Research Journal of Medicine and Medical Sciences, 2(1): 29-34, 2007 © 2007, INSInet Publication
Comparison of Long-term Antihyperglycemic and Hypolipidemic Effects Between Coccinia cordifolia (Linn.) And Catharanthus roseus (Linn.) In Alloxan-induced Diabetic Rats Most. Afia Akhtar, Mamunur Rashid, Mir Imam Ibne Wahed, Md. Robiul Islam, Sharif Mohammad Shaheen, Md. Ariful Islam, Md. Shah Amran and Maruf Ahmed Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh. Abstract: The antihyperglycemic and hypolipidemic effects of ethanol-extract from the leaves of Coccinia cordifolia (C. cordifolia) and Catharanthus roseus (C. roseus) were investigated and compared in alloxaninduced diabetic rat (AIDR). Single doses (150 and 300 mg/kg, i.p.) of both the C. cordifolia and C. roseus leaf extracts were given to diabetic rats. The fasting blood glucose (FBG), serum triglyceride (TG) and serum total cholesterol (TC) levels were investigated in AIDRs on 0 th , 7 th and 14 th days of treatment. Single dose treatment with extracts of C. cordifolia (300 mg/kg), C. roseus (150 mg/kg of body weight) significantly reduced blood glucose by 47.4 and 37.1 % (P < 0.01) on 7 th day and by 59.7 and 48.5 % on 14 th day. A significant reduction (P < 0.01) in serum total cholesterol of 31.7 and 43.3 % and serum triglycerides of 45.5 and 39.4 % was observed on the 14 th day with a single dose of the extracts of C. cordifolia (300 mg/kg) and C. roseus (150 mg/kg of body weight), respectively. Their hypoglycemic and hypolipidemic activities were comparable to glibenclamide (0.05 mg/kg) and metformin HCl (100 mg/kg). In contrast, C. cordifolia extract at the dose of 150 mg/kg did not show any significant effect and C. roseus extract at the dose of 300 mg/kg killed all the rats in 2-5 days. C. cordifolia was found to be better than C. roseus in lowering blood glucose and serum triglycerides but has lower activity for lowering serum cholesterol on long-term treatment basis in AIDRs. Key words: Coccinia cordifolia, Catharanthus hypolipidemic, diabetic rat INTRODUCTION
long-term
effects, antihyperglycemic,
access to the conventional treatment of diabetes is not adequate [7 ]. There is an increased demand to use natural products with antidiabetic activity due to the side effects associated with the use of insulin and oral hypoglycemic agents [8 ][9 ]. The available literature shows that there are more than 400 plant species showing hypoglycemic activity [1 0 ][1 1 ][1 2 ]. Though some of these plants have great reputation in the indigenous system of medicine for their antidiabetic activities, many remain to be scientifically established. Hypercholesterolemia and hypertriglyceridemia are common complications of diabetes mellitus in addition to h y p e r g lyc em ia [ 1 3 ] [ 1 4 ] [ 1 5 ] . T he fre q ue nc y o f hyperlipidemia in diabetes is indeed very high, depending on the type of diabetes and its degree of control[1 6 ]. Ivy gourd or Coccinia cordifolia (C. cordifolia) is an aggressive vine in the Cucurbitaceae (cucumber) family. It is widely cultivated and has escaped to become a vigorous pest in Hawai'i, Australia, Saipan, Texas, and Florida. In Southeast Asia, ivy gourd is cultivated for its edible young shoots and edible
Diabetes mellitus is a metabolic disorder characterized by hyperglycemia, abnormal lipid and protein metabolism along with specific long-term complications affecting the retina, kidney and nervous system [1 ]. Hyperglycemia is an important factor in the development and progression of the complications of diabetes mellitus [2 ]. The pathogenesis of diabetes mellitus is managed by insulin and oral administration of hypoglycemic drugs such as sulfonylureas and biguanides [3 ].Unfortunately, apart from having a number of side effects, none of the oral synthetic hypoglycemic agents has been successful in maintaining euglycaemia a n d c o n tr o lling lo ng-term micro vascula r a nd macrovascular complications [3 ][4 ][5 ]. The toxicity of oral antidiabetic agents d iffers wid ely in clinical manifestations, severity, and treatment[6 ]. The use of herbal medicines for the treatment of diabetes mellitus has gained importance throughout the world. The W orld Health Organization also recommended and encouraged this practice especially in countries where Corresponding Author:
roseus,
Maruf Ahmed, Department of Pharmacy, University of Rajshahi, Rajshahi-6205, Bangladesh. E-mail:
[email protected] 29
Res. J. Medicine & Med. Sci., 2(1): 29-34, 2007 fruits [1 7 ]. It has been classified as one of the medicinal herbs in the traditional practice of the Bangladeshi as well as Indian medicine. The juice of the roots and leaves is used to treat diabetes while the leaves are also used as poultice to treat skin eruptions. In addition, the aqueous and ethanolic extracts of the plant have shown hypoglycemic action [1 8 ]. Another study has shown improvement in glucose tolerance of C. cordifolia in patients with maturity onset diabetes [1 9 ]. However, hypolipidemic properties of C. cordifolia have not been investigated so far. C a th a ra n th u s ro seus (C . ro seu s) Linn. (Apocyanaceae) is an herbaceous subshrub also known as Madagascar periwinkle, Vinca rosea, or Lachnera rosea worldwide. The leaves are used traditionally in various regions of the world including India, W est Indies as well as Nigeria to control diabetes [20 ] . The leaves have been known to contain 150 useful alkaloids among other pharmacologically active compounds. Significant antihyperglycemic and hypotensive activity of the leaf extracts (hydroalcoholic or dichloromethanemethanol) have been reported in laboratory animals [2 1 ]. Fresh leaf juice of C. roseus has been reported to reduce blood glucose in normal and alloxan diabetic rabbits[2 2 ]. The leaf juice of C. roseus produced a significant decrease in serum total cholesterol and triglyceride of rats [23 ] . However, prolong effects (up to 14 days) after a single dose have not been investigated. In this study the prolonged effect (up to 14 th day) of ethanolic extracts of C. cordifolia and C. roseus leaves on fasting blood glucose (FBG) and lipid biochemical parameters such as serum total cholesterol (TC) and serum triglyceride (TG) were compared in alloxaninduced diabetic rats (AIDR). The comparative antihyperglycemic and antihyperlipidemic properties between C. cordifolia and C. roseus have also been investigated.
and DMSO were purchased from Loba Chemie, Bombay, India. Preparation of Ethanol-Extracts: Dried leaves of C. cordifolia and C. roseus were soaked for 5-7 days in 2 liter of 95% ethanol with occasional shaking and stirring. Then, they were passed through cotton and then filtered through filter paper. The remaining parts were filtered again under the same procedure. The collected filtrates were concentrated under atmospheric pressure below 45 °C and yielded an ethanol-extract (yield 50.0 g; 5.0%). Animal Experiments: A total number of 54 albino rats of either sex (20-30 g) obtained from ICDDR,B, Dhaka were used in the study. They were divided into nine groups of six each and were fed with standard rat diet and water ad libitum. They were kept in cages and maintained in well-ventilated room under conditions of natural light and dark cycle. Animals were fasted for 16 h prior to drug administration allowing access only to water and were deprived of both food and water during the experiment. Induction of Diabetes: After fasting 16 h, rats of group (II-IX) were rendered diabetic by injecting a freshly prepared aqueous solution of alloxan monohydrate (110 mg/kg i.p.) after a base-line glucose estimation was done. After 2 weeks, blood glucose content was measured by using BioLand G-423 glucose test meter (BioLand, Germany) using a blood sample from the tail vein of rats. W hen the condition of diabetes was established animals with blood glucose levels above 11.1 mmol/L were selected for the study. Effect on Diabetic Rats: Group I served as a nondiabetic control while groups II to IX were rendered diabetic. Group II served as diabetic control while group III received the vehicle (99% DMSO) and served as the negative control group. Group IV and V were treated with metformin HCl (100 mg/kg, i.p.) and glibenclamide (0.05 mg/kg, i.p.) respectively for consecutive 14 days. Group VI and VII were treated with the extract of C. cordifolia 300 mg/kg and 150 mg/kg respectively for 14 consecutive days. Group VIII and IX were treated with the extract of C. roseus 300 mg/kg and 150 mg/kg respectively for 14 consecutive days. The reference drugs, ethanol-extracts and the vehicle were administered intraperitonially to the rats.
M ATERIALS AND M ETHODS Plant M aterial: Fresh leaves of C. cordifolia and C. roseus were collected from our University campus and were dried under shadow for several days. The dried leaves were then grinded to a coarse powder. The authenticity of the C. cordifolia and C. roseus were identified by M r. A.H.M . M ahbubur Rahman, Department of Botany, University of Rajshahi. Voucher specimens, collection # 33, dated 4/25/2002 for C. cordifolia and collection # 37, dated 10/30/2000 for C. roseus, were kept in the Department of Botany, University of Rajshahi, Rajshahi.
Collection of Blood and Serum and Determination of Blood Glucose, Serum Total Cholesterol (TC) and Serum Triglycerides (TG): Blood samples were collected from tail vein of each rat of a group before and also at 8 th and 15 th day after treatment. The
Reagents: Metformin HCl and glibenclamide were the generous gift samples from Square Pharmaceuticals Ltd., Pabna, Bangladesh. Both alloxan monohydrate
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Res. J. Medicine & Med. Sci., 2(1): 29-34, 2007 samples were analyzed for blood glucose content by using BioLand G-423 glucose test meter (BioLand, Germany). Then the rats were sacrificed and about 1-2 ml of blood was collected directly from the heart by syringes, centrifuged at 4000 rpm for 10 minutes and the serum was obtained for the determination of TC and TG. Serum TC and TG concentrations were a n a ly z e d b y m e a su rin g ab so rb a n c e b y U V spectrophotometer (Shimidzu UV-1200, Tokyo, Japan), using wet reagent diagnostic kits (B oehringer Mannheim, GmbH) according to manufacturer’s protocol. Fig. 1: Effects of C. cordifolia and C. roseus on fasting blood glucose levels after intraperitoneal administration in alloxan-induced diabetic rats. Each bar indicates the mean blood glucose levels (± S.E.M.) of six animals. Significant difference from control in the mean glucose levels at corresponding time intervals: § P < 0.01. Met and Glib denotes metformin HCl and glibenclamide respectively.
Statistical Analysis: Data were expressed as mean ± standard error of mean (S.E.M). Statistical comparisons were performed by one-way ANOVA followed by Dunnett's Multiple Comparison Test (DMCT) and the values were considered statistically significant when P < 0.01. Statistical calculations and the graphs were prepared using G raphPad Prism version 4.00 for W indows (GraphPad Software, San Diego, CA, USA, www.graphpad.com). RESULTS AND DISCUSSIONS The effects of the extracts of C. cordifolia and C. roseus on the FBG, serum TC and serum TG levels were investigated in the control and alloxan-induced diabetic rats using metformin HCl and glibenclamide as standard antidiabetic agents. Effect of C. Cordifolia and C. Roseus on Fasting Blood Glucose Level in Alloxan-induced Diabetic Rats: The mean blood glucose concentration of control and extract of C. cordifolia - and C. roseus -treated animals (after intraperitoneal administration of a single dose) on the 7 th and 14 th day intervals are shown in Fig. 1. Hypoglycemia was observed in animals treated with C. cordifolia and C. roseus extracts. To determine whether there was a statistically significant difference in hypoglycemia achieved by the extracts on the 7 th and 14 th day, one-way ANOVA followed by DM CT was applied and compared with the control group. A significant reduction (P < 0.01) in blood glucose of 47.4, 37.1, 62.7 and 65.5 % was observed on the 7 th day and 59.7, 48.5, 75.9 and 71.4 % was observed on the 14 th day with a single dose of the extracts of C. cordifolia (300 mg/kg), C. roseus (150 mg/kg), metformin HCl (100 mg/kg) and glibenclamide (0.05 mg/kg of body weight), respectively. The leaf juice of C. cordifolia and C. roseus produced reduction in blood glucose of diabetic rats and comparable with that of the standard drugs, metformin HCl and glibenclamide. The results indicate a prolonged action in reduction of blood glucose by
Fig. 2: Effects of C. cordifolia and C. roseus on s e r u m t o t a l c h o l e s t e r o l le v e l s a ft e r intraperitoneal administration in alloxan-induced diabetic rats. Each bar indicates the mean total cholesterol levels (± S.E.M.) of six animals. Significant difference from control in the mean cholesterol levels on corresponding day: § P < 0.01. Met and Glib denotes metformin HCl and glibenclamide respectively. C. roseus and C. cordifolia and the mode of action of the active compound(s) of these plant materials is probably mediated through enhance secretion of insulin from the b-cells of Langerhans o r through extrapancreatic mechanism. However, the blood glucose lowering efficiency of C. cordifolia was found higher than that of C. roseus. W ith respect to the standards, the plant extracts were not found to be, nevertheless, better. Effects of C. Cordifolia and C. Roseus on Total Cholesterol and Triglyceride Levels in Alloxan-
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Res. J. Medicine & Med. Sci., 2(1): 29-34, 2007 induced Diabetic Rats: The mean serum total cholesterol and triglyceride levels of control and extract of C. cordifolia - and C. roseus -treated animals (after intraperitoneal administration of a single dose) on the 14 th day are shown in Fig. 2 and Fig. 3 respectively. Hypolipidemia was observed in animals treated with C. cordifolia and C. roseus extracts. To determine whether there was a statistically significant difference in hypolipidemia achieved by the extracts on the 14 th day, one-way ANOVA followed by DMCT was applied and compared with the control group. A significant reduction (P < 0.01) in serum total cholesterol of 31.7, 43.3, 34.7 and 56.3 % was observed on the 14 th day with a single dose of the extracts of C. cordifolia (300 mg/kg), C. roseus (150 mg/kg), metformin HCl (100 mg/kg) and glibenclamide (0.05 mg/kg of body weight), respectively. Similarly, a significant reduction (P < 0.01) in serum triglycerides of 45.5, 39.4, 48.2 and 56.2 % was observed on the 14 th day with a single dose of the extracts of C. cordifolia (300 mg/kg), C. roseus (150 mg/kg), metformin HCl (100 mg/kg) and glibenclamide (0.05 mg/kg of bo d y weight), respectively. However, the serum cholesterol lowering efficiency of C. roseus was found higher than that of C. cordifolia. On the contrary, C. cordifolia has the higher efficiency of lowering serum triglycerides with respect to C. roseus. Compared to the standards, the plant extracts were found to be similar in activity as antihyperlipidemic in AIDRs. Alloxan, a beta-cytotoxin, induces chemical diabetes in a wide variety of animal species through damage of insulin secreting cell[2 4 ]. It is well established that sulphonylureas like glibenclamide, produce hypoglycemia by increasing the secretion of insulin from the pancreas [2 5 ][2 6 ] and these compounds are active in mild alloxan-induced diabetes whereas they are inactive in intense alloxan diabetes (nearly all b-cells have been destroyed). However, since our results showed that glibenclamide reduced the blood glucose levels in hyperglycemic animals, the state of diabetes is not severe. Alloxan-treated animals receiving the extracts of C. cordifolia and C. roseus showed rapid normalization of blood glucose levels in comparison to the control and this could be due to the possibility that some b-cells are still surviving to exert their insulin releasing effect by C. cordifolia and C. roseus. No histological studies were carried out to prove this and it is not possible to explain the detailed mechanism of antidiabetic action of C. cordifolia and C. roseus. This suggests that the mode of action of the active constituent (s) of C. cordifolia and C. roseus is probably mediated by an enhanced secretion of insulin, like sulphonyl ureas. Metformin is a biguanide drug which inhibits gluconeogenesis in the liver, increases
Fig. 3: Effects of C. cordifolia and C. roseus on serum triglyce ride lev els after intraperitoneal administration in alloxan-induced diabetic rats. Each bar indicates the mean triglyceride levels (± S.E.M .) of six animals. Significant difference from control in the mean triglyceride levels on corresponding day: § P < 0.01. Met and G lib denotes metformin HCl and glibenclamide respectively. low-affinity and high-affinity receptors of insulin, and improves insulin resistance [2 7 ]. It is also possible that the plant extracts might have been taken up by the liver cells and the process of gluconeogenesis was inhibited or these might have improved insulin resistance. However, the possibility that enhanced tissue uptake by C. cordifolia and C. roseus cannot be ruled out. Further experiments are needed to identify the active component(s) of the extracts of C. cordifolia and C. roseus to determine its mechanism of action. In the present study, there was a significant reduction in the levels of total cholesterol and triglycerides. This reduction could have resulted from the antioxidant effect of the ethanol extract of C. roseus, whose phytochemical components include flavonoid, which is known for antioxidant effect[2 8 ].. Through chemical analysis, C. cordifolia is known to be rich in b-carotene, a major precursor of vitamin A from plant sources. Besides, b-carotene, C. cordifolia is a good source of protein, fiber and a moderate source of calcium [2 9 ]. Further investigations are warranted to identify the hypolipidemic active principles in C. cordifolia and to elucidate their mechanism of action. Conclusion: Our results have shown that the ethanol extract of C. cordifolia and C. roseus possess blood g lu c o s e lo w e r in g e ffe c t in a llo x a n -in d u c e d hyperglycemic rats. Thus, the folk use of C. cordifolia and C. roseus for the control of diabetes may be validated by this study. The leaves seem to have a promising value for the development of potent
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Res. J. Medicine & Med. Sci., 2(1): 29-34, 2007 phytomedicine for diabetes. The leaves of C. cordifolia and C. roseus are also useful in reducing the serum total cholesterol and triglyceride levels. Sulphonylureas act by closing the K+-ATP dependent channel, opening Ca2+ channels, increasing intracellular Ca2+, and accelerating insulin secretion [3 0 ]. Sulphonylureas produce hypoglycemia in non-diabetic animals which have retained some normal pancreatic bcells, but sulphonylureas do not significantly reduce hyperglycemia in alloxan-induced diabetic mice because their pancreatic b-cells were damaged seriously by alloxan [3 1 ]. Therefore, it is not surprising that the plant extracts studied in this protocol might have bound to insulin receptors to act as insulin secretagogue. Other probable mechanisms by which the ethanolic extracts of C. cordifolia and C. roseus lowered blood glucose levels in diabetic rats might be by increasing glycogenesis, inhibiting gluconeogenesis in the liver, or inhibiting the absorption of glucose from the intestine. Further comprehensive pharmacological investigations are needed to elucidate the exact mechanism of the antihyperglycemic and antihyperlipidemic effect of C. cordifolia and C. roseus.
8.
REFERENCES
15.
1.
2.
3.
4.
5.
6.
7.
9.
10.
11.
12. 13.
14.
16.
David, M.N., M. James., E.S. Daniel, 1997. The epidemiology of cardiovascular disease in type 2 diabetes mellitus, how sweet it is . . . or is it? Lancet, 350(Suppl. 1): S14-S19. Luzi, L., 1998. Pancreas transplantation and diabetic complications. New England Journal of Medicine, 339: 115-117. Larner, J., 1985. Insulin and oral hypoglycaemic drugs, glucagon. In The Pharmacological Basis of Therapeutics, Eds., Gilman, A.G., L.S. Goodman, T.W . Rall, F. Murad, Macmillan, New York, pp: 1490-1516. Momin, A., 1987. Role of indigenous medicine in primary health care. In Proceedings of First International Seminar on Unani Medicine. New Delhi, India, pp: 54. Stenman, S., P.H. Groop, K. Laakkonen, E. W ahlin-Boll, E. Melander, A., 1990. Relationship between sulfonylurea dose and metabolic effect. Diabetes, 39: 108A. Spiller, H.A., T.S. Sawyer, 2006. Toxicology of oral antidiabetic medications. American Journal of Health-System Pharmacy, 63: 929-938. W HO Expert Committee on Diabetes mellitus, 1980. Second report, Technical report series 646. W orld Health Organization, Geneva, pp: 1-80.
17.
18.
19.
20. 21.
22.
23.
33
Holman, R.R., R.C. Turner, 1991. O ral agents and insulin in the treatment of NIDDM. In Textbook of Diabetes. Blackwell, Eds., Pickup, S. and G. W illiams, Oxford, pp: 467-469. Kameswara Rao, B., R. Giri, M.M . Kesavalu, , Ch. Appa Rao, 1997. Herbal medicine in the management of diabetes mellitus. Manphar Vaidhya Patrika 1: 33-35. Mukherjee, S.K ., 1981. Indigenous drugs in diabetes mellitus. Journal of the Diabetes Association of India 21, 97-106. Oliver-Bever, B., 1986. Oral hypoglycemic action of medicinal plants in tropical W est Africa. Cambridge University Press. Rai, M.K., 1995. A review on some antidiabetic plants of India. Ancient Science of Life, 14: 42-54. Riyad, A., S. Abdul-Ghani Abdul-Salam, S.M. Suleiman, 1988. Effect of fenugreek and lupine seeds on the development of experimental diabetes in rats. Planta Medica, 54: 286. Tarfa, S.P., P.K. Joseph, K.T. Augusti, 1988. Preliminary studies on the antidiabetic effects of cabbage (Brassia var. Capiata L.) oil on streptozotocin diabetic rats. Current Science, 57: 31-32. Sharma, S.R., S.K. Dwivewdi, D. Swarup, 1996. Indian Journal of Experimental Biology, 34: 372. Balassa, E.O., 1985. Hyperlipidaemia in diabetes. Medicographia, 7: 11-14. Linney, G. 1986. Coccinia grandis (L.) Voight: A new cucurbitaceous weed in Hawai'i. Hawaii Botanical Society Newsletter, 25: 3-5. Chopra, R.N., S.L. Nayar, I.C. Chopra, 1986. Glossary of Indian Medicinal Plants (Including the Supplement). Council of Scientific and Industrial Research, New Delhi. Azad Khan, A.K., S. Akhtar, H. Mahtab, 1979. Coccinia indica in the treatment of patients with diabetes mellitus. Bangladesh Medical Research Council Bulletin, 5: 60-66. Cowley, R.C., F.C. Bennett, 1928. Vinca rosea. Australian Journal of Pharmacy,9: 61. Pillay, P.P., C.P.M. Nair, T.N. Santi Kumari, 1959. Lochnera rosea as a potential source of hypotensive and other remedies. Bulletin of Research Institute of the University of Kerala, 1: 51-54. Nammi, S., M.K. Boini, S. Lodagala, R.B.S. Behara, 2003. T he juice of fresh leaves of Catharanthus roseus Linn. reduces blood glucose in normal and alloxan diabetic rabbits. BM C complementary and Alternative Medicine, 2: 3-4. Antia, B.S., J.E. Okokon, 2005. Effect of leaf juice of Catharanthus roseus Linn on cholesterol, triglyceride and lipoproteins levels in normal rats. Indian Journal of Pharmacology, 37: 401-402.
Res. J. Medicine & Med. Sci., 2(1): 29-34, 2007 24. Rerup, C.C., 1970. Drugs producing diabetes through damage of insulin secreting cells. Pharmacological Reviews, 22: 485-520. 25. Yallow, R.S., H.Black, M . Villazan, S.A. Berson, 1960. Comparison of plasma insulin levels following administration of tolbutamide and glucose. Diabetes, 9: 356-362. 26. Grodsky, G.M., G.H. Epstein, R. Fanska, J.H. Karam, , 1971. Pancreatic action of sulphonylureas. Federation Proceedings, 36: 2719-2728. 27. Lord, J.M., T.W . Atkins, C.J. Bailey, 1983. Effect of metformin on hepatocyte insulin receptor binding in normal, streptozotocin diabetic and g e ne tic a lly o b e s e d ia b e tic (o b /o b ) m ic e . Diabetologia, 25: 108-113. 28. Afanas'ev, I.B., E.A. Ostrachovitch, N.E. Abramova, L.G . K o rkina, 1 9 9 5. D ifferent antioxidant activities of biflavonoid rutin in normal a n d ir o n o v e rlo a d i n g r a ts . B io c h e m ic a l Pharmacology, 80: 627-635.
29. W asantwisut, E., T. Viriyapanich, 2003. Ivy gourd (Coccinia grandis Voigt, Coccinia cordifolia, Coccinia indica) in human nutrition and traditional applications. W orld Review on Nutrition and Diet, 91: 60-66. 30. Fuhlendorff, J., P. Rorsman, H. Kofod, C.L. Brand, , B. Rolin, P. MacKay, R. Shymko, R.D. Carr, 1998. Stimulation of insulin release by repaglinide and glibenclamide involves both common and distinct processes. Diabetes ,47: 345-351. 31. Goth, M.D., 1978. Medical Pharmacology. Mosby Company.
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