Page1391 Indo American Journal of Pharmaceutical Research, 2014 ISSN NO: 2231-6876 Journal home page: http:///index.php/en/ INDO AMERICAN JOURNAL OF PHARMACEUTICAL RESEARCH ANTIDIABETIC EFFECT OF HYDROALCOHOLIC COMBINED PLANT EXTRACT OF PORTULACA OLERACEA AND CARALLUMA ATTENUATA IN STREPTOZOTOCIN INDUCED DIABETIC RATS Virendra Singh 1 and Mohan Lal Kori 2 1 Research Scholar, Institute of Pharmaceutical Science and Research Centre, Bhagwant University, Sikar Road, Ajmer, Rajasthan, India- 305004 2 Vedica College of B. Pharmacy, Constituent Institute of R.K.D.F. University, Bhopal, Madhya Pradesh, India- 462033 ARTICLE INFO Article history Received 02/03/2014 Available online 25/03/2014 Keywords Antidiabetic, Streptozotocin, Portulaca Oleracea and Caralluma Attenuata. ABSTRACT The objective of present study is to evaluate the antidiabetic activity of hydroalcoholic combined plant extract of Portulaca oleracea and Caralluma attenuata in streptozotocin induced diabetic wistar albino rats. Hypoglycemic agents from natural and synthetic sources are available for treatment of diabetes. Indian medicinal plants have been found to be useful to successfully manage diabetes. The effect of hydroalcoholic combined plant extract containing seeds of Portulaca oleracea, and whole plant of Caralluma attenuata was investigated in streptozotocin induced diabetic rats. Diabetes was induced in rats by single intraperitoneal injection of streptozotocin (50 mg/kg b.wt.). After 72 hrs rats with marked hyperglycaemia (blood glucose above 220 mg/dl) were selected and used for the study. Antidiabetic effect was evaluated by oral administration of hydroalcoholic combined plant extract at doses of 100 and 200 mg/kg b.wt. for 28 days. Streptozotocin induced diabetic rats showed marked hyperglycemia, hypertriglyceridemia and hypercholesterolemia. Significant decrease in body weight and liver glycogen levels were observed with diabetic control, which was partially restored upon administration of hydroalcoholic combined plant extract. The treatment with hydroalcoholic combined plant extract at the dose of 100 mg/kg and 200 mg/kg significantly improve the alterations in blood glucose levels, serum triglyceride, serum cholesterol, liver glycogen, glycosylated haemoglobin and body weight in streptozotocin induced diabetic rats. In conclusion, the present study indicates that hydroalcoholic combined plant extract which is a mixture of hydroalcoholic extract of Portulaca oleracea and Caralluma attenuata have significant antidiabetic activity to treat diabetes mellitus and its complications. Corresponding author Dr. Mohan Lal Kori Vedica College of B. Pharmacy, Constituent Institute of R.K.D.F. University, Bhopal, Madhya Pradesh, India- 462033 +91-9893968611 mlkori.research@gmail.com Please cite this article in press as Mohan Lal Kori et al. Antidiabetic Effect of Hydroalcoholic Combined Plant Extract of Portulaca oleracea and Caralluma attenuata in Streptozotocin Induced Diabetic Rats. Indo American Journal of Pharm Research.2014:4(03). Copy right 2014 This is an Open Access article distributed under the terms of the Indo American journal of Pharmaceutical Research, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Page1392 INTRODUCTION Diabetes mellitus is a metabolic disorder caused by complete or relative insufficiency of insulin secretion and/or insulin action [1]. Hyperglycemia is the main cause of complications related to coronary artery disease, cerebrovascular disease, renal failure, blindness, limb amputation, neurological complications and premature death [2]. Hyperlipidemia contributes to the development of cardiovascular complications related to diabetes [3]. Biguanides, sulphonylureas and thiozolidinediones became available for treatment of type 2 diabetes and have been effective hypoglycemic agents. However they cause some side effects. Hence search for new drugs without adverse effects is going on in several laboratories around the world [4]. Insulin-dependent diabetes mellitus or type 1 diabetes is an autoimmune disorder caused by destruction of insulin producing β-cells when auto aggressive T-lymphocytes infiltrate the pancreas. This leads to hypoinsulinaemia and thus hyperglycemia [5]. Hyperglycemic condition causes increased glycosylation leading to biochemical and morphological abnormalities due to altered protein structure which over a period of time develops diabetic complications such as nephropathy, retinopathy, neuropathy, and cardiomyopathy [6]. Traditional medicines derived mainly from plants play major role in the management of diabetes mellitus [7]. World Health Organization (WHO) has recommended the evaluation of traditional plant treatments for diabetes as they are effective, non-toxic, with less or no side effects and are considered to be excellent candidates for oral therapy [8]. Recently, many medicinal plants possessing experimental and clinical antidiabetic activity that have been used in traditional systems of medicine [9]. Diabetes mellitus has been considered a major health problem in the world today. Diabetes mellitus is a metabolic disorder of carbohydrate, fat and protein metabolism characterized by elevation of both fasting and postprandial blood glucose levels. Although different types of synthetic oral hypoglycemic agents and insulin are available for the treatment of diabetes mellitus, insulin cannot be taken orally and the synthetic agents in use can produce serious side effects and toxicity [10]. Hence, the demand for safer and more effective oral hypoglycemic agents is on the rise. In many parts of the world, traditional medicinal plants have been used for the treatment of diabetes and therein exists a hidden wealth of potentially useful natural products for diabetes control [11]. The aim of therapy in type II diabetes has always been to reduce hyperglycaemia using several approches: Sulphonylureas (increase insulin release from pancreatic islets); Metformin (reduces hepatic glucose production); Thiazolidines (enhance insulin actions); α-glucosidase inhibitors (interfere with gut glucose absorption) and Insulin (Suppresses glucose production and augments glucose utilization). These therapeutic approaches have limited efficacy and tolerability; and most have tendency to cause weight gain. Also, several of these cause hypoglycaemia and very few address underlying defects such as obesity and insulin resistance. Sulphonylurea use causes secondary failure or refractoriness. Therefore, there is need for newer therapeutic approaches. Medicinal plants play important roles in the management of type II diabetes especially in resource-limited countries. In India, herbal medicines including polyherbal therapy is widely practiced. The combination of various types of agents from different plant sources could have synergistic, potentiative, antagonistic pharmacological and therapeutic effects with minimum side effects [12]. Portulaca oleracea L., commonly known as purslane, belongs to the family Portulacaceae, is a warm climate, annual, green herb, with branched and succulent stems which are decumbent near the base and ascending near the top to a height of 15-30 cm. The flowers are small, yellow, and sessile in clusters of 3-5 on the forks and tips of the branches, opening in the morning only. Seeds are formed in a tiny pod, which opens when the seeds are mature. The seeds are orbicular and 0.5 mm in diameter. Purslane is considered helpful medicinally in treating and/or preventing a wide range of conditions, including scurvy, cataracts, heart disease, asthma, cardiac arrhythmia, depression, gingivitis, multiple sclerosis, and psoriasis, as well as boosting the immune system [13, 14]. Caralluma attenuata W., also known as Caralluma fimbriata, belongs to the family Asclepiadaceae. C. attenuata is known as Dugdha in hindi which is a thick, succulent perennial herb growing wild in dry hill slope regions of Rajasthan, India. It is eaten raw as a cure for diabetes and the juice of the plant along with black pepper is suggested in the treatment of migraine [15]. The purpose of this research was to evaluate the antidiabetic activity of hydroalcoholic combined plant extract of Portulaca oleracea and Caralluma attenuata by estimation of blood glucose levels, body weight measurements and plasma lipid profile such as total cholesterol, triglyceride, high density lipoprotein, very low density lipoprotein and low density lipoprotein in streptozotocin induced diabetic rats. MATERIALS AND METHODS Collection of plant material Seeds of Portulaca oleracea and whole plant of Caralluma attenuata were collected from the Jadaav Nursery, Udaipur and authenticated by Botanist of Rajasthan College of Agriculture, Udaipur, Rajasthan. Plants were preserved in herbarium of the institution. Chemicals Streptozotocin was obtained from Sisco Pharmaceuticals Limited, Mumbai. Glibenclamide was obtained as a gift sample from Zydus Cadila, Ahmedabad. All the other chemicals used for the present study were of analytical grade. All the diagnostic kits were procured from Lab-care diagnostics Ltd., India. Preparation of extract The plants materials were air dried under shade at 25±2 C and then pulverized by a mechanical grinder and sieved through 120 meshes separately. Each plants material was defatted with petroleum ether to remove all the fatty substances. The defatted material was further extracted with 50% aqueous alcohol. Both extracts were concentrated to dry mass using rotary evaporator under controlled temperature (25-40 C). Dried powder of both extract of Portulaca oleracea and Caralluma attenuata were mixed in the equal ratio.
Page1393 Diabetic Screening Methods Experimental animals Wistar albino rats (150 200 gm) of either sex were obtained from the Geetanjali Medical College and Hospital, Udaipur. They were kept in the departmental animal house at temperature 26 2 C and relative humidity 44-56%, light and dark cycles of 10 and 14 hrs respectively for one week before and during the experiments. Animals were provided with standard rodent pellet diet and the food was withdrawn 18-24 hrs before the experiment though water was allowed ad libitum. All studies were performed in accordance with the guide for the care and use of laboratory animals, as adopted and promulgated by the Animal Care committee, CPCSEA, India. Preparation of doses Suspension of finally powdered plant material were prepared by using Tween 80 (2% v/v) as suspending agent and administered orally at the doses of 100 and 200 mg/kg. Acute toxicity studies Acute oral toxicity study was performed as per Organization for Economic Co-operation and Development (OECD) guidelines 420. Wistar albino rats of either sex (150-200gm) were used for the study and administered a limit dose of 2000 and 5000 mg/kg of the hydroalcoholic combined plant extract and animals were observed for mortality and clinical signs for the first hour, then hourly for 3 hrs and finally periodically until 48 hrs. All of the experimental animals were maintained under close observation for 14 days, and the number of rats that died within the study period was noted. The LD50 was predicted to be above 2000 or 5000 mg/kg, if three or more rats survived [16]. Behavioural and neurological changes such as tremors, convulsions, salivation, diarrhoea, sleep, lacrimation and feed behaviour in drug treated rats were observed for sign of acute toxicity. Experimental induction of diabetes in rats Diabetes was induced by using streptozotocin as diabetogenic agent. Streptozotocin (50 mg/kg b.wt.) was dissolved in ice cold citrate buffer (ph 4.3) immediately before use. The solution was injected intraperitoneally in the dose of 50 mg/kg b.wt. in rats. 5% glucose solution was administered orally for 24 hrs to prevent mortality due to initial hypoglycemia induced by streptozotocin. After 72 hrs of streptozotocin injection, blood glucose levels were tested using commercially available kit (Accu-Chek Active Test Meter).The rats with blood glucose level above 220 mg/dl were considered to be diabetic and were used in the experiment. Experimental design The antidiabetic effect of hydroalcoholic combined plant extract of P. oleracea and C. attenuata were studied in streptozotocin induced diabetic rats. Rats were divided into five groups of six rats in each group. I Normal Control; rats were treated with 2% v/v Tween 80 (2 ml/kg/day, p.o.) II Diabetic Control; rats were treated with 2% v/v Tween 80 (2 ml/kg/day, p.o.) III Diabetic rats treated with glibenclamide at a dose of 5 mg/kg/day, p.o. IV Diabetic rats treated with hydroalcoholic combined plant extract at a dose of 100mg/kg/day, p.o. V Diabetic rats treated with hydroalcoholic combined plant extract at a dose of 200mg/kg/day, p.o. Antidiabetic activity of hydroalcoholic combined plant extract was evaluated by estimation of blood glucose levels and body weight measurement on the day 0, day 7, day 14, day 21 and day 28 of the study by using commercially available kit (Accu-Chek Active Test Meter). Serum lipid profile estimation at the end of 28 days, blood was collected from inferior vena cava, serum separated for determination of parameters like total cholesterol, HDL- cholesterol and triglycerides. VLDL cholesterol and LDL-cholesterol were calculated using the Friedewald's formula [17]. VLDL = Triglycerides/5 LDL = Total cholesterol-(hdl-ch+vldl-ch) Liver glycogen estimation Liver of individual rats was homogenized in 5% w/v trichloroacetic acid and its glycogen content was determined at the end of 28 days by the method of Carrol [18]. Glycosylated haemoglobin determination After 28 days of treatment, blood was collected from retro-orbital plexus and subjected for the determination of glycosylated haemoglobin. STATISTICAL ANALYSIS The values are expressed as mean ± standard error of mean (SEM) obtained from the number of experiments. Difference was done by using one-way analysis of variance (ANOVA) followed by Dunnett s multiple comparison tests. The difference in mean P value p<0.05 was considered as statistically significant.
Page1394 RESULTS On the basis of acute toxicity study there is no abnormal behavioural, neurological changes and death was observed till the end of the 14 day. Hence, the median lethal dose (LD50) of the extract was then greater than 2000 mg/kg. Hydroalcoholic combined plant extract was found to be safe up to the dose of 2000 mg/kg. Therefore, dose of 100 and 200 mg/kg b.wt. of were selected for all the experiments. Treatment with hydroalcoholic combined plant extract daily up to 28 days exhibited significant antidiabetic activity in streptozotocin induced diabetic rats, whilst there was no significant effect observed on normoglycaemic rats. However, at the end of 28 days of treatment, there was a significant (P<0.01) decrease of blood glucose levels with the glibenclamide and hydroalcoholic combined plant extract (100 and 200 mg/kg) respectively when compared with diabetic control group (Table 1). Table 1: Antidiabetic effect of hydroalcoholic combined plant extract on blood glucose level in streptozotocin induced diabetic rats Blood Glucose Levels (mg/dl) Day 0 Day 7 Day 14 Day 21 Day 28 I 97.25 ± 1.47 97.88 ± 2.66 98.50 ± 1.85 99.56 ±1.72 101.56 ± 2.33 II 256.23 ± 1.56 274.20 ± 2.87 294.10 ± 1.36 311.71 ± 1.62 338.21 ± 3.65 III 251.30 ± 4.24 236.28 ± 3.22 178.76± 1.52* 139.54 ± 2.25* 121.32 ± 0.86* IV 252.19 ± 3.29 230.78 ± 4.30 171.70 ± 1.80* 130.63 ± 2.56* 113.20 ± 3.43* V 255.13 ± 1.69 229.47 ± 2.70 178.37 ± 1.06* 128.78 ± 3.50* 110.36 ± 2.77* Table 2 shows the level of plasma lipid profile such as total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), very low density lipoprotein (VLDL) and low density lipoprotein (LDL). Plasma TC, TG and LDL level were significantly elevated and HDL level was decreased in diabetic rats when compared with control. After the treatment with hydroalcoholic combined plant extract, a significant reduction of TG, TC and LDL and increase in HDL level was observed. Table 2: Antidiabetic effect of hydroalcoholic combined plant extract on lipid profile in streptozotocin induced diabetic rats Total Cholesterol Triglycerides HDL VLDL LDL (mg/dl) (mg/dl) (mg/dl) (mg/dl) (mg/dl) I 69.53±2.77 66.74±3.43 27.32±2.56 13.63±0.30 22.28±2.26 II 103.20±2.20 105.14±2.19 18.20±2.72 32.22±0.76 76.73±1.20 III 82.34±3.42* 80.28±2.14* 21.20±1.30* 19.27±0.66* 38.20±3.35* IV 77.22±2.36* 76.66±2.52* 25.82±1.46* 15.32±0.29* 27.23±2.23* V 73.75±1.23* 74.38±1.32* 26.23±0.50* 14.12±0.45* 24.44±3.52* There was a significant elevation in the blood glycosylated haemoglobin and a decrease in liver glycogen levels in streptozotocin diabetic rats as compared to normal rats. Oral administration of the hydroalcoholic combined plant extract at the dose of 100 and 200 mg/kg significantly (p<0.01) restored the increased glycosylated haemoglobin and decreased liver glycogen level in streptozotocin-diabetic rats as comparable to glibenclamide (Table 3). Table 3: Antidiabetic effect of hydroalcoholic combined plant extract on liver glycogen and glycosylated haemoglobin in streptozotocin induced diabetic rats Liver Glycogen Glycosylated (mg/g) Haemoglobin (mg/g) I 12.52±0.52 6.35±0.43 II 3.36±0.34 10.44±0.89 III 6.80±0.22 8.26±0.28* IV 9.78±0.20* 7.86±0.21* V 10.10±0.50* 7.64±0.08*
Page1395 Streptozotocin induced diabetic rats showed significant (p<0.01) reduction in body weight as compared to normal group. At the end of 28 days treatment, the body weight of normal rats, treated with hydroalcoholic combined plant extract and standard drug treated group increased significantly, whereas body weight of diabetic control group rats decreased (Table 4). Table 4: Antidiabetic effect of hydroalcoholic combined plant extract on body weight in streptozotocin induced diabetic rats Body Weight (gm) Initial Final I 176.14±2.47 219.83±5.58 II 174.20±2.24 129.17±4.19 III 171.17±4.33 186.66±2.47* IV 171.32±1.28 191.41±2.48* V 172.11±4.37 197.72±3.48* DISCUSSION Diabetes mellitus is one of the common metabolic disorders with micro and macro vascular complications that results in significant morbidity and mortality. In the treatment of diabetes mellitus, non-pharmacologic measures remain a critical component of therapy. Streptozotocin is a β-cytotoxin, induces chemical diabetes in a wide variety of animal species including rat by selectively damaging the insulin-secreting β-cells of the pancreas. The purpose of choosing streptozotocin as diabetes-inducing agent was known to produce diabetes mellitus irreversibly with a single dose of intraperitoneal administration by relative necrotic action on the β-cells of pancreas leading to insulin deficiency. Insulin deficiency leads to various metabolic aberrations in animals viz., increased blood pressure level, decreased protein content, increased level of cholesterol and triglycerides were reported [19]. The WHO expert committee has aptly suggested that research should be aimed at investigating the traditional methods of treatment for refractory diseases like diabetes [20]. The hydroalcoholic combined plant extract at the dose of 100 and 200 mg/kg showed significant antidiabetic activity against streptozotocin induced diabetic rats and the effect was comparable with that of the standard drug glibenclamide. After the treatment with hydroalcoholic combined plants extract significant increase in the liver glycogen and a significant decrease in blood glucose level and glycosylated haemoglobin levels. The total cholesterol and triglycerides levels, low density lipoprotein, and very low density lipoprotein were also significantly reduced and the high density lipoprotein level was significantly increased upon treatment with the hydroalcoholic combined plant extract. Induction of diabetes with streptozotocin is associated with a characteristic loss of body weight, which is probably due to muscle wasting [21], and due to loss of tissue proteins [22]. Diabetic rats treated with the hydroalcoholic combined plant extract showed a significant increase in body weight when compared to the untreated diabetic rats which may be due to its protective effect in controlling muscle wasting i.e. by reversal of gluconeogenesis and may also be due to the improvement in glycemic control [23]. CONCLUSION In conclusion, the present study indicates that hydroalcoholic combined plant extract which is a mixture of hydroalcoholic extract of Portulaca oleracea and Caralluma attenuata have significant antidiabetic activity in streptozotocin induced diabetic rats. The antidiabetic activity in streptozotocin induced diabetic rats is due to decrease in elevated blood glucose level, restored the increased glycosylated haemoglobin and decreased liver glycogen level, reduction of TG, TC and LDL and increase in HDL level. This confirmation justifies its use in ethnomedical medicine for the treatment of diabetes when treated with hydroalcoholic combined plant extract and standard drug glibenclamide. ACKNOWLEDGEMENT The authors are thankful to Zydus Cadila, Ahmedabad for providing gift sample of glibenclamide. REFERENCES 1. Balkau B, Charles MA, Eschwege E. Discussion epidemiologique des nouveaux criters du diabete. Mt Endocrinol 2000; 2: 229 234. 2. Lpoz-Candales A. Metabolic syndrome X: a comprehensive review of the pathophysiology and recommended therapy. J Med 2001; 32(5-6): 283-300. 3. Elizabeth G, Nabel MD. Cardiovascular disease. N Engl J Med 2003; 349: 60-72. 4. Kumar GPS, Arulselvan P, Kumar DS, Subramanian SP. Anti-diabetic activity of fruits of Terminalia chebula on streptozotocin induced diabetic rats. J Health Sc 2006; 52(3): 283 291. 5. Bach JF. Insulin-dependent diabetes mellitus as a β-cell targeted disease of immunoregulation. J Autoimmun 1995; 8: 439 463. 6. Arky RA. Clinical correlates of metabolic derangements of diabetes mellitus in: Kozak GP. (Ed.), Complications of Diabetes mellitus, Saunders WB. Philadelphia 1982; 16-20. 7. Ahmed I, Adeghate E, Cummings E, Sharma AK, Singh J. Beneficial effects and mechanism of action of Momordica charantia juice in the treatment of streptozotocin-induced diabetes mellitus in rat. Mol Cell Biochem 2004; 261(1-2): 63-70.
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