P Sailaja Rao et al. / Journal of Pharmacy Research 2014,8(9),1274-1278 Research Article Available online through ISSN: 0974-6943 http://jprsolutions.info Beneficial effects of laurifolia seed on lipid profile status in streptozotocin induced diabetic rats *P Sailaja Rao1, G. Krishna Mohan2

1*Assistant Professor, Department of Pharmacology, SriVenkateshwara College of Pharmacy & Research Centre, Madhapur, Hyderabad-81, Telangana, India. 2Professor, Centre for Pharmaceutical Sciences, JNTUH, Hyderabad, Telangana, India.

Received on:21-05-2014; Revised on: 17-06-2014; Accepted on:25-07-2014

ABSTRACT Introduction: Diabetes mellitus is one of the widespread and severe metabolic disorders in human all over the world. The effect of the disease on lipid metabolism is very well established. The association of hyperglycaemia with altered lipid parameters presents a major risk for all cardiovascular diseases. Various medicinal have effectively been used to conquer this health problem. Objective:The present study was designed to evaluate the potential anti- hyperlipidemic efficacy of Hydnocarpus laurifolia seeds in Streptozotocin (STZ) induced diabetic rats. Materials and Methods: Diabetes was induced by giving Streptozotocin intraperitoneally, rats which showed blood glucose levels >250 mg/dl were selected for the study. The petroleum ether and ethylacetate extracts of Hydnocarpuslaurifolia seeds were prepared and administered at a dose of 100 mg/kg and 300 mg/kg each orally. Metformin was given as a standard drug at a dose of 50 mg/kg orally. Results: The fasting and post-prandial blood glucose levels were estimated by glucose-oxidase method. The plasma levels of cholesterol (CH), triglycerides (TG) low density lipoproteins (LDL) and very low density lipoproteins (VLDL) were estimated and found to be signifi- cantly (p<0.0001) lowered in extract treated diabetic rats. The results showed that the present study provided a rationale for the use of Hydnocarpus laurifolia seed extract as an anti-diabetic agent and anti-hyperlipidemic agent.

KEY WORDS: Diabetes mellitus, Hydnocarpus laurifolia, Streptozotocin, anti-hyperlipidemic agent. INTRODUCTION: Diabetes mellitus is one of the most major metabolic diseases and defence mechanism which promote the development of late complica- cause of death in modern society. Diabetes is a group of chronic tions. To reduce the late complications and negative outcomes of the disorders characterised by disarrangement in carbohydrates, proteins disease, it is necessary to control the blood glucose levels and lipid and fat metabolism caused by hyperglycemia (or) reduced insulin levels 2. Therefore, successful management depends upon diminished secretion (or) both. There are various severe diabetic complications glucose levels in the blood and lipid homeostasis, which combats such as retinopathy, neuropathy, nephropathy, cardiovascular com- with the cardiovascular obstacles. plications and ulceration. Apart from the above complications, car- diovascular risk is most important with altered lipid profile. It is Many side effects have been reported with oral synthetic hypoglyce- characterised by increased levels of Cholesterol (CH), triglycerides mic agents and they were not successful in maintaining euglycemia (TG), low density lipoproteins (LDL) and very low density lipopro- and controlling long-term microvascular and macrovascular compli- teins (VLDL) 1. An unusual deposition of fat in the liver and muscle is cations. According to W.H.O, 300 million people will have diabetes by observed which elicits the insulin resistance that terminates in ß-cell the year 2025 3. The use of herbal medicines for the treatment of reduction in type 2 diabetes. In Diabetes, biochemical alterations of diabetes has gained importance throughout the world. Hence, there glucose and lipid metabolism occur which leads to an elevated pro- is an increased demand to use natural products with anti-diabetic duction of Reactive oxygen (ROS), a decline in anti-oxidative activity due to the side effects associated with the use of insulin and oral hypoglycemic agents. Hypercholesterolemia and *Corresponding author. hypertriglyceridemia are common complications of diabetes in addi- P Sailaja Rao, tion to hyperglycemia. Hyperlipidemia contributes significantly in Assistant Professor, the manifestation and development of atherosclerosis and coronary Department of Pharmacology, heart disease 4. SriVenkateshwara College of Pharmacy & Research Centre, Madhapur, Hyderabad-81, Telangana, India. India is a country with rich natural resources with a wide variety of

Journal of Pharmacy Research Vol.8 Issue 9. September 2014 1274-1278 P Sailaja Rao et al. / Journal of Pharmacy Research 2014,8(9),1274-1278 medicinal plants. The plants which show significant pharmacological Animals which did not develop more than 250 mg/100 ml of blood activity and low toxicity need extensive screening. The plants which glucose levels were rejected 8. show significant pharmacological activity and low toxicity need ex- Experimental groups: The animals were divided into eleven groups tensive screening.Hydnocarpus laurifolia belongs to family; of 6 animals each. () is one of the ancient plants in the world, Group I: Normal untreated rats (Control) which is used in the traditional system for Diabetes. It is a tree found Group II:Normal rats given with Petroleum ether seed extract (PESE) in tropical forestsand Western Ghatsof south India. It is useful in the (100mg/kg) (o) treatment of Intestinal worms, Helminthiasis, infected wounds, skin Group III: Normal rats given with Petroleum ether seed extract (PESE) diseases, fever, bloating, piles and wounds with inflammation 5,6. The (300mg/kg) (o) chemical constituents contain Hydnocarpic acid and its homologues. Group IV:Normal rats given with Ethylacetate seed extract (EASE) It also contains oleic acid and palmitic acid. Chalmoogra oil obtained (100mg/kg) (o) from this is used in the treatment of skin diseases including Group V: Normal rats given with Ethlyacetate seed extract (EASE) leprosy 7. The preliminary phytochemical screening of Hydnocarpus laurifolia was done and revealed the presence of alkaloids, pheno- (300mg/kg) (o) lics, flavonoids, carbohydrates, saponins and glycosides. Group VI: Diabetic rats Group VII:Diabetic rats given with Metformin 50 mg/kg (o) MATERIALS AND METHODS: Group VIII: Diabetic rats given with Petroleum ether seed extract(PESE) (100mg/kg) (o) Collection of Plant Material: Group IX :Diabetic rats given with Petroleum ether seed extract (PESE) Hydnocarpus laurifolia seeds (5 kg) were collected from Tirumala (300mg/kg) (o) Hills, Tirupathi, Andhra Pradesh during July-Aug 2011. The Authen- Group X :Diabetic rats given with Ethylacetate seed extract (EASE) tication was done by S.V. Univerisity, Botany Department, Tirupathi. (100mg/kg) (o) Group XI: Diabetic rats given with Ethylacetate seed extract (EASE) Preparation of Extract: (300mg/kg) (o) The seeds obtained were powdered in electric grinder, placed in closed vessels; the selected solvents petroleum ether and ethylacetate were Animals of group I were given with 0.9% saline and served as control added and allowed to stand for seven days shaking occasionally. and groups II, III, IV and V were given with petroleum ether and ethyl The liquid was strained off; solid residue was pressed, clarified by acetate extract of doses of 100 & 300 mg/kg dissolved in 0.25% Carboxy filtration and then subjected to evaporation. Methyl Cellulose orally. Group VI were diabetic control rats. Group VII was given with Metformin, a standard oral hypoglycaemic agent Animals: at a dose of 50mg/kg. Diabetes induced groups VIII, IX, X and XI Animal Protocol was approved by IAEC (Institutional Animal Ethical were given with the Petroleum ether and ethylacetate extracts at dose Committee) of CPCSEA (Committee for Purpose of Control and Su- of 100 and 300 mg/kg dissolved in 0.25% Carboxy Methyl Cellulose pervision of Experimentation on Animals) through its reference no: orally. Petroleum ether and ethylacetate seed extract of Hydnocarpus IAEC/SVCP/2011/006, dated: 26/7/11. laurifolia were administered to groups II to V and VIII to XI for a period of 15 days, and on 16th day blood was collected byretro-orbital Male Wistar rats, weighing (180-250 gms) were obtained from NIN sinus puncture. Blood withdrawn was centrifuged and serum was (National Institute of Nutrition), Hyderabad. The animals were housed separated for biochemical study. Serum lipid profile (total cholesterol, with free access to food and water for at least one week in an air triglycerides, HDL-cholesterol, LDL cholesterol and VLDL-choles- conditioned room (250C) under a 12 hr light:dark cycle prior to the experiment. They were fed with standard diet (Hindustan Lever) and terol), was measured by using ERBA reagents and ERBA kit in Semi- water ad libitum. Auto Analyser.

Antidiabetic activity: The Fasting blood glucose (FBS) and Post-Prandial glucose (PLBS) 9 Induction of Experimental Diabetes: Diabetes was induced by a single levels were estimated by Glucose-oxidase method . intraperitoneal injection of a freshly prepared Streptozotocin (STZ) solution (Sisco Research laboratories pvt ltd. Mumbai-93, India. Batch Statistical Analysis: No: T-835796) (Dose: 30-50mg/kg) in citrate buffer 0.1 M, pH 4.5 to The results of the estimation were reported as Mean ± SEM. Student’s overnight fasted rats. Diabetes was identified by polydipsia, polyuria t-test was applied when two groups amongst were compared. The and by measuring blood glucose levels 48 hafter injection of STZ. values were considered significant when p < 0.05, p < 0.001, p < 0.0001. Statistical calculations were done using Graph Pad Prism.

Journal of Pharmacy Research Vol.8 Issue 9. September 2014 1274-1278 P Sailaja Rao et al. / Journal of Pharmacy Research 2014,8(9),1274-1278 RESULTS: cantly decreased (p<0.0001) in diabetic treated group when compared A continuous treatment of petroleum ether and ethylacetate extract to diabetic group. In a present study, Metformin was used as a stan- of Hydnocarpus laurifolia (100 & 300 mg/kg) was given for a period dard oral hypoglycaemic agent which showed a significant reduction of 15 days and blood glucose levels were evaluated. Table 1 showed in post-prandial blood glucose as compared to diabetic rats. the levels of the blood glucose levels i.e., FBS and PLBS in control and experimental animals. Diabetic rats showed a significant increase Anti-hyperlipideamic activity was evaluated by the results in blood glucose levels compared to corresponding control rats. Fol- obtained.Table 2 showed that the levels of serum cholesterol, triglyc- lowing the oral administration of petroleum ether extract (100 mg/kg) erides, LDL, VLDL, HDL in the normal and diabetic rats. and ethylacetate extract (100 & 300 mg/kg), the PLBS levels signifi- A marked increase in the level of serum cholesterol, triglycerides, Table 1: Effect of Hydnocarpus laurifolia on blood glucose levels in LDL, VLDL and a decrease in HDL levels were found in diabetic rats. control and Experimental rats Administration of petroleum ether extract of Hydnocarpuslaurifolia Groups Treatment FBS (mg/dl) PLBS(mg/dl) (100 mg/kg) showed a significant reduction (p<0.0001) in the level of (Fasting (Post-Prandial serum cholesterol, triglycerides, LDL and VLDL. It was also observed blood sugar) blood sugar) that the HDL levels were increased significantly and the values were I Normal (control) 94.4±3.80 132.5±2.5 almost near to the values of normal rats. Similarly, administration of NS NS II Normal + PESE (100) 87.5±3.09 94±2.76 petroleum ether extract of Hydnocarpuslaurifolia (300 mg/kg) also III Normal + PESE (300) 91±2.22NS 94±1.91NS IV Normal + EASE (100) 94±2.26NS 99.83±2.24NS showed a significant reduction (p<0.0001) in the level of serum cho- V Normal +EASE(300) 94.67±3.73NS 294.16±2.24a lesterol, triglycerides, LDL and VLDL when compared to the corre- VI Diabetic Control 300±13.27a 294.17±14.17a sponding diabetic rats. VII Diabetic + Metformin 280.07±15.36NS 153.34±2.47a,NS VIII Diabetic + PESE (100) 280.83±13.57NS 152.75±2.16a,NS IX Diabetic+PESE(300) 270.83±4.36NS 148.95±1.38NS A potent hypolipidaemic effect of ethylacetate extract of X Diabetic+EASE(100) 276.67±2.47NS 185.1±4.42a,a Hydnocarpuslaurifolia (100 mg/kg) was evident by a significant re- NS a,a XI Diabetic+EASE(300) 275.83±3.51 197.5±7.27 duction (p<0.0001) in the level of serum cholesterol, triglycerides, LDL and VLDL in diabetic rats and also marked increase in the HDL Values were reported as Mean±SEM. Diabetic control compared with Nor- mal, ap<0.0001; Normal rats compared to all test drug treated normal groups, levels of extract treated rats. Similarly, ethylacetate extract of NS-Non-significant; Diabetic control compared to all test drug treated Hydnocarpuslaurifolia (300 mg/kg) also showed a significant reduc- diabetic rats,ap<0.0001; Diabetic+Metformin group compared to diabetic tion (p<0.0001) in the level of serum cholesterol, triglycerides, LDL control,ap<0.0001; Diabetic+PESE (100 mg/kg) compared to diabetic group and VLDL in diabetic rats and also marked increase in the HDL levels a p<0.0001; Diabetic+EASE(100 mg/kg & 300 mg/kg) compared to diabetic of extract treated rats. group, ap<0.0001. Table 2: Effect of Hydnocarpus laurifolia on Lipid Profile in control and Experimental rats

Groups Treatment CH (mg/dl) TG (mg/dl) HDL (mg/dl) LDL (mg/dl) VLDL (mg/dl)

I Normal (control) 127.67±3.16 137.83±5.49 39.50±0.76 93.5±0.85 23.5±0.43 II Normal + PESE (100) 96.67±11.70NS 118.33±2.10NS 41.67±0.99NS 90.67±1.56NS 25.17±0.60NS III Normal + PESE (300) 108.83±3.27NS 120.83±3.00NS 39.67±0.67NS 92.83±2.08NS 28±0.57NS IV Normal + EASE (100) 135.83±6.11NS 136.33±2.67NS 40.17±0.47NS 107.83±0.70NS 28±0.30NS V Normal +EASE(300) 138.33±6.67NS 144.67±2.46NS 37.83±0.87NS 98.33±2.78NS 28.83±0.60NS VI Diabetic Control 216.33±1.56a 155±1.82a 20±0.58a 138.83±0.98a 43.67±0.88a VII Diabetic + Metformin 192.5±0.89a 143.83±1.30NS 44.5±0.5a 75±2.58a 26±0.63a VIII Diabetic + PESE (100) 122.82±1.52a 97.38±1.00a 37.4±0.51a 84.5±3.19a 27.5±0.43a IX Diabetic+PESE(300) 106.93±1.48a 87.85±1.22a 43.7±1.04a 81.17±2.70a 29.17±1.58a X Diabetic+EASE(100) 135.3±1.63a 106.08±2.51a 33.57±1.01a 89.33±2.10a 27.83±0.94a XI Diabetic+EASE(300) 125.55±0.81a 97.77±3.25a 40.68±0.68a 78.33±2.47a 26.17±0.48a Values were reported as Mean±SEM. Diabetic control compared with Normal ap<0.0001; Normal (control) group compared to all extract treated normal groups, NS-Not significant; Diabetic control compared to all extract treated diabetic rats, ap<0.0001; Diabetic+Metformin compared to diabetic control group,ap<0.0001; Diabetic+PESE (100mg/kg) and diabetic+PESE(300mg/kg) compared to diabetic control, ap<0.0001; Diabetic+EASE (100mg/kg) and diabetic+EASE(300mg/kg) compared to diabetic control, ap<0.0001.

Journal of Pharmacy Research Vol.8 Issue 9. September 2014 1274-1278 P Sailaja Rao et al. / Journal of Pharmacy Research 2014,8(9),1274-1278

DISCUSSION: reduce hyperlipidaemia in diabetes. Preliminary phytochemical screen- Diabetes mellitus, a third leading cause of death. Prolonged ing revealed the presence of saponins in the extract. Thus, the anti- hyperglycaemia ends with severe complications. Hyperglycaemia oxidants present in Hydnocarpus laurifolia extract may be respon- generates more oxidative stress. Free radicals react with lipids and sible, in part, for the antihyperlipidaemic effect. The hypoglycaemic causing lipid peroxidation. Dyslipidaemia is a frequent complications effect of the extract may be implicated as the major reason for the noted in chemical induced diabetes 10,11,12 and presents a serious risk observed antihyperlipidaemic effect of the extract. The work showed of vascular disease. that Hydnocarpus laurifolia seed extract may be used for the control and management of diabetes. Historical literatures reveal that knowledge regarding diabetes ex- isted since Brahmic period as this was mentioned in Ayurvedic text REFERENCES: books-SushrutaSamhita written in fourth and fifth centuries B.C. 13. 1. Ahmadi S.A., Boroumand M.A., Moghaddam K.Get al.(2008) Phytochemical screening is an essential and very important part of The impact of low serum triglyceride on LDL-cholesterol medicinal plants research 14. After the completion of extraction pro- estimation Arch Iranian Med. 11:318-321. cess the obtained Petroleum ether extract and Ethylacetate extract 2. Baynes J. W. (1991) Role of oxidative stress in development were identified for the presence of phytochemicals. The preliminary of complications in diabeteics. 40:405-12. phytochemical screening of the extract revealed the presence of alka- 3. WHO and IDF. (1999) The economics of diabetes and dia- loids, phenolics, flavonoids, carbohydrates, saponins and glycosides. betic care: A report of the diabetes lealth economics study In the present study, anti-hyperlipidaemic activity of different ex- group International Diabetes Federation, World Health tracts of Hydnocarpus laurifolia seeds have been evaluated in nor- Organisation, Geneva, Switzerland. mal and strptozotocin induced diabetic rats. In this study, it was 4. Balassa E. O. (1985). Hyperlipidaemia in diabetics. observed that there was an increase in cholesterol, triglycerides, LDL Medicographia, 7:11-14. and VLDL in streptozotocin induced diabetic rats. Diabetes induced 5. Kondal Reddy J, Rao B S , Shravani Reddy T, Priyanka B hyperlipidemia is attributable to excess mobilization of fat from the (2013) Anti-diabetic activity of ethanolicextract of Hydno- adipose tissue due to the under utilization of the glucose 15. carpus wightiana Blume using STZ induced Diabetes in Sd rats. IOSR Journal of Pharmac 3(1):29-40. Most of the hypolipidaemic drugs do not decrease serum triglyceride 6. H Sini, P V Mohanan, K S Devi (2005) Insecticidal activity, level but petroleum ether and ethylacetate extracts lowered it signifi- cytogenecity and metabolism of fatty acid rich fraction of cantly since under normal condition, insulin activates the enzyme Hydnocarpus laurifolia Toxicological and Environmental lipoprotein lipase and causes hydrolysis of triglycerides. Chemistry. 87(1):91-98. 7. The useful plants of India (1992) Ed: Publication and Infor- HDL cholesterol exerts a role in the prevention of atherosclerosis by mation Directorate, CSIR, New Delhi, India 379. transporting the cholesterol from peripheral tissues to liver for excre- 8. Vogel H G, Vogel W H. (2002) Chapter K: Anti-diabetic activ- tion. HDL decrease was restored in the extract treated diabetic rats. ity, Drug Discovery and Evaluation, Pharmacological As- The total lipid profile (CH, TG, LDL and VLDL) in the serum of diabetic says. 2nded. New York: Springer Publication 535-538. rats treated with petroleum ether and ethylacetate extracts of Hydno- 9. Trinder P. (1969). Enzymatic determination of blood glu- carpus laurifolia showed a significant reduction as compared to cose. Ann Clin Biochem.6: 24-28. diabetic control rats. This suggests that Hydnocarpuslaurifolia can 10. Qiong L, Yizhong C, Jun Y, Mei S, Harold C (2004) Hypogly- prevent (or) be helpful in reducing the complications of abnormal cemic and hypolipidemic effects and antioxidant activity of lipid profile observed in some diabetics with hyperlipidaemia. fruit extracts from Lycium barbarum Life science 76:137- 149. CONCLUSION: 11. 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Boerhavia diffusa Effect on hepatic key enzymes in experi- Preparation on Lipid Profile, Glycated Haemoglobin and mental diabetes Journal of Ethnopharmacol 91:109-113. Glucose Tolerance in Streptozotocin induced diabetes in 13. Dhanukar S and Thatte U, (1989). Ayurveda Revisited. Popu- Rats. Indian J. Exp. Biol 33:798-800. lar prakashan, Bombay. 15. Krishna kumar K, Augustti K T, Vijayammal P L (2000) 14. Mitra S K, S Gopumadhavan, T S Murlidhar, S Anturlikar Hypolipedemic effect of Solacia oblanaga wall. Root bark and M B Sujatha (1995) Effect of D-400, a Herbo mineral in Streptozotocin diabetic rats Med Sci 28:65-67.

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Journal of Pharmacy Research Vol.8 Issue 9. September 2014 1274-1278