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Bangladesh J Pharmacol 2006; 1: 35-41 Copyright © 2006 by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

Minireview

Anticancer agents from medicinal plants Mohammad Shoeb Department of Chemistry, University of Dhaka, Dhaka 1000, Bangladesh.

[Received 29 November 2006]

Abstract Cancer is a major public health burden in both developed and developing countries. Plant derived agents are being used for the treatment of cancer. Several anticancer agents including taxol, vinblastine, vincristine, the camptothecin derivatives, topotecan and irinotecan, and etoposide derived from epipodophyllotoxin are in clinical use all over the world. A number of promising agents such as flavopiridol, roscovitine, combretastatin A-4, betulinic acid and silvestrol are in clinical or preclinical development. Key words: anticancer, cytotoxicity, medicinal plants, natural products, taxol

Introduction Natural Products, especially plants, have been used for the treatment of various diseases for thousands of years. Terrestrial plants have been used as medicines in Egypt, China, India and Greece from ancient time and an impressive number of modern drugs have been developed from them. The first written records on the medicinal uses of plants appeared in about 2600 BC from the Sumerians and Akkaidians (Samuelsson, 1999). The “Ebers Papyrus”, the best known Egyptian pharmaceutical record, which documented over 700 drugs, represents the history of Egyptian medicine dated from 1500 BC. The Chinese Materia Medica, which describes more than 600 medicinal plants, has been for correspondence: Mohammad Shoeb, Phone: 880-2-9661920 Ext. 7143; E-mail: [email protected]

well documented with the first record dating from about 1100 BC (Cragg et al., 1997). Documentation of the Ayurvedic system recorded in Susruta and Charaka dates from about 1000 BC (Kappor, 1990). The Greeks also contributed substantially to the rational development of the herbal drugs. Dioscorides, the Greek physician (100 A.D.), described in his work “De Materia Medica” more than 600 medicinal plants (Samuelsson, 1999). The World Health Organization estimates that approximately 80% of the world’s inhabitants rely on traditional medicine for their primary health care (Farnsworth et al., 1985). Cancer is a major public health burden in both developed and developing countries. It was estimated that there were 10.9 millions new cases, 6.7 million deaths, and 24.6 million persons living with cancer around the world in 2002 (Parkin et al., 2005). Cancer is the second

leading cause of death in the United States (Hoyert et al., 2005), where one in four deaths is due to cancer. Plants have long been used in the treatment of cancer (Hartwell, 1982). The National Cancer Institute collected about 35,000 plant samples from 20 countries and has screened around 114,000 extracts for anticancer activity (Shoeb, 2005). Of the 92 anticancer drugs commercially available prior to 1983 in the US and among world wide approved anticancer drugs between 1983 and 1994, 60% are of natural origin (Cragg et al., 1997). In this instance, natural origin is defined as natural products, derivatives of natural products or synthetic pharmaceuticals based on natural product models (Jaspars and Lawton, 1998).

Plant derived Anticancer agents in clinical use The isolation of the vinca alkaloids, vinblastine (1) and vincristine (2) from the Madagascar periwinkle, Catharanthus roseus G. Don. (Apocynaceae) introduced a new era of the use of plant material as anticancer agents. They were the first agents to advance into clinical use for the treatment of cancer (Cragg and Newman, 2005). Vinblastine and vincristine are primarily used in combination with other cancer chemotherapeutic drugs for the treatment of a variety of cancers, including leukemias, lymphomas, advanced testicular cancer, breast and lung cancers, and Kaposi’s sarcoma (Cragg and Newman, 2005). The discovery of paclitaxel (Taxol®, 3) from the bark of the Pacific Yew, Taxus brevifolia Nutt. (Taxaceae), is another evidence of the success in natural product drug discovery. Various parts of Taxus brevifolia and other Taxus species (e.g., Taxus Canadensis Marshall, Taxus baccata L.) have been used by several Native American Tribes for the treatment of some non-cancerous cases (Cragg and Newman, 2005) while Taxus baccata was reported to use in the Indian

36 Shoeb Anticancer agents from medicinal plants

Ayurvedic medicine for the treatment of cancer. The structure of paclitaxel was elucidated in 1971 and was clinically introduced to the US market in the early 1990s (Wani et al., 1971; Rowinsky et al., 1992). Paclitaxel is significantly active against ovarian cancer, advanced breast cancer, small and non-small cell lung cancer (Rowinsky et al., 1992). Camptothecin (4), isolated from the Chinese ornamental tree Camptotheca acuminate Decne (Nyssaceae), was advanced to clinical trials by NCI in the 1970s but was dropped because of severe bladder toxicity (Potmeisel, 1995). Topotecan (5) and irinotecan (6) are semi-synthetic derivatives of camptothecin and are used for the treatment of ovarian and small cell lung cancers, and colorectal cancers, respectively (Creemers et al., 1996; Bertino, 1997). Epipodophyllotoxin is an isomer of podophyllotoxin (7) which was isolated as the active anti-tumor agent from the roots of Podophyllum species, Podophyllum peltatum Linnaeus and Podophyllum emodi Wallich (Berberidaceae) (Stahelin, 1973). Etoposide (8) and teniposide (9) are two semi-synthetic derivatives of epipodophyllotoxin and are used in the treatment of lymphomas and bronchial and testicular cancers (Cragg and Newman, 2005; Harvey, 1997). Homoharringtonine (10), isolated from the Chinese tree Cephalotaxus harringtonia var. drupacea (Sieb and Zucc.) (Cephalotaxaceae), is another plant-derived agent in clinical use (Itokawa et al., 2005; Powell et al, 1970). A racemic mixture of harringtonine and homoharringtonine has been used successfully in China for the treatment of acute myelogenous leukemia and chronic myelogenous leukemia (Cragg and Newman, 2005; Kantarjian et al., 1996). Elliptinium (11), a derivative of ellipticine, isolated from a Fijian medicinal plant Bleekeria vitensis A.C. Sm., is marketed in France for the treatment of breast cancer (Cragg and Newman, 2005).

Vol. 1, No. 2, December 2006

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Figure 1: Plant-derived anticancer agents in clinical use

Plant-derived anticancer agents for future development Numerous types of bioactive compounds have been isolated from plant sources. Several of them are currently in clinical trials or preclinical trials or undergoing further investigation. Flavopiridol (12) is a synthetic flavone, derived from the plant alkaloid rohitukine, which was isolated from Dysoxylum binectariferum Hook. f. (Meliaceae) (Kellard et al., 2000). It is currently in phase I and phase II clinical trials against a broad range of tumors, including leukemia, lymphomas and solid tumors (Christian et al., 1997). Synthetic

Bangladesh J Pharmacol

agent roscovitine (13) which is derived from natural product olomucine, originally isolated from Raphanus sativus L. (Brassicaceae), is in Phase II clinical trials in Europe (Cragg and Newman, 2005; Meijer et al., 2003). Combretastatins were isolated from the bark of the South African tree Combretum caffrum (Eckl. & Zeyh.) Kuntze (Combretaceae) (Pettit et al., 1987). Combretastatin A-4 (14) is active against colon, lung and leukemia cancers and it is expected that this molecule is the most cytotoxic phytomolecule isolated so far (Ohsumi et al., 1998; Pettit et al., 1995).

37

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Figure 1 (Cont.): Plant-derived anticancer agents in clinical use

Betulinic acid (15), a pentacyclic triterpene, is a common secondary metabolite of plants, primarily from Betula species (Betulaceae) (Cichewitz et al., 2004). Betulinic acid was isolated from Zizyphus species, e.g. mauritiana, rugosa and oenoplia (Pisha et al., 1995; Nahar et al., 1997) and displayed selective cytotoxicity against human melanoma cell lines (Balunas et al., 2005). The development of systemic and topical formulations of the agent for potential clinical trials by the NCI is ongoing (Cragg and Newman, 2005). Pervilleine A (16) was isolated from the roots of Erythroxylum pervillei Baill. (Erythroxylaceae) (Silva et al., 2001). Pervilleine A was selectively cytotoxic against a multidrug resistant (MDR) oral epidermoid cancer cell line (KB-V1) in the presence of the anticancer agent vinblastine (Mi et al., 2001). Pervilleine A is currently in preclinical development (Mi et al.,

38 Shoeb Anticancer agents from medicinal plants

2003). Silvestrol (17) was first isolated from the fruits of Aglaila sylvestre (M. Roemer) Merrill (Meliaceae) (Hwang et al., 2004). Silvestrol exhibited cytotoxicity against lung and breast cancer cell lines (Cragg and Newman, 2005). Biological studies are ongoing to determine the mechanism(s) of action for silvestrol. Two novel alkaloids, schischkinnin (18) and montamine (19) have been isolated from the seeds of Centaurea schischkinii and Centaurea montana (Shoeb et al., 2005; 2006). Both of the alkaloids exhibited significant cytotoxicity against human colon cancer cell lines. The unique structural features of 18 and 19 can be exploited as a template for generating compounds with enhanced anticancer activity. However, further investigations are necessary for their use as anticancer agents.

Vol. 1, No. 2, December 2006

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Figure 2. Plant-derived anticancer agents for future development

Conclusion Natural products discovered from medicinal plants have played an important role in the treatment of cancer. Natural products or natural Bangladesh J Pharmacol

product derivatives comprised 14 of the top 35 drugs in 2000 based on worldwide sales (Butlet, 2004). Two plant derived natural products, paclitaxel and camptothecin were estimated to 39

account for nearly one-third of the global anticancer market or about $3 billion of $9 billion in total annually in 2002 (Oberlines and Kroll, 2004). There are more than 270,000 higher plants existing on this planet. But only a small portion has been explored phytochemically. So, it is anticipated that plants can provide potential bioactive compounds for the development of new ‘leads’ to combat cancer diseases.

References Balunas MJ, Kinghorn AD. Drug discovery from medicinal plants. Life Sci. 2005; 78: 431-41. Bertino JR. Irinotecan for colorectal cancer. Semin Oncol. 1997; 24: S18-S23. Butlet MS. The role of natural product chemistry in drug discovery. J Nat Prod. 2004; 67: 2141-53. Cichewitz RH, Kouzi SA. Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection. Med Res Rev. 2004; 24: 90-114. Cox PA. Ethnopharmacology and the search for new drugs. In: Bioactive compounds from plants. Ciba Foundation Symposium 154. Chichester, England, John Wiley and Sons, 1990, pp 40-55. Christian MC, Pluda JM, Ho TC, Arbuck SG, Murgo AJ, Sausville EA. Promising new agents under development by division of cancer treatment, diagnosis, and centers of the National Cancer Institute. Semin Oncol. 1997; 13: 2643-55. Cragg GM, Newman DJ, Snader KM. Natural products in drug discovery and development. J Nat Prod. 1997; 60: 52-60. Cragg GM, Newman DJ. Plants as source of anticancer agents. J Ethnopharmacol. 2005; 100: 72-79. Creemers GJ, Bolis G, Gore M, Scarfone G, Lacave AJ, Guastalla JP, Despax R, Favalli G, Kreinberg R, VanBelle S, Hudson I, Verweij J, Huinink WWT. Topotecan, an active drug in the second-line treatment of epithelial ovarian cancer: results of a large European phase II study. J Clin Oncol. 1996; 14: 3056-61. Farnsworth NR, Akerele O, Bingel AS, Soejarto DD, Guo Z. Medicinal plants in therapy. Bull World Health Organ. 1985; 63: 965-81. Hartwell JL. Plants used against cancer: a survey. Lawrence, MA. Quarterman Publications, 1982, pp 43839. Harvey AL. Medicines from nature: are natural products still relevant to drug discovery. Trends Pharmacol Sci. 1999; 20: 196-98.

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Hoyer DL, Kung HC, Smith BL. Natl. Vital. Stat. Rep. 2005; 53: 1-48. Hwang BY, Su BN, Chai H, Mi Q, Kardono LB, Afriastini JJ, Riswan S, Santarsiero B D, Mesecar AD, Wild R, Fairchild CR, Vite GD, Rose WC, Farnsworth NR, Cordell GA, Pezzuto JM, Swanson SM, Kinghorn AD. Silvestrol and episilvestrol, potential anticancer rocaglate derivatives from Aglaila silvestris. J Org Chem. 2004; 69: 3350-58 (ibid. 69 (18), 6156). Itokawa H, Wang X, Lee K-H. Homoharringtonine and related compounds. In: Cragg GM, Kingston, DGI, Newman D, (eds). Anticancer agents from natural products. Boca Raton, Florida, Brunner-Routledge Psychology Press, Taylor & Francis Group, 2005, pp 47-70. Jaspars M, Lawton LA. Cyanobacteria as a novel source of pharmaceuticals. Curr Opin Drug Discovery Develop. 1998; 1: 77-84. Kantarjian HM, O’Brien S, Anderlini P, Talpaz M. Treatment of chronic myelogenous leukemia: current status and investigational options. Blood. 1996; 87: 3069-81. Kappor LD. CRC Handbook of ayurvedic medicinal plants. Boca Raton, Florida, CRC Press, 1990, pp 416-17. Kelland LR. Flavopiridol, the first cyclic-dependent kinase inhibitor to enter the clinic: current status. Expert Opin Investig Drugs. 2000; 9: 2903-11. Meijer L, Raymond E. Roscovitine and other purines as kinase inhibitors. From starfish oocytes to clinical trials. Accounts Chem Res. 2003; 36: 417-25. Mi Q, Cui B, Silva GL, Lantvit D, Lim E, Chai H, You M, Hollingshead MG, Mayo JG, Kinghorn AD, Pezzuto JM. Pervilleine A, a novel tropane alkaloid that reverses the multidrug-resistance phenotype. Cancer Res. 2001; 61: 842-50. Mi Q, Cui B, Silva GL, Lantvit D, Reyes-Lim E, Chai H, Pezzuto JM, Kinghorn AD, Swanson SM. Pervilleine F, a new tropane alkaloid aromatic ester that reverses the multidrug-resistance phenotype. Anticancer Res. 2003; 23: 3607-16. Nahar N, Das RN, Shoeb M, Marma MS, Aziz MA, Mosihuzzaman M. Four triterpenoids from the bark of Zizyphus rugosa and Z. oenoplia. J Bangladesh Academy Sci. 1997; 21: 151-56. Oberlines NH, Kroll DJ. Camptothecins and taxol: historic achievement in natural products research. J Nat Prod. 2004; 67: 129-35. Ohsumi K, Nakagawa R, Fukuda Y, Hatanaka T, Morinaga Y, Nihei Y, Ohishi K, Suga Y, Akiyama Y, Tsuji T. New combretastatin analogues effective against murine solid tumors: design and structure-activity relationship. J Med Chem. 1998; 41: 705-06. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, 2002. CA Cancer J Clin. 2005, 55; 74-108. Pettit GR, Singh SB, Niven ML, Hamel E, Schmit JM. Isolation, structure, and synthesis of combretastatins A-1 and B-1, potent new inhibitors of microtubule assembly, derived from Combretum caffrum. J Nat Prod. 1987; 50: 119-20.

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Pettit GR, Singh SB, Boyd MR, Hamel E, Pettit R, Schmit JM, Hogan F. Antineoplastic agents. 291. Isolation and synthesis of combretastatins A-4, A-5 and A-6. J Med Chem. 1995; 38: 1666-72. Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell GA, Beecher CW, Fong HH, Kinghorn AD, Brown DM, Wani MC, Wall ME, Hieken TJ, Das Gupta TK, Pezzuto JM. Discovery of betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat Med. 1995; 1: 1046-51. Potmeisel M, Pinedo H. Camptothecins: new anticancer agents. Boca Raton, Florida, CRC Press, 1995, pp 149150.

Shoeb M, Celik S, Jaspars, M, Kumarasamy Y, MacManus S, Nahar L, Kong TLP, Sarker SD. Isolation, structure elucidation and bioactivity of schischkiniin, a unique indole alkaloid from the seeds of Centaurea schischkinii. Tetrahedron. 2005; 61: 9001-06. Shoeb M, MacManus SM, Jaspars M, Trevidadu J, Nahar L, Thoo-Lin PK, Sarker SD. Montamine, a unique dimeric indole alkaloid, from the seeds of Centaurea montana (Asteraceae), and its in vitro cytotoxic activity against the CaCo2 colon cancer cells. Tetrahedron. 2006; 62: 1117277.

Powell RG, Weisleder D, Smith CRJr, Rohwedder WK. Structures of harringtonine, isoharringtonine, and homoharringtonine. Tetrahedron Lett. 1970; 11: 815-18.

Silva GL, Cui B, Chavez D, You M, Chai HB, Rasoanaive P, Lynn SM, O’Neill MJ, Lewis JA, Besterman JM, Monks A, Farnsworth NR, Cordell GA, Pezzuto JM, Kinghorn AD. Modulation of the multidrug-resistance phenotype by new tropane alkaloids aromatic esters from Erythroxylum pervillei. J Nat Prod. 2001; 64: 1514-20.

Rowinsky EK, Onetto N, Canetta RM, Arbuck SG, Taxolthe 1st of the texanes, an important new class of antitumor agents. Semin Oncol. 1992; 19: 646-62.

Stahelin H. Activity of a new glycosidic lignan derivative (VP 16-213) related to podophyllotoxin in experimental tumors. Eur J Cancer. 1973; 9: 215-21.

Samuelsson G. Drugs of natural origin: a textbook of pharmacognosy. 4th ed., Stockholm, Swedish Pharmaceutical Press, 1999.

Wall ME, Wani MC. Camptothecin and taxol: from discovery to clinic. J Ethnopharmacol. 1996; 51: 239-53.

Shoeb M. Cytotoxic compounds from the Genus Centaurea. PhD Thesis. Aberdeen, UK, The Robert Gordon University, 2005.

Wani MC, Taylor HL, Wall M E, Coggon P, McPhail AT. Plant anti-tumor agents. VI. The isolation and structure of taxol, a novel anti-leukemic and anti-tumor agent from Taxus brevifolia. J Am Chem Soc. 1971; 93: 2325-27.

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Bangladesh J Pharmacol 2006; 1: 42-50 Copyright © by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

Arsenic-safe drinking water and antioxidants for the management of arsenicosis patients 1

Salamat Khandker, 2Ranjit Kumar Dey, 3AZM Maidul Islam, 4 Sheikh Akhtar Ahmad and 1Ifthaker-Al-Mahmud

1

WHO Arsenic Cell, Kakrail, Dhaka; 2Directorate of Health services, Mohakhali, Dhaka; 3Department of Dermatology, Bangabnadhu Sheikh Mujib Medical University, Shahbag, Dhaka; 4Department of Occupational Health, National Institute for Preventive and Social Medicine, Mohakhali, Dhaka, Bangladesh [Received 10 July 2006]

Abstract The role of arsenic-safe drinking water and antioxidants in the management of arsenicosis patients were observed. Two hundred and fifty patients of arsenicosis from an arsenic-affected area of Bangladesh were included and divided into five groups based on the source of drinking water (greenor red-marked tube well) and intake of antioxidants (vitamin A, C and E). Melanosis improved in 43 patients of the group who took arsenic-safe drinking water from green-marked tube well and antioxidants regularly. Patients of the group who took green-marked tube well water regularly but not the antioxidant showed improvement in melanosis in 22 cases. The respondents who were using redmarked tube well water and antioxidants, only two of them improved; and all other respondents either deteriorated or did not improve. The respondents who were using red-marked tube well water but not the antioxidant, none did show any improvement of their illness. The respondents who took antioxidants irregularly and had irregular intake of safe water, were not considered to compare the prognosis of skin lesions. Regarding keratosis, the respondents who took green-marked tube well water regularly and antioxidant regularly, 8 of them improved, 1 case didn’t change; while the respondents who took green-marked tube well water regularly but not the antioxidant, 8 cases didn’t improve much but majority of them remain unchanged. Among the respondents of other groups, keratosis deteriorated. This study suggests that both arsenic-safe drinking water and use of antioxidants gave good result in improvement of the arsenicosis.

Key words: antioxidant, arsenic, arsenicosis, drinking water

Introduction Access to safe water supply is one of the most important detriments of health and socioeconomic development. In the first half of the 1970s, Bangladesh faced an infant mortality rate of around 140 per 1000 with diarrhea being a major determinant. For young children 1-4 years, diarrhea accounted for nearly half of all deaths. for correspondence: Salamat Khandker; e-mail: [email protected]

A solution was to provide tube wells sunk 20-30 meter was free of harmful bacteria. At the initial stage government and other international agencies assisted the tube wells sinking. At present there are about 7-8 million tube wells to tap better quality water and 97% of rural drinking water supply is obtained from ground water sources (Ahmed and Ahmed, 2002). These tube well installation initiatives have contributed significantly to the halving of infant mortality

over 36 years from 161 per 1000 in 1960 to 83 per 1000 in 1996. The situation became particularly complex, in 1993 when it was discovered that, substantial proportion of the tube wells were yielding water contaminated with high level of soluble arsenic compounds (WHO, 2000) to threat health and epidemiology of arsenicosis. The detection of arsenic in the ground water was first made in West Bengal in 1983 and in Bangladesh in 1987. Chowdhury et al., (2000) states that until 1995 tube well contaminated by arsenic was not widespread across central and southern Bangladesh. The most recent information comes from a testing project involving the collaboration of the Department of International Development (UK), the British Geological Survey, and the Government of the Peoples Republic Bangladesh. This project tested one tube well in every 36 Km2 in the two thirds of the countries most affected areas and found that, 51% of the tube wells were contaminated with at least 0.01 mg arsenic/l, 35% with at least 0.05 mg arsenic/l, 25% with at least 0.1 mg arsenic/l, 8% with 0.3 mg arsenic/l or more and 0.1% with 1.0 mg arsenic/l or more. About 24.5 million people are currently chronically exposed to high levels of arsenic (above 50 µg/l) in drinking water derived from ground water supplied by millions of hand pump tube wells. The health hazards due to this contamination have raised the serious concern to public health. The first case of arsenicosis was detected in Chomogram village of Chapai Nowabgonj District in 1994. Since then, the number of areas and number of patients were gradually increaseing. To date, 38,430 individuals with visible signs of arsenic toxicity have reported from different health sources. The arsenic poisoning in Bangladesh is chronic in nature and is caused due to contamination of ground water. The time taken to develop clinically evident effects from drinking arsenic contaminated water is generally thought to be about 10 years, but early manifestations are not

Bangladesh J Pharmacol

uncommon (Caussy, 2005). Most of the time, the victims do not complain of symptoms until they are detected through screening survey. The present experiment to identify the arsenic cases are by external manifestations especially with the presentation on the skin called melanosis and keratosis with the history of consuming arsenic contaminated water for a prolonged period. Gangrene of peripheral organs and ulceration due to toxic effect on the small blood vessels may also be found. Cancer of skin along with cancer of some internal organs- liver, kidney, urinary bladder is not uncommon (Kaufman et al., 2001). It is also reported that even if a person having no manifestation after chronic exposure of contaminated water the chance of having cancer cannot be ruled out. The only way to save lives is to identify patients at an earlier stage before the conditions become irreversible and to provide them arsenic-safe water and supportive management. The present knowledge about management of arsenicosis is far from satisfactory. The drugs used for chelating arsenic in acute poisoning have proved to be ineffective in chronic arsenicosis (Guha Mazumder et al., 2001). The use of arsenic-safe water may prevent progression of effects, but it is not clear whether reversal of effects is possible. Supportive therapy with nutritional improvement may play some role in diminishing symptoms and may reverse some cases of melanosis. Use of antioxidants like vitamins A, C and E, along with discontinuation of consuming arsenic contaminated water have shown evidences of improvement and some success in arresting the progression of symptoms (Talukder, 1999). Consequently, Directorate General of Health Services (DGHS) recommended these antioxidants in use for the management of patients suffering from arsenicosis. Some experts had advocated for the use of spirulina extracts which are rich in antioxidants in treatment of arsenicosis (Sikder et al., 2000). But, conclusive scientific evidences are still not available to make

43

recommendation in this regard. Numbers of studies are progressing on this issue, but enough evidences are yet to confirm or to reject their effectiveness (Verret et al., 2005). Considering the above facts, the present observational study has been designed to evaluate effectiveness of the antioxidants in management of arsenicosis patients. Factors considered in this study were quality of water intake and intake of antioxidants. The purpose of the study was to see the effectiveness of antioxidant therapy in groups of arsenicosis patients taking arsenic-safe and arsenic-contaminated water, and to compare the result with groups of patients who were not receiving antioxidant therapy.

Materials and Methods Study area: Bhanga is an Upazilla (sub-district) under Faridpur District. It is 70 Km southwest from the capital city, Dhaka. It is a low land area and the river Padma borders a major portion of this Upazilla. The Sitalakha and the Kumar River pass through this Upazilla too. The area covers 216.3 sq km. There are 221 Villages, 12 Unions and one Paurashava in Bhanga Upazilla. The total number of populations is 230,300 (116,560 male and 113,740 female). Sanitation and water supply of this Upazilla are not satisfactory. Majority people depend on tube well water for drinking and cooking purposes. According to Bangladesh Rural Advancement Committee (BRAC) survey report (2001), above 92% tube wells of this Upazilla are contaminated with more than permissible arsenic concentration to Bangladesh standard. Dhaka Community Hospital (DCH) undertook active case detection in Bhanga and so far identified 488 arsenicosis patients (DCH, 2001). List of patients was available with the health service providers and some of those listed patients were receiving treatment with antioxidants (vitamin A- 50,000 i.u; alpha-tocopherol- 200 mg; ascorbic acid- 500 mg) continuously for three months with two months interval for another three months. Moreover, mapping of shallow tube wells and

their water quality were also recorded by local Directorate of Public Health and Engineering (DPHE) people, and the resulted marking in the tube wells made patients as well as other concerned people to know the quality of water in terms of arsenic contamination in those tube wells. Duration of study: The study was conducted during February to September 2003. Initial two months were spent for preparation of the study, collecting socio-economic and epidemiological data and baseline clinical status of selected patients. Study population: For the selection of study population every odd number was chosen from the list purposively. Thus a list of 244 patients was made. During our field interview we found 86 new patients. From the new patients list we selected 16 more patients randomly. In this way we prepared a list of 260 patients for interview. The patients were divided into 5 observation groups on the basis of their quality of water intake and receiving antioxidant therapy. If there were less than 50 patients for particular group new participants randomly selected to fulfill the target similarly rejected to interview patients of a particular group once the desired number was attained. Thus in each group there were 50 patients and the groups were as follows (Table I):

44 Khandker et al Arsenic-safe drinking water and antioxidant

Group I: Drinking water from green-marked tube wells (safe water) and receiving antioxidant treatment, Group II: Drinking water from greenmarked tube wells (safe water) but were not receiving antioxidant treatment, Group III: Drinking water from red-marked tube wells (high arsenic contaminated water) and receiving antioxidant treatment, Group IV: Drinking water from red-marked tube wells (high arsenic contaminated water) but were not receiving antioxidant treatment, Group V: Drinking water from both red and green-marked tube wells (irregular quality of drinking water) and/or irregular intake of antioxidants. Vol. 1, No. 2, December 2006

Table I: Source of drinking water and antioxidants in different groups Group

I II

Source of drinking water Green-marked Red-marked tube well tube well + +

Antioxidants

+

-

-

III

-

+

+

IV

-

+

-

V

irregular

irregular

irregular

Development of questionnaire: A questionnaire was developed containing general information of patients, information about source of drinking water and the duration of tube well used by the study population. The questionnaire was pretested and finalized after incorporation of feedback. Interviewers: Socio-economic and water intake related data were collected by trained interviewer. Fourteen interviewers were locally recruited for the purpose, and were given one day orientation training on different techniques to fill up the questionnaire. The interviewers were divided into two groups headed by one team leader and administered a structured questionnaire to all the selected patients. The interviewers submitted filled up forms to the project manager, recruited for the purpose, who then divided the patients into different groups mentioned above based on their statements about quality of drinking water and antioxidant therapy. Ethical issues: The main ethical problem of this study was that a group of the study subject continued to drink arsenic contaminated tube well water. It was not possible to prevent this group to collect potable water from arseniccontaminated water options although alternative arsenic-safe water options were installed in their neighborhoods. Even in this situation DGHS recommended antioxidants treatment for arsenicosis patients (Talukder, 1999). Local health authority used to supply the medicine free of cost. Therefore, we did not face any ethical problem for drug intervention. Data collection: The medical officer performed clinical examinations of all those patients and Bangladesh J Pharmacol

recorded findings in the checklist. Thus baseline and monthly clinical examination data were recorded in the structured checklist. The medical officer was not aware which group did a particular patient belong. A group of dermatologists evaluated the skin lesions at the beginning and at the end of the study. The evaluation was done in terms of regression of melanosis and keratosis through comparing the state of lesion of previous visit. Patients' perception was also considered for evaluation of improvement. The improvement was evaluated as + = improvement, 0 = no improvement, and = deterioration. Body mass index (BMI) was calculated as weight (kg)/height (meter square). Analysis of data: Data were entered and analyzed using the SPSS software package. Only univariate analysis was done using appropriate formula. Standard deviation and mean value were also determined for each frequency distribution.

Results Among the respondents, 59.6% were male and 40.4% were female. The age of the respondents varied from 13-75 years. The mean age of the respondents of different groups was 37.7, 33.3, 38.0, 36.7 and 32.1 years respectively. Maximum respondents were in the age group of 20-49 years. Mean age of the respondents of group V and group II were comparatively lower. Common occupations of the respondents were service (37.7%), agriculture (25.5%) and business (22.7%) Others were teachers and housewives. Most (95.6%) of the respondents of all the groups used tube well water for drinking and cooking purposes. However, the source of domestic water differed among the respondents. Of the different sources, pond water was the major (57.7%) source of domestic purpose. Other sources for domestic water were tube well water (24.2%) and river water (15.3%). Comparatively the respondents of group V (49.0%) and group I (48.0%) used pond water less than that of other groups (Table 2). 45

Table II: Distribution of respondents according to domestic use and drinking water sources Source of water Shallow tube well

Deep tube well

Dug well

Pond

River

For domestic purpose* Group I

17

2

0

24

7

Group II

6

0

0

32

12

Group III

13

0

0

32

5

Group IV

13

0

1

31

4

Group V

11

3

1

24

10

60

5

2

143

38

Group I

46

4

0

0

0

Group II

48

2

0

0

0

Total

For drinking and cooking

Group III

50

0

0

0

0

Group IV

49

0

0

0

0

Group V

45

3

0

1

1

238

9

0

1

1

Total

*Use of water for bathing, wash of utensils and feed animals

The daily drinking water requirement per person per day varied from 1-12 liters. The mean water intake was 3.97 liters/person/day. Among the group V respondents the mean water intake was lower in comparison to the respondents of other groups which was 2.66 liters/person/day). The water intake per person per day was comparatively higher amongst the group III respondents. The age of the tube wells used by the respondents varied from 3 months to 50 years. Average age of tube well was 10.3 years. The mean years of age of the tube wells used by the group V (7.5 years), group I (6.1 years) and group II (8.2 years) was comparatively lower than the tube wells used by group III (14.0 years) and group IV (15.9 years). Most (97.8%) of the respondents could not inform about the method used for measuring arsenic concentration of their tube wells. But majority of the study subjects replied correctly regarding painted color of the tube wells. Above 96% of the respondents of each group reported that health professional advised them to abstain from drinking water of red-marked tube wells. Of them 100% of the group I respondents

and 98% of the group II respondents didn’t use the water of red-marked tube wells for drinking and cooking purposes; while 100% respondents of group III and group IV still using red-marked tube wells for drinking purpose (Table 2). Almost all the respondents knew about arsenicosis. Regarding a supplemen-tary question on sources of information the participant reported that doctors and health workers made them aware about the disease. Twenty five percent of the respondents were suffering from general weakness, cough, peptic ulcer, hypertension, diabetes mellitus, etc. The mean BMI of the patients of 5 groups was in between 19 to 21. Table 3 shows that all the patients had melanosis. Different forms of melanosis, e.g. round/hypopigmented, diffuse and both were almost homogeneously distributed in all the groups’ except group V. The Ninety four respondents (38.8%) had keratolytic skin lesion. Of them 14 were from group V, 9 from group I, 31 from group II, 13 from group III and 27 from group IV

46 Khandker et al Arsenic-safe drinking water and antioxidant

Vol. 1, No. 2, December 2006

majority of respondents (38 out of 50) of group II also felt that they are improving by green arsenic -safe water only. On the other hand, a few respondents (11) of group III felt that they are improving but all other respondents felt that they

respectively. Eighty percent of the melanosis was defuse-melanosis, and 74% of keratosis belongs to mild stage. The perception of all the respondents of group I felt that they are improving from the illness by taking safe water and antioxidants regularly. The Table III: Distribution of respondents according to skin lesions status Group

Melanosis Diffuse

Both

44

1

5

3

1

Group I Group II

5

40

5

20

9

2

Group III

1

44

5

11

2

0

Group IV

2

40

8

22

5

0

Group V

1

31

18

12

2

0

13

199

37

70

21

3

Total

Mild

Keratosis Moderate

Round/ hypopigmented 4

Severe

not the antioxidant (group IV), none did show any improvement of their illness. The respondents who took antioxidants irregularly and had irregular intake of safe water, were not considered to compare the prognosis of skin lesions.

are not improving at all. It was observed that the respondents who took arsenic-safe drinking water from green-marked tube well and antioxidants regularly (group I), melanosis improved in 43 respondents, 6 didn’t improve and none was found to have deteriorated (Table 4). The respondents who took green-marked tube well water regularly but not the antioxidant (group II), melanosis improved in 22 respondents, 24 did not improve and 4 deteriorated. The respondents who were using red-marked tube well water and antioxidants (group III), only two of them improved; and all other respondents either deteriorated or did not improve. The respondents who were using red-marked tube well water but

Regarding keratosis, the respondents who took green-marked tube well water regularly and antioxidant regularly (group I), 8 of them improved, 1 didn’t change; while the respondents who took green-marked tube well water regularly but not the antioxidant (group II), 8 out of 31 improved much, 21 remained unchanged and 2 deteriorated. Among the respondents of other groups, keratosis deteriorated.

Table IV: Prognosis of respondents of different groups according to skin lesions Group Group I

Improved 43

Melanosis Not improved 6

Deteriorated 0

Improved 8

Keratosis Not improved 1

Deteriorated 0

Group II

22

24

4

8

21

2

Group III

2

24

24

0

4

9

0

8

42

0

0

27

67

62

70

16

26

38

Group IV Total

Discussion The highest proportions of affected people were service holder. Usually service holders live in the

Bangladesh J Pharmacol

Upazilla headquarter and almost 100% tube wells of sadar Upazilla are arsenic contaminated. This may be a reason why highest percent of

47

arsenic affected people of our observation study are service holder. As expected, more than 95% of patients were reported to drink tube well water. From 1975 to 1997, DPHE with co-operation of UNICEF was struggling to reduce mortality and morbidity of children due to waterborne diseases by ensuring bacteriological safe drinking water supply through installation of shallow tube well. At present there are about 6-8 million shallow tube well in the country (Ahmed and Ahmed, 2002) and 97% of the rural drinking water supply in Bangladesh is obtained from ground water. Therefore majority of study population are using shallow tube well for drinking and cooking purposes. Massive tube well water testing started from 1997. The field staff tested tube well water for arsenic, marked red in case of high (above 50 µg/l) arsenic concentration and provided health education on consequences of drinking water of red-marked tube well. As a result people are aware of the effects of using red-marked tube well. Therefore, it was logical that 96.4% of the study subject reported that they have been informed about adverse effect of drinking water from red marked tube well. The respondents of group III and group IV are still drinking arsenic conta-minated water although they are aware about the consequences. This behavioral aspect indicates the gap between knowledge, attitude, practice and scope for using arsenic-safe drinking water. Moreover, more than 32% of the patients could not correctly interpret the message of red- and green-painted tube wells. The mean BMI of the patients of 5 groups was in between 19 to 21 that correspond to normal value of Bangladeshi people (Jahan and Hossain, 1998). Based on the finding it can be said that, on an average the study populations are maintaining a normal nutrition. Although some researcher stated that poor nutritional status may increase an individual’s susceptibility to chronic arsenic toxicity, or alternatively that arsenicosis may contribute to poor nutritional status (Milton et al., 2004). Further intensive research is recommended in this field.

All the respondents had melanosis and 38% had both keratosis and melansis. The respondents who took green-marked tube well water and antioxidant (group I) regularly, regression of melanosis was observed, while among the respondents who took green-marked tube well water regularly but no antioxidant (group II) did not show much regression of melanosis and majority remain unchanged. Among the respondents of other categories, deterioration of melanosis was observed. Similarly, among the respondents who took green-marked tube well water and antioxidant regularly regression of keratosis was observed after 6 months. Kosnet et al., (1990) opined on the basis of study findings that use of vitamin A analogues (retinoids) might be useful in treating precancerous arsenical dermatosis. However, recovery from chronic arsenic toxicity particularly the resulting peripheral neuropathy may take months and may not be complete. Kosnet (1999) suggested that treatment with retinoids might have promise in the treatment of chronic cutaneous arsenism. It is observed that, the skin condition of group III and group IV deteriorated and group II have improved but improvement was less than I group. These finding coincides with the study report of Oshikawa et al., (2001) that drinking piped or bottled water increased the probability of regression in subjects with mild stage of arsenical lesion. Therefore, early preventive measure is recommended to control deterioration of disease. Bangladesh Arsenic Control Society (2003) conducted a double-blind placebo-controlled trial among 319 arsenicosis patients. They treated the patients with beta-carotene, ascorbic acid, alphatocopherol, selenium, zinc and folic acid in standard recommended doses for 6 consecutive months and then every alternative month for a total period of 12 months. They found significant clinical improvement of skin lesion among the treatment group. The severities of palmer and plantar keratosis were reduced after 12 months. It revealed from this study that, patients, who were taking arsenic-safe water as well as antioxidants,

48 Khandker et al Arsenic-safe drinking water and antioxidant

Vol. 1, No. 2, December 2006

the improvement was more. The improvement of the disease could be observed which was evident from the skin color of the pre- and postintervention photographs. It was observed that a combination of arsenic-safe water and antioxidant was the most effective for the management of arsenicosis patients. Ahmad et al., (1998) stated that the manifestations of chronic arseniccosis, melanosis and keratosis showed clinical improvement on treatment with vitamin A, E and C, withdrawal of arsenic contaminated drinking water and use of keratolytic agent where applicable. Amongst those who had used both arsenic safe drinking water and regular medications 90.5% showed improvement of melanosis, 86.4% showed improvement of keratosis but none showed deterioration. The study further stated that the observed clinical improvements almost coincided with patients' perceptions of subsidence of signs of disease. This finding is correlated to this observation study. Comparatively only arsenic-safe water was more effective than only antioxidant treatment. Therefore, for the management of arsenicosis it could be strongly recommend for ensuring arsenic-safe water as immediate preventive measure. DGHS recommended arsenic-safe water and intervention with antioxidant e.g. vitamin A, E and C for a period of 6 months with two months interval in between. Use of antioxidant for longer than 6 months should be cautiously recommended (Talukder, 1999). Verret et al., (2005) reported that supplementation with vitamin E and selenium, either alone or combination, slightly improved skin lesion status although the improvement was not statistically significant. Recently, success in the treatment of arsenicosis with indigenous medicine ‘Spirulina’ has been claimed. Several investigators (Sikder et al., 2000; Khan et al., 2000; Choudhury et al., 2000; Huq et al., 2000) reported that spirulina, a natural microalIe, is found to be effective in the treatment of chronic arsenic poisoning. Misbahuddin et al., (2006) reported that alcohol extracted spirulina in combination with zinc is effective for the management of arsenicosis patients.

Bangladesh J Pharmacol

Ahmad et al., (2006) stated that 204 newly diagnosed patients have been included in the old list of Bhanga Upazilla. They identified 18 old patients those were asymptomatic. Among them 12 were subject of this study. Ahmad et al., (2006) opined that the asymptomatic patients might have recovered due to judicious usages of treatment options. In conclusion, the present study shows that combination of drinking arsenic-safe water and use of antioxidants give good result in improvement of the arsenicosis. It is also evident that both of the interventions have positive impact on arsenicosis patients, but further studies are required to evaluate objective prognosis as well as the individual potentiality. Considering the gap between knowledge and practice about drinking arsenic contaminated water in a considerable number of respondents, interventions are needed to motivate the people with an aim to improve the practice of patients for drinking safe water.

References Ahmad SA, Faruquee MH, Sayed MHSU, Khan MH, Jalil MA. Ahmed R, Hadi SA. Chronic arsenicosis: management by vitamin A, E, C regimen. JOPSOM. 1998; 17: 19-26. Ahmad SA, Rahman MS, Ullah MHSS, Khan MH, Faruquee MH, Karim N. Re-screening of arsenicosis patients in UNF funded 3 Upazillas. WHO-EH Unit; 2006, p 27. Ahmed MF, Ahmed CM (eds). Arsenic mitigation in Bangladesh. Ministry of Local Government, Dhaka, 2002, pp 20-25. Bangladesh Arsenic Control Society. Double-blind, randomized, placebo-controlled trial of antioxidant vitamins and minerals in the treatment of chronic arsenic poisoning in Bangladesh. BACS, Dhaka, 2003, pp 1-103. Bangladesh Rural Advancement Committee (BRAC). Tube well screening report in Bhanga Upazilla, BRAC, Dhaka, 2001, pp 1-22. Caldwell BK, Caldwell JC, Mitra SN, Smith W. Searching for an optimal solution to the Bangladesh water crisis. Social-Science Med. 2003; 56: 2089-96. Caussy D. Field guide for arsenicosis case detection, disease surveillance and patient management. WHO-SEARO, New Delhi, 2005, pp 1-24. Choudhury SAR, Khan AK, Misbahuddin M., Islam AZMM, Sahjahan M. Role of spirulina in the treatment of

49

chronic arsenic poisoning. Bangladesh Armed Forces Med J. 2000; 27: 181-85. Chowdhury UK et al. Ground water arsenic contamination in Bangladesh and West Bengal, India. Environ Health Perspect. 2000; 108: 393-97. Debra SM, Stephen EW, David MC. n-Acetylcysteine in the treatment of human arsenic poisoning. J Am Board Fam Pract. 1990; 3: 293-96. Dhaka Community Hospital (DCH). Arsenicosis patient list of Bhanga Upazilla. Arsenic patient identification and management in 7 Upazilla, DCH, Dhaka, 2001, pp 1-11. Guha Mazumder DN, De BK, Santra A, Ghosh N, Das S, Lahiri S, Das T. Randomized placebo-controlled trial of 2,3-dimercapto-1-propanesulfonate (DMPS) in therapy of chronic arsenicosis due to drinking arsenic contaminated water. Clin Toxicol. 2001; 39: 665-74. Huq MA, Misbahuddin M, Choudhury SAR. Spirulina in the treatment of chronic arsenic poisoning. Bangladesh J Physiol Pharmacol. 2000; 16: 15-16.

Kosnet JM. Clinical approaches to the treatment of chronic arsenic intoxication: from chelation to chemoprevention. Arsenic exposure and health effects, Elsevier Science, 1999, pp 349-54. Milton AH, Hassan S, Shahidullah SM, Sharmin S, Jakariya M, Rahman M, Keith D, Smith W. Association between nutritional and arsenicosis due to chronic arsenic exposure in Bangladesh. Int J Environ Health Res. 2004; 14: 99-108. Misbahuddin M, Islam AZMM, Khandker S, Ifthaker-AlMahmud, Islam N, Anjumanara. Efficacy of spirulina extract plus zinc in patients of chronic arsenic poisoning: a randomized placebo-controlled study. Clin Toxicol. 2006; 44: 135-41. Oshikawa S, Geater A, Chongsuvivatwong V, Piampongsan T, Chakraborti D, Samanta G, Mandal B, Hotta N, Kojo Y, Hironaka H. Long-term changes in severity of arsenical skin lesions following intervention to reduce arsenic exposure. Environ Sci. 2001; 8: 435-48.

Jahan K, Hossain M. Nature and extent of malnutrition in Bangladesh. Bangladesh National Nutrition Survey 199596. University of Dhaka, Dhaka, 1998, p 143.

Sikder MS, Islam AZMM, Khan MAK, Huq MA, Choudhury SAR, Misbahuddin M. Effect of spirulina in the treatment of chronic arsenicosis. Bangladesh J Dermatol Venereal Leprol. 2000; 17: 9-13.

Kaufmann RB, Sorensen BH, Rahman M, Streatfield K, Persson LA. Addressing the public health crisis caused by arsenic contamination of drinking water in Bangladesh. World Bank, Dhaka, 2001, pp 1-30.

Talukder KR. The diagnosis and management of arsenicosis cases, Learning module, environmental and occupational health (including arsenic) unit, Directorate General of Health Services, Dhaka, 1999.

Khan MAK, Choudhury SAR, Misbahuddin M, Islam AZMM, Shahjahan M. Effects of spirulina in the treatment of chronic arsenic poisoning in Bangladesh. Bangladesh J Med Sci. 2001; 7: 223-31.

Verret WJ, Chen Y, Ahmed A, Islam T, Parvez F, Kibriya MG, Graziano JH, Ahsan H. A randomized, double-blind placebo-controlled trial evaluation the effects of vitamin E and selenium on arsenic-induced skin lesions in Bangladesh. J Occup Environ Med. 2005; 47: 1026-35.

Kosnet JM, Kreiss K. Agency for toxic substances and disease registry (ATSDR). Case studies in environmental medicine. Arsenic toxicity, 1990, p-18.

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Vol. 1, No. 2, December 2006

Bangladesh J Pharmacol 2006; 1: 51-57 Copyright © 2006 by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

A comparative study on the adverse effects of two anti-tuberculosis drugs regimen in initial two-month treatment period 1

Begum Lutfun Nahar, 1A.K.M. Mosharrof Hossain, 2M. Monirul Islam and 1Dipti Rani Saha 1

Department of Pharmacology and Therapeutics, Sylhet MAG Osmani Medical College, Sylhet 3100, Bangladesh; 2Chest Disease Hospital, Sylhet 3100, Bangladesh [Received 4 July 2006]

Abstract Tuberculosis (TB) is a leading cause of death throughout the world and Bangladesh stands 4th among high burden countries. Treatment of TB hampered with poor patient compliance and intolerance at least partly due to the adverse drug reactions. A prospective longitudinal non-randomized case study was conducted on 64 Hospital admitted patients diagnosed as primary (category I) and resistant or treatment failure (category II) to compare adverse effects between two anti-TB drug treatment regimen based on diagnostic category. Category I received four drug (rifampicin, isoniazid, ethambutol, pyrazinamide) and category II received five drug (rifampicin, isoniazid, ethambutol, pyrazinamide, sparfloxacin) combination treatments for initial 2 months under DOTS during the period of July 2004 to July 2005. Adverse effect parameters e.g. gastrointestinal disturbances, arthralgia, hepatic dysfunction and renal impairment were estimated before, two and eight weeks after initiation of treatment. Predisposing risk factors for adverse effects e.g. age, sex, nutritional status, associated disease, habits were also analyzed. In our study, 76.47% of total patients experienced some sorts of adverse effects. In 4- and 5-drug regimen groups’ adverse reaction were observed in 50% and 95% of patients respectively. Serum bilirubin, SGPT, creatinine did not change in neither of the treated group while alkaline phosphatase tended to decrease and uric acid to increase. No disease was established to be risk factor for drug intolerance. Key words: Bangladesh, isoniazid, pyrazinamide, rifampicin, tuberculosis

Introduction Tuberculosis (TB) causes three million death each year (Wright et al., 1998), that is declared to be 'global health emergency' by World Health Organization (WHO, 1996). Bangladesh is one of the top five high burden countries in the world where TB is the second killer infectious disease next to diarrhea and about 0.6 million people are estimated to suffer from TB (Benoor et al., for correspondence: AKM Mosharrof Hossain, Department of Pharmacology and Therapeutics, Sylhet MAG Osmani Medical College, Sylhet 3100, Bangladesh

1998). Despite the availability of affective chemotherapy TB is still a major health problem in most countries (Grange, 1990). The poor outcome was attributed to poor patient compliance, to primary multidrug resistance and to interruption partly due to adverse drug reaction (WHO, 1997). The most important step to ensure treatment is the patients’ adhere to treatment are introduction of direct observation treatmentshort course (DOTS) and use of fixed dose combination, as recommended by WHO (WHO, 1998; Blomberg et al., 2001).

Anti-TB chemotherapy exhibit greater level of efficacy with an acceptable degree of toxicity, however combination treatment may produce severe adverse events (Schaberg et al., 1996). Important adverse effects are hepatitis, rash, gastrointestinal upset, hyperuricemia, peripheral neuropathy, visual disturbances (Pande et al., 1996; Hussain et al., 2003; Bass et al., 1994; Scott, 1995; Solangi et al., 2004; Pereira et al., 2000; Tsai and Lee, 1997). A few studies are done on adverse effects of antiTB drugs related to specific regimen of drug and risk factors for those adverse effects. So, we aimed to study the adverse drug reaction and to analyze risk factors for intolerance and adverse effects of two anti-TB drug regimen in initial two month phase of treatment.

Material and Methods A prospective longitudinal non-randomized case study was carried out on diagnosed pulmonary TB patients admitted in the Sylhet Chest Disease Hospital and Sylhet Shaheed Shamsuddin Ahmed Hospital during the period of July 2004 to July 2005. Inclusion criteria were pulmonary tuberculosis patients diagnosed as primary treatment failure (category I) or relapse case or resistance TB (category II). Cases of either sex with age above 14 years and patients who were under DOTS. While patients with history of hepatitis, renal or hepatic impairment; pregnant women and age below 14 years were excluded from the study. In total 34 patients were included in this study, of which 14 had received category I, 4 anti-TB drugs that include rifampicin, isoniazid, ethambutol, pyrazinamide. Another 20 patients received category II: 5 anti-TB drugs that included rifampicin, isoniazid, ethambutol, pyrazinamide and sparfloxacin. All the patients received the drugs under direct observation therapy shortcourse (DOTS)-treatment program.

52

Nahar et al Adverse effects of two anti-tubercular drugs

Primary variables were organ related adverse effects e.g. signs, symptoms and function tests. Secondary variables were predisposing risk factors e.g. age, sex, nutritional status, socio-economic condition and habits. Organ-related adverse effects and biochemical parameters were estimated before (0 week) and 4 and 8 weeks after initiation of treatment. Data was analyzed by x², One-way ANOVA with repeated measure design and post hoc test of significance was done by pair t test by using SPSS 11.

Results A total number of 64 patients were included of which 34 completed the study. Among the 30 patients who did not complete the study, 4 patients expired and 26 patients dropped out. Among the patients completed the study, 27 (79.42%) were male and 7 (20.58%) were female. In 4-drug treatment group 14 male and female patients of age 38.00 ± 9.56 years (mean ± SD) and in 5-drug treatment group 20 male and female patients of age 38.55 ± 12.78 years were included. The mean age of male and female patients was 38.37 ± 11.59 years and 38.14 ± 13.95 years respectively. Male and female patients did not differ in their age (t = 0.04, NS). All of 27 (100%) male patients and out of 7 female patients 5 (71.42%), yielding a total number of 32 (94.12%) were in under nutritional group and lived in low socio-economic condition. In habitual status 10 (37.03%) of males were smoker, while none of female had that habit. Eight (29.62%) of male and 4 (57.14%) female patients making a total number of 12 (35.28%) were habituated with betel nuts. Out of the treated patients 76.47% experienced some sorts of unwanted effects (Table I). Adverse effects were observed in 7 patients (50%) in 4-drug regimen group and in 19 patients (95%) in 5-drug regimen group. Notable adverse effects were abdominal pain experienced by 1

Vol. 1, No. 2, December 2006

patient (7.14%), muscle weakness by 2 patients (14.28%) and joint pain by 5 (35.71%) in 4-drug regimen group. In 5-drug regimen group abdominal pain was experienced by 6 (30%) patients, muscle weakness by 5 (40%) and joint pain by 6 (30%) patients respectively. Table I: Adverse effects observed in course of two antituberculosis drug treatment regimen recorded during initial two month of treatment Symptoms Nausea Vomiting Anorexia Abdominal pain Joint pain Joint swelling Muscle weakness Visual disturbances Rash Vestibular disturbances Peripheral neuropathy

4-drug group n = 14 1 (7.14) 1 (7.14) 1 (7.14) 1 (7.14) 5 (35.71) 0 2 (14.28) 4 (28.56) 0 1 (7.14) 00

5-drug group n = 20 1 (5) 1 (5) 2 (10) 6 (30) 6 (30) 3 (15) 5 (40) 5 (40) 3 (15)) 0 1 (5)

Values in parenthesis are expressed in percentage

In the 4-drug treatment group before initiation of treatment regimen the estimated serum glucose level was 93.60 ± 20.88 mg/dl. After 2 weeks and 8 weeks of inception of treatment regimen glucose level was detected as 99.18 ± 20.70 mg/dl and 85.23 ± 13.97 mg/dl respectively (Table II). These differences were not statistically significant. In the 5-drug treatment group before initiation of treatment regimen the estimated serum glucose level was 91.88 ± 25.46 mg/dl. After 2 weeks and 8 weeks of inception of treatment regimen creatinine level was detected as 85.98 ± 27.21 mg/dl and 84.48 ± 16.61 mg/dl respectively (Table III). In the 4-drug treatment group before implementing the treatment regimen bilirubin was 0.48 ± 0.20 mg/dl. After 2 and 8 weeks of inception of treatment regimen bilirubin level was estimated 0.63 ± 0.80 mg/dl and 0.37 ± 0.23 mg/dl respectively (Table II). One way ANOVA shows no changes in course of treatment in this group [F (2, 39) = 0.952, NS].

Bangladesh J Pharmacol

In the 5-drug treatment group before initiation of the treatment regimen bilirubin (mean ± SD) was 0.58 ± 0.72 mg/dl. After 2 and 8 weeks of inception of treatment regimen bilirubin level was estimated 00.72 ± 1.28 mg/dl and 0.43 ± 0.30 mg/dl respectively (Table III). One way ANOVA shows no changes in course of treatment in this group [F (2, 57) = 0.547, NS]. In the 4-drug treatment group before administration of the treatment regimen SGPT was 04.85 ± 2.17 U/l. After 2 and 8 weeks of treatment regimen SGPT level was estimated 07.50 ± 6.02 mg/dl and 05.92 ± 3.09 U/l respectively (Table II). One way ANOVA shows no changes in SGPT in this group [F (2, 39) = 1.46, NS]. After two weeks of treatment SGPT tended to increase but was neither statistically significant nor clinically appreciable [t = 1.41; df 13, NS]. After 8 weeks of treatment SGPT level was almost same as that of pre-treatment level [t = 1.11; df 13; NS]. There observed no difference between SGPT level estimated 2 and 8 weeks of treatment [t = 0.85; df 13; NS]. In the 5-drug treatment group before initiation of the treatment regimen SGPT was 11.10 ± 16.63 U/l. After 2 and 8 weeks of inception of treatment regimen SGPT level was estimated 11.50 ± 8.87 U/l and 8.05 ± 4.47 U/l respectively (Table III). One way ANOVA shows no changes in course of treatment in this group [F (2, 57) = 0.569, NS]. There observed no difference between SGPT level estimated 2 weeks and 8 weeks of treatment [t = 1.64; df 19; NS]. No differences in SGPT level was observed in the group of patients (4-drug versus 5-drug) when estimated after 2 weeks [t = 1.46; NS] and 8 weeks [t = 1.53; NS]. In the 4-drug treatment group before implementing treatment regimen serum alkaline phosphatase was 102.80 ± 51.15 U/l. After 2 weeks and 8 weeks of inception of treatment regimen alkaline phosphatase tended to decrease to 78.49 ± 24.14 U/l and 70.66 ± 41.59 U/l respectively (Table II). However, One way ANOVA shows no signifi-

53

cant differences in alkaline phosphatase in course of treatment [F (2, 39) = 2.392, NS]. In the 5-drug treatment group before administration of treatment regimen serum alkaline phosphatase was 115.51 ± 85.71 U/l. After 2 weeks and 8 weeks of inception of treatment regimen alkaline phosphates was estimated to be as 106.29 ± 55.85 U/l and 79.18 ± 26.13 U/l respectively (Table III). However, One way ANOVA shows no significant differences in alkaline phosphatase in course of treatment [F (2, 57) = 1.91, NS]. In the 4-drug treatment group before initiation of treatment regimen the estimated creatinine level was 1.11 ± 0.39 mg/dl. After 2 weeks and 8 weeks of inception of treatment regimen creatinine level was detected as 1.08 ± 0.35 mg/dl and 1.04 ± 0.22 mg/dl respectively (Table II). In the 5-drug treatment group before initiation of treatment regimen the estimated creatinine level was 00.99 ± 0.27 mg/dl. After 2 weeks and 8 weeks of inception of treatment regimen creatinine level was detected as 1.08 ± 0.23 mg/dl and 1.07± 0.21 mg/dl respectively (Table III).

In the 4-drug treatment group before implementing the treatment regimen serum uric acid level was 4.86 ± 2.12 mg/dl. After 2 weeks and 8 weeks of inception of treatment regimen uric acid level was detected 6.22 ± 2.70 mg/dl and 6.34 ±1.52 mg/dl (Table II). After 2 weeks of treatment uric acid tended to increase but was not statistically significant [t = 2.05; df 13, NS]. After 2 months of treatment uric acid level significantly increased in this treatment group [t = 2.33, df 13; NS]. There observed no difference between uric acid level estimated 2 weeks and 8 weeks of treatment [t= 0.16, df 13; NS]. In the 5-drug treatment group before administration of the treatment regimen serum uric acid level was 4.48 ± 1.57 mg/dl. After 2 weeks and 8 weeks of inception of treatment regimen uric acid level was detected 5.57 ± 2.12 mg/dl and 5.74 ± 2.49 mg/dl (Table III). After 2 weeks of treatment uric acid tended to increase but was not statistically significant [t=2.07; df 19, NS]. After 2 months of treatment uric acid level did not change [t=1.87, df 19; NS].

Table II: Biochemical organ function tests of 4-drug treatment regimen group recorded during initial two month of treatment Biochemical tests Serum bilirubin Serum ALT (SGPT) Serum Alkaline phosphatase Serum creatinine Serum uric acid Serum glucose

0 week 0.48 ± 0.20 4.85 ± 2.17 102.80 ±51.15 1.11 ± 0.39 4.86 ± 2.12 93.60 ± 20.88

4 weeks 0.63 ± 0.80 7.5 ± 6.02 78.49 ± 24.14 1.08 ± 0.35 6.22 ± 2.70 99.18 ± 20.70

8 weeks 0.37 ± 0.23 5.92 ± 3.09 70.66 ± 41.59 1.04 ± 0.22 6.34 ± 1.52 85.23 ± 13.97

Table III: Biochemical organ function tests of 5-drug treatment group recorded during initial two month of treatment Biochemical tests Serum bilirubin Serum ALT(SGPT) Serum Alkaline phosphatase Serum creatinine Serum uric acid Serum glucose

0 week 0.53 ± 0.72 11.1 ± 16.63 111.51 ± 85.71 0.9 ± 0.27 4.48 ± 1.57 91.88 ± 25.46

Discussion In 1998 the global TB program of WHO established Global TB Research initiative to support related research. Research has been done by NGOs on Health System and Service Research 54

Nahar et al Adverse effects of two anti-tubercular drugs

4 weeks 0.72 ± 1.28 11.5 ± 8.87 106.29 ± 55.85 1.08 ± 0.23 5.57 ± 2.12 85.98 ± 27.21

8 weeks 0.43 ± 0.30 8.05 ± 4.47 79.18 ± 26.13 1.07 ± 0.21 5.74 ± 2.49 84.48 ± 16.61

that include studies on DOTS by community health workers (Chowdhury et al., 1997), on microscopic diagnosis (Vandeun and Portalels, 1998) and drug resistance surveillance (Haque et al., 1995). Vol. 1, No. 2, December 2006

Compliance with anti-TB medication is essential to effective management. Two strategies to ensure compliance are DOTS and fixed dose combination (FDC) (Van leuven et al., 1997). Our study fully applied on DOTS strategy. Generally these drugs are well tolerated (Dutt et al., 1983), may be associated with unwanted effects of different origin. Adverse drug reactions had been extensively studied and reviewed (Haque and Golam Nabi, 1999; Mohan et al., 2004; Resi et al., 2004; Maddrey, 2005). The present prospective study was undertaken to demonstrate the extent of adverse effects of two anti-tuberculosis drug treatment regimens under DOTS. Adverse effects were assessed according to sign and symptoms of the patients and the systemic organ-related adverse effects were estimated by serum glucose, serum bilirubin, serum alanine aminotransferase, alkaline phosphatase, creatinine, uric acid. As predisposing risk factors for adverse effects we observed age, sex, nutritional status, socioeconomic condition and habit. Serum glucose was estimated before the treatment initiation with the aim to assess hyperglycemia as predisposing risk factor for TB or adverse drug events involving hepatic or renal dysfunction. Subsequently in course of treatment serum glucose level was monitored with the view of effects of these drugs on carbohydrate metabolism of liver. In our observation in course of treatment there were no significant changes in glucose level estimated neither before nor after two month of treatment. Most of the TB patients complete treatment without any significant adverse effects, however data (Ormerod and Horsfield, 1996) showed that side effects occur in 57.8% patients, while we observed 76.47% of patients experienced some sort of unwanted effects. Unwanted effects were minor in nature, only 2.94% exhibited major adverse effects e.g. hepatitis in our study, while Shakya et al., (2004) reported an 8% incidence of hepatotoxicity. Side effects related to the digestive system were observed in 29.41% of patients that was consistent with the observation of others (Ormerod and Horsfield, 1996). We observed no

Bangladesh J Pharmacol

evidence of allergic reactions, neurotoxicity, cardiovascular-related adverse effects as observed by other (Ormerod and Horsfield, 1996). It is important that patients should be monitored during treatment so that adverse effects can be detected promptly and managed properly (WHO, 2003). Serious adverse effects require documented change in therapy or hospitalization. If minor adverse effects develop patients should continue with reduced doses and receive symptomatic treatment, if major side effects develop the offending drug should be stopped (WHO, 2003). The withdrawal of treatment due to major adverse effect e.g. hepatitis in 1.8 to 6.0% patients (Girling, 1978; Smith and Zirk, 1961). Ormerod and Horsfield (1996) reported that 51% had reaction to anti-tuberculosis drug requiring modification of treatment while none of our patients required modification of treatment. Termination of treatment either with INH, rifampicin or pyrazinamide because of severe side effects was necessary in 23% as reported by Schaberg et al., (1996). Baseline studies of complete blood counts, hepatic and renal function studies were suggested to be performed at the initiation of treatment (Dutt et al., 1983). In our study hepatic and renal function tests were done with the view to assess the effects of the used drugs on these organs. We observed no substantial abnormality in baseline liver and renal function tests in course of treatment regimens. It is very uncommon to have adverse reactions to a single anti-TB drug, but such reaction with more than one is very small (Smith and Zirk, 1961), while adverse reactions to multiple drugs were reported (Mathus et al., 1979; Thamari and Gupta, 1981). In our observation, major side effects as hepatitis developed in 2.94%, that was similar to the report of Dutt et al., 1983). During the initial phase of 4-drug combination treatment regimen side effects were reported in 22.4% of patients (Haque and Golam Nabi, 1999).

55

In our 4 drug treatment regimen study group, 50% of patients developed side effects during the initial phase. Of them 35.7 % complained of arthralgia, 28.57% experienced GIT upset, muscle weakness evidenced in 14.28%, vestibular disturbances in 7.14% persons and sign of peripheral neuropathy was absent. Our observation differed to some extent from observation of others (Dutt et al., 1983; Ohkawa et al., 2002). In 5-drug treatment regimen study group, adverse effects observed in 90% of patients. Of them 30 % developed arthalgia, 50% experienced GIT upset, 40% complained of muscle weakness. Adverse effects showed commoner in 5-drug treatment group compared to 4-drug group was not statistically significant. In our study 21.42% patients exhibited a transient and symptomless increase of SGPT that was in agreement with other reports (Lee, 1995; Lee et al., 2002). Fattinger et al. (1997) reported eight times increase of SGPT, while we observed 2-3 times increase in a few patients. Incidence of hepatitis was reported in 7.7% with combination of use of rifampicin and pyrazinamide (Lee et al., 2002), compared to drug used alone viz. with pyrazinamide 15%, with isoniazid 7%, with rifampicin 1.5% and hepatitis was suggested due to pyrazinamide rather than isoniazid or rifampicin (Schaberg et al., 1996). Isoniazid and pyrazinamide combination administration was associated with an increased mortality in patients with hepatitis. Hepatotoxic potential of rifampicin or pyrazinamide is far less in the doses used in the modern day short-course regimen. We did not observe such results in our study. We analyzed serum uric acid and creatinine level to assess kidney functions. Increase of uric acid level was reported (Solangi et al., 2004; Zierski and Bek, 1980). In our study 55.88% of patients showed increased uric acid level, that confirms other studies. Ethambutol is also reported to increase uric acid level (Postlethwaite et al., 1971). We did not observe any change in serum creatinine level, while Solangi et al. (2004) observed

56

Nahar et al Adverse effects of two anti-tubercular drugs

non-significant elevation. Our observation suggests no appreciable effects of anti-TB drugs on kidneys. Risk factors for anti-TB drugs-induced hepatitis had been studied (Shakya et al., 2004; Fauzi et al., 2004; Marti et al., 2005; Tost et al., 2005). Case control studies were undertaken to assess the risk factors for hepatotoxicity with variable like age, sex, BMI, acetylator status, extent of disease, protein, alcohol intake (Pande et al., 1996). In our study hepatotoxicity developed in younger males who were given PZA that also is observed by Ohkawa et al. (2002). As predisposing risk factors there is no suggestive finding in our observation related to age and habit, however we observed TB more common in male; in poor nutritious and low socio-economic status. Our study applied on DOTS of both 4-drug and 5 drug–regimen patients. Due to time constrains and limitation of facilities the obtained results may not reflect the actual effects of drugs on population. Further prospective longitudinal interventional randomized studies with larger sample size of different subject group are necessarily recommended.

References Bass JB, Farer LS, Hopewell PC, O’Brien R, Jacobs RF, Ruben F, Snider DEJ, Thornton G. Treatment of tuberculosis and tuberculosis infections in adults and children. Am J Respir Crit Care Med. 1994: 149: 1359-74. Benoor KS, Mahmood AM. Huq AKMS. Multidrug resistant tuberculosis: problem arise. Abstract, The National Conference of Chest; 1998, p 15. Blomberg B, Spinaci S, Fourie B, Laing R. The rationale for recommending fixed-dose combination tablets for treatment of tuberculosis. WHO 2001; 79: 61-68. Chowdhury AM. Chowdhury S. Islam MN, Islam A, Vaughan JP. Control of tuberculosis by community health workers in Bangladesh. Lancet. 1997; 350: 169-72. Dutt AK. Moers D, Stead WW. Undesirable side-effect of isoniazid and rifampicin in largely twice-weekly shortcourse chemotherapy for tuberculosis. Am Rev Respir Dis. 1983; 128: 419-24. Fattinger K, Braunschweig S, Reichen J, Meier-Abt PJ, Krahenbuhl S. Liver injury under tuberulostatic treatment. Schweiz Rundsch Med Prax. 1997; 86: 626-29.

Vol. 1, No. 2, December 2006

Fauzi AR, Shah A, Rathor MY, Satwi S, Risk factors for antituberculous drugs-induced hepatitis: a prospective survey from a chest clinic in a general hospital. Med J Malaysia. 2004; 59: 72-77.

Resi D, Gangliotti C, Moro ML. Side effects of anti-tuberculosis therapy. Am J Respir Crit Care Med. 2004; 169: 542.

Girling DJ. The hepatictoxicity of antituberculosis regimens containing isoniazid, rifampicin and pyrazinamide. Tubercle. 1978; 59: 13-32.

Schaberg T, Rebhan K, Lode H. Risk factors for side-effect of isoniazid, rifampicin and pyrazinamide in patients hospitalized for pulmonary tuberculosis. Eur Respir J. 1996; 9: 2026-30.

Grange JM. Drug resistance and tuberculosis elimination. Bull Int Union Tuberc Lung Dis. 1990; 65: 57-59.

Scott JT. Drug-induced gout. Baillieres Clin Rheumatol 1991; 5: 39-60.

Haque MA, Golam Nabi PD. Achievements of national TB control program. Bangladesh Med Res Counc Bull. 1999; 25: 71-82.

Shakya R, Rao BS, Shrestha B. Incidence of hepatotoxicity due to anti-tubercular medicines and assessment of risk factors. Ann Pharmacother. 2004; 38: 1074-79.

Haque ME, Yousuf M, Haque AKMS. A study on side effects of thiacetazone. Chest Heart Bull. 1995; 2: 63-65.

Smith JM, Zirk MH. Toxic and allergic drug reaction during the treatment of tuberculosis. Tubercle. 1961; 42: 287.

Hussain Z, Kar P, Hussain SA. Antituberculosis druginduced hepatitis; risk factors, prevention and management. Indian J Exp Biol. 2003; 41: 1226-32.

Solangi GA. Zuberi BF, Shaikh S, Shaikh WM. Pyrazinamide-induced hyperuricemia in patients taking antituberculosis therapy. J Coll Physicians Surg (Pak) 2004; 14: 136-38.

Lee AM, Mennone JZ, Jones RC, Paul WS. Risk factors for hepatotoxicity associated with rifampicin and pyrazinamide for the treatment of latent tuberculosis infection: experience from three public health tuberculosis clinics. Int J Tuberc Lung Dis. 2002; 6: 995-1000. Lee WM. Drug-induced hepatotoxicity. N Engl J Med. 2003; 349: 474-85. Maddrey WC. Drug-induced hepatotoxicity. J Clin Gastroenterol. 2005; 39: S83-89. Marti L, Del Olmo JA, Tosca J, Ornia E, Garcia-Torres ML, Serra MA, Rodriguez F, Lluch P, Escudero A, Rodrigo JM. Clinical evaluation of drug-induced hepatitis. Rev Esp Enferm Dig. 2005; 97: 258-65.

Thamari JP, Gupta VK. Hypersensitivy of multiple drug in treatment of pulmonary tuberculosis. Indian J Tuberc. 1981; 28: 143. Tost JR, Vidal R, Cayla J, Diaz-cabanela D, Jimenez A, Broquetas JM. Study group for severe hepatotoxicity due to anti-tuberculosis Drugs. Int J Tuberc Lung Dis. 2005; 9: 534-40. Tsai RK, Lee YH. Reversibility of ethambutol optic neuropathy. J Ocul Pharmacol Ther. 1997; 13: 473-77. Van leuven M. De Groot M, Shean K. Pulmonary resection as an adjunct in the treatment of multidrug resistant tuberculosis. Ann Throac Surg. 1997; 63: 1370-72.

Mathus KC, Jain VK, Gupta PP, Jain A. Multiple drug reaction during chemotherapy of tuberculosis. Indian Med Gazette. 1979; 8: 274.

Vandeun A. Portalels F. Limitation and requirement for quality control of sputum smear microscopy for AFB. Int J Tuberc Lung Dis. 1998; 2: 756-57.

Mohan A, Sharma SK. Side effects of anti-tuberculosis drugs. Am J Respir Crit Care Med. 2004; 169: 882-83.

WHO. Guide line for the management of drug resistant tuberculosis. 1998, pp 5-8.

Ohkawa K, Hashiguchi M, Ohno K, Kiuchi C, Takahashi S, Kondo S, Echizen H, Ogata H. Risk factors for antituberculosis chemotherapy-induced hepatotoxicity in Japanese pediatric patients. Clin Pharmacol Ther. 2002; 72: 220-26.

WHO. Standardized treatment regimens. Treatment of tuberculosis. Guide lines for national programs, WHO/ TB. 2nd ed. 1997, pp 23-31. WHO. TB deaths reach historic levels. Press release WHO; 1996, pp 1-3.

Ormerod LP, Horsfield N. Frequency and type of reactions to anti-tuberculosis drugs: observation in routine treatment. Tuberc Lung Dis. 1996; 77: 37-42.

WHO. Treatment of tuberculosis guidelines for national program 3rd ed. Geneva, WHO/CDS/TB/ 2003, p 313.

Pande JN, Singh SP, Khilnani GC, Khilnani S, Tandon RK. Risk factors for hepatotoxicity from anti-tuberculosis drugs: a case-control study. Thorax. 1996; 51: 132-36.

Wright PW, Wallace RJ Jr, Write NW, Brown BA, Griffith DE. Sensitivity of flurochrome microscopy for detection of mycobacteria. J Clin Microbial. 1998; 4: 1046-49.

Pereira RM, Tresoldi AT, Hessel G. Isoniazid-induced hepatic failure report of a case. Arq Gastroenteral. 2000; 37: 72-75.

Zierski M, Bek E. Side-effect of drug regimens used in short course chemotherapy for pulmonary tuberculosis a controlled clinical study. Tubercle. 1980; 61: 41-49.

Postlethwaite AE, Bartel AG, Kelley WN. Hyperuricemia due to ethambutol. N Engl J Med. 1972; 286: 761-62.

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Bangladesh J Pharmacol 2006; 1: 58-63 Copyright © 2006 by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

Condensation reaction of benzil with resorcinol and the establishment of the spectral data as well as cytotoxicity study A. Kashem Liton and M. Rabiul Islam Department of Chemistry, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh

[Received 23 September 2006]

Abstract Condensation of benzil, 1 with resorcinol in the presence of potassium carbonate at 110-120˚C gave the mixture of the compounds. Using various kinds of separation technique only five types of the polymeric products, a1, a2, a3, a4 and a5 were isolated. The compounds a1, a4 showed high cytotoxic activity and the compounds a2, a3 and a5 showed relatively low cytotoxic activity against the brine shrimp lethality bioassay. Key words: benzil, resorcinol, cytotoxic

Introduction

Methods and Materials

Various types of substituted heterocyclic compounds were synthesized and cytotoxic activity of these compounds have been found by screening tests (Islam et al., 2001a; Islam et al., 2001b; Lingcon et al., 2001). To compare the cytotoxic activity of the titled compounds in scheme 1.2 have been isolated and sent for screening tests whether they can show reasonable lethal effect on brine shrimp (Anderson et al., 1999) or not. The compounds that are mentioned have been furnished in Scheme 1.2 with flow chart 1.1.

Melting points were not corrected. IR spectra were recorded on a Shimadzu DR 8001 FT-IR spectrometer, NMR spectra on a WP 200 spectrometer using TMS as internal standard and mass spectra on an MS Kratas mass spectrometer.

for correspondence: M. Rabiul Islam; e-mail: [email protected]

Reaction: Benzil (0.01 moles or 2.10 gm), resorcinol (0.005 moles or 0.55 gm) and potassium carbonate (0.015 moles or 2.07 gm) were mixed and heated in three necked quick-fit round bottom flask in the oil bath at 110-120˚C. Heat was continued until all the solids were melted to liquid and this liquid was heated for half an hour. CO2 was evolved and this liquid was converted into colored solid mass. This solid contains mixture of compounds (Hans et al., 1905), PM that was examined on TLC.

Separation of mixture of the compounds, PM

Mixture of compounds, PM Washed with distilled water

Water insoluble portion

Water soluble portion, Pw

Washed with 10% acetic acid

Acetic acid insoluble portion

Acetic acid soluble portion

Washed with ethanol

Ethanol soluble portion, PETS

Final product, PETR (Ethanol insoluble)

After evaporation, 100% DCM, room temperature

Soluble portion, PDCMS

Insoluble portion, PDCMS

12 hours in Freeze Filtration

Filtrate, PLDM

Product (precipitate), a2

Flow Chart 1.1

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59

1 a1

a2

a3

a4

a5 Scheme 1.2

Extraction of the compound (a1): Chloroform was mixed to the water-soluble portion and shaken half an hour. Two layers were formed. Lower layer was organic layer and upper layer was aqueous layer. Thus yellow organic layer was collected in several times and dried over anhydrous Na2SO4. This organic layer was evaporated and it gives a single product, a1, m.p. 110-113ºC. Its color is red. IR: ν Nujol (cm-1) 3500-2500 (b, H bonded OH of COOH); 2955, 2926 (νC-H); 1605 (b, νC=O); 1207 (b, νC-O stretching). 1H-NMR (DMSO): δ 10.20-9.80 (b, COOH), δ 8.50-8.20 (b, OH), 8.00-6.00 (m, C-H, aromatic protons). 13C-NMR (DMSO): δ 195.20 (C-1); δ 135.93 (C-10), δ 132.59 (C-8), δ 131.11 (C-7), δ 129.97 (C-11), δ 129.89 (C-3), δ 129.62 (C-15), δ 129.33 (C-6), δ 129.21 (C-4), δ 128.90 (C-5), 128.00 (C-12) and δ 70.56 (C-9). Mass:

873.0 (15%), 677.0 (55%), 225.1 (6%), 154.0 (10%) and 105.0 (100%). The molecular ion peak appears at m/z 1256 due to C80H56O15. Separation of the product (a2): The product, a2 was dried in desiccator and gave single spot on TLC examination, m.p. 258-260ºC. The color of the compound was light brick red. IR: ν Nujol (cm1 ) 3200-3600 (s, b, νOH); 3087, 3056 (s, νC-H, aromatic); 1732, 1600 (νC=O); 1565, 1547 (νC=C, aromatic rings); 1212 (νC-O). Isolation of the compound (a3): After collecting the PLDM product, this portion was separated by column chromatography on silica gel using pet ether and ethyl acetate. 0.639 gm was taken for separation. Different fractions were recorded and then later were evaporated. TLC examinations were shown in Table I.

+ .

m/z (% of relative intensity) 1256.0 (M , 2%), Table I: Several fractions that were recorded during column chromatography Fractions st

1 fraction (1-5) 2nd fraction (6-13) 3rd fraction (14-18) 4th fraction (19-29) 5th fraction (30-34) 6th fraction (35) 7th fraction (36-42) 8th fraction (43-48)

60

Color ---light yellow light orange orange yellow light green slightly yellow yellow orange Light yellow orange

Liton and Islam Reaction of benzil with resorcinol

TLC examination and Rf value ---One spot, 0.73, benzil (reactant) Three spots having reactant one spot, 0.35 one spot with tailing two spot, 0.35, 0.81 Three spots with tailing Three spots having too much tailing

Vol. 1, No. 2, December 2006

Identification of the 4th fraction: This portion was orange yellow in color yielded 0.080 gm and m.p 140-142ºC. This was designated as a3. IR: ν Nujol (cm-1) 3600-3200 (b, νOH); 3025 (νC-H, aromatic); 1684, 1672 (νC=C, aromatic rings); 1277 (b, νC-O).

Table II: Different bands in TLC

Isolation of the compound (a4): Further separation of the 6th fraction: The 6th fraction in Table I was taken for PTLC method for further separation and pet ether and ethyl acetate were used as solvent (EA:PE=3:2). Three bands were found in Table II.

3rd band that collected was designated as a4. The product was solid (light pink color). IR: ν Nujol (cm-1) 3600-3200 (b, νOH (H bonded of COOH) group); 3060, 3028 (νC-H, aromatic); 1799, 1714 (b, νC=O); 1601 (νC=C, aromatic rings); 1277 (b, νC-O stretching).

Amount

Rf value

st

Trace amount

Discarded

nd

2 band

3 mg

Three spots with tailing

3rd band

4 mg

One spot, 0.81

Name 1 band

Table III: Different fractions that were recorded during column chromatography Fraction no

Test tube no

1st fraction

1-4

Color

Amount

----

----

TLC exam. & Rf value ----

nd

5-10

Light yellow

0.05 gm

rd

3 fraction

11-18

Reddish

0.08 gm

4th fraction

19-21

Brown

0.03 gm

5 fraction

22-28

Red

0.10 gm

too many spots

6th fraction

29-50

Deep red

0.15

too many spots

7 fraction

51-70

Yellow

0.17

too many spots

8th fraction

71-90

Yellow

0.21

too many spots

2 fraction

th

th

Isolation of the compound (a5): After getting the PETR product this portion was separated by column chromatography on silica gel using ethyl acetate and pet ether. 1.0 gm was taken for separation. Different fractions were collected and evaporated for the TLC examination, which were given in Table III. Further separation of the 4th fraction: The 4th fraction was taken for PTLC method for further separation and pet ether and ethyl acetate were used as solvent (EA:PE=5:95). Three bands were found in Table IV. Table IV: Different bands in TLC Name st

1 band nd

Amount Trace amount

Rf value

0.95, 0.90 0.90, one spot with tailing

2nd band was collected and was designated as a5. The product was solid, m.p. 287-288ºC. IR: ν Nujol (cm-1) 3600-3300 (b, νOH, stretching); 3063, 3050 (νC-H, aromatic); 2980 (νC-H, aliphatic); 1809, 1740 (b, νC=O asymmetric and symmetric stretching); 1597, 1581 (νC=C, aromatic rings); 1211 (νC-O). 1H-NMR (DMSO): δ 8.20-8.00 (b, OH, phenol), δ 7.907.10 (m, C-H, aromatic protons), δ 6.40-6.20 (s, OH, alcohol), δ 2.30 (s, 3H, CH3, aliphatic). Mass: m/z (% of relative intensity) 677.0 (18%), 483.1 (25%), 307.1 (22%) and 154.0 (100%). The molecular ion peak appears at m/z 1150 due to C78H54O10.

discarded

2 band

8 mg

0.90 (one spot)

3rd band

5 mg

0.90, 0.88 tailing

Bangladesh J Pharmacol

0.95, it may reactant (benzil)

61

Results and Discussion The mixture of the products, PM was washed with water and then this water soluble portion was extracted by chloroform and TLC examination of the product, a1 showed the single spot. In the IR spectrum of the compound a1, the broad band at 3500-2500 cm-1 points out the presence of H-bonded OH of COOH (i.e., presence of COOH group and OH group of aromatic in H-bonding). The signal at 2955 cm-1 indicates the presence of C-H group in aromatic ring. The value of δH is lowered due the Hbonded. The weak band at 1605 cm-1 identifies C=O group in COOH and the value at 1288 cm-1 indicates the presence of C-O group. In 1H-NMR spectrum, the broad signal (hum) at δ 10.20-9.80 is assigned for COOH and the other broad value at δ 8.50-8.20 indicates the presence of OH group in aromatic. The multiplet at 8.00-6.00 shows the presence of the rest of C-H protons in aromatic ring. In 13C-NMR spectrum, the peak at δ 195.20 (C-1) is assigned for the carbonyl group, C=O in COOH. The aromatic carbons are designated by the following values δ 135.93 (C10), δ 132.59 (C-8), δ 131.11 (C-7), δ 129.97 (C11), δ 129.89 (C-3), δ 129.62 (C-15), δ 129.33 (C-6), δ 129.21 (C-4), δ 128.90 (C-5), 128.00 (C12) and δ 70.56 (C-9). In the mass spectrum, + .

molecular ion peak (M 2%) appears at m/z 1256 that corresponding to the molecular formula C80H56O15. In this spectrum the base peak is formed at m/z 105. The product, a2 was obtained from the cold precipitate of DCM soluble portion and this DCM soluble portion was found from ethanol soluble portion of PM. This product gave the single spot on TLC examination. In the IR spectrum of the compound, a2 the broad band at 3600-3200 cm-1 indicates the presence of OH group. The signals at 3087 cm-1, 3056 cm-1 indicate the presence of C-H group in aromatic ring. The values at 1732 cm-1, 1600 cm-1 show the presence of C=O group in CO-O-CO. The bands at 1565 cm-1 and 1547 cm-1 indicate the presence of C-H group of aromatic ring. The 62

Liton and Islam Reaction of benzil with resorcinol

weak band at 1212 cm-1 also identifies the existence of C-O group. After collecting the cold filtrate, PLDM of DCM soluble portion that obtained from ethanol soluble portion of PM and this portion (PLDM) was separated by column chromatography on silica gel using pet ether and ethyl acetate. 0.639 gm was taken for separation. Different fractions were recorded and from these fractions, the 4th and 6th fractions were collected. The 4th portion was orange yellow colored and this portion was designated as a3. In the IR spectrum of the compound, a3 the broad band at 3600-3200 cm-1 indicates the presence of OH group. The signal at 3025 cm-1 indicates the presence of C-H group in aromatic ring. The bands at 1684 cm-1 and 1672 cm-1 indicate the presence of C-H group in aromatic ring. The weak band at 1277 cm-1 also identifies the existence of C-O group. The 6th fraction was taken for PTLC method for further separation. Pet. ether and ethyl acetate were used as solvents (EA:PE=3:2). Here 3 bands were found. 3rd band that collected was designated as a4. The product was light pink color. In the IR spectrum of the compound a4 the broad band at 3600-3200 cm-1 indicates the presence of OH group. The signals at 3060 cm-1, 3028 cm-1 indicate the presence of C-H group in aromatic ring. The values at 1799 cm-1 and 1714 cm-1 show the presence of C=O group in COOH. The band at 1601 cm-1 indicates the presence of C-H group of aromatic ring. The weak band at 1277 cm-1 also identifies the existence of C-O group. After getting the ethanol insoluble portion, PETR this portion was separated by column chromatography on silica gel using ethyl acetate and pet ether. 1.0 gm was taken for separation. Different fractions were recorded, from these fractions only 4th fraction was collected and it was taken for PTLC method for further separation. Pet.ether and ethyl acetate were used as solvents (EA:PE=5:95). Here 3 bands were found. 2nd band was collected that designated as a5. In the IR spectrum of the compound, a5 the Vol. 1, No. 2, December 2006

broad band at 3600-3300 cm-1 indicates the presence of OH group. The signals at 3063 cm-1, 3050 cm-1 indicate the presence of C-H group in aromatic ring. The value at 2980 cm-1 shows the presence of C-H in saturated aliphatic. The bands at 1809 cm-1 and 1740 cm-1 indicate the presence of C=O group (asymmetric and symmetric stretching). The values at 1597 cm-1 and 1581 cm-1 exhibit C=C group in aromatic ring. The sharp band at 1211 cm-1 also identifies the existence of C-O group. In 1H-NMR spectrum the broad peak shows the singlet at δ 8.20-8.00 which would be assigned for OH protons of phenol type compounds. The multiplet at δ 7.907.10 indicates the presence of C-H protons in aromatic rings. The two singlets at δ 6.30 and δ 6.20 indicate the presence of two OH groups. The singlet at 2.30 indicates the presence of CH3 group in CH3-CO-O. In the MS spectrum of the + .

compound, a5 molecular ion peak (M 1%) appears at m/z 1150 that corresponding to the molecular formula C78H54O10. In this spectrum the base peak is formed at m/z 154.

Cytotoxicity Cytotoxicity of all the compounds was measured by brine shrimp lethality bioassay method (Anderson et al., 1999) Cis-platin, a recognized anticancer drug was used as reference to compare the efficacy of the synthesized compounds. Compounds a1, a2, a3, a4, and a5 showed

Bangladesh J Pharmacol

significant cytotoxicity. Compounds a1 and a4 showed high cytotoxicity. Compounds, a2, a3 and a5 showed relatively low cytotoxicity. So these compounds may show anti-microbial, antitumor etc. activity.

Acknowledgement The authors express their sincere thanks to Khurshida Khayer Mamun, Department of Chemistry, Jahangirnagar University, Savar, Dhaka, Bangladesh for supplying 1HNMR, 13C-NMR and Mass spectra of the compounds from Germany.

References Anderson JE, Goetz CM, McLaughlin JL, Suffness M. Phytochemical analysis. Oxford University Press, 4th ed. 1991, pp 107-10. von Liebig H. Condensation reaction of benzil with resorcinol. J Prac Chem. 1905; 74: 345-59. Islam MR, Khayer K, Mahmud MI. Reaction of Isatin with 2-aminothiophenol leading to spiroheterocyclic having anticancer activity. Jahangirnagar Univ J Sci. 2001a; 24: 17-22. Islam R, Khayer K, Abedin MJ, Islam MR. Synthesis of (5spiro(5´-methylisatin)-4-acetyl-2-(acetylamino)∆2-,3,4thiadiazoline and 5-spiro (5´-methylisatin)-4-acetyl-2(5´-methylisatin-3´-hydrazineo)-∆2-1,3,4-thiadiazoline, Indian J Chem. 2001b; 40B: 240-42. Lingcon MH, Islam R, Khayer K, Islam MR. Cyclization of substituted indole-2-one-3-thiosemicarbazones to noble heterocyclic systems. J Bangladesh Chem. Soc. 2001; 14: 127-32.

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Hypolipidemic effects of fenugreek seed powder Abu Saleh M. Moosa, Mamun Ur Rashid, A.Z.S. Asadi, Nazma Ara, M. Mojib Uddin and A. Ferdaus Department of Pharmacology, Rajshahi Medical College, Rajshahi, Bangladesh

[Received 18 July 2006]

Abstract Effects of fenugreek (Trigonella foenugraecum Linn) on serum lipid profile in hypercholesteremic type 2 diabetic patients were studied. Administration of fenugreek seed powder of 25 gm orally twice daily for 3 weeks and 6 weeks produces significant (P<0.001) reduction of serum total cholesterol, triacylglyceride and LDL-cholesterol in hypercholesteremic group but the change of serum HDLcholesterol was not significant. On other hand, changes of lipid profile in hypercholesteremic type 2 diabetic patients without fenugreek were not significant (P<0.001). The present study suggests that fenugreek seed powder would be considered as effective agent for lipid lowering purposes. Key words: fenugreek, hypolipidemic effect

Introduction Hyperlidaemia is the current medical as well social problem, specially associated with diabetes mellitus leading to increasing morbidity and mortality. The major risk factors of hyperlipidemia are associated with atherosclerosis which predisposes ischemic heart disease and cerebrovascular disease (Brown and Goldstein, 1990). In type 2 diabetic patients there is mild to moderate hypertriglyceridemia, low level of high density lipoprotein (HDL) and over production of very low density lipoprotein (VLDL) (Foster, 1991). Serum total cholesterol is also increased (Florey et al, 1973). In the present century modern medicine draws its nourishment from the rich legacy of traditional medicine. Fenugreek (Trigonella foenumgraecum) is one of the oldest medicinal plants, dating back to Hippocrates and ancient Egyptian times (Jensen, 1992). The antihyperlipidemic properties of oral fenugreek seed powder has been suggested (Basch, 2003). Al-Habori M. et for correspondence: Abu Saleh M. Moosa, Department of Pharmacology, Rajshahi Medical College, Rajshahi, Bangladesh e-mail : [email protected]

al., (1998) showed the effect of fenugreek seeds and its extracts on plasma lipid profile on rabbits. Studies have also shown that fenugreek seeds reduce serum lipids in experimental animals. Sharma (1984, 1986) demonstrated that fenugreek administration increased excretion of bile acids and neutral sterols in feces, thus depleting the cholesterol stores in the body in experimental rats. Awal et al. (1999) has studied the effect of fenugreek and karela on lipid profile in hypercholesterolemic diabetic patients and shown that fenugreek significantly reduces the lipid level. The present study was undertaken to demonstrate the effect of whole fenugreek seed on lipid profile.

Materials and Method Thirty patients of type 2 diabetes mellitus with hyperlipidemia of either sex, aged 30-65 years, weighing 58-84 kg were selected for the study from Diabetic Center, Rajshahi. Patients having history of coexisting liver, kidney or thyroid dis-

order, etc. were not included in the study. Patients were well controlled with hypoglycemic drug and not on any other hypolipidemic medications. Counseling of the patients about the study was done and informed consents were taken from the patients. Three fasting blood samples of 5 ml were collected on three separate days to estimate lipid profile. The fenugreek seeds were collected from the local market. These were washed with clean water and dried in sun light. After drying seeds was crushed in an electric grinder to make powder. The powder was then stored in a clean, oven-dried stopper plastic container. The patients were divided into two groups: one group received only their usual anti-diabetic treatment (control group) and another group received fenugreek seed powder with their usual treatment (experimental group). Patients were advised to come after overnight fasting and blood samples were collected in early morning. The control group on the day 1, blood sample was taken as baseline record and advised to continue their usual diabetic treatment (i.e. drug or diet control plus exercise). Another two blood samples were taken on day 21 and day 42 for the study of serum lipid levels. In experimental group on day 1, blood sample was taken as baseline record and advised to continue their usual diabetic treatment (i.e. drug or diet control plus exercise). They were advised to swallow 25 gm of fenugreek seed powder twice daily (after breakfast and after dinner). Again two blood samples were taken on day 21 and day 42 day as study group serum level. All the parameters of lipid profile i.e. serum total cholesterol, LDLcholesterol, triacylglyceride and HDL-cholesterol were done. All values expressed as mean in mg/dl ± SEM (standard error of mean). Statistical significance of difference between the base line serum level i.e. control (day 1) serum level and after 3 weeks (day 21) of treatment serum level and again base line serum level i.e. control (day 1) serum level and after 6 weeks (day 42) of treatment serum

Bangladesh J Pharmacol

level was performed. The ‘p’ values of 0.05 or less were regarded as significant.

Results The mean serum total cholesterol, LDL-cholesterol, triacylglyceride and HDL-cholesterol level of control group on the first day, after 3 weeks (day 21) and after 6 weeks (day 42) was compared with serum total cholesterol, LDLcholesterol, triacylglyceride and HDL-cholesterol level of experimental group, on the first day, after 3 weeks (day 21) and after 6 weeks (day 42). The results are shown in Table I. Table I: Serum lipid profile in patients with type 2 diabetes mellitus treated with or without fenugreek Day

Without fenugreek

With fenugreek

Total cholesterol (mg/dl) Day 1

285.00 ± 1.45

285.12 ± 2.40

Day 21

288.00 ± 2.24 NS

279.12 ± 2.36*

Day 42

289.87 ± 1.89 NS

278.37 ± 2.31*

LDL-cholesterol (mg/dl) Day 1

157.25 ± 0.38

158.71 ± 0.61

Day 21

156.00 ± 1.26 NS

155.71 ± 0.52NS

Day 42

160.71 ± 4.21 NS

152.00 ± 6.42*

Triacylglyceride (mg/dl) Day 1

201.50 ± 1.56

202.62 ± 4.14

Day 21

201.12 ± 4.39 NS

191.87 ± 3.68*

Day 42

201.12 ± 4.39 NS

189.37 ± 4.06*

HDL-cholesterol (mg/dl) Day 1

35.37 ± 0.60

35.00 ± 0.21

Day 21

35.50 ± 0.32 NS

36.50 ± 0.46

Day 42

34.76 ± 0.41 NS

37.37 ± 0.67

Data are expressed as mean ± SEM; NS means not significant; * means p<0.001

The fenugreek seed powder significantly (p < 0.001) reduced serum total cholesterol, serum triacylglyceride level and serum LDL-cholesterol level in hyperlipidemic type 2 diabetic patients. The serum HDL-cholesterol level increased but not significantly by the fenugreek seed powder. No significant changes found in control group i.e. hyperlipidemic patients not taking fenugreek.

65

Discussion The present study has been undertaken to demonstrate the effect of fenugreek (local name: methy) seed powder on lipid profile in hyperlipidemic type 2 diabetic patients. In this study parameter of lipid profile was done for all hyperlipidemic patients. Estimation of lipid profile was done in all the patients after 3 and 6 weeks. No significant changes were observed in all the parameters of lipid profile in control group. But significant changes were observed in serum total cholesterol, LDL-cholesterol and triacylglyceride level in experimental group. Changes of serum HDL-cholesterol level were not significant. Similar observations were made by number of workers, demonstrated hypolipidemic effect of fenugreek powder in experimental animals like rabbit, rat, etc (Al-Habori et al., 1998). Some researchers also demonstrated the hypolipidemic effect of fenugreek seeds in hyperlipidemic type 2 diabetic patients (Sharma, 1986; Awal et al, 1999; Prasanna, 2000). Modern lipid lowering agents i.e. statins (atrovastatin, cimvastatin, rosuvastatin etc.) are expensive. The most important adverse effects of statins are liver and muscle toxicity. Other risk factors are: hepatic dysfunction, renal insufficiency, hypothyroidism, advanced age and serious infections (Stancu and Sima 2001). Limitations of the use of synthetic statins are pregnancy and lactations, etc (James, 2004). Liver and kidney functions may be modified. On the other hand herbal agents like fenugreek, are cheap easily available in many countries like Bangladesh, India, Nepal, Pakistan and Mediterranean region and south African countries. There is no toxic or adverse effect shown by any researcher worked on fenugreek cited above. In addition to its high fiber content (total fiber content 48%), fenugreek also contains a biologically significant level of saponins. Saponins are known to have hypocholesterolemic effects (Sharma, 1986; Sharma and Raghuram, 1990).

66 Moosa et al. Hypolipidemic effects of fenugreek

The quality and quantity of protein in the diets have a direct effect on the levels of cholesterol. Generally plant protein appears to lower cholesterol level (James, 2004). The plant protein in fenugreek is 26%, so it might exert a lipid lowering effect (Sharma, 1986). Further, since a high proportion of diabetic patients in tropics and subtropics suffer from malnutrition, fenugreek which is in rich protein (26%), has an added advantage in that it is a good source of protein as well as fiber (48%) (Sharma, 1986). From the results it can be concluded that fenugreek seeds exhibits significant hypolipideic effect in hyperlipidemic persons. Synthetic statins has some adverse effects and costly. In the light of these comparative findings, it can be stated that fenugreek seeds may be useful in hyperlipidemic states of patients with hypertension, atherosclerosis, ischemic heart diseases etc. In conclusion, the present study fenugreek seed powder significantly reduced serum total cholesterol, triacylglyceride and LDL-cholesterol but serum HDL-cholesterol level elevation is not significant. So, it can be suggested that fenugreek may be used for lipid lowering purposes and needs extensive comparative study with the modern lipid lowering agents. Further study on the fenugreek seeds in this aspect and isolation of active principles from the extract is suggested.

References Al-Habori M, Al-Aghban AM, Al-Mamary M. Effect of fenugreek seeds and its extracts on plasma lipid profile: a study on rabbits. Phytotherapy Res. 1998; 12: 572-75. Awal MA, Rashid MU, Ahmed KW, Asadi ZS, Islam K. Effect of karela and fenugreek on lipid profile in hypocholesterolemic diabetic patients. Bangladesh J Physiol Pharmacol. 1999; 15: 6-8. Basch E, Ulbricht C, Kuo G, Szapary P, Smith M. Therapeutic applications of fenugreek. Altern Med Rev. 2003; 8: 20-7. Mahley RW, Bersot TP. Drug therapy for hypercholesterolemia and dyslipidemia. In: Goodman & Gilman's The pharmacological basis of therapeutics. Hardman JG and Limbird LE (eds). 10th edi, New York, McGraw-Hill, 2001, pp 971-1002. Florey CD, Medonald H, Miall W. Serum lipids and their relations to blood glucose in cardiovascular measure-

ments in rural population of Jamaican adults. J Chronic Dis. 1973; 26-85. Powers AC. Diabetes mellitus. In: Harrison’s principles of internal medicine. Kasper et al. (eds). 16th ed, New York, McGraw-Hill, 2005, pp 2152-80. James H, Atrovastatin reduces remnant lipoproteins and small, dense low-density lipoprteins regardless of the baseline lipid pattern. Prev Cardiol. 2004; 7: 154-60. Jensen R. Fenugreek, overlooked but not forgotten. UCLA Lactation Alumni Newsletter. 1992; 1: 2-3. Prasanna M. Hypolipidemic effect of fenugreek: a clinical study. Indian J Pharma. 2000; 32: 34-6.

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Sharma RD. Effects of fenugreek seeds and leaves on blood glucose and serum insulin responses in human subjects. Nutr Res. 1986; 6: 1353-64. Sharma RD, Raghuram TC. Hypoglycaemic effect of fenugreek seeds in non-insulin dependent diabetic subjects. Nutr Res. 1990; 10: 731-39. Sharma RD. Hypocholesterolemic activity of fenugreek an experimental study in rat. Nutr Rep Int. 1984; 30: 221-31. Stancu C, Sima A. Statin: mechanism of action and effects. “Nicolae Simionescu” Institute of Cellular Biology and Pathology, Bucharest, Romania, 2001

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Effect of ascorbic acid on reduced glutathione level in arsenic-loaded isolated liver tissues of rat Beauty Saha Department of Pharmacology, Bangabnadhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh [Received 18 Octobery 2006]

Abstract Isolated liver tissues of rat were loaded with trivalent arsenic and were exposed in presence and absence of ascorbic acid. The amount of reduced glutathione (GSH) in normal liver tissue was 52.0 ± 0.2 µg/g protein. Addition of arsenic to the tissues reduced the amount of GSH to 11.5 ± 0.3 µg/g protein. But when the arsenic loaded liver tissues were incubated with ascorbic acid at the concentration of 20 µg/ml, the amount of GSH was 14.2 ± 0.1 µg/g protein. There was 22.6% increase of GSH level which was statistically significant (p<0.001) when compared with arsenic alone. This study suggests that ascorbic acid increased the GSH level in arsenic-treated rat's liver. Key words: arsenic, ascorbic acid, gluthathione, liver, rat

Introduction Inorganic arsenic contamination of drinking water is found in Bangladesh. The source of arsenic in drinking water is the earth's crust where arsenic occurs naturally; industrial source s are also important in man made pollution. In Bangladesh, arsenic concentration in drinking water are alarmingly high than the standard set by World Health Organization (WHO) which is 10 µg/l. about 90% of the tube well providing 97% of the countries drinking water supply were found to contain unacceptable levels of arsenic ranging from 50-3000 µg/l. in 61 out of 64 districts of Bangladesh, water samples were found to have arsenic above 50 µg/l, the maximum permissible limit recommended by WHO. About 57 million people of Bangladesh are exposed to harmful concentration of arsenic through drinking water. More than 40,000 patients of chronic arsenic poisoning are identified. for correspondence: Beauty Shah, Department of Pharmacology, Rangpur Medical College, Rangpur, Bangladesh

Together with poverty and poor nutritional status of the population, chronic arsenic exposure to arsenic in drinking water is causing wide spread health hazard in Bangladesh (Rabbani et al., 2002). There is no specific drug in the treatment of chronic arsenic poisoning (Khan et al., 1997; WHO, 2003). There remains some controversy in the use of chelating agents such as penicillamine, 2,3-dimercaptopropanesulphonate sodium (DM PS) and meso-2,3-dimercaptosuccinic acid (DMSA) in the treatment of chronic arsenic poisoning (WHO, 1997). These are not easily available in our country, costly and too toxic for administration. The main mechanism of arsenic toxicity is the inhibitory effects on cellu-lar respiration at the level of mitochondria. Exposure of cells to arsenicals cause oxidative damage to essential proteins perhaps secondary to uncoupling or disruption of mitochondrial oxidative phosphorylation and increased production of hydrogen peroxide and subsequent production of reactive oxygen species (Barchowsky et al.,

1999). These reactive species produced by endothelial cells after arsenic exposure stimulate cell signaling and activate transcription factors. In health, there is a balance between free radical production and an endogenous antioxidative defense in the body. If the free radical production is very high overwhelming the endogenous defense of the body toxic injury to cells increases, known as endogenous antioxidant acts as a reducing agent for arsenic during biotransformation. Maintenance of normal glutathione concentration is necessary for continuation of this biotransformation mechanism. Not only for biotransformation of arsenic, glutathione is also necessary to detoxify free radicals and therefore, necessary to decrease the free radical-induced lipid peroxidation. In addition to converting hydrogen peroxide to water and oxygen, the remarkable property of GSH lies in its ability to destroy the radical nature of lipid peroxide by transforming it into the hydroxyl fatty acids (PUFA-OH). The selenium dependent glutathione peroxidase first converts reduced glutathione (GSH) to oxidized glutathione (GSSG) at the expense of hydrogen peroxide (Rabbani et al., 2002). Various nutrients and vitamins can support the body's defense system to neutralize the toxic substances. Ascorbic acid is a natural antioxidant and water soluble, which can suppress free radical formation in the body. It also protects tissue and blood components. Antioxidant plays an important role in preventing free radical mediated damages by directly scavenging those (Ramanathan et al., 2002). Ascorbic acid can restore the antioxidant propery of oxidized tocopherol, suggesting that a major function of ascorbic acid is to recycle the tocopheroxyl radical (Ramanathan et al., 2002). Availability of appropriate levels of antioxidants may help in the overall balance of the degenerative effects and the survival mechanism (Chattopadhyay et al., 2002). Arsenic administration significantly increased the serum TBARs, nitric oxide and decreased the whole blood GSH

Bangladesh J Pharmacol

levels in rabbits indicating oxidative stress in vivo. Ascorbic acid, alpha-tocopherol, betacarotene are more effective in increasing the GSH of intoxicated rabbits as compared to placebo indicating its arsenic detoxification properties. The protective effects of antioxidant vitamins on the activities of glutathione cycle enzymes were observed in chicken (OzturkUreket et al., 2001). Ascorbic acid is easily available in foodstuff and cheap. It is present in citrus fruits, tomatoes, and green leafy vegetables. Ascorbic acid may be clinically useful to treat patients with arsenic toxicity. The aim of the present study was to see the effect of ascorbic acid on GSH level in arsenic-loaded isolated liver tissues of rat.

Materials and Methods Chemicals: Arsenic trioxide was purchased from BDH. DTMB (5,5-dithiol-2-nitrobenzoic acid), glutasthione and ascorbic acid were obtained from Sigma Chemicals, USA. Isolation of liver tissues: The study was carried out on isolated liver tissues of Long Evans Norwegian adult healthy male rats weighing 160200 g obtained from the animal house. The rats were housed in standard plastic cages with a light/dark cycle 12/12 hours at room temperature in a well-ventilated room. Rats were sacrificed under chloroform anesthesia. By giving a midline incision, abdomen was opened and the liver was taken out and it was immerged immediately in a beaker containing modified Tyrode solution, which was placed in the ice bath. It was then placed in a petri dish containing Tyrode solution and chopped into small pieces approximately 2 x 2 x 2 mm size. Each tube contained 10-12 small pieces of liver in presence of Tyrode solution and loaded with arsenic. Arsenic loaded tissues was then incubated in presence or absence of ascorbic acid at 37°C. The reaction was stopped by immersing the test tube into 0-4°C. Tissues were then homogenized by a hand homogenizer and

69

estimated the amount of GSH using the method described by Beulter et al., (1963).

Results The effect of different concentrations of ascorbic acid on GSH level in arsenic loaded isolated liver tissues of rat were shown in Table 1. The amount of GSH in normal liver tissue was 52.0 ± 0.2 µg/g protein. Addition of arsenic to the tissues reduced the amount of GSH to 11.5 ± 0.3 µg/g protein. That is, arsenic caused 77.7% reduction of GSH level. But when the arsenic loaded liver tissues were incubated with ascorbic acid at the concentration of 20 µg/ml, the amount of GSH was 14.2 ± 0.1 µg/g protein. There was 23.5% increase of GSH level which was statistically significant (p<0.001) when compared with arsenic alone. Ascorbic acid at concentration of 200 µg/ml increased the amount of GSH to 20.9 ± 0.1 µg/g protein. That is, there was 80.5% increase of GSH level. Two milligram of ascorbic acid per milliliter increased the GSH level to 26.8 ± 0.2 µg/g protein. Table I: Effect of different concentrations of ascorbic acid on GSH level in arsenic loaded liver tissues First incubation (40 min)

Second incubation (40 min)

Amount of GSH (µg/g protein)

% Increased

Arsenic (100 µg/ml) Arsenic (100 µg/ml) Arsenic (100 µg/ml)

None

11.5 ± 0.3

-

Ascorbic acid (20 µg/ml) Ascorbic acid (200 µg/ml)

14.2 ± 0.1

22.6

20.9 ± 0.1

80.5

Arsenic (100 µg/ml)

Ascorbic acid (2 mg/ml)

26.8 ± 0.2

131.8

n = 6; Data are expressed in mean ± SE

Next the time course of the effect of ascorbic acid on GSH level in arsenic loaded liver tissues of rat was studied (Figure 1). The concentration of ascorbic acid used was 2 mg/ml and liver tissues were incubated for different periods. The amount of GSH in arsenic untreated tissues was 54.2 ± 0.3 µg/g protein. After 20, 40, 60 and 80 minutes

70

Saha Effect of ascorbic acid on reduced glutathione level

Amount of GSH (mcg/g protein)

Statistical analysis: Data were analyzed using unpaired Students' t test. The level of significance was set at p value of 0.05 to 0.001.

60

Arsenic + Ascorbic acid

50 40 30 20

Arsenic 10 0

20

40

60

80

Time (min)

Figure 1: Time course of the effect of ascorbic acid on GSH level in arsenic loaded liver tissues

incubation of arsenic loaded tissues, the amount of GSH were 12.9 ± 0.4, 10.9 ± 0.4, 8.2 ± 0.1, 7.5 ± 0.3 µg/g protein. But in presence of ascorbic acid the amount of GSH were 14.1 ± 0.1, 26.5 ± 0.1, 39.0 ± 0.1 and 49.4 ± 0.1 µg/g protein after 20, 40, 60 and 80 minutes incubation of arsenic loaded tissue. The amount of GSH increased linearly up to 80 minutes of incubation.

Discussion Results of the present study showed that GSH level decreased in arsenic loaded isolated rat liver tissues could be reversed by addition of ascorbic acid. The effect of ascorbic acid was both dose-dependent and time-dependent. In this study ascorbic acid was used at concentrations of 20, 200 and 2000 µg. It was found that GSH level increased linearly even at concentrations of 2 mg. Arsenic intoxication of rabbits significantly reduced the blood GSH level (Rabbani et al., 2002). It also inhibits the enzyme that catalyze GSH synthesis such as gluthatione reductase (Styblo and Thomas, 1995). The recovery of the depleted GSH was significantly greater in presence of vitamins such as β-carotene, ascorbic acid, α-tocopherol (Rabbani et al., 2002). Rats exposed to arsenic showed increased generation of nitric oxide and reactive oxygen species (ROS), loss of glutathione content, increase in

Vol. 1, No. 2, December 2006

lipid peroxidation and decrease superoxide dismutase (Chattopadhyay et al., 2002: Shi et al., 2004). The administration of ascorbic acid or αtocopherol showed partial reversal of the effects indication possible protection from arsenic toxicity. Ascorbic acid is a major antioxidant and water soluble. It neutralizes the toxicity and enhances the antioxidative system. It protects cells from free radical in the body. Ascorbic acid and GSH are among the most active reducing substances in living tissues. Both of these chemicals undergo redox cycling in vivo, and there seems to be a significant interrelationship in this cycling. For example, the toxic effects of GSH deficiency can be prevented by administrating ascorbic acid, indicating that ascorbic acid has a sparing effect on GSH (Meister, 1994). The mechanism of excretion of arsenic depends on the presence of antioxidants and thiols that aid arsenic methylation and both arsenic and cadmium metallothionein binding (Patrick, 2003). In conclusion, this study may give an evidence that ascorbic acid may be used for the treatment of chronic arsenic poisoning in order to increase the antioxidant effect which reduced following arsenic exposure.

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Acknowledgement I am grateful to Prof. Mir Misbahuddin, Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh for his guidance due the study.

References Chattopadhyay S, Bhaumik S, Purkayastha M, Basu S, Chaudhuri AN, Gupta SD. Apoptosis and necrosis in developing brain cells due to arsenic toxicity and protection with antioxidants. Toxicol Lett. 2002; 136: 6576. Meister A. Glutathione-ascorbic acid antioxidant system in animals. J Biol Chem. 1994; 269: 9397-00. Patrick L. Toxic metals and antioxidants. Altern Med rev. 2003; 8: 106-28. Rabbani GH, Saha SK, Marni F, Akhtar M, Alauddin M, Mitra AK, Nasir M, Chowdhury AKA. Antioxidants in detoxification of arsenic-induced oxidative injury in rabbits. Bangladesh arsenic control society, Dhaka, 2002, pp 69-77. Shi H, Shi X, Liu KJ. Oxidative mechanism of arsenic toxicity and carcinogenesis. Mol Cell Biochem. 2004; 255: 67-78. Styblo M, Thomas DJ. In vitro inhibition of glutathione reductase by arsenotriglutathione. Biochem Pharmacol. 1995; 49: 971-07.

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Efficacy of nebulized magnesium sulfate in the treatment of acute exacerbation of asthma in children Mohammed Ataul Haqq, Md. Hamidur Rahman, Selina Khanam and M.A. Mannan Department of Pediatrics, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka, Bangladesh [Received 17 October 2006]

Abstract A double-blind randomized controlled trial was conducted to examine the efficacy of nebulized magnesium sulfate in the treatment of acute exacerbation of asthma in children. Thirty patients received nebulized salbutamol (0.15 mg/kg; minimum dose 2.5 mg) with 2.0 ml of isotonic magnesium sulfate solution and another 30 patients received same dose of salbutamol with 2.0 ml of normal saline on 3 occasions at 20 minute intervals. Mean percent of predicted peak expiratory flow rate (PEFR) detected in both group at 0 minute were not significantly different. But from 10 minute up to 60 minute, the values were significantly different among the groups. Mean respiratory rate at 0 and 10 minute were similar in both groups and from 20 up to 60 minute, respiratory rate improvement were significantly different. Arterial oxygen saturation (SaO2) at presentation was not significantly different. But from 10 min up to 60 minute differences were significant. With single dose of nebulization, in the magnesium sulfate with salbutamol group, by 20 minute almost all (29 out of 30) patient achieved at least 60% of predicted PEFR. Within this 20 minute, from control group none could achieve 60% of predicted PEFR. After 2nd dose of nebulization control group started achieving 60% PEFR value. Regarding response criteria, with 2nd dose of nebulization, at 30 and 40 minute 9 (30.0%) and 17 (56.7%) patient from magnesium sulfate with salbutamol group showed good response (PEFR ≥ 70% predicted). But within this first 40 minutes time, none could show good response in control group. With 3rd dose of nebulization all from magnesium sulfate group showed good response but even at 60 minute, 4 (13.3%) patient in control group failed to be included as good responder. So, it is evident that nebulization by isotonic magnesium sulfate solution with salbutamol provide early and better response as compared to conventional approach (salbutamol plus normal saline) in acute exacerbation of asthma in children. Key words: asthma, salbutamol, nebulization, PEFR

Introduction In patients with acute asthma exacerbation, the use of isotonic magnesium as a vehicle for nebulized salbutamol produced a significantly greater increase in peak expiratory flow compared with salbutamol in saline. The more severe the baseline obstruction, the greater the response to for correspondence: M. Ataul Haqq; e-mail: [email protected]

the combined magnesium sulfate and salbutamol, which supports the observation that magnesium is particularly effective in acute exacerbations of asthma(Nannini et al., 2000). The anticholinergic has little role if added to the repeated dose of β2-agonist; use of xanthine derivatives (methylxanthine) in acute asthma is controversial and steroid takes at least a few hours to act (Expert panel report-2, 1997). The

use of nebulized magnesium sulfate, in addition to a β2-agonist, in the treatment of acute exacerbation of asthma appears to produce benefits with respect to improved pulmonary function (Blitz et al., 2005). Bronchodilating effect of intravenous magnesium sulfate and its use and efficacy were suggested (Bloch et al., 1995; Boonyavorakul et al., 2000; Santana et al., 2001). On the contrary, data on nebulized magnesium sulfate have been scant. The effect of nebulized magnesium sulfate as a vehicle for salbutamol has been less evaluated (Kokturk et al., 2005). In pediatric population this type of study is very rare. Quickly achieving at least 60% predicted peak expiratory flow rate (PEFR) reduces the likelihood of relapse and hospitalization rate. One of the treatment goals in acute exacerbation of asthma is to achieve as rapidly as possible a safe value for the percentage predicted peak flow of about 60% (Nannini et al., 2000). Initial assessment of pulmonary function before treatment and repeat assessment after three dose of nebulization is necessary to categorize response (good, incomplete or poor) of individual patient (Expert panel report-2, 1997). At present, regarding assessment of response, no published study is known in pediatric population. Most of the studies (Nannini et al., 2000; Mollick, 2003) measured 1st outcome at 10 minute after completion of the nebulization. Some studies also measured 1st outcome at least 30 minute after completion of nebulization. Nebulization itself may continue for 5-10 minutes depending upon the volume of fluid/ drug and properties of the nebulizer. In practical, their 1st measurement lies at not earlier than 1520 minute from the start point. No known study done measuring outcome before 10 minutes after completion of nebulization. In some study, it is found that magnesium sulfate nebulization has shown better response as compared to normal saline. But no known study completed recommended schedule of 3 doses of nebulization.

Bangladesh J Pharmacol

The present study was carried out to see the effect of nebulized magnesium sulfate in presence of salbutamol in the treatment of acute exacerbation of asthma in children.

Materials and methods Duration of study: A double-blind randomized controlled trial was carried out from January to July 2006. Study population: A total of 60 children with acute exacerbation of asthma presented to the Pediatric Pulmonology Follow-up Clinic and Asthma Centre, BSMMU were included. Children (age range 7-16 years) of either sex with acute exacerbation of asthma and capable of measuring PEFR were included. Exclusion criteria included a) severely ill patient requiring immediate hospital care, b) any evidence of respiratory tract infection or suppurative lung diseases, c) any history or evidence of cardiac, renal or hepatic dysfunction, d) use of short acting bronchodilator within 8 hours or long acting bronchodilator within 24 hours, and e) use of steroid within 7 days. They were distributed randomly as 30 patients in nebulized isotonic magnesium sulfate with salbutamol group and the rest 30 in nebulized salbutamol with normal saline i.e., control group. Ethical issue: The protocol of the study was approved by the Ethical Committee, Bangabandhu Sheikh Mujib Medical University. Before enrolment, the aims and objectives of the study along with its procedure and benefits or drawbacks were explained to the guardian and patient (where applicable) in details in an understandable way. In case of any query, they were answered. When the guardian became satisfied, informed consent was obtained from the guardian and the patient was included in the study. They also had freedom to withdraw from the study at any time they would like. Materials/instruments used: a) salbutamol respirator solution containing 5 mg of salbutamol per ml (Ventolin respirator solution, Glaxo Well73

come Bangladesh Ltd.); b) normal saline (0.9% NaCl, Institute of Public Health, Bangladesh); c) isotonic magnesium sulfate solution (7.5 g/100 ml, 286 mOsm; d) 60 sets of identical eppendorf tube (each set containing 3 eppendorfs); e) nebulizer with face mask (medel AEROFAMILY®); f) Pulse oxymeter (BIONICS® BPM-200); and g) Peak Flow Meter (MiniWright™, Clement Clarke International Ltd, UK). Preparation for double-blind study: 60 sets of identical eppendorf tube (each set containing 3 eppendorf tube) were taken and labeled serially from 1 to 60. In any set all the 3 eppendorf tube had the same label number. By following random table, prepared a coding sheet to distribute the eppendorf randomly within the two groups each containing 30 sets. According to the grouping based on label number, each set of eppendorf tube was filled with appropriate solution i.e.; isotonic magnesium sulfate solution in magnesium sulfate group and normal saline in control group. In any set, each eppendorf tube contained 2.0 ml of same fluid (either isotonic magnesium sulfate or normal saline solution). Coding sheet was returned back to and preserved by the guide till the time to analyze the data. Clinical procedure: Clinical examinations of the patients were done with regards to the vital signs of acute attack as well as the chest findings as per requirements of the study. Questionnairecum-data sheet filled in. Objective measurement of airway obstruction was recorded with a peak flow meter. The procedure of using the peak flow meter was demonstrated to the patient. The use of peak flow meter was repeated at 10, 20, 30, 40, 50 and 60 minutes from the start point. Starting from 0 minute, every patient was under continuous monitoring with pulse oxymeter and readings regarding SaO2. Pulse and respiratory rates were recorded every 10 minute starting from 0 min up to 60 minute. In this step, at 0 minute, each patient was given salbutamol (0.15 mg/kg; minimum dose 2.5 mg) along with the content solution of an eppendorf tube (containing 2.0 ml of either isotonic magnesium sulfate or 74 Haqq et al Efficacy of nebulized magnesium sulfate

normal saline solution), from a set based on serial, by air driven nebulizer with face mask. Emptiness of the nebulizer was guided by lack of any further mist. When the nebulizer was sputtered it was gently shaken and then driven again until there was no more mist. First dose of nebulization done at 0 minute. Immediately after recording the information/data on data sheet, this step was repeated with 2nd dose of nebulization at 20 minute and 3rd dose of nebulization at 40 minute from start point using the eppendorf of same set. Statistical analysis: Coding sheet was collected from the guide, data decoded and analyzed. Results were expressed as mean ± SD. Most of the analyses were done by SPSS 12.0 for Windows (Statistical Package for Social Sciences) software. Unpaired Student's t-tests were used to compare means between two groups. Chi-square analysis was done to compare distribution of age, sex, family history of asthma, history of smoking, medication and presenting symptoms and signs. Confidence interval was set at 95% level. Results were considered to be statistically significant at P value <0.05.

Results Table I shows the baseline characteristics of patients of two groups. Age of the patients in magnesium sulfate group was 11.83 ± 2.39 years whereas in control group it was 10.83 ± 2.78 years. In the isotonic magnesium sulfate with salbutamol group, 12 patients were male and 18 female. On the other hand in the control group, 13 were male and 17 female. In magnesium sulfate group and control group height was 57.21 ± 5.37 inches and 54.87 ± 6.17 inches respectively. Weights of the patient in magnesium sulfate group and control group were 35.77 ± 8.92 kg and 33.88 ± 10.14 kg respectively. The duration of asthma was 1.60 ± 1.00 years in the magnesium sulfate group and 1.30 ± 0.54 years in the control group. In all cases the differences between the groups were not statistically significant. Vol. 1, No. 2, December 2006

Parameters

Table I: Baseline characteristics of patients Parameters

Magnesium sulfate Salbutamol with salbutamol (n=30) (n=30)

Age (years)

p value

>0.10ns

11.83 ± 2.39

10.83 ± 2.78

Male

12

13

Female

18

17

Height (inch)

57.21 ± 5.37

54.87 ± 6.17

>0.10ns

Weight (kg)

35.77 ± 8.92

33.88 ± 10.14

>0.10ns

Duration of asthma (years)

1.60 ± 1.00

1.30 ± 0.54

>0.10ns

26

25

ns

Sex

Family history of asthma

Data are mean ± SD ; ns = not significant

Table II shows that there was history of taking oral short acting β2-agonist in 26 cases in the magnesium sulfate group and 24 cases in the control group. Short acting inhaled β2-agonist was taken by 2 and 4 patients in the magnesium sulfate group and control group respectively. The differences were not statistically significant. Table II: Beta-2 agonist taken within seven days of presentation β2-agonist

Magnesium sulfate with salbutamol group (n=30)

Salbutamol (control) group (n=30)

No.

(%)

No.

(%)

Oral short acting

26

(86.7)

24

(80.0)

Oral long acting

2

(6.7)

2

(6.7)

Inhaler short acting

2

(6.7)

4

(13.3)

Inhaler long acting

0

(0.0)

0

(0.0)

All the children were conscious and not cyanosed at presentation. Table III shows that only 1 patient from magnesium sulfate group and 2 from control group were exhausted during presentation. Twenty eight patients talked in phrases and 2 in words in the magnesium sulfate group whereas 26 patients talked in phrases and 4 in words in the control group. In the magnesium sulfate group, loud and very loud wheeze was present in 25 and 5 patients respectively while in the control group they were found in 27 and 3 patients. The differences were not statistically significant in all

Symptoms Breathlessness during Talking Resting Physical exhaustion Yes No Talks in Phrases Words Signs Wheeze Loud Very loud Use of accessory muscle No Yes Prominent Pulse (per minute) 100-160 PEFR (%) 40-60 SaO2 94% - 90% <90%

Magnesium sulfate with salbutamol (n=30) No. (%)

Salbutamol (n=30)

No.

(%)

28 2

(93.3) (6.7)

26 4

(86.7) (13.3)

1 29

(3.3) (96.7)

2 28

(6.7) (93.3)

28 2

(93.3) (6.7)

26 4

(86.7) (13.3)

25 5

(83.3) (16.7)

27 3

(90.0) (10.0)

2 28 0

(6.7) (93.3) (0.0)

2 27 1

(6.7) (90.0) (3.3)

30

(100.0)

30

(100.0)

30

(100.0)

30

(100.0)

29 1

(96.7) (3.3)

28 2

(93.3) (6.7)

the parameters in both the group. In all cases pulse rate was within 100-160 per minute and PEFR was 40-60 percent of predicted value. In magnesium sulfate group and control group 28 and 26 patients respectively were breathless during talking while 2 and 4 patients were breathless during resting. Table IV shows baseline PEFR (l/min) between the two groups both in absolute value and percent of predicted were similar. Absolute value (mean ± SD) for magnesium sulfate group was 164.17 ± 35.04 l/min and for control group was 146.67 ± 40.94 l/min. P value was reached by unpaired Student's 't' test and was >0.05 which was not significant. Mean of percent predicted value for magnesium sulfate group and control group were 48.87 ± 2.37 and 48.03 ± 2.18 respectively and the differences were also non-significant (p value >0.10).

Table III: Presenting symptoms and signs Table IV: PEFR (l/min) status at different times

Bangladesh J Pharmacol

75

Parameters

Magnesium sulfate with salbutamol group (n=30), PEFR(l/min) mean ± SD 336.23 ± 71.49

Predicted 0 min (baseline) Mean % of predicted 10 min

Salbutamol (control) group (n=30) PEFR (l/min) mean ± SD 305.10 ± 82.09

p value >0.10ns

164.17 ± 35.04

146.67 ± 40.94

>0.05ns

48.87 ± 2.37

48.03 ± 2.18

>0.10ns

192.33 ± 40.30

166.83 ± 43.97

<0.05*

Mean % change from 0 min

17.42 ± 5.48

14.22 ± 5.74

<0.05*

Mean % of predicted

57.28 ± 1.69

54.77 ± 1.99

<0.001*

20 min

209.17 ± 41.73

174.50 ± 46.62

<0.01*

Mean % change from 0 min

28.01 ± 7.04

19.38 ± 6.03

<0.001*

Mean % of predicted

62.42 ± 1.75

57.24 ± 1.53

<0.001*

30 min

229.17 ± 46.11

189.00 ± 49.80

<0.01*

Mean % change from 0 min

40.20 ± 8.21

29.44 ± 5.70

<0.001*

Mean % of predicted

68.35 ± 1.95

62.07 ± 1.57

<0.001*

40 min

235.33 ± 48.44

194.83 ± 52.68

<0.01*

Mean % change from 0 min

43.76 ± 7.06

33.19 ± 6.00

<0.001*

Mean % of predicted

70.10 ± 1.03

63.87 ± 1.51

<0.001*

250.33 ± 52.21

211.67 ± 56.85

<0.01*

52.88 ± 8.41

44.78 ± 7.07

<0.001*

50 min Mean % change from 0 min Mean % of predicted

74.53 ± 1.40

69.41 ± 1.73

<0.001*

257.83 ± 52.88

216.50 ± 57.52

<0.01*

Mean % change from 0 min

57.58 ± 9.06

48.16 ± 6.96

<0.001*

Mean % of predicted

76.82 ± 1.84

71.04 ± 1.54

<0.001*

60 min

Unpaired Student's 't' test; ns = not significant, * = significant

At 10 minute, mean PEFR, mean percent change from 0 minute and mean percent of predicted value for magnesium sulfate group and control group respectively were 192.33 ± 40.30 and 166.83 ± 43.97 (p value <0.05); 17.42 ± 5.48 and 14.22 ± 5.74 (p value <0.05); 57.28 ± 1.69 and 54.77 ± 1.99 (p value <0.001) respectively. So, at 10 minute, there were significant differences between the groups regarding peak flow rate (absolute value), percent improvement from baseline and improvement in percent predicted value. Similar types of results were found up to 60 minute of the study period. Table V shows, at presentation, all the patient had PEFR <60% of predicted value. In the magnesium sulfate group at 10 minute from the start point only 2 (6.7%) patient and at 20 min 29 (96.7%) patient achieved at least 60% of predicted PEFR. So with single dose of nebulization almost all children (29 out of 30) achieved at least 60% Table V: Comparison of patients achieving at least 60% of predicted value

76 Haqq et al Efficacy of nebulized magnesium sulfate

Parameters

Magnesium sulfate with salbutamol group (n=30)

Salbutamol (control) group (n=30)

At 0 min PEFR ≥60% of predicted

0

0

<60% of predicted

30

30

≥60% of predicted

2

0

<60% of predicted

28

30

At 10 min PEFR

At 20 min PEFR ≥60% of predicted

29

0

<60% of predicted

1

30

≥60% of predicted

30

28

<60% of predicted

0

2

≥60% of predicted

30

30

<60% of predicted

0

0

≥60% of predicted

30

30

<60% of predicted

0

0

≥60% of predicted

30

30

<60% of predicted

0

0

At 30 min PEFR

At 40 min PEFR

At 50 min PEFR

At 50 min PEFR

Vol. 1, No. 2, December 2006

Table VI: Response on percent predicted PEFR PEFR (% predicted)

Magnesium sulfate with salbutamol group (n=30)

Salbutamol (control) group (n=30)

At 10 minute Good response Incomplete response Poor response

0

0

30

30

0

0

At 20 minute Good response

0

0

30

30

0

0

9

0

21

30

0

0

Good response

17

0

Incomplete response

13

30

0

0

30

9

Incomplete response

0

21

Poor response

0

0

30

26

Incomplete response

0

4

Poor response

0

0

Incomplete response Poor response At 30 minute Good response Incomplete response Poor response At 40 minute

Poor response At 50 minute Good response

At 60 minute Good response

PEFR ≥ 70% of predicted value- good response; 50% to <70% of predicted value- incomplete response; < 50% of predicted value- poor response. (National guidelines, 2005).

predicted PEFR in magnesium sulfate group. Within this 20 minute, in the control group none could achieve PEFR at least 60% of the predicted value.

Immediately after recording data as per schedule, second dose of nebulization done at 20 minute (from start point). After another 10 minute i.e.; at 30 minute from start point 30 (100.0%) and 28 (93.3%) patient from magnesium sulfate group and control group respectively achieved at least 60 percent predicted value. From 40 minute, all patient from both the group achieved PEFR at least 60 percent predicted. Table VI shows that within 20 minutes, none could show the good response. Second dose of nebuliza-tion done at 20 minute and after that, at 30 and 40 minute only 9 (30.0%) and 17 (56.7%) patients respectively in the magnesium sulfate group showed good response. Despite of using two doses of nebulization, within this first 40 minutes time, from control group none could show good response (PEFR ≥ 70.0% of predicted). Immediately after recording data at 40 minute 3rd dose of nebulization done. At 50 minute, 30 (100.0%) patient from magnesium sulfate group and 9 (30.0%) patient from control group showed good response. Finally at 60 minute 4 (13.3%) patient from the control group failed to be included as good responder. As shown in Table VII, respiratory rate, pulse rate and SaO2 were also recorded at 0, 10, 20, 30, 40, 50 and 60 minute. At 0 minute, regarding all the parameters, differences between two groups were

Table VII: Respiratory rate, pulse rate and SaO2 % at different times Parameter 0 min

10 min

20 min

30 min

Magnesium sulfate with salbutamol group Salbutamol group p value Magnesium sulfate with salbutamol group Salbutamol group p value Magnesium sulfate with salbutamol group Salbutamol group p value Magnesium sulfate with salbutamol group

Bangladesh J Pharmacol

Respiratory rate / min

Pulse rate / min

SaO2 %

34.03 ± 1.92

121.57 ± 6.27

90.77 ± 0.73

34.97 ± 1.96 >0.05ns 31.23 ± 1.68

123.23 ± 6.70 > 0.10ns 113.07 ± 8.02

90.40 ± 0.72 >0.05ns 92.23 ± 1.10

32.00 ± 1.56 >0.05ns 28.60 ± 1.13

115.67 ± 7.12 > 0.10ns 101.47 ± 5.49

91.67 ± 0.84 < 0.05* 92.60 ± 0.97

29.23 ± 1.07 <0.05* 27.23 ± 0.97

102.90 ± 5.74 > 0.10ns 95.97 ± 3.77

91.97 ± 1.00 < 0.05* 93.70 ± 0.88

77

40 min

50 min

60 min

Salbutamol group p value Magnesium sulfate with salbutamol group Salbutamol group p value Magnesium sulfate with salbutamol group Salbutamol group p value Magnesium sulfate with salbutamol group Salbutamol group p value

27.73 ± 0.87 < 0.05* 25.67 ± 0.84

97.13 ± 3.63 > 0.10ns 91.73 ± 1.95

93.17 ± 0.87 < 0.05* 94.23 ± 1.01

26.33 ± 0.80 < 0.01* 24.90 ± 0.85

92.60 ± 2.06 > 0.05ns 89.10 ± 2.04

93.73 ± 0.87 < 0.05* 95.03 ± 0.67

25.57 ± 0.77 < 0.01* 24.13 ± 1.04

90.53 ± 2.30 < 0.05* 86.97 ± 2.27

94.57 ± 0.73 < 0.05* 95.50 ± 0.73

25.40 ± 0.81 < 0.001*

88.57 ± 2.67 < 0.05*

95.07 ± 0.58 < 0.05*

Unpaired Student's 't' test; ns = not significant, * = significant

not significant. From 10 to 60 minute, differences of mean PEFR percent of predicted were always significant between the groups (p value < 0.001).

maintained up to 60 minute from the start point. This finding is consistent with that of Mollick, (2003).

Respiratory rate at 0 and 10 minute were similar in both groups and from 20 up to 60 minute, respiratory rate improvement were significantly different. Pulse rate differences were not statistically significant up to 40 minutes. At 50 and 60 minute, mean pulse rate differences were significant. Only 3 patient (1 in magnesium sulfate group and 2 in control group) had SaO2 89% (within <90% range) at presentation and within 10 minute, SaO2 was ≥ 90% in all cases. So as per protocol, oxygen was not used. Mean oxygen saturation at presentation was not significantly different. But from 10 min up to 60 minute, improvement in mean SaO2 was significantly different and magnesium sulfate group showed superiority.

PEFR expressed as percentage predicted value eliminated gender, age, weight and height bias and mean percent improvement in PEFR from baseline (0 minute) eliminated the bias introduced by difference in the degree of initial airflow obstruction. Mean percent of predicted PEFR detected in both group at 0, 10, 20, 30, 40, 50 and 60 minutes. Results in both groups at base line were similar but from 10 minute up to 60 minute, the values were significantly different. Mean percent improvement in PEFR from baseline was always significantly different from 10 up to 60 minute and magnesium sulfate group showed superiority which is also consistent with findings of Nannini et al., (2000).

At the end of 60 minutes, magnesium sulfate group showed better and significant differences in clinical improvement and all the patients from both the groups were given step care management.

Discussion This double-blind randomized controlled study revealed that combining nebulized isotonic magnesium sulfate with salbutamol results in early and better response in peak flow as compared with the standard approach (salbutamol plus normal saline) for nebulization in the initial treatment of acute exacerbation of asthma in children. The effect was evident at 10 minute and was

78 Haqq et al Efficacy of nebulized magnesium sulfate

In this study, in the magnesium sulfate with salbutamol group at 10 minute from the start point only 2 (6.7%) patient and at 20 minute almost all (29 out of 30) patients achieved at least 60% of predicted PEFR. Following 1st dose of nebulization at 0 minute, within this 1st 20 minutes, from the control group none could achieve PEFR at least 60% of the predicted value. Second dose of nebulization done at 20 minute and after another 10 minutes i.e, at 30 minute from start point, 30 (100.0%) and 28 (93.3%) patient from magnesium sulfate group and control group respectively achieved at least 60 percent predicted value. From 40 minutes, all patient from both the group achieved PEFR at least 60 percent predicted. In acute exacerbation of asthma, to reduce the

Vol. 1, No. 2, December 2006

likelihood of relapse and hospitalization rate, achieving as rapidly as possible a safe value for the percentage predicted peak flow of about 60% is needed which is suggested as the cut-off point between discharge from emergency department and admission into the hospital (Nannini, 1995). According to GINA (2005), patient with post treatment lung function (FEV1/PEF) in the range of 40%-60% of predicted value can potentially be discharged, assuming adequate follow-up is available in the community and compliance is assured. Patients with objective evidence of lung function 60 percent predicted or greater can usually be discharged (GINA, 2005). Ten minute after 2nd dose of nebulization i.e, at 30 and 40 minute only 9 (30.0%) and 17 (56.7%) patients respectively in the magnesium sulfate group showed good response (PEFR ≥ 70% predicted). Despite of using two doses of nebulization, within this first 40 minutes time, from control group none could show good response. With three doses of nebulization, at 50 minute, 30 (100.0%) patients from magnesium sulfate with salbutamol group and 9 (30.0%) patient in the control group showed good response. Finally at 60 minute 4 (13.3%) patient in the control group failed to be included as good responder. At present no known study done before to see this type of response. In the present study, all patient of magnesium sulfate group achieved 70% PEFR after 3 dose of nebulization. Nannini et al., (2000) conducted study with patient aged 18 years and over and found significant benefit after single dose of nebulization with isotonic magnesium sulfate. But the effect is much better in the present study. Hughes et al., (2003) undertaken a double blind placebo controlled study and found significantly greater improvement in FEV1 with nebulized salbutamol plus isotonic magnesium sulfate solution than salbutamol plus normal saline. In that study 3 doses of nebulization were done and observations were made at 30 minute interval and the response was similar as 10 min interval in the present study. Mollick, (2003), carried out prospective controlled study in adult population Bangladesh J Pharmacol

and also found the similar response after 20 minute of completion of treatment. The current recommendation for initial treatment of acute asthma is 3 doses of nebulization at 20 min interval for 1 hour but most of the studies (Mangat et al., 1998; Nannini et al., 2000; Hughes et al., 2003; Mollick, 2003) did not follow. Moreover, they recorded peak flow at 10 and/or 20 and/or 30 minutes after completion of nebulization. But nebulization itself continues for 5 - 10 minutes depending upon the amount of fluid and properties of nebulizer. The outcome could be different if those protocols were adopted to see the effect of 3 doses of nebulization from the start point. Though intravenous magnesium sulfate can be used as an adjunct to conventional nebulization and other therapy but if nebulization of salbutamol with isotonic magnesium sulfate can exert the same effect, use of this combination nebulization may be convenient both for the physician and for the patient (Mollick, 2003). So the nebulized magnesium sulfate is preferable to IV magnesium. Acute exacerbation of asthma requires start of treatment even at home and in the protocol of home management of asthma, oxygen administration is not recommended (Expert panel report2, 1997). Acute hypoxia has no effect on short acting β2-agonist (salbutamol) induced bronchodilatation in patients with asthma (Dagg et al., 2001). Improvements in oxygen saturation following bronchodilator administration documents the presence of relative preexisting hypoxemia which is reversed to some degree with bronchodilators (Yamamoto et al., 1992). In our study, oxygen saturation also raised to a safe value in all patients. Following current recommendation for initial treatment of acute exacerbation of asthma, in this study, nebulization was done as one dose every 20 minutes for 1 hour and found that patient gets comfort earlier if salbutamol nebulization done with isotonic magnesium sulfate solution.

79

In conclusion, this study suggest that a) combining isotonic magnesium sulfate solution 2.0 ml with salbutamol for nebulization results in early response and greater improvement in PEFR as compared with the standard approach (salbutamol nebulization with normal saline) in the initial treatment of acute exacerbation of asthma in children and b) patient treated by nebulized isotonic magnesium sulfate solution with salbutamol quickly achieves a safe value for the percentage predicted peak flow and at the end of initial treatment shows good response.

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Hughes R, Goldkorn A, Masoli M, Weatherall M, Burgess C, Beasley R. Use of isotonic nebulised magnesium sulfate as an adjuvant to salbutamol in treatment of severe asthma in adults: randomised placebo-controlled trial. Lancet. 2003; 361: 2114-17. Kokturk N, Turktas H, Kara P, Mulloglu S, Yilmaz F, Karamercan A. A randomized clinical trial of magnesium sulfate as a vehicle for nebulized salbutamol in the treatment of moderate to severe asthma attacks. Pulm Pharmacol Ther. 2005; 18: 41621. Mangat HS, D'Souza GA, Jacob MS. Nebulized magnesium sulfate versus nebulized salbutamol in acute bronchial asthma: a clinical trial. Eur Respir J. 1998; 12: 341-44. Mollick MKA. Comparison of nebulized magnesium sulfate plus salbutamol vs saline plus salbutamol in the treatment of acute asthma. MD Thesis, National Institute of Diseases of the Chest & Hospital, Mahakhali, Dhaka, Bangladesh, 2003. Nannini LJJ. Which PEF value is the best? Chest. 1995; 107: 1475-76. Nannini LJJ, Pendino JC, Corna RA, Mannarino S, Quispe R. Magnesium sulfate as a vehicle for nebulized salbutamol in acute asthma. Am J Med. 2000; 108: 193-97.

Boonyavorakul C, Thakkinstian A, Charoenpan P. Intravenous magnesium sulfate in acute severe asthma. Respirology. 2000; 5: 21-25.

Santana JC, Baretto SSM, Piva JP, Garcia PCR. 2001, Controlled study on intravenous magnesium sulfate or salbutamol in early treatment of severe acute asthma attack in children. J Pediatr (Rio J). 2001; 77: 279-87.

Dagg KD, Clayton RA, Thomson LJ, Chalmers GW, McGrath JC, Thomson NC. The effect of acute alteration in oxygen tension on the bronchodilator response to salbutamol in vitro and in vivo in man', Pulm Pharmacol Ther. 2001; 14: 99-05.

Silverman RA, Osborn H, Runge J, Gallagher EJ, Chiang W, Feldman J, Gaeta T, Freeman K, Levin B, Mancherje N, Scharf S. IV magnesium sulfate in the treatment of acute severe asthma. A multicenter randomized controlled trial. Chest. 2002; 122: 489-97.

Expert panel report-2: Guidelines for the diagnosis and management of asthma 1997, National Institutes of Health, National Heart, Lung and Blood Institute, USA.

Yamamoto LG, Wiebe RA, Rosen LM, Ringwood JW, Uechi CM, Beardsly ES, Toshi AS, Sugimoto SP, MacPherson KA, Young-Sasaki WM. 1992, Oxygen saturation changes during the pediatric emergency department treatment of wheezing. Am J Emerg Med. 1992; 10: 274-84.

GINA. Global strategy for asthma management and prevention. 2005. Available at: http://www. ginasthma. org.

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