THE EVALUATION OF THE ANTIDIABETIC EFFECT OF COCCINIA INDICA LEAVES EXTRACT IN STREPTOZOTOCIN INDUCED DIABETES IN RATS

Transcription

1 Journal of Cell and Tissue Research Vol. 14(2) (2014) (Available online at ISSN: ; E-ISSN: Original Article THE EVALUATION OF THE ANTIDIABETIC EFFECT OF COCCINIA INDICA LEAVES EXTRACT IN STREPTOZOTOCIN INDUCED DIABETES IN RATS? RAO, S., RAO, S., MANJUNATHA, K. P., SATYANARAYAN, M. L., BYREGOWDA, S. M., RAMACHANDRA, G., SUDHARANI, R., SHETTAR, M., MUNDAS, S. AND KALGE, R. Department of Veterinary Pathology, Veterinary College, Hebbal, KVAFSU, Bangalore. E. Mail : drraoiahvb@gmail.com, Cell: Received: March 8, 2014; Accepted: March 31, 2014 Abstract: An alcoholic extract of coccinia indica leaves (200 mg/kg b w / day) was tested for its biochemical and pathomorphological effects in streptozotocin induced diabetes in rats. Experimental diabetes mellitus was induced in wister female rats with single buffer intraperitonial at 45 mg/kg. The rats were separated into five groups as follows: group 1 served as a normal control, grpoup-2 served as a diabetic control group-3 serves as a glibenclamide control, group-4 and group-5 serves as a diabetic rats administered with 200mg/kg Coccinia indica extract and Coccinia indica extract along with half dose of glibenclamide 300 ìg /kg body weight once daily for 90 days respectively. In diabetic animals there was a increase in the mean (± SE) serum levels of glucose, cholesterol, triglycerides, ALT and AST and a significant reduction in body weight and serum insulin. Coccinia indica leaves extract alone and in combination with glibenclamide half dose treated rats showed significant (Pd 0.001) reduction in the levels of serum glucose, cholesterol, triglycerides, ALT and AST from Day 3 post treatment till the end of the study with significant improvement in body weight and serum insulin levels (Pd 0.001) in diabetic animals. Coccinia indica leaves extract alone and with glibenclamide half dose also alleviated the damage caused by STZ morphologically in beta cells of islets of Langerhans and hepatocytes. However the combined treatment of Coccinia indica with glibenclamide half dose revealed a very good antidiabetic effect with reference to improvement in insulin level and beta cell number which indicated a synergistic effect between Coccinia indica and glibenclamide half dose. Key words: Diabetes, Coccinia indica, INTRODUCTION Diabetes is the most common endocrine disorder characterized by hyperglycemia and a predisposition to chronic complications like retinopathy, nephropathy, neuropathy and macrovascular disease [1]. It is chrome disease caused by inherited or acquired deficiency in production of insulin by the pancreas of by ineffectiveness of insulin produced, such a deficiency results in increased concentration of glucose in the blood, which in turn damages many of the body s systems in particular the blood vessels and nerves. According to WHO projections, the prevalence of diabetes is likely to increase by 35%. Currently, there are over 150 million diabetic patients worldwide and this is likely to increase to 300 million or more by the year Statistical projection about India suggests that the number of diabetics will rise from 15 million in 1995 to 57 million in the year 2025, the highest number of diabetics in the world [2]. 4259

2 J. Cell Tissue Research Reasons for this rise include increase in sedentary lifestyle, consumption of energy-rich diet, obesity, higher life span, etc. Other regions with greatest number of diabetics are Asia and Africa, where diabetes mellitus rates could rise to twofold to threefold than the present rates [3]. The world health organization (WHO) has declared India as the country with the largest number of diabetic subjects in the world. Management of diabetes in humans with conventional hypolycemic drugs and hormonal supplementation has been proved to be expensive and associated with secondary complications. Recently, anti-diabetic herbal therapy has become very popular in conjunction with conventional anti-diabetic treatment which is cost effective with no side effects. Coccinia indica (Cucurbitaceae) is found in warmer and humid part of India. It is also known as Kundru, Bimbi. The fruits, leaves, roots are used for medicinal purpose by folklore like fresh juice of roots to treat diabetes, tincture of leaves to treat gonorrhoea and the paste of leaves to the skin diseases. Dried bark has been reported to be a good cathartic, leaves and stem as antispasmodic and expectorant and green fruits to cure sores on the tongue [4]. The phytochemicals constituents of this plant include saponin, flavonoid, glycosides and polysaccharides, xyloglucan, taraxerol, carotenoids, cryptoxanthin, presence of these phytochemicals constituent help in either repair or regeneration of islets of Langerhans in diabetes. To establish a systematic scientific support over the effect of Coccinia indica on the diabetic pathology, the study was designed and the results were compared with glibenclamide, a sulphonylurea antidiabetic drug. MATERIAL AND METHODS Animals: Normal adult healthy female Wistar albino rats weighing g were procured from RRL Instruments and Animals supplier, Bangalore for the study purpose. They were maintained under standard laboratory conditions and offered ad lib of standard commercial rat feed (Amruth Feeds, Bangalore) and clean drinking water. The experiment was carried out for a period of 90 days upon permission from Institutional Animal Ethics Committee. Streptozotocin: To induce diabetes in rats, streptozotocin (Sigma Chemicals, St.Louis, USA) was used intraperitonially in ice-cold citrate buffer (ph ) at the dose of 45 mg/kg. Coccinia indica: The alcoholic extract of Coccinia indica leaves was procured from PLANTEX, Vijayawada, India. The extract was administered at the dose rate of 200mg/kg body weight as aqueous solution. Glibenclamide solution: Glibenclamide (Daonil, 5 mg) an oral hypoglycaemic drug was administered orally at a dose of 600 µg / kg. Administration of plant extract and glibenclamide: The plant extract, glibenclamide and combined plant extract with glibenclamide were administered orally to their respective groups by using clean rat gavaging needle attached to an appropriate disposable syringe every day for a period of 90 days. Experimental design: The rats were divided into five different groups of twelve animals each based on body weight. Care was taken to maintain the intra group weight variation to be less than 25g and intergroup weight variation by 30 g. Group-I was normal control, group-ii- diabetic control, group-iii was diabetic animals treated with glibenclamide (600 µg/ kg b w), group-iv was diabetic rats supplemented with alcoholic extract of Coccinia indica leaves (200mg/kg b w) and the group-v was diabetic rats supplemented with alcoholic extract of Coccinia indica leaves (200mg/kg b w) and glibenclamide half dose (300 µg/kg b w). Experimental induction of diabetes: Freshly prepared streptozotocin at the dose rate of 45 mg/kg intraperitonially was injected to the rats fasted for 16 hours [5]. The normal control animals received citrate buffer alone. The diabetic state was confirmed by estimating the serum glucose level at 72 hours post STZ injection using Span Diagnostic kit with Semi- Automatic Biochemical Analyser (ARTOS, Bangalore). The animals that showed the serum glucose level above 200 mg/dl were considered diabetic and selected for the study. Rats of all the groups were observed clinically for the feed and water intake, general behaviour, alertness, urine output, diarrhoea and for the development of clinical symptoms and recorded. Collection of serum samples: About 2 ml of blood from the retro-orbital plexus of the rats of all the 4260

3 Rao et al. groups was collected under light ether anaesthesia separately in clean test tubes at different time intervals of the study such as 3 rd, 15 th, 30 th, 45 th and 90 th day post STZ injection. The collected blood was allowed to clot for 30 min and then centrifuged at 3000 rpm for 10 min. The serum samples collected at various intervals were subjected to biochemical estimation of glucose, cholesterol, triglycerides, ALT and AST using semi-automatic biochemical analyzer with commercial biochemical kits. For the estimation of serum insulin concentrations, radio-immunoassay was performed using iodine labelled insulin assay kit (RIAK-1) obtained from Board of Radiation and Isotope Technology (BRIT), BARC, Mumbai, India. Collection of tissue samples: To study the progressive effects of the treatments given to different groups, two rats from each group were sacrificed under light ether anaesthesia on Day 15 and 30, 6 animals on Day 45 and remaining two rats on Day 90 of the experiment. Sacrificed animals were subjected for detailed post mortem examination and gross change if any, were recorded. Further, representative tissue samples from pancreas, liver, kidney, lungs, heart, intestine, brain and muscle were collected in 10 % neutral buffered formalin (NBF) for the pathomorphological evaluation. Statistical analysis: Statistical analysis was performed using the statistical software Graph pad Prism, version 5. Mean values and standard error of mean were calculated and all values were expressed as Mean (± SE). The data were analysed by Two Way ANOVA. RESULTS AND DISCUSSION The groups IV and V rats in the present study following induction of diabetes with STZ and treatment with Coccinia indica alone and in combination with gibenclamide revealed a significant increase in the body weight compared to diabetic rats from 15 th day onwards and on 90 th day it was comparable to that of control rats and did not differ significantly from those of glibenclamide alone and other treatment groups (Table A). The improvement in the body weight by feeding Coccinia indica also has been reported by many earlier workers [5.6].The increase in the body weight could be attributed to the alleviation of diabetic symptoms and hyperglycemia by insulinomimetic effect of triterpene of C. indica [6] and also to insulin stimulated glucose transport and anabolic effects of insulin [7] (Table 1). In the present study the mean serum glucose values of group IV and group V reduced significantly and progressively in comparison with diabetic group from 15 th day onwards. On 90 th day the mean glucose values were comparable between groups IV and V but were significantly higher compared to control group and glibenclamide alone group (Table 2). Presence of alkaloids, tannin, saponins, flavonoids and pectin of C. indica has been shown to improve secretion of insulin by β-cells by promoting their regeneration and there by controlling hyperglycaemia [8]. Coccinia indica has been rated A while assessing the quality of the herbs for glycemic control. A maximum decline in the blood glucose level in STZ induced diabetes has been recorded by Mishra et al. [9]. The hypoglycaemic activity of leaf extract of C. indica which could be attributed to the insulinomimetic effect of triterpene of the plant which act like insulin by decreasing the elevated enzymes, glucose 6-phosphatase and lactate dehydogenase in the glycolytic pathway and restoring the lipoprotein lipase activity in the lipolytic pathway with the control of hyperglycaemia in diabetes [5] (Table 3). The serum cholesterol and triglyceride levels showed a significant (Pd 0.001) decreasing trend from 15 th day onwards in the present study in Coccinia indica alone (group IV) and Coccinia indica in combination with glibenclamide half dose (group V) groups in comparison with diabetic control group throughout the experiment. On 90 th day the mean serum cholesterol level was comparable with that of control group and did not differ between group IV and group V though numerically the values were lesser in combined group than Coccinia alone group. This indicated that there was no synergistic effect between Coccinia indica and glibenclamide in alleviating the hypercholestrolaemia in induced diabetes. The triglyceride level in combined group on 90 th day was significantly lesser compared to C. indica alone group which indicated that combination of glibenclamide with herbal extract has beneficial effect in controlling the triglyceride level in diabetes and Coccinia and glibenclamide have synergistic effect which could be attributed to glibenclamide induced alleviation of hypertriglyceridemia by the acute reduction in triglyceride of intestinal origin [10] in addition to the hypolidaemic effect of C. indica (Table 4). 4261

4 J. Cell Tissue Research Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig

5 Rao et al. Fig. 7 Fig. 8 The ALT and AST levels in group IV and group V were significantly improved in comparison with that of diabetic control. On 90 th day the AST values did not differ between groups IV and V and were comparable to that of glibenclamide group. However the values were significantly higher compared to those of normal control rats. The ALT values on 90 th day in group IV was comparable with that of glibenclamide group but the mean value in group V was significantly reduced which indicated synergistic effect and was statistic-ally comparable with that of control group. The improvement could be attributed to the combined effect of C. indica with glibenclamide for their hypoglycaemic, insulin secretogogue and antioxidant activities (Table 5). Serum insulin values in the present investigation showed a progressive increase in C. indica alone and in combination with glibenclamide treated animals from 15 th day post treatment onwards in comparison with those of diabetic control rats. The mean insulin value on 90 th day was significantly higher in group V compared to group IV and group III. This indicated a synergistic effect in elevating the insulin level between C. indica and glibenclamide. Both C. indica and glibenclamide alone elevated the insulin level only moderately but in combination elevated the insulin level significantly which was comparable to that of control group (Table 6). An another effect of C. indica is that it preserves the cells of islets of Langerhans of beta cell function which results in a significant increase in insulin activity [10]. The antioxidant property of C. indica which scavenge the free radicals produced by oxidative stress during STZ diabetes also could prevent beta cell damage and stimulate the secretion of insulin from beta cells [11]. The improvement in insulin level in the present study is well supported by an increase in the number of beta cells microscopically. Grossly, a significantly appreciable improvement was observed in the appearance of pancreas and liver in both IV and V groups which could be attributed to Explanation of figures Fig. 1: Section of pancreas of normal control showing a normal islet with round to oval shape and compact arrangement of beta cells at the centre and alpha cells at the periphery.h&e X 200. Fig. 2: Pancreas from normal control animal showing large sized islet with more number of beta cells and compact arrangement of beta cells at the centre and alpha cells at the periphery.gomori s X 200. Fig. 3: Section of liver from control rat on 45th day showing central vein and well formed hepatic cords with normal appearing hepatocytes. H&E X 200. Fig. 4: Pancreas of diabetic control animal showing loss of normal architecture, degeneration and necrosis of islet cells. H&E X 200. Fig. 5: On Day 90 diabetic rat treated with Coccinia indica showing improvement in the overall architecture of islet of Langerhans however, with persistence of occasional beta cells with STZ effect. H&E X 200. Fig. 6: Pancreas from a diabetic rat treated with combination of Coccinia indica and glibenclamide showing large and well formed islet on Day 90 of the treatment. H&E X 200. Fig. 7:Pancreas from a diabetic rat treated with combination of Coccinia indica and glibenclamide showing increase in number of beta cells on Day 90 of the study. Note distribution of beta cells at the centre. Gomori s X 200. Fig. 8: Normal appearance of liver from a diabetic rat treated with the combination of Coccinia indica and glibenclamide on 90th day of treatment H&E X

6 J. Cell Tissue Research Group I ±1.53 a ±1.94 a ±1.62 a ±1.48 a ±3.00 a Group II ±2.44 b ±2.00 b ±2.40 b ±5.43 b ±9.00 b Group III ±2.39 b ±3.97 b ±3.21 a ±2.99 a ±3.00 a Group IV ±2.50 b ±3.77 b ±3.57 a ±3.88 a ±2.00 a Group V ±2.10 b ±2.36 b ±2.88 a ±3.96 a ±4.50 a Table A: The mean (±SE) body weight in (gm) values of different Group I 89.25±2.86 c 86.00±4.21 d 84.20±5.67 e 85.87±4.22 e 77.50±3.50 d Group II ±40.52 ab ±21.74 a ±48.80 a ±45.65 a ±28.50 a Group III ±5.57 b ±12.73 c ±15.37 bcd ±13.39 bcd ±11.60 c Group IV ± ab ±17.18 bc ±21.06 bc ±19.50 bc ±15.40 bc Group V ±15.28 a ±16.95 c ±7.27 cd ±3.48 cd ±5.00 bc Group I 80.88±3.58 b 81.15±3.63 c 84.65±5.52 c 81.68±3.07 c 79.70±2.39 b Group II ±7.61 a ±5.28 a ±6.69 a ±6.64 a ±12.40 a Group III ±7.65 a ±5.36 b ±4.01 b ±4.88 b 90.20±8.00 b Group IV ±4.81 a ±3.77 b ±3.16 b ±3.20 b ±4.75 b Group V ±7.19 a ±5.95 b ±5.59 b ±5.65 b 88.85±0.25 b Table 1: The mean (±SE) Serum Glucose (mg/dl) values of different groups at different intervals of time Table 2: The mean (±SE) Serum Cholesterol (mg/dl) values of different groups at different intervals of time Group I 95.95±0.99 c 96.70±1.07 d 96.03±1.13 f 96.20±1.23 g 97.95±1.45 d Group II ±1.34 b ±2.13 a ±2.67 a ±2.39 a ±0.10 a Group III ±0.93 a ±1.96 b ±2.58 d ±3.35 ef ±6.50 bcd Group IV ±1.05 a ±0.64 b ±1.47 b ±1.14 b ±0.90 b Group V ±1.06 ab ±0.99 c ±1.69 de ±0.94 d ±1.95 d Table 3: The mean (±SE) Triglyceride values of different Group I 53.07±1.64 c 51.81±1.70 b 51.52±1.90 c 52.61±1.83 c 56.40±2.10 e Group II ±7.34 b ±8.74 a ±13.60 a ±21.51 a ±6.44 a Group III ±3.55 a ±2.00 bc ±3.54 b 90.11±4.22 b 95.40±3.000 b Group IV ±1.59 ab ±8.57 b ±2.46 b ±2.82 bi 94.45±1.95 b Group V ±1.93 a ±1.76 c 94.95±1.90 b 86.36±1.53 bc 76.39±1.79 dc Table 4: The mean (± SE) (ALT) (IU/L) values of different Group I 67.06±2.24 b 67.91±2.60 c 67.53±1.80 c 69.78±2.22 dc 71.70±3.10 dc Group II ±10.20 a ±9.23 a ±21.23 ac ±23.35 a ±5.35 a Group III ±3.05 a ±2.13 b ±2.24 b 94.73±2.22 bcd 94.15±2.25 b Group IV ±2.87 a ±2.15 b ±1.68 b ±2.48 b 90.73±2.48 b Group V ±2.54 a ±1.86 b ±1.58 b 87.60±2.37 dc 88.80±2.40 b Group I 53.85±3.21 a 52.85±3.16 a 54.15±2.78 a 52.08±5.35 a 53.95±4.51 a Group II 17.58±1.70 b 15.66±0.87 d 13.61±1.37 c 14.05±1.81 d 12.76±0.94 c Group III 15.42±2.06 b 18.44±2.41 bcd 25.73±2.07 b 31.01±2.91 bc 35.19±5.63 b Group IV 17.96±1.36 b 19.87±1.99 bc 21.76±2.60 b 25.04±2.53 c 32.03±9.15 b Group V 17.17±1.34 b 20.52±0.92 bc 24.62±2.08 b 35.38±2.13 bc 48.10±7.29 b Table 5: The mean (±SE) (AST) (IU/L) values of different groups at different intervals of time Table 6: The mean (±SE) RIA Insulin μu/ ml values off different 4264

7 Rao et al. the alleviation of the diabetic effect by improvement in the insulin level.microscopically, C. indica treatment for a period of 90 days showed an improvement in the architecture of pancreas, liver and other organs (Figs. 6,8). In pancreas there was an improvement in the number, size and shape of the islets, increase in the number of granulated β-cells and attainment of almost normal distribution of alpha and beta cells. These changes were progressive in nature from 15 th day to 90 day post treatment (Fig. 6) The improvement in architecture of pancreatic islets could be attributed to the effect of antioxidant property of the C. indica which prevented damage to the beta cells and resulted in promotion of regeneration of β-cell subsets or repair of damaged cells, there by restored insulin concentration to almost normal basal level [6,13] The improvement in the architecture of the pancreas in combination group with Coccinia indica and glibenclamide was better than that of Coccinia indica alone group which could be attributed to the combined effect in regenerating beta cells and repairing the damaged cells by the antioxidant effect. This result was well substantiated by the increase in the number of beta cells by Gomori stain in the combined group (Fig. 7). Microscopically the STZ induced liver damage in the form of necrosis and vacuolar degeneration persisted in a subtle manner on Day 15 in the present study, but the liver attained almost normal architecture which was comparable to that of control liver by 90 th day post-treatment in both groups IV and V (Fig. 8). successfully as an alternative treatment in the management of diabetes. REFERENCES [1] Chandra, A., Singh, R.K. and Tewari, L.: SFRR-India. Bull., 3: (2004) [2] Satyanarayana, T.: Pharmacog, Mag., 2: (2006). [3] Eidi, A.: Phytomedicine., 13: (2006). [4] Chandrasekar, B., Bajpai, M.B. and Mukherjee, S.K.: Indian J. Exp. Biol., 28 (7): (1990) [5] Kuriyan, Rajendran, R., Bantwal, G. and Kurpad, A. V.: Diabetes Care., 31(2): (2008). [6] Rafiq, K., Sherajee, S.J., Nishiyama, A., Sufiun, M.A. and Mostofa, M.: Afr. J. Pharm. Pharmaco., 3(12): (2009). [7] Kanetkar, P., Singhal,R. and Kamat, M.: J. Clin. Biochem. Nut., 41: (2007). [8] Ramakrishnan, M., Bhuvaneshwari, R., Duraipandiyan, V. and Dhandapani, R.: Indian J. Natural Products and Resour., 2(3): (2011). [9] Mishra, A., Jaitly. A.K. and Srivastava, A.K.: Indian J. Sci. Technol., 2(9): (2009). [10] Yadav, G., Mishra, A. and Tiwari, A.: Int. J. Pharma Res. Develop., 2(4): (2010). [11] Kamiya, K., Hamabe, W., Harada, S., Murakami, R., Tokuyama, S. and Satake, T.: Biol. Pharma. Bull., 31: (2008). [12] Venkateswaran, S. and Pari, L.: J. Ethnopharmacol., 84: (2003). [13] Singh, P.K., Baxi, D.B., Mukherjee, R. and Ramachandran, A.V.: J. Herbal Med. Toxicology., 4(2): (2010). CONCLUSION Diabetes mellitus can be effectively induced by using streptozotocin at the dose rate of 45 mg/kg intraperitonially in laboratory rats. C. indica along with half dose of glibenclamide has a synergistic effect in improving serum levels of glucose, cholesterol, triglycerides, ALT and AST and a significant elevation in serum insulin with improvement in the number of beta cells in bringing about regeneration of beta cells and in increasing the insulin production and secretion. Lastly the present study high lights that the indigenous medicinal plants can be used 4265