No title

Bangladesh J Pharmacol 2006; 1: 1-4 Copyright © by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

Early days of Pharmacology education in Bangladesh*

Mohammad Shamsuzzoha (born- December 1, 1923)

Honorary Professor of Pharmacology, Bangladesh Medical College, Dhanmondi, Dhaka, Bangladesh.

Honorable chairperson Prof. A.K.M. Nurul Anwar and respected participants of this excellent program, Assalamualaikum.

thanks to the Almighty is being granted today, the 10th of March, 2006, although delayed. It is better late than never.

A heartfelt thanks you to all for having selected me as the chief guest of this event. I am honored. It is delightful to think that the younger generation of which the majority is constituted of has not forgotten the old. They have taken care to even remember an ancient person like me.

Pharmacologists, it is now your duty and responsibility to make this society flourish and blossom before everybody’s eyes. It is my belief that every physician should have some idea as to how the subject Pharmacology evolved. Pharmacology is a branch of medical science that had long been neglected not only in this country but throughout the world. It had no identity of its own but had always functioned under the supervision of the Physiology Department.

The best part of this occasion is the fact that the Pharmacological Society is being launched, a wish that pharmacologists had long cherished in their hearts. This special wish, with all our *This speech was delivered at the first annual meeting of Bangladesh Pharmacological Society at Dhaka on March 10, 2006.

To the extent of my knowledge, there used to be no medical college in Bangladesh before 1946. Only a meager number of medical schools in the

country were involved in teaching medical science. After successfully completing a four years’ course, graduates used to be awarded with an LMF diploma. Among these institutions, Mitford Medical School of Dhaka and Campbell Medical School of Calcutta were renowned. After the partition (1947), established 5 medical schools were located in Dhaka, Chittagong, Sylhet, Khulna and Mymensingh. Pharmacology was taught in the second year of the course under the name of MATERIA MEDICA. Dose, identification, composition, incompatibility, and prescription writing of drugs were taught as the part of practical lessons while the theoretical part consisted of a summary of the syllabus as constructed in those days. It was in the year 1946 when we were students at Calcutta Medical College when it came to our knowledge that a medical college had been established in Dhaka. The present building of Dhaka Medical College was then a part of Dhaka University. A portion of the northern wing of this building first served as the Dhaka Medical College and Hospital. After completing my MBBS and pre-registration clinical assistant within the November of 1948, I joined Dhaka Medical College and Hospital in the June of 1949. At that time, the Principal and Superintendent was the respectable eye specialist Dr. T. Ahmed. He was someone who would always try his utmost for the development and success of Dhaka Medical College and Hospital in spite of various technical difficulties that he had to encounter. Consequently I had to work part time in the pharmacology department for the first six months in spite of my being assigned to the hospital. At that time Pharmacology, Pathology, Jurisprudence and Hygiene were taught in the 3rd and 4th year. Dr. Hafizuddin who was the Resident Physician of the hospital, earnestly requested me to teach pharmacology to the students. I began to teach the subject during gaps in my hospital schedule. After facing a lot of problems, the most of which involved noncooperation from the clinical staff, Dr. Hafizuddin, Dr. Yusuf Ali, Dr. Nesaruddin and I endeavored to establish the Department of Pharmacology together. At the end of the year, 2

Shamsuzzoha Early days of Pharmacology

Dr. A.K.S. Ahmed (a double MRCP) was assigned as the Professor of the Department. Although relieved, we wondered whether he would be able to look over the Department or whether he would be interested at all. This is exactly what happened. He got completely busy with the hospital. Dr. Hafizuddin became full Resident Physician, Dr. Yusuf Ali full House Physician, and I received papers assigning me as the Demonstrator of Pharmacology. Being a non-Bengali, Dr. Nesaruddin was not interested in working for East Pakistan. Henceforth, the entire responsibility of the Pharmacology Department was bestowed upon me unawares. Then the staffing of the department began. The entire education planning and curriculum of the then east Pakistan’s Department of Pharmacology, Dhaka Medical College was assigned to a newly passed MBBS graduate, Dr. Zoha. With the exception of one or two lectures all the lectures, practical and theoretical classes had to be taken by me. The students were in 4th year in 1949. Since it was not known whether the students had learnt any pharmacology in the 3rd year, the entire course had to be covered in their 4th year. The greatest of help then were Mr. Hamid Ali and Mr. Naimuddin (Class IV employees), Pharmacist Mr. Ali Shaheb (Class III employee) whose sincerity and devotion I still reminisce with earnest respect. The topics which then used to be given maximum preference were practical and applied education. The chief preparations in the practical classes used to be emulsions constituting of castor oil, cod liver oil, glycerin suppository, quinine mixtures, etc. Students had to learn to calculate via the metric, Apothecaries’ and Avoirdssepois system. At that time, 50 to 60 years from now, the medical students were very eager to learn for which they usually came out with flying colors. It is not as though there weren’t kids who had not interest. There had been situations when students had passed Medicine, Surgery, Gyne-Obs, however, were failing Pharmacology year after year in succession. Consequently, they were not being able to acquire their final MBBS certificate.

Vol. 1, No. 1, June 2006

During then, the rule was that a student had to sit for the final professional three years after taking their second professional irrespective of whether they had passed every subject or not. After all this and successive reshufflings, it was finally decided that Pharmacology would be taught in the 3rd and 4th year along with Pathology, Forensic Medicine and Community Medicine. According to the latest curriculum, preclinical subjects are taught for one and a half years while clinical and para-clinical subjects are taught for three and a half years. I rejoined Dhaka Medical College in the March of 1958 after completing my PhD. The college had then acquired its very own building, which is the present building we have, with good adequate space for the accommodation of every department. We succeeded in enabling the students to carry out the common pharmacological experiments from the early 1959. They were: blood pressure of anaesthetized cats, isolated heart perfusion, and effects of acetylcholine, adrenaline, noradrenaline, vasomotor reversal, isolated auricle (guinea pig/ rabbit), effects of drug on isolated guinea pig ileum, rabbit duodenum, frog rectus abdominis muscle, effect of drug on blood vessels, etc. The students were first allowed demonstrations that we carried out after which they had to carry out the experiments themselves. Everything was running with enthusiasm and vigor, when at the end of the year, I was transferred to Chittagong Medical College as an Associate professor. The then Professor of Dhaka Medical College was Dr. Mir Monsur Ali who never voluntarily participated in such things, but never discouraged anything either. Prof. Kamaluddin had tried to continue the classes, however, he too left for Glasgow University after a while for his PhD. Prof. Mazharul Imam took over the charge but the entire process began to lag. To my dismay, I discovered that the Pharmacology department at Chittagong Medical College was anything but developed. I sent over the requisitions for necessary equipments, which I was granted after one year. It was then that experimental pharmacology was established at Chitta-

Bangladesh J Pharmacol

gong Medical College. Next, I was transferred to Rajshahi Medical College as a full Professor. During that time I traveled to Pakistan to attend a meeting of the Pakistan Pharmacopoea Committee. There I raised a question to the then health minister of the Central government Lt. General Barki about why West Pakistan had so many more medical colleges compared to East Pakistan who admitted that I was right and offered to look into the matter. The pharmacology department of RMC in 1964 was almost non-existent, not to mention, non functional. It took me great hours of toil to set out requests and requisitions till at long last, all the required space, staff, equipment, and everything necessary was obtained and a full-fledged good department of experimental pharmacology was established. It is worth mentioning here that the first fully equipped and facilitated experimental pharmacology department was pioneered from there at Rajshahi Medical College, complete with an animal house. I was retransferred to Dhaka Medical College in 1966 and then to Chittagong in 1969 as Principal, superintendent, as well as the Head of the department of Pharmacology. There I found that the construction of the huge new hospital complex as well as the medical college was complete. To my great disappointment I discovered that no space had been allotted for the Pharmacology department. Great was my sorrow at this especially because this was a subject devoid of which neither could any drugs be correctly prescribed, nor any maladies rightly cured. At the inauguration ceremony of the new hospital building, I held up this matter directly to the Health secretary who insisted that I take full charge of the matter. Being the Principal as well as the superintendent I then indeed had the right to do anything I wanted. So I proceeded to take over the old hospital building and renovate it to turn it into a big flourishing department devoted wholly to Pharmacology. Later on the departments of Community Medicine and Jurisprudence were accommodated in that building also. I also had to bear with the realization that the department had

3

remained absolutely static and devoid of any development since I had left it and all the equipments for which I had previously laid out requisitions were sealed up in packets. Gradually by and by, these requisitions were set out, animals and animal houses bought, lab technicians and Class IV employees hired till the department was established and running. By observing the rapid development occurring at Dhaka Medical College, Chittagong Medical College and Rajshahi Medical College, the rest of the medical colleges began to develop more or less. Today it is very disheartening to see that experimental pharmacology is no more practically carried out by the government as well as the non-government medical colleges. Expensive equipments that were acquired via the foreign exchange had either become useless due to lack of use or have been dumped at the corners of storerooms. The

4

Shamsuzzoha Early days of Pharmacology

current teaching staffs of Pharmacology are much more qualified and there are plenty of opportunities. In addition, there is a special section allotted for experimental pharmacology in the syllabus of Bangladesh Medical and Dental Council. In spite of all these, this subject is not paid much attention, the reason of which I cannot understand. I am of the opinion that everywhere outside Bangladesh, medical students themselves carry out experiments in experimental pharmacology, and in our country, the little that used to be practiced have ceased. Teaching and research are interconnected for which I believe that paying as much heed to research as is paid to teaching the theory, is a must. In spite of all limitations I believe that someone in the country has interest in developing the research sector.

Vol. 1, No. 1, June 2006

Bangladesh J Pharmacol 2006; 1: 5-9 Copyright © by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

α-Tocopherol reduces oxidative stress in perinatal asphyxia Rashidul Karim and M. A. Mannan Department of Pediatrics, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka 1000, Bangladesh [Received 13 December 2005]

Abstract Twenty asphyxiated neonates were studied by estimating reduced gluthathione (GSH) level in red blood cell (RBC) to assess the level of oxidative stress. Neonates were randomly divided into two groups. One group received α-tocopherol (10 mg/kg body weight) once orally daily for 5 days and other group received only vehicle. The mean (± SD) value of GSH in RBC within 24 hrs of age of asphyxiated neonates was 12.26 ± 4.29 mg/dl in untreated group and 11.97 ± 2.34 mg/dl in treated group. After 5 days of asphyxiated neonates (in α-tocopherol untreated group) the GSH level increased to 14.45 ± 3.46 mg/dl whereas in asphyxiated neonates treated with α-tocopherol, it increased to 25.65 ± 4.99 mg/dl indicating 5 days treatment with α-tocopherol among asphyxiated neonates caused approximately two fold increase in GSH level which was statistically significant (P<0.001). This study suggests that α-tocopherol may be useful to reduce the oxidative stress in patients of perinatal asphyxia. Key words: α-tocopherol; oxidative stress; perinatal asphyxia

Introduction Perinatal asphyxia, all over the world, remains an important cause of perinatally acquired brain injury in full term infants. In developing countries, perinatal asphyxia appears to be more common. During perinatal asphyxia, hypoxia and ischemia cause primary neuronal injury because of cell necrosis (Hossman, 1983). Neonatal resuscitation results in oxygenation and reperfusion, which in turn, leads to delayed, or secondary neuronal injury. The mechanisms believed to be important in this secondary phase of neuronal injury include oxygen free radical production (McCord, 1985), intracellular calcium entry and apoptosis (Evans and Levene, 1999). Numerous studies have suggested that free radicals could have a for correspondence: Rashidul Karim

key role in causing hypoxic ischemic damage to the brain, especially during the reoxygenation /reperfusion phase (Ogihara et al., 2003). Vitamin E is a fat-soluble vitamin that exits in eight different forms, four tocopherols (α-, β-, γand δ-) and four tocotrienols (also α-, β-, γ- and δ-). α-tocopherol is the name of the most active form of vitamin E in humans. Vitamin E or tocopherol is a membrane bound, chain breaking antioxidant. Once lipid peroxidation is initiated, peroxyl radicals react with vitamin E instead of an adjacent fatty acid, thus terminating the process (Palmer and Vanucci, 1993). Perinatal asphyxia is a leading cause of morbidity and mortality of neonates in our country. It is seen that infants with asphyxia have elevated oxidative stress, and leads to delay or secondary

neuronal injury. This problem may cause death or subsequent severe neurodevelopemental disability/handicap in future. In present study, administrating α-tocopherol acts as a free radicals scavenger to infants following perinatal asphyxia with primary aims of prevention of death or subsequent severe neurodevelopemental disability by reducing secondary neuronal injury from free radicals.

Materials and methods Chemical: α-tocopherol was a gift from Square Pharmaceuticals Ltd (Bangladesh). DTNB was purchased from Sigma Chemicals (USA). Design and place of study: This prospective study was conducted at the Department of Pediatrics (Neonatal unit), Bangabandhu Sheikh Mujib Medical University and Dhaka Medical College Hospital. The study was carried out from July 2004 to January 2005. Subjects: A total number of 30 newborns were selected randomly of which 20 were asphyxiated neonates and the rest 10 were nonasphyxiated normal neonates. Asphyxiated neonates were equally divided into two groups. One group was supplemented with α-tocopherol and another group received vehicle only. Term neonates, who did not require respiratory support, were clinically stable, not suffering from perinatal asphyxia or sepsis, having no major congenital malformations were taken as the control. All the cases of this study were infants, irrespective of male and female, having gestational age of 37 completed weeks to 42 weeks with the birth weight of more than 2,500 g, whose postnatal age were within 24 hrs. Asphyxia neonataram was diagnosed by performing apgar score 3 or less at the end of 1st min of life, delayed onset of first cry, infants who required resuscitation with positive pressure ventilation to establish spontaneous respiration after birth and also showed overt clinical signs of asphyxia, such as hypotonia, apnea, non responsive to external stimuli, pallor and also bradycardia (<80 6

Karim and Mannan a-tocopherol reduces oxidative stress

/min). Any major congenital anomalies, neonatal sepsis, hemolytic disease or any other symptomatic hematologic disorders were excluded from the study. Study procedure: After enrolment in this study relevant information from history, physical findings were recorded on a pre-designed questionnaire form. The purpose and procedure of the study were explained to the parents and their consents were taken. α-tocopherol 10 mg/kg body weight/dose was given to 10 asphyxiated neonates as daily single dose in the morning for 5 days. Under all precaution minimal 1 ml of blood samples were drawn from a peripheral vein. The blood was collected into heparinized tubes. Blood samples were drawn twice, within 24 hrs of age and after 5 days of age. Laboratory Procedure: The amount of GSH in RBC was estimated spectrophotometrically by the method described elsewhere and expressed as mg/dl of RBC (Beutler et al., 1963). In brief, 1 ml of heparinized blood was centrifuged at 4,000 rpm for 5 min. Upper clear solution (plasma) was removed and erythrocytes were washed twice with phosphate buffer solution. 1 ml of TCA (trichloroacetic acid) was added, mixed thoroughly and again centrifuged for 5 min at 4,000 rpm. After centrifuged, 0.25 ml of clear fluid was taken into another test tube and 2 ml of buffer solution (Na2HPO4) and 0.25 ml of DTNB reagent were added and mixed thoroughly. 40 µl of glutathione standard (1 mg/ml) solution and 210 µl of deionized water was taken in another test tube as standard, 2 ml of Na2-HPO4 and 0.25 ml of DTNB were also added in a test tube and mixed thoroughly, and a blank reagent obtained by replacing blood with deionized water, were included. The absorbance was measured by spectrophotometer (UV-Vis 1201, Shimadzu, Japan) at 412 nm after 20 minutes of adding DTNB solution. The absorbance of the reagent blank was subtracted from those of the standard and samples. Hematocrit value of whole blood was measured. Vol. 1, No. 1, June 2006

Ethical consideration: Prior to commencement of this study, the University Ethical Committee approved the research protocol. The aims and objectives of this study along with its procedure and benefits of this study were explained to the parents of the newborns in detail in easily and perfectly understandable language and informed consent was taken. Statistical Analysis: Collected data were checked for correctness and editing and coding was done and then data were entered into computer. The numerical data obtained from the study were analyzed and significance of difference was estimated by using the statistical methods. Data were analyzed by using computer based SPSS (Statistical Package for Social Science) program (version 11.5). Data were expressed in percentage, mean and standard deviation as applicable. Comparison between groups was done by unpaired student’s t test and paired t test as applicable. Probability less than 0.05 was considered as significant.

Results GSH level in RBC according to causes of perinatal asphyxia. It was evident that among the asphyxiated patients, frequent causes of perinatal asphyxia were obstructed labor (55.0%) followed by eclampsia (40.0%) and forcep delivery (5.0%). The mean value of GSH in RBC of obstructed labor was 13.73 ± 3.4 mg/dl and of eclampsia was 9.70 ± 1.1 mg/dl and the mean difference was statistically significant (p<0.001). GSH level in RBC of normal and asphyxiated neonates The mean value of GSH in RBC of normal baby within 24 hrs of age was 65.61 ± 12.62 mg/dl. But in asphyxiated neonates, it was reduced to 12.26 ± 4.29 mg/dl (α-tocopherol untreated group) and 11.97 ± 2.34 mg/dl (α-tocopherol treated group) respectively. Asphyxia reduced the level of GSH to 18.70% and 18.13% respecBangladesh J Pharmacol

tively. This difference was not statistically significant (P>0.05). After 5 days of treatment with conventional therapy, the level of GSH increased from 12.26 ± 4.29 to 14.45 ± 3.46 mg/dl (17.9%), which was not statistically significant (P>0.05). On the other hand, asphyxiated baby treated with α-tocopherol increased the level of GSH from 11.97 ± 2.34 to 25.65 ± 4.99 mg/dl (114.3%), which was statistically significant (P<0.001). GSH improvement was found only 22.02% in asphyxiated neonates treated without α-tocopherol, whereas 39.09% improvement was evident in neonates treated with α-tocopherol compared to that of the normal neonates, which was considered as 100.0%.

Discussion Oxidative stress in vivo is a degenerative process caused by the overproduction and propagation of free radical reactions (Buonocore et al., 2001). Oxidative stress may be assessed by determining GSH, GSSG and antioxidant enzyme activities. Blood GSH/GSSG ratio may be an indicator of oxidative stress. Decrease in GSH and increase in GSSG reflects oxidative stress (Vento et al., 2003). Table 1: The mean (± SD) concentration of GSH in RBC according to causes of perinatal asphyxia Causes Obstructed labor (n=12) Eclampsia (n=8)

GSH level (mg/dl) in RBC within 24 hrs of age* 13.73 ± 3.4

P value <0.001

9.70 ± 1.1

* Data are expressed as Mean ± SD; P value reached from unpaired student’s test

Table II: GSH level in RBC of normal and asphyxiated neonates Study subjects

Normal neonates Asphyxiated neonates Without α-tocopherol With α-tocopherol**

GSH level (mg/dl) in RBC* Within 24 hrs of age 65.61 ± 12.62

After 5 days

12.26 ± 4.29 11.97 ± 2.34

14.45 ± 3.46a 25.65 ± 4.99 b

-

* Data are expressed as Mean ± SD; **Dose of α-tocopherol 10 mg/kg body weight/day a b for 5 days; p value >0.05; p value<0.001

7

In this study, degree of oxidative stress assayed in the newborns was performed by the measurement of GSH level only could the other parameters, as mentioned above, be measured in this study; stress conditions would definitely be better assessed. However, those measurements could not be done because of lack of necessary laboratory supports. This study showed the mean ± SD concentration of GSH in RBC within 24 hrs of age of normal baby was 65.61 ± 12.62 mg/dl. But in asphyxiated neonates, it was reduced to 12.26 ± 4.29 mg/dl (α-tocopherol untreated group) and 11.97 ± 2.34 mg/dl (α-tocopherol treated group). This finding is consistent with the finding of Vento et al., (2003). This result suggests that newborn undergoes oxidative stress in perinatal asphyxia. Perinatal asphyxia causes oxidative stress, can be explained by the observation of Ogihara et al., (2003). As they viewed, free radicals could have a key role in causing hypoxic-ischemic damage to the brain, especially during neonatal resuscitation results in reoxigenetion and reperfusion phase in perinatal asphyxia Palmer, (1995) also explained that oxygen in paradoxically the basis of most free radical species generated during reperfusion in perinatal asphyxia. During reperfusion of the previously ischemic brain, potentially damaging amounts of superoxide, hydrogen peroxide, and the even more reactive species such as the hydroxyl radical can be produced by free fatty acid and prostaglandin metabolism. During cerebral hypoxia-ischemia, mitochondrial oxidative phosphorylation is impaired, causing ATP degradation and accumulation of hypoxanthine. Hypoxanthine is metabolized by xanthine oxidase to xanthine and uric acid in reactions that produce superoxide and hydrogen peroxide. This study also showed the mean (± SD) concentration of GSH in RBC after 5 days of age (αtocopherol untreated group) was increased from 12.26 ± 4.29 mg/dl to 14.45 ± 3.46 mg/dl whereas in asphyxiated neonates treated with α-tocopherol, it was increased from 11.97 ± 2.34 mg/dl 8

Liton and Islam Synthesis of hydantoin

to 25.65 ± 4.99 mg/dl indicating 5 days treatment with α-tocopherol among asphyxiated neonates showed approximately two fold increased GSH level which was statistically significant (P <0.001). There is no comparable data regarding the role of α-tocopherol in the treatment of oxidative stress in perinatal asphyxia. This favorable role of α-tocopherol in improving the oxidative stress conditions in perinatal asphyxia in the term newborns can be explained by the observations of Conner and Grisham (1996). As they viewed, Vitamin E or tocopherol is a membrane bound, chain breaking antioxidant may protect against reactive oxygen metabolites mediated cellular damage through free radical scavenging properties. It prevents the oxidation of low density lipoprotein, inhibits the propagation of lipid per-oxidation, and reduces toxicity associated with glutathione depleting agents Palmer and Vanucci (1993) also explained that vitamin E or tocopherol reduces the damaging effects of oxidative stress by once lipid peroxidation is initiated, peroxylradicals react with vitamin E instead of an adjacent fatty acid, thus terminating the process. In this study, as α-tocopherol was administered, the condition of oxidative stress improved. Although GSH level rose significantly higher following supplementation of α-tocopherol in asphyxiated newborns yet the rise of GSH was not as high as that of a normal baby. Inappropriate duration α-tocopherol treatment could be one of the factors responsible for that. In this respect, another point of concern should be the use of lipid soluble α-tocopherol. It was reported previously, that the use of water-soluble α-tocopherol could improve its absorption from GI tract (Jansson et al., 1984). Because of nonavilability of the preparation of water-soluble α-tocopherol, we did not use that preparation. In conclusion, oxidative stress was observed in asphyxiated term neonates and α-tocopherol administration was seen to produce some favorable impact in the reduction of oxidative stress in perinatal asphyxia. Therefore, α-tocopherol may Vol. 1, No. 1, June 2006

be useful to reduce the oxidative stress in patients of perinatal asphyxia.

Jansson L, Linroth M, Tyopponen J. Intestinal absorption of vitamin E in low birth weight infants. Acta Paediatr Scand. 1984; 73: 329-32.

This study recommends more prospective studies with bigger sample size to determine the duration of α-tocopherol therapy in asphyxiated newborn.

McCord JM. Oxygen derived free radicals in post-ischemic tissue injury. N Engl J Med. 1985; 312: 159-63.

References Beutler E, Duron O, Kelly BM. Improved method for the determination of blood glutathione. J Lab Med. 1963; 61: 882-88. Buonocore G, Perrone S, Bracci R. Free radicals and brain damage in the newborn. Biol Neonate. 2001; 79: 18086. Conner EM, Grisham MB. Inflammation, free radicals and anti-oxidants. J Nutr. 1996; 12: 274-76.

Ogihara T, Hirano K, Ogihara H, Misaki K. Non-protein bound transition metals and hydroxyl radical regeneration in cerebrospinal fluid of newborn infants with hypoxic ischemic encephalopathy. Pediatr Res. 2003; 53: 594-99. Palmer C, Vanucci RC. Potential new therapies for perinatal cerebral hypoxia ischemia. Clin Perinatol. 1993; 20: 411-32. Palmer C. Hypoxic ischaemic encephalopathy (Therapeutic approaches aganist microvascular injury, and role of Neutro-phils, PAF, and free radicals). Clin Perinatol. 1995; 22: 481-95.

Evans DJ, Levene MI. Hypoxic-ischemic injury. In: Rennie JM and Roberton NRC, editors. Textbook of neonatology, 3 rd edi. London: Churchill Livinstone, 1999, pp 1233-34.

Vento M, Asensi M, Sastre J, Fernando GS, Federico V, Pallardo, Vina J. Resuscitation with room air instead of 100% oxygen prevents oxidative stress in moderately asphyxiated term neonates. Pediatrics. 2001; 107: 64247.

Gitto E, Reiter RJ, Karbownik M, Dunxian, Gitto P. Causes of oxidative stress in the pre- and perinatal period. Biol Neonate. 2002; 81: 146-57.

Vento M, Asensi M, Sastre J, Lloret A, Vina J. Oxidative stress in asphyxiated term infants resuscitated with 100% oxygen. J Pediatr. 2003; 142: 240-46.

Hossman KA. Neuronal survival and revival during and after cerebral ischemia. Am J Emerg Med. 1983; 1: 19197.

Bangladesh J Pharmacol

9

Bangladesh J Pharmacol 2006; 1: 10-15 Copyright © 2006 by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

Synthesis of hydantoin and thiohydantoin related compounds from benzil and study of their cytotoxicity A. Kashem Liton and M. Rabiul Islam Department of Chemistry, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh [Received 15 Dec 2005]

Abstract Condensation of benzil (1) with urea, monophenyl urea and diphenyl urea in the presence of absolute ethanol using 30% aqueous NaOH gave the products 1a, 1b and 1c respectively and also with thiourea, monomethyl thiourea, dimethyl thiourea and diethyl thiourea the products 2a, 2b, 2c and 2d were obtained. Methylation of the product, 2a in the presence of dimethyl formamide (DMF) using K2CO3 formed 2. The compounds 1b, 1c, 2b, 2c and 2 showed highly cytotoxic activity and the compounds 1a, 2a, 2d showed relatively low cytotoxic activity against brine shrimp lethality bioassay. Key words: benzil; cytotoxicity; hydantoin; thiohydantoin

Introduction

Methods and Materials

Several types of substituted isatin heterocyclic derivatives were synthesized and found cytotoxic activity of these compounds by screening tests (Islam et al., 2001a; Islam et al., 2001b; Lingcon et al., 2001). Methyl and bromine groups in the ben-zene ring of isatin, ∆2-1,3,5-thiadiaozolines show more cytotoxic activity. For this interest, the title compounds have been synthesized for screening tests whether they show reasonable lethal effect on brine Shrimp or not. We report to see herein the results of the synthesis of the mentioned compounds (Scheme 1), spectral characterization and their cytotoxic effects by brine shrimp lethality bioassay (Anderson et al.,

Melting points are not corrected. IR spectra 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.

1999). for correspondence: A. Kashem Liton e-mail: [email protected]

Preparation of mono- and diphenyl urea: Monophenyl urea and diphenyl urea were prepared from aniline hydrochloride and urea following the reported procedure (Furniss et al., 1998). Preparation of 5,5-Diphenylhydantoin (1a) (Muccioli et al., 2003): Benzil, 1 (0.80 gm, 3.81 mmol/l) was placed in a 100-ml round-bottomed flask with urea, (0.40 gm, 6.69 mmol/l) in the molar ratio of ca 1:2. Absolute ethanol (12 ml) and 30% aqueous sodium hydroxide (2.50 ml) were added to these reactants. Boiling chips were also added to this solution and a condenser was

attached after wrapping the ground glass joint with Teflon tape, and was heated (110-1200C) the mixture under reflux (2 hrs). The reaction mixture was cooled before adding 15 ml of water. The solution was not clear, so the suspended solids were removed by filtration. Then the clear solution was cautiously acidified with concentrated hydrochloric acid and the product was collected by vacuum filtration and washed thoroughly with water.

CO-NH-CO); 3204 (s, νΝ-H amide, CO-NHCPh2); 3055 (m, νC-H aromatic); 1772 (s, νC=O amide, NH-CO-CPh2); 1741 (s, νC=O amide, NH-CO-NH); 1597, 1541, 1508 (s, νC=C, aromatic). 1H-NMR (DMSO): δ 10.10 (s, 1H, NH, CO-NH-CO); δ 9.10 (s, 1H, NH, CO-NHCPh2); δ 7.80-7.00 (m, 10H, C-H, aromatic). 13CNMR (DMSO): δ 175.19 (C-4); δ 156.36 (C-2); δ 140.27 (C-6); δ 128.87 (C-7); δ 128.39 (C-8); δ 126.93 (C-9); δ 70.56 (C-5). Mass: m/z (% of

Then the product, 1a was recrystallized by ethanol, dried, the m.p. 294-295ºC and yields 0.750 gm (80%). IR: ν Nujol (cm-1) 3271 (s, νΝ-H amide,

relative intensity) 252 (M 3%), 176 (30%), 154 (100%), 128 (3%), 77 (15%), 51 (8%), and

H N N N

O

O

O

+ .

O

N

H N

1b

N H

O 1a

1c

ethanol, NaOH, reflux O H2N C NH2 + S + CH3CH2HN C NHCH2CH3

O O H2N C NHPh PhHN C NHPh + + OO

S + H2N C NH2

H N O 2a

N H

CC 1 +

H2N C NHCH3

S

CH3 N

O 2b

N

N O H3C 2d

S

DMF, CH3I

O

S CH3HN C NHCH3

S

H3C

N

+

H N

H N

O

N

O S

2c

N

S CH3

CH3

S CH3

2

Scheme 1

Bangladesh J Pharmacol

11

OH Ph O

O

NaOH H2N

NH

H2N

H OH

O NH

Ph

-

OH

-

O

Ph

as before

O

O +

N H

-

Step 2: Nucleophilic attack on carbonyl carbon

Ph

H N

HN

Ph

O

NH2

Ph

NH2

O H2N O

O Ph O-

N H

H

OH

Ph OH

N H

Step 3: Protonation of the OH group OH Ph

H N O

Ph OH

H

Ph

+

OH2 H N O

N H

Ph

N H

OH

Step 4: Elimination of water Ph

+ OH 2 H N O

Ph OH

Ph

-H2O

H N

+

O

N H

Ph

N H

OH

Step 5: Rearrangement and deprotonation Ph

H N

+

O Ph

-H

Ph

+

Ph O

N H

O

H N O N H

H

Scheme 2

39 (10%). The molecular ion peak appears at m/z 252 due to C15H12N2O2. Preparation of 3,5,5-Triphenyl hydantoin (1b): Benzil, 1; (0.148 gm, 0.70 mmol/l) and the monophenylurea; (0.298 gm, 1.40 mmol/l) in the molar ratio of ca 1:2 were refluxed in the absolute ethanol and the procedure for 1b was similar to that of the compound, 1a. The product, 1-a1 was colorless solid; m.p. 172-173ºC having yields 0.077 gm, 50%. IR: ν Nujol (cm-1) 12

Liton and Islam Synthesis of hydantoin

3220 (s, νΝ-H amide); 3055 (m, νC-H aromatic); 1716 (s, νC=O amide, NPh-CO-C-Ph2); 1637 (s, νC=O amide, NH-CO-NPh); 1595, 1541, 1508 (νC=C, aromatic). 1H-NMR (DMSO): δ 9.40 (s, 1H, NH, amide); δ 7.80-7.40 (m, 15H, C-H, aromatic). 13C-NMR (DMSO): δ 173.06 (C-4); δ 154.32 (C-2); δ 141.99 (C-10); δ 140.60 (C-6); δ 129.05 (C-7) δ 128.30 (C-8); δ 127.97 (C-9); δ 127.33 (C-12); δ 121.53 (C-13); δ 117.70 (C-11); 68.70 (C-5). Mass: m/z (% of relative intensity) Vol. 1, No. 1, June 2006

+ .

328 (M 3%), 176 (25%), 154 (100%), 136 (68%), 120 (12%), 55 (27%), 43 (26%). The molecular ion peak appears at m/z 328 due to C21H16N2O2. Preparation of 1,3,5,5-Tetraphenyl hydantoin (1c): Benzil 1; (0.148 gm, 0.70 mmol/l) and the above 1,3-diphenylurea, a3; (0.298 gm, 1.40 mmol/l) in the molar ratio of ca 1:2 were refluxed in the absolute ethanol and the procedure of 1c was similar to that of the compound, 1a. The product, 1c was off white powder. It was recrystallized in methanol, dried and m.p. 163-165ºC and yields were 0.070 gm, 42%. IR: ν Nujol (cm-1) 3110 (w, νC-H aromatic); 1716 (s, νC=O amide, NPh-CO-CPh2); 1635 (s, νC=O amide, NPh-CONPh); 1595, 1541, 1508 (νC=C, aromatic). 1HNMR (DMSO): δ 7.20-6.70 (m, 20H, C-H, aromatic). 13C-NMR (DMSO): δ 173.10 (C-4); δ 154.25 (C-2); δ 141.88 (C-10); δ 140.60 (C-6); δ 139.88 (C-14) δ 129.33 (C-7) δ 128.71 (C-8); δ 127.99 (C-9); δ 127.38 (C-12); δ 127.26 (C-13); δ 127.12 (C-16) δ 121.60 (C-17); δ 117.73 (C11); δ 109.48 (C-15) δ 69.50 (C-5). Mass: m/z + .

(% of relative intensity) 404 (M 3%), 369 (75%), 347(68%), 302 (15%), 259 (7%), 233 (12%), 211 (100%), 182 (92%), 136 (29%), 93 (41%). The molecular ion peak appears at m/z 404 due to C27H20N2O2. Preparation of 5, 5-Diphenyl-2-thiohydantoin (2a): The compound, 2a was prepared from Benzil, 1; (0.80 gm, 3.81 mmol/l) and thiourea; (0.58 gm, 7.63 mmol/l) following the procedure of 1a. The product was recrystallized in ethanol, m.p. 234-235ºC yielded 0.595 gm, 94%. IR: ν Nujol (cm-1) 3255 (s, b, νN-H amide, CO-NH-CS); 3135 (b, νN-H, amide, CPh2-NH-CS); 3010 (s, C-H aromatic); 1749 (s, νC=O amide); 1558, 1541, 1508 (νC=C, benzene); 1215 (s, νC=S). 1 H-NMR (DMSO): δ 7.80-7.60 (m, 10H, C-H, aromatic), δ 1.10-1.12 (1H, SH). 13C-NMR (DMSO): δ 181.65 (C-4); δ 175.54 (C-2); δ 138.70 (C-6); δ 129.13 (C-7); δ 128.80 (C-8); δ 126.92 (C-9); 73.92 (C-5). Mass: m/z (% of + .

relative intensity) 268 (M 12%), 182 (18%), 154 (100%), 136 (59%), 120 (12%), 107 (19%), 77 Bangladesh J Pharmacol

(17%), 57 (27%), 43 (23%). The molecular ion peak appears at m/z 268 due to C15H12N2OS. Preparation of 3-Methyl-5, 5-diphenyl-2-thiohydantoin (2b): Benzil, 1; (0.84 gm, 4.00 mmol/l) with methylthiourea (0.72 gm, 8.00 mmol/l) in the molar ratio of ca 1:2 were refluxed in ethanol and the procedure for the compound, 2b is similar to that of the compound, 1a. The product was recrystallized in ethanol; m.p. 182-183ºC having yields 1.0 gm, 94%. IR: ν Nujol (cm-1) 3250 (b, νN-H thiamide); 3110 (s, w, νC-H aromatic); 2950, 2900 (s, νC-H, aliphatic), 1716 (s, νC=O amide); 1595, 1541, 1508 (νC=C, aromatic); 1215 (s, νC=S). 1H-NMR (DMSO): δ 7.20-6.70 (m, 10H, C-H, aromatic), δ 3.50 (s, 3H, C-H, aliphatic), δ 1.30 (s, 1H, SH). 13C-NMR (DMSO): δ 181.94 (C-2); δ 173.93 (C-4); δ 138.55 (C-6); δ 129.13 (C-7) δ 128.89 (C-8); δ 127.44 (C-9); δ 71.76 (C-5); δ 27.79 (C-10). Mass: m/z (% of relative intensity) 282 + .

(M 100%), 205 (22%), 180(68%), 165 (50%), 121 (7%), 104 (72%), 77 (49%), 59 (6%), 51 (17%), 39 (3%). The molecular ion peak appears at m/z 282 due to C16H14N2OS. Preparation of 1,3-Dimethyl-5, 5-diphenyl-2thiohydantoin (2c): Compound, 2c was obtained from benzil, 1; (1.00 gm, 4.76 mmol/l) and 1,3dimethylthiourea; (1.00 gm, 9.61 mmol/l) in ethanol following the procedure of 1a. Compound 2c was obtained as white crystalline solids, m.p. 154-156ºC having 0.950 gm, yielded 85%. IR: ν Nujol (cm-1) 3030 (s, νC-H aromatic); 2930, 2858 (s, νC-H, aliphatic); 1643 (s, νC=O amide); 1470, 1448 (s, νC=C, aromatic); 1296 (s, νC-N); 1126 (s, νC=S stretching vibration). 1HNMR (DMSO): δ 7.00-6.60 (m, 10H, C-H, aromatic), δ 3.30 (bs, 6H, CH3). 13C-NMR (DMSO): δ 184.08 (C-2, C-4); δ 137.90 (C-6); δ 127.97 (C-7) δ 127.74 (C-8); δ 127.12 (C-9); δ 95.39 (C-5); δ 33.41 (C-10); δ 30.54 (C-11). + .

Mass: m/z (% of relative intensity) 296 (M 100%), 224 (23%), 209(13%), 176 (10%), 154 (31%), 136 (45%), 105 (56%), 91 (13%), 77 (14%), 43 (12%). The molecular ion peak appears at m/z 296 due to C17H16N2OS. 13

Preparation of 1,3-Diethyl-5, 5-diphenyl-2-thiohydantoin (2d): Refluxing of benzil, 1; (0.84 gm, 4.00 mmol/l) and 1,3-diethylthiourea; (1.06 gm, 8.00 mmol/l) and the following procedure of 1a gave the product 2d. The product was recrystallized in ethanol, m.p.116-117ºC having yielded 0.297 gm, 45%. IR: ν Nujol (cm-1) 3110 (w, νC-H aromatic); 2990, 2830 (s, νC-H, aliphatic); 1716 (s, νC=O amide, NPh-CO-C-Ph2); 1635 (s, νC=O amide, NPh-CO-NPh); 1595, 1541, 1508(νC=C, aromatic). H-NMR (DMSO): δ 7.00-6.80 (m, 10H, C-H, aromatic), δ 3.60 (q, 2H, 11CH2), δ 3.30 (q, 2H, 13 CH2), δ 1.20 (t, 6H, CH3). 13C-NMR (DMSO): δ 183.31 (C-2); δ 183.21 (C-4); δ 140.07 (C-6); δ 128.17 (C-7); δ 127.97 (C-8); δ 127.83 (C-9); δ 99.62 (C-5); δ 96.54 (C-11); δ 96.28(C-13); δ 14.85 (C-12); δ 14.43 (C-14). Mass: m/z (% of 1

+ .

relative intensity) 324 (M 12%), 194 (28%), 165(19%), 150 (55%), 121 (12%), 105 (100%), 86 (9%), 77 (50%), 51 (5%), 29 (21%). The molecular ion peak appears at m/z 324 due to C19H20N2OS. Preparation of S-Methyl-5, 5-Diphenyl-2-thiohydantoin (2) (Muccioli et al., 2003): The compound, 2a (0.200 gm, 0.71 mmol/l) was dissolved in anhydrous DMF (0.70 ml) and K2CO3 (0.41 gm, 0.31 mmol/l) then iodomethane (0.70 ml, 0.71 mmol/l) were added to this mixture and stirred overnight at room temperature. The mixture was poured into distilled water. The resulting precipitate was collected, dried and recrystallized from ethanol (If some of K2CO3 are nonreacted then hot ethanol solution of product is filtered to separate K2CO3 from the product) the product, 2. The yields of the product, 2 were 0.75 gm, 38% having m.p.172–174ºC. IR: ν Nujol (cm1 ) 3240 (w, νN-H, amine); 3010 (s, νC-H, aromatic); 2900, 2855 (s, νC-H, aliphatic); 1725 (s, νC=O); 1684 (s, νC=N); 1585, 1506, 1489 (νC=C, aromatic). 1H-NMR (DMSO): δ 7.407.20 (m, 10H, C-H, aromatic), δ 3.20 (s, 1H, NH), δ 2.70 (s, 3H, C-H, aliphatic). 13C-NMR (DMSO): δ 180.36 (C-2); δ 162.42 (C-4); δ 140.80 (C-6); δ 128.72 (C-7); δ 127.97 (C-8); δ 127.14 (C-9); δ 77.94 (C-5); δ 26.87 (C-10). 14

Liton and Islam Synthesis of hydantoin

+ .

Mass: m/z (% of relative intensity) 282 (M 2%) 176 (27%), 165(12%), 154 (100%), 136 (70%), 120 (13%), 107 (19%), 88 (18%), 77 (15%), 51 (5%), 43 (8%). The molecular ion peak appears at m/z 282 due to C16H14N2OS.

Results and Discussions Simple base catalyzed condensation of benzil, 1 with urea, monophenyl urea and diphenyl urea in absolute ethanol furnished 1a, 1b, 1c whereas with thiourea, methyl thiourea, dimethyl thiourea and diethyl thiourea gave 2a, 2b, 2c and 2d. The formation of these products follows the pinacolepinacoline type (Shukla and Trivedi, 1997) rearrangement that is shown in the Scheme 2 (e.g., compound, 1a). The IR spectrum of the compound, 1a at 3271 cm-1 indicates the presence of NH group of amide moiety. The other band at 3204 cm-1 also indicates the presence of NH in Ph2C-NH-CO. The clear band at 3055 cm1 shows the presence of C-H stretching vibration of phenyl ring. The lower absorption at 1772 cm1 assigns C=O group of amide in Ph2-CO-NH-, on the other hand the band at 1741 cm-1 corresponds to other C=O group in NH-CO-NH. The bands at 1597 cm-1, 1541 cm-1, 1508 cm-1 give the strong support of C=C stretching vibration of phenyl ring. 1H-NMR spectrum shows two singlets at δ 10.10 and δ 9.10, which would be assigned for NH proton of amide. The aromatic protons appear as multiplet at δ 7.80-7.00. 13 C-NMR spectrum at δ 175.19 (C-4) and δ 156.36 (C-2) is for the carbonyl groups of amide. The aromatic carbons are designated by the following values δ 140.27 (C-6), δ 128.87 (C-7), δ 128.39 (C-8), δ 126.93 (C-9) and at δ 70.56 (C5). In the MS spectrum of the compound, 1a the + .

molecular ion peak (M 3%) appears at m/z 252 that corresponding to the molecular formula C15H12N2O2. In this spectrum the base peak is formed at m/z 154. Methylation of 2a with CH3I in the presence of K2CO3 in dry DMF gave the product (2): In the IR spectrum of the compound, 2 the sharp band at 3240 cm-1 indicates the presence of NH group Vol. 1, No. 1, June 2006

of amide. The signal at 3010 cm-1 locates the presence of C-H stretching vibration in aromatic. The bands at 2900 cm-1 and 2855 cm-1 indicate the presence of C-H group for CH3. The clear and sharp band at 1725 cm-1 is assigned for C=O group. The band at 1684 cm-1 indicates the presence of C=N group. The bands at 1585, 1506 and 1489 give the strong support for the presence of C=C stretching vibration in the aromatic ring. In 1H-NMR spectrum the aromatic protons appear as multiplet at δ 7.40-7.20. The singlet at δ 3.20 indicates the presence of NH group and the singlet at δ 2.70 is observed for CH3 group in S-CH3. In 13C-NMR spectrum the signals at δ 180.36 (C-2) and δ 162.42 (C-4) appears for both C=S and C=O groups respectively. The aromatic carbons are designated by the following values) δ 140.80 (C-6), δ 128.72 (C-7), δ 127.97 (C-8), δ 127.14 (C-9). The value at 77.94 (C-5) appears 1.4 1.2

+ .

spectrum the molecular ion peak (M 2%) appears at m/z 282 that corresponding to the molecular formula C16H14-N2OS. 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. Cisplatin, a recognized anticancer drug was used as reference to compare the efficacy of the synthesized compounds. Compounds 1a, 1b, 1c, 2a, 2b, 2c, 2d and 2 showed potential cytotoxicity but compounds 1b, 1c, 2b, 2c and 2 that showed highly cytotoxic activity and compounds 1a, 2a, 2d showed relatively low cytotoxic activity. The LC50 values of the titled compounds are represented in the Figure 1.

1.25 1.07

1.05

1 LC500

for the aliphatic carbon with various substituted groups. The signal at δ 26.87 (C-10) is concerned for simple aliphatic carbon CH3. In the MS

0.95

1.05

1.03

2b

2c

1.1

1.03

0.8 0.6 0.4 0.2 0 1a

1b

1c

2a

2d

2

Sample no

Figure 1: Comparative graphical representation of the LC50 of toxicity for the synthesized compounds against brine shrimp lethality test References Anderson JE, Goetz CM, McLaughlin JL, Suffness M. Phytochemical Analysis, Oxford University press, 4th ed. 1991, p 107.

1,3,4-thiadiazoline and 5-spiro (5´-methylisatin)-4acetyl-2-(5´-methylisatin-3´-hydrazineo)-∆2-1,3,4thiadiazoline, Ind J Chem. 2001b; 40B, 240-42.

Furniss BS, Hannaford AJ, Rogers V, Smith PWG, and Tatehell AR. Vogel’s Test Book of Practical Organic Chemistry, Longman Press, 4th ed., 1998, pp 734-35.

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.

Islam MR, Khayer K, Mahmud MI. Reaction of Isatin with 2-Aminothiophenol Leading to Spirohe-terocyclic having Anticancer Activity, Jahangirnagar University J Sci. 2001a, 24, 17-22.

Muccioli GG, Poupaert JH, Woulers J, Norberg B, Poppitz W, Scriba GKE, Lambert DM. A rapid and efficient microwave-assisted synthesis of hydantoins and thiohydantoins. Tetrahedron. 2003; 59: 1301-07.

Islam R, Khayer K, Abedin MJ, Islam MR. Synthesis of (5spiro(5´-methylisatin)-4-acetyl-2-(acetylamino)∆2-

Shukla SP, Trivedi GL. Modern Organic Chemistry, S. Chand and Company Ltd., 1st ed., 1997, p 40.

Bangladesh J Pharmacol

15

Bangladesh J Pharmacol 2006; 1: 16-20 Copyright © by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

Effect of n-Hexane extract of Nigella sativa on gentamicin-induced nephrotoxicity in rats Nasim A. Begum, Zesmin F. Dewan, Nilufar Nahar,1 and MI Rouf Mamun1 Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka 1000, Bangladesh; 1 Department of Chemistry, University of Dhaka, Dhaka 1000, Bangladesh [Received 9 January 2006; Revised form received 1 February 2006]

Abstract The present study investigated whether the administration of the n-hexane extract of the Nigella sativa Linn. (kalajira) ameliorates gentamicin-induced nephrotoxicity in rats. Gentamicin (100 mg/kg/day for 7 days) was administered and nephrotoxicity was evaluated biochemically (significantly decreased reduced glutathione in renal cortex and significantly increased serum creatinine and serum urea) and histologically (moderate degree of proximal tubular damage). The n-hexane extract of N. sativa (5 ml/kg/day) was administered as pre-, post- and concomitant treatment for 7 days in the nephrotoxic rats. Statistically significant amelioration in all the biochemical parameters supported by significantly improved renal cortical histology was observed in the n-hexane extract of N. sativa treated nephrotoxic rats, which was more evident in the post-treatment group than the pre- treatment and the concomitantly-treated group. It is suggested that some ingredients contained in the n-hexane extract of N. sativa effected in ameliorating the signs of nephrotoxicity and that the specific active principle of the nhexane extract of N. sativa responsible for this amelioration if obtained, would be more useful.

Key words: gentamicin, nephrotoxicity, n-hexane extract, Nigella sativa

Introduction Gentamicin is an important aminoglycoside antibiotic commonly used in treating life-threatening gram-negative infections (Ali, 1995). However its usefulness is limited by signs of nephrotoxicity, which may occur in 13-30% of treated patients (Mathew, 1992). Lipid peroxidation may occur in the course of gentamicin administration (Ramsammy et al., 1985), giving rise to free radicals (Yang et al., 1995), which are highly toxic to tissue (Feldman et al., 1982). Oxidation and necrosis by apoptosis may occur. Antioxidants have been shown to ameliorate signs of gentamicin-induced nephrotoxicity (Ali and for correspondence: Nasim A. Begum, Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka 1000, Bangladesh; Phone: 8802-8013804; e-mail: [email protected]

Mousa, 2001). A recent study suggested that N. sativa oil (Ali, 2004) given prophylactically was able to alleviate gentamicin nephrotoxicity in rats. N. sativa (kalajira) occupies a unique position among the herbal products of Southeast Asia as a natural remedy for a number of illnesses. Its antibacterial, hypolipidaemic, antidiabetic and antihypertensive properties have been reported (Ara, 1999; Saha, 2004; Uddin, 2002; Rashid et al., 1987). The seeds or compounds isolated have been found to be useful in a number of models of nephrotoxicity. The phytochemical, pharmacological and toxicological properties of N. sativa have recently been reviewed (Ali and Blunden, 2003). Attempts were made to obtain agents that can ameliorate or potentiate the nephrotoxicity of gentamicin (Ali, 2003; Mingeot-Leclercq and Tulkens, 1999). Among these agents, extract of

medicinal plants like garlic (Pedraza-Chaverri et al., 2000), and N. sativa oil (Ali, 2004) have been reported to possess properties to ameliorate gentamicin-induced nephrotoxicity. One common feature of the herbal agents is that they all have antioxidant properties (Prasad et al., 1996; Burits and Bucar, 2000). A potential therapeutic approach to ameliorate gentamicin-induced renal damage would have very important clinical consequence (Mengeot-Leclercq and Tulkens, 1999). In the present work, we have attempted to test and compare the possible pre-, post- and concomitant action of n-hexane extract of N. sativa on gentamicin-induced nephrotoxicity in rats.

Materials and methods Chemicals and reagents: Gentamicin (80 mg/ml) was obtained from the Essential Drug Company Ltd. (Bangladesh). N. sativa was purchased from the local market. Chemicals and reagents of estimation of serum creatinine and serum urea were obtained from Human GmbH (Germany). Reduced glutathione was purchased from Loba Cheme (India). 5,5-dithiobis-2-nitrobenzoic acid (DTNB) was from Sigma Chemicals (USA). n-hexane extract of N. sativa: The seeds of N. sativa (800 g) were soaked in 3 liters of n-hexane for 48-72 hr. They were filtered and the filtrate was concentrated in a rotary evaporator. The concentrate was freeze-dried, and a concentrated dark brown oily n-hexane extract of N. sativa was obtained. Animals: Adult male rats aged between 8-12 weeks, weighing 200-230 g were obtained from the animal house of Bangabandhu Sheikh Mujib Medical University. Normal rat-feed, water ad libitum was provided under 12-hour light and 12-hour dark schedule at room temperature (2428oC). Rats were divided into 12 groups, 6 rats in each control group and 10 rats in each experimental group. Experimental design: Rats were treated with distilled water (2.5 ml/kg/day orally), gentamicin (100 mg/kg/day by subcutaneous injection), Bangladesh J Pharmacol

soybean oil (5 ml/kg/day orally) or extract of N. sativa (5 ml/kg/ day orally) for 7 days followed by none or above concentration of distilled water, gentamicin, soybean oil or extract of N. sativa for another 7 days. Rats were sacrificed on either day 8th or 15th. 2 ml of blood was collected in clean test tubes and centrifuged (4000 x g for 5 min). The serum obtained was stored at 0-4oC for estimation of creatinine and urea concentrations. The kidneys were excised, blotted on a filter paper and weighed in pairs. The cortex was dissected out and a portion was placed in formalin for subsequent histological processing. Another portion of the renal cortex was processed for biochemical estimation of reduced glutathion (GSH) concentration. Biochemical measurements: GSH concentration was measured in homogenates of the renal cortex spectrophotometically (Sedlak and Lindsay, 1968). Serum creatinine concentration was measured by Jaffè reaction (Spencer, 1986). Serum urea concentration was measured by enzymatic colorimetric method (Fawcett and Scott, 1960). Histological procedure: Small portion of cortex of the representative kidneys were fixed in 10% formalin, dehydrate in graded alcohol and embedded in paraffin wax, sectioned at 5 µm thickness and stained with Hematoxylin and Eosin (H&E) for light microscopic examination. Renal proximal tubular damage was assessed on the basis of arbitrary score (Teixeira et al., 1982) as follows: 0 for no cell necrosis; 1 for mild, usually single cell necrosis in sparse tubules; 2 for moderate, sparse tubules showing more than one cell involvement; 3 for marked, tubules in almost every power field exhibiting total necrosis; and 4 for massive, total necrosis. Protein analysis: Protein content was determined by Biuret method (Weichselbaum et al., 1946) Statistical analysis: The results obtained from the experiments are represented as mean ± SEM of the number of samples. Data were analyzed by Student's unpaired 't' test, and significant difference between means ± SEM of the different groups were estimated using one-way analysis of 17

variance (ANOVA) followed by Student's unpaired 't' test.

Results The groups of rats injected subcutaneously with gentamicin for 7 days, sacrificed on day 8 and 15 had serum creatinine and serum urea concentrations significantly (P<0.001) increased while the renal cortical reduced glutathione concentrations of these groups of rats were significantly reduced (P<0.001) compared to those in the control rats. This would suggest that these rats were made model for nephrotoxicity (Table I). This assumption was supported by histological observation of the H&E stained transverse sections through the renal cortex, which suggested massive damage to the proximal tubules (score 3) of these groups of rats (Fig. 1b). The transverse sections through the renal cortex of the vehicle control groups of rats showed almost identical architecture with no damage to proximal tubules (Fig. 1a). The n-hexane extract of N. sativa treated rats differed from the vehicle control rats by an elevated

(P<0.001) concentration of renal cortical reduced glutathione, while the other biochemical parameters were identical (no statistical difference could be obtained) almost to those of the control rats, and the histology did not differ from those of the control groups of rats. Treatment of nephrotoxic rats with the n-hexane extract of N. sativa as pre-, post- and concomitantly mitigated the increases in serum creatinine and urea, and the decreases in reduced glutathione (GSH) while compared to those of the gentamicin-treated group sacrificed on day 15 (Table I). But none of the parameters in any of the groups were identical or closer to those of the control groups (Table I). However, the post-treatment group (gentamicin followed by n-hexane extract of N. sativa) demonstrated significant reduction in serum creatinine and serum urea concentrations (P<0.001) and also a significant elevation (P<0.001) of renal cortical glutathione concentrations compared to those observed in the pre treatment group (nhexane extract of N. sativa followed by gentamicin) and the concomitantly treated (gentamicin

Table I: Effects of gentamicin and n-hexane extract (N. sativa) on biochemical parameters and histology Duration of treatment

n

Cortical GSH Serum crea- Histological (mg/g protein) tinine (mg/dl) score

P value, Group vs Group

Sacrificed on day 8 Group 1 2 3 4

First week Distilled water Gentamicin Soybean oil Extract of N. sativa

Second week -

6 10 6 10

2.21 ± 0.01 1.01 ± 0.01 2.20 ± 0.02 5.71 ± 0.01

0.39 ± 0.01 3.09 ± 0.11 0.39 ± 0.01 0.38 ± 0.01

0 3 0 0

First week Distilled water Gentamicin

Second week None None

6 10

2.21 ± 0.01 1.09 ± 0.03

0.39 ± 0.01 2.90 ± 0.05

0 3

7 8 9 10

Soybean oil Extract of N. sativa Distilled water Gentamicin

Distilled water 6 Gentamicin 10 Soybean oil 6 Extract of N. sativa 10

2.21 ± 0.01 1.68 ± 0.02 2.20 ± 0.02 2.01 ± 0.02

0.39 ± 0.01 0.75 ± 0.02 0.39 ± 0.01 0.57 ± 0.02

0 2 0 0

11

Distilled water and soybean oil Gentamicin and extract of N. sativa

None

6

2.21 ± 0.01

0.39 ± 0.01

0

None

10

1.75 ± 0.02

0.68 ± 0.02

2

<0.001, 1 vs 2 <0.001, 3 vs 4

Sacrificed on day 15 Group 5 6

12

<0.001, 5 vs 6 >0.05, 2 vs 6 <0.001, 6 vs 8; 7 vs 8 <0.001, 6 vs 10; 8 vs 10; 9 vs 10

<0.001, 6 vs 12; 10 vs 12; 11 vs 12; <0.05, 8 vs 12

Dosage: distilled water (2.5 ml/kg/day orally); gentamicin (100 mg/kg/day by subcutaneous injection); soybean oil (5 ml/kg/day orally); n-hexane extract of N. sativa (5 ml/kg/kg/day orally); Values are means ± SEM;

and n-hexane extract of N. sativa concomitantly treated). Histological picture obtained in the post 18

treatment was also apparently improved (score 1). However, the histological score obtained in both

Begum et al Hexane extract on gentamicin-induced nephrotoxicity

Vol. 1, No. 1, June 2006

the pre treatment and concomitantly treated group were score 2. The post-treatment, therefore, remained as the most alleviated treatment group from toxic damage of gentamicin (Fig. 1c).

Discussion In the present study, nephrotoxicity induced by gentamicin was evidenced by depletion of renal cortical GSH and increases in serum creatinine and serum urea concentrations. This was supported by proximal tubular histology suggestive of gross tubular damage. These observations were similar to those of Ali (2004) who reported similar biochemical and histological changes suggestive of nephrotoxicity. The n-hexane extract of N. sativa possess strong antioxidant properties (Badary et al., 2000; Burtis and Bucar, 2000) that is why they were used in the present study to expect that the action of the toxic free radicals (Feldman et al., 1982) in the course of gentamicin

administration, causing oxidative damage to the renal cortex would be antagonized. Reports about similar ameliorating action of antioxidants upon gentamicin nephrotoxicity is available (Ali and Mousa, 2001; Naidu et al., 2000; Ali, 2004). We have used three treatment groups to ameliorate gentamicin nephrotoxocity e.g., the n-hexane extract of N. sativa followed by gentamicin (pretreatment), gentamicin followed by n-hexane extract of N. sativa (post treatment) and lastly, concomitant administration of gentamicin and the nhexane extract of N. sativa (concomitant treatment). The results of biochemical and histologycal observations indicate that the groups of rat which received gentamicin followed by n-hexane extract of N. sativa better overcome the toxic actions when compared to the other two groups. Gentamicin treatment in rats gives rise to free radicals (Walker and Shah, 1988; Yang et al., 1995) that induces oxidative damage at the cellular level of the renal cortex (Feldman et al.,

Figure 1: Representative photograph of sections of renal cortex under light-microscope of rats treated with either distilled water (a), gentamicin (100 mg/kg/day for 7 days (b) and gentamicin plus extract of N. sativa (5 ml/kg/day for next 7 days)(c). H&E 400 x

1982). Agents with antioxidant action could antagonize the depletion of the reduced glutathione (Sandhya and Varalakshmi, 1997; PedrazaChaverri et al., 2000; Ali, 2004). Probably the antioxidant action of the n-hexane extract of N. sativa prevents the oxidation of renal tissue. The cellular GSH content was therefore not depleted.

Bangladesh J Pharmacol

In conclusion the present study suggests that the n-hexane extract of N. sativa was able to produce considerable alleviation from the nephrotoxic action of gentamicin in the adult male rats. The antioxidant action perhaps better exerted in presence of oxidative damage and prior supply of antioxidant may not protect the tissue to the expected degree in absence of free radicals. The 19

mechanism of alleviation and the specific dose and ingredients of the n-hexane extract of N. sativa for complete nephroprotection remains to be identified.

Pedraza-Chaverri J, Maldonado PD, Medina-Champos N, Olivares-Corichi IM, Granados-Silvestre ML, Hernandez-Pando R, et al. Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes. Free Rad Biol Med. 2000; 29: 602-11. Prasad K, Laxdal VA, Yu M, Raney BL. Evaluation of hydroxyl radical-scavenging property of garlic. Mol Cell Biochem. 1996; 154: 55-63.

References Ali BH. Gentamicin nephrotoxicity in humans and animals: Some recent research. Gen Pharmacol. 1995; 26: 147787. Ali BH. Agents ameliorating or augments the nephrotoxicity of gentamicin: Some recent research. Food Chem Toxicol. 2003; 41: 1434-39. Ali BH. The effect of Nigella sativa oil on gentamicin nephrotoxicity in rats. Am J Chin Med. 2004; 32: 49-55 Ali BH, Blunden G. Pharmacological and toxicological proper-ties of Nigella sativa. Phytother Res. 2003; 17: 299-305. Ali BH, Mousa HM. Effect of dimethyl sulfoxide on gentamicin-induced nephrotoxicity in rats. Human Exp Toxicol. 2001; 20:199-203. Ara N. Antimicrobial activity of the volatile oil of Nigella sativa Linn. Seeds. MPhil thesis. Bangabandhu Sheikh Mujib Medical University, Dhaka. 1999. Badary OA, Abdel-Naim AB, Abdel-Wahab MH, Hamada FMA. The influence of thymoquinone on doxorubicinindu-ced hyperlipidemic nephropathy in rats. Toxicology 2000; 143: 219-26. Burits M, Bucar F. Antioxidant activity of Nigella sativa essen-tial oil. Phytother Res. 2000; 14: 323-08. Fawcett JK, Scott JE. A rapid and precise method for the determination of urea. J Clin Path. 1960; 13:156. Feldman S, Wang M, Kaloyanides GJ. Aminoglycosides induce a phospholipidosis in the renal cortex of the rat: An early manifestation of nephrotoxicity. J Pharmacol Exp Therp. 1982; 220: 514-20. Mathew TH. Drug-induced renal disease. Med J Aust. 1992; 156: 724-28. Mingeot-Leclercq M, Tulkens PM. Aminoglycosides: nephrotoxicities. Antimicrobial Agents Chemother. 1999; 43: 1003-12.

Ramsammy L, Ling KY, Josepovitz C, Levine R, Kaloyanides GJ. Effect of gentamicin on lipid peroxidation in rat renal cortex. Biochem Pharmacol. 1985; 34: 3895-900. Rashid MA, Rahman S, Chowdhury SAR, Ahmed M, Chowdhury AKA. Primary screening of some herbal products of Bangladesh for antihypertensiv activity. Bangladesh J Neuro. 1987; 3: 56-58. Saha RR, Dewan ZF, Uddin N. Effect of Nigella sativa Linn (kalajira) on serum lipid profile of hyperlipidemic rats. Bnagladesh J Physiol Pharmacol. 2004; 20: 36-38. Sandhya P, Varalakshmi P. Effect of lipoic acid administration on gentamicin-induced lipid per oxidation in rats. J Appl Toxicol. 1997; 17: 405-08. Sedlak J, Lindsay RH. Estimation of total, protein-bound and non-protein sulfhydryl groups in tissue with Elman's reagent. Anal Biochem. 1968; 25: 192-205. Spencer K. Analytical reviews in clinical biochemistry: the estimation of creatinine. Ann Clin Biochem. 1986; 23:1-25. Teixeira RB, Kelley J, Alpert H, Pardo V, Vaamonde CA. Com-plete protection from gentamicin-induced acute renal failure in diabetes mellitus rats. Kidney Int. 1982; 21: 600-12. Uddin N, Dewan ZF, Zaman M, Saha RR, Sultana M. Effects of Nigella sativa Linn. (kalajira) on serum glucose concentration in streptozotocin-induced diabetic rats. Bangladesh J Physiol Phamacol. 2002; 18: 6-9. Walker PD, Shah SV. Evidence suggesting a role of hydroxyl radical in gentamicin-induced acute renal failure in rats. J Clin Invest. 1988; 81:334-41. Weichselbaum TE. Estimation of serum total protein by Biruet method. Am J Clin Path. 1946; 16: 40-48. Yang C, Du X, Han Y. Renal cortical mitochondria are the source of oxygen free radicals enhanced by gentamicin. Renal Fail. 1995; 17: 21-26.

Naidu MU, Shifow AA, Kumar KV, Ratnaker KS. Ginkgobiloba extract ameliorates gentamicin-induced nephrotoxocity in rats. Phytomedicine 2000; 7: 191-97.

20

Begum et al Hexane extract on gentamicin-induced nephrotoxicity

Vol. 1, No. 1, June 2006

Bangladesh J Pharmacol 2006; 1: 21-26 Copyright © 2006 by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

Synthesis of some bis-triazole derivatives as probes for cytotoxicity study Mohammad Al-Amin and M. Rabiul Islam Department of Chemistry, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh [Received 19 December 2005]

Abstract A series of bis–[4-N-amino-5-mercapto-1,2,4-triazol-3-yl] alkanes (1a-e) and their Schiff bases with 2adamanta-none (2a-d) and bis – [1, 2, 4-triazolo [3, 4-b] - 1, 3, 4-thiadiazol-4-yl] alkanes (3a-e) have been synthesized with high yields. The cytotoxicity study of these newly synthesized compounds against brine shrimp lethality test as well as Structure-Activity-Relationship (SAR) has been discussed. Key words: bis-triazole, cytotoxicity, shrimp

Introduction

Triazoles are five membered heterocyclic compounds having three nitrogen atoms. They are of two types: NH

N

N

N

N

N H

1, 2, 3-Triazole

N

N

N

N

N H

NH

1, 2, 4-Triazole

for correspondence: Mohammad Al-Amin, Department of Chemistry, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh Phone: 088-2-7708624; E-mail: [email protected]

If two triazole units are linked by carbon atoms, then they form bis-triazole. Various 1,2,4-triazols are found to be linked with diverse pharmacological activities (Hirota et al., 1991; Yale and Piala, 1966; Andotra and Sharma, 1988). The 1,2,4-triazol nucleus has been incorporated into a wide variety of therapeutically interesting drug candidates including H1/H2 histamine receptor blockers, cholinesterase active agents, CNS stimulants, antianxiety agents and sedative (Heindel and Reid, 1980). It was also found that the thiadiazoline nucleus which incorporates a toxophoric N-C-S linkage exhibits a large number of biological activities. A number of 1,3,4-thiadiazoline possessed antibacterial properties comparable with sulphonamide drugs (Omar and AboulWafa, 1986). Subsequently, thiadiazole derivatives have found applications as antitumour

agents, pesticides, dyes, lubricants and analytical reagents (Lubrizol Corp, 1981).

cytotoxicity properties by brine shrimp lethality bioassay (Meyer et al., 1982).

Encouraged by the varied biological activities of 1,2,4-triazoles and in continuation of our work on the synthesis of N-bridged heterocycles derived from bis-triazoles (Shivarama Holla et al., 1988), a series of bis-[4-N-amino-5-mercaptotriazol-3-yl] alkanes were synthesized and their Schiff bases were prepared by condensing them with 2-adamantanone in DMF-ethanol in the presence of concentrated H2SO4. Further, the some of the bis-triazoles were cyclised with various amino acids using phosphorous oxychloride (POCl3) (Scheme 1). All of these newly synthesized compounds were characterized with the help of spectral data analysis and all of the synthesized compounds were screened for their

The obligatory bis-[4-N-amino-5-mercapto-1, 2, 4-triazol-3-yl] alkanes, 1a-e were synthesized by the direct fusion of dicarboxylic acids (n= 1-5) with thiocarbohydrazide (Scheme 1). Then these bis-triazolylalkanes, 1a-e were converted into their respective Schiff bases, 2a-d by condensing them with 2-adamantanone in the presence of few drops of concentrated sulfuric acid. Finally, the cyclisation of bis-triazolylalakanes with various amino acids using phosphorous oxychloride afforded bis–[1, 2,4-triazolo [3,4-b]1,3,4-thiadiazol-4-yl] alkanes, 3a-e. The structures of compounds 1a-e, 2a-d and 3a-e were confirmed on the basis of IR, 1H-NMR and 13CNMR spectral data analysis. S

HOOC

(CH2)n COOH + H2N

Dicarboxylic acid

NH

C

NH

NH2

Thiocarbohydrazide

[n= 1-5] Fusion

N

HS

N

N

(CH2)n

N NH2

N

SH

N

[n = 1-5]

NH2

1a-e

O (i) H2C

(i)

HS

N

N

N

COOH

NH2 R (ii) POCl 3 /

(ii) H2SO4 /

N

HC

(CH2)n

N

N

N

N

SH

S

N H2C

[n = 1,2,3&5]

R

HC NH2

2a-d

N

N

N

N

(CH2)n

N

N

[n = 1-5]

N

S

3a-e 3a : R =

& n =1

3b : R =

& n =5

CH

CH2

NH2

R

& n =5

3c : R = 3d : R =

N H OH

& n =1

3e : R =

OH

& n =4

Scheme 1

22

Al-Amin and Islam Synthesis of bis-triazole

Vol. 1, No. 1, June 2006

Materials and Methods All melting points were recorded by thin disk method on a “Fischer Johns” electrothermal melting point apparatus and are not corrected. Infrared spectra were recorded on DR-8001, SHIMADZU FT-IR spectrophotometer as a solid which was finely grounded in a small agate mortar with a drop of nujol (liquid hydrocarbon) as a mull and also in KBr disk. 1H-NMR spectra were measured by WP 400-NMR spec-trometer, deuterated solvents such as dimethyl-sulphoxide (DMSO-d6), methanol (CD3OD) and also chloroform (CDCl3) were used as solvents and the chemical shifts were quoted as δ-value relative to tetramethyl silane (TMS, δ =O) as an internal stan-dard. The 13C-NMR spectra were measured by WP 50 NMR spectrometer. The purity of compounds was checked by TLC on silica gel plates and iodine was used as a visualizing agent.

Bis-(4-N-amino-5-mercapto-1,2,4-triazol-3-yl) alkanes, 1a-e:A mixture of dicarboxylic acids (malonic acid, succinic acid, gluteric acid, adipic acid, palmilic acid) and thiocarbohydrazide in the ratio of 1:2 contained in a 100 ml. round-bottom flask was heated in an oil bath until the contents melted. The mixture was maintained at melting temperature for 15-20 minutes. The product obtained on cooling was treated with sodium bicarbonate solution to dissolve the unreacted dicarboxylic acid if any. It was then washed with water and collected by filtration. The product was recrystallised from a mixture of dimethylformamide and water to afford the title compounds 1a-e and characterized spectroscopically. The melting points, yields and IR data of the compounds, 1a-e are given in Table 1.

Table 1 – Physical and spectral data of compounds, 1a-e, 2a-d and 3a-e Compounds

n

Yield (%)

m.p. (°C)

Nature of compounds

1-a

1

80

240-245

1-b

2

85

280-282

1-c

3

75

240-242

1-d

4

80

250-252

1-e 2-a

5 1

78 60

215-217 265-270

2-b

2

65

255-257

2-c

3

75

208-210

2-d

5

79

235-237

White crystals White crystalline solid White crystalline solid White crystalline solid White crystalline solid White crystalline solid White crystalline solid White crystalline solid White crystalline solid

3-a

1

60

210-215

Gray crystalline solid

3-b

5

65

165-168

Gray crystalline solid

3-c

5

74

250-252

Brown crystalline solid

3-d

1

60

210-212

Gray crystalline solid

3-e

4

73

220-222

Gray crystalline solid

Bangladesh J Pharmacol

IR(cm-1) 3315&3298(υN-H),2926 & 2855 (υC-H,aliphatic),2359(υS-H),1600 (υC=N). 3315&3155(υN-H),2924 & 2855(υCH,aliphatic),2361(υS-H),1595 (υC=N). 3310&3295(υN-H),2925 & 2855(υCH,aliphatic),2359(υS-H),1598 (υC=N). 3325&3285(υN-H), 2924& 2855 (υC-H,aliphatic),2361(υS-H),1590 (υC=N). 3244&3200(υN-H), 2928& 2856 (υC-H,aliphatic),2360(υS-H),1600 (υC=N). 2926&2855(υC-H,aliphatic),2359 (υSH),1608(υC=N). 2924&2855(υC-H,aliphatic),2359 (υSH),1635(υC=N). 2926&2855(υC-H,aliphatic),2359 (υSH),1608(υC=N). 2928&2853(υC-H,aliphatic),2361 (υS-H), 1615(υC=N). 3315(υN-H),3100(υC-H,aromatic), 2924&2855(υCH,aliphatic), 1593(υC=N),1508 (υC=C, aromatic). 3240( υN-H), 3092 (υC-H,aromatic), 2952&2865(υCH,aliphatic),1599 (υC=N),1600&1508(υC=C,aromatic). 3240 (υN-H), 3100 (υC-H, aromatic), 2924&2855(υCH,aliphatic),1687 (υC=N),1600&1500(υC=C, aromatic. 3240(υN-H/OH),3092(υC-H, aromatic),2952&2860(υC-H, aliphatic),1599(υC=N),1600&1500 (υC=C, aromatic). 3395(υN-H/OH),3065(υC-H, aromatic),2945&2865(υC-H, aliphatic),1651(υC=N),1600,1558 &1506 (υC=C, aromatic).

23

Table 2. Cytotoxicity study of newly synthesized compounds Tested compounds N

N

N

N

1a-e ( CH2)n

N

HS

SH

N

[ n=1,2,3,4,5]

NH2

NH2

2-a (n=1) N HS

N

N (CH2)n

N N

N

2-b (n=2)

SH

N N

2-c (n=3) 2-d (n=5) 3-a (n=1, R=C6H5)

N

S

H2C CH R

NH2

N

N

N N

( CH2)n

3-b (n=5, R=C6H5)

N

N

S

N CH

CH2

3-c (n=5, R=C8H5NH)

NH2 R

3-d (n=1, R=C6H4OH) 3-e (n=4, R=C6H4OH)

Bis-[4-N-(adamantyl) imino-5-mercapto-1,2,4triazol-3-yl] alkanes, 2a-d: A mixture of bis-[4N-amino-1,2,4-triazol-3-yl] alkanes, 1-a, 1-b, 1-c and 1-e and 2- adamantanone in the ratio of 1:2 in dimethylformamide + ethanol (5 + 15 ml) media was heated under reflux on an oil bath for 4-5 hours after the addition of a few drops of concentrated sulfuric acid. The solid mass obtained on cooling the reaction mixture was collected by filtration and recrystallised from dimethylformamide to obtain Schiff bases, 2a-d and characterized spectroscopically. Bis- (6-phenylalanino/tryptopheno/tyrosino-1, 2, 4- triazolo-[3,4-b]-1,3,4-thiadiazol-4-yl) alkanes, 3a-e: A three-necked quick fit flask was fitted with a dropping funnel and a condenser. To a mixture of 1-a/phenylalanine, 1-e/phenylalanine, 1-e/tryptophan, 1-a/tyrosine, 1-d/tyrosine and 24

Al-Amin and Islam Synthesis of bis-triazole

Concentration (µg/ml) 50 100 150

Percentage of mortality 16.66 42.86 50.00

50 100 150

86.15 81.82 92.86

50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150 50 100 150

56.25 85.71 92.86 69.23 87.50 100.00 91.66 93.33 100.00 42.85 66.67 100.00 73.33 100.00 100.00 66.67 100.00 100.00 63.64 91.67 100.00 53.33 86.67 100.00

LC50 (µg/ml) 2.40

1.20

1.15

1.10

1.05

1.40

1.05

1.05

1.10

1.10

phosphorus oxychloride was added and the contents were heated under reflux for 2 hours on an oil bath. Excess of phosphorus oxychloride was then distilled off and the residue was poured onto crushed ice and stirred well. These were then washed with sodium bicarbonate solution (5%) and the resulting solids were then washed with water and recrystallised from dimethylformamide to obtain the compounds 3a-e respectively and characterized spectroscopically. Screening test: Because of the continuing interest of bis-triazoles derivatives, we conducted cytotoxicity investigation of the newly synthesized compounds by brine shrimp lethality bioassay and the test results show significant activity, as recorded in Table 2.

Vol. 1, No. 1, June 2006

(1)The compounds, 1a-e having free amino (NH2) groups showing very little cytotoxic activity.

LC50: The LC50 of an agent is the concentration, which will kill, or inactive 50% of the test animal. LC50 is inversely proportional to the toxicity of a compound, i.e. the lower is the LC50, the higher is the cytotoxicity.

N

HS

N

SH

NH2

[ n=1,2,3,4,5]

LC50 : 2.40

Sample: 1a-e

(2)The Schiff bases like 2-b, 2-c and 2-d have very good cytotoxic activity. That is, these compounds may act as potent cytotoxic agents. It should be mentioned here that, as the chain length increases, the cytotoxic activity also increases, i.e., the big molecule can easily interact with the DNA molecule.

N

N

N

N

N

NH 2

Structural-Activity-Relationship (SAR) according to the brine shrimp lethality test: The chemical structure of a drug is important as the relatively minor modification in the drug molecule may result a major change in pharmacological properties. This does not mean that changes in molecular configuration always alter all actions and effects of drug. So we have been able to recognize the functional groups/ring and determine which one is important. By synthesizing different compounds, one particular group of the molecule is removed or altered, to find out which groups are essential for biological activity and which are not. In this study we have the following results: N

( CH2)n

N

HS

N

N

(CH2)n

N

SH

N N

n =2,3 &5

2-b: LC50 =1.15 2-c: LC50 =1.10 2-d: LC50 = 1.05

(3) The compounds containing amino acid moiety such as, compounds 3b-e have very high cytotoxic activity.

N N

N

N

H2 C CH

N

( CH2)5

N

( CH2)5

NH2

S

NH2 N H

N H

3-b: LC50 =1.05

N N

N NH2

CH CH2

NH2

N

S

N

N

N

N

S S

N

N

3-c: LC50 =1.05 N

S

N

N

N

( CH2)n

N

N

S NH2

N

N n = 1&4

HO

OH

NH2

3-d (n=1): LC50 =1.10 3-e (n=4): LC50 =1.10 Bangladesh J Pharmacol

25

Results and Discussions All the newly synthesized compounds analyzed satisfactory for their nitrogen content. Characterization of the compounds was done on the basis of spectral analysis, The IR spectrum of compound 1-b shown absorption bands at 3315 and 3155 cm-1 indicating the presence of primary amino (R-NH2) group in the molecule. The band at 1685 cm-1 indicating the presence of C=N in the ring and the band at 2361 cm-1 corresponded the SH functional group in the molecule. In 1H-NMR spectrum of the compound, 1-b the methylene protons (CH2) appear as a singlet at δH 5.6, relatively a higher value which may be due to the anisotropic effect of the C=N group. The singlet at δH 3.4 arises due to labile protons in NH2. The singlet at δH 2.5 appear due to the proton in SH. In 13C-NMR spectrum of the synthesized compound 1-b, clearly indicated the three signals at δ 13C 21.223, 151.220 and 165.348 corresponded to the nonequivalent carbons respectively. The IR spectrum of the Schiff base, 2-b did not show any absorption bands corresponding to the NH stretching frequencies of the parent triazolylalakane, 1-b. However, a sharp absorption band was seen around 1576 cm-1, corresponding to the C=N linkage. The band at 2359 cm-1 corresponded to the υS-H stretching. In the 1H-NMR spectrum, the two labile protons in SH appeared as a singlet at δH 3.4, relatively a higher value which may be due to the electron withdrawing effect of nitrogen atoms (electronegativity 3.5). The CH2 protons appeared as a triplet at δH 2.5 which may be due to the coupling with neighboring CH2 protons. The adamantyl protons appear as multiplets at δH 2.0-1.6. In 13C-NMR spectrum of the synthesized compound 2-b, clearly indicated the signals at δ 13C 20.397, 150.187, 165.520 and 161.626 and also δ 13C 26-34 corresponded to the nonequivalent carbons respectively. In the IR Spectrum of the synthesized compounds 3a-e, the absorption band around at 1593

26

Al-Amin and Islam Synthesis of bis-triazole

cm-1 come into sight due to the presence of υC=N stretching in the ring. At 2360 cm-1 did not show any absorption bands corresponding to the SH stretching frequencies of the parent triazolylalkanes, 1a-e, confirmed the involvement of the SH groups of the parent bis-triazoles in the cyclisation. In the IR spectra of the cyclised products, 3a-e, the absorption band corresponding to the carbonyl stretching frequency (due to COOH groups) was absent, which again gave a conclusive evidence for the cyclisation. In our present research work, the synthesized compounds were investigated for their property as cytotoxic agents by brine shrimp lethality bioassay. Among these compounds 2-b, 2-c, 2-d, 3-b, 3-c, 3-d and 3-e were found to be very active and compounds 2-a, 3-a were moderately active and compounds 1a-e shown very poor activity against brine shrimp.

References Andotra CS, Sharma SK. In the synthesis of new 1,3,4oxazdiazole and 1,2,4-triazole derivatives. Ind J Pharm Sci. 1989; 51: 107 Heindel ND, Reid JR. 4-Amino-3-mercapto-4H-1,2,4,triazoles and propargyl aldehydes: a new route to 3-R-8Aryl-1,2,4-triazolo [3,4-b]-1,3,4-thiadiazepines. J Heterocycl Chem 1980; 17: 1087-8. Hirota T, Sasaki K, Yamamoto H, Nakayama T. Polycyclic N-hetero compounds. 91XVI. Syntheses and antidepressive evaluation of 11,13,15,17-tetraazasteroids and their 17-oxides. J Heterocycl Chem 1991; 28: 257-61. Shivarama Holla B, Gonsalves R, Shenoy S. Studies on some N-bridged heterocycles derived from bis-[4-amino5-mercapto-1,2,4-triazol-3-yl]alkanes. Farmaco 1988; 53: 574-78. Lubrizol Corp, US Pat, 4, 246, 126 (1981). Meyer BN, Ferringni NR, Putnam JE, Jacobsen LB, Nichols DE, Mclaughlin JL. Brine shrimp: a convenient general bioassay for bioactive plant constituents. Planta Medica 1982; 45, 31-34. Omar AMME, AboulWafa OM. Synthesis and in vitro antimicro-bial and antifungal properties of some novel 1,3,4,thiadiazole and s-triazole[3,4-b][1,3,4]thiadiazole derivatives. J Heterocycl Chem 1986; 23: 1339-41. Yale HL, Piala JJ. Substituted s-triazoles and related compounds. J Med Chem 1966; 9: 42-46.

Vol. 1, No. 1, June 2006

Bangladesh J Pharmacol 2006; 1: 27-32 Copyright © by Bangladesh Pharmacological Society

Available online at www.bdjpharmacol.com

Effect of alpha-lipoic acid on the removal of arsenic from arsenic-loaded isolated liver tissues of rat Noor-E-Tabassum Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka 1000, Bangladesh. [Received 26 May 2006]

Abstract The patient of chronic arsenic toxicity shows oxidative stress. To overcome the oxidative stress, several antioxidants such as beta-carotene, ascorbic acid, α-tocopherol, zinc and selenium had been suggested in the treatment of chronic arsenic toxicity. In the present study universal antioxidant (both water and lipid soluble antioxidant) α-lipoic acid was used to examine the effectiveness of reducing the amount of arsenic from arsenic-loaded isolated liver tissues of rat. Isolated liver tissues of Long Evans Norwegian rats were cut into small pieces and incubated first in presence or absence of arsenic and then with different concentrations of α-lipoic acid during the second incubation. α-Lipoic acid decreases the amount of arsenic and malondialdehyde (MDA) in liver tissues as well as increases the reduced glutathione (GSH) level in dose dependent manner. These results suggest that α-lipoic acid remove arsenic from arsenic-loaded isolated liver tissues of rat. Key words: arsenic; isolated liver tissues; α-lipoic acid.

Introduction About half of the total populations (more than 50 millions) of Bangladesh, at present, are consuming arsenic through drinking and cooking (Misbahuddin, 2003; Mudur 2000). Among them more than 40,000 people have already developed the signs and symptoms of chronic arsenic toxicity. The basic treatment is to stop drinking arsenic contaminated water and allow the patient to use arsenic-safe water (Smith et al., 2000). Some authors suggest the use of beta-carotene, vitamin A, ascorbic acid, α-tocopherol, zinc, selenium and spirulina for the treatment of chronic arsenic toxicity (Ahmad et al., 1998; Misbahuddin et al., 2006). These are antioxidants in nature. Although some are water-soluble for correspondence: Noor-E-Tabassum, Department of Pharmacology, Medical College for Women and Hospital Dhaka 1230, Bangladesh. e-mail: [email protected]

antioxidants and some are lipid soluble. Duration of treatment ranges from 4-12 months. Prolong duration of treatment affects the patients’ compliance as well as treatment cost. Our body also contains α-lipoic acid, which is a short chain fatty acid with sulfhydryl (-SH) groups that has potent antioxidant property (Packer et al., 1995). Antioxidant properties of αlipoic acid are due to its ability to scavenge hydroxy radicals, hypochlorous acid and singlet oxygen (Biewenga et al., 1997). α-Lipoic acid is present in our diet such as spinach, broccoli and tomatoes. Naturally occurring R-enantiomer of α-lipoic acid is an essential cofactor in α-ketoacid dehydrogenase complexes and the glycine cleavage system (Jones et al., 2002). It is readily absorbed from the gut and the mean peak plasma concentration of α-lipoic acid following a single oral administration of 200 mg was 3.1 µM. The mean plasma half-life of α-lipoic acid was about

30 min (Teichert et al., 1998). Within the tissue, it is rapidly converted into dihydrolipoic acid (DHLA). Both α-lipoic acid and DHLA may chelate or bind metal ions and facilitating their removal from the cell (Ou et al., 1995). Exogenous administration of α-lipoic acid has been found to be effective in many pathological condition associated with oxidative stress, diabetic neuropathy (Zeigler et al., 1999), metal toxicity (Muller and Menzel, 1990), hypertension (Midaoui and Champlain, 2002), diabetic complication and cataracts (Packer, 1994). Recently it has been found that α-lipoic acid suppressed the free radicals initiated by arsenic in different parts of rat brain regions (Shila et al., 2005a). α-Lipoic acid also causes an increase in intracellular GSH in vitro as well as in vivo (Busse et al., 1992). Simultaneous administration of lipoate (α-lipoic acid) to arsenic-treated rats has been shown to decrease arsenic content and increase glutathione status remarkably in discrete brain areas (Shila et al., 2005b). Glutathione and glutathione related enzyme play an important role in the cell against the effect of reactive oxygen species (ROS). GSH also stimulates the arsenic detoxification process by modulating arsenic speciation (Scott et al., 1993). Therefore, this study was designed to evaluate the effect of α-lipoic acid on the removal of arsenic from the arsenic-loaded isolated liver tissue of rat.

Materials and Methods Chemicals and reagents: Arsenic trioxide (As2O3), reduced glutathione (GSH), 5,5-dithiobis-2-nitro-benzoic acid (DTNB) and thiobarbituric acid were purchased from Sigma Chemical Company (St. Louis, MO, USA). Chemicals and reagents to measure lactase dehydrogenase (LDH) and total protein were from Human Gmbh (Germany). α-Lipoic acid was a gift from Opsonin Pharma Limited, Bangladesh. All other reagents and solvents were highest analytical grade available. Preparation of isolated liver tissues: The study was carried out on isolated liver tissues of Long

28

Tabassum α-Lipoic acid on removal of arsenic

Evans Norwegian adult healthy male rats weighing about 150-180 g. The rats were housed in standard plastic cages in a clean rodent room where a 12-h light/dark cycle was maintained. On the day of experiment, rats were sacrificed under chloroform anesthesia and the abdomen was opened by giving a midline incision. The liver was dissected out and immersed immediately into the physiological solution (NaCl 150 mM, KCl 5.6 mM, NaHCO3 25 mM, NaH2PO4 2.5 mM, Glu-cose 10 mM), placed in ice bath. The liver tissues were chopped into small pieces (approximately 2 mm in size). Research Design: Isolated liver tissues of rat were incubated with in presence or absence of arsenic (50 µg) at 37oC for 45 minutes. After the first incubation, tissues were washed twice with physiological solution. The purpose of this incubation was to load the liver tissues with arsenic. Then during the second incubation (at 37oC for another 45 minute), liver tissues were treated with different concentrations of α-lipoic acid (1 µM, 10 µM, 100 µM). Several test tubes were taken and each test tube contains 250 mg small pieces of liver tissues immersed in 5 ml of physiological solution. The test tubes were marked as groups: Group IControl; Group II- arsenic (50 µg); Group IIIarsenic (50 µg) + α-lipoic acid (1 µM); Group IV- arsenic (50 µg) + α-lipoic acid (10 µM); Group V- arsenic (50 µg) + α-lipoic acid (100 µM). Number of the test tubes (samples) in each group was six. After second incubation, tissues were homogenized and used for the estimation of total arsenic, total protein, malondialdehyde (MDA), and GSH level. Estimation of arsenic: The amount of total arsenic was measured using Atomic Absorption Spectrophotometer with Hydride Generator (Buck Scientific, USA). In brief (Wang et al., 1994): the sample, at first, digested with nitric acid (3 ml), sulfuric acid (2 ml) and perchloric acid (2 ml) for 2 hours by Bunsen burner. Following digestion, each sample was introduced into the hydride generator by continuous flow of

Vol. 1, No. 1, June 2006

10% hydrochloric acid, 3% sulfuric acid and 1.5% sodium borohydride into a gas-liquid separator. The arsine vapor produced by arsenic and the hydrogen gas (produced by sodium borohydride and acid) was carried out by flowing argon gas into quartz T-tube. The tube was heated in an air-acetylene flame (2300° C), serve as atomization cell. The current of the Hollow Cathode Lamp for arsenic was 10 mA. The wavelength and spectral band-width were 193.7 nm and 0.7 nm respectively. Estimation of MDA: The extent of lipid peroxidation was estimated by using the thiobarbituric acid method to determine MDA levels described by Wilber et al. (1949). Briefly, 1 ml of tissues homogenate was reacted with 4.5 ml of 5.5% trichloroacetic acid. The mixture was vortexes and centrifuged at 4,000 x g for 10 minutes. 1 ml of 0.67% thiobarbituric acid was added to supernatant and heated at 100ºC for 10 minutes, forming a pink colored solution. After cooling of the mixtures, absorbance was measured by Spectrophotometer (UV-Vis 1201; Shimadzu, Japan) at 532 nm. The results were expressed as nmol MDA per mg of protein. Estimation of GSH: GSH level was assayed by the method of Ellman (1959). In brief, 1 ml of tissues homogenate was added to 1 ml of 5% trichloroacetic acid and the mixture was vortexes and centrifuged at 4,000 x g for 5 minutes. To 250 µl of supernatant, 2 ml Na2HPO4 (4.25%) and 250 µl of DTNB (0.04%) were added. The mixture was allowed to stand for approximately 15 minutes, and forming a yellow substance. The absorbance was measured at 412 nm using a Spectrophotometer. Cell viability test: The tissues were incubated with different strengths of arsenic (25, 50, 100, 200 µg) at 37oC for 45 minutes to determine the concentration of arsenic that would not induce tissues damage. Cytotoxicity was determined by the release of LDH into the medium. After the incubation of tissues with arsenic, the supernatant (medium) were removed and analyzed for LDH content using Human Gmbh diagnostic

Bangladesh J Pharmacol

LDH assay based on the technique of Schumann et al. (2002). Estimation of protein: Protein concentration of tissues was estimated by 'Biuret' method described by Weichselbaum (1949). Bovine serum albumin (8 g/dl) was used as standard. Statistical analysis: Statistical analyses were carried out using Statistical Package for Social Science (SPSS), version 9.0, USA. The values were expressed as mean ± SEM for results obtained with six samples in each group and the significant of differences between values was determined by one way analysis of variance (ANOVA) F-test coupled with the Dunnets’s multiple comparison test. Statistical significance was determined by p value less than 0.05.

Results The amount of total arsenic in arsenic-loaded isolated liver tissues of rat after second incubation was 91.87 ± 2.04 µg/g of protein (Table I). But when the arsenic-loaded tissues were incubated with 1, 10, and 100 µM concentration of αlipoic acid during the second incubation, the amounts of total arsenic in tissues were decreased to 67.72 ± 2.52, 50.09 ± 1.74, and 27.18 ± 1.66 µg/g of protein respectively. The removals of accumulated arsenic from tissues were 26.3%, 45.5% and 70.5% respectively. These effects of removing arsenic were dose dependent and statistically significant (P <0.001). As shown in Table II, the mean (± SEM) concentration of GSH in arsenic-untreated tissues was 2.83 ± 0.12 µg/mg of protein and following incubation of tissues with 50 µg of arsenic, the concentration of GSH decreased to 1.37 ± 0.04 µg/mg of protein which was statistically significant (P<0.001) in comparison to the control group. In arsenic plus α-lipoic acid (1 µM) treated group, GSH concentration averaged 1.74 ± 0.05 µg/mg of protein. With increasing the doses of α-lipoic acid (10 and 100 µM), GSH concentration significantly (P<0.001) increased to 2.05 ± 0.07 and 2.52 ± 0.14 µg/mg of protein

29

respectively. This result also indicate that GSH level significantly (P<0.001) decreased in arsenic -treated groups by 52% compared to control while administration of α-lipoic acid in arsenicloaded isolated liver tissues significantly (P< 0.001) restored the GSH level by 25%, 47% and 79% at 1, 10 and 100 µM concentration respectively. The mean (± SEM) concentration of MDA in arsenic-untreated group was 3.08 ± 0.11

nmol/mg protein. When the tissues were treated with 50 µg of arsenic, the concentration of MDA increased to 9.30 ± 0.14 nmol/mg protein which was statistically significant (P<0.001) in comparison to the control group. Treatment of arsenicloaded isolated liver tissues with different concentrations of α-lipoic acid (1, 10, and 100 µM)

Table I. Effect of α-lipoic acid on the removal of arsenic from arsenic-loaded isolated liver tissues of rat Group

Incubation of liver tissues at 37oC for 45 minutes First incubation Second incubation

n

I II III IV V

6 6 6 6 6

None Arsenic (50 µg) Arsenic (50 µg) Arsenic (50 µg) Arsenic (50 µg)

None None lipoic acid (1 µM) lipoic acid (10 µM) lipoic acid (100 µM)

Tissues concentration of arsenic after second incubation (µg/g protein; mean ± SEM) 1.09 ± 0.13 91.87 ± 2.04 67.72 ± 2.52 50.09 ± 1.74 27.18 ± 1.66

P value

P<0.001 P<0.001 P<0.001

n = number of samples in each group; None means tissues were incubated with physiological solution without arsenic

Table-II. Effect of α-lipoic acid on the levels of GSH and MDA in arsenic-loaded isolated liver tissues of rat Parameter GSH MDA

Control 2.83 ± 0.12 3.08 ± 0.11

Arsenic (50 µg) 1.37 ± 0.04 9.30 ± 0.14

Arsenic (50 µg) + Lipoic acid (1 µM) 1.74 ± 0.05 6.77 ± 0.17

Arsenic (50 µg) + Lipoic acid (10 µM) 2.05 ± 0.07 5.18 ± 0.16

Arsenic (50 µg) + Lipoic acid (100 µM) 2.52 ± 0.14 3.86 ± 0.17

P value P<0.001* P<0.001

Values were expressed as mean ± SEM of six samples in each group; GSH refers to reduced glutathione level expressed as µg sulfhydryl groups/mg protein; MDA refers to malondialdehyde level expressed as nmol/mg protein.* Significance based on Dunnet’s multiple comparison tests for comparison of treated groups with standard (only arsenic-treated) group.

significantly (P<0.001) decreased the MDA concentration thus bring back the changes to near normalcy (6.77 ± 0.17, 5.18 ± 0.16, 3.86 ± 0.17 nmol/mg protein respectively). The increased production of MDA by exposure to arsenic and its recovery by different concentrations of αlipoic acid treatment were shown in Table II. Results indicate that MDA production, an indicator of lipid peroxidation, was increased (P<0.001) in arsenic-treated groups and it was 201% compared to control while administration of different concentrations of α-lipoic acid (1, 10, and 100 µM) in arsenic-loaded tissues could significantly (P< 0.001) reduce the production of MDA by 40%, 66% and 88% respectively.

whereas, the LDH activity was not detected in tissues loaded with 25 and 50 µg of arsenic. These result suggested that 25 and 50 µg of arsenic preserve cell viability.

The effect of different concentrations of arsenic (25, 50, 100 and 200 µg) on cell viability was examined. The LDH activity after incubation of tissues with 100 and 200 µg of arsenic were 58.46 ± 0.90 and 139.19 ± 3.09 U/I respectively,

In this study, viability of the tissues was determined by the activity of LDH. The activity of LDH was not showed in a concentration of 25 and 50 µg of arsenic. These data not only suggested that the preparations were viable but also suggested

30

Tabassum α-Lipoic acid on removal of arsenic

Discussion Oxidative stress due to enhanced production of free radicals has been incriminated as one of the several mechanisms involved in arsenic-induced toxic effects in different organs. In view of the antioxidant properties of α-lipoic acid, the present work was conducted to evaluate its effects on the removal of arsenic from arsenic-loaded isolated liver tissues of rat.

Vol. 1, No. 1, June 2006

that arsenic exposure at least at the 50 µg concentrations used in this experiments was not induce tissue damage.

lipid peroxidation and GSH levels in the liver on arsenic exposure was suggested by Ramos et al. (1995).

In the present work, a substantial increase in the level of arsenic was observed in the tissues treated with arsenic. However, treatment with different concentrations of α-lipoic acid remarkably brings down the level of arsenic (P<0.001) in a dose-dependent manner.

However, treatment with different concentrations of α-lipoic acid significantly (P<0.001) increase the levels of GSH. Enhancement of intracellular availability of cysteine, by passing the rate limiting cystine transport system may be the underlying mechanism of α-lipoic acid-induced elevation of GSH levels observed in this study. This finding was in agreement with previous in vitro research work (Busse et al., 1992).

Arsenic binds to the −SH group of dihydrolipoate and inhibits pyruvate dehydrogenase consequently prevents oxidation of dihydrolipoate to lipoate, which is needed in the formation of acetyl-CoA from pyruvate (Miller et al., 2002). Dose-dependent protection offered by α-lipoic acid might be attributed to the ability of α-lipoic acid to protect the –SH groups in the reduced form or compete with mitochondrial lipoamide for availability of arsenic, which thus prevents the binding of arsenic to proteins in isolated liver tissues of rat. Arsenic exposure in this experiment resulted in a significant (P<0.001) reductions in the level of the GSH and associated with increases in lipid peroxidations in comparison to control group. Arsenic content causes extensive oxidation of intramitochondrial NADPH by inhibition of αketoacid dehydrogenase (Shi et al., 2004). The shortage of NADPH production during arsenic exposure would suppress the reduction of GSSG subsequently decrease the GSH content. The increase in the levels of MDA was due to the increased release of iron that was believed to be involved in the Fenton type of reaction. Arsenic is shown to stimulate the release of iron from ferritin and through the activation of heme oxygenease the rate-limiting enzyme in heme degradation (Ahmad et al., 2000). The free iron is considered as a potent enhancer of ROS formation, as exemplified by the reduction of H2O2 with the generation of highly aggressive hydroxyl radical (Farber, 1994). Therefore, this may be the possible reason for elevation of the levels of MDA with a concomitant fall in the GSH content. The inverse relationship between

Bangladesh J Pharmacol

In the present study, the decrease levels of MDA observed in the α-lipoic acid treatment group may be attributed to its capacity to regenerate the reduced glutathione pool and / or may be directly react with ROS. These findings were consistent with the results of previous studies (Shila et al., 2005b). In conclusion, the finding of the present study suggests that exposure of liver tissues with arsenic was associated with a depletion of GSH and increased lipid peroxidation. α-Lipoic acid administration offered a significant removal of arsenic, which was associated with its antioxidant activity that combine free radical scavenging and metal chelating properties. However, further studies with α-lipoic acid need to be carried out in vivo to ascertain their therapeutic efficacy in modifying chronic arsenic toxicity.

Acknowledgement I am grateful to Prof. Mir Misbahuddin, Department of Pharmacology, Bangabandhu Sheikh Mujib Medical University, Shahbag, Dhaka 1000, Bangladesh for his support and encouragement throughout this work.

References Ahmad S, Kitchin KT, Cullen WR. Arsenic species that causes release of iron from ferritin and generation of activated oxygen. Arch Biochem Biophys. 2000; 382: 195-02.

31

Ahmad SA, Faruquee MH, Sayed MHSU, Khan MH, Hadi SA, Khan AW. Chronic arsenicosis: Management by vitamin A, E, C regimen. J Pre Social Med (JOPSOM). 1998; 17: 19-26.

Ramos O, Carrizales L, Yanez L, Mejia J, Batres L, Ortiz D, Barriga DF. Arsenic increased lipid peroxidation in rat tissues by a mechanism independent of glutathione levels. Environ Health Perspect. 1995; 103: 85-88.

Biewenga G, Haenen GRMM, Bast A. The pharmacology of antioxidant lipoic acid. Gen Pharmacol. 1997; 29: 31531.

Schumann G, Bonora R, Ceriotti F, Ferard G, Ferrero CA, Franck PFH, Gella FJ, Hoelzel W, Schimmel HG, Weidemann W, Siekmann L. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37°C. Clin Chem Lab Med. 2002; 40: 725-33.

Busse E, Zimmer G, Schopohl B, Kornhuber B. Influence of alpha-lipoic acid on intracellular glutathione in vitro and in vivo. Arzneim-Forsch. 1992; 42: 829-31. Ellman LG. Tissues sulfhydryl groups. Arch Biochem Biophys. 1959; 82: 70-77. Farber LJ. Mechanisms of cell injury by activated oxygen species. Environ Health Perspect. 1994; 102: 17-24. Jones W, Li X, Qu ZC, Perriott L, Whitesell RR, May JM. Uptake, recycling, and antioxidant action of α-lipoic acid in endothelial cells. Free Rad Bio Med. 2002; 33: 83-93. Midaoui AE, Champlain JD. Prevention of hypertension, insulin resistance, and oxidative stress by α-lipoic acid. Hypertension. 2002; 39: 303-07. Miller WH, Schipper HM, Lee JS, Singer J, Waxman S. Mechanisms of action of arsenic trioxide. Cancer Res. 2002; 62: 3893-03. Misbahuddin M, Maidul Islam AZM, Khandker S, Mahmud IA, Islam N, Anjumanara. Efficacy of spirulina extract plus zinc in patients of chronic arsenic poisoning: A randomized placebo-controlled study. Clinical Toxicol. 2006; 44: 1-7. Misbahuddin M. Consumption of arsenic through cooked rice. Lancet. 2003; 361: 435-36. Mudur G. Half of Bangladeshi population at risk of arsenic poisoning. Br Med J. 2000; 359:1127. Muller L, Menzel H. Studies on the efficacy of lipoate and dihydrolipoate in the alteration of cadmium2+ toxicity in isolated hepatocytes. Biochim Biophys Acta 1990; 1052: 386-91. Ou P, Trischler HJ, Wolff SP. Thiotic (Lipoic) acid: A therapeutic metal-chelating antioxidant? Biochem Pharmacol. 1995; 50: 123-26. Packer L, Witt EH, Tritschler HJ. Alpha lipoic acid as a biological antioxidant. Free Rad Bio Med. 1995; 19: 22750. Packer L. Antioxidant properties of lipoic acid and its therapeutic effects in prevention of diabetes complications and cataracts. Ann N Y Acad Sci. 1994; 738: 25764.

32

Tabassum α-Lipoic acid on removal of arsenic

Scott N, Hatlelid KM, MacKenzie NE, Dean CE. Reactions of arsenic (III) and arsenic (V) species with glutathione. Chem Res Toxicol. 1993; 6: 102-06. Shi H, Shi X, Liu J. Oxidative mechanism of arsenic toxicity and carcinogenesis. Molecul Cell Biochem. 2004; 255: 67-78. Shila S, Kokilavani V, Subathra M, Panneerselvam C. Brain regional responses in antioxidant system to alpha-lipoic acid in arsenic intoxicated rat. Toxicology. 2005a; 210: 25-36. Shila S, Subathra M, Devi MA, Panneerselvam C. Arsenic intoxication-induced reduction of glutathione level and of the activity of related enzymes in rat brain regions: Reversal by DL α-lipoic acid. Arch Toxicol. 2005b; 79: 140-46. Smith AH, Lingas EO, Rahman M. Contamination of drinking-water by arsenic in Bangladesh: A public health emergency. Bull W H O. 2000; 78: 1093-03. Teichert J, Kern J, Trischler HJ, Ulrich H, Preiss R. Investigations on the pharmacokinetics of α-lipoic acid in healthy volunteers. Int J Clin Pharmacol Ther. 1998; 36: 625-28. Wang CT, Chang CT, Huang CW, Chou SS, Lin CT, Wang RT. Studies on the concentrations of arsenic, selenium, copper, zinc and iron in the hair of blackfoot disease patients in different clinical stages. Eur J Clin Biochem. 1994; 32: 107-11. Weichselbaum TE. Estimation of serum total protein by Biuret method. Am J Cli Path. 1946; 16: 40-48. Wilber KM, Baerheim F, Shapiro OW. The thiobarbituric acid reagent as a test for the oxidation of unsaturated fatty acid by various reagents. Arch Biochem Biophys. 1949; 24: 304-11. Ziegler D, Reljanovic M, Mehnert H, Gries FA. α-lipoic acid in the treatment of diabetes polyneuropathy in Germany: Current evidence from clinical trials. Exp Clin Endocrin Diab. 1999; 107: 421-30.

Vol. 1, No. 1, June 2006

LETTER TO THE EDITOR Evaluation of medical teachers and traditional teaching in Pharmacology Medical teaching is a noble profession in the field of medical science. Everybody is aware of the fact that Health is Wealth. People want to live in good health. This can be achieved with the help of service provided by medical practitioners. Medical practitioners however can only provide good medical health facilities after passing the full MBBS course and acquiring a registration from BMDC. In western countries, the facilities for medical care are widely available as well as of fine quality. In underdeveloped countries however, the story is quite different. Proper facilities are hardly available and the quality is poor and steadily declining. The patients are flying abroad for treatment, the most common destinations being India and Singapore. Since ‘Health for all’ is our slogan, we must rise to take measures against this and ensure that medical health in our country depends solely on the medical practitioners of our country. Our doctors acquire their MBBS degree after passing three professional examinations. The full course, along with the training takes six years. A paper has been published in Journal of Chittagong Medical College Teachers Association (1996; 7: 59-62) from which it is clear that they are hardly interested in acquiring knowledge; rather they are interested in getting their MBBS certificates in the easiest ways possible. Simultaneously, the medical teachers are the ones held responsible for producing doctors who prove to be national assets. Pharmacology is one of the vital branches of medical science that acknowledges the doctor on how to write out a prescription rational. Sadly though, in my lifetime of teaching I have discovered a mere 5% of the students to be interested in Pharmacology. The students somehow manage to pass their examinations after which they write out drugs in their prescriptions with help from medical representatives. This leads to me another Bangladesh J Pharmacol

thought- whether it is the students or rather the teachers who are actually responsible for this lack of interest. There must be some lacunae in the teachers. We should try to make pharmacology understandable and interesting too, because whichever field in medical science our students undertake, they will always require some knowledge of pharmacology if they are to fulfill the moral and legal duties they have towards their patients. All in all, I conclude that both students and teachers ought to be evaluated: one by the other. Admittedly, it is very difficult to evaluate teachers. I, however, have taken upon the challenge to evaluate teachers with the help of postgraduate medical students. The study was carried out at the Department of Pharmacology, Chittagong Medical College during the years 1992, 1993 and 1994. Postgraduate students were the subjects, those studying for DA, DCH and DGO. The total number of students present was forty-five, out of which only twenty-five agreed to take part in the survey. Six students out of ten participated in the study carried out in 1992, while ten out of fifteen participated in 1993. In 1994, nine out of fifteen were interested. Area of evaluation

Mean scoring* 1992

1993

1994

Selection of topic

3.5

3.7

4.2

Clear audible voice

3.6

4.1

3.8

Able to make the subject clear

3.1

3.4

3.3

Interest in teaching

4.3

4.4

4.2

Modern concept in

3.0

3.3

2.8

Using audio-visual aids

2.5

3.8

3.3

Behavior of the teacher with the

5.0

4.8

4.8

4.8

4.8

5.0

2.6

3.6

3.5

pharmacology

student Teacher- regular, sincere and punctual Any other comment

*below average- 1; average- 2; good- 3; very good- 4; excellent- 5

A questionnaire was prepared and supplied to the students each year after they completed their 33

course of Pharmacology. After completion of the questionnaires, the data collected was analyzed. The teachers were evaluated on the basis of parameters like, the topics they chose to teach, whether they possessed a clear, audible voice, whether they were capable of clarifying a topic, how interested they were in teaching the students, whether their conceptions of Pharmaco-

logy were contemporary and whether the teacher was regular, sincere and punctual.

Shamsun Nahar Department of Pharmacology Mymensingh Medical College Mymensingh, Bangladesh

You can submit your opinion related to the pharmacology by e-mail: [email protected]

34

Letter to the editor

Vol. 1, No. 1, June 2006