/kg, sc.) showed depressed pentagastrin-stimulated gastric acid secretion (Dugani, Glavin, 1986). On the contrary, adult systemic capsaicin pretreatment with 300 mg/kg sc. did not modify gastric acid secretion elicited by pentagastrin, carbachol or by a small dose of histamine (0.1 mg/kg). However, the gastric acid secretory response to 0.5 and 5.0 mg/kg histamine was greatly reduced in capsaicin-desensitized rats. It was suggested that the histamine-induced increase in gastric acid secretion involves a capsaicin-sensitive mechanism, while these mechanisms are not required for the effects of pentagastrin and cholinergic stimulation of gastric acid secretion (Alfoldi et al., 1986, 1987). Similar data were reported by Raybould and Tache (1989) using topical capsaicin application into the cervical vagus. The gastric acid secretory response to distension (5 ml for 6 min) was reduced in capsaicin-treated rats. This mechanism by which capsaicin-sensitive vagal afferent fibres play a role in the secretory response to histamine was explained by histamine acting in part by increasing vagal C-fibres discharge resulting in a vago-vagal reflex increase in gastric acid secretion or by that histamine stimulates vagal afferent C-fibres which results in the release of peptides from sensory nerves terminals. However, the authors admit that up to date a peptide that increases gastric acid secretion and that is localized in vagal afferent nerves has not been identified. Adult rats treated neonatally with systemic capsaicin (300 mg/kg, sc.) did not show any reduction in their gastric acid secretory to histamine, pentagastrin or carbachol, while acid secretion in response to distension was abolished. On the other hand, capsaicin desensitization (neonatal treatment) substantially reduced the gastric acid secretion to 2-deoxy-D-glucose (Evangelista et al., 1989), while it did not modify that stimulated by insulin (Esplugues et al., 1990). The conflicting observations regarding the effect of capsaicin continued to be seen when it was given into the stomach. It was reported that intraduodenal (but not intragastric) instillation of capsaicin (1.0 mg in 2 ml saline solution) in pylorus-ligated rats induced a significant rise in total acidity 12 hours later (Makara et al., 1965). The results with capsaicin in the gastrointestinal tract were contradictory, as mentioned above. Small attention was paid to the applied doses of capsaicin. Szolcsanyi and Bartho (1981), however, emphasized that capsaicin protects against the chemical-induced gastric ulcer formation, when the capsaicin was given in 5 and 50 pg doses (10 pg/ml concentration) intragastrically, meanwhile capsaicin in high dose aggravated the ulcer formation by the production of desensitization. We carried out systematic observations with capsaicin concerning its small and high concentrations (or pretreatment produced desensitization of capsaicin sensitive afferent nerves) on different experimental models (aspirin, HC1, indomethacin, ethanol, cysteamine). The changes of gastric acid secretion, gastric mucosal damage, gastric H back-diffusion, gastric mucosal blood flow were measured and calculated, when the capsaicin was applied in small doses and in high doses producing +

106

capsaicin desensitization (Mozsik, et al., 1997a,b). The results of these observations clearly demonstrated that: 1. capsaicin, given in small doses, dose-dependently inhibited all of the parameters in all experimental models; 2. the gastric mucosal protective effects of capsaicin remained besides those of the other drugs (acting at the level of efferent nerves, e.g. atropine, cimetidine or topically, such as sucralfate, retinoids). Consequently, capsaicin enhanced the other drugs-induced gastric mucosal protective effects (Mozsik et al., 1997a,b); 3. after denervation of capsaicin-sensitive afferent nerves (by pretreatment with high dose of capsaicin) the gastric mucosal ulcer formation was enhanced. These results offered to conclude that the gastric mucosal protective effects can be obtained only with capsaicin, when it is given in small doses, but not by its application in higher doses (Abdel-Salam et al., 1994, 1995a,b,c,d,e,f,g, 1996a,b, 1997a,b,c,d; Mozsik etal., 1993, 1996a,b, 1997a,b,c).

8.1.7. Molecular-pharmacological studies Recently, the molecular-pharmacological observations were carried out (and calculated, based on the dose-response curves of drugs) with capsaicin, atropine, cimetidine, omeprazole, PGI , vitamin A, P-carotene, studying their effects on the gastric acid secretion in 2 and 4 hours pylorus-ligated rats alone, or in combination of betanechol (7.6 and 15.4 nmol/kg), histamine (2.7 and 13.6 pmol/kg) and pentagastrin (65.1 and 325.6 nmol/kg) and on the gastric mucosal damage produced by intragastrically applied ethanol, HC1, acidified aspirin and subcutaneously applied indomethacin (alone and in combination with application of 7.6 and 15.2 pmol/kg betanechol, 13.6 and 54.3 pmol/kg histamine and 6.51 and 325.6 nmol/kg pentagastrin) (calculating the number and severity of gastric mucosal damage) in rats (Figs 18-21, Tables 18-25). The necessary doses for producing 50% inhibition on the gastric acid secretion and gastric mucosal damage were calculated in molar values/kg body weight (ED ). The values for affinity (pD) and intrinsic (a-values) were calculated according to standard procedures employed in molecular pharmacology (Csaky, 1969). The values of the pD (necessary dose to inhibit the gastric acid secretion and gastric mucosal damage in 50%) and pA (necessary dose to produce 50% decrease in gastric acid secretion and on gastric mucosal damage) were calculated from the affinity and intrinsic activity curves. The doses of drugs (compounds) were calculated as molar values for the determination of their biological effects. The affinity (pD values) and intrinsic activity (revalues) are shown as molar values. The intrinsic activity of atropine (a) was taken as 1.00 for comparing the effects of agents on gastric acid secretion and gastric mucosal damage (Figs 18-21, Tables 18-25) (Mozsik et al., 2006). The results obtained allowed the following conclusions: 1. Only the values of pD and pA (expressed in [-] molar doses) can be used for the evaluation of physiological and pharmacological regulations of the target organ(s) in animal experiment(s) (Mozsik et al., 2006); 2. The following pD (ED %) values were obtained for the different drugs (compounds) for actions in inhibiting gastric acid secretion: atropine 5.75; omeprazole 2

50

2

2

2

2

2

2

50

107

means±SEM

Fig.

"i

1

9

8

—

7

1

1

1

1

6

5

4

3

- g l l |o

M

18. Affinity c u r v e s for different drugs a n d c o m p o u n d s i n h i b i t i n g t h e gastric a c i d s e c r e t i o n of 4 h pylorus-ligated rats ( M o z s i k , G y . , D o m o t o r , A . , A b d e l - S a l a m , O . M . E . , 2 0 0 6 : M o l e c u l a r p h a r m a c o l o g i c a l a p p r o a c h to d r u g a c t i o n s o n t h e afferent a n d efferent fibres of t h e v a g a l n e r v e in t h e gastric m u c o s a l p r o t e c t i o n in rats. I n f l a m m o p h a r m a c o l o g y 1 4 : 2 4 3 - 2 4 9 ) (with permission)

i.ooH

9

8

7

6

5

4

3

-loglM] Fig. 19. Intrinsic affinity c u r v e s for different drugs a n d c o m p o u n d s i n h i b i t i n g t h e gastric a c i d s e c r e t i o n of 4 h pylorus-ligated rats e x p r e s s e d in relation to that of a t r o p i n e ( 1 . 0 0 ) ( a

a t r o p i n e

)

(Mozsik, Gy., Domotor, A., Abdel-Salam, O . M . E . , 2 0 0 6 : M o l e c u l a r pharmacological a p p r o a c h to d r u g a c t i o n s o n t h e afferent a n d efferent fibres of t h e v a g a l n e r v e in t h e gastric m u c o s a l p r o t e c t i o n in rats. I n f l a m m o p h a r m a c o l o g y 1 4 : 2 4 3 - 2 4 9 ) ( w i t h permission)

108

Fig. 20. Affinity c u r v e s for different drugs a n d c o m p o u n d s i n h i b i t i n g t h e gastric m u c o s a l d a m a g e p r o d u c e d b y v a r i o u s c h e m i c a l agents in rats ( M o z s i k , C y . , D o m o t o r , A . , A b d e l - S a l a m , O . M . E . , 2 0 0 6 : M o l e c u l a r p h a r m a c o l o g i c a l a p p r o a c h to d r u g a c t i o n s o n t h e afferent a n d efferent fibres of t h e v a g a l n e r v e in t h e gastric m u c o s a l p r o t e c t i o n in rats. I n f l a m m o p h a r m a c o l o g y 14: 2 4 3 - 2 4 9 ) (with permission)

Fig. 21. Intrinsic a c t i v i t y c u r v e s for different drugs a n d c o m p o u n d s i n h i b i t i n g t h e gastric m u c o s a l d a m a g e p r o d u c e d by c h e m i c a l agents e x p r e s s e d relative to that of a t r o p i n e (oc tropine 1 -00) a

=

in rats ( M o z s i k , G y . , D o m o t o r , A . , A b d e l - S a l a m , O . M . E . , 2 0 0 6 : M o l e c u l a r p h a r m a c o l o g i c a l a p p r o a c h to d r u g a c t i o n s o n t h e afferent a n d efferent fibres of t h e v a g a l n e r v e in t h e gastric m u c o s a l p r o t e c t i o n in rats. I n f l a m m o p h a r m a c o l o g y 1 4 : 2 4 3 - 2 4 9 ) (with permission)

109

T a b l e 1 8 . I n h i b i t o r y effects of c a p s a i c i n a n d resiniferatoxin ( R T X ) o n basal gastric a c i d s e c r e t i o n in rats.* Compounds

Models

ED

values

Capsaicin

2 h p y l o r u s ligated rats

3.27 nmol/kg

Capsaicin

4 h p y l o r u s ligated rats

3.27 nmol/kg

RTX

4 h p y l o r u s ligated rats

0 . 9 5 4 nmol/kg

5 0

* Mozsik, Cy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats. Inflammopharmacology 14: 243-249 (with permission)

T a b l e 1 9 . I n h i b i t o r y effects of resiniferatoxin o n t h e s t i m u l a t e d gastric a c i d s e c r e t i o n in rats* Compounds

ED

Models

values

5 0

Capsaicin

I h pylorus-l igated rats + b e t a n e c h o l ( 7 . 6 u.mol/kg)

0 9 5 4 nmol/kg

Capsaicin

1 h pylorus- i gated rats + b e t a n e c h o l (15.2 umol/kg)

0 9 5 4 nmol/kg

Capsaicin

1 h pylorus-l igated rats + h i s t a m i n e ( 1 3 . 6 nmol/kg)

0 9 5 4 nmol/kg

Capsaicin

1 h pylorus-l igated rats + h i s t a m i n e (54.3 pmol/kg)

0 9 5 4 nmol/kg

Capsaicin

1 h pylorus- igated rats + pentagastrin (65.1 nmol/kg)

0 9 5 4 nmol/kg

pentagastrin ( 3 2 5 . 6 nmol/kg)

0 9 5 4 nmol/kg

Capsaicin

1 h pylorus- igated rats

RTX

4 h pylorus-l igated rats + b e t a n e c h o l ( 7 . 6 nmol/kg)

0 9 5 4 nmol/kg

RTX

4 h pylorus-l igated rats + b e t a n e c h o l (15.2 nmol/kg)

0 9 5 4 nmol/kg

RTX

4 h pylorus-l igated rats + h i s t a m i n e (2.7 pmol/kg)

0 9 5 4 nmol/kg

RTX

4 h pylorus-l igated rats + h i s t a m i n e ( 1 3 . 6 pmol/kg)

0 9 5 4 nmol/kg

RTX

4 h pylorus-l igated rats + pentagastrin (65.1 nmol/kg)

0 9 5 4 nmol/kg

RTX

4 h pylorus-l igated rats

0 9 5 4 nmol/kg

pentagastrin ( 3 2 5 . 6 nmol/kg)

* Mozsik, Gy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats. Inflammopharmacology 14: 243-249 (with permission)

4.88; cimetidine 3.00; capsaicin 8.50, whereas no effects were observed with PGI , vitamin A and P-carotene; 3. The intrinsic activity values obtained in relation to atropine 1.00 were cimetidine 0.64, capsaicin 0.75, omeprazole 1.00; no effects were observed for PGI , vitamin A and P-carotene; 4. The following p D (ED ) values were obtained for the different drugs or compounds inhibiting the gastric mucosal damage produced by chemicals: capsaicin 8.48, PGI 7.45, atropine 5.75, cimetidine 3.00, omeprazole 4.88, vitamin A 5.45 and P-carotene 5.73; 5. The intrinsic activity values ( c % r o p i n e 1-00) obtained: capsaicin 0.76, cimetidine 0.64, those of other components were 1.00 on the gastric mucosal damage; 6. The values of pA were obtained as follows: capsaicin 8.50, PGI 7.44, atropine 5.80, cimetidine 3.00, omeprazole 4.90, vitamin A 5.44 and p-carotene 5.70. 2

2

2

50

2

at

2

110

=

w e r e

2

T a b l e 2 0 . P r o t e c t i v e effects of c a p s a i c i n a n d resiniferatoxin o n t h e gastric m u c o s a l d a m a g e c a u s e d b y e x o g e n o u s agents i n rats* Compounds

Models

Capsaicin

0.6 M H C I (2 ml) (4 h)

0.135 pmol/kg

Capsaicin

a s p i r i n ( 2 0 0 mg/kg) + 0 . 1 5 M H C I (4 h)

0.13 p m o l / k g

Capsaicin

e t h a n o l ( 9 6 % in 1 m l ) (1 h)

0.13 p m o l / k g

Capsaicin

4 h pylorus-ligated rats + I N D ( 2 0 mg/kg)

1.98 n m o l / k g

RTX

4 h pylorus-ligated rats + I N D ( 2 0 mg/kg)

0.95 nmol/kg

Capsaicin

4 h pylorus-ligated rats + I N D ( 2 0 mg/kg) + 0 . 1 5 N H C I (2 m l )

0.25 pmol/kg

RTX

4 h pylorus-ligated rats + I N D ( 2 0 mg/kg)+ 0 . 1 5 N H C I (2 m l )

0.95 n m o l / k g

Capsaicin

4 h pylorus-ligated rats + I N D ( 2 0 mg/kg) 0.3 N H C I (2 m l )

2.78 p m o l / k g

RTX

4 h pylorus-ligated rats + I N D ( 2 0 mg/kg) 0.3 N H C I (2 m l )

0.15 p m o l / k g

ED

5 0

values

* Mozsik, Gy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats. Inflammopharmacology 14: 243-249 (with permission)

T a b l e 2 1 . P r o t e c t i v e effects of resiniferatoxin o n gastric m u c o s a l d a m a g e c a u s e d b y e x o g e n a m a n d e n d o g e n o u s agents in rats* Compounds

Models

RTX

4 h pylorus-ligated rats +

RTX

4 h pylorus-ligated rats +

RTX

4 h pylorus-ligated rats +

RTX

4 h pylorus-ligatedrats +

RTX

4 h pylorus-ligated rats +

RTX

4 h pylorus-ligated rats+

b e t a n e c h o l (7.6 p m o l / k g ) + I N D ( 2 0 mg/kg) b e t a n e c h o l (15.2 p m o l / k g ) + I N D ( 2 0 mg/kg) h i s t a m i n e ( 1 3 . 6 p m o l / k g ) + I N D ( 2 0 mg/kg) h i s t a m i n e (54.3 pmol/kg) + I N D ( 2 0 mg/kg) pentagastrin (6.51 nmol/kg) + I N D ( 2 0 mg/kg) pentagastrin ( 3 2 5 . 6 nmol/kg) + I N D ( 2 0 mg/kg)

ED

5 0

values

0.20 p m o l / k g 0.20 p m o l / k g 0.13 p m o l / k g 0.13 p m o l / k g 0.13 p m o l / k g 0.13 p m o l / k g

* Mozsik, Gy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats. Inflammopharmacology 14: 243-249 (with permission)

The results of these molecular pharmacological observations clearly proved that the capsaicin-sensitive afferent nerves have an essential role both in the regulation of gastric acid secretion and in the defense of the gastric mucosal damage produced by the different chemical agents. It has been emphasized that capsaicin exerts its gastric acid inhibitory and gastric mucosal protective effect in smaller molar concentrations than other compounds (atropine, cimetidine, omeprazole and other compounds with-

111

T a b l e 2 2 . T h e p D , intrinsic a c t i v i t y ( a , 2

a

r o p i n

e=1.00) and p A

v a l u e s for different drugs

2

( c o m p o u n d s ) i n h i b i t i n g t h e gastric a c i d o u t p u t s in 4 h pylorus-ligated rats* Compounds

pD

Capsaicin

2

Intrinsic activity

pA

2

8.48

0.76

8.50

Atropine

5.75

1.00

5.80

Cimetidine

3.00

0.64

3.20

Omeprazole

4.88

1.00

4.90

* For further information, see Figs 18 and 19. * Mozsik, Cy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats. Inflammopharmacology 14: 243-249 (with permission)

T a b l e 2 3 . D o s e r a n g e s of t h e tested n u t r i t i o n a l c o m p o u n d s ( v i t a m i n A a n d (3-Carotene), P G I PGE

2

2

and

o n t h e gastric a c i d s e c r e t i o n of 4 h pylorus-ligated rats, w i t h o u t p r e s e n c e of a n y

gastric i n h i b i t o r y a c t i o n s * V i t a m i n A : 3.49 x 10

8

to 3.49 x 10~ M ( 0 . 0 1 - 1 0 . 0 mg/kg): n o i n h i b i t o r y a c t i o n 5

o n t h e gastric a c i d s e c r e t i o n P-Carotene: 1.86 x 1 0

8

to 1.86 x 1 0 " M ( 0 . 0 1 - 1 0 . 0 mg/kg): n o i n h i b i t o r y a c t i o n 5

o n t h e gastric a c i d s e c r e t i o n P G I : 2.8 x 10-'' to 1.42 x 1 0 2

8

M ( 1 . 0 - 5 . 0 pg/kg): n o i n h i b i t o r y a c t i o n o n t h e gastric

a c i d secretion P G E : 1.33 to 1.99 x 1 0 " M ( 5 0 . 0 - 1 5 0 . 0 pg/kg): n o i n h i b i t o r y a c t i o n o n t h e gastric 7

2

a c i d secretion

** Mozsik, Gy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats. Inflammopharmacology 14: 243-249 (with permission)

T a b l e 2 4 . T h e p D , intrinsic a c t i v i t y ( a 2

a t r o p i n e

=1.00) and p A

2

v a l u e s for different drugs,

c o m p o u n d s i n h i b i t i n g t h e gastric m u c o s a l d a m a g e p r o d u c e d by c h e m i c a l agents in rats* Intrinsic activity

pA

8.48

0.76

8.50

7.45

1.00

7.44 5.80

Compounds

pD

Capsaicin PGI2

2

2

5.75

1.00

Cimetidine

3.00

0.64

3.20

Omeprazole

4.88

1.00

4.90

Vitamin A

5.45

1.00

5.44

P-Carotene

5.73

1.00

5.73

Atropine

* For further information, see Figs 18-21. (*M6zsik, Gy, Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats. Inflammopharmacology 14: 243-249 (with permission)

112

T a b l e 2 5 . S u m m a r y of t h e affinity ( p D ) a n d intrinsic a c t i v i t y ( e x p r e s s e d in v a l u e of 2

^atropine

1 . 0 0 ) ( p A ) v a l u e s of c a p s a i c i n , a t r o p i n e , p i r e n z e p i n e , c i m e t i d i n e , r a n i t i d i n e , 2

f a m o t i d i n e , o m e p r a z o l e a n d e s o m e p r a z o l e o n t h e gastric basal a c i d o u t p u t ( B A O ) in healthy h u m a n subjects* Compounds

M.W.

pD

2

Intrinsic activity

pA

2

Capsaicin

305.4

5.88

0.76

5.87

Atropine

289.38

5.40

1.00

5.40

Pirenzepine

424.34

3.93

0.89

3.93 2.23

Cimetidine

252.34

2.23

1.00

Ranitidine

314.41

3.33

1.00

3.33

Famotidine

337.43

3.77

1.00

3.77

Nizatidine

331.47

3.34

1.00

3.34

Omeprazole

345.42

3.97

1.00

3.97

Esomeprazole

345.42

3.97

1.00

3.97

* Mozsik Cy., Szolcsanyi, J . , Domotor, A. (2007b): Capsaicin research as a new tool to approach of the human gastrointestinal physiology, pathology and pharmacology (review). Inflammopharmacology 15: 232-245 (with permission) (For further information, see Figs 26-27)

out any gastric acid inhibitory effects). That is a clear explanation for why the essential role of capsaicin-sensitive afferent nerves in the physiological and pharmacological regulation of gastrointestinal tract is emphasized.

8.1.8. Capsaicin actions in healthy human subjects

and in patients with different gastrointestinal disorders

Early works regarding the effect of peppers on the human stomach have yielded conflicting results. In peptic ulcer patients, Schneider et al. (1956) studied the influence of a variety of spices on sensation of pain and healing peptic ulcer. Ulcer patients placed on established treatment with anticholinergics and diet were given different pungent species in capsules that contained the average quantity habitually consumed by Americans with three of their daily meals for six weeks. With the exception of black pepper, which resulted in distressing pain after one or two days, none of the tested spices have modified pain sensation or delayed healing of ulcers. In another study, instillation of red chilli powder was reported to be associated with a significant increase of DNA from gastric aspirates (Desai et al., 1973). Viranuvatti et al. (1972) studied the local effect of capsicum in twenty human subjects by instillation of a 3% capsicum solution through an intragastric tube or via the lumen of gastrofiberscope. There was no change in 13 cases, edema and/or hyperemia developed in three cases, hemorrhagic spots occurred in another three cases, and bleeding occurred in one case. Capsaicin was reported to increase the gastric acidity in human subjects receiving intragastrically aqueous suspensions of hot peppers (Berkesy, 1934; Varga, 1936; 113

Ketusinh et al., 1966; Solanke, 1973). Solanke (1973) studied the effect of red pepper suspension (200 ml of 4% solution) instilled through a nasogastric tube on gastric acid secretion in patients with proven duodenal ulcer and non-duodenal ulcer subjects. Patients were allocated into two treatments: fresh red pepper suspension and a red pepper suspension with pH adjusted to 7.4 with 0.01 N sodium hydroxide. They found a significant increase in gastric acid secretion after treatment with either form of the red pepper suspension. Many other observations were carried out in human beings with different extractions of chilli, paprika. The Good Clinical Practice (GCP) was introduced in the clinical research of human beings and patients. From 1997, our clinical studies with capsaicin were carried out in prospective and randomized, multiclinical conditions as officially accepted in the multiclinical pharmacological studies over the World. The following principles were accepted in these human observations: 1. Pure capsaicin (Sigma, U.S.A., later on Sigma-Aldrich, U.S.A.) was used in the studies with healthy human subjects and in patients with different diseases (instead of different extractions of different capsaicin containing plants); 2. The clinical observations were carried out according to the medical rules of clinical pharmacology (in random allocation and in prospective and randomized studies); 3. The clinical observations were carried out according to the criteria of GCP; 4. All of the persons participating received all the different doses of capsaicin, however, in a random allocation. We have to emphasize that the classical pharmacological studies were carried out with capsaicin obtained from Sigma (U.S.A.), or later on from Sigma-Aldrich (U.S.A.). The main results of the human multiclinical pharmacological studies with capsaicin: 1. Capsaicin (in the dose range of 100 to 800 pg given orally to one person) dosedependently inhibited the gastric basal acid output (BAO) in healthy human subjects (Fig. 22) (Mozsik et a l , 1999, 2005a); 2. Capsaicin dose-dependently enhanced the gastric transmucosal potential difference (GTPD) in healthy human subjects (Fig. 23) (Mozsik et al., 2005a); 3. The ethanol-induced decrease of GTPD can be dose-dependently reversed by topical application of capsaicin (given in doses of 100, 200, 400 and 800 pg orally) (Fig. 24) (Mozsik et al., 2005a); 4. Indomethacin (3x25 mg/day given orally, plus 25 mg given immediately before measuring of gastric blood loss) produced a significant increase of gastric microbleeding in comparison to control (untreated) conditions (Fig. 25) (Mozsik et al., 2005a, 2007b); 5. The extent of basal and indomethacin-induced gastric microbleeding remained unchanged before and after 2-weeks treatment with capsaicin (3 x 400 pg/ person/day) (Mozsik, et al., 2005a, 2007b); 6. The dose-dependent gastroprotective effect of capsaicin on the indomethacininduced gastric microbleeding remained the same after two-weeks capsaicin treatment (3 x 400 pg given orally /day/person) (Mozsik et al., 2005a, 2007b). 114

These observations proved that capsaicin (dose-dependently) prevents the ethanoland indomethacin-induced gastric mucosal damage in healthy human subjects (Mozsik et al., 2005a, 2007b) before and after two-weeks treatment with capsaicin (3x400 pg orally /day/ person) (Mozsik et al., 2005a, 2007b), however, we have to emphasize that indomethacin (3x25 mg given orally, plus 25 mg given immediately before the measuring of gastric microbleeding) produced the same extent of gastric microbleeding. We have also to emphasize that the extent of baseline of gastric microbleeding remained the same before and after two-weeks treatment with capsaicin (Mozsik et al., 2004, 2005a, 2007b). Effect of capsaicin on gastric basal acid output ( B A O ) in healthy h u m a n subjects mean±SEM

7

6

5

Doses of capsaicin (-iog[M]) Fig. 22. D o s e - r e s p o n s e c u r v e of t h e effect of c a p s a i c i n o n t h e gastric basal a c i d o u t p u t ( B A O ) in healthy h u m a n subjects ( M o z s i k , C y . , R a c z , I., S z o l c s a n y i , J . , 2 0 0 5 a : G a s t r o p r o t e c t i o n i n d u c e d by c a p s a i c i n in h e a l t h y h u m a n s u b j e c t s . W o r l d J . G a s t r o e n t e r o l . 1 1 : 5 1 8 0 - 5 1 8 4 ) (with permission)

8.1.8.1. Results of the comparative molecular-pharmacological studies of capsaicin, atropine, omeprazole, famotidine, ranitidine and cimetidine on the gastric basal acid output (BAO) in human subjects The affinity (pD values) and intrinsic activity (pA values) curves of capsaicin, atropine, omeprazole, famitidine, ranitidine and cimetidine (applied in their physiological and human therapeutic doses) were determined in patients with gastrointestinal disorders, according to the method of Csaky (1969) (Figs 26-27, Table 25). 115

Effect of capsaicin on G T P D (-AmV) in healthy human subjects

Doses of capsaicin (-log[M|) Fig. 23. D o s e - r e s p o n s e c u r v e of t h e effect of c a p s a i c i n o n t h e gastric t r a n s m u c o s a l potential d i f f e r e n c e ( G T P D ) in h e a l t h y h u m a n s u b j e c t s . M o z s i k , G y . , R a c z , I., S z o l c s a n y i , J . , 2 0 0 5 a : G a s t r o p r o t e c t i o n i n d u c e d by c a p s a i c i n in h e a l t h y h u m a n s u b j e c t s . W o r l d J . Gastroenterol. 1 1 : 5180-5184) (with permission)

mean±SEM

6

5 Doses of capsaicin (-log[M])

Fig. 24. D o s e - r e s p o n s e c u r v e of t h e effect of c a p s a i c i n o n t h e gastric t r a n s m u c o s a l potential d i f f e r e n c e after intragastric a d m i n i s t r a t i o n of e t h a n o l in h e a l t h y h u m a n subjects. M o z s i k , G y . , R a c z , I., S z o l c s a n y i , J . , 2 0 0 5 a : G a s t r o p r o t e c t i o n i n d u c e d by c a p s a i c i n in healthy h u m a n subjects. W o r l d J . Gastroenterol. 1 1 : 5180-5184) (with permission)

116

mean±SEM

= P<0.01 = P<0.001

6

5 Doses of capsaicin (-Iog[M])

Fig. 25. D o s e - r e s p o n s e c u r v e of t h e effect of c a p s a i c i n o n t h e i n d o m e t h a c i n ( 3 x 2 5 + 2 5 m g g i v e n orally) i n d u c e d gastric m i c r o b l e e d i n g in h e a l t h y h u m a n s u b j e c t s . M o z s i k , G y . , R a c z , I., S z o l c s a n y i , J . , 2 0 0 5 a : G a s t r o p r o t e c t i o n i n d u c e d by c a p s a i c i n in h e a l t h y h u m a n subjects W o r l d J . Gastroenterol. 1 1 : 5180-5184) (with permission)

Fig. 26. Affinity c u r v e s for t h e i n h i b i t o r y a c t i o n s of different drugs o n t h e gastric basal a c i d o u t p u t ( H + output/h) in h e a l t h y h u m a n subjects [ M o z s i k G y . , S z o l c s a n y i , J . , D o m o t o r , A . , 2 0 0 7 b : C a p s a i c i n research as a n e w t o o l to a p p r o a c h of t h e h u m a n gastrointestinal physiology, p a t h o l o g y a n d p h a r m a c o l o g y ( r e v i e w ) . I n f l a m m o p h a r m a c o l o g y 1 5 : 2 3 2 - 2 4 5 ] (with permission)

117

Fig. 27. Intrinsic a c t i v i t y c u r v e s for t h e i n h i b i t o r y effects of different drugs o n t h e gastric basal a c i d o u t p u t ( H + output/h) in h e a l t h y h u m a n subjects, c o m p a r e d to t h e a c t i o n of a t r o p i n e (1.00)(a

a t r o p i n e

) [ M o z s i k G y . , S z o l c s a n y i , J . , D o m o t o r , A . , 2 0 0 7 b : C a p s a i c i n research

as a n e w t o o l to a p p r o a c h of t h e h u m a n gastrointestinal p h y s i o l o g y , p a t h o l o g y a n d p h a r m a c o l o g y (review). I n f l a m m o p h a r m a c o l o g y 1 5 : 2 3 2 - 2 4 5 ] (with permission)

8.1.9. Side effects of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) in the gastrointestinal tract of patients NSAIDs treatment is necessary for the patients with myocardial infarction, thromboembolic episodes, stroke, cancers, for persons in whom the development of different diseases should be prevented (reinfarction after myocardial infarction, thromboembolic episodes produced by atrial fibrillation, cancers, after different surgical interventions and immobilization), and for healthy subjects in order to prevent the development of colorectal cancers. The number of these groups of patients reaches up to 50-60% of the total population in Hungary, an extremely large number of patients with these diseases is involved in the different countries of the World. The patients who underwent cardiac surgeries have to treated permanently with aspirin (in doses of 100 mg/day/person). This is the basic stand-point of the different consensus meetings of Europe and the World (Todd, Clissold, 1990; Expert Consensus Document on the Use of Antiplatelet Agents 2004; Mcgettigan, Henry 2006). The administration of aspirin is absolutely indicated from medical points of view in patients mentioned above, accepting the opinion of cardiologists; however, we have to emphasize that aspirin very frequently produces gastrointestinal bleeding. Consequently there is a contradictory medical (and evidence-based proved) standpoint between the cardiologists and gastroenterologists (during the treatment of only one patient, as well as in the treatment of populations of patients mentioned above). Another large population of patients suffers from different degenerative joint diseases, trauma, acute and chronic pain producing states. These patients have to receive 118

permanently treatment with NSAIDs. The NSAIDs are not gastrointestinal protective agents either in healthy person or in patients with these diseases. The patients appearing at the ambulance of Gastrointestinal Units are suffering from the druginduced side effects in the gastrointestinal tract. The number of patients with NSAIDs-induced gastrointestinal disorders (blood loss, bleeding, peptic ulcer) also represents a significant fraction of the population. Furthermore, these patients have to be treated permanently by different NSAIDs. The actions of NSAIDs are associated with the selective and non-selective inhibitory properties of the cyclooxygenase system (emphasizing the key role of COX-1 and COX-2). Aspirin is a specific COX-1 inhibitor, meanwhile the other NSAIDs applied in the clinical practice represent the compounds acting as non-selective COX-1 and COX2 inhibitors. Recently the specifically acting compounds, inhibiting the COX-2 enzyme, have been produced, however, it was also observed that during the treatment an extremely large number of patients suffered myocardial infarction (Couzin, 2004a,b; Lenzer, 2004; McGettigan, Henry, 2006). We have to emphasize that the small doses of capsaicin are able to prevent gastric mucosal bleeding in humans, produced by the inhibition the COX-1 and COX-2 enzymes. This discovery opened a new pathway in the physiological and pharmacological regulation of different tissues (see later).

119

9. Toxicological studies

with capsaicin

9 . 1 . Animal observations 9.1.1. Acute toxicology studies of capsaicin in animal experiments The only reported acute toxicity study with capsaicin was in ears which were administered four increasing s.c. doses of capsaicin to a cumulative amount of 21.0-66.0 mg/rat, eight of 17 rats died (Cabanac et al., 1976). Additionally, Molnar (1965) and Molnar and Gyorgy (1967) reported that capsaicin aministered i.v. at a dose higher than 10 pg/kg to cats caused a rapid fall in the mean arterial pressure which was followed either by a pressor phase or by death. These results called our attention to methods of administration of capsaicin (intravenously or orally in animal experiments9. Capsaicin had an i.p. L D (7.65 mg/kg) in adult female and male mice (please note that capsaicin was not given as pure agent, but as an extract) (Glinsukon et al., 1980; Ato, Yamamoto, 1996). The toxicity of capsaicin present in the capsicum extract was approximately fourfold higher than that of pure capsaicin given intraperitoneally to mice. Capsaicin had a slightly elevated L D in pregnant female rats when administered in propylene glycol as compared to that administered intraperitoneally in dimethylsulfoxyde (DMSO) (P< 0.05) (Glinsukon et al., 1980). Guinea pigs are the most susceptible species to capsaicin toxicity with L D of 1.10 mg/kg, whereas hamsters and rabbits are less susceptible (Glinsukon et al., 1980). The relative lethality caused by capsaicin administered by various routes in the mouse (Glinsukon et al., 1980): 0.56 (0.36-0.87) mg/kg intravenously, 1.60 (1.03-2.48) mg/kg intratracheally, 7.65 (5.28-11.09) mg/ kg intraperitoneally, 7.80 (5.53-10.99) mg/kg subcutaneously, 60 to 190 mg/kg intragastrically, > 218 mg/kg intrarectally and > 512 mg/kg dermally. At the autopsy, only hyperemia without hemorrhage was observed in the visceral organs and the muscular wall of the peritoneal cavity with a slight increase in the amount of peritoneal fluid in the rats treated intraperitonally with capsaicin. A similar observation was also found in mice treated intraperitoneally with capsaicin. Histopathological changes seen in the gastric mucosa of mice treated intragastrically with capsaicin were desquamatic necrosis with increased mucous material (PAS stain). Some of the chief and parietal cells showed pale basophilic cytoplasm and vacuolization. No significant histopathological changes were observed in the other organs. 50

50

50

120

The pattern of the electrocardiogram and heart rate did not change for 5 min after capsaicin administration. Respiratory rates were slightly increased during the first min, whereas a small increase of the tidal volume was also observed. The tidal volume then decreased to 10 to 20% of the control within 3-4 min, and the respiration stopped. During this time, the heart rate gradually decreased, the electrocardiographic signals disappeared much later (in about 6-14 min). Mean arterial pressure was somewhat variable in the rats treated with capsaicin. At the beginning, capsaicin caused a transient hypotension and then hypertension. Mean arterial pressure gradually decreased along with the decrease in the tidal volume. Convulsions were not observed in these rats anesthetized with a lethal dose of capsaicin. This finding was confirmed in mice anesthetized with sodium barbital and subsequently given a single lethal dose of capsaicin. The L D values indicate a high susceptibility in guinea pigs, rats and mice, whereas hamsters and rabbits are less susceptible to capsaicin. Capsaicin is a highly toxic compound when administered by all routes except gastric, rectal and dermal. Cabanac et al. (1976) published a report on the acute toxicity of capsaicin in which adult male rats were given four increasing subcutaneuos doses of capsaicin (cumulative amount of 21.0 to 66.0 mg/kg). The lethality caused by capsaicin administered gastrically to the mouse is much less than that caused by the intraperitoneal administration route. The minimum lethal dose of capsaicin per kg was 100 mg, which would be contained in 32.4 g dry weight of fruits. For a 60 kg person, this toxic level would be comparable to the consumption of about 1.94 kg of dry weight of capsicum fruits (Molnar, 1965). 50

9.1.2. Acute toxicity studies with pure trans-capsaicin derivates in dogs after intravenous administration The trans-geometric isomer of capsaicin, or trans-8-methyl-N-vanillyl-6-nonenamide, is the most abundant pungent molecule in chilli peppers and thus represents the most important ingredient in spicy foods. Although there are two geometric isomers of capsaicin, only trans-capsaicin occurs naturally (Cordell, Araujo, 1993). The capsaicin content of chilli peppers ranges from 0.1 to 1.0% w/w (Govindajarajan, Sathyanarayna, 1991). Furthermore, this food additive has been widely used to evaluate the different physiological or pathological regulatory mechanisms in the human observation in the form of non-prescription (in U.S.A.) or prescription (in Europe) topical analgetics, and self-defense products (e.g. pepper spray). The trans-geometric isomer of capsaicin is a highly selective agonist for the transient receptor potential vanilloid receptor 1 (TRVP1 or VR1, according to older nomenclature) (Caterina et al., 1997). TRVP1 is a ligand-gated, non-selective, cation channel preferentially expressed in small-diameter, primary afferent neurons (C-fibres and A8-fibres), especially nociceptive sensory nerves. TRPV1 responds to noxious stimuli including capsaicin, heat and extracellular acidification, and integrates simultaneous exposures to these stimuli (Tominata et al., 1998). Based on the highly selective agonistic property of 121

capsaicin toward TRVP1 receptors, drug products containing pure synthetic transcapsaicin are under evaluation as topical and injectable therapies (Bley, 2004). Former studies of the toxicological potential of capsaicin in vivo began in 1935, when De Lille and Ramirez (1935) reported that administration of a capsaicin extract into dogs produced a fall in blood pressure accompanied by variable effects on the respiration, an increase in salivary secretion, and a relatively small increase in gastric secretion. Capsaicin really can increase the buffering ("non-parietal component") of the gastric secretion, in association with the decrease of "parietal component" of gastric secretion by the application of pure capsaicin (Sigma, U.S.A.) in healthy human subjects (Mozsik et al., 2004, 2007b). The capsaicin materials tested in the studies cited above were either natural extracts or racemic mixtures, and may not exhibit the same toxicological profile as pure trans-capsaicin. Although the exact content and nature of impurities in the test articles used in these studies are often not explicitly stated, a typical capsaicin extract is a mixture of trans-capsaicin (cis-capsaicin does not occur naturally) and other capsaicinoids (including capsaicin, nordihydrocapsaicin, dihydrocapsaicin, homocapsaicin and homodihydrocapsaicin). Earlier Buck and Burks (1986) clearly proved that there is no physiological difference between the application of capsaicin or dihydrocapsaicin in animal experiments. The actual percentage of capsaicin and other capsaicinoids will vary depending on the peppers used and method of extraction: In fact, the United States Pharmacopeia defines capsaicin as a product which contains > 55% capsaicin and in combination of capsaicin and dihydrocapsaicin > 75%; total capsaicinoid content may be as little as 90% (United States Pharmacopoeia, 2005). Additionally, extracts are expected to contain chemical entities other than vanilloid compounds. Chanda et al. (2004, 2005) made observations with pure trans-capsaicin in dogs. The objectives of this study were to evaluate the possible cardiovascular and respiratory effects of pure capsaicin, and to evaluate the potential of any target organ toxicity that might occur as a result of introduction of pure trans-capsaicin given into the systemic circulation in dogs. These studies were carried out in approximatively 10-17 months old dogs weighing between 19-21.8 kg at the time of the observations. Capsaicin for this study was dissolved in 10% v/v hydroxypropyl B-cyclodextrin (Aldrich Chemical Gillingham, U.K.). The studies were carried out in acute observations, and after two weeks of capsaicin treatment (in doses of 0.03, 0.1 and 0.3 mg/ kg given intravenously). Different biochemical (glucose, urea nitrogen, creatinine, total protein, albumin, albumin/globulin ratio, cholesterol, alanine aminotransferase, alkaline phosphatase, calcium, gamma glutamyltransferase, inorganic phosphorus, sodium, potassium, chloride, total bilirubin) and hematological parameters (red blood count, hemoglobin, hematrocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, leukocyte count, differential blood cell count, blood smear, prothombin time, activated partial thromboplastin time) were determined. Urine samples were tested for appearance/color parameters, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, blood, microscopic examination of urine sediments, and urobilinogen. At the necropsy, the macroscopic observations were recorded, the organs were weighed, and selected tissues 122

were collected and preserved. Microscopic examinations were carried out from all the tissue samples. The capsaicin concentration of the plasma samples was determined by high performance liquid chromatography (HPLC). All of these observations were carried out in acute experiments and after two weeks of capsaicin treatment. The studies reported were conducted according to the principles of Good Laboratory Practice (GLP). The trans-capsaicin (CAS 404-86-4) used in both studies decribed was manufactured under the Current Good Manufacturing Practice (cGMP) conditions. The two batches of trans-capsaicin used for the studies had > 99% agreement. The main results of these observations with trans-capsaicin in acute administration (before a chronic capsaicin treatment) are as follows.

9.1.2.1. Acute effects on cardiovascular and respiratory parameters Administration of capsaicin (given in vehicle, 0.03 and 0.1 mg/kg) had no detectable effect on the cardiac and respiratory systems. No effect was observed on arterial blood pressure, rate change of the force of ventricular contraction (dP/dt ), heart rate ECG waveform, and femoral blood flow. However, administration of 0.3 mg/kg capsaicin elicited a rise in mean arterial blood pressure from a base line of 96±7 to 138±21 mmHg within 2 min of starting the infusion. This effect peaked at 146±17 mmHg at the end of infusion (15 min). The hypertensive effect was accompanied by increases in heart rate (from 71 ±3 at baseline to 119±25 bpm), d P / d t (from 4050±91 at baseline to 6679±1027 mmHg/s) and femoral blood flow (from 117+27 at baseline to 174±34 ml/min). These changes were statistically significant {P<0.05) in comparison with the results obtained after giving vehicle, 0.03 or 0.1 mg/kg capsaicin injection (Chanda et al., 2005). The change in heart rate was also associated with decrases in the RR and QT intervals of the ECG. However, the corrected QT intervals (both Q T and Q T ) were unchanged. After administration the vehicle elicited a decrease in the depth of respiration. This was reflected as decreases at 30 min post-infusion in tidal volume (TV) (from 143±19 to 118+20 ml), peak inspiratory flow (PIF) (from 253 ±12 to 198+10 ml/s) and peak expiratory flow (PEF) (from 297±47 to 246±24 ml/s). The rate of respiration was unaffected. The administration of 0.03 and 0.1 mg/kg trans-capsaicin did not elicit any further changes in respiration. The administration of high (0.3 mg/kg) dose of trans-capsaicin (given intravenously) elicited increases in PIF, PEF and TV. The increase of in PIF and PEF following 0.3 mg/kg trans-capsaicin is significantly different from the decrease in these parameters following the vehicle treatment (P<0.05). However, these increases were transient, lasting only 5 to 10 min after the end of infusion. max

max

C F

CB

123

9.1.2.2. Plasma levels of capsaicin No detectable levels of capsaicin were found 5 min after administration of 0.05 mg/kg capsaicin. Following the administration of the intermediate dose (0.1 mg/kg intravenously), 2-A dogs showed detectable levels of capsaicin (in ranges of approximatively 17 and 11 ng/ml). The high dose (0.3 mg/kg) produced an increase in the plasma levels in all dogs (ranging from 32.2 to 65.6 ng/ml, mean 47.9±6.4 ng /ml).

9.1.3. Results of subacute toxicology of capsaicin in dogs 9.1.3.1. Two weeks treatment with trans-capsaicin All dogs survived to the scheduled termination on Day 15. The only capsaicin solution-related sign observed during the study was vacuolization during dosing, which was noted in all dogs. In general, the observation was made more frequently in male than in female dogs. Clear nasal discharge was seen in all groups, and the daily incidence was slightly higher in males than in females given 0.3 mg/kg trans-capsaicin (intravenously), although the daily incidence in males given 0.1 or 0.3 mg/kg/day of trans-capsaicin in females 0.3 mg/kg/day was slightly higher than in controls. Slight tremors (head, limbs, and/or body) were seen in all groups during this study. The majority of these observations were noted during the study. As the study progressed, dogs given 0.3 mg/kg trans-capsaicin demonstrated an apparent tolerance to the general anesthetic and analgesic, as indicated by a general vacuolization during the dosing period. There were no statistically significant differences in the body weights and food consumption values between the groups of dogs treated with capsaicin chronically. The body weights, however, were slightly lower (in about 7%) in males treated with 0.3 mg/kg trans-capsaicin for 14 days. Over the duration of the study, males in this group lost approximately 0.4 kg, whereas the control gained 0.1 kg. Though this value was statistically not significant, the food consumption of males and females given 0.3 mg/kg/ day was slightly lower than that of controls (approximately 11 and 12%, respectively).

9.1.3.2. Clinical chemistry and hematology The only difference considered related to test article was the minimally higher ALT for males and females given 0.3 mg/kg/day trans-capsaicin intravenously after 14 days' treatment. Other statistically significant differences for clinical chemistry test results were considered incidental because they exhibited no dose relationship or were present before initiation of the treatment. In hematology, in female dogs in the 0.3 mg/kg/day treated group WBC was statistically significantly (P<0.05) lower. A few animals, including controls, had notably high neutrophil counts, which were likely secondary to inflammated lesions at injection sites. 124

9.1.3.3. Organ weights, macroscopic Snd microscopic observations There were no capsaicin solution-related organ weight changes or macroscopic or microscopic observations. The statistically significant differences of prostate, brain and adrenal weight values with respect to controls were considered incidental because there were no correlating macroscopic and microscopic findings. Thrombosis, due to administration of vehicle, was noted at the intravenous injection sites in all groups. Other lesions observed were thrombosis-induced inflammation, fibrosis, edema and hemorrhage.

9.1.3.4. Pharmacokinetic data after 14-days treatment with trans-capsaicin in dogs After intravenous administration, the peak of the plasma concentration ( C ) was reached in all cases immediately at the end of infusion. Capsaicin was rapidly eliminated and measurable values were only obtained immediately after the end of infusion (0.25 h) in 0.03, 0.1 and 0.3 mg/kg/day. In the 0.3 mg/kg/day group, measurable values were obtained up to 0.5 h in all dogs on Day 1, but they were very close to the limit of quantitation (10 ng/ml). On Day 15, only one dog had still a measurable value. Females generally had higher than, or similar, mean C values compared to males, but the largest difference did not exceed 44%. The increases in mean C for males and females were proportional to the increase of dose levels from 0.03 to 0.3 mg/kg/day. max

m a x

m a x

9.1.3.5. Absorption and metabolism of oral application of capsaicinoids in animal experiments Because of their increasing experimental use and planned drug production in a very wide field of medical research and medical treatment, we have to gain correct knowledge on the absorption, metabolism and excretion of capsaicinoids. It is known that capsaicin given directly into the stomach of rats has only minimal excitatory effects visible on immediate blood pressure responses (Lippe et al., 1989) in contrast to intravascular or subcutaneous administration (Donnerer, Lembeck, 1983). On the other hand, it has been shown that capsaicin disappears within a rather short time from the intestinal lumen (Kawada et al., 1984; Kawada, Iwai, 1985) and should therefore reach the circulatory system. Since biotransformed products of capsaicin are difficult to detect, the use of [ H]labelled dihydrocapsaicin ([ H]-DHC) allows to determine the percentage of unchanged compound in the total extracted radioactivity. Dihydrocapsaicin (DHC) has been shown to display pharmacodynamic and pharmacokinetic properties comparable with those of capsaicin (Burk, Bucks, 1982; Kawada et al., 1984). [ H]-Dihydrocapsacin ([ H]-DHC and unlabelled capsaicin were readily absorbed +

+

3

3

125

from the gastrointestinal tract but were almost completely metabolized before reaching the general circulation. A certain degree of biotransformation already took place in the intestinal lumen. Unchanged compounds (identified by chromatography) were present in the portal vein blood. There seems to be a saturable absorption and degradation process in the gastrointestinal tract and a very effective metabolism limited to the liver (Donnerer et al., 1990). Less than 5% of the total amount of extracted radioactivity consisted of unchanged [ H]-DHC in blood and brain 15 min after gastrointestinal application. On the other hand, approximately 50% unchanged [ H]-DHC was detected 3 min after intravenous application or 90 min after subcutaneous application of capsaicinoids (Donnerer et al., 1990). Dihydrocapsaicin (DHC) or [ H]-DHC were metabolized when incubated in vitro with liver tissue but not with brain tissue (Donnerer et al., 1990). The metabolic product(s) did not show capsaicin-like biological activity (Donnerer et al., 1990). These results clearly indicate that the rapid hepatic metabolization limits the systemic pharmacological effects of enterally absorbed capsaicin in rats (Donnerer et al., 1990). Mean C values for males increased 1: 3.3: 10- fold on Day 1 and 1: 2.9: 9.2 fold on Day 14-fold increase in administered dose. These results clearly indicate that no accumulation exists for capsaicin after multiple dosing by this route of its administration (Chanda et al., 2005). 3

3

3

m a x

9.1.3.6. Summary and conclusions of the administration of different doses of trans-capsaicin in acute and subacute experiments in dogs In the acute study, surface Lead II ECG was monitored to determine the QT intervals and the duration of cardiac repolarization. However, there were no observable changes in the QT F (Fridericia's correlation, Q T F = Q T / A T (RR interval). Such a change would have been theoretically possible, as capsaicin has been reported to block voltage-activated potassium channels in rat ventricular myocytes (Castle, 1992). Because many drugs able to block voltage-activated channels actually shorten the duration of cardiac action potentials, there are limited correlations between the potassium channel blocking activity and QT interval prolongation (Martin et al., 2004). The lack of measurable effects on the cardiac action potential makes it likely that the hemodynamic effects of capsaicin measured during the acute study with 0.3 mg/kg/day capsaicin are due to agonist activity at TRVP1 receptors; this is because the reported potency of capsaicin appears to be several fold higher than that of either calcium and potassium ion channels (Castle, 1992; Cheng et al., 2003). Thus, it is likely that capsaicin receptors expressed on pericardiac sensory nerves induced the transient increases in the heart rate observed during the acute dosing study, in the course of performing their role to sense cardiac ischemia (Pan, Chen, 2004). For longer administration of capsaicin, it is possible that the putative antihypertensive actions of capsaicin result from either prolonged desensitization of pericardiac sensory fibres, including the release of vasoactive peptides (CGRP and SP) from the C

3

C

126

C

perivascular C-fibres, or activating an endogenous system which counterbalances hypertension caused by sodium salt loading (Vaishnava, Wang, 2003). Aside from the cardiac effects, capsaicin has also been studied in animals for other possible target organ toxicity. Almost all of these studies used pepper plant extracts, which are likely to display varying degrees of capsaicinoids content and possible diverse impurity profiles. These impurities may be the contributing factor in some of toxicities observed. Chanda et al. (2004) observed that the pure capsaicin displays a different genotoxicity profil than that described in some previous literature. Additionally, in contrast to the high dose levels used in toxicological studies, human exposure to dietary capsaicinoids (a mixture of capsaicin, dihydrocapsaicin, nordihydrocapsaicin, homocapsaicin and homodihydrocapsaicin) in U.S.A. and in European countries is about 1.5 mg/day, which translates into, at most, 0.025 mg/day/ day (Chanda et al., 2005). When capsaicin was dissolved in diethylene glycol monoethyl ether and Dulbecco's phosphate-buffered saline and administered in intravenous infusion for 14 days (in a 15 min time period to anesthetized dogs), the vehicle itself caused marked vascular irritation at the administration sites. There were no deaths, no test article-related organ weight changes, and no macroscopic and microscopic observations. The only test article-related clinical sign observed in the study was vacuolization in the dogs treated with 0.3 mg/kg /day. The only test article-related clinical pathology finding was higher ALT in male and female dogs receiving 0.3 mg/kg/day capsaicin. This may indicate the liver as a possible target organ when capsaicin is delivered at high doses directly into the systemic circulation. When capsaicin was given in a dose of 0.1 mg/kg/day for 14 days, it was rapidly eliminated in dogs. Capsaicin dissolved in dimethyl sulfoxide (DMSO) has been studied by Glinsukon et al. (1980) for determination of L D values by several administration routes in mice. The authors also determined the L D values of capsaicin for one administration route (intraperitoneal) in different species. The order of sensitivity ( L D values) for species, from the least to most, by the intraperitoneal route using DMSO as the delivery vehicle was reported to be: hamsters (> 120 mg/kg), rabbit (> 50 mg/kg), rat (9.5 mg/kg), mouse (6.5 to 7.65 mg/kg) and guinea pig (1.1 mg/kg). This study and those described above were mixed with respect to gender. Electrocardiograms (ECGs), mean arterial pressure and resporiratory rates were also measured by Glinsukon et al. (1980) in anesthetized rats after treatment with a lethal intraperitoneal dose of capsaicin. Saito and Yamamoto (1996) reported the oral L D values for capsaicin extract for both genders in mice and rats, when propylene glycol was used as vehicle. They did not find any significant difference in the L D values of the two genders. The major toxic signs in mice and rats included salivation, staggering gait, bradypnoe and cyanosis. Tremors, clonic convulsions, dyspnoe, lateral or prone position was observed, then the animals died 4 to 26 min after oral dosing by gavage. The cause of death was proposed to be due to hypotension and respiratory paralysis in rats and mice although the authors noted that the pathophysiology of these deaths was not clearly understood. In a study with Monsereenusorn (1983), 50 mg /kg capsaicin was given orally by gavage to rats for up to 60 days. The effects of capsaicin on body weight, rectal 50

50

50

50

50

127

temperature, food and water consumption, hematological parameters, plasma biochemistry, urine concentration and dilution tests were evaluated at 10, 20, 30, 40, 50 and 60 days. The major finding was decreased body weight gain starting at 40 days, despite an increase in food consumption. Minor changes in the clinical chemistry (reduced plasma urea nitrogen, glucose, phospholipids, triglycerides, total cholesterol, free fatty acids, glutaminic pyruvic transaminase, and alkaline phosphatase) were noted after one-month treatment; however, these differences were not biologically significant, The results of observations done by Chanda et al. (2005) indicate that trans-capsaicin, given into the sytemic circulation, induces transient increases in heart rate and blood pressure which are rapidly eliminated, and do not cause alterations in cardiac repolarization.

9.1.3.7. Chronic toxicity studies in animals Chronic toxicological studies are absolutely required to test drug candidates in species (one from the rodents and dog) for a period of 6 months. No observations of these types could be found in the literature, consequently these studies should be done in the forthcoming time with our preparation.

9.1.4. Metabolism of capsaicin Early studies by Lee and Kumar (1980) showed that phenobarbital-induced rat liver microsomes converted capsaicin and dihydrocapsaicin to corresponding catechol metabolites, N-(4,5-dihydroxyl-3- methoxybenzyl)-acidamides via hydroxylation on the vanillyl moiety. This finding was further confirmed by Miller et al. (1983) who demonstrated the covalent binding of [ H]-dihydrocapsaicin to hepatic microsomal proteins following in vitro incubation or administration to rats. Based on these results, it has been postulated that capsaicin is activated by the liver mixed-function oxidase system to an electrophilic intermediate, most likely a ring epoxide, capable of covalently interacting with nucleophilic sites of hepatic protein (Fig. 28). This irreversible interaction of capsaicin with liver microsomal protein may account for the binding of capsaicin which was observed in the spinal cord or brain, and it was concluded that capsaicin-induced neuropathy in rodents might be mediated by mechanisms other than covalent interaction (Miller et al., 1983). The alkyl side chain of capsaicin is also considered to be susceptible to enzymatic oxidation. Thus, when capsaicin was incubated with NADPH and the liver S9 fraction from phenobarbital pretreated rats, it was hydroxylated at the terminal carbon of the side chain (Surh, Lee, 1995, 1996; Surh et al., 1995). 3

128

CH NHCO-R-OH

CH NHCO-R

2

CH NH

2

2

COVALENT INTERACTION W I T H CRITICAL CELLULAR

(e.g.,

PROTEINS, DNA,

2

NUCLEOPHILES

RNA...)

TOXICITY (NECROSIS, MUTAGENESIS, CARCINOGENESIS,

ETC.)

R = (CH ) CH = CHCH 2

2

^ C H

3

Fig. 28. M e t a b o l i s m of c a p s a i c i n ( S u r h , Y.J., L e e , S.S., 1 9 9 5 : C a p s a i c i n a d o u b l e - e d g e d s w o r d : toxicity, m e t a b o l i s m , a n d c h e m o p r e v e n t i v e p o t e n t i a l . Life S c i . 5 6 : 1 8 4 5 - 1 8 5 5 ) (with permission)

9.1.4.1. The potential routes of metabolism of capsaicin 9.1.4.1.1. Enzymatic oxidative metabolism of capsaicin

One-electron oxidation of capsaicin has been investigated by means of electrochemical, enzymatic and chemical procedures (Lawson, Gannett, 1989; Boersch et al., 1991). Lawson and Gannett (1989) reported that incubation of capsaicin with micro129

somes or its non-enzymatic reaction with potassium ferricyanide resulted in the formation of a dimer, 5,5'-bis-capsaicin. A phenoxy radical was proposed to be involved in the mutagenesis by capsaicin. Formation of phenoxy radical intermediates has been often observed with certain plant phenolics (Newmark, 1984, 1987), which plays a critical role in lignin biosynthesis in the process of wood formation (Freudenberg, 1962). Formation of dimeric tyrosine by oxidation of tyrosine with horseradish peroxidase-catalyzed coupling reaction has been proposed as a mechanism for the dimerization of two diiodotyrosyl residues in thyroglobin to form the thyroid hormone, thyroxine (Taurog et al., 1994). Boersch et al. (1991) have demonstrated that incubation of capsaicin with peroxidase and hydrogen peroxide produced a fluorescent dimer analogue to that previously reported by Lawson and Gannett (1989). The formation of this fluorescent oxidation product was also observed by chemical or electrochemical oxidation of capsaicin (Lawson, Gannett, 1989). Gannett et al. (1997) have shown that the liver cytochrome P450 2E1 (CYP2E1) activity is responsible for conversion to the reactive phenoxy radical which, in turn, dimerizes or covalently binds to CYP2E1, thereby interacting with the enzyme (Fig. 28). 9.1.4.1.2. Non-oxidative metabolism of capsaicin Cell-free extracts of various tissues of rats contained enzyme activity for hydrolyzing capsaicin or its dihydro analogue at the acid-amide bond to produce vanillylamine and the corresponding fatty acyl moieties (Kawada et al., 1984; Kawada, Iwai, 1985; Oi et al., 1992). The highest enzyme activity was found in the liver followed by such extrahepatic tissues as kidney, lung and small intestine (Kawada, Iwai, 1985), after oral administration of capsaicin (Oi et al., 1992). The splitting of the side chain of dihydrocapsaicin also occurred in vivo (Kawada, Iwai, 1985), which is considered to be the rate-limiting step in the overall metabolism of this compound. Hydrolysis of the amide linkage of capsaicinoids will thus lead to the formation of vanillylamine as a common metabolite regardless of the type of fatty acid functional in their side chain. Indeed, the systemic vanilloid, olvanil [N-(3-methoxy-4-hydroxybenzyl)oleamide] which has a longer side chain than capsaicin, has been found to be susceptible to hydrolysis of the amide bond as determined in various metabolic model systems including cell-free extracts of liver and intestine, isolated hepatocytes and enterocytes, and isolated perfused intestine, and also in whole animal studies (Wehmeyer et al., 1990). Oxidative deamination of the resulting vanillylamine produces the aromatic alcohol for excretion as a free form or a glucoronic conjugate (Kawada, Iwai, 1985; Wehmeyer et al., 1990). Capsaicin hydrolyzing enzymes have been purified from the rat hepatic microsomes (Park, Lee, 1994), and identified as previously known isoenzymes of carbylesterase based on such biochemical and biophysical parameters as Mr (relative mass), pi (inhibitory concentration in logarithmic scale), pH-dependency, mode of inhibition and subcellular toxicity. The enzymes are likely to be present either free in the lumen of endoplasmatic reticulum or loosely bound to the terminal surface of the membrane (Park, Lee, 1994). Capsaicinoids, when administered to rats intragastrically, were readily absorbed from the gastrointestinal tract but were further metabolized to a great extent in the 130

liver before reaching the general circulation (Park, Lee, 1994). As a result, gastrointestinally absorbed capsaicinoids are expected to reach the central nervous system or other extrahepatic organs almost exclusively as degradation products (Donnerer et al., 1990).

9.1.4.2. Role of metabolic activation in capsaicin-induced toxicity There is no clear-cut mechanism which can solely explain the toxicity exerted by capsaicin. Bioactivation to an electrophilic intermediate with subsequent covalent modification of critical cellular macromolecules such as DNA, RNA and proteins has been thought to play a role in cell death (Miller et al., 1983; Anonymous, 1986), fueling interest in the role of metabolism in these observed processes. Based on the results of previous metabolism studies, the following activation pathways can be postulated which may account for the capsaicin-induced cellular damages (Fig. 29): a) cytochrome P450-catalyzed epoxidation of the vanillyl ring to produce an arene oxide; b) one-electron oxidation of the hydroxy 1 group to form a phenoxy radical; c) O-demethylation at the aromatic ring and subsequent oxidation of the resulting catechol to the semiquinone and quinone derivates. The possible involvement of an electrophilic epoxide is not proven (Miller et al., 1983), but the presumed oxirane epoxidation of capsaicin is expected to occur because of the formation of arene oxide. Nonetheless, ring epoxidation of capsaicin is expected to occur since the formation of arene oxide intermediates is the most general phenomenon in the monooxygenase-catalyzed metabolism of aromatic compounds. The best evidence for the epoxidation of capsaicin will be the actual isolation of the presumed arene oxide, but the chemical reactivity of such species precludes its direct isolation from incubation mixtures or from the biological fluids or tissues of treated animals. The advances in the development of novel mild oxidating agents such as dimethyl dioxirane have made it possible to prepare the extremely reactive epoxides of certain chemical carcinogens including aflatoxin B1-8-9 oxide. A similar synthetic approach could be applied to the synthesis of the benzoepoxide derivate of capsaicin for testing its biological activity as well as chemical reactivity. The covalent binding of tritium-labeled capsaicin to hepatic microsomal protein was significantly inhibited by reduced glutathione, which implies the formation of a reactive intermediate (epoxide) during the metabolism of capsaicin (Miller et al., 1983). Since glutathione is relatively nonspecific in terms of interacting with reactive intermediates including not only oxiranes but also capsaicin this does not necessarily suggest that the study of the aforementioned arene oxide as a sole electrophilic epoxide hydrolase might be a more sensitive approach in exploring the possible involvement of an epoxi metabolite in the toxification processes induced by capsaicin. The intermediacy of the phenoxy radical of capsaicin has been investigated by using the electrochemical or chemical methods (Lawson, Gannett, 1989; Boersch et al., 1991; Gannett et al., 1997). Furthermore, horseradish peroxidase plus the phenoxy radical intermediate have to be taken into account (Boersch et al., 1991; 131

Catechol OH

CH NHCOCH CH CH CH CH=CHCH(CH ) 2

2

2

2

2

3

2

Capsaicin

CH CH(NH)COOH 2

DOPA

Dopamine

•

CH(OH)CH NHCH 2

2

Epinephrine OH OH Fig. 29. S t r u c t u r e s of s e l e c t e d c o m p o u n d s c o n t a i n i n g c a t e c h o l m o i e t i e s , i n c l u d i n g c a p s a i c i n ( C h a n d a , S., E r e x s o n , G . , R i a c h , C , I n n n e s , D., S t e v e n s o n , F., M u r l i , H . , B l e y , K., 2 0 0 4 : C e n o t o x i c i t y studies w i t h p u r e t r a n s - c a p s a i c i n . M u t a t . R e s . 5 5 7 : 8 5 - 9 7 ) ( w i t h permission)

Gannett et al., 1997). Likewise, hepatic microsomal cytochrome P450 (particularly CYP2E1) might generate the same reactive radical species that is capable of attacking the nucleophilic sites of the enzyme or the target cell protein (Gannett et al., 1997), which may lead to the loss of catalytic activity in other crucial biological functions. A quinone-type intermediate also represents a potential ultimate electrophilic metabolite of capsaicin. The formation of such intermediate could proceed via Odemethylation of the 3-methoxy group on the vanillyl ring with concomittant oxidation to the semiquinone and ortho quinone derivates. The same O-quinone metabolite could be generally obtained through O-demethylation of the aforementioned phenoxy radical intermediate of capsaicin (Fig. 29). This process will also lead to the formation of an extremely readable group of CH , which is well known to alkylate cellular nucleic acids and proteins. The above-mentioned reactions are likely to 3

132

occur in view of the presence of microsomal O-demethylase activity and relatively high reactivity of catechol or antineoplastic agent, epoxiside, has been known to exert its cytotoxic effect by enzymic O-demethylation of one of its methoxy groups to the ortho-quinone derivative capable covalently binding to cellular macromolecules (Haim et al., 1987a,b; Mans et al., 1991). Similarly, initial ring epoxidation of capsaicin and subsequent NIH shift in the resulting benzoepoxide derivate would generate a catechol intermediate without O-demethylation.

9.1.5. Effects of capsaicin on xenobiotic metabolism

and chemically induced mutagenesis and carcinogenesis

Capsaicin has been suggested to exert chemopreventive effects through the modulation of metabolism of carcinogens and their interactions with target cell DNA. It has been reported that capsaicin displays a dose-related inhibition of the activity of rat epithelial aryl hydrocarbon hydroxylase (Modly et al., 1986), a marker enzyme for metabolism of polycyclic aromatic hydrocarbons such as benzol(a)pyrene. Furthermore, capsaicin suppressed the metabolism and covalent DNA binding of benzol(a)pyrene in human and murine keratocytes (Modly et al., 1986). The results of different studies suggested the interaction of capsaicinoids with microsomal mixedfunction oxidases. Thus, the pretreatment of rats with subcutaneous dosages of capsaicin resulted in pronounced prolongation of phenobarbital or hexobarbital sleeping time (Miller et al., 1983; Damhoeri et al., 1985; Rauf et al., 1985; Surh et al., 1995). Capsaicin also competitively inhibited the ethylmorphine demethylase activity in rat liver microsomes and produced a type I spectral change (Miller et al., 1983). Oral administration of capsaicin (50 mg/kg) together with 10% ethanol in drinking water increases xenobiotic metabolizing enzyme activity to a much greater extent that induced by ethanol alone (Iwama et al., 1990). Capsaicin pretreatment also induced 4-hydroxylation of biphenyl groups in the rat liver (Rauf et al., 1985). Yagi (1990) reported that capsaicin and dihydrocapsaicin repressed the energytransducing NADH-quinone oxidoreductase activity. This finding confirms an earlier observation by investigations of inhibitory effects of capsaicin on rat hepatic mitochondrial energy metabolism through supression of energy flow from NADH to coenzyme Q (Chudapongse, Jathanasoot, 1981). Capsaicin was shown to inhibit calmodulin-mediated oxidative burst in the rat macrophages as determined by the attenuation of Ca ionophore-triggered production of superoxide anion and hydrogen peroxide (Savitha et al., 1990). Joe and Lokesh (1994) have also shown that capsaicin can strongly block the generation of reactive oxygen species by rat peritoneal macrophages in vitro. Capsaicin fed to animals was also inhibitory in these macrophages (Joe, Lokesh, 1994). Pretreatment of rats with capsaicin (1.68 mg/kg, intraperitoneally) for three consecutive days resulted in enhancement of activities of pulmonary anti-oxidant enzymes such as superoxide dismutase, catalase and peroxidase while long-term treatment caused an opposite effect on the latter two enzymes (De, Ghosh, 1990). Since reactive oxygen species are known to play an important role in phorbol-12-myristate-13-acetate (PMA)-mediated tumor promotion as well 2+

133

as in inflammation (Kensler et al., 1986), it would be worth determinining if capsaicin with potential anti-inflammatory activity (Flynn, Rafferty, 1986; Joe, Lokesh, 1994) could act as an anti-tumor promoter. It is noteworthy that the inhibition of prostaglandin synthesis by curcumin, the complex of tumeric, correlates well with its protective activity; it was reported for curcumin to inactivate xanthine dehydrogenase/oxygenase, which may account for its anti-promotion activity. Capsaicinoids have also been found to retain inhibitory effects on liver microsomal CYP2E1 activity (Gannett el al., 1997; Lee et al., 1997; Satyanarayana 2006). Capsaicin inhibits the metabolism and mutagenicity (Guengerich et al., 1991; Koop, 1992; Espinosa-Aguirre et al., 1993), which is known to be activated by CYP2E1. CYP2E1-mediated mutagenicity and tumorigenicity of vinyl carbamate have also been shown to be reduced by capsaicin (Lee et al., 1997). The initiation of papillomas in mouse skin by benzol(a)pyrene was also significantly lessened by topical application of capsaicin prior to the carcinogen (Lee et al., 1997). This finding is in agreement with in vitro inhibition of rodent epidermal arylhydrocarbon hydroxylase activity by capsaicin as previously reported by Modly et al. (1986). Chili extracts have been found to modulate the mutagenic activity of particulate organic matter in urban air samples (Espinosa-Aquuirre et al., 1993). In a series of observations, it has been observed that capsaicin has a protective effect on the metabolism, DNA binding, and/or mutagenicity of some carcinogens [aflatoxin Bl, tobacco-specific nitrosoamine and 4-(methylnitroso)-l-(3-pyridyl)-lbutanone)]. These findings suggest that capsaicin might act as a chemoprotective agent by modulating the activities of microsomal mixed-function oxidases which play key roles in metabolic activation as well as detoxication of a wide array of chemical carcinogens and mutagens (Surh, Lee, 1995).

9.1.6. Hepatoprotection of capsaicin in rats •

Recently Abdel-Salam et al. (2006) observed in detail the dose-dependent hepatoprotective effect of capsacin in rats treated with carbon tetrachloride (CC1 ), which was given orally (2 ml/kg followed by 1 ml/kg after one week). Capsaicin at three dose levels (10, 100 and 1000 pg/kg) or silymarin (22 mg/kg) was administered orally for 10 days, starting at the time of administration of CC1 . The daily administration of capsaicin conferred significant protection against the hepatotoxic effects of CC1 in rats. It decreased the increase of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and also prevented the development of histological hepatic necrosis caused by CC1 as determined 10 days after drug administration. Thus, compared with CC1 control group, serum ALT decreased by 39.3, 59.3 and 71.1%, while AST decreased by 14.3, 21.5 and 23.3%, after the capsaicin administration (given in doses mentioned above), respectively. Serum bilirubin was decreased by 10 and 100 pg/kg (46.4 and 66.5% reduction, respectively), but an increased bilirubin and ALP were observed after the highest dose of capsaicin. Meanwhile, silymarin reduced serum ALT by 65.3%, AST by 18.9%, ALP by 22% and bilirubin by 13.4%, compared to CC1 control. Serum proteins were significantly increased by 16.9 to 22.9% after treatment with capsaicin, 4

4

4

4

4

4

134

whilst a marked, 66.9%, increase in serum glucose was observed after the highest dose of capsaicin compared with vehicle-treated group. Quantitative analysis of the area of damage by image analysis technique showed a reduced area of damage from 13.6% to 7.5, 4.3 and 2.8% by application of capsaicin (used in the above-mentioned doses), respectively (Abdel-Salam et al., 2006). Haematoxylin-eosin staining indicated markedly less hepatic necrosis in rats treated with capsaicin or silymarin. Histochemical alterations such as decreased nuclear DNA, cell glycogen and protein contents caused by CC1 in hepatocytes were prevented by capsaicin as well as by silymarin. It has been concluded from these results that orally applied capsaicin exerts beneficial effects on liver histopathologic changes and enzymatic release caused by CC1 in rats, but its higher doses result in cholestasis (Abdel-Salam et al., 2006). 4

4

9.1.7. Genotoxicity studies with capsaicin or trans-capsaicin Published information on the potential genotoxicty of capsaicin is inconsistent, both positive and negative effects have been found in classic genetic toxicology assays (Azizan, Blevins, 1995; Surh, Lee, 1995). Eight bacterial point mutation tests (including Ames assays) were performed between 1981 and 1995 on capsaicin of varying origins, using various strains of S. typhimurium. Various forms of S9 activation were provided in seven of the eight assays. Four of these tests resulted in a positive response and four in a negative response, respectively. Point mutation tests in Chinese hamster V79 cells were conducted twice, resulting in one positive and one negative response. The in vivo micronucleus test was conducted once in mice and it was positive (Nagabhushan, Bhide, 1985, 1986). Data from one micronucleus and sister chromatid exchange study in human lymphocytes was interpreted to show that capsaicin is genotoxic (Marques et al., 2002). Capsaicin was also reported to induce DNA strand breaks in human neuroblastoma cells SHHY5Y (Richeux et al., 1999). Most of these studies were carried out with natural extracts, and they may not exhibit the same toxicological profile with pure capsaicin. Recently different studies were carried out to evaluate the genotoxic potential of pure frafts-capsaicin using different genotoxic assay used by international regulatory agencies to evaluate drug product safety (Chanda et al., 2004). These included Ames assay, mouse lymphoma cell mutations assay, mouse in vivo bone marrow micronucleus assay and chromosomal aberration assay in human peripheral blood lymphocytes (HPBL). All studies were conducted according to the Organization of Economic Cooperation and Development (OECD) principles of Good Laboratory Practice (GLP) (Table 26).

135

T a b l e 2 6 . G e n o t o x i c i t y of c h i l l i extracts a n d their m a j o r p u n g e n t constituents c a p s a i c i n a n d d i h y d r o c a p s a i c i n u p to 1 9 9 5

Test C o m p o u n d

A n i m a l / C e l l s tested

H e p a t i c S 9 for metabolic activation

CAP, Chilli

5.

typhimurium

Aroclor 1 2 5 4 - i n d u c e d rat

CAP, Chilli

C h i n e s e hamster V 7 9

Aroclor 1 2 5 4 - i n d u c e d rat

CAP, Chilli CAP

Swiss mice 5.

typhimurium!A98

In v i v o Aroclor 1 2 5 4 - i n d u c e d rat

CAP, D H C , Chilli

5.

typhimuriumlA98,

TA1535 CAP, D H C , Chilli

Aroclor 1 2 5 4 - i n d u c e d rat

C h i n e s e hamster V 7 9

Endpoint

Response

His

+

+

reversion

Azaguanine resistance Micronuclei

5.

typhimurium

only)

His

+

reversion

+

His

+

reversion

-

Azaguanine Phenobarbitali n d u c e d rat

CAP, Chilli

S.

typhimurium

+ (CAP

formation

resistance CAP, Chilli

-

+

reversion

-

Streptomycin-

+

His

+

resistance

(Chilli); - (CAP)

CAP, D H C , Chilli

S.

TA100 Chilli

typhimuriumTA98,

Mouse bone marrow

+ (source unclear)

His

In v i v o

Micronuclei

+

reversion

- or + (CAP) +

formation CAP

Albino mice

In v i v o

Pregnancy frequency

CAP

M o u s e epididymis

In v i v o

Sperm abnormality

CAP

H u m a n lymphocytes

Chromosome

+

aberrations CAP=capsaicin; DHC=dihydrocapsaicin; Chilli=plant extract. After Surh, Y.J., Lee, S.S (1995): Capsaicin a double-edged sword: toxicity, metabolism, and chemopreventive potential. Life Sci. 56: 1845-1855 (with modification)

9.1.7.1. Ames assay Ames assay described in the paper of Chanda et al. (2004) was based on the method described by Ames et al. (1975). The thymidine kinase (TK) heterozygote system, where tk+/tk- is mutated to tk-/tk-, was described by Clive et al. (1972, 1979) and it is based upon the L5178Y mouse lymphoma cell line established by Fischer (1958). In this assay, cells deficient tk+/tk- to tk-/tk- are resistant to cytotoxic effects of pyrimidine analogue trifluorothymidine (TFT). Thymidine kinase proficient cells are sensitive to TFT, which causes the inhibition of cellular metabolism and halts further cell devison. Thus the mutant cells are able to proliferate in the presence of TFT, whereas normal cells which contain thymidine kinase are not (Moore et al., 2003). 136

Salmonella typhimurium TA 1535, TA 1537, TA 98 and TA 100 were used. The assays were performed in the presence and absence of S9, using the direct method and preincubation method. Capsaicin did not induce mutagenic activity in any of the bacterial strains used, in either activation condition.

9.1.7.2. Mouse lymphoma cell mutation assay The tk+/tk-3.7.2C heterozygote of L5178Y mouse lymphoma cells were applied in these studies (for details of the methods used, see paper of Chanda et al, (2004). In the presence of S9 mix both assays gave weak mutagenic responses. Both assays contained at least two treatment groups of capsaicin that tested significant (for log mutant fraction) at the level of 5%. Both assays had a linear trend (of mutant fraction with concentration) that was significant at P< 0.001. The increases in the mutant fraction obtained were small, the largest being 190 mutants per million above the control value, obtained at near the maximum acceptable level of toxicity (12% relative total growth). In absence of S9 mix, the 4-hour exposure assay gave a very weak mutagenic response, while the 24-hour assay gave no significant responses at dose levels giving acceptable levels of genotoxicy though the test for for linear trend was significant (P = 0.022). Colony size distribution patterns are, in instances of weak positives, difficult to assess due to the very small samples. The same situation occurs for vehicle control groups, which show a high level of variation between and within experiments. The numbers of mutant colonies assessed in the capsaicin treatments giving significant increases were very low.

9.1.7.3. Mouse in vivo micronucleus assay For details of this methodology, see the paper of Chanda et al. (2004). At least 2000 polychromatic erythrocytes (PCE) were scored for frequency of micronucleated cells. The numbers of micronucleated normochromic erythrocytes (NCE), which were observed within the same microscope fields, were similarly recorded. The PCE/NCE ratio was assessed by scoring a total of at least 500 PCE+NCE. Each assessment was performed on coded slides. In preliminary toxicity tests the maximum tolerated dose of capsaicin was determined to be around 800 mg/kg per day in males and around 200 mg/kg per day in females. Three groups of male mice were dosed with 200, 400 and 800 mg/kg per day and one group of females was dosed with 200 mg/kg per day of capsaicin at 0 and 24 hours. Five mice per sex from each test material dose group were selected to provide the normal assessment base. Current vehicle and positive groups were included. Treatment-related animal deaths and clinical signs were observed in the middleand high-dose level groups. One death occurred in the male vehicle control group, immediately after dosing and was considered as a result of a dosing error.

137

The frequencies of micronucleated polychromatic erythrocytes (MN-PCE) in the capsaicin-treated groups were 0.04, 0.10, 0.09% (males) and 0.05% (females). All of these frequencies were within the historical control range for negative responses (0.01-0.23% for a group of five mice). The frequencies of MN-PCE in the concurrent vehicle control groups were 0.07% (males) and 0.11% (females), whereas the MN-PCE frequency in the positive control group was 1.57%, demonstrating the sensitivity of the test system.

9.1.7.4. Chromosomal aberration in human peripheral blood lymphocytes ( H P B L ) Human venous blood from healthy, adult donors (non-smokers without any history of radiotherapy, chemotherapy or drug usage and lacking current viral infections) was used. The whole blood cultures were initiated in 15 ml centrifuge tubes by adding 0.6 ml of fresh heparinized blood, and the final volume of culture medium and test arcticle was 10 ml. Cultures were incubated with loose caps at 37±2°C in a humidified atmosphere of 5±1.5°C in air. The medium was RPMI 1640 supplemented with HEPES buffer (25 mM), about 20% heat-activated fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin 100 pg/ml9, L-glutamine (2 mM) and 2% phytohemagglutinin M (PGA-M). Negative (untreated controls) and vehicle controls (cultures treated with 10 pi of DMSO/ml) were used. The positive control agents used in the assays were mitomycin-C (MMC) for the nonactivation series and CP (cyclophosphamide) in the metabolic activation series. The in vitro metabolic activation system (Maron, Ames, 1983) consisted of liver post-mitochondrial fraction (S9) and an energy-producing system (NADP at 1.5 mg/ml (1.8 mM) and isocitric acid at 2.7 mg/ml (10.5 mM). S9 was prepared 5 days after a single dose of 500 mg/kg of Aroclor® 1254. Two trials were conducted. In the initial trial, cultures were treated for about 3 hours with and without S9 and harvested about 22 hours after initiation of treatment. In the second trial, cultures were treated for about 22 hours without S9 and about 3 hours with S9 and harvested about 22 hours after initiation of treatment. This harvesting time corresponds to 1.5 times a cell cycle time (Galloway et al., 1994). The period of cell cycle is approximately 15 h after the lymphocytes are induced to divide by the addition of PHA-M. At harvest, cells were swollen with 75 mM KC1 hypotonic solution and fixed with absolute methanol-glacial acetic acid (3:1 v/v). Cells selected from each duplicate culture were analyzed for the different types of chromosomal aberrations (Evans, 1962a,b, 1996). Mitotic index was evaluated from the negative control, vehicle control and a range of test article concentrations and this was used for the measurement of toxicity and selection of doses for analysis. Percent polyploidy and endoreduplication were also analyzed. For control of bias, all slides were coded prior to analysis and read blind. In the first trial, 6.78, 9.69, 13.8, 19.8, 28. 2, 40.4, 57.6, 82.4, 118, 168, 240, 343, 490, 700 and 1000 pg/ml of capsaicin were evaluated with and without metabolic activation by S9. The highest concentration was limited due to the presence of a precipitate dosing. In the first trial, a precipitate was observed at >240 pg/ml, and 138

hemolysis was observed prior to washing and harvesting of these cultures. Only dead cells were present on slides prepared from cultures treated with >343 pg/ml due to excessive toxicity (Chanda et al., 2004). Chromosomal aberrations were analyzed from the cultures treated with 82.4,118, 168 and 240 pg/ml. The high concentration had >50% reduction in mitotic index. No increase in structural or numerical chromosomal aberrations was observed (Chanda et al., 2004). Based on the results from the initial assay, the second trial was conducted at concentrations of 6.15, 12.3, 24.6, 49.2, 98.4, 154, 192, 240 and 320 pg/ml without metabolic activation and 49.2, 98.4, 123, 154, 192, 240 and 320 pg/ml with metabolic activation. Treatment periods were about 22 and about 3 h without and with metabolic activation, respectively, and the cultures were harvested about 22 h after the initiation of treatment. In the assay without metabolic activation, a precipitate was observed after >92 pg/ml and hemolysis was observed prior to harvest of the cultures treated with >240 g/ml. Only dead cells were present on slides prepared from cultures treated with > 192 pg/ml, due to excessive toxicity. Severe toxicity was observed also at 98.4 pg/ml (92% reduction in mitotic index). Chromosomal aberrations were analyzed from the cultures treated with 24.6, 49.2 and 123 pg/ml. Due to toxicity, <100 metaphases were available for analysis in the duplicate cultures treated with 123 pg/ml. The highest tested concentration (240 pg/ml) had >50% reduction in mitotic index. No increase in structural or numerical chromosomal aberrations was observed. In the assay with metabolic activation, a precipitate was observed after dosing at >192 pg/ml and hemolysis was observed prior to harvest of the cultures treated with 320 pg/ml. Since the cultures treated with 240 pg/ml had excessive toxicity (100% reduction in mitotic index) the slides prepared from the cultures treated with 192 pg/ml were selected as the highest concentration for analysis. Chromosomal aberrations were analyzed from the cultures treated with 98.4, 123, 154 and 192 pg/ml. No increase in structural or numerical chromosomal aberrations was observed (Chanda et a l , 2004). Although there are a number of publications focusing on the genotoxic potential of capsaicin or spicy pepper extracts, the test substances used in these studies were various according to source, purity and impurity profile. Consequently, there has not been any systemic observation of the genotoxic potential of pure trans-capsaicin. The majority of the studies found in the literature (Fischer, 1958; Ames et al., 1975; Richeux et al., 1999; Marques et al., 2002) performed the Ames assay. There was one study each with the micronucleus assay, the sister chromatoid exchange assay and the assay invetigating DNA strand breaks in human neuroblastoma cells (Ames et al., 1975; Nagabhushan, Bhide, 1985, 1986; Azizan, Blevins, 1995; Surh, Lee, 1995; Marques et al., 2002). None of the studies were conducted using systematically pure capsaicin, though it would be very important to be able to attribute the results to capsaicin alone, not the impurities. Chanda et al. (2004) applied synthetic capsaicin alone (purity >99%) to perform the genotoxicity studies. In the Ames assay with pure synthetic capsaicin, no increase in mutation frequency was observed in any assays, with and without S9. It was concluded from these studies that capsaicin is not genotoxic in the bacterial assay, with and without metatabolic activation (at the highest concentrations that could be tested). 139

Capsaicin was found to be weakly mutagenic in mouse lymphoma L5178Y cells, in the presence of S9, when it was dissolved in DMSO at concentrations that extended into the toxic range. The lowest positive concentration in the presence of S9 in any individual test was 12 pg/ml. Limited evidence of very weak activity was also noted in the absence of S9. The lowest positive concentration in the absence of S9 was 65 pg/ml. Although criteria for the determinations for positive response in the lymphoma assay remained controversial, these tests are used by different laboratories to evaluate the toxicity (Clive et al., 1979; Moore et al., 2003). The results of the above-mentioned studies were interpreted as weak toxicity. With longer exposures (about 24 hours), higher capsaicin concentrations were too toxic for analysis and the mutant fractions were not significantly different from the controls at the usable concentrations. Thus it can be concluded that capsaicin is non-mutagenic at concentrations up to 28 pg/ml after 24 h exposure in the absence of S9. In the in vivo micronucleus study in mice, the frequency of MN-PCEs was 0.04-0.10% in the treated mice compared to 0.07-0.11% in the vehicle treated control mice. Both were within the historical control range, which was stated to be 0.07±0.08%. The frequency of MN-PCE for the positive control (50 mg/kg of cyclophosphamide) was 1.5%. However, in this study a difference was observed between the maximum tolerated doses estimated for males (800 mg/kg) and females (200 mg/kg). These values are higher than those of previous reports in the literature (Glinsukon et al., 1980; Saito, Yamamoto, 1996), which probably may reflect the quality of the current drug substance rather than the sex difference. Capsaicin was evaluated for its ability to induce clastogenicity in cultured human lymphocytes with and without an exogenous metabolic activation system. Clastogenecity was evaluated at concentrations that induced severe toxicity to no toxicity. Cultures were harvested about 22 hours from the initiation of treatment. Capsaicin did not induce structural and numerical chromosomal aberrations. It can be concluded from the results of genotoxicity observations with pure transcapsaicin that its genotoxicity potential is very limited, differing from that when impure capsaicin or chilli extracts were used in genotoxicity assays. These data have important implications for analysis of risks associated with dietary or environmental capsaicin exposures. Although the majority of epidemiological data suggests that dietary capsaicin consumption is not associated with enhanced risk of cancer (Surh, Lee, 1995), it is true that a positive mathematical correlation was observed between the intake of Chilli pepper and gastric cancer. In Mexico, other factors than capsaicin should be investigated as the causal link in such epidemiological evaluations (Lopez-Carrillo et al., 1994, 2003).

9.1.7.5. Brief summary of the main results of observations with capsaicin in animals Capsaicin is a very active compound acting at the level of capsaicin sensitive afferent nerves. It has dual action, namely in small doses it produces a biologically significant gastrointestinal mucosal protective effect, while in larger doses it enhances the gastrointestinal mucosal damages to different chemical, osmotic and pressure stimuli. 140

It is important to note that the doses (10 to 100 pg/kg) producing gastrointestinal mucosal defensive actions are significantly lower than those (100-200 mg/kg) which produce gastrointestinal mucosal damage. Capsaicin is absorbed well from the animal gastrointestinal tract, its metabolization is carried out by the liver (in pathways of enzymatic and non-enzymatic oxidation). The production of epoxy (arene) by the rat liver was suggested, however, it was not proved clearly. There is a specific and important observation that capsaicin dose-dependently prevents the CCl -induced hepatic injury during a one-week treatment. The genotoxicity studies indicated a very limited positivity, depending on the degree of purification of capsaicin of the plants. 4

9.2. Human observations with capsaicin 9.2.1. Observations with capsaicin in healthy human subjects The capsaicin studies have been carried out since 1997, by the permission of the Regional Ethical Committee of Pecs University, Hungary. These studies were carried out in a randomized, prospective manner, respecting the Helsinki Declaration, according to the Good Clinical Practice (GCP), with the methods required for classical drug (or drug candidate) studies. The First Department of Medicine, Medical and Health Science Centre, University of Pecs, Hungary, is one of the Hungarian Accredited Centres for the human phase I—II examinations. This Institute has been participating in drug development since 1968 up to now.

9.2.1.1. Dose-response curves of capsaicin in the human stomach acute observation The dose-response curves were determined on the gastric basal acid secretion (BAO) in healthy human subjects, and on the measurements in gastric transmucosal potential difference (GTPD) without and with topically (intragastrically) applied ethanol (Mozsik et al., 2004 2005a, 2007b). We tested the effects of 100, 200, 400 and 800 pg/ 100 ml saline solutions given intragastrically via a nasogastric tube on the gastric BAO values, meanwhile the same doses of capsaicin given intragastrically (via endoscopic channel of gastrofiberoscope) were tested on GTPD. In other series of observations, the gastric microbleeding was produced by oral (3x25 mg, plus 25 mg given at the start of examinations) indomethacin in healthy human subjects. Indomethacin was given alone, or in combination with capsaicin (200, 400 and 800 pg). The results were compared with the results obtained without application of Indomethacin (baseline). Capsaicin given in a dose smaller than 100 pg had no effect. The E D value was obtained at the 400 pg dose on the gastric basal acid output (BAO), gastric transmucosal potential difference (GTPD) (without and with topically applied 5 0

141

ethanol), and Indomethacin-induced gastric microbleedings (Mozsik et al., 2005a, 2007b). It was also observed that gastric microbleeding produced by inhibition of both COX-1 and COX-2 was completely prevented by the application of 400 pg capsaicin (Mozsik et al., 2007b; Sarlos et al., 2003). When capsaicin was given in a dose of E D intragastrically, the "parietal component" of the gastric acid secretion in healthy human subjects decreased (P<0.001), meanwhile its "non-parietal components" (buffering secretion) increased significantly (P<0.001) (Mozsik et al., 2004; 2005a; 2007b); gastric emptying was increased (Debreceni et al., 1999). Recently it was observed that capsaicin (in a dose of 400 pg orally) enhanced glucose absorption and glucagon release during the regular glucose loading test in healthy human subjects (Domotor et al., 2006). 50

9.2.1.2 Changes in laboratory parameters and complaints

of healthy human subjects during the study with capsaicin

No systemic laboratory changes were noted in the biochemical parameters, with the exception of special observations with gastric juice (Mozsik et al., 2004; 2005a), glucose loading test (Domotor et al., 2007) where specific changes were obtained in the examined biochemical parameters. No subjective complaints (pain, diarrhoea, vomiting) were observed.

9.2.2. Subchronic observations with capsaicin in healthy human subjects 9.2.2.1. Two weeks treatment with capsaicin ..

•

The group of healthy human subjects received capsaicin treatment for two weeks (3x400 pg given orally) in a prospective, randomized study. Gastric microbleeding was produced by Indomethacin application before and two weeks after capsaicin treatment. The baseline of blood loss, Indomethacin-induced gastric microbleedings (without and with different doses of capsaicin) were measured before and two weeks after the capsaicin treatment. No changes were obtained in the baseline, Indomethacin-induced gastric microbleeding, and on the other hand, the gastric mucosal protective effects of capsaicin remained the same after the two-week capsaicin treatment as those at the beginning of the two-weeks capsaicin treatment (Mozsik et al., 2005a, 2007b).

9.2.2.2. Biochemical measurements and complaints in healthy human subjects during two weeks capsaicin treatment No changes were noted in the biochemical parameters and no complaints were reported in the healthy human subjects. 142

9.2.3. Human chronic observations with capsaicinoids In a case-control study in Mexico City which included 220 cases of gastric cancer and 752 controls randomly selected from the general population, chilli pepper consumers were at a 5.5-fold greater risk for gastric cancer than non-smokers. Persons who held themselves as heavy consumers of chilli peppers were at a 17-fold greater risk. However, when chilli consumption was measured as frequency per day, a significant dose-response relationship was not observed (Lopez-Carrillo et al., 1994, 2003). In another case-control study in India, red chilli powder was found to be a risk factor for cancers of the oral cavity, pharynx, esophagus and larynx compared with population controls, but not with hospital controls (Notani, Jayant, 1987). In an Italian case-control study, chilli was briefly mentioned as being protective against stomach cancer (Buiatti et al., 1989). Chilli pepper, however, is not heavily consumed in Northern Italy, where this study was conducted, and it is possible that chilli consumption was correlated with other protecting spices such as onions and garlic that are consumed in large quantities in Italy. The Committee of Experts on Flavoring Substances of the Council of Europe concluded that the available data not allow it to establish a safe exposure level of capsaicinoids in foods (Opinion of the Scientific Committee on Food on Capsaicin, adopted on 26 February, 2002). It was also observed that the non-selective COX-1 and COX-2 inhibiting nonsteroidal anti-inflammatory drugs-induced gastric microbleeding can be completely prevented by 400 pg capsaicin administrations in healthy human subjects (Mozsik et al., 2007b).

9.2.4. Preventive effects of capsaicin against the selective and non-selective inhibitory actions produced by nonsteroidal anti-inflammatory drugs on COX-1 and COX-2 enzymes Indomethacin (as a non-selective COX-1 and COX-2 inhibitor) was used to provoke gastric microbleeding in healthy human subjects, and capsaicin itself, as a specific stimulator at the capsaicin (TRVP1) receptor (given in small doses) was applied in healthy human subjects. The capsaicin treatment was carried out for two weeks, when it was given in 3x400 pg orally (400 pg capsaicin dose was found to be equal to the E D value in different human observations) (Mozsik, 2006; Mozsik et al., 1999; 2004; 2005a, 2007b) (Tables 27-28). 50

143

T a b l e 2 7 . C o m p a r i s o n o f i n h i b i t o r y effects ( I C ) of C O X - 1 a n d of v a r i o u s N S A I D s using h u m a n 5 0

platelet C O X - 1 a n d s y n o v i a l c e l l C O X - 2 * NSAID

Ratio COX-1 : COX-2

Aspirin

0.12

Diclofenac

38.00

Etodolac

179.00

Ibuprofen

0.86

Indomethacin

0.30

Loxoprofen-SRS

3.20

NS-398

1263.00

Oxaprozm

0.061

Zaltoprofen

3.80

* Kawai, S., Nishida, S., Kato, M., Furumaya, Y., Okomoto, T, Mizushima, Y. (1998): Comparison of cyclooxigenase-1 and-2 inhibitory activities of nonsteroidal anti-inflammatory drugs using humai'n platelets and synovial cells. Eur. J . Pharamcol. 347: 87-94 (with permission)

T a b l e 2 8 . C o r r e l a t i o n b e t w e e n t h e c a p s a i c i n a c t i o n s , C O X - 1 a n d C O X - 2 systems a n d gastric m i c r o b l e e d i n g s p r o d u c e d b y i n d o m e t h a c i n in h u m a n h e a l t h y subjects b e f o r e a n d after 2 w e e k s c a p s a i c i n (3x400 pg orally) treatment*

IC

5 0

v a l u e of i n d o m e t h a c i n t o ratio of C O X - 1 / C O X - 2 = 0.30 (1:3.25) M i c r o b l e e d i n g in t h e s t o m a c h «- 2 w e e k s c a p s a i c i n treatment —>

Before

After

Baseline

2.1 ± 0 . 1 ml/day

2.0 ± 0.1 ml/day

After I N D

8.25 ± 0 . 2 5 ml/day

7.8 ± 0.3 ml/day

A I N D - i n d u c e d 6 . 1 5 ± 0.2 ml/day

5.8 ± 0.3 ml/day

( = inhibition on C O X - 1 + COX-2) ( = 1 0 0 % )

COX-1:

1.447±0.1

COX-2:

4 . 7 0 ± 0 . 2 ml/day

ml/day

1.364±0.1 ml/day 4.44±0.2 ml/day

• 4 0 0 p g c a p s a i c i n ( I G g i v e n ) i n d u c e d d e c r e a s e of I N D - i n d u c e d gastric m i c r o b l e e d i n g

6±0.2 m L / d a y

5.9±0.2 mL/day

means±SEM in 14 healthy human subjects. * Mozsik Gy, Szolcsanyi, J . , Domotor, A. (2007b): Capsaicin research as a new tool to approach of the human gastrointestinal physiology, pathology and pharmacology (review). Inflammopharmacology 15: 232-245 (with permission)

144

9.2.5. Summary of the observations with capsaicin alone or in combination with selective and non-selective inhibition of COX-1 and COX-2 enzymes by nonsteroidal anti-inflammatory compounds (drugs) in animal experiments and in human observations Capsaicin does not represent only one chemically well identified compound, but covers the same biological, physiological, and pharmacological actions as capsaicinoids (capsaicin, dihydrocapsaicin, norcapsaicin, nordihydrocapsaicin). Capsaicin (capsaicinoids) has (have) been widely used in the nutrition of the population of different countries in the last 7500 years. It was an important internationally accepted discovery that capsaicin (capsaicinoids) significantly stimulates (stimulate) a subgroup of the afferent nerves (named as "capsaicin-sensitive afferent nerves") to different chemical agents, heat, pH gradients in animal experiments and human observations. The doses of capsaicin (capsaicinoids) are significant in the biological actions, because when given in small doses it causes tissue protection (including the gastrointestinal tract), however, in larger doses enhances the organ's damage (including the gastrointestinal tract). Capsaicin (capsaicinoids) in larger doses produces organ damaging effects as a consequence of a capsaicin-induced desensitization process. The existence of these principal observations was scientifically proved in animal experiments and human observations. The application of capsaicin has been carried out as a tool to approach the different physiological and pharmacological regulations of different diseases and their prevention. The study of the afferent nerves has been carried out since 1970. The results of this research clearly proved the principal and important role of the afferent nerves in the physiological regulation of different organs as well as in the development of damage and its prevention in these organs. The human observations with capsaicin were carried out in randomized, prospective studies by the permission of Regional Ethical Committee of the University of Pecs, Hungary, in accordance with the Good Clinical Practice (GCP) respecting the Helsinki Declaration. The results of the animal experiments and human observations clearly proved the gastrointestinal protection by application of small doses of capsaicin. The anti-inflammatory drugs (acting as selective and non-selective inhibitors of COX-1 and COX-2 enzymes) are widely used in the prevention of thromboembolic episodes, in the prevention of reinfarction in patients who underwent myocardial infaction, in the treatment of patients with acute and chronic pains with degenerative chronic joint diseases, malignant diseases and in healthy persons for the prevention of gastrointestinal cancers, etc. Capsaicinoids can be absorbed well from the gastrointestinal tract. They are metabolized in the pathways of enzymatic as well as non-enzymatic oxidation of the liver. It is very important that capsaicin alone dose-dependently prevents the hepatic damage produced by carbon tetrachloride (in a significantly higher level than sily145

marin). The studies with genotoxicity clearly indicated that pure capsaicin has a very limited toxicity (this value is higher if the capsaicin preparation is not chemically clear and is contaminated with different toxicological agents of plants) (aflatoxin, pesticides, etc.). The human observations also clearly indicated that capsaicin actions can be reproduced well in healthy human subjects. Capsaicin application produces a dose-dependent increase in gastric transmucosal potential difference (without and with combined ethanol), decreases the gastric acid basal acid output (BAO), and completely prevents the Indomethacin-induced gastric mucosal microbleedings (in a range of 100 to 800 pg given intragastrically). Furthermore, two-weeks treatment with capsaicin (3x400 pg given intragastrically) did not modify the sensitivity of the capsaicin-sensitive afferent nerves in the gastric mucosa, and the gastric mucosal protective effects against Indomethacin remained as dose-dependent as those before and after 2-weeks capsaicin treatment (Mozsik et al., 2007b). The conclusions of these animal and human observations clearly proved that the application of capsaicin given in small doses is able to completely prevent the nonsteroidal anti-inflammatory drugs-induced gastrointestinal side effects. In other words, capsaicin is able to inhibit the functions of the selective and non-selective COX-1 and COX-2 enzymes in animal experiments and in human observations. Further scientific research may offer an absolutely new pathway(s) for the development of drug(s) by the stimulation of capsaicin-sensitive afferent nerves with small doses of capsaicinoids for patients, who have to be treated with nonsteroidal antiinflammatory drugs and for those with diseases in which the COX-1 and COX-2 enzymes are in over- (inflammations, tumors) or under- (after application of the different nonsteroidal anti-inflammatory drags) expressed.

146

10. Nature and characteristics of the innovative drug research

10.1. Characterization of the innovative drug research Our attention was focused on producing new drug combinations [aspirin, naproxen, and diclofenac with capsaicin (capsaicinoids)] and capsaicin (capsaicinoids) alone for the treatment of patients with different diseases. It has been proven earlier that the natural capsaicin (capsaicinoids) - given in small doses (nano and microgram/kg bw in animals) - prevents (prevent) the aspirin, acidified aspirin (and other chemical agents, such as 96% ethanol 0.2 M NaOH, 25% NaCl)-induced gastric mucosal injury, indomethacin (when it was given in basal and in betanechol, pentagastrin and histamine-stimulated gastric secretory conditions), acidified indomethacin-produced gastric mucosal damage in animal experiments (Mozsik et al., 1997a; Mozsik 2006), and that the capsaicin (capsaicinoids) given in hundred micrograms orally decreases (decrease) the orally applied indomethacininduced gastric microbleedings in healthy human subjects (Mozsik et al., 2005a, 2007b; Sarlos et al., 2003). It is also known that aspirin as a selective cyclooxygenase-1 (COX-1) inhibitor has to be administered for a long time or for the whole life time in patients with myocardial infarction and later on for the prevention of their reinfarction (see guidelines: Fox et al., 2006). The beneficial effect of aspirin is based on the inhibition of platelet aggregation. Myocardial infarction and reinfarction is a leading cause of the mortality in the Hungarian patients. It is also known that many people suffer from the different degenerative diseases of the locomotor system, who have to be treated with NSAIDs also for a long time. The number of these patients is also very high. There is a great medical problem in the treatment that the very wide application of NSAIDs is absolutely indicated from the points of cardiology, rheumatology, neurology (stroke prevention), however, the application of these compound(s) is (are) contraindicated from the view point of gastroenterology (based on the laws of evidence based medicine, EBM). There is a general dilemma (and failure) to apply the evidence based medicine in the patients' treatment plan. Furthermore, these diseases occur frequently together in one patient. The H2RA and proton pump inhibitors are widely used in the prevention of NSAIDs-induced gastrointestinal side effects (GI mucosal bleeding and ulceration, liver injury etc.). Platelet aggregation can be produced by selective and non-selective COX-1 and COX-2 inhibitors, but not by selective COX-2 inhibitors. These facts give the phar147

macological basis of the primary and secondary prevention of myocardial and brain infarction. The discovery of capsaicin-sensitive afferent nerves opened an absolutely new gate in the physiology, pharmacology, pathology, when Szolcsanyi and Bartho (1982) had clearly proved that the stimulation of capsaicin-sensitive afferent nerves by application of small doses (pg/body weight) of capsaicin prevented the gastrointestinal mucosal damage produced by different chemical and noxious agents (for the details see Chapter 8). Capsaicinoids are essential compounds from the spices, which have been widely used in the cuisine from the old ancient time up to now. The mechanisms of gastrointestinal mucosal protection, produced by capsaicinoids, have been extensively studied in animal experiments (Mozsik et al., 1997, 2007b; Domotor et al., 2005, 2007). The application of capsaicin (orally given in micrograms doses) prevented the indomethacin- and ethanol-induced gastric mucosal damage in healthy human subjects (Mozsik et al., 2005a). The results of studies with capsaicin in animal models and healthy human subjects offered a possibility to conclude that the beneficial effect of application of small doses of capsaicin can be probably used (as therapeutic tool) against the different noxious agents-induced gastrointestinal mucosal damage in healthy humans beings and patients. Our aim(s) was (were) to produce new pharmaceutical preparations (capsaicin alone) or drug combination^) (capsaicin plus aspirin, diclofenac and Naproxen). The physiological and pharmacological regulatory mechanisms of actions became more clear for medical research people, however, we had not enough knowledge in the fields of pharmaceutical technology [industrial production of drugs; necessary circumstances around the new drug (or drug combinations)] research approach of the necessity the new drug (or drug combinations) composition; production of the preclinical dossier; production of drug master file (DMF); organization and carrying out of stability tests of a new component (or drug combinations); preparation of documentation to receive the permission of the National Institute of Pharmacy of Hungary and National Clinical Pharmacological and Ethical Committee to start with the human Phase I—II studies]. We had a special challenge in this field, because capsaicin (as mentioned earlier, in the chapter on research) is not uniform chemically. The successful solution of these scientific, technical, industrial questions requires very complicated and administrative work. This type of work suggests the a priori establishment of a multidisciplinary research team. None from our research team described below could have surveyed alone all the aspects of the above-mentioned scientific, drug industrial laws, many national and international regulatory laws and administrative fields. 21 researchers have participated in this "innovative drug research" from 2005; they are the members of the Department of Pharmaceutical Chemistry, First Department of Medicine, Department of Ophthalmology, Medical and Health Centre, University of Pecs, Hungary and PannonPharma Ltd., Co., Pecsvarad, Hungary, respectively. The university people represent the basis of research work, while the participants from the industrial partners (PannonPharma Ltd., Co, Pecsvarad) give the basis of technological and industrial research, including the final production of new drug or drug compositions and their marketing. 148

The ages of researchers are between 23 to 69 years (in average 36 years). There are 11 women and 10 men among them. They are physicians (internist, ophthalmologist), chemists, chemical engineers, biologist, bioengineers, pharmacologist, and horticultural engineer. Some of them have different scientific qualifications [Ph.D., Sc.D. (med), Ph.D. (chemists, physicians, pharmaceutists)], and two Ph.D. students joined our work team. This "multidisciplinary team" has enough practice in clinical pharmacology, medicine, ophthalmology, drug research and industrial research. The participants of this team never worked together before receiving this grant. The time for the execution a successful study in this "innovative drug research" covers only a four-year period. Consequently we had a very short time to acquire the necessary knowledge from the medical, chemical, industrial, pharmaceutical chemical fields and from their experts We had no other possibility as: 1. To identify the scientific research organization, the main steps of necessary studies to be carried out by our "innovative drug research" (from the basic research to reach to the drug production for human beings) during the four-year period; 2. To receive correct information on the depth of the special knowledge from the different members of participants; 3. To create a "functional map" on the knowledge of the participants, which is used to give information for the others; 4. To pick up the basic and related knowledge from all the fields involved in the study; 5. To establish a well functioning equilibrium between the researchers working in the different university institutes and their industrial partners; 6. To follow permanently the new results of the international research; 7. To study the national (Hungarian) and international (mainly European Union) laws existing in the field of new drug production; 8. To read many scientific papers; 9. To organize regular (monthly) meetings for the evaluation of the scientific problems together with all the participants, who are involved in the study; 10. To meet the main research people every week (before and after the above mentioned meetings), to discuss the further necessary steps; 11. To collect the necessary knowledge from the neighboring fields for our "innovative drug research"; 12. To learn the main lines and the main results of the national and international research connected to our fields; 13. To find the different pathways, which are suitable to stimulate the work of the participants; 14. To give absolutely new information from the different fields to all the members of the study participants; 15. To prepare ourselves to solve creatively problems of earlier unsuccessful scientific (and perhaps other) activities to receive results for innovative research. The "innovative drug research" is an extremely hard interdisciplinary work, which cannot be done successfully without the acceptance of the national and international 149

trends and laws. We had to recognize our real possibilities (including the spiritual, technical and economical aspects). No clear internationally accepted definition exists for the "innovative research". The main point is probably to summarize the possible pathways in the research. Our aim was to find the pathways for solving the different (interdisciplinary) problems successfully in a relative short time, and the participants of innovative research who are able to help the works of each others. We never worked together, consequently we had no common experience on how we can solve these types of current scientific, technical, industrial, marketing challenges. So, we had to pick up the necessary knowledge from the different fields, and to learn from each other permanently, from day to day. The author of this chapter (Gy. M.) participated in many national and international research programs (in the establishment of clinical pharmacology, clinical nutrition, peptic ulcer research, drug research, medicine, producing new foods, molecular and biochemical pharmacology) and gained significant experience in the fields of experimental and clinical research. Nevertheless, he learned more than he did earlier during the "innovative drug research" (furthermore in a very short time) (pharmaceutical chemistry, general chemistry, plant physiology, technological problems, questions of publication, industrial research, research organization, harmonization between the academic and industrial research, the national and international new results and laws, etc.). There was a special feeling in the organizer(s) and participants during our innovative drug research. We learned the different pathways of problem- and success-oriented works (under the different, and in some instances difficult, national and international conditions). We had about half a year to learn and accept this line of the "innovative drug research". The following main steps were carried out in this "innovative drug research" from 2005 to 2008: 1 .Preparation of experts' opinion; 2. Introduction of the necessary chemical, pharmaceutical, drug industrial methods for the quantitative measurements of the chemical components; 3. Plant origin capsaicin (capsaicinoids) is (are) used as drug compound(s), so preparation of Drug Master File (DMF) is basically necessary, and it has to be used in the preparation of the preclinical documentation(s) of the planned drug (or drug combinations); 4. We read many original printed papers or written information; 5. We studied very carefully the new scientific results in our field, which were published in other parts of the World; 6. We had many consultations with the National Institute of Pharmacy (Hungary) on the chemical purity of the capsaicin (capsaicinoids), on the possibility of its use as an effective material to produce a new orally applicable drug (or drug combination) in humans; 7. We became acquainted with a lot of pharmaceutical laws in the production of new drugs (or drug combinations), which finally will be used during the industrial production of the "ready drug (or drug combinations)";

150

8. We had to learn the national and international difficulties of the production of Drug Master File (DMF) (including the problems existing in the USA, Europe, and India); 9. The patent research was carried out by our industrial partner in this study, the Technological and Transfer Office of the Consortium. 10. We prepared three Hungarian (P0700779, P0800602 for itraconazole and P0700755 for capsaicin) and two PCT-safe (Patent Cooperation Treaty) (PCT/HU 2008/000144 for intraconazole and PCT/HU 2008/000136) patents from our work, which were accepted by the Technological and Transfer Office of our University, while the final acceptance of these three patents by Patent's Office is in progress; 11. The Clinical Pharmacological Unit of our Department (First Department of Medicine, Medical Faculty, Pecs University, Hungary) was qualified by the National Institute of Pharmacy (Hungary); 12. We permanently wrote our appraisement of new results, documentation, notes on the meetings or other new important information. We noticed the actual state of the problems, and we indicated continuously the forthcoming steps and their responsible persons in a register; 13. We prepared the overview of the annual works in Hungarian and English languages; 14. We had permanent contact to the National Patent' Office in order to obtain the final acceptance of our three patents; 15. We carried out many consultations with the different experts and national (or international) authorities on our actual problems; 16. We were open to all new problems and all of the possible solutions; 17. We considered the possibilities of our survival after the closing of this grant (we planned to establish different spin-off firms); 18. We published the results of the new experimental and clinical research; 19. We produced a plan to the end of 2010 for the participation in the international science (by the organization of different international symposia, writing books upon international invitation). In the forthcoming part, we will emphasize some interesting and important problems, which appeared during our "innovative drug research": 1. We had many problems with learning and understanding the details of the scientific knowledge of the individuals participating in this project, who received significantly different scientific education and participated in different practices (at different levels) on their fields. A period of nearly half a year was necessary for understanding each other. After this period, our correspondences and personal communication represented the main sources of our new knowledge. These events offered a new pathway for the continuous learning for all of us; 2. We recognized that the international use of "capsaicin" in the research does not represent a chemically uniform entity (because it means a mixture of a few compounds). The "capsaicin" preparations, which were mainly used in the research, contained at least three components: capsaicin, dihydrocapsaicin and nordihydrocapsaicin.

151

The references in the different scientific papers mention that the "capsaicin" was obtained from Sigma, Aldrich or Sigma-Aldrich. Interestingly the scientific papers (except for a few ones) never mentioned that the researchers use a chemically not uniform "capsaicin" preparation in their experiments. Capsaicin transforms into dihydrocapsaicin chemically, which produces the same physiological responses as capsaicin in the different experimental conditions (Burk, Burks, 1986). We registered this interesting event, and we initiated special consultations with the experts of the National Institute of Pharmacy (Hungary) on the official chemical composition of "capsaicin"(capsaicinoids) from the aspects of "innovative drug research". Since capsaicin (capsaicinoids) is (are) prepared from the plants, we asked the "official authorities" how we can use it (them) as pharmaceutical sources for the production of orally applicable drug(s) in the human medical therapy. The constant composition of "capsaicin" is a basic requirement independently from sources of "capsaicin preparation". That was the answer of the experts of the National Institute of Pharmacy (Hungary). We respected the opinion of the Hungarian authorities (as national) and international laws regarding the chemical purity of "capsaicin preparation". 3. Capsaicin can be obtained from the plants of Capsicum (see Chapter 2) over the World. The main producers of capsaicin preparations are India, China, Central and SouthAmerica, and in some extent the European Countries (including Hungary). The cultivation of domestic Capsicum and the wild Capsicum is done in significantly different ways in the different parts of the World. The application of different chemical compounds for increasing the quantity of capsaicin from Capsicum plants is widely and internationally accepted. We obtained Internet information about the pesticide contamination of sweet bell pepper (see on the website www.ewg.org the "Report Card: Pesticides in Sweet Bell Peppers") (Table 29): Table 2 9 . S w e e t Bell Peppers w i t h the most pesticide residues (Report card)

(www.ewg.org)

S w e e t B e l l Peppers w i t h t h e most pesticide residues Sample 1 Acephate Dicofol

Sample 2 •

•

•

A z i n p h o s methyl T

• •

Dimethoate

•

• •

Carbaryl

•

•

•

Metalaxyl Methamidophos Methomyl

•

T

Malathion •

Acephate • T

• • T •

T •

Carbaryl •

•

• •

Dimethoate • • Endosulfans •

T

T •

Methamidophos T

Methomyl •

O-Phenylphenol

•

•

•

Methamidophos T

Oxamyl

•

• •

Endosulfans •

T

Diphenylamine (DPA) Fenvalerate

Sample 3

• •

Oxamyl

•

P e r m e t h r i n Total •

•

• - Animal Carcinogen; • - Causes Birth Defects in Animals; V- Damages Reproductive System; • - Interferes with Hormones; T- Damages Brain and Nervous System; • - Damages Immune System

•

T

• Pesticides were found on 68% of the sweet bell peppers tested. There were 39 pesticides found in sweet bell peppers: Acephate, Azinphos methyl, Benomyl, Bifenthrin, Captan, Carbaryl, Carbofuran, Chlorothalonil, Chlorpropham, Cyfluthrin, DDT, Diazinon, Dichlorvos (DDVP), Dicloran, Dicofol, Dimethoate, Diphenylamine (DPA), Disulfoton, Endosulfans, Ethoprop, Fenvalerate, Folpet, Iprodione, Lambda cyhalothrin total, Lindane (BHC gamma), Malathion, Metalaxyl, Methamidophos, Methomyl, Metribuzin, Mevinphos Total, Myclobutanil, O-Phenylphenol, Oxamyl, Oxydemeton methyl, Permethrin Total, Piperonyl butoxide, Pronamide, Thiabendazole. The three pesticides found most often in sweet bell peppers were Methamidophos, Acephate, and Endosulfans (Fig. 30). Number of pesticides

Percent of Samples

per sample 0

22%

Fig. 30. M o s t S w e e t B e l l P e p p e r s a r e c o n t a m i n a t e d w i t h m o r e t h a n o n e p e s t i c i d e . Foodnews Enviromental W o r k i n g Group//foodnews.org http://www.drgreene.org/body.cfm?xyzpdqabc=0&id=21 &action=detail&ref=l 9 2 0 "

Furthermore, the national and international controls of the applicable chemical components are also significantly different. For example, the use of organic phosphate components was prohibited in the European countries, but not in India or China. So, the exact elucidation of the use of different components during the cultivation of Capsicum plants is a key question. The Department of National Chemical Safety (Bordas, 2006) published a book on the classification of dangerous abatement means and their dose limits. Furthermore, this book summarized the chemical compounds used in the cultivation of the plants. 153

Looking at this information concerning the toxicological conditions in the cultivation of plants, we had a new challenge to understand the conditions of cultivation of Capsicum plants in the different countries (India, China, U.S.A., Hungary, etc.). We received correct information on the official requirements of the use of the different chemicals (including the national and international recommendations) during plant cultivation. However, we had no correct information on the usually applied chemicals in the plant cultivation abroad. The National Institute of Pharmacy (Orszagos Gyogyszereszeti Intezet, OGYI) asked us to repeat the toxicological examinations (see toxicological chapter) with our capsaicin preparation (including the chronic toxicological studies in rats and beagle dogs). This request of the National Institute of Pharmacy surprised us, because this meant extra work from the point of both using capsaicin for drug production and repeating the of toxicological examinations carried out earlier. We performed, of course, theses studies in the last year successfully. How did we control these problems? The control of our activities is a very complicated and hard job. One of the most important pathways is to follow the different steps of plant cultivation, and thereafter the steps used in the processing of the plants. The collection and qualification of the main steps is an essential requirement, and these factors are the key-points in the Good Manufactorial Practice (GMP). The collection of this information in these questions seems to be theoretically very simple, but in the practice we have no real possibility to follow and to collect all the written documentation given by nationally (or internationally) accredited authorities. How did we meet with these problems in our "innovative drug research"? Earlier we tried to collect these data from the international trade firm "SigmaAldrich". We have no correct and sure information on the capsaicin supplying countries for this trade firm. The mentioned data were absolutely necessary to prepare Drug Master File (DMF). The DMF is absolutely necessary for the preparation of the preclinical dossier before the preparation of the protocols for carrying out the human phase I—II clinical pharmacological studies. We realized with surprise that Sigma-Aldrich had no DMF for capsaicin. We received the same information on these questions from the other trade firms concerning capsaicin supplying. We studied thoroughly the circumstances of the cultivation of the Capsicum in Hungary. The exclusion of organic phosphate compounds from the agricultural and horticultural practices was absolutely necessary. Concerning this, we could not obtain correct data from the circumstances of cultivation of Capsicum. We had the same problem with the capsaicin coming from India. The DMF of capsaicin originated from India is signed in the documentation of Food and Drug Administration (FDA) in the United States ("17856 A II26.10.2004 Asian Herbex Ltd: Capsaicin USP as manufactured in Andhra Pradesh, India") as existing registration, which can be applied to the use of capsaicin as basic source for the capsaicin-containing drug. We could not understand that the results of capsaicin research increased extremely in the last years, while only one registration of capsaicin DMF was signed by the date 26.10.2004. over the World, registration number: 17856 A II. We followed closely the different pharmaceutical firms over the World in the literature, however, we could not find an 154

answer as to why these firms did not use this source DMF from the Food and Drug Administration of the USA. It has been concluded that we need to find another pathway of the capsaicin source for basic drug material for the production of the capsaicin containing drug (or drug combinations). Thereafter, we returned to receiving the opinion from the experts of the National Institute of Pharmacy (Hungary) on the possibility to surmount these problems. 4. New consultation was done with the experts of the National Institute of Pharmacy (Hungary) on the acceptance of capsaicin compound as basic drug material without existing DMF of the capsaicin preparation obtained from Hungary. We followed the application of the agricultural chemistry during the cultivation of Capsicum plants (e.g. the application of organic phosphorus compounds, as pesticide agents, was prohibited from the last decades). Other chemicals are also used in plant cultivation and processing. The list of the chemicals, permitted by the Institute of National Chemical Safety was collected and published (Bordas, 2006). Up to this moment we could not receive a reliable DMF from the workers of Capsicum cultivation. We mentioned that the ordering of the basic drug compound is the task of the industrial partner. We tried together to discover different firms supplying us with the basic drug compound for our research work. We found a firm from India for supplying us with capsaicin, but we did not receive from them the correct DMF. Of course, the registration of DMF was electronically found in the collection of DMF by the Food and Drug Administration in the USA. We received a new possibility to obtain capsaicin from this Indian firm. An extremely important law was in the "innovative drug research" as follows: we had carry out all the observations (chemical analysis, stable observation, toxicological studies, drug formulation, human phase I—II clinical pharmacological studies) with the same capsaicin preparation (charge). In the meantime, we obtained 3 to 4 kg capsaicin (without DMF) from a Hungarian firm. This amount of capsaicin was obtained from different charges of Hungarian paprika (Capsicum), however, it was supplemented with oleoresin imported from India. The chemical composition of this amount of capsaicin (as basic drug component) can be chemically and toxicologically studied by the different accredited University Institutes. We received permission from the experts of the National Pharmaceutical Institute for preparation of DMF of the Hungarian capsaicin by the pathways above, however, we were not able to follow the different steps of Capsaicin cultivation. Why did we start this way? In the original agreement it was finalized that the Hungarian cultivated Capsicum will be used by our research team in March of 2006, and at the end of 2006 we had no capsaicin drug basic component. In the meantime, the capsaicin supplying basic drug compound (capsaicin or capsaicinoids) changed from the Hungarian firm to the Indian firm in October, 2006. On the other hand, we lost two years' time from our grant. We had only two further years, and nearly one year was necessary for doing the chronic toxicological examinations in animals, moreover, we could not write the protocols for human phase I—II studies because of the absence of international DMF for capsaicin. 155

The official permission from the National Institute of Pharmacy (Hungary) and of the National Clinical Pharmacological and Ethical Committee of Hungary is basically necessary before starting the human phase I—II clinical pharmacological studies. The mentioned events clearly demonstrated that we would be having many different works in the same time period. 5. We met new events practically every day. On the other hand, all of us gave new information to the others orally and in writing. There was a very effective regular consultation between the university researchers and industrial researchers. Written notes were prepared on all of consultations. We informed all of the members of our research team permanently. This type of research is very exciting, multidisciplinary, responsible work. Consequently, we had to help all the participants in this research work independently of our primary place and education. Because the researchers are 23 to 69 years old, our common meetings represent a special education form for the younger generation. 6. We learned at an early working stage of this grant that we have to ask help from other experts. 7. There was a special occasion when we performed so-called "research of the patent" from the original Hungarian and foreign languages. It was a specially exciting work, because our idea on the preparation of new drug (or drug combination) is a really original discovery. 8. How can we characterize the behaviors of participants, who were involved the innovative drug research"? We try to collect the most important characteristic steps of our "innovative drug research": - The research is very quick and timed; - The participants are intellectually at a high level; - The participants are able to pick up a significant amount of knowledge from the other experts of the participants, as well as from other experts; - The participants are able to accept the opinions of others; - They are able work quickly and use foreign language(s); - They have good personal contacts with the people working at different levels of research, national scientific societies, national offices or institutes which are responsible for making decisions; - T h e y have good contacts with foreign experts; - They have enough practice to stimulate the work of all of us; - They have a responsibility to accept the results of the work of others; - They want to receive good results together; - They are able to win acceptance for the ideas necessary to achieve our goals; - They are able to keep their perseverance in accepting the opinion and ideas of each other; - They are able to learn from each other from day to day; - They have a good dynamism in their personal contacts and in discussions; - They have to be suitable persons for doing creative and innovative research works (including the establishment of new ideas, problems orientated field, realization of the research, organization, realization of the animal observations in the human studies). 156

11. Pharmaceutical industrial research and development

Different special terminologies are widely used in the everyday industrial pharmaceutical research and development (Table 30). T a b l e 3 0 . A b b r e v i a t i o n s u s e d in t h e e v e r y d a y p h a r m a c e u t i c a l industrial r e s e a r c h and development API

A c t i v e P h a r m a c e u t i c a l Ingredient

BMR

Batch Manufacturing Record

BSE/TSE

B o v i n e S e r u m Encephalitis/Transmissible S e r u m Encephalitis

CAS

C h e m i c a l Abstracts S e r v i c e

CU

Content Unformity

FP

Finished Product

CLP

G o o d Laboratory Practice

cGMP

current G o o d Manufacturing Practice

ICH

International C o n f e r e n c e o n Harmonisation

IHS

In H o u s e S t a n d a r d

MBD

Master Batch D o c u m e n t

MQD

Master Quality D o c u m e n t

NMT

Not M o r e Than

NSAID

N o n Steroid Antiinflammatory D r u g

PH EUR

European Pharmacopoeia

PMF

P r o d u c t M a s t e r File

QA

Quality Assurance

RET

R e g i o n a l C e n t r e of S c i e n c e

RS

Relative Substances

USP

U n i t e d States P h a r m a c o p e i a

Preamble In the RET cooperation Pannonpharma's role is the industrial partnership, joining in at the start of the project and being active continuously to the end. We assume that scientific basic research ends when its results become common practice. We think 157

that the result will be a registered medicinal product for the cure of patients who expect their recovery from new medicines. Although this partnership is quite new and even seemed to be a historical step in the region, all members thought of the project as an innovative drug-research - thus, a useful and path-finder job. Pannonpharma Ltd. (Pecsvarad, Hungary) has realized its importance quite enough, taking into account especially the company's young background. Not only the business, but the challenge of the project impressed all of us, while we believed in the chance of our first regional cooperation.

1 1 . 1 . Product design and development As the references and partners' scientific results certified the pharmacological activity of capsaicin, confirmed in several promising and convincing animal tests (Ref. former sections), a detailed evaluation was decided by the team in order to start a new product development. The main aspects of the decision were clarified by pharmacologists and clinicians aiming at a clear pharmacological intention, while the details were worked out together.

11.1.1. General goals • Principle of the research and development: - QUALITY - should cover conformance of all chemical and pharmaceutical data and requirements (reference in this section). - SAFETY - should cover conformance of all non-clinical data and requirements. - EFFICACY- should cover conformance of all clinical data and requirements. • All activities should be done in conformance of QA requirements (cGMP), • Legal protection of the product should be required, • Oral and written publications should be taken into account.

11.2. Chemical-pharmaceutical aspects of the product development The selected formulation (e.g. pharmaceutical form) is summarized in Table 31. Having the form, questions have been grouped for clarification as follows:

158

11.2.1 Pharmaceutical form Details are presented in Table 31. T a b l e 3 1 . S u m m a r y of d e t a i l s of p h a r m a c e u t i c a l industrial data yes

no

comment

1 . a c o m m o n tablet n e e d e d , or retard b y a c t i o n

common

2 . t h e tablet s h o u l d b e c o a t e d or u n c o a t e d

coated

3. if c o a t i n g is n e e d e d , w h a t t y p e of c o a t i n g :

•

sugarcoated filmcoated

V

4. w h a t is targeted by c o a t i n g : aesthetic f i l m colored film

red

other f u n c t i o n a l role, s u c h as: m a s k i n g taste/odor p r o t e c t i o n against s t o m a c h d e c o m p o s i t i o n

•

i n c r e a s e stability 5. Tablet size average weight

medium appr. 3 0 0 ^ 0 0 m g

diameter

10-11 m m

shape

round, biconvex

In consultation with clinicians a tablet form has been agreed, which disintegrates in the stomach, and acts in place. It was made clear that the disintegration time should be not more than 30 minutes. On the other hand, film-coating had to be selected because of capsaicin's pungent taste. The color of the film-coating tablet could be selected practically freely, and the coating film was expected even to serve for enhancing the stability. The size of the tablet size was determined not only simply by its active content, but also taking into account combination variations of the NSAIDs' active pharmaceutical ingredients.

11.2.2 Composition and quality of starting materials Details of these questions are presented in Table 32. The quality of the starting materials was required to meet pharmacopoeial standards of the European Pharmacopoeia (2007): Fifth Edition, Council of Europe Strassbourg (PH EUR 5.7) and The United States Pharmacopeial Convention, INC. (2003): 12601 Twinbrook Parkway, Rockwille (USP 27-NF 22).

159

T a b l e 3 2 . C o m p o s i t i o n a n d q u a l i t y of starting m a t e r i a l s ( p r o d u c t s ) * yes 1

Disintegrant Lubricant Clidant 3 C o a t i n g m a t e r i a l s (mixture)

quality USP27-NF22

API

PH EUR 5

2 Excipients Binder

no

•

PH EUR 5 PH EUR 5 PH EUR 5 PH EUR 5 IHS

* see Table 30, for additional explanation

11.2.2.1. Active Pharmaceutical Ingredient (API) of the product The API of the product, capsaicin, is not a homogenous substance, thus it is better to call it capsaicinoids. By chemical structure it is often called as pseudo alkaloid or proto alkaloid too. The basic part of the 5 components' chemical structure is vanillyl-amide, and the differences of the capsaicinoid components' structure are in the substituents. {Chemical coterminus of capsaicin: 6-Nonenamide, (£> Af-[(4-Hydroxy-3-methoxy-phenyl) methyl]-8-methyl. (£)-8-Methyl- Af-vanillyl-6-nonenamide, (Ref. USP, Ref. 6.2.3).} In the formulation we used USP quality of capsaicin ("capsaicin natural", CAS Number: 404-86-4), since PH EUR does not involve capsaicin paragraph (USP 27NF 22, THE UNITED STATES PHARMACOPEIAL Convention, INC. 12601 Twinbrook Parkway, Rockwille , 2003). API supplier was audited and they released a copy of PMF for capsaicin. The pharmaceutical characteristics of the substance: Properties: - Characteristics: - pale brownish-yellow, amorphous powder - intensive and irritating odor even in very low air-volume concentration - Morphology: - particle size is not homogeneous, it is varying in a wide range between 0.05-2 mm (like a usual vacuum-dried extract) - Polymorphism: - no polymorphism of the substance is known - Solubility: - good in methanol, ethanol and acetone, practically insoluble in water Chemical properties: testing and specification is upon USP 83/2001 EC Guide (Directive of the European Parliament and of the Council on the Community code relating to medicinal products for human use) (Handbook of Pharmaceutical Excipients 5th Edition, edited by Raymond Rowe, Paul Sheskey and Paul Weller, USA, October 2005).

160

11.2.2.2. Excipients' quality of P H EUR requirements Excipients' quality was meeting PH EUR requirements, involving the BSE/TSE aspect, when relevant, as well. (Directive of the European Parliament and of the Council on the Community code relating to medicinal products for human use (2001) (83/2001 EC Guide). Excipients used in formulation - as above -were expected to be chemically - indifferent, - to serve the solid dosage form requirement (Handbook of Pharmaceutical Excipients 5th Edition (2005): Eds: Rowe, R., Sheskey, P. and Weller, P., USA, October 2005) - not to involve lactose in order of protection of lactose-sensitive patients As from view of formulation, the excipients must have proper physical-, physicalchemical characteristics for powder rheology in order to assure good homogenity, fluidity and lubrication for compression.

11.2.2.3. Coating powder mixture quality Coating powder mixture quality is by IHS, formulated for serving the above-mentioned purposes. Its red color was aiming at some association with the Hungarian paprika. Additionally, for suspension phase or solvent of capsaicin purified water was used exclusively in order to decrease the risk of organic residual.

11.2.3. Packaging Details of the packaging are shown in Table 33. T a b l e 3 3 . S u m m a r y of a s p e c t s of p a c k a g i n g yes

no

1. Primary blister

V

PVC/ALU PVC/PVdC/ALU

•

C o l d blister

•

vial 2. Secondary carton box

V

p a c k a g e insert booklet

•

3. D e s i g n design i n v o l v i n g b l i n d - c o d i n g

•

* abbreviations: PVC, Polyvinyl Chloride PVdC, Polyvinylidene Chloride ALU, Aluminium

161

A good packaging retains the products' quality, is easy to handle by patients and externally serves some aesthetic aspects. Blister packaging has been selected, because it was an individual packaging by dosage form, and the indicated packaging materials were resistant against humidity, thus supporting the stability of the tablet.

11.3. Formulation 11.3.1. Formulation steps In the first step a lab-scale batch (appr. 3 kg) was planned to be produced, in order to confirm the planned composition, then a pilot batch (100 000 tablets) was targeted. As we have worked out the process in detail, FP Specification has been set up after the complex testing of the tablets.

11.3.2. FP Specification The details of the FP are listed in Table 34. T a b l e 3 4 . F i n i s h e d p r o d u c t ( F P ) of t h e s p e c i f i c a t i o n s a r e s u m m a r i z e d in this T a b l e . * Aspect

Requirement

Description

Characteristics

R e d c o l o r e d , b i c o n v e x f i l m tablet w i t h s m o o t h surface

Dosage form

- Dimensions: - height - diameter - shape - average weight - individual weight - disintegration time - friability -CU

Composition

Practical average ± 1 0 % (mm) 10 ± 3 %

(mm)

biconvex T h e o r e t i c a l a v e r a g e ± 1 0 % (g) P r a c t i c a l a v e r a g e ± 1 0 % (g) 30 min. NMT 1 % 85-115%

- Identity: -API

positive

- binder

positive

- lubricant - glidant

positive positive

- assay: - capsaicin I m p u r i t y test

85-115%

- RS

Total R S N M T

- Microbiology

III.A. - P H

* see Table 30, for other additional explanation

162

2%

EUR

11.3.3. Samples for clinical trial After manufacturing and testing the samples taken from the pilot batch and evaluation of the BMR, Master Batch Documents and Master Quality Documents have been created for approval of QA. All the other SOPs, safety rules involving any other aspect of producing a clinical sample have been created formerly and authorized to achieve quality assurance requirements as well. Then, a new pilot batch was planned to be produced for two purposes: - t o take samples for clinical trial (11.3.3.1) - to take samples from the same batch for stability testing of the formulation (11.3.3.2)

11.3.3.1. Preparation of samples for clinical trial Samples for clinical trial were planned to be taken after the release of the batch. The number of the samples was calculated on the basis of the Clinical Protocol, their packaging and coding were compliant with GMP regulation (Eudralex Volume 4 (2007): Good Manufacturing Practice, European Comission).

11.3.3.2. Exposing, stability and packaging of the planned final product Samples for exposing and stability testing were packed in the same packaging as the planned final packaging. The product's stability is a main quality requirement. It means that the product packed in its final form retains chemical, pharmaceutical stability and pharmacological activity within a certain range which was set up in the finished product specification. (If there is any reasonable reason, some requirements of the release and expiry specification may be different within a certain range.) A complex stability project has been created for exposition and testing by ICH Guides in order to determine expiry specification and expiry date of the product as well (International Conference on Harmonization (ICH) (2003) (Ql, Q8), Brussels).

11.4. Plant batch manufacturing and process validation Scaling up for plant batch is almost the final step of a development project. It was planned and achieved by the collected information and data of the former steps. On the other hand, planning of the process involves the site manufacturing machinery and technical supplying system too. Taking into account all of the above, batch size could be determined easily. After plant batch manufacturing and quality control of the taken samples a final report evaluates the conclusions of scaling up. Process validation and cleaning validation must be achieved upon written plans, then evaluated in a written report for 163

justification of the process and its parameters. Validation batches are also involved in stability project. Cleaning validation is the last important plant activity belonging to the validation procedures, while certifying the proper method of cleaning after operation. Approved validation reports usually confirm not only the processes, but the MBDs and MQDs belonging to the as well.

1 1 . 5 . Summary of the Chemical-Pharmaceutical development The chemical-pharmaceutical part of the products' dossier is one of the main 3 detailed sections. This is for certification and documentation of all activities of the product development from pre-formulation to plant batch production on the basic principle of QUALITY. For certification of the above, we assume the next points to be relevant: - written plans are required (involving formulation and processes) for the different steps of development, - standard quality and test methods of the used starting materials are required, - standard methods are required for quality control of products at different levels of development, - development activity must follow QA rules, especially in documentation: - basic documents must have stability control, - plans must be performed, and evaluated in correct reports, - consecutive steps must follow an approved report and conclusion of the former step, - records must be controlled and approved, - the plant batch size production and the process belonging to it must be validated, - a required number of batches must be subject to stability testing, - in the final evaluation, i.e. in the Chemical and pharmaceutical expert report, expiry date, storing condition and other notes must be clarified and declared unambiguously. The planned appearance of capsaicinoids containing pills is seen in Fig. 31.

Fig. 31. T h e v i s u a l c h a r a c t e r i z a t i o n of c a p s a i c i n o i d s c o n t a i n i n g pills (see t h e p i c t u r e in c o l o r s o n p. 2 5 5 )

164

12. Clinical pharmacological studies with capsaicinoids alone and with combination of capsaicinoids with nonsteroidal anti-inflammatory drugs 12.1. Main aims of clinical pharmacology and its relation to the evidence-based medicine The evidence-based medicine (EBM) was established as the basis of the activity of medical treatment, in the everyday medical practice. The general aims of this EBM are to give exact and clinically well proved evidences for making correct diagnoses and for the treatment of patients suffering from different diseases. In the last 3-4 decades (since 1970) only the "problem orientated medicine (POM)" has been emphasized in the graduate and postgraduate medical education. This internationally well accepted trend has been applied in the medical education and in the diagnostics of diseases in patients. The final aims of our medical activity are to give scientifically well proven medical treatments (including the surgical treatment, treatment with different drugs, etc.) for the patients. The drugs have a key role in the medical treatment. It was earlier suggested that the medical treatment can be based on the: 1. classical "old" medical experiments (e.g. Chinese medicine) and 2. classical (theoretically well planned) pharmacological research. The pharmacological studies were (and are) based on the results obtained from: 1. various animal experiments; 2. results obtained on the human isolated cell cultures [especially from that time, when the Leagu(ag)e for Animal Defense (LAD) emphasized the defense of animals to be involved into the research activity]; and 3. human pharmacological studies. The animal observations played essential roles in the discoveries of different physiological and pharmacological facts and events. The researchers used mice, rats, guinea pigs, dogs, chicken, etc. in their pharmacological research. Probably mice and rats are the most accepted animals for physiological and pharmacological studies. The different human cell cultures were also involved in medical research to clear up the exact details of different physiological and pharmacological mechanisms of different compounds. There is, however, a significant problem with the exclusive use of the isolated human cell lines for the "human medical research", because these cell lines have no innervation, no correct hormonal regulation, etc. Of course, the results of these types of observations are basically necessary to establish the human pharmacotherapy. Without denying the importance of these types of research on the animal observations and on isolated human cell line, the most important physiologi165

cal and pharmacological regulatory mechanisms remain to be unclear in the human body. The results of the research on cell cultures cannot be adapted directly in the production of antibodies against the suggested mediators, in patients with inflammatory bowel diseases. The human pharmacotherapy has undergone significant changes in the last decades. Earlier the efficacy of drug therapy was based only on the observation made by the physicians. These observations can be done retrospectively and prospectively (e.g. the efficacy of antituberculotic treatment was easily acceptable in Hungary, because earlier we had no good antituberculotic drugs, such as Streptomycin; isonicotinic hydrazine, INH; rifampicin, Tubocin®, etc). The Hungarian people died before the discoveries of these drugs, however, the Hungarian patients were healed (practically in their whole number) after the introduction of these drugs in the everyday medical treatment. Consequently the "Hungarian Disease" ("Morbus Hungaricus") disappeared in the last decades. This process was a clear medical proof for the efficacy of medical treatment in patients. Penicillin was accidentally discovered by Fleming (1922). Its efficacy could be proved clearly in patients with different bacterial infections (the fever disappeared and the patients got better in a short time after the introduction of penicillin). A. Fleming received Nobel prize in 1945. When the different drugs are used in the treatment the patients with different diseases, the efficacies of different drugs can be evaluated by parametric (measureable) and nonparametric (subjective) parameters, which change from time to time. Clinical pharmacology was established in the years of 1960 over the World (at the same time in Hungary). Professor Tibor Javor (Second Department of Medicine, Medical University of Debrecen, Hungary) and his research group (the writer of this chapter personally participated in the establishment of clinical pharmacology of anticholinergic drugs) were pioneers in the establishment of human clinical pharmacology in Hungary. The results of the human clinical pharmacology offered the most principal research argument for the "classical human pharmacology", e.g. medical treatments. The main aims of the human clinical pharmacology are: 1. To give objective data on drug absorption from the gastrointestinal tract (in case of orally applicable drugs), metabolism in the human body (dominantly by the liver) and excretion of drugs (or their metabolites) by the urine or by the stool; 2. To observe the main pharmacodynamic actions of drugs; 3. To measure the drug (or metabolites) in the serum, urine, stool, after the application of the drug given in different doses; 4. To identify the oral and parenteral dose rate of the drug; 5. To identify the correlation between the pharmacodynamic actions and pharmacokinetic parameters in humans; 6. To identify the similarities (and differences) of these parameters obtained in healthy human persons (volunteers) and patients; 7. To examine these parameters before and after chronic treatment with drug(s); 8. To find a correlation between the results of the clinical pharmacological studies and those of classical human pharmacological treatment (which finally represent the everyday medical treatments). 166

Clinical pharmacology represents a classical field in the medical sciences. It involves a classical multidisciplinary knowledge, which can be given only by experts working in the different fields of medical sciences (chemists, mathematicians, physicians, drug technologists, drug industrial experts, clinical pharmacologists, etc.). Of course, the special laboratories and medical wards (clinical pharmacological units) are basically necessary for carrying out these studies. There is no doubt about that the human clinical pharmacology has developed extremely in the last decades in the World (including Hungary). The classical clinical pharmacology plays an essential role in the innovative drug research. Furthermore, the innovative drug research and clinical pharmacology can be evaluated by the internationally accepted parameters (it was true already before Hungary joined the European Union, 2004).

12.2. Our special scientific problems in the human clinical pharmacology of capsaicinoids alone and together with the application of aspirin, diclofenac and Naproxen We mentioned earlier that "capsaicin" (which name is only used in the classical physiological and pharmacological research) does not represent only one chemical structure. The capsaicin of plant origin consists of capsaicin, dihydrocapsaicin, norcapsaicin and nordihydrocapsaicin as main components. Capsaicin is transformed by the metabolism in human and animals into dihydrocapsaicin. Physiological and pharmacological studies indicated clearly that there is no difference between the chemically very similar capsaicinoids in animal experiments (Buck, Burks, 1986). We had the following main scientific problems: 1. There was as important question as to how many components can be put into the planned drug products (capsaicinoids + aspirin; capsaicinoids + diclofenac and capsaicinoids + Naproxen). The measured parameters (chemical measurements of the chemical compound in human biological samples) in the human pharmacological studies inform us on the efficacy of the drugs or drug combinations from the point of clinical pharmacology. Capsaicin and dihydrocapsaicin give the most important part of the chemical components of "classical capsaicin preparate". According to special consultations with the experts of the Hungarian Institute of Pharmacy, capsaicin and dihydrocapsaicin can be measured as main components in the human clinical pharmacological studies. Furthermore, dihydrocapsaicin is the main metabolite of capsaicin. Consequently the measurements of dihydrocapsaicin can be used as one of the main chemical components for the pharmacokinetic approach to capsaicinoids in the different human clinical pharmacological studies; 2. When we tried to establish new drug combinations (capsaicinoids + aspirin; capsaicinoids + diclofenac and capsaicinoids + Naproxen) we had to measure two chemical compounds from the two main components of capsaicinoids (capsaicin and dihydrocapsaicin) during the clinical pharmacological studies (together with aspirin, diclofenac and Naproxen ) from the human sera; 167

3. The acceptance of this standpoint resulted very important consequences for our clinical pharmacological studies: - the pharmacokinetic measurements of capsaicinoids during the pharmacokinetic studies in healthy human subjects and in patients with different disorders (absorption rate, metabolisation, excretion of capsaicinoids) can be characterized by the results of measurements of capsaicin and dihydrocapsacin, - i f we do not have to measure all capsaicinoid components (having the same physiological mechanisms) the carrying out of these studies becomes considerably cheaper; 4. We have to perform "classical pharmacodynamic" and "pharmacokinetic" studies with capsaicinoids alone in healthy human subjects, because there have been no similar observations published in the world literature up to now (either in healthy human subjects or in patients with different disorders); 5. Since we will use different combinations of capsaicinoids plus NSAIDs (aspirin, diclofenac, Naproxen), consequently we have to carry out the human Phase I study with capsaicinoids alone and together with aspirin, diclofenac and Naproxen.

12.3. Principal schedules for the human Phase l-ll studies with capsaicinoids alone and together with aspirin, diclofenac and Naproxen 12.3.1. Preparation of protocols for the human clinical pharmacological studies (including Phase I to IV) 12.3.1.1. Medical points of the preparation of the study protocols Very carefully carried out preparation of the medical protocols is the basis for prospective, randomized and multicentric studies (including one or more countries of the World). These protocols describe the medical points of clinical pharmacological studies [clear identification of the aim(s) of the study(studies) in the healthy human subjects or patients with different diseases involved in the study, their exact inclusion and exclusion criteria, schedule of the well-planned examinations, clinical controlling examinations and their time, drop-out, determination of end points of the studies]. The protocols prepared for the clinical study (studies) have to be accepted by nationally or internationally well known experts only from the medical points of views. The place and the names of person(s) responsible for performing these clinical pharmacological studies should be given. Details of the whole documentation of the clinical pharmacological studies (informed consent, health insurance, case report sheet, circumstances of the data, preparation for the chemical pharmacological studies, storage, transfer of biological samples, the registration and archivation of the obtained results and finally to write a 168

final summary of the whole clinical pharmacological study) are clearly indicated and regulated. The final medical control is given by the National Institute of Pharmacy, and this Department gives the final permission (from the medical points of views) for carrying out the studies.

12.3.2. Control of the protocols by the National or Regional Clinical Pharmacological and Ethical Committees After the acceptance of different protocols by the National Institute of Pharmacy (including the necessary preliminary decisions by the national and internationally well known experts), the protocols will be controlled by the National Clinical Pharmacological and Ethical Committee of Hungary (in case of Phase I to III, and in some extent in case of Phase IV studies) from the ethical points of views, looking for the health and juristic defense of participants involved in the different studies. These Committees include medical experts from the different fields of medical sciences, lawyers, ethical experts, and nurses. If the National Clinical Pharmacological and Ethical Committee of Hungary will accept the correct circumstances (including the medical and ethical aspects of the study planned to be carried out) of the planned clinical pharmacological study (studies), then the examinations can be commenced.

12.3.3. Pharmacokinetic and pharmacodynamic effects of capsaicinoids only 12.3.3.1. Human Phase I clinical pharmacological study The results of different animal experiments clearly demonstrated that the pharmacodynamic effects of capsaicinoids depend on the applied doses. Four different doses of capsaicinoids are able to produce four different pharmacological effects (Szolcsanyi, 1996; Mozsik et al., 2000): 1. small doses of capsaicinoids stimulate the capsaicin-sensitive afferent nerves (these actions are reversible ones); 2. small doses of capsaicinoids, but in a little higher than those mentioned above, produce the inhibition of the same capsaicin sensitive afferent nerve fibres (which is also a reversible process); 3. higher doses of capsaicinoids (than those mentioned under point 2) produce an injury of the capsaicin sensitive afferent nerves (which is probably a reversible one); 4. the extremely high doses of capsaicinoids produce an irreversible injury of these nerves. These pharmacological studies were carried out in animal experiments. In healthy human subjects we applied the very small doses of capsaicinoids (Mozsik et al., 169

2005a), which stimulate the capsaicin-sensitive afferent nerves. The capsaicinoidsinduced side effects (diarrhoea, development of malignant diseases) were observed and published in the world literature, in connection with the administration of extremely high doses of capsaicinoids. We have to emphasize that the capsaicinoids were applied in different capsaicin extracts in these studies (and not in a chemically pure form). The pharmacokinetic and pharmacodynamic Phase I observations were planned to be carried out only with small doses of capsaicinoids (up to 1.0 to 1.2 mg/person/day). The primary aims of these studies are to identify the tolerability, safety and pharmacodynamic aspects of capsaicinoids in healthy human subjects and in patients with different diseases. The capsaicinoids are orally given in doses 400 to 800 pg/person/day.

12.3.3.2. Human clinical pharmacological Phase I study with capsaicinoids plus nonsteroidal antiinflammatory drugs in healthy human subjects 12.3.3.2.1. Human clinical pharmacological phase I study with capsaicinoids plus aspirin

The dose of aspirin is 100 mg/day/person, in case of aspirin resistance 300 mg/day/person, respectively, estimated by many international studies, and these doses have been accepted recently by the European and American Societies of Cardiology (2006) in patients with myocardial infarction. Consequently the doses of aspirin are internationally well established. We planned to carry out human Phase I studies with three arms: 1. E D dose of capsaicinoids (400 pg in oral dose); 2. aspirin (100 mg and 300 mg), given orally; 3. aspirin (in doses mentioned above) plus capsaicinoids (ED ), given orally. The observations will be carried out in healthy human subjects (volunteers). Every subject will undergo the different observations, however, the actually applied dose will be selected according to randomization. 50

50

12.3.3.2.2. Human clinical pharmacological Phase I study with capsaicinoids plus diclofenac

We planned also a human clinical pharmacological phase I study with capsaicinoids plus diclofenac with three arms: 1. capsaicinoids (in dose of E D ) , (400 pg oral dose); 2. diclofenac (25, 50 and 75 mg), given orally; 3. diclofenac (25, 50 and 75 mg) plus capsaicinoids (in dose of ED ), given orally. The clinical pharmacological observations will be done in healthy persons (volunteers). 500

50

170

12.3.3.2.3. Human clinical pharmacological Phase I study with capsaicinoids plus Naproxen

Healthy human subjects (volunteers) are included in these studies. The persons receive all the treatments mentioned below: 1. capsaicinoids in dose of E D (400 pg oral dose); 2. Naproxen (250, 375 and 500 mg), given orally alone; 3. capsaicinoids (in dose of ED ) plus Naproxen (in the above-mentioned doses) given together orally. 50

50

12.3.3.3. Human clinical pharmacological Phase II studies in patients 12.3.3.3.1. Human clinical pharmacological Phase II study in patients with thromboembolic diseases (myocardial infarction, stroke, thromboembolic events)

The patients selected for the study were treated with aspirin (in doses of 100 or 300 mg given orally) without and with co-administration of capsaicinoids (orally given ED ). The general laboratory parameters, ECC, platelet aggregation were registered dayly, together with the subjective complaints of patients. Of course, the pharmacodynamic examinations were also carried out in the patients (treated with capsaicinoids alone, aspirin alone and capsaicinoids plus aspirin). 50

12.3.3.3.2. Human clinical pharmacological Phase II studies in patients with different degenerative locomotor diseases

Similar types of the human Phase II examinations are carried out in these patients as those mentioned in Section 12.3.3.2.2. These laboratory examinations (plus others related to the degenerative locomotor system) and semi-quantitative parameters (complaints of patients) were registered according to the protocols. The patients were treated with diclofenac (25, 50 and 75 mg given orally), capsaicinoids (given orally in doses of E D ) and diclofenac (in the above-mentioned doses) plus capsaicinoids (in dose of ED ) given orally. Another group of patients received Naproxen (in doses of 250, 375 and 500 mg orally) instead of diclofenac. The further details of this study were the same as in the case of diclofenac. The pharmacodynamic examinations were carried out during the clinical observation. 50

50

171

Note We received absolutely new information from the chronic toxicological studies in Beagle dogs (2008). These animals were treated with different doses (0.1, 0.3 and 0.9 mg/kg bw/day orally given) of capsaicin(s) for one month. No toxicological side effects were observed in these dogs during the whole treatment periods. We noticed surprisingly that capsaicin (capsaicin and dihydrocapsaicin) could not be detected in the sera of Beagle dogs, either by High Pressure Chromatography (HPLC)* or by Liquid Chromatography-Mass Spectrometry (LC-MS)** at any time after the oral application of capsaicin (in doses of 0.1; 0.3; 0.9 mg/kg bw/day). The limit of detection by HPLC is 20 nanogram/ml serum and its value is 26 fg for capsaicin and 20 fg for dihydrocapsaicin by LC-MS. These results suggest that we will not be able to produce a classical pharmacokinetic study for capsaicin and dihydrocapsaicin in healthy human subjects and in patients with different diseases because the dose range of capsaicin is 0.4-1.2 (400-1200 microgram)/ adult persons/ day.

* Mozsik, Gy., Past, T , Perjesi, P., Szolcsanyi, J.: Determination of capsaicin and dihydrocapsaicin content of dog's plasma by HPLC-FLD method. In: Mozsik, Gy., Past, T., Perjesi, P., Szolcsanyi, J.: Original Reports on Toxicology of Capsaicin VII. 8-Day Oral Toxicity Study of Test Item Capsaicin Natural USP 27 in Beagle Dogs (Final Report). LAB International Research Centre Hungary Ltd. Veszprem by the date of final report 13 June 2008. Study Code: 07/496-100K pp. 1-35 in text and 190 pages in Appendices. (Appendix 2.11) pp. 1-37 (2008) ** Boros, B., Dornyei, A., Felinger, A.: Determination of capsaicin and dihydrocapsaicin in dog plasma by Liquid Chromatography-Mass Spectrometry (Analytical method report) PTE TTK Analitikai Kemiai Tanszek, Pecs, Hungary (2008) 172

Acknowledgements

The study was supported by the grant of the National Office for Research and Technology, "Pazmany Peter program" (RET-II 08/2005). The authors express their sincere thanks to Mrs. Judit Szabo for her excellent help in the preparation of this monograph. They are also very grateful to Akademiai Kiado (Budapest, Hungary), especially to Mrs. Judit Kerpel-Fronius for her precious corrections during the copy editing and proofreading process.

173

References

Abdel-Salam, O.M.E., Szolcsanyi, J., Bartho, L., Mozsik, Gy. (1994): Sensory nerve-mediated mechanisms, gastric mucosal damage and its protection: A critical overview. Gastroprotection 2: 4-12 Abdel-Salam, O.M.E., Bodis, B., Karadi, O., Nagy, L., Szolcsanyi, J., Mozsik, Gy. (1995a): Stimulation of capsaicin-sensitive sensory peripheral nerves with topically applied Resiniferatoxin decreases salicylate-induced gastric H back-diffusion in the rat. Inflammopharmacology 3: 121-133 Abdel-Salam, O.M.E, Bodis, B., Karadi, O., Szolcsanyi, J., Mozsik, Gy. (1995b): Modification of aspirin and ethanol-induced mucosal damage in rats by intragastric application of Resiniferatoxin. Inflammopharmacology 3: 135-147 Abdel-Salam, O.M.E., Bodis, B., Karadi, O., Szolcsanyi, J., Mozsik, Gy. (1995c): Nature of gastric H back-diffusion approached by cimetidine, vagotomy, RTX and sucralfate. Ceska Slovenska Gastroenterologie 49: 175-185 Abdel-Salam, O.M.E., Mozsik, Gy., Szolcsanyi, J. (1995d): The effect of intragastrically administered capsaicin analogue in HCI and non-HCl dependent models of gastric mucosal injury. Z. Gastroenterol. 33: 80 Abdel-Salam, O.M.E., Mozsik, Gy., Szolcsanyi, J. (1995e): Capsaicin and its analogue Resiniferatoxin inhibit gastric acid secretion in pylorus-ligated rats. Pharmacol. Res. 31: 341-345 Abdel-Salam, O.M.E., Mozsik, Gy., Szolcsanyi, J. (1995f): Studies on the effect of intragastric capsaicin on gastric ulcer and on the prostacyclin-induced cytoprotection in rats. Pharmacol. Res. 32: 209-215 Abdel-Salam, O.M.E., Mozsik, Gy., Szolcsanyi, J. (1995g): Effect of Resiniferatoxin on stimulated gastric acid secretory responses in the rat. J. Physiol., Paris 88: 353-358 Abdel-Salam, O.M.E., Szolcsanyi, J., Mozsik, Gy. (1996a): Differences in action of topical and system cystamine on gastric blood flow, gastric acid secretion and gastric ulceration in the rat. J. Physiol., Paris 90: 63-73 Abdel-Salam, O.M.E., Szolcsanyi, J., Porszasz, R., Mozsik, Gy. (1996b): Effect of capsaicin and resiniferatoxin on gastrointestinal blood flow in rats. Eur. J. Pharmacol. 305: 127-136 Abdel-Salam, O.M.E., Mozsik, Gy., Szolcsanyi, J. (1997a): The effect of capsaicin and Resiniferatoxin on the indomethacin-induced gastric mucosal damage in rats. In: Mozsik, Gy., Nagy, L., Kiraly, A. (eds): Cell Injury and Protection in the +

+

175

Gastrointestinal Tract. From Basic Science to Clinical Perspectives. Kluwer Academic Publisher, Dordrecht, pp. 95-105 Abdel-Salam, O.M.E., Mozsik, Gy., Szolcsanyi, J. (1997b): The role of afferent sensory nerve in gastric mucosal protection. In: Mozsik, Gy., Nagy, L., Kiraly, A. (eds): Twenty Five Years of Peptic Ulcer Research in Hungary. From Basic Science to Clinical Practice 1971-1995. Akademiai Kiado, Budapest, pp. 295-308 Abdel-Salam, O.M.E., Szolcsanyi, J., Mozsik, Gy. (1997c): The effect of Resiniferatoxin on experimental gastric ulcer in rats. In: Gaginella, T., Mozsik, Gy., Rainsford K.D. (eds): Biochemical Pharmacology as Approach to Gastrointestinal Disease. From Basic Science to Clinical Perspectives. Kluwer Academic Publisher, Dordrecht, pp. 269-285 Abdel-Salam, O.M.E., Szolcsanyi, J., Mozsik, Gy. (1997d): The indomethacininduced gastric mucosal damage in rats. Effect of gastric acid, acid inhibition, capsaicin-type agents and prostacyclin. J. Physiol., Paris 97: 7-19 Abdel-Salam, O.M.E., Sleem A.A., Hassan N.B., Sharaf H.A., Mozsik Gy. (2006). Capsaicin ameliorates hepatic injury caused by carbon tetrachloride in the rat. J. Pharmacol. Toxicol. 1:147-156 Acosta, Jose de (1985): Historia naturaly moral de las Indias. Mexico. Agrawal, R.C., Bhide, S.V. (1987): Biological studies on carcinogenicity of chillies in balb/c mice. Indian J. Med. Res. 86: 391-396 Agrawal, R.C., Bhide, S.V. (1988): Histopathological studies on toxicity of Chilli (Capsaicin) in Syrian golden hamsters. Indian J. Exp. Biol. 26: 377-382 Agrawal, R.C., Wiessler, M., Heckers, E., Bhide, S.V. (1986): Tumour-promoting effect of chilli extract in balb/c mice. Int. J. Cancer 38: 689-695 Akagi, A., Sano, N., Uehara, H., Minami, T , Otsuka, H., Izumi, K. (1998): Noncarcinogenicity of capsaicinoids in B6C3F1 mice. Food Chem. Toxicol. 36: 1065-1071 Alfoldi, P., Obal, F. Jr., Toth, E., Hideg, J. (1986): Capsaicin pretreatment reduces the gastric acid secretion elicited by histamine but does not affect the responses to carbachol and pentagastrin. Eur. J. Pharmacol. 123: 321-632 Alfoldi, P., Toth, E., Obal, F., Hideg, J. (1987): Capsaicin treatment reduces histamine-induced gastric acid secretion in the rat. Acta Physiol. Hung. 69: 509-512 Ames, B.N., McCann, J., Yamasaki, E. (1975): Methods for detecting carcinogens and mutagens with the Salmonella/microsoma test assay of 300 chemicals. Mutat. Res. 31: 347-364 Anonymus (1986): Metabolism and toxicity of capsaicin. Nutr. Rev. 44: 20-22 Anu, A., Peter K.V. (2000): The chemistry of Paprika. Indian Species 37: 15-18 Ascherio, A., Stampfer, M.J., Colditz, G.A. (1992): Correlations of vitamin A and E intakes with the plasma concentration of carotenoids and tocopherols among American men and women. J. Nutr. 122: 1792-1801. ASTA (1968): Method No 20, Extractable color in paprika, In: Official Anal. Meth. Englewood Cliffs, N.J. Atkinson, D.E. (1968): The energy charge of adenylate pool as a regulatory parameter. Interaction with feedback modifiers. Biochemistry 7: 4030-4034 Ato, A., Yamamoto, M. (1996): Acute oral toxicity of capsaicin in mice and rats. J.Toxicol. Sci. 21: 195-200 176

Augustin, B. (1907): Historisch-kritische und anatomisch-entwicklungsgeschichtliche Untersuchungen iiber den Paprika. Rosner, Nemetbogsan. Azizan, A., Blevins, R.D. (1995): Mutagenecity and antimutagenecity testings of six chemicals associated with the pungent properties of specific spices as revelated by the Ames salmonella microsomal assay. Arch. Environ. Contam. Toxicol. 28: 248-258 Babbs C.F. (1990): Free radicals and the etiology of colon cancer. Free Rad. Biol. Med. 8: 191-200 Bajaj, K.L. (1980): Colorimetric determination of capsaicin in Capsicum fruits. J. Assoc. Off. Anal. Chem. 63: 1314-1320 Balint, S. (1962): A Szegedi Paprika (The Paprika of Szeged). Akademiai Kiado, Budapest. Baranyai, M., Szabolcs, J. (1976): Determination by reduction of the red and total pigments content in paprika products. Acta Aliment. 5: 87-105. Baranyai, M., Matus, Z., Szabolcs, J. (1982): Determination by HPLC of carotenoids in paprika products. Acta Aliment. 11: 309-323. Bargen, J.A., Parker, N.W. (1936): Extensive arterial and venous thrombosis complicating chronic ulcerative colitis. Arch. Intern. Med. 58: 17-31 Bartley, G.E., Scolnik, P.A., Beyer, P. (1999): Two Arabidopsis thaliana carotene desaturases, phytoene desaturase and zeta-carotene desaturase, expressed in Escherichia coli, catalyze a poly-cis pathway to yield pro-lycopene. Eur. J. Biochem. 259: 396-403 Basu, S.K., De, A.K. (2003): Capsicum: Historical and Botanical Perspectives. Taylor and Francis Ltd., London, pp. 1-15 Benbrook, D.M., Lu, S., Flanagan, Wilson, G. (1997): Biological assay for activity and molecular mechanisms of retinoids in cervical tumor cells. Gynecol. Oncol. 66: 114-121 Benedek, L. (1958): Determination of pigment material contents in ground paprika. Z. Lebensm. Unters.-Forsch. 107: 228-232 Benedek, L. (1959): Determination of capsaicin in red peppers (paprika). Kfserletiigyi Kozl. C. Kertesz. 52: 33 Berkesy, L. (1934): Effect of paprika on gastric secretion, (in Hungarian). Orv. Hetil. 78: 397-399 Bertina, R.M., Koeleman, B.P., Koster, T, Rosendaal, F.R., Dirven, R.J., de Ronde H., van der Velden P.A., Reitsma, PH. (1994): Mutation in blood coagulation factor V associated with resistance to activated protein C resistance resistance. Nature 369: 64-67 Bertram, J.S., Pung, A., Churley, M., Buring, S. (1991): Diverse carotenoids protect against chemically induced neoplastic transformation. Carcinogenesis 12: 671-678 Best, W., Bectel, J.W., Singleton, J.W., Kern, F. (1976): Development of a Crohn's disease activity index. Gastroenterology 70: 439^144. Bevan, S., Szolcsanyi, J. (1990): Sensory neuron-specific actions of capsaicin: mechanisms and application. Trends Pharmacol. Sci. 11: 330-333 Bevan, S., Yeats, J. (1990): Protons activate a cation conductance in a subpopulation of rat dorsal root ganglion neurons. J. Physiol. (London) 433: 145-161 177

Bieri, J.G., Brown, E.D., Smith, Jr. J.C. (1985): Determination of individual carotenoids in human plasma by high performance liquid chromatography. J. Liquid Chromatogr. 8: 473-484 Blaner, W.S. (1993): Biochemistry and pharmacology of retinoids. In: Hong, W.K., Lotan, R. (eds): Retinoids in Oncology. Marcel Dekker, New York, pp. 1-41 Blaner, W.S., Olson, J.A. (1994): Retinol and retinoic acid metabolism. In: Sporn, M.B, Roberts, A.B., Goodman, D.S. (eds): The Retinoids: Biology, Chemistry and Medicine. 2 ed. Raven Press, New York, pp. 229-255 Bley, K.R. (2004): Recent developments in transient receptor potential vanilloid 1 agonist-based therapies. Expert Opin. Invest. Drugs 13: 1445-1456 Block, G. (1991): Dietary guidelines and the results of food consuption surveys. Am. J. Clin. Nutr. 53: 356-357 Block, G. (1992): The data support a role for antioxidants in reducing cancer risk. Nutr. Rev. 50: 207-213 Block, G. (1996): Dietary giudelines and the results of food consumptions surveys. Am. J. Clin. Nutr. 53: 356-357 Block, G., Patterson, B., Subar, A. (1992): Fruit, vegetable and cancer prevention: a review of epidemiological evidence. Nutr. Cancer 18: 1-29 Boersch, A., Calligham, B.A., Lembeck, F , Sharman, D.F. (1991): Enzymatic oxidation of capsaicin. Biochem. Pharmacol. 41: 1863-1969 Bordas, I. (2006): Veszelyes anyagok keszftmenyek peszticidek (Dangerous compounds, products pesticides). Orszagos Kemiai Biztonsagi Intezet (National Chemical Safety Institute), Budapest Bosland, P.W. (1994): Chiles: history, cultivation, and uses. In: Charalambous, G. (ed.): Spices, Herbs, and Edible Fungi. Elsevier Publ., New York, pp. 347-366 Bosland, P.W., Votava, E.J. (2000): Peppers: Vegetable and Spice Capsicums. CABI Publishing, Oxon New York, S 3 Bousvaros, A., Zurakowski, D. (1998): Vitamins A and E serum levels in children and young adults with inflammatory bowel disease: effect of disease activity. J. Pediatr. Gastroenterol. Nutr. 22: 29-35 Bouvier, F , Suire, C , Mutterer, J., Camara, B. (2003): Oxidative remodeling of chromoplast carotenoids: Identification of the carotenoid dioxygenase CsCCD and CsZCD genes involved in Crocus secondary metabolite biogenesis. Plant Cell 15: 47-62 Brash, A.R., Baertschi, S.W., Ingram, C D . , Harris, T.M. (1988): Isolation and characterization of natural allene oxides: unstable intermediates in the metabolism of lipid hydroperoxides. Proc. Natl. Acad. Sci. USA 85: 3382-3386 Brawer, O., Schoen, J. (1962): Determination of flavour constituents of paprika. Angew. Botan. 36: 25-30 British Standards Institution (1979): Methods for testing spices and condiments: Determination of Scoville index of chillies. BS 4548 (Part 7), BSI, London Britton, G. (1991): The biosynthesis of carotenoids: a progress report. Pure Appl. Chem. 63: 101-108. Bruggeman, T.M., Wood, J.G., Davenport, H.W. (1979): Local control of blood flow in the dog's stomach: vasodilatation caused by acid back-diffusion following topical application of salicylic acid. Gastroenterology 77: 736-744 n d

178

Buchanan, R.L., Goldstein, S., Budroe, J.D. (1981): Examination of chili pepper and nutmeg oleoresins using the salmonella/mammalian microsome mutagenicity assay. J. Food Sci. 47: 330-333 Buck, S.H., Burks, T.F. (1986): The neuropharmacology of capsaicin: review of some recent observation. Pharmacol. Rev. 38: 179-226 Buck, S.H., Miller, M.S., Burks, T.F. (1982): Depletion of primary afferent substance P by capsaicin and dihydrocapsaicin without altered thermal sensitivity. Brain Res. 233:216-220 Buiatti, E., Palli, D., Dacarli, A., Amaddori, D., Avelini, C , Biachi, S., Biserni, S., Cipriani, E., Cocco, P., Giacoso, A., Marubini, E., Puntoni, J., Blot, Jr., W. (1989): A case-control study of gastric cancer and diet in Italy. Int. J. Cancer 44: 611-616 Bussey, H.J.R., DeCosse, J.J., Deschner, E.E., Eyes, A.A., Lesser, M.L., Morson, B.C., Ritchie, S.M., Thomson, J.P.S., Wadsworth, J. (1982): A randomized trial of ascorbic acid in polyposis coli. Cancer 50: 1434-1439 Buttery, R.G., Seifert, R.M., Guadagni, D.G., Ling, L.C. (1969a): Characterisation of some volatile constituents of bell peppers. J. Agric. Food Chem. 17: 1322-1327 Buttery, R.G., Seifert, R.M., Lundin, R.E., Guadagni, D.G., Ling, L.C. (1969b): Characterisation of an important aroma component of bell peppers. Chem. Ind. 490 Cabanac, M., Cormareche-Leyder, M., Poirior, L.J. (1976): The effect of capsaicin on the temperature regulation of the rat. Pflugers Arch. ges. Physiol. 366: 217-221 Cahill, R.J., O'Sullivan, K.R., Mathias, P.M., Beattie, S., Hamilton, H., O'Morain, C. (1991): Cell kinetic effects of vitamin E and selenium supplementation in patients with adenomatous polyps. Eur. J. Cancer Prev. 1: 27 Cahill, R.J., O'Sullivan, K.R., Mathias, P.M., Beattie, S., Hamilton, H., O'Morain, C. (1993): Effects of vitamin antioxidant supplementation on cell kinetics of patients with adenomatous polyps. Gut 34: 963-967 Camara, B. (1980): Carotenoid biosynthesis: in vitro conversion of violaxanthin to capsorubin by a chromoplast enriched fraction of Capsicum fruits. Biochem. Biophys. Res. Commun. 93: 113-117 Camara, B., Monegar, R. (1981): Carotenoid biosynthesis: in vitro conversion of antheraxanthin to Capsicum fruits, Biochem. Biophys. Res. Commun. 99: 1117— 1122 Capristo, E., Mingrone, G., Addolorato, G., Greco, A.V., Gasbarrini, G. (1998): Metabolic features of inflammatory bowel disease in a remission phase of the disease activity. J. Intern. Med. 243: 339-347 Capsicum Oleoresin: Capsicum Oleoresin. The USP30-NF25 2006 Page 1611 Castle, N.A. (1992): Differential inhibition of potassium currents in rat ventricular myocytes by capsaicin. Cardiovasc. Res. 26: 1137-1144 Caterina, M.J., Schumacker, M.A., Tomigana, M., Rosen, T.A., Levine, J.D., Julius, D. (1997): Capsaicin receptor: a heat activated ion channel in the pain pathway. Nature 389: 816-624 Chahl, L.A., Szolcsanyi, J., Lembeck, F. (eds) (1984): Antidromic Vasodilatation and Neurogenic Inflammation. Akademiai Kiado, Budapest Chanda, S., Erexson, G , Riach, C , Innnes, D., Stevenson, F , Murli, H., Bley, K. (2004): Genotoxicity studies with pure trans-capsaicin. Mutat. Res. 557: 85-97 179

Chanda, S., Mould, A., Esmail, A., Bley, K. (2005): Toxicity studies with pure trans-capsaicin delivered to dogs via intravenous administration. Reg. Toxicol. Pharmacol. 43: 66-75 Chard, P.S., Bleakman, D., Saridge, J.R. (1995): Capsaicin-induced neurotoxicity in cultured dorsal root ganglion neurons: involvement of calcium-activated proteases. Neuroscience 65: 1099-1108 Chem. Abstr. (1958): 52: 20903 Chem. Abstr. (1963a): 58: 11166 Chem. Abstr. (1963b) 59: 8055 Cheng, Y.P., Wang, Y.H. Cheng, L.P., He, R.R. (2003): Electrophysiologic effects of capsaicin on pacemaker cells in sinoatrial nodes of rabbits. Acta Pharmacol. Sin. 24: 826-830 Chitwood, R.L., Pangborn, R.M., Jennings, W. (1983): GC-MS and sensory analysis of volatiles from three cultivars of Capsicum. Food Chem. 11: 201-216 Chudapongse, P., Janthasoot, W. (1981): Mechanism of the inhibitory action on capsaicin on energy metabolism by rat liver mitochondria. Biochem. Pharmacol. 30: 735-740 Clive, D., Flamm, W.G., Machesko, M.R., Bernheim, N.J. (1972): A mutational assay system using the thymidine kinase locus in mouse lymphoma cells. Mutat. Res. 16:77-87 Clive, D., Johnson, K.O., Spector, J.F., Batson, A.G., Brown, M.M. (1979): Validation and characterization of the L5178Y/TK+/- mouse lymphoma and mutagens assay system. Mutat. Res. 59: 61-108 Collins, M., Bosland, P. (1994): Capsicum eggplant. Newsletter 13: 48-51 Constant, H.L., Cordell, G.A., West, P.D., Johnson, J.H. (1995): Separation and quantification of capsaicinoids using complexation chromatography. J. Nat. Prod. 58:1925-1928 Cordell, G.A., Araujo, O.D. (1993): Capsaicin: identification, nomenclature, and pharmacology. Ann. Pharmacother. 27: 330-336 Corpet, D.E., Stamp, D., Messer, A. (1990): Promotion of colonic microadenoma growth in mice and rats fed cooked sugar or cooked casein and fat. Cancer Res. 50: 6955-6958 Council of Europe (2001): Committee of experts on flavouring substances. Datasheet on capsaicin Couzin, J. (2004a): Nail-biting time for trials of COX-2 drug. Science 306: 1673-1675 Couzin, J. (2004b): Withdrawal of vioxx casts shadow over COX-1 inhibitors. Science 306: 3844-3850 CREDOC/OCA (Observatoire des Consommations Alimentaires) (1998): Estimation des niveaux d'ingestion de substances aromatisantes Safrole, Estragole, Coumarine et Capsaicine. Note technique No. 98.25 Csaky, T.Z. (1969): Introduction to General Pharmacology. Appleton CenturyCraft Educational Division, Meredith Corporation. New York, pp. 17-34 Csapo, J. (1775): Uj fuves es viragos kert. (New grassy and flower garden) (in Hungarian) Landerer, Pozsony

180

Cunningham, EX., Jr, Pogson, B., Sun, Z., McDonald, K.A., DellaPenna, D., Gantt, E. (1996): Functional analysis of beta and epsilon lycopene cyclase enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid formation. Plant Cell 8: 1613-1626 Dahlback, B. (1995): New molecular insights into genetics of thrombophilia. Resistance to activated protein C caused by Arg 506 to Glu mutation of factor V as a pathogenic risk factor for venous thrombosis. Thromb. Haemost. 74: 139-148 Dahlback, B., Carlsson, M., Svensson, P.J. (1993): Familiar thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C. Proc. Nat. Acad. Sci. USA 90: 1004-1008 Damhoeri, A., Hosono, A., Itoh, A., Matsuyama, A. (1985): In vitro mutagenicity tests on capsicum pepper, shallot and nutmeg oleoresins. Agric. Biol. Chem., 49: 1519-1520 Daoud, A.H., Griffin, A.C. (1980): Effect of retinoic acid, butylated-hydroxytoluene, selenium and sorbic acid on azo dye hepatocarcinogenesis. Cancer Lett. 9: 299-304 Davenport, H.V. (1970): Backdiffusion of acid through the gastric mucosa and its physiological consequences. Dig. Dis. Sci. 21: 141-143 Davenport, H.V. (1973): Backdiffusion of acid through the gastric mucosa and its physiological consequences II. Gastroenterology 65: 619-624 Davies, B.H. (1980): Carotenoid biosynthesis, In: Czygan, F.C. (ed): Pigments in Plants. 2nd ed. Gustav Fischer, Stuttgart, pp. 31-56 De, A.K. (1992): Capsicum: the wonder drug from chilli. Everyman's Science 27: 47 De, A.K. (1993): The wonders of chilli. Indian Spices 29: 15 De, A.K. (1994): Chilli. Book for All, Delhi. De, A.K. (ed.) (2000): Recent Trends in Spices and Medicinal Plant Research. Associated Publishing Company, Delhi. De, A.K., Ghosh, J. J. (1990): Inflammatory responses induced by substance P in rat paw. Indian J. Exp. Biol. 28: 946-948 De Cosse, J.J., Adams, M., Kuzma, J.F., Logerto, P., Condon, R.E. (1975): Effect of ascorbic acid on rectal polyps in patient with familial polyposis. Surgery 78: 608-612 De Cosse, J.J., Miller, H.H., Lesser, M.L. (1989): Effect of wheat fiber and vitamins C and E on rectal polyps in patients with familial adenomatous polyposis. J. Natl. Cancer Inst. 81: 1290-1297 De Lille J., Ramirez, E. (1935): Pharmacodynamic action of the active principle of chilli (Capsicum annuum). Chem. 4836 (abstract) Debreceni, A., Juricskay, I., Figler, M., Abdel-Salam, O.M.E., Szolcsanyi, J., Mozsik, Gy. (1999): A direct stimulatory effect of small dose of capsaicin on gastric emptying rate in healthy human subjects measured by C labeled octanoid acid breath test. J. Physiol., Paris 93: 455^*60 Deli, J., Molnar, P. (2002): Paprika carotenoids: analysis, isolation, structure elucidation, Curr. Org. Chem. 6: 1197-1219 Deli, J., Osz, E. (2004): Carotenoid 5,6-, 5,8- and 3,6-epoxides, ARKIVOK (vii) 150-168 Derera, N.F. (2000): Recent developments in condiment paprika research. Australian Agri-Food, Research Forum, Melbourne (17th) 181 13

Desai, H.G., Venugopalam, K., Anita, F.P. (1973): Effect of red chili powder on NDA content of gastric aspirates. Gut 14: 974-976 Diaz Barriga Arceo, S., Madrigal-Bujaidar, E., Calderon Montellano, E., Ramirez Herrera, L., Diaz Garcia, B.D. (1995): Genotoxic effects produced by capsaicin in mouse during subchronic treatment. Mutat. Res. 345: 105-109 Di Cecco, J.J. (1976): Gas-liquid chromatographic determination of capsaicin, J. Assoc. Off. Anal. Chem. 59: 1-9 Directive of the European Parliament and of the Council on the Community code relating to medicinal products for human use (2001). (83/2001 EC Guide) Doll, R. (1992): The lessons of life: Keynote Address to the Nutrition and Cancer Conference. Cancer Res. 52: 2024-2029 Domenichi, P. (1983): The correct way to spell chile. Congressional Record 129 Domotor, A., Peidl, Zs., Vincze, A., Hunyady, B., Szolcsanyi, J., Kereskay, L., Szekeres, Gy., Mozsik, Gy. (2005): Immunohistocemical distribution of vanilloid receptor (TRPV1), calcitonin-gene related peptide (CGRP) and substance-P (SP) in gastrointestinal mucosa of patients with different gastrointestinal disorders. Inflammopharmacology 13: 161-177 Domotor, A., Szolcsanyi, J., Mozsik Gy. (2006): Capsaicin and glucose absorption and utilization in healthy human subjects. Eur. J. Pharmacol. 534: 208-283 Domotor, A., Kereskay, L., Szekeres, Gy., Hunyady, B., Szolcsanyi, J., Mozsik, Gy. (2007): Participation of capsaicin-sensitive afferent nerves in the gastric mucosa of patients with Helicobacter pylori-positive or -negative chronic gastritis. Dig. Dis. Sci. 52: 411-417 Donnerer, J., Lembeck, F. (1983): Capsaicin-induced reflex fall in rat blood pressure is mediated by afferent substances P-containing neurons via a reflex centre in the brain stem. Naunyn-Schmiedeberg's Arch. Pharmacol. 324: 293-295 Donnerer, J., Amann, R., Schuligoi, R., Lembeck, F. (1990): Absorption and metabolism of capsaicinoids following intragastric administration in rats. NaunynSchmiedeberg's Arch. Pharmacol. 342: 357-361 Dugani, A.M., Galvin, G.B. (1986): Capsaicin effects on stress pathology and gastric acid secretion in rats. Life Sci. 39: 1531-1538 EC (1999): Commission Decision 1999/217/EC adopting a register of flavouring substances used in or on foodstuffs drawn up in application of Regulation (EC) N° 2232/96 of the European Parliament and of the Council of 28 October 1996. Official Journal of European Communities 27. 3. 1999, L 84/1-137 EC (2002): Commission Decision 2002/113/EC amending Decision 1999/217/EC as regards the register of flavouring substances used in or on foodstuffs. Official Journal of European Communities 20. 2. 2002, L 49/1. Eisvale, R.J. (1981): Oleoresins Handbook. 3rd ed. Dodge and Elcott, Inc., New York, NY, USA Endoh, K , Leung, F.W. (1990): Topical capsaicin protects the distal but not the proximal colon against acetic acid injury. Gastroenterology 98: A 446 Espinosa-Aquire, J.J., Reyes, R.E., Rubio, J., Ostrosky-Wegman, P., Martinez, G. (1993): Mutagenic activity of urban air samples and its modulation by chilli extracts. Mutat. Res. 303: 55-61

182

Esplugues, J.V., Whittle, B.J.R. (1990): Morphine potentiation of ethanol-induced gastric mucosal damage in the rat. Role of local sensory afferent neurons. Gastroenterology 98: 82-89 Esplugues, J.V., Whittle, B.J.R., Moncanda, S. (1989): Local opioid-sensitive afferent sensory neurons in the modulation of gastric damage induced by PAF. Br. J. Pharmacol. 97: 579-585 Esplugues, J. V., Ramos, E.G, Gil, L., Esplugues, J. (1990): Influence of capsaicinsensitive afferent neurons on the acid secretory responses of the rat stomach in vivo. Br. J. Pharmacol. 100: 491^196 Esplugues, J.V., Whittle, B.J.R., Moncanda, S. (1992): Modulation by opioids and by afferent sensory neurons of prostanoids protection of the rat gastric mucosa. Br. J. Pharmacol. 106: 846-852 Essential Oil Association (1975): Specification of oleoresin paprika - EOA No. 239, New York Essential Oil Association (1975): Oleoresin Capsicum - EOA No. 244, New York Essential Oil Association (1975): Oleoresin red pepper - EOA No. 245, New York Eudralex Volume 4 (2007): Good Manufacturing Practice, European Commission European Pharmacopoeia (2007): Fifth Edition, Council of Europe, Strassbourg (PH EUR 5.7) Evangelista, S., Meli, A. (1989): Influence of capsaicin-sensory fibres on experimentally-induced colitis in rats. J. Pharm. Pharmacol. 41: 574-576 Evangelista, S., Santicioli, P., Maggi, C.A., Meli, A. (1989): Increase in gastric acid secretion induced by 2-deoxy-D-glucose is impaired in capsaicin pretreated rats. Br. J. Pharmacol. 98: 35-37 Evans, H.J. (1962a): Chromosomal aberrations produced by ionizing radiation. Int. Rev. Cytol. 13:221-231 Evans, H.J. (1962b): Cytological methods for detecting chemical mutagens. In: Hollander A. (ed.): Chemical Mutagens, Principles and Methods for their Detection. Vol. 4. Plenum Press., New York and London, pp. 1-29 Evans, W.E. (1996) Treatise and Evans' Pharmacognosy. 14th ed. Harcourt Brace & Co. Asia, Ltd., Singapore Expert Consensus Document on the Use of Antiplatelet Agents. ESC Eur. J. 2004 Farmer, R.G., Michener, W.M. (1986): Association of inflammatory bowel disease in families. Front. Gastrointest. Res. 11: 17-26 Felt, L., Pacakova, V, Stulik, K., Volka, K. (2005): Reliability of carotenoid analyses: A review. Curr. Anal. Chem. 1: 93-102 Fernandez-Banares, F , Abad-Lacrus, A., Xiol, X., Gine, J.J., Dolz, C , Cabre Esteve, M., Gonzalez-Huix, F , Gassul, M.A. (1989): Vitamin status in patients with inflammatory bowel disease. Am. J. Gastroenterol. 84: 744-748 Fischer, G.A. (1958): Studies of the culture of leukemic cells in vitro. Ann. N. Y. Acad. Sci. 76: 673-680 Fitzgerald, M. (1983): Capsaicin and sensory neurons. A review. Pain 15: 109-130 Flynn, D.L., Rafferty, M.F. (1986): Inhibition of human neutrophil 5-lipoxygenase activity by ginerdione, shogaol, capsaicin and related pungent compounds. Prostagland. Leukotrien Med. 24: 195-198 183

Fontham, E.T.H. (1997): Prevention of gastrointestinal tract cancers. In: Bendich, A., Deckelbaum, R. (eds): Preventive Nutrition. Humana Press, New Jersey, pp. 33-55 Fox, K., Garcia, M.A.A., Ardissino, D., Buszmann, P., Camici, P.G., Crea, F , Dly, C , De Backer, G., Hjemdahl, P., Lopez-Sendon, J., Marco, J., Leiria, J. M., Pepper, J., Sechtem, U., Simoons, M., Thygesen, K. (2006): Guidelines on the management of stable angina pectoris: The Task Force on the Management of Stable Angina Pectoris of the European Society of Cardiology. Eur. Heart J. 27: 1341¬ 1381 Freudenberg, K. (1962): Research on lignin. Fortschr. Chem. Org. Naturst. 20: 41-72 Fujiwake, H., Suzuki, T., Oka, S., Iwai, K. (1980): Enzymatic formation of capsaicinoids from vanillylamine and iso-type fatty acids by cell free extracts of Capsicum annuum var. annuum cv. Karayatsubusa. Agric. Biol. Chem. 44: 2907-2915 Fung, T, Jeffery, W, Beveridge, A.D. (1982): The identification of capsaicinoids in tear-gas spray. J. Forensic Sci. 27: 812-821 Galloway, S.M., Aardema, M.J., Ischidate, M., Ivett, Jr. J.L., Kirkland, D.J., Morita, T., Moscesso, P., Sofuni, T. (1994): Report from working group on in vitro tests for chromosomal aberrations. Mutat. Res. 312: 241-261 Gannett P.M., Shi X., Lawson T , Kolar C , Toth B. (1997): Aryl radical formation during the metabolism of arylhydrazines by microsomes. Chem. Res. Toxicol. 10: 1372-1377 Gislason, H., Guttu, K., Sorbye, H., Schifter, S., Waldum, L.H., Svanes, K. (1995): Role of histamine and calcitonin gene-related peptide in the hyperemic response to hypertonic saline and H back-diffusion in the gastric mucosa of cats. Scand. J. Gastroenterol. 30: 300-310 Glinsukon, T , Stitmunnaitnum, Y., Toskulkao, C , Buranawuth, T , Tandkrisanavinont, V. (1980): Acute toxicity of capsaicin in several animal species. Toxicon 18: 215-220 Gnayfeed, M.H., Daood, H.G., Biacs, P.A., Alcaraz, C.F. (2001): Content of bioactive compounds in pungent spice red pepper (paprika) as affected by ripening and genotype. J. Sci. Food Agric. 81: 1580-1585 Goettler, D., Rao, A.V., Bird, R.P. (1987): The effects of a "low-risk" diet on cell proliferation and enzymatic parameters of preneoplastic rat colon. Nutr. Cancer 10: 149-162 Goodman, D.S. (1984): Biosyntesis, absorption and hepatic metabolism of retinol. In: Sporn, M.B., Roberts, A.B., Goodman, D.S. (eds): The Retinoids. Vol. 2. Academic Press, New York, 1-39 Goso, C , Evangelista, S., Tramontana, N., Manzini, S., Blumberg, P.M., Szallasi, A. (1993): Topical capsaicin protects against trinitrobenzene sulfonic acid-induced colitis in the rat. Eur. J. Pharmacol. 249: 185-190 Govindarajan, VS. (1985): Capsicum - Production, technology, chemistry, and quality - Part I: History, botany, cultivation, and primary processing. CRC Critical Rev. Food Sci. Nutr., CRC Press, Boca Raton 22: 109-176 Govindarajan, V.S. (1986a): Capsicum - Production, technology, chemistry, and quality - Part II: Processed products, standards, world production and trade. CRC Critical Rev. Food Sci. Nutr., CRC Press, Boca Raton 23: 207-288 +

Govindarajan, V.S. (1986b): Capsicum - Production, technology, chemistry, and quality - Part III: Chemistry of the color, aroma, and pungency stimuli. CRC Critical Rev. Food Sci. Nutr., CRC Press, Boca Raton 24: 245-355 Govindarajan, V.S. (1986c): Capsicum - Production, technology, chemistry and quality. Part II. Processed products, standards, world production, and trade. In: Furia, T.E. (ed): CRC Crit. Rev. Food Sci. Nutr., CRC Press, Boca Raton 23: 207-288 Govindarajan, V.S. (1986d): Capsicum - Production, technology, chemistry, and quality - Part II: Processed products, standards, world production and trade. CRC Critical Rev. Food Sci. Nutr., CRC Press, Boca Raton 23: 28-34 Govindarajan, V.S. (1986e): Capsicum - Production, technology, chemistry, and quality - Part III: Chemistry of the color, aroma, and pungency stimuli. CRC Critical Rev. Food Sci. Nutr., CRC Press, Boca Raton 24: 270-276 Govindarajan, V.S. (1986f): Capsicum - Production, technology, chemistry, and quality - Part III: Chemistry of the color, aroma, and pungency stimuli. CRC Critical Rev. Food Sci. Nutr., CRC Press, Boca Raton 24: 327-330 Govindarajan, VS., Sathyanarayana, M.N. (1991): Capsicum - Production, technology, chemistry, and quality. Part V. Impact on physiology, pharmacology, nutrition, and metabolism: structure, pungency, pain, and desensitization sequences. CRC Crit. Rev. Food Sci. Nutr. CRC Press, Boca Raton 29: 435-474 Govindarajan, V.S, Narasimhan, S., Dhanraj, S. (1977): Evaluation of spices and oleoresins. II. Pungency of Capsicum by Scoville heat units - a standardized procedure. J. Food Sci. Technol. 14: 28-34 Govindarajan, VS., Rajalakshmi, D., Chand, N. (1987): Capsicum - Production, technology, chemistry, and quality - Part IV: Evaluation of quality. CRC, Critical Rev. Food Sci. Nutr. 25: 266; Gray, J.L., Bunnet, N.W., Orloff, S.L., Mulvihill, S.J., Debas, H.T. (1994): Role for calcitonin gene-related peptide in protection against gastric ulceration. Ann. Surg. 219: 58-64 Greenberg, E.R., Baron, J.A., Tosteson, T.D., Freeman, D.H. Jr, Beck, G.J., Bond, J.H., Colacchio, T.A., Coller, J.A., Frankl, H.D., Haile, R.W., Mandel, J.S., Nierenberg, D.W., Rothstein, R., Snover, C D . , Stevens, M.M., Summers, R.W., van Stolk, R.U. (1994): A clinical trial of antioxidant vitamins to prevent colorectal adenoma. N. Engl. J. Med. 331: 141-147 Guengerich, F.P, Kim, D.H., Iwasaki, M. (1991): Role of human cytochrome P450 HE 1 in the oxidation of many low molecular weight cancer suspects. Chem. Res. Toxicol. 4: 168-179 Gulbekian, S., Merighi, A., Wharton, J., Varndell, I.M., Polak, J.M, (1986): Ultrastructural evidence for the coexistence of calcitonin gene-related peptide and substance P in secretory vesicles of peripheral nerves in the guinea-pig. J. Neurocytol. 15: 535-542 Haim, N., Nemec, J., Roman, J., Sinha, BK. (1987a): In vitro metabolism of etoposide (VP-16-213) by liver microsomes and irreversible binding of reactive intermediates to microsomal proteins. Biochem. Pharmacol. 36: 527-536 Haim, N., Nemec, J., Roman, J., Sinha, BK. (1987b): Peroxidase-catalyzed metabolism of etoposide (VP-15-213) and covalent binding of reactive intermediates to cellular macromolecules. Cancer Res. 47: 5835-5840 185

Hartman, K.T. (1970): A rapid gas-liquid chromatographic determination for capsaicin in capsicum species. J. Food Sci. 35: 543-574 Hawer, W.S., Ha, J., Hwang, J., Nam, Y. (1994): Effective separation and quantitative analysis of major heat principles in red pepper by capillary gas chromatography. Food Chem. 49: 99-103 Hawkes, J.G., Lester, R.N., Skelding, A. (1979): The Biology and Taxonomy of the Solanacea. Academic Press, London Haymon, L.W., Aurand, L.W. (1971): Volatile constituents of tabasco peppers, J. Agric. Food Chem. 19: 1131-1134 Heiser, C.B. (1967): Peppers Capsicum (Solanaceae) In: Simmonds, N. W. (ed): The Evolution of Crops Plants. Longman Press, London, pp. 265-268 Hendriks, H.F.J., Verhoofstad, W.A.M.M., Brouwer, A., de Leeuw, A.M., Knook, D.L. (1985): Perisinusoidal fat-storing cells are the main vitamin A storage sites in the rat liver. Exp. Cell. Res. 160: 138-149 Hennekens, C.H., Buring, J.E., Peto, R. (1994a): Antioxidant vitamins - benefit not yet proved. N. Engl. J. Med. 330: 1080-1081 Hennekens, C.H., Buring, J.E., Peto, R. (1994b): Alpha-Tocopherol, (3-Carotene Cancer Prevention Study Group. The effect of vitamin A and P-carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med. 330: 1029-1035 Hinds, T.S., West, W.L., Knight, E.M. (1997): Carotenoids and retinoids: a review of research, clinical, and public health applications, J. Clin. Pharmacol. 37: 551-558 Hoch-Ligeti, C. (1951): Production of liver tumours by dietary means: Effect of feeding chillies to rats. Acta UICC 7: 606-611 Hoffman, P.G., Lego, M.C., Galetto, W.G. (1983): Separation and quantitation of red pepper major heat principles by reverse-phase high pressure liquid chromatography. J. Agric. Food Chem. 31: 1326-1330 Hoffman, V.L., Schadle, E.R., Villalon, B. Burns, E.E. (1978): Volatile components and pungency in fresh and processed jalapeno peppers. J. Food Sci. 43: 1809-1811 Hollo, J. Gal, I., Suto, J. (1957): Chromatographic determination of capsaicin in paprika oil and paprika products. Fette Seifen Anstrichm. 59: 1048-1055 Holzer, P. (1988): Local effector functions of capsaicin-sensitive sensory nerve endings: Involvement of tachykinins, and their neuropeptides. Neuroscience 24: 739-768 Holzer, P. (1990): Capsaicin-sensitive nerves in the control of vascular effector mechanisms. In: Green, B.G., Mason, J.R., Kare, M.R. (eds): Chemical Senses. Irritation. Marcel Dekker, New York, 2: 191-210 Holzer, P. (1991a): Capsaicin: cellular targets, mechanisms of action, and selectivity for thin sensory neurons. Pharmacol. Rev. 43: 144-202 Holzer, P. (1991b): Afferent nerve-mediated control of gastric mucosal blood flow and protection. In: Costa, M., Surrenti, C , Gorini, S., Maggi, C.A., Meli, A. (eds): Sensory Nerves and Neuropeptides in Gastroenterology. From Basic Science to Clinical Perspective. Plenum Press, New York, pp. 97-108

186

Holzer, P. (1992a): Capsaicin: selective toxicity for thin primary sensory neurons. Selective neurotoxicity. In: Herken, H., Hucho, F. (eds): Handbook of Experimental Pharmacology. Springer Verlag, Berlin, pp. 419^481 Holzer, P. (1992b): Peptidergic sensory neurons in the control of vascular functions: mechanisms and significance in the cutaneous and splanchnic vascular beds. Rev. Physiol. Biochem. Pharmacol. 121: 50-146 Holzer, P. (1998): Neural emergency system in the stomach. Gastroenterology 114: 823-839 Holzer, P., Lippe, I.T. (1988): Stimulation of afferent nerve endings by intragastric capsaicin protects against ethanol-induced damage of gastric mucosa. Neuroscience 27: 981-987 Holzer, P., Sametz, W. (1986): Gastric mucosal protection against ulcerogenic factors in the rat mediated by capsaicin-sensitive afferent neurons. Gastroenterology 91:975-981 Holzer, P., Pabst, M.A., Lippe, I.T. (1989): Intragastric capsaicin protects against aspirin-induced lesion formation and bleeding in the rat gastric mucosa. Gastroenterology 96: 1425-1433 Hori, T. (1984): Capsaicin and central control of thermoregulation. Pharm. Ther. 26: 389-416 Hornero-Mendez, D., Gomez-Ladron, R., Minguez-Mosquera, M.I. (2000): Carotenoid biosynthesis changes in five red pepper (Capsicum annuum L.) cultivars during ripening. Cultivar selection for breeding. J. Agric. Food Chem. 48: 3857¬ 3864 Hugueney, P., Badillo, A., Chen, H.C., Klein, A., Hirschberg, J., Camara, B., Kuntz, M. (1995): Metabolism of cyclic carotenoids: A model for the alteration of this biosynthetic pathway in Capsicum annuum chromoplasts. Plant J. 8: 417-424 Hungarian Standard. Examination of Ground Paprika Spice. Determination of Pigment Content, (MSZ 9681/5-76) (MSZ 9681-5:2002). Hunter, D.J., Manson, J.E., Colditz, G.A. (1993): A prospective study of the intake of vitamin C, E, and A and the risk of breast cancer. N. Engl. J. Med. 329: 234-240 Hunyady, B., Vincze, A., Garamszegi, M., Cziraki, A., Mozsik, Gy. (1991): Effect of ranitidine, famotidine and vitamin A on gastric acidity studied by a new computerized intragastric pH-metry system. Exp. Clin. Gastroenterol. 1: 365-369 Hussain, M.M., Mahley, R.W., Boyles, J.K., Fainaru, M., Brecht, W.J., Lingquist, PA. (1989): Chylomicron-chylomicron remnant clearance by liver and bone marrow in rabbits. J. Biol. Chem. 264: 9571-9582 International Conference on Harmonisation (ICH) (2003) (Ql, Q8), Brussels International Standards Organization (1981): Spices and condiments - chillies: Determination of capsaicinoids content, draft proposal DP 7543, ISO, Geneva International Standards Organization (1997): Spices and condiments - chillies: Determination of Scoville index, ISO 3513:1977E, ISO, Geneva) Ishikawa, K. (2003): Biosynthesis of capsaicinoids in Capsicum. Chapter 5. In: De, A.K. (ed.): Capsicum. The Genus of Capsicum, Taylor and Francis, London, New York, pp. 87-95 IUPAC-IUB (1982): Joint Commission on Biocemical Nomenclature. Eur. J. Biochem. 129: 1-5 187

Iwai, K., Suzuki, T., Fujiwake, H., Oha, S. (1979): Simultaneous microdetermination of capsaicin and its four analogues by using high-performance liquid chromatography and gas chromatography-mass spectrometry. J. Chromatogr. 172: 303-311 Iwama, M., Tojima, T., Iitol, Y., Takahashi, N., Kanke, Y. (1990): Effects of capsaicin and ethanol on hepatic drug-metabolizing enzymes in rat. Int. J. Vit. Nutr. Res. 60: 100-103 Jackson, B.P., Snowdon, D. (1974): Powdered Vegetable Drug. Standley Thornes Publishers, London, pp. 14—15 Jaiarj, P., Kitphati, C , Sinchaipanid, N., Lertsin, N., Siripanyachan, P. (2000): Stability testing of capsaicinoid cream. Mahidol University Annual Research Abstracts, p. 371 Jancso, G., Kiraly, E., Joo, I., Such, G., Nagy, A. (1985): Selective degeneration by capsaicin of a subpopulation of primary sensory neurons in the adult rat. Neurosci. Lett. 59: 200-214 Jancso, G., Kiraly, E., Such, G., Joo, I., Nagy, A. (1987): Neurotoxic effect of capsaicin in mammals. Acta Physiol. Hung. 69: 295-313 Jancso, N., Jancso-Gabor, A. (1959): Dauerausschaltung der chemischen Schmerzempfindlichkeit durch Capsaicin. Naunyn-Schmiedebergs Arch. Exp. Path. Pharmacol. 236:142-145 Jancso, N., Jancso-Gabor, A., Szolcsanyi, J. (1968): The role of sensory nerve endings in neurogenic inflammation induced in human skin and in the eye and paw of the rat. Br. J. Pharmacol. 33: 32-41 Jancso-Gabor, A., Szolcsanyi, J. (1972): Neurogenic inflammatory response. J. Dental Res. 41: 264-269 Jang, J.J., Kim, S.H. (1988): The promoting effect of capsaicin on the development of diethylnitrosamine-induced enzyme altered hepatic foci in male SpragueDawley rats. J. Korean Cancer Assoc. 20: 1-7 Jang, J. J., Kim, S. H., Yun, T.K. (1992): A 4-week feeding study of ground red chilli (Capsicum annuum) in male B6C3F1 mice. Food Chem. Toxicol. 30: 783-787 Jankowsky, J. (1994): Genelteresek a vastagbelrakban. (Genetic abberations in colorectal carcinoma). In: Ujszaszy, L., Simon, L. (eds): Colorectalis carcinoma. Valogatott fejezetek a vastagbelrakrol. (Colorectal Carcinoma) (In Hungarian) Medicom, Budapest, pp. 159-199 Javor, T, Bata, M., Lovasz, L., Moron, F., Nagy, L., Patty, I., Szabolcs, J., Tarnok, F., Toth, Gy., Mozsik, Gy. (1983): Gastric cytoprotective effects of vitamin A and other carotenoids. Int. J. Tiss. React. 5: 289-296 Jensen, K., Gluud, C. (1994): The Mallory body: theories on development and pathological significance. Hepatology 20: 1330-1342 Jentzsch, K., Kubelka, W., Pock, H. (1969): A method for determination of capsaicinoids content in capsicum fruits and preparations, (in German) Sci. Pharm. 37: 153-162 Jewell, D.P. (1995): Pathogenesis of Crohn's disease: the environment revisited. Eur. J. Gastroenterol. Hepatol. 7: 383-384 Joe, B., Lokesh, B.R. (1994): Role of capsaicin, curcumin and dietary n-3 fatty acids in lowering the generation of reactive oxygen species in rat peritoneal macrophages. Biochim. Biophys. Acta 1224:255-263

Johnson, E.L., Majors, R.E., Werum, L., Reiche, R (1979): The determination of naturally occurring capsaicins by HPLC. In: Charalambous, G. (ed.): Liquid Chromatographic Analysis of Food and Beverages. Vol. 1. Academic Press, New York 17-29 Joint Committee for Pharmaceutical Society and Society for Analytical Chemistry on Methods of Assay of Crude Drugs (1964): The determination of capsaicin content of Capsicum and its preparations. II. Analyst 89: 377-385 Jurenitsch, J. (1981): Pungent principle composition in fruits of defined strains of Capsicum. Consequences relating to quality requirements and taxonomic aspects, (in German). Sci. Pharm. 49: 321-328 Jurenitsch, J., Kampelmuehler, I. (1980): Rapid determination of nonylic acid vanillylamide and other capsaicinoids in capsicum fruit extracts by means of A g complexation high performance liquid chromatography, (in German) J. Chromatogr. 193: 101-110 Jurenitsch, J., Leinmueller, R. (1980): Quantification of nonylic acid vanillylamide and other capsaicinoids in the pungent principle of Capsicum fruits and preparations by gas-liquid chromatography on glass capillary columns, (in German) J. Chromatogr. 189:389-397 Jurenitsch, J., Kubelka, W., Jentzsch, K. (1978): Gas chromatographic determination of the content of individual and total capsaicinoids in Capsicum fruits after thinlayer chromatographic separation, (in German) Sci. Pharm. 46: 307-318 Jurenitsch, J., Bingler, E., Becker, H., Kubelka, W. (1979a): Simple HPLC method for determination of total and single capsaicinoids in Capsicum-fruits, (in German) Plant. Med. 36: 54-60 Jurenitsch, J., David, M., Heresch, F , Kubelka, W. (1979b): Detection and identification of new pungent compounds in fruits of Capsicum. Planta Med. 36: 61-67 Jurenitsch, J., Kubelka, W., Jentzsch, K. (1979c): Identification of cultivated taxa of Capsicum - Taxonomy, anatomy and composition of pungent principles. Plant Med. 35: 174-183. Kandaaswami, C , Perkins, E., Solonink, D.S., Drezewiwki, G., Middleton, E. (1983): Ascorbic acid-enhanced antiproliferative effect of flavonoids on squamous cell carcinoma in vitro. Anti-Cancer Drugs 4: 91-96 Karadi, O., Mozsik, Gy. (2000): Surgical and Chemical Vagotomy on the Gastrointestinal Mucosal Defense. Akademiai Kiado, Budapest Karnka, R., Rayanakorn, M., Wtanesk, S., Vaneesorn, Y. (2002): Optimization of high-performance liquid chromatographic parameters for the determination of capsaicinoid compounds using the simplex method. Anal. Sci. 18: 661-665 Kawada, T, Iwai, K. (1985): In vivo and in vitro metabolism of dihydrocapsaicin, a pungent principle of hot pepper, in rat. Agric. Biol. Chem. 49: 441^-48 Kawada, T , Suzuki, T , Takahashi, M., Iwai, K. (1984): Gastrointestinal absorption and metabolism of capsaicin and dihydrocapsaicin in rats. Toxicol. Appl. Pharmacol. 72: 449-456 Kawai, S., Nishida, S., Kato, M., Furumaya, Y, Okomoto, T , Mizushima, Y. (1998): Comparison of cyclooxygenase-1 and -2 inhibitory activities of nonsteroidal anti-inflammatory drugs using human platelets and synovial cells. Eur. J. Pharamacol. 347: 87-94 +

189

Kawaromi, T., Tanaka, T., Suzui, M. (1995): Chemoprevention of azoxymethaneinduced intestinal carcinogenesis by a novel synthesised retinoidal butenolide, 5-hydroxy-4-(2-phenyl-(E)-enthenyl)-2(5H)-furanone in rats. Carcinogenesis 16: 795-800 Keller, U., Flath, R.A., Mon, R.A., Teranishi, R. (1981): Volatiles from red pepper (Capsicum spp.), In: Teranishi, R., Barrera-Benitez, H. (eds): Quality of Selected Fruits and Vegetables of North America. ACS Symposium Series, No. 170, American Chemical Society, Washington, D.C., Chapter 12, pp. 137-146 Kensler, T.W., Egner, P.A., Davidson, N.E., Roebuck, B.D., Pikul, A., Groopman, J.D. (1986): Modulation of aflatoxin metabolism, aflatoxin-N7-guanine formation, and hepatic tumorigenesis in rats fed ethoxyquin: role of induction of glutathione Stransferases. Cancer Res. 46: 3924-3931 Ketusinh, O., Dhorranintra, B., Juengjareon, K. (1966): Influence of capsaicin solution on gastric acidities. Am. J. Protocol. 17: 511-515 Kim, J.P., Park, J.G., Lee, M.D., Han, M.D., Park, S.T., Lee, B.H., Jung, S.E. (1985): Cocarcinogenic effects of several Korean foods on gastric cancer induced by N-methyl-N'-nitro-N-nitrosoguanidine in rats. Jap. J. Surg. 15: 427^137 Kliewer, S.A., Umesono, K., Magelsdorf, D.J., Evans, R.M. (1992): Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone anvitamin D signalling. Nature 355: AA6-A69 Koop, D.R. (1992): Oxidative and reductive metabolism by cytochrome P450 2E1.FASEBJ. 6: 724-730 Kopec S.E., DeBellis R.J., Irwin R.S. (2002): Chemical analysis of freshly prepared and stored capsaicin solutions: implications for tussigenic challenges. Pulm. Pharmacol. Ther. 15: 529-534 Kopp, B., Jurenitsch, J. (1982): Biosynthese der Capsaicinoide in Capsicum annuum L. var. annuum. III. Zur Frage der Bildung von Capsaicin und Dihydrocapsaicin. Sci. Pharm. 50: 150-157 Korel, F , Bagdatlioglu, N., Balaban, M.O., Hisil, Y. (2002): Ground red peppers: Capsaicinoid content, Scoville scores, and discrimination by an electronic nose. J. Agric. Food Chem. 50: 3257-3261 Kosuge, S., Inagaki, Y. (1959): Studies on pungent principles of red pepper. III. Determination of pungent principles. Nippon Nogei Kagaku Kaishi, 33: 470; Chem. Abstr. (1960): 54: 12404h Kozukue, N., Han, J-S., Kozukue, E., Lee, S-J., Kim, J.A., Lee, K-R., Levin, C.E., Friedman, M. (2005): Analysis of eight capsaicinoids in peppers and pepper-containing foods by high-performance liquid chromatography and liquid chromatography-mass spectrometry. J. Agric. Food Chem. 53: 9172-9181 Krajewska, A.M., Powers, J.J. (1987): Gas chromatographic determination of capsaicinoids in green capsicum fruits. J. Assoc. Off. Anal. Chem. Int. 70: 926-928 Kramer, A. (1966): Parameters of quality. Food Technol. 20: 1147-1180 Krukonis, V.J. (1988): Processing with supercritical fluids. Overview and applications. In: Charpentier, B.A., Sevenants, M.R. (eds): Supercritical Fluid Extraction and Chromatography. Techniques and Applications, ACS Symposium Series 366; American Chemical Society, Washington, DC, pp. 26-43 3

190

Kulka, K. (1967): Aspects of functional groups and flavour. J. Agric. Food Chem. 15: 48-52 Kuzma, M., Molnar, Sz., Perjesi, P. (2006): Development and application of a gas chromatographic method for determination of capsaicinoids, In: Abstracts of Papers, Symposium of Drug Research Committe of Hungarian Pharmaceutical Society, November 24-25, Debrecen, Hungary, p. 51 Ladefoged, K , Hessov, I., Jarnum, S. (1996): Nutrition in short-bowel syndrome. Scand. J. Gastroenterol. Suppl. 216: 122-131 Lakatos, L. (1994): A vastagbelrak epidemiologiaja. (Epidemiology of Colorectal Carcinoma.). In: Ujszaszi, L., Simon, L. (eds): Colorectalis carcinoma. Valogatott fejezetek a vastagbelrakrol. (Colorectal Carcinoma.) (In Hungarian). Medicom, Budapest, pp.11-31 Lawson, T., Gannett, P. (1989): The mutagenicity of capsaicin and dihydrocapsaicin in V79 cells. Cancer Lett. 49: 109-113 Lee, H.Y., Dawson, M.I., Claret, EX., Holzer, S. (1997): Evidence of retinoid signaling alteration involving the activator protein 1 complex in tumorigenic human bronchial epithelial cells and small cell lung cancer cells. Cell Growth Differ. 8: 283-291 Lee, K.R, Suzuki, T , Kobashi, M., Hasegawa, K , Iwai, K. (1976): Quantitative micro analysis of capsaicin, dihydrocapsaicin, nordihydrocapsaicin using mass fragmentography. J. Chromatogr. 123: 119-128 Lee, S.O. (1963): Studies on the influence of diets and lipotropic substances upon the various organs and metabolic changes in rabbits on long term feeding with red pepper. I. Histopathologic changes of the liver and spleen. Taehan Naekwa Hakhoe Chapchi 143: 383-400 Lee, S.S., Kumar, S. (1980): Microsomes, Drug Oxidations, and Chemical Carcinogenesis. Vol. 2. In: Coon, M.J., Conney, A.H., Estabrook, R.W., Gelboin, H.V., Gillette, J.R., O' Brien, P.J. (eds): Microsomes, Drug Oxidations and Chemical Carcinogenesis. Academic Press, New York, pp. 1009-1012 Lenzer, J., (2004): FDA is incapable of protecting of US "against another Vioxx". Br. Med. J. 329:1253 Li, D.S., Raybould, H.E., Quintero, E., Guth, PH. (1992): Calcitonin gene-related peptide mediates the gastric hyperemic response to acid back-diffusion. Gastroenterology 102: 1124-1128 Lichtenthaler, H.K., Schwender, J., Disch, A., Rohmer, M. (1997): Biosynthesis of isoprenoids in higher plant chloroplasts proceeds via mevalonate-independent pathway. FEBS Lett. 400: 271-274 Linden, H., Misawa, N., Chamnovitz, D., Pecker, I., Hirschberg, J., Sandmann, G. (1991): Functional complementation in Escherichia coli of different phytoene desaturase genes and analysis of accumulated carotenes. Z. Naturforsch. 46: 1045-1051 Lippe, I.T., Holzer, P. (1992): Participation of endothelium-derived nitric oxide but not prostacyclin in the gastric mucosal hyperaemic response due to acid backdiffusion. Br. J. Pharmacol. 105: 708-714 Lippe, I.T., Pabst, M.A., Holzer, P. (1989): Intragastric capsaicin enhances rat gastric acid elimination and mucosal blood flow by afferent nerve stimulation. Br. J. Pharmacol. 96: 91-100 191

Lok, E., Ratnayake, W.M.N., Scott, F.W., Mongeau, R., Ferine, S., Nera, E.A., Malcolm, S., McMathew, E., Jee, R, Clayson, D.B. (1992): Effect of varying type of fat in a semi-purified AIN76A diet on cellular proliferation in the mammary gland and intestinal crypts in female Swiss Webster mice. Carcinogenesis 13: 1735-1741 Longnecker, D.S., Curphey, T.J., Kuhlman, E.T., Roebuck, B.D. (1982): Inhibition of pancreatic carcinogenesis by retinoids in azaserine treated rats. Cancer Res. 42: 19-24 Lopez-Carrillo, L., Avila, H. M., Dubrow, R. (1994): Chili pepper consumption and gastric cancer in Mexico: A case-control study. Am. J. Epidemiol. 139: 263-271 Lopez-Carrillo, L., Lopez-Cervantes, M., Bobles-Diaz, G., Ramilez-Espitia, A., Mohar-Betancourt, A., Menses-Gratia, A., Lopez-Vidal, Y., Blair, A. (2003): Capsaicin consumption Helicobacter pylori positivity and gastric cancer in Mexico. Int. J. Cancer. 106: 277-282 Lopez-Hernandez, J., Oruna-Concha, M.J., Simal-Lozano, J., Gonzales-Castro, M.J., Vazquez-Bianco, M.E. (1996a): Determination of capsaicin and dihydrocapsaicin in cayenne pepper and padron peppers by HPLC. Deutsche LebensmittelRundschau 92: 393-395. Lopez-Hernandez, J., Oruna-Concha, M. J., Simal-Lozano, J., Vazquez-Bianco, M. E., Gonzales-Castro, M.J. (1996b): Chemical composition of Padron peppers (Capsicum annuum L.) grown in Galicia (N.W. Spain). Food Chemistry 57: 557-559 MacNeish, R.S. (1964): Ancient Mesoamerican civilization. Science 143: 531¬ 537 Macrae, R. (ed.) (1993): Encyclopedia of Food Science. Food Technology and Nutrition (Peppers and Chillies). Academic Press, London, pp. 3496-3505 Maga, J.A. (1975): Capsicum. Crit. Rev. Food. Sci. Nutr. 177-199 Maggi, C.A., (1995): Tachykinins and calcitonin gene-related peptide (CGRP) as co-transmitters released from peripheral endings of sensory nerves. Prog. Neurobiol. 45: 1-98 Maggi, C.A., Meli, A. (1988): The sensory-efferent function of capsaicin-sensitive sensory neurons. Gen. Pharmacol. 19: 1-43 Maggi, C.A., Santicioli, P., Geppetti, P., Parlani, M., Astolfi, M., DelBianco, E., Patacchini, R., Giuliani, S., Meli, A., (1989): The effect of calcium free medium and nefidipine on the release of substance P-like immunoreactivity and contractions induced by capsaicin in the isolated guinea-pig bladder. Gen. Pharmacol. 40: 445^156 Maillard, M.-N. Giampaoli, P., Richard, H.M.J. (1998): Analysis of eleven capsaicinoids by reversed-phase high performance liquid chromatography. Flavour Fragrance J. 12: 409-413 Makara, G.B., Frenkl, C.R., Somfai, Z., Szepeshazi, K. (1965): Effect of capsaicin on the experimental ulcer in the rat. Acta Med. Sci. Hung. 21: 213-216 Manirakiza, P., Covaci, A., Shepens, P. (1999): Solid-phase extraction and gas chromatography with mass spectrometric determination of capsaicin and its analogues from chilli peppers (Capsicum spp.) J. Assoc. Off. Anal. Chem. Int. 82: 1399-1405

192

Manirakiza, P., Covaci, A., Shepens, P. (2003): Pungency principles in Capsicum - analytical determination and toxicology, In: De, A.K. (ed.): Capsicum. The genus of Capsicum. Chapter 4. Taylor and Francis, London, New York Mans, D.R., Lafleur, M.V., Westmijze, E.J., van Maanen, J.M., van Schaik, M.A., Lankelma, J., Retel, J. (1991): Formation of different reaction products with singleand double-stranded DNA by the ortho-quinone and the semi-quinone free radical of etoposide (VP-16-213). Biochem. Pharmacol. 42: 2131-2139 Maron, D.M., Ames, B.N. (1983): Revised methods for the Salmonella mutagenecity test. Mutat. Res. 113: 173-215 Marques, S., Oliveira, N.G., Chaveca, T , Rueff, J. (2002): Micronuclei and sister chromatid exchanges induced by capsaicin in human lymphocytes. Mutat. Res. 517: 39^16 Martin, R.L., McDermott, S.J., Salmen, H.J., Palmatier, J., Cox, B.F., Gintant, G.A. (2004): The utility of hERG and repolarization assays in evaluating delayed cardiac reporalization: influence of multi-channel block. J. Cardiovasc. Pharmacol. 43:369-379 Matsushima, N.R., Shidoji, Y, Nishiwaki, S., Kawasaki, R. (1996): Aberrant metabolism of retinoid X receptor proteins in human hepatocellular carcinoma. Mol. Cell. Endocrinol. 121: 179-190 Mcgettigan, P., Henry, D. (2006): Cardiovascular Risk and Inhibition of Cyclooxygenase. A systematic review of the observational studies of selective and nonselective inhibitors of cyclooxygenase 2. JAMA 296: 1633-1644 McHugh, M.A., Krukonis, V.L. (1986): Supercritical fluid extraction: principles and practice. Butterworth, Stoneham, MA, pp. 13-22 Mckeown-Eyssen, G., Holloway, C , Jazmaji, V., Bright-See, E., Dion, P., Bruce, W.R. (1988): A randomized trial of vitamin C and E in the prevention of recurrence of colorectal polyps. Cancer Res. 48: 4701^1705 Menguy, R., Master, YF. (1974a): Mechanisms of stress ulcer II. Differences between the antrum, corpus and fundus with respect to the complete ischemia on gastric mucosal energy metabolism. Gastroenterology 66: 509-614 Menguy, R., Master, Y.F. (1974b): Mechanisms of stress ulcer III. Effect of hemorrhagic shock on metabolism in the mucosa of the antrum, corpus and fundus of the rabbit stomach. Gastroenterology 66: 1168-1176 Menguy, R., Master, Y.F. (1974c): Mechanisms of stress ulcer IV. Influence of fasting on the tolerance of gastric mucosal energy metabolism to ischemia and on the incidence of stress ulceration. Gastroenterology 66: 1177-1186 Menguy, R., Desbaillets, L., Master, Y.F. (1974): Mechanisms of stress ulcer I. Influence of hypovolemic shock on energy metabolism in the gastric mucosa. Gastroenterology 66: 46-55 Merighi, A., Polak, J.M., Gibson, S.J., Gulbekian, S., Valentino, K.L., Peirone, S.M. (1988): Ultrastructural studies on calcitonin gene-related peptide-, tachykininand somatostatin-immunoreactive neurons in rat dorsal root ganglia: Evidence for colocalization of different peptides in single secretory granules. Cell Tiss. Res. 254: 101-109 Micko, K. (1898): Zur Kenntnis des Capsaicins, Z. Unters. Nahr. Genussm. 1: 818-829 193

Miller, M.S., Brendel, K., Burks, T.F., Sipes, I.G. (1983): Interaction of capsaicinoids with drug-metabolizing systems. Relationship to toxicity. Biochem. Pharmacol. 32:547-551 Modly, C.E., Das, M., Don, P.S., Marcelo C.L., Mukhtar, H., Bickers, D.R. (1986): Capsaicin as an in vitro inhibitor of benzo(a)pyrene metabolism and its DNA binding in human and murine keratinocytes. Drug Metab. Dispos. 14: 413-416 Molnar, J. (1965). Die pharmakologischen Wirkungen des Capsaicins, des scharf schmeckenden Wirkstoffes im Paprika. Arzneimittel-Forsch. 15: 718-727 Molnar, J., Gyorgy, L. (1967). Pulmonary hypertensive and other haemodynamic effect of capsaicin in the cat. Eur. J. Pharmacol. 1: 86-92 Moon, R.C., McCormick, D.L. (1982): Inhibition of carcinogenesis by retinoids. J. Am. Acad. Dermatol. 6: 809-814 Monsereenusorn, Y. (1983): Subchronic toxicity studies of capsaicin and capsicum in rats. Res. Commun. Chem. Pathol. Pharmacol. 41: 95-110 Monsereenusorn, Y, Kongsamut, S., Pezalla, PD. (1982): Capsaicin - A literature survey. CRC Critical Rev. Toxicol. 10: 321-339 Monterio, E, Tavarela Valoso, F. (1995): Inflammatory Bowel Disease. New Insight into Mechanism of Inflammation and Challengers in Diagnosis and Treatment. Kluwer Academic Publishers, Lancaster Moon, R.C. (1993): Retinoids in experimental oncology. In: Hong, W.K., Lotan, R. (eds): Retinoids in Oncology. Marcel Dekker, New York, pp. 109-123 Moor, A. (1969): A paprika hibridmag termesztese. (Hybrid seed production of paprika) Thesis. Hort. Univ. of Budapest Moore, M.M., Honna, M., Clements, J., Bolcsfoldi, G., Cifone, M., Delondchamp, R., Fellows, M., Gollapudi, B., Jenkinson, P., O'Kirby, P., Kirchner, S., Muster W., Myhr, B., O'Donovan, M., Oliver, J., Ouldelhkim, M.C., Thakur, A., Wakuri, S., Yoshimura, I. (2003): Mouse thymidine kinase gene mutation assay: International workshop on genotoxicity test. Workgroup Report-Plymouth, U.K. 2002. Mutat. Res. 540: 127-140 Moron, F , Cuesta, E., Bata, M., Mozsik, Gy. (1983): Cytoprotecting effect of cimetidine: experimental evidence in the rat gastric mucosal lesions induced by inragastric administration of necrotizing agents. Arch. Int. Pharmacodyn. Ther. 265: 310-319 Moron, F , Mozsik, Gy., Nagy, L., Patty, I., Tarnok, F , Javor, T. (1984): Evidence for the existence of gastric cytoprotective effect of atropine and cimetidine in humans. Acta Physiol. Hung. 64: 181-186 Morrison, J.I. (1967): Gas chromatographic method for measuring pungency in capsicum spices. Chem. Ind. (London) 1785 Mozsik, Gy. (1969): Some feed-back mechanims by drugs in the interrelationship between the active transport system and adenyl-cyclase system localized in the cell membrane. Eur. J. Pharmacol. 7: 319-327 Mozsik, Gy. (2004): Neural, hormonal and pharmacological regulation of retinoid-induced gastrointestinal mucosal protection. Recent Res. Devel. Life Sci. 3: 131-207 Mozsik, Gy. (2006): Molecular pharmacology and biochemistry of gastroduodenal mucosal damage and perotection. In: Mozsik, Gy. (ed.): Discoveries in Gas194

troenterology: from Basic Research to Clinicla Perspectives (1960-2005). Akademiai Kiado, Budapest, pp. 139-224 Mozsik, Gy., Figler, M. (2005): Metabolic Ward in Human Clinical Nutrition and Dietetic. Research Signpost, Kerala, India Mozsik, Gy., Javor, T. (1988): A biochemical and pharmacological approach to the genesis of ulcer disease. I. A model study of ethanol-induced to gastric mucosa in rats. Dig. Dis. Sci. 33: 92-105 Mozsik, G , Javor, T. (1991): Therapy of ulcers with sulfhydryl and nonsulfhydryl antioxidants. In: Swab, A., Szabo, S. (eds): Ulcer Disease, Investigation and Basis for Therapy. Marcel Dekker, New York, pp. 321-341 Mozsik, Gy., Pfeiffer, C.J. (1992): A biochemical and pharmacological approach to the genesis of ulcer disease. III. A model study of epinephrine-induced injury to gastric mucosa in rats. Exp. Clin. Gastroenterol. 2: 1-12 Mozsik, Gy, Vizi, F. (1976): Examination of stomach wall Mg -Na -K - dependent ATPase, ATP and ADP in pylorus-ligated rats. Am. J. Dig. Dis. 21: 449-454 Mozsik, Gy., Figler, M., Nagy, L., Patty, I., Tarnok, F. (1981): Gastric and small intestinal energy metabolism in mucosal damage. In: Mozsik Gy., Hanninen O., Javor T, (eds): Advances in Physiological Sciences. Vol. 29. Gastrointestinal Defense Mechanisms. Pergamon Press, Oxford, Akademiai Kiado, Budapest, pp. 213-276 Mozsik, Gy., Moron, F. Nagy, L., Ruzsa, Cs., Tarnok, F , Javor, T. (1986): Evidence of the gastric cytoprotective effects of vitamin A, atropine and cimetidine on the development of gastric mucosal damage produced by administration of indomethacin in healthy subjects. Int. J. Tiss. React. 8: 85-90 Mozsik, Gy., Garamszegi, M., Javor, T , Nagy, L., Patty, I., Siito, G., Vincze, A. (1990): A biochemical and pharmacological approach to the genesis of ulcer disease. II. A model study of stress induced injury to gastric mucosa in rats. Ann. N. Y. Acad Sci. 517: 264-281 Mozsik, Gy., Kiraly, A., Garamszegi, M., Javor, T., Nagy, L., Siito, G., Toth, Gy., Vincze, A. (1991): Failure of prostacyclin, P-carotene, atropine and cimetidine to produce gastric cyto- and general mucosal protection in surgically vagotomized rats. Life Sci. 49: 1383-1389 Mozsik, Gy., Kiraly, A., Siito, G., Vincze, A. (1992): ATP breakdown and resynthesis in the development of gastrointestinal mucosal damage and its prevention in animals and human (an overview of 25 years ulcer research studies). Acta Physiol. Hung. 80: 39-80 Mozsik, Gy., Kiraly, A., Siito, G., Vincze, A. (1993): ATP breakdown and resynthesis in the development of gastrointestinal mucosal damage and its prevention in animals and human (an overview of 25 years ulcer research studies). In: Mozsik, Gy., Par, A., Kitajima, M., Kondo, M., Pfeiffer, CJ., Rainsford, KD., Sikiric, P., Szabo, S. (eds): Cell Injury and Protection in the Gastrointestinal Tract: From Basic Science to Clinical Perspectives. Akademiai Kiado, Budapest, pp. 39-80 Mozsik, Gy., Bodis, B., Garamszegi, M., Karadi, O., Kiraly, A., Nagy, L., Siito, G., Toth, Gy., Vincze, A. (1995): Role of vagal nerve in the development of gastric mucosal injury and its prevention by atropine, cimetidine, P-carotene and prostacyclin in rats. In: Szabo, S., Tache, Y. (eds): Neuroendocrinology of Gastrointestinal Ulceration. Plenum Press, New York, pp. 175-190 2+

+

+

195

Mozsik, Gy., Abdel-Salam, O.M.E., Bodis, B., Karadi, O., Kiraly, A., Siito, G , Rumi, Gy., Szabo, I., Vincze, A. (1996a): Gastric mucosal protective effects of prostacyclin and P-carotene, and their biochemical backgrounds in rats treated with ethanol and HCI in dependence of their doses and of their time after administration of necrotizing agents. Inflammopharmacology 4: 361-378 Mozsik, Gy., Abdel-Salam, O.M.E., Bodis, B., Karadi, O., Nagy, L., Szolcsanyi, J. (1996b): Role of vagal nerve in defense mechanisms against NSAIDs-induced gastrointestinal mucosal damage. Inflammopharmacology 4: 151-172 Mozsik, Gy., Abdel-Salam, O.M.E., Szolcsanyi, J. (1997a) Capsaicin-Sensitive Afferent Nerves in Gastric Mucosal Damage and Protection. Akademiai Kiado, Budapest Mozsik, Gy., Nagy, L., Kiraly, A. (eds) (1997b): Twenty-Five Years of Peptic Ulcer Research in Hungary. From Basic Science to Clinical Practice 1971-1995. Akademiai Kiado, Budapest Mozsik, Gy., Nagy, L., Par, A., Rainsford, K.D. (eds) (1997c): Cell Injury and Protection in the Gastrointestinal Tract: From Basic Science to Clinical Perspectives. Kluwer Academic Publisher, Boston, Dordrecht Mozsik, Gy., Bodis, B., Karadi, O., Nagy, L., Rumi, Gy., Siito, G., Szabo, I., Vincze, A. (1998): Cellular mechanisms of P-carotene-induced gastric cytoprotection in indomethacin-treated rats. Inflammopharmacology 6: 27^4-0 Mozsik, Gy., Debreceni, A., Abdel-Salam, O.M.E., Szabo, I., Figler, M., Ludany, A., Juricskay, I., Szolcsanyi, J. (1999): Small doses of capsaicin given intragastrically inhibit gastric secretion in healthy human subjects. J. Physiol. Paris 93: 433-436 Mozsik, Gy., Figler, M., Rumi, Gy. (2000): Membrane-bound ATP dependent energy system and tissue hypoxia in human gastric mucosa. (Editorial) J. Gastroenterol. Hepatol. 15: 105-108 Mozsik, Gy., Karadi, O., Kiraly, A., Debrecni, A., Figler, M., Nagy, L., Par, A., Par, G., Siito, G., Vincze, A. (2001a): The key-role of vagal nerve and adrenals in the cytopotection and general gastric mucosal integrity. J. Physiol. Paris 95: 229-237 Mozsik, Gy., Nagy, Zs., Nagy, A., Rumi, Gy, Karadi, O , Czimmer, J., Matus, Z., Toth, Gy., Par, A. (2001b): Leiden mutation (as genetic) and environmental (retinoids) sequences in acute and chronic inflammatory and premalignant colon disease in human gastrointestinal tract. J. Physiol. Paris 95: 489^194 Mozsik, Gy., Belagyi, J., Szolcsanyi, J. (2004): Capsaicin-sensitive afferent nerves and gastric mucosal protection in the human healthy subjects. A critical overview. In: Takeuchi, K., Mozsik, Gy., (eds): Mediators in Gastrointestinal Protection and Repair. Research Signpost Kerala, India, pp. 43-62 Mozsik, Gy., Racz, I., Szolcsanyi, J. (2005a): Gastroprotection induced by capsaicin in healthy human subjects. World J. Gastroenterol. 11: 5180-5184 Mozsik, Gy., Rumi, Gy., Domotor, A., Figler, M., Gasztonyi, B., Papp, E., Belagyi, J., Matus, Z., Melegh, B. (2005b): Involvement of serum retinoids and Leiden mutation in patients with esophageal, gastric, liver, pancretic, and colorectal cancers in Hungary. World J. Gastroenterol. 11: 7646-7650 Mozsik, Gy., Domotor, A., Abdel-Salam, O.M.E. (2006): Molecular pharmacological approach to drug actions on the afferent and efferent fibres of the vagal nerve in the gastric mucosal protection in rats. Inflammopharmacology 14: 243-249 196

Mozsik, Gy., Domotor, A., Rumi, Gy., Szekeres, Gy. (2007a): Gastrointestinal cytoprotection: from basic science to clinical pespectives. (Review). Inflammopharmacology 15: 49-60 Mozsik Gy., Szolcsanyi, J., Domotor, A. (2007b): Capsaicin research as a new tool to approach of the human gastrointestinal physiology, pathology and pharmacology. (Review) Inflammopharmacology 15: 232-245 Muralidhara, T., Narasimhamurthy, K. (1988): Non-mutagenicity of capsaicin in albino mice. Food Chem. Toxicol. 26: 955-958 Murray, K.E., Whitfield, F.B. (1975): The occurrence of 3-alkyl-2-methoxypyrazines in raw vegetables. J. Sci. Food Agric. 26: 973-986 Nagabhushan, M., Blide, S.V. (1985): Mutagenecity of chili extract and capsaicin in short term tests. Environ. Mutagen. 7: 881-888 Nagabhushan, M., Bhide, S. V. (1986): Nonmutagenicity of curcumin and its antimutagenic action versus chili and Capsaicin. Nutr. Cancer 8: 201-210. Nagy, L., Mozsik, Gy., Feledi, E., Ruzsa, Cs., Vezekenyi, Zs., Javor, T. (1984): Gastric microbleeding measurements during one day treatment with indomethacin and indomethacin plus sodium salicylate (1:10) in patients. Acta Physiol. Hung. 64: 187-191 Nagy, Zs., Nagy, A., Karadi, O., Par, A., Mozsik, Gy. (2000): The high prevalence of factor V. Leiden mutation in Central European inflammatory bowel disease patients. Am. J. Gastroenterol. 95: 3013-3014 Nagy, Zs., Nagy, A., Karadi, O., Figler, M., Rumi, Gy., Jr., Siito, G., Vincze, A., Par, A., Mozsik, Gy. (2001): Prevalence of factor V. Leiden mutation in human inflammatory bowel diseases with different activity. J. Physiol. Paris 95: 483-^187 National Institute for Occupational Safety and Health (NIOSH). Manual of Analytical Methods (NMAM). 4th Edition, May 15, 1996. Capsaicin and dihydrocapsaicin: Method 5041 Nayini, J.R., Sugie, S., El-Bayoumy, K., Rao, V., Rigotty, J., Sohn, O., Reddy, B.S. (1991): Effect of dietary benzylselenocyanate on azoxymethane-induced colon carcinogenesis in male F344 rats. Nutr. Cancer 15: 129-139 Nelson, E.K. (1919): The constitution of capsaicin - the pungent principle of capsicum. J. Am. Chem. Soc. 41:1115-1121 Nelson, E.K., Dawson, L.E. (1923): The constitution of capsaicin - the pungent principle of capsicum. III. J. Am. Chem. Soc. 45: 2179-2181 Neumann, D. (1966): On the biosynthesis of capsaicin. (In German) Naturwissenschaften 53: 131-132 Newmark, H.L. (1984): A hypothesis for dietary components as blocking agents of chemical carcinogenesis: plant phenolics and pyrole pigments. Nutr. Cancer 6: 58-70 Newmark, H.L. (1987): Plant phenolics as inhibitors of mutational and precarcinogenic events. Can. J. Physiol. Pharmacol. 65: 461^166 Nopanitaya, W. (1974): Effects of capsaicin in combination with diets of varying protein content on the duodenal absorptive cells of the rat. Am. J. Dig. Dis. 19: 439-449 Nopanitaya, W., Nye, S.W. (1974): Duodenal mucosal response to the pungent principle of hot pepper (capsaicin) in the rat: Light and electron microscopic study. Toxicol. Appl. Pharmacol. 30: 149-161 197

Notani, P.N., Jayant, K. (1987): Role of diet in upper aerodigestive tract cancers. Nutr. Cancer 10: 203-113 Noy, N., Blaner, W.S. (1991): Interactions of retinol with binding proteins: studies with rat cellular retinol-bindign protein. Biochemistry 30: 6380-6386 Oi, Y., Kawada, T., Watanabe, T., Iwai, K. (1992): Induction of capsaicinhydrolyzing enzyme activity in rat liver by continuous oral administration of capsaicin. J. Agric. Food Chem. 40: 467-470 Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keough, J.R, Meyskens, F.L., Jr., Valanis, B., Wiliams, J.H., Jr., Barnhart, S., Hammar, S. (1996): Effects of a combination of P-carotene and vitamin A on lung cancer and cardiovascular disease. N. Engl. J. Med. 334: 1150-1155 Pabst, M.A., Schoninkle, E., Holzer, P. (1993): Ablation of capsaicin-sensitive afferent neurons impairs defense but not rapid repair of rat gastric mucosa. Gut 34: 897-903 Paganelli, G.M., Biasco, G., Brandi, G., Santucci, R., Gizzi, G., Villani, V., Cianci, M., Miglioli, M., Barbara L. (1992): Effect of vitamin A, C and E supplementation on rectal cell proliferation in patients with colorectal adenomas. J. Natl. Cancer Inst. 84: 47-51 Palacio, J.J.R. (1977): Spectrophotometric determination of capsaicin. J. Assoc. Off. Anal. Chem. 60: 970-972 Pan, H.L., Chen, S.R. (2004): Sensing tissue ischemia: amother new function for capsaicin receptors? Circulation 110: 1826-1831 Papa, A., Danese, S., Grillo, A., Gasbarrini, G., Gasbarrini, A. (2003): Review article: inherited thrombophilia in inflammatory bowel disease. Am. J. Gastroenterol. 98:1247-1251 Park, Y.H., Lee, S.S. (1994): Identification and characterization of capsaicinhydrolyzing enzymes purified from rat liver microsomes. Biochem. Mol. Biol. Int. 34:351-360 Parrish, M. (1996): Liquid chromatographic method of determining capsaicinoids in capsicums and their extractives: Collaborative study. J. Assoc. Off. Anal. Chem. Intern. 79 738-745 Patty, I., Benedek, S., Deak, G., Javor, T., Nagy, L., Simon, L., Tarnok, F , Mozsik, Gy. (1982): Controlled trial of vitamin A therapy in gastric ulcer. Lancet 2: 876 Peto, R., Doll, R. (1981): The Causes of Cancer. Oxford University Press, Oxford Pfander, H. (ed.) (1987): Key to Carotenoids. 2nd ed. Birkhauser, Basel Pickersgill, B. (1986) Evolution of hierarchical variation patterns under domestication and their taxonomic treatment. In: Styles, B.T. (ed.): Intraspecific Classification of Wild and Cultivated Plants. Systematic Association. Vol. 29. Clarenden, Oxford, pp. 191-209 Pickersgill, B. (1989): Genetic Resources of Capsicum for Tropical Regions. AVRD Publication No. 89-317, pp. 1-9 Pique, J.M., Esplugues, J.V., Whittle, B.J.R. (1990): Influence of morphine or capsaicin pretreatment on rat gastric microcirculatory response to PAF. Am. J. Physiol. 258: G352-357 Poppel, G. von Goldbohm, R.A. (1995): Epidemiologic evidence for beta-carotene and cancer prevention. Am. J. Clin. Nutr. 62: S1393-S1402 198

Potter, J.D. (1992): Reconciling the epidemiology, physiology, and molecular biology of colon cancer. JAMA 268: 1573-1577 Potter, J.D, Slattery, M.L., Bostick, R.M., Lowry, A. (1993): Colon cancer: a review of the epidemiology. Epidemiol. Rev. 15: 499-545 Pruthi, J.S. (2003): Chemistry and quality control of Capsicums and Capsicum products. Chapter 3. In: De, A.K. (ed.): Capsicum. The genus of Capsicum. Taylor and Francis, London, New York, pp. 25-70 Rahman, F.M.M., Buckle, K.A. (1980): Pigment changes in capsicum cultivars during maturation and ripening, J. Food Technol. 15: 241-249 Rainsford, K.D. (1996): Emerging research in gastrointestinal disease. In: Mozsik, Gy., Nagy, L., Par, A., Rainsford, K.D. (eds): Cell Injury and Protection in the Gastrointestinal Tract. From Basic Sciences to Clinical Perspectives. Kluwer Academic Publishers, Dordrecht, Boston, London, pp. 1-10 Rainsford, K.D. (2006): The Discovery and development of anti-inflammatory analgesic drugs. In: Mozsik, Gy. (ed.): Discoveries in Gastroenterology: from Basic Research to Clinical Perspectives (1960-2005). Akademiai Kiado, Budapest, pp. 363-392 Rajpoot, N.C., Govindarajan, V.S. (1981): Paper chromatographic determination of total capsaicinoids in capsicum and their oleoresins with precision, reproducibility and validation through correlation with pungency in Scoville units. J. Assoc. Off. Anal. Chem. 64: 311-318. Rauf, M., Bachmann, E., Metwally, S.A. (1985): Interaction of capsaicin with mixed function oxidases: ex-vivo and in-vivo studies. J. Drug Res. Egypt 16: 29-36 Ravishankar, G.A, Suresh, B., Giridhar, P., Ramachandra Rao, S., Sudhakar Johnson, T. (2003): Biotechnological studies on Capsicum for metabolite production and plant improvement. Chapter 6. In: De, A.K. (ed.): Capsicum. The genus of Capsicum. Taylor and Francis, London, New York, pp. 99-128 Raybould, H.E., Tache, Y. (1989): Capsaicin-sensitive vagal afferent fibres and stimulation of gastric acid secretion in anesthetized rats. Eur. J. Pharmacol. 167: 237-243 Reilly, C.A., Crouch, D.J, Yost, G.S. (2001a): Quantitative analysis of capsaicinoids in fresh peppers, oleoresin capsicum, and pepper spray products. J. Forensic Sci. 46: 502-509 Reilly, C.A., Crouch, D.J., Yost, G.S., Fatah, A.A. (2001b): Determination of capsaicin, dihydrocapsaicin and nonivamide in self-defense weapons by liquid chromatography-mass spectrometry. J. Chromatogr. A 912: 259-267 Reineccius, G. (1994): Source Book of Flavors. 2nd ed. Chapmann and Hall, New York, pp. 267-273 Reinshagen, M., Pate, A., Sottili, M., Nast, C , Davis, W., Mueller, K , Eysselein, V.E. (1994): Protective functions of extrinsic sensory neurons in acute rabbit experimental colitis. Gastroenterology 106: 1208-1214 Richeux, F , Cascante, M., Ennamary, I., Sabourcau, D., Creppy, E.E. (1999): Cytotoxicity and genotoxicity of capsaicin in human neuroblastoma cells SHSY-5Y. Arch. Toxicol. 73: 403-409 Ritchie, W.P., Jr. (1991): Mediators of bile acid induced alterations in gastric mucosal blood flow. Am. J. Surg. 161: 126-129 199

Robert, A., Nezamis, J, Lancaster, L A . , Hanchar, J. (1979): Cytoprotection by prostaglandin in rats. Prevention of gastric necrosis produced by alcohol, HCI, NaOH, hypertonic NaCl, and thermal injury. Gastroenterology 77: 433^143 Robert, A., Olafsson, A.S., Lancaster, C , Zhang, W. (1991): Effects of capsaicin and of capsaicin denervation on gastric secretion and gastric lesions produced by ulcerogenic agents. Exp. Clin. Gastroenterol. 1: 5 (abst.) Rodriguez-Amaya, D.B. (1997): Carotenoids and food preparation: The retention of provitamin A carotenoids in prepared, processed, and stored foods. John Snow, Inc./OMNI Project, 1997 Roncucci, L., Di Donato, P., Carati, L., Ferrari, A., Perini, M., Bertoni, G., Bedogni, G., Paris, B., Svanoni, E, Girola, M., Ponz de Leon, M. (1993): Antioxidant vitamins or lactulose for the prevention of the recurrence of colorectal adenomas. Dis. Colon Rectum 36: 227-234 Roosterman, D., George, T , Schneider, S. W., Bunnett, N., Steinhoff, M. (2006): Neural control of skin function: the skin as a neuroimmunoendocrine organ. Physiol. Rev. 86: 1309-1379 Ross, A.C., Ternus, M.E. (1993): Vitamin A as a hormone: recent advances in understanding the actions of retinol, retinoic acid and (3 carotene. J. Am. Diet. Assoc. 93:1285-1290 Rowe, R., Sheskey, P., Weller, P. (eds) (2005): Handbook of Pharmaceutical Excipients. 5th ed. USA Rozin, P. (1990): Getting to like the burn of chili pepper. Biological, physiological, and cultural perspective. In: Green, B.G., Manson, J.R., Kare, M.R. (eds): Chemical Senses. Irritation. Vol 2. Marcel Dekker, New York, pp. 231-269 Rumi, Gy., Jr., Szabo, I., Matus, Z., Vincze, A., Toth, Gy., Rumi, Gy., Mozsik, Gy. (1999): Decrease in serum levels of vitamin A and zeaxanthin in patients with colorectal polyp. Eur. J. Gastroenterol. Hepatol. 11: 305-308 Rumi, Gy., Jr., Szabo, I., Vincze, A., Matus, Z., Toth, Gy., Rumi, Gy., Mozsik, Gy. (2000): Decrease in serum carotenoids in Crohn's disease. J. Physiol. Paris 94: 159-161 Rumi, Gy., Jr., Par, A., Matus, Z., Rumi, Gy., Mozsik, Gy. (2001): The Defensive Effects of Retinoids in the Gastrointestinal Tract (Animal Experiments and Human Observations). Akademiai Kiado, Budapest Russel, L.C, Burchiel, K.J. (1984): Neurophysiological effects of capsaicin. Brain Res. Rev. 8: 165-176 Saari, J . C (1991): Enzymes and proteins of the mammalian visual cycle. In: Osborne, N.N., Charder, G.J. (eds): Progress in Retinal Research. Vol. 9. Pergamon Press, Oxford, pp. 363-381 Saito, A., Yamamoto, M. (1996): Acute oral toxicity of capsaicin in mice and rats. J.Toxicol. Sci. 21: 195-200 Salonen, J.T., Salonen, R., Lappetelainen Maepaa, PH., Alfthan, G., Puska, J. (1985): Risk of cancer in relation to serum concentration of selenium and vitamin A and E: matched case-control analysis of prospective data. Br. Med. J. 290: 417^42 Sandmann, G. (2001): Carotenoid biosynthesis and biotechnological application. Arch. Biochem. Biophys. 385: 4-12 200

Sarlos, P., Rumi, Gy., Szolcsanyi, J., Mozsik, Gy., Vincze, A. (2003): Capsaicin prevents the indomethacin-induced gastric mucosal damage in human healthy subjects. Gastroenterology 124, Suppl. 1, A-511 Satsangi, J., Jewell, D.P. (1996): Genetic markers in inflammatory bowel disease. Curr. Opin. Gastroenterol. 12: 322-326 Satyanarayana, M.N. (2006): Capsaicin and gastric ulcers. Crit. Rev. Food. Sci. Nutr. 46: 275-328 Savitha, G , Panchanathan, S, Salimath, B.P. (1990): Capsaicin inhibits calmodulin-mediated oxidative burst in rat macrophages. Cell Signal 2: 577-585 Schneider, M.A., de Luca, V, Gray, S.J. (1956): The effect of spice ingestion upon the stomach. Am. J. Gastroenterol. 26: 722-732 Schweiggert, U., Carle, R., Schieber, A. (2006a): Characterization of major and minor capsaicinoids and related compounds in chili pods {Capsicum frutescens L.) by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry. Anal. Chim Acta 557: 236-244 Schweiggert, U., Schieber, A., Carle, R. (2006b): Effects of blanching and storage on capsaicinoid stability and peroxidase activity of hot chili peppers (Capsicum frutescens L.). Innov. Food Sci. Emerg. Technol. 7: 217-224 Scoville, W.L. (1912): Note on Capsicum. J. Am. Pharm. Assoc. 1: 453^154 Semba, R.D. (1997): Impact of vitamin A on immunity in developing countries. In: Benedich, A., Deckelbaum, R.J. (eds): Preventive Nutrition. Humana Press, New Jersey, pp. 337-351 Sherman, J.A., Partidge, M. (1997): Expression of retinoic acid receptors in normal, dysplastic and malignant oral epithelia. Br. J. Oral Maxillofac. Surg. 35: 260-266 Simon, J.E. (1990): Essential oils and culinary herbs. In: J. Janick, J., Simon, J.E. (eds): Advances in New Crops. Timber Press, Portland, OR, pp. 472-483 Singh, S., Asad, A. E, Ahmad, A., Khan, N.U., Hadi, S.M. (2001): Oxidative DNA damage by capsaicin and dihydrocapsaicin in the presence of Cu(II). Cancer Lett. 169: 139-146 Smith, J.E., Milch, P.O., Muto, Y., Goodman, D.S. (1973): The plasma transport and metabolism of retinoic acid in the rat. Biochem. J. 132: 821-827 Solanke, T.F. (1973): The effect of red pepper (Capsicum frutescens) on gastric acid secretion. J. Surg. Res. 15: 385-390 Somogyi, N., Moor, A., Pek, M. (2003): The preservation and production of Capsicum in Hungary. In: De, A. K. (ed.): Capsicum. The genus Capsicum. Taylor and Francis Ltd., London, New York, pp. 144-162 Somos, A. (1981): A paprika. (The paprika). (In Hungarian) Akademiai Kiado, Budapest Soprano, D.R., Blaner, W.S. (1994): Plasma-retinol-binding protein. In: Sporn, M.B., Roberts, A.B., Goodman, D.S. (eds): The Retinoids: Biology, Chemistry, and Medicine. 2nd ed. Raven Press, New York, pp. 257-281 Spanyar, P., Blazovich, M. (1969): Thin-layer chromatography method for the determination of capsaicin. Analyst 94: 1084-1090 Spanyar, P., Kevei, E., Kiszel, M. (1957): Determination of capsaicin. Acta Chim. Acad. Sci. Hung. 11: 137-142 201

Spurgeon, S.L., Porter, J.W. (1983): Biosynthesis of carotenoids. In: Porter, J.W., Spurgeon, S.L. (eds): Biosynthesis of Isoprenoid Compounds. Vol. 2. Wiley, New York, pp. 1-122 Stahelin, H.B., Gey, K.F., Eichholzer, Z., Liidin, E., Bernasconi, E, Thurneysen, J., Brubacher, G. (1991): Plasma antioxidant vitamins and subsequent cancer mortality in the 12-year follow-up of the Prospective Basle Study. Am. J. Epidemiol. 133:766-775 Starlinger, M., Schiessel, R., Hung, C.R. (1981): H back-diffusion stimulating mucosal blood flow in the rabbit fundus. Surgery 89: 232-236 Sticher, O., Soldati, E, Joshi, R.K. (1978): High performance liquid chromatography separation and quantitative determination of capsaicin, dihydrocapsaicin, nordihydrocapsaicin and homodihydrocapsaicin in natural capsaicinoid mixtures of Capsicum fruits. (In German) J. Chromat. 166: 221-231 Strike, D.J., Meijerink, M.G.H., Koudelka-Hep, M. (1999): Electronic noses - A mini review. Fresenius' Anal. Chem. 364: 499-505 Surh, Y.J., Lee, S.S. (1995): Capsaicin, a double-edged sword: toxicity, metabolism, and chemopreventive potential. Life Sci. 56: 1845-1855 Surh, Y.J., Lee, S.S. (1996): Capsaicin in hot chili pepper: Carcinogen, co-carcinogen or anticarcinogen? Food Chem. Toxicol. 34: 313-316 Surh, Y.J., Ahn, S.H., Kim, K.C., Park, J.B., Sohn, Y.W., Lee, S.S. (1995): Metabolism of capsaicinoids: evidence for alipharic hydroxylation and its pharmacological implications. Life Sci. 56: PL305-311 Suzuki, J.I., Tausing, F , Morse, R.E. (1957): Some observations on red pepper. I. A new method for the determination of pungency in red pepper. Food Technol. 11: 100-104 Suzuki, T , Iwai, K. (1984): Constituents of red pepper species: Chemistry, biochemistry, pharmacology and food science of the pungent principle of Capsicum species. In: Brossi, A. (ed.): The Alkaloids. Academic Press Inc., New York, pp. 227-299 Szolcsanyi, J. (1977): A pharmacological approach to elucidation of the role of different nerve fibres and receptor endings in mediation of pain. J. Physiol. Paris 73: 251-259 Szolcsanyi, J. (1982): Capsaicin type pungent agents producing pyrexia. In: Milton, A.S. (ed.): Handbook of Experimental Pharmacology. Vol. 60. Pyretics and Antipyretics. Springer-Verlag, Berlin, pp. 437-478 Szolcsanyi, J. (1983a): Disturbances of thermoregulation induced by capsaicin. J. Therm. Biol. 8: 207-212 Szolcsanyi, J. (1983b): Tetrodotoxin-resistant non-cholinergic neurogenic contraction evoked by capsaicinoids and piperine on the guinea-pig trachea. Neurosci. Lett. 42: 83-88 Szolcsanyi, J. (1984a): Capsaicin and neurogenic inflammation: History and early findings. In: Chahl, L. A., Szolcsanyi, J., Lembech, F. (eds): Antidromic Vasodilatation and Neurogenic Inflammation. Akademiai Kiado, Budapest, pp. 7-26 Szolcsanyi, J. (1984b): Capsaicin-sensitive chemoceptive neural system with dual sensory-efferent function. In: Chahl, L.A., Szolcsanyi, J., Lembech, F. (eds): +

202

Antidromic Vasodilatation and Neurogenic Inflammation. Akademiai Kiado, Budapest, pp. 27-56 Szolcsanyi, J. (1985): Sensory receptors and the antinociceptive effect of capsaicin. In: Hakanson, R., Sundler, F. (eds): Tachykinin Antagonists. Elsevier, Amsterdam, pp. 45-54 Szolcsanyi, J. (1990a): Capsaicin, irritation, and desensitization: Neurophysiological and future perspectives. In: Green, B.G., Mason, J.R., Kare, M.R. (eds): Chemical Senses. Irritation. Vol. 2. Marcel Dekker, New York, pp. 141-169 Szolcsanyi, J. (1990b): Effect of capsaicin, rediniferatoxin and piperine on ethanol-induced gastric ulcer of the rat. Acta Physiol. Hung. 75: 267-268 Szolcsanyi, J. (1993): Actions of capsaicin on sensory receptors. In: Wood, J.N. (ed.): Capsaicin in the Study of Pain. Academic Press, London, pp. 1-33 Szolcsanyi, J. (1996): Capsaicin-sensitive sensory nerve terminals with local and systemic efferent functions: facts and scopes of unorthodox neuroregulatory mechanisms. In: Kumazawa, T , Kumazawa, L., Mizumura, K., (eds): The Polymodal Receptor - A Gateway to Pathological Pain. Progress in Brain Research. Vol.113. Elsevier, Amsterdam, pp. 343-359 Szolcsanyi, J. (2004): Forty years in capsaicin research for sensory pharmacology and physiology. Neuripeptides 38: 371-384 Szolcsanyi, J., Bartho, L. (1981): Impaired defense mechanisms to peptic ulcer in the capsaicin-desensitized rat. In: Mozsik, Gy., Hanninen, O., Javor, T. (eds): Advances in Physiological Sciences. Vol. 29. Gastrointestinal Defense Mechanisms. Pergamon Press and Akademiai Kiado, Oxford and Budapest, pp. 39-51 Szolcsanyi, J., Porszasz, R., Petho, G. (1994): Capsaicin and pharmacology of nociceptors. In: Besson, J.M., Besson, G., Ollat, H. (eds): Peripheral Neurons in Nociception. Physicopharmacological Aspects. John Libby Eurotext, Paris, pp. 109-124 Sziits, E.Z., Harosi, F.I. (1991): Solubility of retinoids in water. Arch. Biochem. Biophys. 287: 297-304 Takeuchi, K., Ohuchi, T , Okabe, S. (1994): Capsaicin-sensitive sensory neurons in healing of gastric lesions induced by HCI in rats. Dig. Dis. Sci. 39: 2543-2546 Takeuchi, K , Kato, S., Takehara, K. (1996): Analyses of pathogenic elements involved in gastric lesions induced by indomethacin in rats. In: Mozsik, Gy., Nagy, L., Par, A., Rainsford, K.D. (eds): Cell Injury and Protection in the Gastrointestinal Tract. From Basic Sciences to Clinical Perspectives. Kluwer Academic Publishers, Dordrecht, Boston, London pp. 1-10 Talbot, R.W., Heppell, J., Dozois, R.R., Beart, R.W., Jr. (1986): Vascular complications of inflammatory bowel disease. Mayo Clin. Proc. 61: 140-145 Tan, B., Chu, FL. (1991): Effects of palm carotenoids in rat hepatic cytocrome P450-mediated benzo(a)pyrene metabolism. Am. J. Clin. Nutr. 53: 1071-1075 Taurog, A., Dorris, M., Doerge, D.R. (1994): Evidence for a radical mechanism in peroxidase-catalyzed coupling. I. Steady-state experiments with various peroxidases. Arch. Biochem. Biophys. 315: 82-87 The United States Pharmacopeia Convention, INC. (2005): 12601 Twinbrook Parkway, Rockwille (USP 27-NF 22)

203

Thomas, B.V., Schreiber, A.A., Weisskopf, CP. (1998): Simple method for quantitation of capsaicinoids in peppers using capillary gas chromatography. J. Agric. Food Chem. 46, 2655-2663 Thomson, R.Q., Phinney, K.W., Sander, L.C, Welch, M.J. (2005): Reversed-phase liquid chromatography and argentation chromatography of minor capsaicinoids, Anal. Bioanal. Chem. 381: 1432-1440 Thresh, L.T. (1846): Isolation of capsaicin. Pharmacol. J. 6: 941 Tirimanna, A.S.L. (1972): Quantitative determination of the pungent principle capsaicin of Ceylon chillies. Analyst 97: 372-380 Todd, P.A., Clissold, S.P. (1990): Naproxen. A reappraisal of its pharmacology, and therapeutic use in rheumatic disease and pain states. Drugs 40: 91-137 Todd, PH., Jr., Bensinger, M., Biftu, T. (1975): TLC screening techniques for qualitative determination of natural and synthetic capsaicinoids. J. Chrom. Sci. 13: 577-580 Todd, PH., Jr., Bensinger, M.G., Biftu, T.J. (1977): Determination of pungency due to capsicum by gas liquid chromatography. J. Food Sci. 42: 660-665 Tominata, M., Caterina, M.J., Malmberg, A.B., Rosen, T.A., Gilbet, H., Skinner, K., Raumann, B.E., Basbaum, A.I., Julius, D. (1998): The cloned capsaicin receptor integrates multiple pain-producing stimuli. Neuron 21: 531-543 Toth, B., Gannett, P. (1992): Carcinogenicity of lifelong administration of capsaicin of hot pepper in mice. In Vivo 6: 59-64 Toth, B., Rogan, E., Walker, B. (1984): Tumorigenicity and mutagenicity studies with capsaicin of hot peppers. Anticancer Res. 4: 117—120 Tramontana, M., Renzi, D., Calabro, A., Panerai, C , Milani, S., Surrenti, C , Evangelista, S. (1994): Influence of capsaicin-sensitive afferent fibres on acetic acidinduced chronic gastric ulcers in rats. Scand. J. Gastroenterol. 29: 406-413 Vaishnava, P., Wang, D.H. (2003): Capsaicin sensitive-sensory nerves and blood pressure regulation. Curr. Med. Chem. Cardiovasc. Hematol. Agents 1: 177-188 Varga, L. (1936): Action of various stimulants on gastric chemistry. (In Hungarian) Orv. Hetil. 80: 702-704 Venkat, J.A., Shami, S., Davis, K., Nayak, M., Plimmer, J.R., Pfeil, R. Nair, P.P. (1995): Relative genotoxic activities of pesticides evaluated by a modified SOS microplate assay. Environ. Mol. Mutagen. 25: 67-76 Veszelszky, A. (1798): A noveny-plantak orszagabol valo eredeti gyujtemeny, vagyis fa es fuszeres konyv. (Original collection from the land of plants, a book of trees and spices.) (In Hungarian) Trattner, Pest Villasenor, I.M., De Ocampo, E. (1994): Clastogenicity of red pepper (Capsicum frutescens L.) extracts. Mutat. Res. 312: 151-155 Villasenor, I.M., De Ocampo, E.J., Bremner, J.B. (1995): Genotoxic acetamide(s) from Capsicum frutescens fruits. Natural Prod. Lett. 6: 247-253 Vincze, A., Garamszegi, M., Javor, T , Siito, G., Toth, Gy., Zsoldos, T., Mozsik, Gy. (1991): The role of oxygen related free redicals in P-carotene and prostacyclin induced gastric cytoprotection in rats. Eur. J. Gastroenterol. 3: 175-179 Vincze, A., Kiraly, A., Siito, G., Karadi, O., Mozsik, Gy. (1997): Role of neurohormonal and local mucosal factors in P-carotene-induced gastroprotection in the

204

rat. In: Mozsik, Gy., Nagy, L., Kiraly, A. (eds): Twenty-Five Years of Peptic Ulcer Research in Hungary. Akademiai Kiado, Budapest, pp. 265-273 Viranuvatti, V., Kalayasiri, C , Chearani, O. (1972): Effect of capsicum solution on human gastric mucosa as observed gastroscopically. Am. J. Gastroenterol. 58: 225-232 Vogelstein, B., Knizler, K.W. (1993): The multi-step nature of cancer. Trends Genet. 9: 138-141 Waliszewski, P., Waliszewska, M.K., Gupta, M. (1997): Expression of retinoidresponsive genes occurs in colorectal carcinoma-derived cells irrespective of the presence of resistance to all-trans retinoic acid. J. Surg. Oncol. 66: 156-167 Wall, M.M., Bosland, P.W. (1998): Analytical methods for color and pungency of chilies (capsicums). In Wetzel, D., Charalambous, G. (eds): Instrumental Methods in Food and Beverage Analysis. Elsevier, Amsterdam, pp. 347-373 Walpole, C.S., Wrigglesworth, R., Bevan, S., Campbell, E.A., Dray, A., James, I.F., Perkins, M.N., Raid, D.J., Winter, J. (1993a): Analogs of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 1. The aromatic "A-region". J. Med. Chem. 36: 2362-2372 Walpole, C.S., Wrigglesworth, R., Bevan, S., Campbell, E.A., Dray, A., James, I.F., Masdin, K.J., Perkins, M.N., Winter, J. (1993b): Analogs of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 2. The amide bond "B-region". J. Med. Chem. 36: 2373-2380 Walpole, C.S., Wrigglesworth, R., Bevan, S., Campbell, E.A. Dray, A., James, I.F., Masdin, K.J., Perkins, M.N., Winter, J. (1993c) Analogs of capsaicin with agonist activity as novel analgesic agents; structure-activity studies. 3. The hydrophobic side chain "C-region". J. Med. Chem. 36: 2381-2389 Wehmeyer, Y, Kasting, G.B., Powell, J.H., Kuhlenbeck, D.L., Underwood, R.A., Bowman, L.A. (1990): Applications of liquid chromatography with on-line radiochemical detection to metabolism studies on a novel class of analgesic. J. Pharm. Biomed. Anal. 8: 177-183 West, K.P., Jr., Sommer. A. (1984): Periodic, large oral doses of vitamin A for the prevention of vitamin A deficiency and xerophthalmia: a summary of experiences. Nutrition Foundation, Washington, DC, pp. 1^44 Whitington, P.F., Freese, D.K., Alonso, E.M., Schwarzenberg, S.J., Sharp, H.L. (1994): Clinical and biochemical findings in progressive familial intrahepatic cholestasis. J. Pediatr. Gastroenterol. Nutr. 18: 134-141 Whittle, B.J.R. (1977): Mechanisms underlying gastric mucosal damage induced by indomethacin and bile salts, and the action of prostaglandins. Br. J. Pharmacol. 60:455-460 Whittle, B.J.R., Lopez-Belmonte, J. (1991): Interactions between the vascular peptide endothelin-1 and sensory neuropeptides on gastric mucosal injury. Br. J. Pharmacol. 102: 950-954 Willett, W.C., (1991): Nutritional Epidemiology. Oxford University Press, New York Winter, J., (1987): Characterization of capsaicin sensitive neurons in adult rat dorsal root ganglion culture. Neurosci. Lett. 80: 134-140

205

Wolbach, S.B., Howe, P.R., (1928): Vitamin A deficiency in the guinea-pig. Arch. Pathol. 5: 239-253 Wood, J.N, Winter, J., James, I.F., Rang, H.Ph., Yeats, J., Bevan, S. (1988): Capsaicin induced ion fluxes in dorsal root ganglion neurons in culture. J. Neurosci. 8:3208-3220 Woodall, A.A., Britton, G., Jackson, M.J. (1997): Carotenoids and protection of phospholipids in solution or in liposomes against oxidation by peroxyl radicals: Relationship between carotenoid structure and protective ability. Biochim. Biophys. Acta 1336: 575-586 Woodbury, J.E. (1980): Determination of capsicum pungency by high pressure liquid chromatography and spectrofluorometric detection, J. Assoc. Off. Anal. Chem. 63: 556-558 Yagi, T. (1990): Inhibition by capsaicin of NADH-quinone oxidoreductases is correlated with the presence of energy-coupling site 1 in various organisms. Arch. Biochem. Biophys. 281: 305-311 Yamada, M., Blaner, W.S., Soprano, D.R., Dixon, J.L., Kjeldbye, H.M., Goodman, D.S. (1987): Biochemical characteristics of isolated rat stellate cells. Hepatology 7: 1224-1229 Zatyko, L. (1979): Paprikatermesztes. (Cultivation of paprika.) (In Hungarian) Mezogazdasagi Kiado, Budapest Zhang, X.K., Liu, Y , Lee, M.O., Sing, Z. (1996): Retinoid receptors in human lung cancer and breast cancer. Mutat. Res. 350: 267-277 Ziegler, R.G. (1991): Vegetables, friuts and carotenoids and the risk of cancer. Am. J. Clin. Nutr. 53: 251-259 Zurakowski, D., Law, T., Goldberg, N.E., Leichtner, A.M. (1997): Fat soluble vitamins in children with malabsorption. J. Pediatr. Gastroenterol. Nutr. 17: 12-16

206

Appendix

1. European Commission (Health and Consumer Protection Directorate General Directorate-C Scientific Opinion): C2 Management of Scientific Committees II Scientific Cooperation and Networks (http://ec.europa.eu/food/fs/sc/scf/out120_en.pdf) 1.1 Opinion of the Scientific Committee on food on capsaicin Terms of reference The Committee is asked to advise the Commission on substances used as flavoring substances or present in flavorings or present in other food ingredients with flavoring properties for which existing toxicological data indicate that restrictions of use or presence might be necessary to ensure safety for human health. In particular, the Committee is asked to advise the Commission on the implications for human health of capsaicin in the diet. Introduction Previous evaluations The Committee of Experts on Flavouring Substances of the Council of Europe evaluated the capsaicinoids in Capsicum preparations used as flavourings. A TDI of 0-0.2 mg/kg bw, expressed as total capsaicinoids, was numerically derived from the results of a population based case-control study conducted in Mexico City, where chilli pepper consumers were at high risk for gastric cancer compared with non-consumers. The daily intake of the chilli pepper consumers was estimated to be 4 mg capsaicinoids/kg bw and a safety factor of 20 was applied. In addition, general limits of 5 ppm for foods and beverages, 10 ppm for hot foods and beverages, 20 ppm for hot ketchup and 50 ppm for tabasco, harissa, hot pimento oils and similar preparations expressed as total capsaicinoids were suggested (Council of Europe, 2001). Current regulatory status Capsaicin is listed in the register of chemically defined flavouring substances laid down in Commission Decision 1999/217/EC (EC, 1999), as last amended by Commission Decision 2002/113/EC (EC, 2002). 207

Chemical characterisation Name: Capsaicin (N-(4-hydroxy-3-methoxybenzyl)-8-methyl-trans-6-nonenamide) Capsaicin in Capsicum preparations is always accompanied by other capsaicinoids: mainly dihydrocapsaicin, but also small amounts of nordihydro-, homo-, homodihydro-, nor-, and nornorcapsaicin. The capsaicinoids present in the Capsicum fruit are predominantly capsaicin and dihydrocapsaicin, making up 80 to 90%. The ratio of capsaicin to dihydrocapsaicin is generally around 1:1 and 2:1 (Govindarajan, Sathyanarayana, 1991). Synonyms: 8-Methylnon-6-enoyl-4-hydroxy-3-methoxybenzylamide; trans-8 methyl-N-vanillyl-6-nonenamide; Isodecenoic acid vanillylamide (Fig. 32) FL No: 16.014 CAS No: 404-86-4 FEMANo: 3404 CoENo: 2299 EINECS: 206-969-8 Structure:

Fig. 32.

8-Methylnon-6-enoyl-4-hydroxy-3-methoxybenzylamide; trans-8-methyl-N-vanillyl-6-nonenamide; Isodecenoic acid v a n i l l y l a m i d e

Exposure assessment Capsaicinoids are mainly ingested as naturally occurring pungency-producing components of capsicum spices (chilli, cayenne pepper, red pepper). They typically range from 0.1 mg/g in chilli pepper to 2.5 mg/g in red pepper and 60 mg/g in oleoresin red pepper (Parrish, 1996). Pepper varieties from Capsicum frutescens, annuum and chinense were found to contain 0.22-20 mg total capsaicinoids/g of dry weight (Thomas et al., 1998). Cayenne pepper samples had mean capsaicin and dihydrocapsaicin contents of 1.32 and 0.83 mg/g dry weight, respectively (Lopez-Hernandez, 1996). The consumption of capsicum spices was reported to be 2.5 g/person/day in India, 5 g/person/day in Thailand (Monsereenusorn, 1983; Monsereenusorn et al., 1982) and 20 g/person (one chilli pepper) per day in Mexico (Lopez-Carrillo, 1994). Assuming a content of capsaicinoids in these spices of about 1%, the daily intake of capsaicinoids in these countries has been estimated to be 25-200 mg/person/day or in the case of a person with 50 kg body weight 0.5-4 mg/kg bw/day (Council of Europe, 2001). The maximum daily intake of capsaicin in the U.S. and Europe from mild chillies and paprika was roughly estimated to be 0.025 mg/kg bw (Govindarajan, Sathyanarayana, 1991), equivalent to 1.5 mg/person/day. According to a recent estima208

tion, the mean and maximum intake of capsaicin from industrially prepared food products containing the recommended general limit of 5 pm would be 0.77 and 2.64 mg/day, respectively (CREDOC/OCA, 1998). Hazard identification/characterisation Capsaicin and other members of the group of capsaicinoids produce a large number of physiological and pharmacological effects such as effects on the gastrointestinal tract, the cardiovascular and respiratory system as well as the sensory and thermoregulation system. These effects result principally from the specific action of capsaicinoids on primary afferent neurons of the C-fiber type. This provides the rationale for their use to treat some peripheral painful states, such as rheumatoid arthritis (Surh, Lee, 1995) In addition, capsaicinoids are powerful irritants, causing burn and pain at low concentrations on the skin and mucous membranes. Given orally, they induce an increase of salivation and gastric secretion, a rapid change of sensation, warm to intolerable burning, and gastrointestinal disorders depending on the dose (Govindarajan, Sathyanarayana, 1991). Absorption, distribution, metabolism and excretion Capsaicinoids, when administered to rats intragastrically are readily absorbed and metabolized to a great extent in the liver before reaching the general circulation and extrahepatic organs (Donnerer et al., 1990). In vitro and in vivo studies have shown that capsaicinoids are metabolized by different pathways: (1) hydrolysis of the acidamide-bond and oxidative deamination of the formed vanillylamine, (2) hydroxylation of the vanillyl ring, possibly via epoxidation, (3) one electron oxidation of the ring hydroxyl forming phenoxy radicals and capsaicinoid dimers, (4) oxidation at the terminal carbon of the side chain (Surh, Lee, 1995). Within 48 hrs after oral administration of dihydrocapsaicin to male rats, 8.7% of the dose were excreted unchanged in urine and 10% in faeces. Metabolites found in urine were vanillylamine (4.7%), vanillin (4.6%) vanillyl alcohol (37.6%) and vanillic acid (19.2%) in free form or as glucuronides (Kawada, Iwai, 1985). Based on results of Miller et al. (1983), who demonstrated the covalent binding of dihydrocapsaicin to hepatic microsomal proteins, the formation of electrophilic intermediates (arene epoxides, phenoxy radicals or quinone type derivatives formed after O-demethylation) and subsequent covalent binding to cellular macromolecules is discussed to play a role in the etiology of capsaicin-induced toxicity including mutagenicity and carcinogenicity (Surh, Lee, 1995). Acute toxicity The acute toxicity of capsaicin shows a large variation depending on the route of administration. In male mice, the LD 50 varies from 0.56 mg/kg bw (i.v.) to 60-75 mg/kg bw (in ethanol) and 190 (122-294) mg/kg bw (in dimethyl sulfoxide), following intragastric intubation. The possible cause of death was considered to be due to respiratory paralysis (Glinsukon et al. 1980). Intraduodenal and intragastric administration of 10% Capsicum as well as 0.014% capsaicin in 0.85% saline to male rats produced morphological damages in the duodenal mucosa (Nopanitaya, Nye, 1974). 209

Subacute/subchronic toxicity A 4-week feeding study with groups of 5 male B6C3F1 mice with 0, 0.5, 1.0, 2.5, 5.0, 7.5, and 10% ground red chilli (Capsicum annuum) in the diet showed slight glycogen depletion and anisocytosis of hepatocytes in the 10% group. Other organs did not reveal any lesions. General health, body weight and food intake were not adversely affected (Jang, Kim 1988; Jang et al., 1992). Groups of 10-14 rats were fed by stomach tube with 50 mg/kg bw/day capsaicin or 0.5 g/kg bw/day Capsicum extract for 10-60 days. There were significant reductions of growth, plasma urea, glucose, phospholipids, triglycerides, total cholesterol, free fatty acids, glutamic pyruvic transaminase, and alkaline phosphatase in both groups with a tendency for Capsicum treated animals to show more adverse effects. No gross pathological changes and no differences in organ weights from control values were observed at autopsy, only a slight hyperemia in the livers and reddening with increasing mucous materials in the gastric mucosa. The organs, however, were not examined histopathologically (Monsereenusorn, 1983). BALB/c mice received an alcoholic chilli extract in drinking water 5 days a week till 16 months of age (27 males, 25 pg capsaicin/week, equivalent to about 0.125 mg/kg bw/d) or on the tongue 2 days a week for 14 months (22 males without and 19 males with 1% atropin solution prior to application, 50 pg capsaicin/week, equivalent to about 0.25 mg/kg bw/d). Compared with 40 untreated mice, the treated animals showed increased mortality and histopathological changes in liver, kidneys, stomach and tongue. The lesions in the liver observed in all treated mice were in the form of focal necrosis with inflammatory cells around, fatty changes and fibrosis (Agrawal, Bhide, 1987). 36 male Syrian hamsters received 20 pi alcoholic chilli extract with 50 pg capsaicin (equivalent to about 0.5 mg/kg bw/d) 5 days a week by cheek pouch application for 14 months. 30 untreated animals and 17 hamsters treated with 20 pi alcohol were used as controls. The animals treated with chilli extract had increased mortality and histopathological lesions in liver, kidneys, stomach and cheek pouch. The main lesions were liver cirrhosis, observed in 49 % of examined livers from exposed hamsters compared to 8 and 17 % in the control groups and glomeruli degeneration in 50% of examined kidneys of exposed animals compared to 8 and 0 % in the control groups, respectively (Agrawal, Bhide, 1988). Capsaicin, administered intraperitoneally to adult male mice at doses of 0.4, 0.8 or 1.6 mg/kg bw/day on 5 consecutive days, did not induce significant alterations in epididymal weights, caudal sperm counts, testicular weights or testicular histology. In the sperm morphology assay, sperms at 1, 3, 5 and 7 weeks did not reveal any treatment-related increase in the incidence of sperm-head abnormalities (Muralidhara, Narasimhamurthy, 1988). In a 13-week study performed to determine the maximum tolerated dose, groups of 10 male and 10 female B6C3F1 mice received a mixture of 64.5% capsaicin and 32.6% dihydrocapsaicin at those levels of 0, 0.0625, 0.125, 0.25, 0.5, and 1% in the diet. Significant reduction of food intake and body weight gain in all dose groups, especially in treated females, and significantly increased liver/body weight ratios of both sexes and renal toxicity (focal tubular dilatation) in the 1 % treated males were observed (Akagi et al., 1998). 210

Capsaicin (purum) was administered at concentrations of 0.0625, 0.125, 0.25, 0.5 and 1% in the diet of groups of 4 male and 4 female Swiss albino mice for 35 days. When the animals died at an age of 62-126 weeks, one adenocarcinoma of the duodenum had developed at each dose level, except for the highest dose, while no such tumours occurred in a historical control group of 100 males and 100 females. There was no concurrent control group and the observed tumour incidence was not doserelated (Toth, Gannett, 1992; Toth et al., 1984). Chronic toxicity/carcinogenicity 15 out of 26 rats fed for seven months with 10% chillies in a semisynthetic diet containing ardein, a purified protein of the ground nut, developed neoplastic changes in the liver (hepatomas, multiple cystic cholangiomas, solid adenomas or adenocarcinomas of the bile duct). Although no tumour developed in rats fed the basic diet without chillies, the authors stress, that it cannot be said whether chillies have a specific carcinogenic effect or whether a deficiency in the diet aggravated by a non-specific irritant caused the tumours (Hoch-Ligeti, 1951). Capsaicin (capsaicin 65%, dihydro- 31%, nordihydro- 0.9%, homo- 1%, homodihydro- 0.6%, nor- 0.5%, nornor- 0.3%), administered in a semisynthetic diet at 0.03125% to 50 male and 50 female Swiss albino mice for their life span from 6 weeks of age, induced benign polypoid adenomas of the caecum in 22% of females (p <0.05) and 14% of males, compared to 8% in the untreated female and male controls (incidence of historical controls not given). The survival rate was not substantially altered (Toth, Gannet, 1992). Groups of 50 male and 50 female B6C3F1 mice were given 0, 0.025, 0.083, and 0.25% capsaicinoid mixture (64% capsaicin and 32.6% dihydrocapsaicin) in the diet for 79 weeks, equivalent to daily doses of up to 220 and 200 mg/kg bw in males and females, respectively. In all dose groups, food intake was significantly reduced, in females also the body weight gain and in males the liver/body weight ratio. No evidence of carcinogenicity was found. Renal cell adenomas developed only in one male mouse of the 0.025 and 0.25% groups. (Akagi et al., 1998). A number of studies have shown that capsaicin or chilli extract can act as tumour promoters (Surh, Lee, 1995, 1996). Thus, capsaicin (0.002% in drinking water for 6 weeks) has been reported to act as a promoter for the development of diethylnitrosamine-initiated enzyme-altered foci in the liver of male rats (Jang, Kim, 1988). Chilli extract has also been shown to have a promoting effect on the development of stomach and liver tumours in BALB/c mice initiated by methyl-acetoxy methylnitrosamine and benzene hexachloride, respectively (Agrawal et al., 1986). In another study, rats fed diets containing hot chilli pepper showed slightly higher incidence of N-methyl-N-nitrosoguanidine-induced gastric cancer (Kim et al., 1985). On the other side, capsaicin has been suggested to exert chemoprotective effects through modulation of metabolism of carcinogens and their interaction with target cell DNA (Surh, Lee, 1995, 1996). Genotoxicity Capsaicin (purum) was found to be mutagenic in Salmonella typhimurium strain TA 98 in the presence of Aroclor induced rat liver S9 fraction (Toth et al., 1984), whereas another study in which S9 from phenobarbital-induced rats was used for metabolic 211

activation was negative (Buchanan et al., 1981). Capsaicin containing 20% dihydrocapsaicin exhibited mutagenicity in Salmonella strains TA 98, TA 100 and TA 1535 in the presence of S9 mixture from Aroclor-induced rats. An alcoholic chilli extract was mutagenic only in strain TA 98 (Nagabhushan, Bhide, 1985, 1986). Capsicum pepper oleoresin was reported to have mutagenic activity in Salmonella strains SD 1018 and SD 7823 without metabolic activation (Damhoeri et al., 1985). In addition, also a modified SOS microplate assay indicated a genotoxic activity of capsaicin (Venkat et al., 1995). Capsaicin containing 20% dihydrocapsaicin and alcoholic chilli extract failed to induce 8-azaguanine resistant mutants in Chinese hamster V79 cells with and without metabolic activation by S9 from Aroclor-induced rats (Nagabhushan, Bhide, 1985). On the other side, synthetic capsaicin, dihydrocapsaicin and a crude mixture of capsaicinoids from Capsicum frutescens activated with hamster hepatocytes were mutagenic in the V 79 assay measured by resistance to ouabain and 6-thioguanine (Lawson, Gannet, 1989). The results of the comet assay and a DNA fragmentation assay show that capsaicin is able to induce DNA damage in human neuroblastoma cells (Richeux et al., 1999). Capsaicin also induced DNA strand breakage with calf thymus and plasmid DNA in the presence of Cu (II) (Singh et al., 2001). Capsaicin containing 20% dihydrocapsaicin induced micronuclei in polychromatic erythrocytes in the mouse-bone-marrow assay at 7.5 mg/kg i.p. (Nagabhushan, Bhide, 1985). It also produced a significant increase of micronucleated normochromatic erythrocytes in the peripheral blood and SCEs in bone marrow cells of male mice at 1.46 and 1.94 mg/kg i.p. (Diaz Barriga Arceo et al., 1995). A fraction of an alcoholic extract from the fruits of Capsicum frutescens, containing 3-acetamido-2methyltetradecane as major component, has been found positive in the mouse-bonemarrow micronucleus assay after i.p. administration (Villasenor, Ocampo, 1994, 1995). Dominant-lethal mutations in mice were not induced by capsaicin (Muralidhara, Narasimhamurthy, 1988). Furthermore, capsaicin inhibited DNA biosynthesis in the testes of Swiss mice injected intraperitoneally (Nagabhushan, Bhide, 1985). Some other studies cannot be evaluated, because important experimental details have not been published. Reproductive and developmental toxicity No data available Human data In a case-control study in Mexico City which included 220 cases of gastric cancer and 752 controls randomly selected from the general population, chilli pepper consumers were at a 5.5-fold greater risk for gastric cancer than non-consumers. Persons who rated themselves as heavy consumers of chilli peppers were even at a 17-fold greater risk. However, when chilli pepper consumption was measured as frequency per day, a significant dose response relationship was not observed (Lopez-Carrillo et al., 1994). In another case-control study in India, red chilli powder was found to be a risk factor for cancer of the oral cavity, pharynx, esophagus, and larynx (2- to 3-fold risk 212

with a dose-response relationship) compared with population controls, but not with hospital controls (Notani, Jayant, 1987). In an Italian case-control study, chilli was briefly mentioned as being protective against stomach cancer (Buiatti et al., 1989). Chilli peppers, however, are not heavily consumed in Northern Italy, where this study was conducted, and it is possible that chilli consumption was correlated with other protecting spices such as onions and garlic that are more heavily consumed in Italy (comment from Lopez-Carrillo et al., 1994). Summary of hazard identification/characterisation Capsaicin, capsaicinoid mixtures, chillies and chilli extracts have been tested toxicologically by oral administration to mice, rats and hamsters. Some of these studies indicated a carcinogenic potential of capsaicin. These studies are regarded, however, as limited. A more recent carcinogenicity study did not show carcinogenic effects in mice. In humans, however, high consumption of chillies has been reported to be a risk factor for cancer of the upper gastrointestinal tract, possibly due to the irritating effect of capsaicinoids. Genotoxic effects of capsaicin and capsaicinoid mixtures have been shown in vitro and in vivo. Risk characterisation The Committee concluded that the available data did not allow it to establish a safe exposure level for capsaicinoids in food. The human intake of capsaicinoids in India, Thailand and Mexico, where capsicum spices are heavily consumed, has been estimated to be 25-200 mg/day. The high consumption of chillies in Mexico and India was reported to be associated with cancer of the upper digestive tract. In contrast, the maximum daily intake from mild chillies and paprika in Europe was roughly estimated to be 1.5 mg/day. In the one study conducted in Europe, no increase in the incidence of gastric cancer was found in association with occasional and lower intakes of chillies.

1.2. European Herbal Practitioners Association Response to EMEA Consultation Document CPMP/QWP/2819/00 REV 1 AKA EMEA/CVMP/814/00 REV 1: Guideline on Quality of Herbal Medicinal Products/Traditional Herbal Medicinal Products (Released 21 July 2001/Consultation Date 3 0 September 2005) th

1. The European Herbal Practitioners Association (EHPA) was founded in 1993. It represents the interests of professional herbal practitioners and their patients across the EU. The EHPA is designated an "interested party" by the EMEA Herbal Medicinal Products Committee, attending its open meetings. 2. The EHPA notes that Footnote 1 of the document states, "Throughout the guideline and unless otherwise specified, the term 'herbal medicinal products' includes 'traditional herbal medicinal product'". It is thus clear that so far as quality assurance is concerned, the Guideline on the Quality of Herbal Medicinal Products 213

regards herbal medicinal products as synonymous with traditional herbal medicinal products. The EHPA contends that quality guidelines for traditional herbal medicinal products should be specifically designed to recognise the characteristics and meet the needs of traditional herbal medicinal products. As explained below, these should not be taken to be exactly the same as those of herbal medicinal products. 3. The Guidelines on Specifications CPMP/QWP/2819 (hereinafter referred to as "the Guidelines") were originally developed by the Herbal Medicinal Products Working Party (HMPWP) in 1999. These guidelines were published to provide guidance in support of a full marketing authorisation for licensed herbal products and were adopted by the Committee for Proprietary Medicinal Products (CPMP) and the Committee for Veterinary Medicinal Products (CVMP) in July 2001. Thus these guidelines were developed and agreed a considerable time before the passage into law of the Traditional Herbal Medicinal Products Directive (THMPD) in April 2004. This Guideline and CPMP/QWP/2820/00 Rev 1, both part of the present consultation process, seek to ensure the appropriateness of the Guidelines for the THMPD. 4. Although the EHPA concurs with the general principle proposed in the Guidelines that "the quality of a medicinal product is independent of its traditional use", nevertheless, it should be recognised that the Guidelines were developed for assuring the quality of products licensed under Directive 2001/83/EC (e.g. the "WellEstablished Use" category), not Directive 2004/24/EC (THMPD). The EHPA submits that these current Guidelines now out for consultation should be redrafted to suit the new class of herbal products destined to be licensed under Directive 2004/24/EC (THMPD). The adoption of specific Guidelines appropriate to this new class of herbal products will enable the THMPD to achieve its objective of bringing traditionally-used herbal medicinal products within the EU medicines licensing system. We submit that this goal will not be achieved if the currently proposed Guidelines are adopted without such revision and that this will lead to an increase by the public of unregulated herbal products available via the Internet. 5. It is a fact that many traditional herbal medical products currently on sale in the EU are multi-herb preparations. It is not unusual for such traditional formulae to comprise 5 or more herbs. In the case of traditional Chinese, Tibetan and Ayurvedic medicines, the number of herbs in a complex herbal formula may frequently number 10 or more. 6. The EHPA submits that the relatively rigid quality standards being implemented by these Guidelines and those also under review in the consultation Guideline entitled Guideline On Quality of Herbal Medicinal Products (CPMP/QWP/2820/00 Rev 1) are proving technically very difficult and in many cases unworkable in practice. The Guideline (2819/00 Rev 1) calls for tests to demonstrate that the known constituents of any herbal medicines in the product are present in the finished product. The Guideline states (vide page 7 "Control Tests on the Finished Product") that if a herbal medicinal product contains a combination of several herbs, "the determination may be carried jointly for several active substances." The Guidance note advises (vide page 6 "control of herbal preparations") that such identification tests should be carried out using "appropriate chromatographic methods ". The problem here is that demonstrating exactly what is present in the finished product by chromatographic means is easier said than done. These quality control measures are rela214

tively easy to carry out for an orthodox drug which contains a single chemical entity or for a single herbal compound for which the Guidelines were originally designed but often impossible to demonstrate when evaluating a complex herbal mixture of several herbs each one containing a multiplicity of chemical signatures. The EHPA contends that the proposed Guidelines must be adapted so that they can apply to complex herbal mixtures. 7. The EHPA submits that quality and safety of multiherb products should be assured by the identification and control of the raw materials of these products rather that by the quantitative testing of the end product. 8. Similarly, the EHPA submits that stability testing of the final product should focus on microbial, physical and finger-print chromatographic testing and only require the quantification of claimed active(s) or marker(s) solely for single-herb products where appropriate. 9. The rationale of the THMPD is that herbal products must demonstrate 30 years of safe traditional use, with 15 years of this usage being within the EU. In this way, by definition, herbal products licensed under the THMPD must have demonstrated a good safety profile over many years so that the quantification of actives is not necessary for establishing a safety profile. Moreover the quantification of actives is unnecessary for the establishment of efficacy since the basis of efficacy for the THMPD is traditional use. The EHPA proposes that this proposed quality and safety model is supported by required pharmacovigilance, providing a clear method of evaluating the safety of traditional herbal medicinal products on the market. This response sent to [email protected] 30/09/2005 should be read in conjunction with the EHPA response to CPMP/QWP/2820/00 Rev 1 sent previously. Contact Information: European Herbal Practitioners Association 8 Lion Yard Tremadoc Road London SW4 7NQ Tel: +44 (0) 20 7627 2680 ENDS

1.3. Commmission Decision of 1 8 M a y 2005 amending Decision 1999/21 7/EC as regards the register of flavouring substances used in or on foodstuffs (notified under document number C(2005) 1437) (Text with EEA relevance) (2005/389/EC) THE COMMISSION OF THE EUROPEAN COMMUNITIES, Having regard to the Treaty establishing the European Community, Having regard to Regulation (EC) No 2232/96 of the European Parliament and of the Council of 28 October 1996 laying down a Community procedure for flavouring substances used or intended for use in or on foodstuffs (OJ L 299, 23.11.1996, p. 1. Regulation as amended by Regulation (EC) No 1882/2003 (OJ L 284, 31.10.2003, p.l)), and in particular Article 3(2) and Article 4(3) thereof,

215

Whereas: 1. Regulation (EC) No 2232/96 lays down the procedure for the establishment of rules in respect of flavouring substances used or intended to be used in foodstuffs. That Regulation provides for the adoption of a register of flavouring substances (the register) following notification by the Member States of a list of the flavouring substances which may be used in or on foodstuffs marketed in their territory and on the basis of scrutiny by the Commission of that notification. That register was adopted by Commission Decision 1999/217/EC (OJ L 84, 27.3.1999, p. 1. Decision as last amended by Decision 2004/357/EC (OJ L 113, 20.4.2004, p. 28).). 2. In addition, Regulation (EC) No 2232/96 provides for a programme for the evaluation of flavouring substances in order to check whether they comply with the general criteria for the use of flavouring substances set out in the Annex to that Regulation. 3. The European Food Safety Authority (the Authority) concluded in its opinion of 13 July 2004 on para-hydroxybenzoates, that propyl 4-hydroxybenzoate (FL 09.915) had effects on sex hormones and the male reproductive organs in juvenile rats. The Authority was unable to recommend an acceptable daily intake (ADI) for this substance because of the lack of clear no-observed adverse-effect-level (NOAEL). The use of propyl 4-hydroxybenzoate as a flavouring substance in food is not acceptable, as it does not comply with the general criteria for the use of flavouring substances set out in the Annex to Regulation (EC) No 2232/96. As a consequence, propyl 4-hydroxybenzoate should be deleted from the register. 4. The Authority concluded in its opinion of 7 December 2004 on aliphatic dialcohols, diketones and hydroxy-ketones, that pentane-2,4 dione (FL07.191) is genotoxic in vitro and in vivo. Accordingly, its use as a flavouring substance is not acceptable, because it does not comply with the general criteria for the use of flavouring substances set out in the Annex to Regulation (EC) No2232/96. As a consequence, pentane-2,4-dione should be deleted from the register. 5. In application of Regulation (EC) No 2232/96 and Commission Recommendation 98/282/EC of 21 April 1998 on the ways in which the Member States and the signatory States to the Agreement on the European Economic Area should protect intellectual property in connection with the development and manufacture of flavouring substances referred to in Regulation (EC) No 2232/96 of the European Parliament and of the Council (OJ L 127, 29.4.1998, p. 32. has adopted this decision), for a number of substances, the notifying Member States requested that they should be registered in such a way as to protect the intellectual property rights of the manufacturer. 6. Protection for these substances, listed in Part B of the register, is limited to a maximum period of five years following the date of receipt of the notification. That period has now expired for 28 substances which should consequently be transferred to Part A of the register. 7. Decision 1999/217/EC should therefore be amended accordingly. 8. The measures provided for in this Decision are in accordance with the opinion of the Standing Committee on the Food Chain and Animal Health,

216

Article 1 The Annex to Decision 1999/217/EC is amended in accordance with the Annex to this Decision. Article 2 This Decision is addressed to the Member States. Done at Brussels, 18 May 2005. For the Commission Markos KYPRIANOU Member of the Commission ANNEX The Annex to Decision 1999/217/EC is amended as follows: 1. Part A is amended as follows: (a) The rows set out in the table for the substances attributed with FL-numbers 07.191 (pentane-2,4-dione) and 09.915 (propyl 4-hydroxybenzoate) are deleted (b) The following rows are inserted in the Table 35: 2. The table of part B is replaced by the following Flavouring substances notified in application of Article 3(2) of Regulation (EC) No 2232/96, for which the protection of the intellectual property rights of the manufacturer has been requested (Table 36) T a b l e 3 5 . C o m m i s s i o n D e c i s i o n of 18 M a y 2 0 0 5 a m e n d i n g 1999/21 7/EC as regards to register of f l a v o u r i n g s u b s t a n c e s u s e d in or o n foodstaffs part A * FL. N o .

Chemical

CAS

Name

Einecs

group

Syno-

Comments

nyms

01.070

31

111-66-0

1 -Octene

203-893-7

01.071

31

111-67-1

2-Octene

203-894-2

01.072

31

544-76-3

Hexadecane

280-878-9

07.251

21

577-16-2

Metylaceto-

CAS N o

phenone

corresponds to 2-methylacetopenone

01.073

31

592-99-4

4-Octene

01.074

31

593-45-3

Octadecane

209-790-3

16.084

30

627-67-8

3-Methyl-

211-008-0

1-nitrobutane 01.075

31

629-78-7

Heptadecane

12.260

20

4131-76-4

Methyl-2-methyl-

211-108-4

3-mercapto propionate

223-949-4

217

T a b l e 3 5 . C o m m i s s i o n D e c i s i o n of 18 M a y 2 0 0 5 a m e n d i n g 1999/21 7/EC as regards to register of f l a v o u r i n g s u b s t a n c e s u s e d in o r o n foodstaffs part A * FL. N o .

Chemical

Name

CAS

Einecs

Syno-

Comments

nyms

group 12.261

20

6725-64-0

Methaneditiol

01.076

31

2099635-4

3,7-Decadiene

16.085

20

27959-66-6

4,4-Dimethyl-

12.262

20

29414-47-9

(Methylthio)

05.210

04

30390-51-3

4-Dodecenal

05.211

02

30689-75-9

6-Methyloctanal

1,3-oxathiane methanethiol

250-174-9

14.166

30

32536-43-9

Indole acetic acid

07.252

05

33655-27-9

4-Octen-2-one

02.244

04

54393-36-1

4-Octen-1-ol

10.070

09

57681-53-5

4-Hydroxy-

05.212

04

76261-02-04

6-Dodecenal

05.213

04

90645-87-7

5-Nonenal

15.124

29

103527-75-9

2-heptenoic acid lactone

260-902-7

3-Methyl-2-butenyl-

Rose

thiophene

thiophene

8-Dodecenal

05.214

04

121052-28-6

05.215

03

134998-59-7

2,3-Decadienal (c,c)

05.216

03

134998-60-0

2,6-Decadienal (t,t)

12.263

20

12.264

20

92585-08-5

4,2-Thiopentanone

03.021

16

142-96-1

Dibutyl ether

3-Mercapto3-methylbutanal 205-575-3

•The rows set out in the table for the substances attributed with FL-numbers 07.191 (pentane-2,4-dione) and 09.915 (propyl 4-hydroxybenzoate) are deleted

T a b l e 3 6 . C o m m i s s i o n D e c i s i o n of 18 M a y 2 0 0 5 a m e n d i n g 1999/21 7/EC as regards t o register of f l a v o u r i n g s u b s t a n c e s u s e d in o r o n foodstaffs part B* Code

D a t e of receipt of the notification by the Commission

Comments

CN065

26.1.2001

CN074

18.4.2003

6

CN075

18.4.2003

6

CN076

18.4.2003

6

* Flavouring substances notified in application of Article 3(2) of Regulation (EC) No 2232/96, for which the protection of the intellectual property rights of the manufacturer has been requested

218

2. Medical products for human use: Common Technical Document (CTD)

2 . 1 . Foreword This Notice to Applicants (NTA) has been prepared by the European Commission in consultation with the competent authorities of the Member States, the European Medicines Agency and interested parties in order to fulfil the Commission's obligations with respect to article 6 of Regulation (EC) No. 726/2004, and with respect to the Annex I to Directive 2001/83/EC as amended (Directive 2003/63/EC, OJ L 159 27.6.2003 p.46 NTA, Vol. 2B-CTD, foreword & introduction, edition June 2006). The first edition of the Notice to Applicants (Volume 2 in the series "The Rules Governing Medicinal Products in the European Union") was published in 1986. A revised and completed version, the second edition, was issued in January 1989. In 1993, the procedures for applications for marketing authorisations were amended, and the centralised and mutual recognition procedures became applicable from 1995. It was decided to separate the procedural and presentational parts of this guidance as Volumes 2A and 2B respectively. In 2000, a need for additional specific regulatory guidelines was recognised and a Volume 2C was prepared. The NTA is now published in the following volumes: Volume 2A dealing with procedures for marketing authorisation Volume 2B dealing with the presentation and format of the application dossier Volume 2C dealing with regulatory guidelines. The latest updates of all of the above-mentioned volumes can be found on the European Commission's pharmaceutical unit's web-site at the following address: http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/homev2.htm.

2.2. Introduction Volume 2B is concerned with the presentation of the application dossier and was first published as a separate volume in 1998. It provides guidance for the compilation of dossiers for applications for European marketing authorisations and is applicable for the centralised procedure and national procedures, including mutual recognition and decentralised procedures. The update takes account of the international agreements on the structure and format of the Common Technical Document (CTD) which were agreed in November 2000 within the International Conference on Harmonisation (ICH) framework and further documents and revised guidelines agreed upon since that time. This introduction should be read together with other published documents (e.g. 219

documents published on the ICH-website: http://www.ich.org, and "questions and answers" published on the Website of the European Commission). The update of the NTA, Vol. 2B: EU-CTD also reflects the revised ICH CTDguidelines (see http://www.ich.org) for Quality, Safety and Efficacy. The revised ICH-guidelines were signed off at the ICH meeting in Washington, September 02. The reasons for revision were minor changes in the numbering and the headings of the CTD, which have been incorporated in the updated Modules 2, 3, 4 and 5 of the EU CTD NTA. Module 1 was updated in April 2006 taking into account the requirements of the new pharmaceutical legislation. To each Module a list of relevant CHMP /ICH-guidelines is annexed, which have to be taken into consideration when preparing an EU Marketing authorisation dossier. These will be updated at regular intervals. The CTD is an internationally agreed format for the preparation of applications to be submitted to regulatory authorities in the three ICH regions of Europe, USA and Japan. It is intended to save time and resources and to facilitate regulatory review and communication. The CTD gives no information about the content of a dossier and does not indicate which studies and data are required for a successful approval. Regional requirements may affect the content of the dossier submitted in each region, therefore the dossier will not necessarily be identical for all regions. The CTD indicates an appropriate format for the data that have been required in an application. Applicants should not modify the overall organisation of the Common Technical Document as outlined in the guideline. However, in the Non-clinical and Clinical Summaries, applicants can modify individual formats if needed to provide the best possible tabulated presentation of the technical information, in order to facilitate the understanding and evaluation of the results. The new EU-CTD-presentation will be applicable for all types of marketing authorisation applications irrespective of the procedure (CR MRR DCP or national) and of type of application (stand alone, generics etc). The CTD-format will be applicable for all types of products (new chemical entities, radiopharmaceuticals, vaccines, herbals etc.) To determine the applicability of this format for a particular type of product, applicants should consult with the appropriate regulatory authorities.

2.3. Terminology The Common Technical Document was developed as an international document, and therefore specific European legal terms such as "active substance", "medicinal product", and "marketing authorisation" were not used in its development. Applicants are reminded that the term "medicinal product" covers both pharmaceutical and biological medicinal products. Unless otherwise indicated, it should be considered to be synonymous with the term "drug product". Similarly, the term "active substance" should be considered as synonymous with "drug substance". The terms used in the ICH documents may be used in the CTD part of the application. 220

2.4. Presentation of European Marketing Authorisation Applications The current requirements for the content of the European application dossier are set out in Annex I to Directive 2001/83/EC as amended, as stated in Article 8.3 "the application shall be accompanied by the following particulars and documents, submitted in accordance with Annex I". Annex I of Directive 2001/83/EC sets out the legal provision for implementation of the CTD-format. The provision of this update of Volume 2B (EU CTD), which take into account the ICH agreements, replaces the previous structure of the European marketing authorisation dossier described in the 1998 edition of Volume 2B. From 1 July 2003, all applications should be made entirely in accordance with the EU-CTD presentation outlined in the July 2003 edition of NTA, Vol. 2B or its subsequent updates. In order to take into account experience with CTD structure and changes of a technical or scientific nature, it is anticipated that NTA, Volume 2B will be updated regularly and additional guidance will be provided in the form of Question & Answer documents as experience is gained (Question & Answer document: Presentation and content of the dossier Common Technical Document (CTD) Volume 2B. Applicants Notice to NTA, Vol. 2B-CTD, foreword & introduction, edition June 2006. Applicants are advised to consult the Commission web-site: http://ec.europa.eu/ enterprise/pharmaceuticals/eudralex/homev2.htm to verify the latest updated information). st

2.5. Presentation of Applications in the Mutual Recognition Procedure or Decentralised Procedure All new applications have to be submitted in accordance with the CTD format. For Mutual Recognition Procedure based on marketing authorisation approved to the old format it is an obligation to reformat the Quality data of the dossiers before starting a new Mutual Recognition Procedure or a Repeat use procedure after 1.5.2005. For Repeat use procedures and duplicate applications/multiple applications there is no need to reformat the non-clinical and clinical data of dossiers for medicinal products for human use, authorised before 1 July 2003. Furthers guidance is given in section "presentation of the application" and the corresponding Annex to the Question & Answer document. If the original Part II contained data on bioequivalence, then this data should be extracted from the Part II and reformatted into the new CTD structure, and annexed in a separate binder as a separate section 5.3.1.2. The applicants are strongly reminded and encouraged to submit the Quality part of a dossier in the EU-CTD format as soon as possible. If a MAH wants to reformat the dossier into the CTD-format, it must first be submitted to the RMS, who has to take this reformat of the dossier into account. For

221

updating the Assessment Report it will be sufficient for the RMS to attach one page to the Assessment Report explaining that the format of the relevant dossier has been changed to the CTD-format but not the content of the dossier.

2.6. Presentation of Follow-up Measures, Specific Obligations and P S U R s Also for the submission of Follow-up Measures, a Specific Obligation dossier or a dossier including post-marketing experience, the CTD structure needs to be used. The CTD structure should always be utilised whereby documents are assigned to the most appropriate sections in Modules 1-5. They should be structured according to the granularity defined in the ICH guidance on CTD Organisation, Annex: Granularity Document (CHM/ICH/2887/99 Rev 2 Correction Organisation CTD) with Tables of Content and tab dividers for paper submissions or as separate files according to eCTD guidance. When submitting via the Centralised Procedure the Post-authorisation Guidance provided on the EMEA website (http://www.emea.eu.int/htms/human/postguidance/list.htm) should also be followed. The PSUR should be located in Module 5.3.6, Reports of Post-Marketing Experience. If a Summary Bridging Report is also to be provided then this should be included in Module 5.3.6 as well.

2.7. Reformatting of dossiers of already authorised products There is no obligation to reformat the dossier of already authorised medicinal products into the new EU-CTD format. If a marketing authorisation holder (MAH) wishes to reformat the documentation, such reformatting will be allowed, although it is not recommended for the Non-clinical and Clinical parts of the documentation. However, for the Quality part of the documentation, companies are encouraged to voluntarily reformat into the CTD-format, especially to facilitate the handling of variations and line extensions after 1 July 2003. Such reformatting must however involve the complete Quality parts, including any Drug Master Files (if applicable) and also including and integrating all approved variations. A signed declaration from the MAH, must also be submitted stating that the content/data of the Quality Module is identical to the currently approved Quality part and that there has been no changes to the dossier as a result of the reformatting. Reformatted Quality documentation submitted in the CTD-format must consist of a new Module 3 in CTD format, but need not necessarily contain the Quality Overall Summary together with the signed template for the Quality Expert. A Module 1 need not be submitted. If the original Part II contained data on bioequivalence, then this data should be extracted from the Part II and reformatted into the new CTD structure, and annexed in a separate binder. st

222

The submission of reformatted documentation should preferably occur simultaneously but separately with the submission of a variation, extension or renewal. A clear distinction between the reformatted (unchanged) information and the documentation supporting the simultaneously submitted variation / line extension or renewal should be made. Any reformatted documentation should also be submitted in electronic format (e-CTD) if available. Reformatting of a dossier does not fall under the legal definition of a variation, because there is no amendment of the dossier's content. For dossiers of products which are already NTA, Vol. 2B-CTD, foreword & introduction, edition June 2006 approved via Mutual Recognition Procedure in more than one Member State, any reformatting has to be made simultaneously in all the Member States concerned. Where products are approved via national procedures in different Member States it is also highly recommended to reformat the dossiers at the same time. The relevant competent authority has to decide whether a fee would be charged or not. In case of Mutual Recognition Procedures the reformatted dossier (new CTD) format of an already approved medicinal product cannot be submitted directly to the Concerned Member States. It must first be submitted to the Reference Member State, who has to take this reformat into account. For the RMS it will be sufficient to attach one page to the Assessment Report explaining that the format has been changed and not the content.

2.8. Presentation of the application The Common Technical Document is organized into five modules. The content of Module 1 is defined by the European Commission in consultation with the competent authorities of the Member States, the European Agency for the Evaluation of Medicinal Products and interested parties. Concerning the structure of Modules 2, 3, 4, and 5 they are common for all ICH regions.

2.9. Administrative, regional or national information is provided in different Modules

Module 1 This module contains the specific EU-requirements for the administrative data (e.g. the application form, the proposed summary of product characteristics, labelling and package leaflet, etc.). Module 2 The module 2 contains high level summaries (the Quality Overall Summary, the Non-clinical Overview / Summaries, and the Clinical Overview / Summaries), which must be prepared by suitably qualified and experienced persons (experts). Although the term "Expert Report" must be maintained for legal reasons, the content is expected 223

to be given in the Quality Overall Summary, the Non-clinical Overview / Summaries, and the Clinical Overview / Summaries documents. Old Expert Reports are now replaced by Module 2. The experts have to sign and add brief information on their educational background and specific expertise in a special section in Module 1.4. Module 3 Chemical, Pharmaceutical and Biological documentation is provided by module 3. This information should be structured as described in Guideline M4Q (M4Q (Rl): QUALITY Module 2 :Quality Overall Summary (QOS) Module 3 : Quality The section of the application covering chemical and pharmaceutical data including data for biological/ biotechnological products). Module 4 The documentation on the Toxicological and Pharmacological Tests performed on drug/active substance and a drug/medicinal product is provided in the Non-clinical Written Summaries (from Module 2) and by the Non-clinical Study Reports. These reports should be presented in the order described in Guideline M4S (M4S (R2): SAFETY Nonclinical Summaries and Organisation of Module 4 The non-clinical section of the application). Module 5 The documentation on the Clinical Trials performed on the drug/medicinal product is provided in the Clinical Written Summaries (from Module 2) and in the Clinical Study Reports. These reports should be presented in the order described in Guideline M4E (M4E (Rl): EFFICACY Module 2: Clinical Overview and Clinical Summary Module 5 Clinical Study Reports The clinical section of the Application) (Fig 33). http://www.ich.org/cache/compo/276-254-1 .html

2.10. Preparing and Organizing the C T D Throughout the CTD, the display of information should be unambiguous and transparent, to facilitate the review of the basic data and to help a reviewer become quickly oriented to the application contents. Text and tables should be prepared using margins that allow the document to be printed on A4 paper. The left-hand margin should be sufficiently large that information is not obscured through binding. Font sizes for text and tables should be of a style and size that are large enough to be easily legible, even after photocopying. Times New Roman, 12-point font is recommended for narrative text. Acronyms and abbreviations should be defined the first time they are used in each module. However when preparing the product information for applications in the centralised procedure (ref. Module 1.3.) it is mandatory to use the "QRD (Quality Review of Documents) convention".

224

Fig. 33. D i a g r a m m a t i c R e p r e s e n t a t i o n of t h e O r g a n i z a t i o n of t h e C T D

2.11. Pagination and Segregation Every document should be prepared in line with the CHMP /ICH/2887/99 Revision 1 Organisation CTD recommendation.

225

2.12. Information about national administrative requirements Information about the addresses of the national authorities, the numbers of copies of dossier-modules required, and further information are published by the EC in the NTA, Vol. 2A, Chapter 7 (http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/vol-2/a/ctdchap7_200603.pdf).

2.13. Special guidance for different kinds of applications This international format is intended to apply to all categories of drug products / medicinal products (incl. NCE's, radiopharmaceuticals, vaccines, herbals, etc.) and all types of applications (stand alone, generics, biosimilar etc.), although some adaptations may be necessary for specific application/product types. It is not designed to indicate what studies are required for successful approval, but to indicate an appropriate organization for the information included in the application. If no information is available or required under a specific heading, that section of the application should be marked "not applicable" or "not relevant" whilst retaining the section title and numbering, and, if necessary, a justification for the absence of a study should be provided in the Quality Overall Summary, the Non-clinical Overview and the Clinical Overview. Applicants are reminded that for bibliographical, generic and biosimilar, "hybrid" and extensions the non-clinical/clinical overviews/summaries should focus on particular issues concerning the basis for the application. Applicants should also consult Chapter 1 of the NTA, Vol. 2A - Marketing authorisation. For generic, biosimilar and "hybrid" applications and extensions cross-references to previous applications in the "old" EU-format will be accepted. No reformatting of already assessed and authorised "old" documentation into the CTD-format is necessary. 1. Bibliographical applications For applications based upon Article 10a of Directive 2001/83/EC, non-clinical/clinical overviews/summaries should demonstrate that the constituent(s) of the medicinal product have a well-established use, with an acceptable level of safety and/or efficacy, as outlined in Annex I to Directive 2001/83/EC. Tabulated clinical and non-clinical summaries in Module 2 shall be provided. Tables may not be necessary for very old, well known substances, but a proper justification will be required. Overviews always have to be provided. 2. Informed consent, Generic, "Hybrid" or Bio-similar applications 2 a) Consent from the marketing authorization holder For applications based upon Article 10c of Directive 2001/83/EC, the original expert reports or quality/non-clinical/clinical overviews/summaries of the original marketing authorization holder may be referred to. 2 b) Applications relating to generic medicinal products and biosimilar medicinal products; For applications according to Article 10 (1), (3) and (4) 226

Directive 2001/83/EC, Module 2 must include the Quality Overall Summary, Non-clinical Overview and Clinical Overview. Non-clinical and Clinical Summaries can be provided, but they are only mandatory if new additional studies have been provided within the documentation. 3. Variation Applications in accordance with Regulation 1084/2003/EC and Regulation 1085/2003/EC After 1 July 2003, all variation applications must be submitted using the EUCTD format. However, cross-references to "old" EU-format documentation will be accepted, because the content is identical. Clear references to any "old" format documentation are essential. Examples: Any new (either additional or revised) data in support of the variation must be submitted using the CTD format. If any data need to be submitted which are unchanged, for example, the Type I variation Guideline might specify the need for submission of a copy of the approved specifiations, then the marketing authorisation holder should update the specifications into the new CTD-format. The marketing authorisation holder must also provide a declaration that the content of any reformatted documents is unchanged. Any future variation applications would then be able to use these "updated" (CTD) specifications. Where only a cross-reference to already authorised data is required, such crossreferences can still be made to the relevant "old" format dossier (Part and section). However, if the marketing authorisation holder prefers to take the opportunity to present the (unchanged) data in the new CTD-format instead, this would be equally acceptable as it would facilitate the handling of future variations. The marketing authorisation holder must also provide a declaration stating that the content of any reformatted documents is unchanged. Type IA/IB Variation Applications and their supportive documentation - where appropriate - should be presented as follows Where a cover letter is provided it should be placed in Module 1, 1.0 Cover Letter. The checklist of conditions to be fulfilled and documentation to be supplied, i.e. the extract of the relevant page from the Guideline on Dossier Requirements for Type IA and Type IB notifications, should be placed in 1.2 Application Form after the Application Form itself. http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/homev2.htm Documents should be assigned, wherever possible, to the relevant CTD section, primarily within Module 3 Quality and 1.3.1 Summary of Product Characteristics, Labelling and Package Leaflet. These would include replacement sections and additional information. Where documents cannot be assigned to specific CTD-defined locations then they should be included in 1.2 Application Form. These might include declarations, certificates, justifications etc. Where possible, they should be organised according to the Annexes for the Application Form for new products (Annexes 1 to 22). Where documents cannot be assigned to one of the annexes then it is appropriate to place them after the annexes. The 'Additional Data' section of Module 1 should not be used except for country-specific information defined in Table 3.2 of Chapter 7, General Information, of the Notice to Applicants. Documents should be presented with tab dividers for paper submissions or as separate files according to eCTD guidances. st

227

Type II Variation Applications and their supportive documentation - where appropriate - should be presented as follows (non-exhaustive list depending on the scope of the variation and supportive data): Module 1 : 1.0 Cover Letter 1.1 Comprehensive Table of Contents 1.2 Application Form 1.3 Product Information 1.3.1 SPC, Labelling and Package Leaflet - where appropriate 1.3.4 Consultation with Target Patient Population (e.g. in case of significant changes) 1.3.6 Braille (when Braille is implemented for an already authorised medicinal product as part of a variation) 1.4 Information about the Experts: The relevant expert declaration(s) and signature^) must be provided, corresponding to the Overview/Summary submitted in Module 2. In cases where MAHs wish to distinguish this declaration from any previous declarations, the Variation Procedure Number of the RMS/EMEA may be included on top. 1.5 Specific Requirements for Different Types of Applications 1.5.3 (Extended) Data/Market Exclusivity In case the applicant wants to claim a oneyear data exclusivity at the time of the application for a new therapeutic indication, a document, justifying that the application concerns "a new therapeutic indication which is claimed to bring a significant clinical benefit" or that significant preclinical or clinical studies have been carried out, has to be provided. Related study reports and supporting literature references shall be placed in the relevant Modules of the dossier and thus cross-referred to accordingly. 1.6 Environmental Risk Assessment (e.g. in case of a new indication with significant increase of the extent of use) 1.7. Orphan Market Exclusivity (in case the indication applied for is the same as an already authorised orphan medicinal product) 1.8.1 Pharmacovigilance system (e.g. in case of changes), where appropriate 1.8.2 Risk-Management System (e.g. in case of a significant change in indication) 1.9 Information relating to clinical trials (in case clinical trials supporting the variation application were carried out outside the EU) Module 2: As mentioned in the Variation Regulation any Type II variation should be accompanied by the relevant Overviews/Summaries updates or addenda (even if a variation is submitted at the request of the Competent Authority/CHMP). Expert details and signature are to be provided in Module 1.4 separated from the actual Overview/ Summary. Modules 3, 4, 5: Supportive data are to be included in Modules 3, 4 and/or 5 as appropriate and in accordance with the EU-CTD structure. 4. New Applications as referred to in Annex II of Regulation 1084/2003/EC and Regulation 1085/2003/EC ("Extensions"). The non-clinical/clinical overviews/summaries should particularly focus on the following elements: an evaluation of the results of the additional studies. The results 228

should be discussed in the perspective of what is known from published literature and previous submissions. Additional studies should also be submitted in tabular formats provided in this Notice to Applicants: - if applicable an update of published literature relevant to the substance and the present application. The documentation may include annotated articles published in "peer review" journals, which may be acceptable for this purpose; - every claim in the Summary of Product Characteristics (SPC) not known from or inferred from the properties of the medicinal product and/or its therapeutic group should be discussed in the non-clinical/clinical overviews/summaries and substantiated by published literature and/or additional studies. After 1 July 2003, all applications for extensions must also be submitted using the new EU-CTD format. However, references can be made to already assessed and authorised "old" Parts of the dossier, but only if no new additional data are submitted in these parts. In such cases, it is not necessary to reformat already assessed and authorised "old" documentation. Modules 1 and 2 always have to be provided. Where no new clinical and/or nonclinical data are submitted the respective overviews/summaries can be replaced by expert-statements. It is however necessary to provide new CTD-format summaries and overviews to cover any new information or data provided in support of the application. In these CTD-format summaries/overviews, all the headings (numbers and titles) must be included, but where cross-references can be made, as the data has not changed, it is sufficient to include a statement such as "Not changed" (or similar). Marketing authorisation holders are also encouraged, in addition to presenting the new extension quality data in the CTD-format, to reformat the entire assessed and authorised "old" format Part II into the new CTD-format, in order to obtain a complete CTD Module 3, covering all strengths/pharmaceutical forms. Exceptionally, in those cases where there are multiple strengths and/or pharmaceutical forms, the Quality module of the extension may include only the data for the new strength/pharmaceutical form and cross-refer to the relevant "old" quality data. At the occasion of the next variation affecting the "old" part, marketing authorisation holders should "reformat" (at least) that part into the CTD format. 5. Renewal Applications: Since 1 July 2003 all applications for renewals must be submitted using the EUCTD format. The guidance stated in the relevant guidelines/recommendations on the processing of renewals, have to be taken into account. See Vol. 2C, http://ec.europa.eu/enterprise/pharmaceuticals/eudralex/homev2.htm st

st

2.14. Special guidance on herbal medicinal products For the purposes of this document the terms "herbal substances and preparations" shall be considered equivalent to the terms "herbal drugs and herbal drug preparations", as defined in the European Pharmacopoeia. For ease of reference the section titles of Modules 2 and 3 have been copied. The text following the section titles is intended to be explanatory and illustrative to herbal 229

medicinal products only. The content of these sections should include relevant information described in Guidelines published by the Agency. Click here to obtain the specific information on Modules 2 and 3 for herbal medicinal products: http://www.ich.org. Information relevant to the Active Substance Master File (ASMF). According to the current EU guideline on the ASMF procedure (Active Substance Master File guideline (CHMP QWP/227/02) (Former European Drug Master File (EDMF) CPMP/QWP/227/02 Guideline on Active Substance Master File procedure; http://www.emea.eu.int/htms/human/qwp/qwpdraft.htm. NTA, Vol. 2B-CTD, foreword & introduction, edition June 2006) it is the responsibility of the applicant for a marketing authorisation for a medicinal product to ensure that the complete ASMF, that is both the applicant's ("open") part and the active substance manufacturer's restricted ("closed") part with original signed Letter of Access is supplied to the authorities directly by the active substance manufacturer in the CTD format, synchronised to arrive at around the same time as the marketing authorisation application. A copy of the "Letter of Access" shall be included in Annex 6.10 of the application form, in Module 1 and addressed to the regulatory authority to where the application is made. The applicant's ("open") part of the ASMF should be included in section 3.2.S of the Quality documentation presented in the CTD-format. The active substance manufacturer's restricted ("closed") part of the ASMF should follow the structure of Module 3.2.S of the CTD. A separate Quality Overall Summary for the information included in the active substance manufacturer's restricted ("closed") part should also be provided, as part of the ASMF. When an ASMF is provided as part of a new application for which the Quality data are submitted in the EU-CTD format, the complete ASMF (open and closed part and the Quality Overall Summary on the ASMF) must also be presented in the EU-CTD format.

2.15. Variation of an A S M F If a change concerns a section of an ASMF, the documentation for this change must be submitted in the CTD-format. The ASMF-holder should be strongly encouraged to reformat the complete ASMF at this occasion. This will facilitate the handling of changes/variations which affect data in the ASMF concerned. The ASMF-holder should make a clear distinction between the: - reformatting of the ASMF data, already assessed by the competent authorities and - the new documentation supporting the change to ASMF-data. If an ASMF is reformatted into the new EU-CTD-format without any change in data, a signed declaration must be provided by the ASMF-holder certifying that the content of that ASMF is identical to the currently held version of the ASMF. After reformatting of the ASMF, the new "open part" of the ASMF in CTD-format has to be sent by the ASMF-holder to the MAHs concerned, in order that the MAH is able to update all marketing authorisations where that ASMF has been used. It will be acceptable to have the ASMF in the CTD format, without reformatting of the corresponding Quality data in the dossier of the medicinal product. 230

If there is a change to the ASMF-data, the corresponding variation has to be submitted to the authorities by the MAH. For extension applications to MAs where an ASMF is used, the ASMF Holder should reformat the complete ASMF into the EUCTD-format so that the "new" format ASMF can be included in the extension application, rather than any cross reference being made to an "old" format ASMF.

2.16. European Certificate of Suitability of monographs of the European Pharmacopoeia (CEP) Applicants may use the CEP-scheme to replace some of the information needed in Module 3 for drug substances described in the European Pharmacopoeia. The Drug Substance section should refer to the Certificate of Suitability in the relevant sections in Module 3.2.S. The Certificates of Suitability are deemed to replace the data of the corresponding sections and therefore no further additional information is necessary except concerning technical characteristics of the substance where not covered by the Certificate of Suitability (e.g. when the Certificate of Suitability does not describe a specific technical grade). A complete copy of the Certificates of Suitability (including any annexes) should be provided in the annex 6.10 of the application form in Module 1 and in Module 3 R. TSE-compliance can also be demonstrated by a CEP.

2.17. European Community Guidelines on Quality, Safety and Efficacy In assembling the dossier for application for marketing authorisation, applicants are required to take into account the Community guidelines relating to the quality, safety and efficacy of drug/medicinal products published by the Commission in "The rules governing medicinal products in the European Community", Volumes 3A, 3B, 3C: Guidelines on the quality, safety and efficacy of drug/medicinal products for human use, and subsequent updates as adopted by the Committee for Human Medicinal Products. The guidelines adopted within the ICH process are considered as Community guidelines once adopted by the CHMP and published. References to the relevant Community or ICH guidelines have been included either within the relevant sections, or as annexes to each part of the dossier. For the latest updates of Community / ICH guidelines, applicants are advised to consult the Website of the EMEA on http://www.emea.eu.int/index/indexhl.htm (Regulatory Guidance and Procedures - Notes for Guidance). With respect to the quality part of the dossier, the monographs and general chapters of the European Pharmacopoeia are also applicable. All materials of ruminant origin have also to comply with the TSE requirements. Correlation EU-CTD (NTA, Vol. 2B, edition May 2006) vs. NTA, Vol. 2B (edition 1998) (Table 37) 231

T a b l e 3 7 . S u m m a r y of t h e parts of C o m m o n T e c h n i c a l D o c u m e n t s ( C T D ) ( E U - C T D ( N T A , V o l . 2 B , e d i t i o n M a y 2 0 0 6 ) v s . N T A , V o l . 2 B ( e d i t i o n 1998) M O D U L E 1 -ADMINISTRATIVE I N F O R M A T I O N A N D PRESCRIBING CTD

E U C T D ( N T A , Vol. 2 B , Edition 2006)

1.0

C o v e r Letter

INFORMATION

N T A , Vol. 2 B (Edition 1998)

NTA

I A

1.1

C o m p r e h e n s i v e t a b l e of c o n t e n t

1.2

Application Form

Administrative Data

1.3

Product Information

S u m m a r y of P r o d u c t C h a r a c t e r i s t i c s ,

1.3.1

S u m m a r y of P r o d u c t C h a r a c t e r i s t i c s ,

S u m m a r y of P r o d u c t C h a r a c t e r i s t i c s

L a b e l l i n g a n d P a c k a g e Leaflet L a b e l l i n g a n d P a c k a g e Leaflet

I B I B 1

P r o p o s a l for p a c k a g i n g , l a b e l l i n g & p a c k a g e leaflet 1.3.2

Mock-up

1.3.3

Specimen

1.3.4

C o n s u l t a t i o n w i t h Target Patient

I B 2 I B 2

Groups 1.3.5 1.3.6

Product Information already

S P C s a l r e a d y a p p r o v e d in t h e

a p p r o v e d in t h e M e m b e r States

M e m b e r States

I B 3

Expert R e p o r t s : S i g n a t u r e of Experts

I C

Braille

1.4

I n f o r m a t i o n a b o u t t h e Experts

1.4.1

Quality

1.4.2

Non-clinical

1.4.3

Clinical

1.5

S p e c i f i c R e q u i r e m e n t s for different t y p e s of a p p l i c a t i o n s

1.5.1

I n f o r m a t i o n for b i b l i o g r a p h i c a l applications

1.5.2

I n f o r m a t i o n for G e n e r i c , " H y b r i d " or B i o - s i m i l a r A p p l i c a t i o n s

1.5.3

(Extended) Data/Market Exclusivity

1.5.4

Exceptional Circumstances

1.5.5

Conditional Marketing Authorisation

1.6

E n v i r o n m e n t a l risk assessment

1.6.1

Non-GMO

E n v i r o n m e n t a l risk assessment E n v i r o n m e n t a l risk assessment / e c o t o x i c i t y (for n o n - G M O s )

1.6.2

GMO

III R

D a t a r e l a t e d to t h e e n v i r o n m e n t a l risk assessment for p r o d u c t s c o n t a i n i n g , or c o n s i s t i n g of g e n e t i c a l l y m o d i f i e d organisms ( G M O s )

232

II H

M O D U L E 1 -ADMINISTRATIVE I N F O R M A T I O N A N D PRESCRIBING CTD

E U C T D ( N T A , Vol. 2 B , Edition 2006)

INFORMATION

N T A , V o l . 2 B (Edition 1998)

NTA

I n f o r m a t i o n relating to O r p h a n

1.7

Market Exclusivity 1.7.1

Similarity

1.7.2

M a r k e t Exclusivity

1.8

I n f o r m a t i o n r e l a t i n g to Pharmacovigilance

1.8.1

Pharmacovigilance System

1.8.2

Risk-management System

1.9

I n f o r m a t i o n relating to C l i n i c a l Trials R e s p o n s e s to Q u e s t i o n s

R e s p o n s e s to Q u e s t i o n s

M O D U L E 2 -ADMINISTRATIVE I N F O R M A T I O N A N D PRESCRIBING CTD

INFORMATION

E U C T D (NTA, Vol. 2 B , Edition 2006)

N T A , Vol. 2 B (Edition 1998)

Additional Data

Additional Data

O v e r a l l C T D T a b l e of C o n t e n t s of

T a b l e of C o n t e n t s for r e m a i n d e r

M o d u l e s 2, 3, 4, a n d 5

of t h e dossier

I.A

2.2

Introduction

Product profile

I.C

2.3

Quality Overall Summary

Expert report o n t h e c h e m i c a l ,

2.1

NTA

pharmaceutical and biological documentation

I C 1

2.4

Non-clinical O v e r v i e w

Expert R e p o r t o n t h e t o x i c o -

2.5

Clinical O v e r v i e w

Expert R e p o r t o n t h e C l i n i c a l

2.6

Non-clinical Summary

A p p e n d i c e s to t h e t o x i c o -

2.6.1

Pharmacology Written Summary

Written Summary

I C 2

2.6.2

Pharmacology Tabulated S u m m a r y

T a b u l a r Formats

I C 2

2.6.3

Pharmacokinetics Written Summary

Written Summary

IC2

2.6.4

Pharmacokinetics Tabulated S u m m a r y

Tabular Formats

I C 2

2.6.5

Toxicology Written S u m m a r y

2.6.6

Toxicology Tabulated S u m m a r y

Tabular Formats

I C 2

2.7

Clinical Summary

A p p e n d i c e s to t h e c l i n i c a l

2.7.1

S u m m a r y of b i o p h a r m a c e u t i c s

Written Summary

pharmacological documentation Documentation p h a r m a c o l o g i c a l Expert R e p o r t

Expert R e p o r t and associated analytical methods

I C 2 I C 3 I C 2

I C 3 I C 3

2.7.2

S u m m a r y of c l i n i c a l p h a r m a c o l o g y studies

Written Summary

2.7.3

S u m m a r y of c l i n i c a l e f f i c a c y

Written Summary

I C 3

2.7.4

S u m m a r y of c l i n i c a l safety

Written Summary

I C 3

2.7.5

S y n o p s e s of I n d i v i d u a l S t u d i e s

T a b u l a r Formats

I C 3

I C 3

233

MODULE 3 - QUALITY CTD

E U C T D (NTA, Vol. 2 B , Edition 2001)

3.1

M O D U L E 3 TABLE O F

3.2

B O D Y O F DATA

3.2.S

DRUG

3.2.S.1

General Information

Scientific Data

II C 1 .2

3.2.S.1.1

Nomenclature

Nomenclature

II C 1 .2.1

3.2.S.1.2

Structure

D e s c r i p t i o n : Structural f o r m u l a

II C 1 .2.2

3.2.S.1.3

G e n e r a l Properties

N T A , Vol. 2 B (Edition 1998)

CONTENTS Chemical, Pharmaceutical, Biological

Documentation

Physico-chemical characterization

II C 1 .2.5 II C 1 .2.3

Manufacture

Manufacture

3.2.S.2.1

Manufacturer(s)

N a m e ( s ) address(es) of t h e

3.2.S.2.2

D e s c r i p t i o n of m a n u f a c t u r i n g

S y n t h e t i c or m a n u f a c t u r i n g

process a n d process controls

route D e s c r i p t i o n of p r o c e s s

3.2.S.2.3

C o n t r o l of m a t e r i a l s

Q u a l i t y control during

3.2.S.2.4

C o n t r o l s of c r i t i c a l steps

Quality control during

and intermediates

manufacture

m a n u f a c t u r i n g source(s)

manufacture

II C 1 .2.3 II C 1 .2.3 II C 1 .2.4 II C 1 .2.4

P r o c e s s v a l i d a t i o n and/or e v a l u a t i o n

3.2.S.2.6

Manufacturing process d e v e l o p m e n t

3.2.S.3

Characterisation

3.2.S.3.1

II

SUBSTANCE

3.2.S.2

3.2.S.2.5

NTA

E l u c i d a t i o n of structure

D e v e l o p m e n t chemistry

a n d other characteristics

II C 1 2.5

3.2.S.3.2

Impurities

Impurities

3.2.S.4

C o n t r o l of d r u g s u b s t a n c e

Specifications a n d routine tests

II C 1 2.6 II C 1 1

3.2.S.4.1

Specification

S p e c i f i c a t i o n s a n d r o u t i n e tests

II C 1 1

3.2.S.4.2

Analytical Procedures

S p e c i f i c a t i o n s a n d r o u t i n e tests

II C 1 1

3.2.S.4.3

V a l i d a t i o n of a n a l y t i c a l p r o c e d u r e s

Development Chemistry: Analytical Validation

II C 1 2.5 II C 1 2.7

3.2.S.4.4

Batch analyses

Batch analysis

3.2.S.4.5

J u s t i f i c a t i o n of S p e c i f i c a t i o n

D e v e l o p m e n t Chemistry: C o m m e n t s o n the c h o i c e of r o u t i n e tests a n d standards

3.2.S.5

R e f e r e n c e S t a n d a r d s or M a t e r i a l s

II C 1 2.5

D e v e l o p m e n t c h e m i s t r y : Full c h a r a c t e r i z a t i o n of t h e p r i m a r y

3.2.S.6

Container

3.2.S.7

Stability

reference material Batch

II C 1 2.5

analysis: R e f e r e n c e material

II C 1 2.7

Closure System Stability Tests o n A c t i v e Substance(s)

234

II F 1

MODULE 3 - QUALITY NTA, Vol. 2 B (Edition 1998)

CTD

E U C T D (NTA, Vol. 2 B , Edition 2001)

3.2.P

DRUG

3.2.P.1

Description and composition

Composition

of t h e d r u g p r o d u c t

(brief d e s c r i p t i o n )

3.2.P.2

Pharmaceutical Development

Development Pharmaceutics

3.2.P.2.4

C o n t r o l s a n d c r i t i c a l steps

NTA

PRODUCT and container

a n d c l i n i c a l trial f o r m u l a e a n d intermediates

II A 1 II A 2 II A 4 II A 3

Manufacturing process ( i n c l u d i n g in-process c o n t r o l and phamraceutical assembly process) C o n t r o l tests on intermediate products

II B 3 II D

3.2.P.3

Manufacture

M e t h o d of P r e p a r a t i o n

II B

3.2.P.3.1

Manufacturer(s)

Administrative Data

I A

3.2.P.3.2

Batch formula

Manufacturing Formula

II B 1

3.2.P.3.3

D e s c r i p t i o n of M a n u f a c t u r i n g

Manufacturing Process

Process and Process Controls

( i n c l u d i n g In-process C o n t r o l and Pharmaceutical Assembly Process)

3.2.P.3.4

C o n t r o l s of c r i t i c a l steps

Manufacturing Process (inclu-

and intermediates

d i n g In-process C o n t r o l

11 B 2

and Pharmaceutical Assembly Process) 3.2.P.3.5

P r o c e s s v a l i d a t i o n a n d / or

II B 2

V a l i d a t i o n of t h e P r o c e s s II B 3

evaluation 3.2.P.4

C o n t r o l of e x c i p i e n t s

Excipients(s)

II C 2

3.2.P.4.1

Specifications

S p e c i f i c a t i o n s a n d r o u t i n e tests

II C 2 . 1

3.2.P.4.2

Analytical procedures

S p e c i f i c a t i o n s a n d r o u t i n e tests

II C 2 . 1

3.2.P.4.3

V a l i d a t i o n of a n a l y t i c a l p r o c e d u r e s

S c i e n t i f i c data

II C 2.2

3.2.P.4.4

Justification of s p e c i f i c a t i o n s

S c i e n t i f i c data

II C 2.2

3.2.P.4.5

E x c i p i e n t s of h u m a n or a n i m a l o r i g i n

3.2.P.4.6

N o v e l E x c i p i e n t s (ref to A 3)

Excipient(s) not d e s c r i b e d in a

II C 2.2.1

p h a r m a c o p o e i a S c i e n t i f i c data

II C 2.2

3.2.P.5

C o n t r o l of d r u g p r o d u c t

C o n t r o l Tests o n t h e F i n i s h e d

3.2.P.5.1

Specification(s)

Product specifications

Product

II E

Q u a l i t y specifications

II E 1.1 II

for t h e p r o p o s e d shelf life

F 2 II E 1.2

3.2.P.5.2

Analytical Procedures

Control Methods

3.2.P.5.3

V a l i d a t i o n of A n a l y t i c a l P r o c e d u r e s

Analytical validation

3.2.P.5.4

Batch analyses

3.2.P.5.5

C h a r a c t e r i s a t i o n of I m p u r i t i e s

3.2.P.5.6

Justification of s p e c i f i c a t i o n ( s )

of m e t h o d s

II E 2 . 1

Batch analysis

II E 2 . 2

C o m m e n t s on the choice of r o u t i n e tests a n d s t a n d a r d s

II E 2.1

235

MODULE

3 - QUALITY

CTD

E U C T D (NTA, Vol. 2 B , Edition 2001)

3.2.P.6

R e f e r e n c e S t a n d a r d s or M a t e r i a l s

N T A , V o l . 2 B (Edition 1998) Batch analysis: Reference

II E 2 . 2

material 3.2.P.7

Container Closure System

NTA

Packaging Material ( I m m e d i a t e Packaging)

II C 3

Stability Tests o n t h e F i n i s h e d

3.2.P.8

Stability

3.2.A

APPENDICES

Product

3.2.A.1

Facilities a n d E q u i p m e n t

3.2.A.2

A d v e n t i t i o u s A g e n t s Safety

II F 2

Evaluation 3.2.A.3

Excipients

3.2.R

REGIONAL

3.3

LITERATURE

INFORMATION REFERENCES

V a l i d a t i o n of t h e p r o c e s s

-II B 3 -

OTHER

II Q

INFORMATION

MODULE 4 - NONCLINICAL STUDY

REPORTS

CTD

E U C T D ( N T A , Vol. 2 B , Edition 2001)

N T A , Vol. 2 B (Edition 1998)

NTA

4.1

M O D U L E 4 TABLE O F

4.2

STUDY

DOCUMENTATION

III

4.2.1

PHARMACOLOGY

PHARMACODYNAMICS

III F

4.2.1.1

Primary pharmacodynamics

P h a r m a c o d y n a m i c s effects

CONTENTS

REPORTS

TOXICO-PHARMACOLOGICAL

relating to t h e p r o p o s e d indications

III F 1

4.2.1.2

Secondary pharmacodynamics

General pharmacodynamics

III F 2

4.2.1.3

Safety p h a r m a c o l o g y

General pharmacodynamics

III F 2

4.2.1.4

P h a r m a c o d y n a m i c drug interactions

D r u g interactions

III F 3

4.2.2

PHARMACOKINETICS

PHARMACOKINETICS

III G

4.2.2.1

Analytical Methods and Validation

O t h e r Information

4.2.2.2

Absorption

IIIQ

Reports P h a r m a c o k i n e t i c s after a single dose

III G 1

P h a r m a c o k i n e t i c s after r e p e a t e d administration

III G 2

D i s t r i b u t i o n in n o r m a l

4.2.2.3

Distribution

a n d pregnant animals

III G 3

4.2.2.4

Metabolism

Biotransformation

III G 4

4.2.2.5

Excretion

Pharmacokinetics

III G 1 , 2

4.2.2.6

P h a r m a c o k i n e t i c D r u g Interactions

4.2.2.7

O t h e r Pharmacokinetic Studies

(nonclinical)

236

MODULE 4 - NONCLINICAL STUDY

REPORTS

CTD

E U C T D (NTA, Vol. 2 B , Edition 2001)

N T A , Vol. 2 B (Edition 1998)

NTA

4.2.3

TOXICOLOGY

TOXICITY

III A

4.2.3.1

Single-dose t o x i c i t y

S i n g l e d o s e t o x i c i t y studies

III A 1

4.2.3.2

Repeat-dose toxicity

R e p e a t e d d o s e t o x i c i t y studies

III A 2

4.2.3.3

Genotoxicity

M u t a g e n i c Potential

III D

4.2.3.4

Carcinogenicity

C a r c i n o g e n i c Potential

III E

4.2.3.5

Reproductive and developmental

Reproductive Function Embryo-

toxicity

foetal a n d P e r i n a t a l T o x i c i t y

III B III C

4.2.3.6

Local tolerance

Local Tolerance

III H

4.2.3.7

O t h e r t o x i c i t y studies

O t h e r Information

IIIQ

LITERATURE

OTHER

IIIQ

4.3

REFERENCES

M O D U L E 5- C L I N I C A L S T U D Y CTD

E U C T D (NTA, Vol. 2 B , Edition 2001)

5.1

M O D U L E 5 TABLE O F

5.2

T A B U L A R LISTINGS O F ALL CLINICAL

INFORMATION

REPORTS

N T A , V o l . 2 B (Edition 1998)

CONTENTS

STUDIES

EXPERT REPORT O N THE CLINICAL

DOCUMENTATION,

A P P E N D I X 2: W R I T T E N SUMMARY -TABULAR

REPORTS

CLINICAL

OVERVIEW

DOCUMENTATION

5.3

CLINICAL STUDY

5.3.1

Reports of B i o p h a r m a c e u t i c S t u d i e s

Pharmacokinetics

Reports of S t u d i e s Pertinent to P h a r m a -

Pharmacokinetics

5.3.2

Reports of h u m a n p h a r m a c o k i n e t i c Reports of h u m a n p h a r m a c o d y n a m i c

Pharmacokinetics

IV A 2

Pharmacodynamics

IV A 1

( P D ) studies 5.3.5

R e p o r t s of e f f i c a c y a n d safety studies

5.3.6

R e p o r t s of post-marketing e x p e r i e n c e

5.3.7

C a s e report forms a n d i n d i v i d u a l

5.4

IV B 1

C l i n i c a l Trials Post-marketing e x p e r i e n c e

IV B 2

(if a v a i l a b l e ) patient listings, w h e n s u b m i t t e d LITERATURE

REFERENCES

IV

IV A 2

( P K ) studies 5.3.4

I C 3

IV A 2

cokinetics using H u m a n Biomaterials 5.3.3

NTA

A p p e n d i x to e a c h c l i n i c a l study report, w h e n s u b m i t t e d ( A p p e n d i x 16.3) PUBLISHED AND

IV B 1 UNPUB-

LISHED EXPERIENCE T H A N 1) O T H E R

(OTHER

INFORMATION

IV B 3 IV Q

237

About the auth

Professor Gyula Mozsik, MD, Sc.D. He was born in Dancshaza, Hungary in 1938. After earning a Medical Doctorate at the University of Debrecen in Hungary in 1962, he served as an assistant at the Second Department of Medicine at the University Medical School of Debrecen in Hungary, where he received the degree of specialist in internal medicine in 1967. He was visiting scientist at the Department of Pharmacology at the University of Oslo in Norway from 1968-1969. The following year he received a Philosophy Doctorate (Ph.D.) in medicine at the University of Pecs. As assistant professor of the First Department of Medicine, at the Medical University of Pecs in 1969, he worked as a trainee in clinical pharmacology at the Medical Universities of Debrecen and Pecs as well as a trainee in experimental and clinical biochemistry at the Medical University of Pecs and the University of Oslo from 1962 to 1970. He received a Science Doctorate (Sc.D.) in medicine in 1977 at the University of Pecs, Hungary. He became a specialist in gastroenterology in 1980, and was visiting scientist in the Chemical Pathology Laboratory of Harvard Medical University of Boston, Massachusetts, USA in 1985. Four years later he became full professor of internal medicine at the First Department of Medicine, Medical University Pecs, he was named head of the department from 1993 to 2003. He was full professor of medicine from 2003 to 2008, and from May 2008 is Professor Emeritus at the First Department of Medicine, University of Pecs, Hungary. Serving on the Advisory Board of the National Institute of Dietetics of the Hungarian Ministry of Health, he is a member of the European Society for Clinical Investigation, Hungarian Society of Physiology, Hungarian Society of Pharmacology, Hungarian Society of Nutrition, Hungarian Society of Gastroenterology, International Brain-Gut Society, International Society of Internal Medicine, International Society of Metabolic Therapy, American Dietetical Association, American Gastroenterological Association, International Union of Pharmacology Gastrointestinal Section's Transition Committee (1989), National Board of Gastroenterology and New York Academy of Sciences. Serving on the Standing Committee of the International Conferences on Ulcer Research, General Secretary of Executive Committee (from 2000). He is the leader of both the Gastroenterological and Clinical Nutrition and Dietetic Sections of the Pecs Committee of the Hungarian Academy of Sciences. He is the President of the International Union of Pharmacology Gastrointestinal Section (from 2002). He is the member of Leadership of the Hungarian 241

Society of Gastroenterology, Hungarian Society of Internal Medicine, Transdanubian Section of the Hungarian Society of the Internal Medicine and he is the President of this Section (from 1998). During his activity five Szechenyi Awards, three Bolyai Awards and three Bekesi Awards were given to the colleagues of the First Department of Medicine. Twentyeight colleagues of the Department received Ph.D. qualification, eleven physicians obtained the title of the habilitation and three physicians were named as Academic Doctor (Sc.D.). Six physicians became full professors of medicine and one of Professor of the Health Sciences from the member of the First Department of Medicine, Pecs University, and two researchers became full professors of Pharmacology at Universities of Habana (Cuba) and of Cairo (Egypt). The Department of Family Medicine and 3 Department of Medicine and Department of Human Nutrition and Dietetics were established by the members of the First Department of Medicine. Professor Mozsik is the author of seventeen textbooks for college students in clinical nutrition and dietetics, 20 textbooks for medical students. He wrote 16 monographs, edited 26 books, 191 book chapters, 350 regular papers (published in English), and he participated in giving 700 lectures. The Hungarian Ministry of Health gave to Professor Mozsik the award of Excellent Worker of Health in 1977, while the Hungarian Ministry of Education named him Excellent Worker of Education in 1983. That year, he received the Hetenyi Geza medal, the highest medal for gastroenterological research, from the Hungarian Society of Gastroenterology. He received the Sos Jozsef medal from the Hungarian Society of Nutrition in 1984, the medal of Pro Optimo Merito in Gastroenterologia from the Hungarian Society of Gastroenterology in 1989, Diploma of the International Brain Gut Society in 1997, the High Quality Price of Hungarian Academy of Sciences (for the book of Mozsik Gy., Abdel Salam OME., Szolcsanyi J.: Capsaicin Sensitive Afferent Nerves in Gastric Mucosal Damage and Protection. Akademiai Kiado, 1998) in 1998, He received Exellent Prize of the Intellectual Work from the Faculty of the Health Science from the Pecs, University for the book of Metabolic Ward in Human Clinical Nutrition and Dietetics 2005 in 2006 and Excellent Prize of the Intellectual Work from the Medical Faculty of Pecs, University for the book Discoveries in Gastroeterology: from Basic Research to Clinical Perspectives 1960-2005 in 2007. Award of the Hungarian Society of Internal Medicine Transdanubian Section in 1999, and the medal of Pro Facultate Sciential Sanitatis from the University of Pecs in 2002. From 1998 he won Szechenyi Scholarship (by the Hungarian Government). In 2003 the Rector of University of Pecs gave him the Pro Facultate MedicinaeGolden Medal. His special fields for the research are: internal medicine, experimental and clinical nutrition (dietetics), gastroenterology, clinical pharmacology, clinical biochemistry and genetics. r d

242

Andras Domotor, MD Date of Birth: Place of Birth: Nationality: Citizenship: Marital Status: Working place:

December 5, 1980 Szekszard, Tolna County, Hungary Hungarian Hungary Single First Department of Medicine, Medical and Health Centre Medical Faculty University of Pecs, Hungary H-7643, Ifjusag u. 13., Pecs, Hungary Home address: Epitok utja 14. H-7030 Paks, Hungary E-mail: domotorjandras @yahoo. com Education: 1995-1999. Vak Bottyan High School, Paks, Hungary 1999-2005. Medical University of Pecs, Hungary. 2005PhD student at the University of Pecs, Hungary. Subject: The mechanisms of gastrointestinal mucosal damage and protection. Supervisor: Prof. Dr. Gyula Mozsik Scientific Student Association: 2000-2002. Biochemistry and Medical Chemistry, University of Pecs Subject: Detection of GSH and GSSG by HPLC Supervisor: Prof. Dr. Robert Ohmacht st

2003-2005. 1 Department of Medicine, Medical and Health Centre of University of Pecs Subject: Capsaicin-sensitive afferent nerves in the development of human gastrointestinal disorders Supervisor: Prof. Dr. Gyula Mozsik

243

Tibor S. Past, PhD Date of Birth: January 05. 1944. Place of Birth: Pecs, Baranya County Citizenship: Hungarian Marital Status: Married Education Graduated Eotvos Lorand University, Faculty of Sciences Subject Chemistry (1968). Hospital and University Appointments: Chem.eng. sci. research worker, 1 Department of Medicine, Med. Univ. Pecs 1969. Chem.eng. senior researcher, 1 Department of Medicine, Med. Univ. Pecs 1982. Invited lecturer of Janus Pannonius University, Faculty of Sciences Subject Chemistry 1995. License Licensed Biology Certified Med.Univ. Pecs, Hungary, 1973. Licensed Isotopic Diagnostics and Therapy Certified Postgraduate Medical Institute Budapest, Hungary, 1973. Licensed Biometry Certified Medical Post-graduate Institute Budapest, Hungary, 1978. Licensed Candidate in Biological Scholarship Certified Hung. Sci. Academy, 1982. Societies Hungarian Pharmacological Society (1970) Hungarian Society of Nuclear Medicine (foundation member) (1972) Hungarian Chemical Society (local secretary (1993)) (1970) Hungarian Gastroenterological Society (1978) Hungarian Humangenetic Society (1980) John von Neumann Computer Society (1984) Hungarian Diabetes Society (1982) Member of Committee of the Hungarian Academy of Sciences on Coordination of Research of Biologically Active Compounds (1975) Member of Management of Polytechnic and Natural Science (local, Baranya County) Associations (1993) Member of Local Committee of the Hungarian Academy of Sciences (1993) Public member of the Hungarian Academy of Sciences (1995) st

st

244

Grants and Contracts Pharmacokinetic, metabolization and bioavailability studies of about 55-60 drugs which were produced by Hungarian and foreign pharmaceutical works, certified by the archives of the National Institute of Pharmacy and the archives of different Hungarian pharmaceutical works, Research Institute for Pharmaceutical Chemistry, Budapest, Tiszavasvari, Debrecen (Hungary) and abroad. Developments of new drugs [Tisacid, Cr(III)-complexes, dietary fibre preps, artificial dental cleaner, oral capsaicinoids contained compositions, antifungal enamel and solution, eye drop], manufacturing process, new analytical instruments (H-tester, electric alcohol-tester, electric gastrointestinal transit time tester, instrument for controlled electric deposition), new analytical methods (quantitative analysis of transuran elements in environmental and power plant samples, new electric-deposition methods, quantitative measurement of iodine micro amounts in complex pharmaceutical samples), new therapeutic method (in vivo dissolution of pigment gallstones). Certified by partly the archives of the Hungarian Patent Office, Home Office, partly in archive of Beres articles of association and different scientific journals.

245

Viktoria Vas She was born in 1984 at Szekszard. She finished secondary school in 2000. She entered the Faculty of Adult Education and Human Resourches, University of Pecs, in 2004. She successfully finished the First Degree of first teaching program of Bologna in this field, and she started her studies for receiving the Master Degree. Her special field is human manager. She joined the work of the Regional South-Danubian Knowledge Centre, as a co-worker to the leader of one subchapter involved in this programme (Gy.M.). She typed the whole text, prepared figures and tables for the book. She carried out all of the necessary corrections on the final form on the manuscript, as well as on the proof of this book (together with Gy.M.). She helped us in the solutions of our scientific and human problems during the research period of 2005-2008 (in the above-mentioned programme) based on her knowledge on Adult Education and Human Resources. Besides this, she participated in the preparation of five books, published in Hungarian and English.

246

Pal Perjesi, PhD He was born in Oroshaza, Hungary in 1956. He received an M.S. degree in Pharmacy at the University of Szeged (Hungary) in 1979. After qualifying he started work as a graduate fellow at the Institute of Pharmaceutical Chemistry of the University Medical School of Szeged. As a result of his work here he got a "University Doctor" degree qualification in Pharmaceutical Chemistry in 1983. Since 1981 he has been working at the University of Pecs (Hungary). He completed and defended his dissertation for the "Candidate" degree in organic/pharmaceutical chemistry at the Hungarian Academy of Sciences in 1994. Based on his "Candidate" degree he received a Doctor of Philosophy (PhD) degree at the University of Szeged in 1996. He got a Specialization Certificate of preparative chemical laboratory investigations (1985) and that of toxicology (1994) from the Postgraduate Medical School (Budapest). In 1990/1991 he worked at the Department of Medicinal Chemistry, University of Washington, Seattle (USA) as a Fogarty International Fellow. In 2001 and 2003 he worked at the University of Florida, Gainesville (USA) as a visiting scientist at the Center for Drug Discovery and the Department of Medicinal Chemistry, respectively. In 2000 he got a Habilitation Certificate from the University of Pecs. At present, he is working as an Associate Professor, the founder and the first head of the Institute of Pharmaceutical Chemistry, School of Pharmacy, University of Pecs. He also serves as the head of the GLP Laboratory of the Institute of Pharmaceutical Chemistry and the MEDIPOLISZ (Regional University Science Center of Pecs), which actively participates in the drug development projects sponsored by the National Office for Research and Technology. His scientific interest is focused on the investigation of structure-to-reactivity and structure-to-biological activity relationship of selected natural products and their synthetic analogs. So far he has been involved in chemical, physico-chemical and biological investigations of chalcones, cyclic chalcone analogues, estrogens and synthetic TRH analogues. Recently, he has started research to investigate molecular pathways of degradation and metabolism of active pharmaceutical ingredients. In connection with these latter studies, his former research activities have been extended by the development of analytical methods to be used during stability as well as preclinical and clinical studies.

247

He is coauthor of more than 80 publications and 90 conference presentations. He is a member of the Hungarian Pharmaceutical Society, the Hungarian Chemical Society, and the Medicinal Chemistry and Pharmaceutical Technology Section of the Hungarian Academy of Sciences. Since 2005 he has been serving as President of the Section of Drug Research of the Hungarian Pharmaceutical Society. He is member of the editorial advisory board of The Open Medicinal Chemistry, and Open Medicinal Chemistry Letters. He is member of the editorial board of Gyogyszereszet (Pharmacy) and Acta Pharmaceutica Hungarica.

248

Monika Kuzma She was born in Nagykanizsa, Hungary in 1982. She received an M.S. degree in Pharmacy at the University of Pecs, Hungary in 2005. After qualifying she started work as a graduate fellow at the Institute of Pharmaceutical Chemistry of the University Medical School of Pecs and she is a qualified pharmacist. At present, she is working as a research fellow of the Institute of Pharmaceutical Chemistry, School of Pharmacy, University of Pecs and participates in the teaching program of Pharmaceutical Chemistry. She also serves as the analytical associate of the GLP Laboratory of the Institute of Pharmaceutical Chemistry and the MEDIPOLISZ (Regional University Science Center of Pecs), which actively participates in the drug development projects sponsored by the National Office for Research and Technology. Her scientific interest is focused on the investigation of non-enzymatic metabolism of non-steroidal anti-inflammatory drugs, especially related to salicylic acid and its hydroxylated derivates. Recently, she has participated in the development of analytical methods to be used during stability as well as preclinical and clinical studies. She is a member of the Hungarian Pharmaceutical Society.

249

Gyula Blazics, PhD H-7720 Pecsvarad, Pannonpharma ut 1., Hungary * Tel: (+36) 72 566 750 E-mail: [email protected]; Personal Date of birth: 10 January, 1953 Nationality: Hungarian Marital status: married Objective To use my educational and practical maximum performance experience in a profes sional business, or management environment while pursuing personal and com pany set goals Education Budapest Medical University - Pharmaceutical Faculty, Hungary, 1972-1977 Training for qualification of Inspector Pharmacist, 1981 Training for GMP production, 1993 Coating course at COLORCON (Orpington-UK) 1995 Profile Eight-year experience in drug control More than 14-year experience in pharmaceutical production Advanced GMP knowledge (involving QC&QA fields) and application in pharma ceutical fields Advanced experiences in planning GMP conform plants - especially for OSD phar maceuticals Advanced experiences in generic development, especially for solid forms Proven ability to initiate, maintain and develop positive business relationships Qualifications Pharmacist 1977 Inspector Pharmacist 1981 PhD (phytochemistry) 1982 Expert of physical, physical-chemical and chemical drug control 1985 Expert of pharmaceutical technology 1994 QP 2004 250

Experience 09.2002- Quality Assuarance Director of Pannonpharma Pharmaceutical Ltd 2002-1999 Managing Director of Pannonpharma Pharmaceutical Ltd 1999-1993 Manager of Production & Development of Pannonpharma Pharmaceutical Ltd 1989-1993 Manager of Pharmaceutical/Galenic Laboratory of Pannonmedicina Co. 1982-1993 Inspector Pharmacist of Baranya Regional Pharmaceutical Co. Honors and activities Adviser member of the Management of the Analytical Session of the Hungarian Pharmaceutical Society International Experience Traveled through thirteen European and Asian countries (involving Egypt and India) over last fifteen years, including GMP audits and some business development discussions at appr. 20 pharmaceutical companies.

251

Janos Szolcsanyi, MD Present position: Professor Emeritus, Department of Pharmacology and Pharmacotherapy Faculty of Medicine, University of Pecs and head of the Neuropharmacology Research Group of the Hungarian Academy of Sciences H-7624 Pecs, Szigeti str. 12. Personal Date of Birth: 24 February, 1938 Place of Birth: Budapest Marital Status: Married, two children

Degrees 1962 M.D. (Univ. Medical School of Szeged "summa cum laude") 1977 C.Sc. (PhD) 1987 D.Sc. (Hungary) 1995 Corresponding member of the Hungarian Academy of Sciences (HAS) 2001 Regular member of HAS Employment 1962-1964 Research Associate: Dept. Pharmacol. Univ. Med. School of Szeged 1965-1966 Warner Research Fellowship in the Department of Pharmacology, University of London, King's College 1966-1970 Research Associate of the Hungarian Academy of Sciences (1966-1967), then Assistant Professor (1968-1970) in the Department of Pharmacology, University of Szeged 1970-1977 Associate Professor in the Department of Pharmacology, University of Pecs, Hungary 1977-1978 Visiting Associate Professor in the Department of Physiology, University of North Carolina at Chapel Hill, USA (Professor Ed Perl) 1981-1994 Visiting scientist in the Max-Planck-Institut fiir physiologische und klinische Forschung W.G. Kerckhoff Institut, Bad Nauheim (BRD) for 14 months

252

19851988-1991 19901994-2003 1991-1995 1995-

Visiting professor in II. Physiologisches Institut, Universitat Heidelberg (BRD) for 7 months (Professor H.O. Handwerker) Consultant of the Sandoz Institute for Medical Research, London (Professor H.P. Rang). Professor in the Dept. of Pharmacology, University of Pecs Head of the Department of Pharmacology, then Pharmacology and Pharmacotherapy, University of Pecs Pro-Rector for Research of the University Medical School of Pecs Head of the Neuropharmacology Research Group of the Hungarian Academy of Sciences in Pecs

Professional associations Scientific committee membership: Hungarian Pain Society (president) Hungarian Society for Experimental and Clinical Pharmacology (president elect) Doctor of Science (D.Sc.) committee of HAS (chairman) Committee of HAS on Drug Research and Pharmacotherapy Neuroscience Research Committee of the Hungarian Ministry of Health Social and Family Affairs (chairman) Hungarian Neuroscience Association (council member) IASP Committee on Research (member 1997-2000) Member of the Scientific Committee of the 3 EFIC Meeting, Prague 2003. and 4th EFIC Meeting, Istanbul, 2006 Councillor of EFIC r d

Other societies Hungarian Physiological Society British Pharmacological Society International Association for the Study of Pain (IASP) Federation of European Neuroscience Societies (FENS) European Neuropeptide Club (founding member) Society for Neuroscience (USA) Honours and awards 1987 Award for Excellence in Teaching (Ministry of Education, Hungary) 1990 Nonvoting member of the Hungarian Academy of Sciences 1993 Medal of the Helsinki University 1994 Szent-Gyorgyi Albert Award (Ministry of Culture and Education, Hungary) 1995 Corresponding member of the Hungarian Academy of Sciences 1996 Plaque of Appreciation (Seoul National University) 1997 Batthyany-Strattmann Award (Ministry of Welfare, Hungary) 1998 Issekutz Medal, Hungarian Pharmacological Society 1999 Ipolyi Arnold Medal of the National Science Foundation (OTKA) 1999 Award of Excellence of the Hungarian Academy of Sciences 2000 Szechenyi Professorship 2001 "Pro Facultate Medicinae" gold medal of the University of Pecs 253

2001 2003 2003 2004 2004

Medal of the Krakow University. Jancso Miklos Award, University of Szeged Szechenyi Award, Hungary Manfred Zimmermann Award (ENC) Valyi-Nagy Tibor Award

Main research field Capsaicin and pharmacology of nociceptors, neurogenic inflammation. Preclinical pharmacology of two new drugs (thymoxamine, setastine), patents on somatostatin analogs. Publications The number of full publications is 179 (121 in international journals, 20 in Hungarian journals in English, 37 book chapters, 1 book, 1 book edition). During the last 5 years one European and one Hungarian Patent. Citation Independent SCI citation is around 5600 (full SCI citations: 6033). The most cited paper received 838 citations and further 16 papers over 100 SCI citations (102-328). Around 70% of the citations are on papers from his own laboratories without foreign coworkers. Keywords of PubMed (first appeared under his name): Thymoxamine (1965), Capsaicin (pharmacological) receptor (1975), "Capsaicin-sensitive" afferents (1979), "Sensory-efferent" or local efferent function of sensory receptors in different organs (gut 1978), gastric ulcer (1981), bronchi (1982), Setastine (1990), "sensocrine" function of sensory receptors (2003), and among the firsts "capsaicin and pain".

254

T h e v i s u a l c h a r a c t e r i z a t i o n of c a p s a i c i n o i d s c o n t a i n i n g pills

255