INTRODUCTION:

Hyperlipidemia has been ranked as one of the greatest risk factors contributing to the prevalence and severity of coronary heart diseases ¹.Coronary heart disease, stroke, atherosclerosis and hyperlipidemia are the primary cause of death ².Hyperlipidemia is characterized by elevated serum total cholesterol, low density, and very low-density lipoprotein and decreased high-density lipoprotein levels. Hyperlipidemia associated lipid disorders are considered to cause atherosclerotic cardiovascular disease ³. Among these hypercholesterolemia and hypertriglyceridemia are closely related to ischemic heart disease ⁴. The main aim of treatment in patients with hyperlipidemia is to reduce the risk of developing ischemic heart disease or the occurrence of further cardiovascular or cerebrovascular disease ⁵. Currently available hypolipidemic drugs have been associated with a number of side effects ⁶.

The consumption of synthetic drugs leads to hyperuricemia, diarrhoea, nausea, myositis, gastric irritation, flushing, dry skin and abnormal liver function ⁷. Medicinal plants are used for various research purposes.

It has been reported that traditional systems have immune potential against various diseases. More than thirteen thousand plants have been studied for various pharmacological properties. An herbal treatment for hypercholesterolemia has no side effects and is relatively cheap, locally available.

They are effective in reducing the lipid levels in the system⁸. Hyperlipidemia is classified into a primary and a secondary type, which indicates the complexities associated with disease. The primary disease may be treated by anti-lipidemic drugs but the secondary type originating from diabetes, renal lipid nephrosis or hypothyroidism demands the treatment of the original disease rather than hyperlipidemia ⁹. Consumption of much fat may lead to the production of extra VLDL, resulting in the formation of large amounts of LDL which may stick to the walls of the blood vessels if the quantity of HDL is insufficient, causing blockages for the normal flow of blood. Therefore, improvement in human diet is highly recommended for disease prevention ¹⁰. Medicinal plants play a major role in hypolipidemic activity. The leaves of Aleurites moluccana, Piper betle suggests that the lipid lowering action is mediated through inhibition of hepatic cholesterol biosynthesis and reduction of lipid absorption in the intestine ¹¹.

Sapindus emarginatus Vahl family Sapindaceae is a medium-sized deciduous tree found in south india. It is commonly called as soap nut tree. Native to South India Sapindus emarginatus is found wild or introduced in tropical and sub-tropical regions, particularly the Indo-Malayan region. Traditionally, Sapindus emarginatus is used as anti-inflammatory and antiprurutic. It is used to purify the blood. The seed is in intoxicant and the fruit rind has oxytropic action. Its powder is used as nasal insufflations. S.emarginatus also showed strong anti-bacterial activity against the tested bacterial strains¹². Antifertility and antiandrogenic activities of S.emarginatus extract have been reported¹³.High content of saponins has been reported in the pericarp¹⁴. Two Pisicidal triterpenoid saponins¹⁵, acetylated triterpene saponins, hederagenin, sweet acyclic sesquiterpene glycoside, Mukurozioside IIb15 have been isolated from the Pericarps of S. emarginatus. Flavonoids have been isolated from the pericarp of Sapindus emarginatus ¹⁶.

However, there are no reports on the Antihyperlipidemic activity of this plant; the present study was undertaken for the first time to investigate Antihyperlipidemic activity of the methanol extract of pericarps pf Sapindus emarginatus.

MATERIALS AND METHODS:

Chemicals:

Triton WR-1335(a non-ionic detergent, iso octyl polyoxy ethylene phenol, formaldehyde polymer) was obtained from Lab Chemicals, Chennai. Fenofibrate was obtained from Moral Labs, Chennai. All other chemicals were of analytical grade and obtained locally.

Plant materials and extraction:

The plant S.emarginatus fruit was collected in March 2007 from the Thiagarajar college campus, Madurai, Tamilnadu, India. The plant material was taxonomically identified by the Botanical survey of India, Coimbatore, Tamilnadu, India and the voucher specimen BSI/SC/5/23/08-09/Tech 895 was retained in our laboratory for future reference. The dried powder material (500 g) of the pericarp of Sapindus emarginatus was extracted with 2000 ml of methanol in a soxhelt apparatus. The methanol extract was then distilled, evaporated and dried in vacuum. The resulted extract yield was 7.45%, and the appearance of the extract was dried gum resin in nature.

Preliminary Phytochemical analysis:

The Methanol extract of the Pericarp of S.emarginatus was subjected to preliminary phytochemical screening ¹⁷.

Experimental Animals:

Wistar albino adult male rats weighing 200-250g were obtained from the animal house, C. L. Baid Metha College of Pharmacy, Thoraippakkam, India. The animals were grouped and housed in polyacrylic cages (38x 23 x 10 cm) with not more than five animals per cage and maintained under standard laboratory conditions (temperature 25+2oC) with dark and light cycle (14/10 hour). They were allowed free access to standard dry pellet diet (Hindustan Lever, Kolkata, India) and water ad libitum. The mice were acclimatized to laboratory condition for 10 days before commencement of experiment. The experimental protocol was approved by Institutional Animal Ethical Committee (IAEC) constituted under CPCSEA. (IAEC ref no: IAEC ref no: IAEC/XIII/04/CLBMCP/2008-2009 dt/16-6-2008)

Acute toxicity studies:

Albino mice weighing 22-25 g selected by random sampling technique were used in the study. Acute oral toxicity was performed as per OECD- 423 guidelines (acute class method) ¹⁸ .The animals were fasted overnight, provided only water after which extract was administered to the groups orally at the dose level of 5 mg/kg body weight by gastric intubation and the groups were observed for 14 days. If mortality was observed in 2 or 3 animals among 6 animals then the dose administered was assigned as a toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for further higher doses such as 50, 300 and 2,000 mg/ kg body weight. The animals were observed for toxic symptoms such as behavioral changes, locomotion, convulsions and mortality for 72 hours.

Antihyperlipidemic studies:

The animals were divided into five groups of five rats each. The first group was given standard pellet diet, water and orally administered with 5% CMC. The second group was given a single dose of triton administered at a dose of 400mg/kg, p.o. After 72 hours of triton injection, this group received a daily dose of 5% CMC (p.o) for 7 days. The third and fourth group was administered a daily dose of SE aqueous extract 100mg/kg and 200 mg/kg suspended in 5%CMC, p.o., for 7 days, after inducing hyperlipidemia. Fifth group was administered with the standard Fenofibrate 65mg/kg, p.o. for 7 days ¹⁹.

Collection of blood:

On the 8thday, blood was collected by retero orbital sinus puncture, under mild ether anaesthesia. The collected samples were centrifuged for 10 minutes. Then serum samples were collected and used for various biochemical experiments. The animals were then sacrificed and the liver collected ²⁰.

Liver lipid extraction:

The liver was homogenized in cold 0.15M KCl and extracted with CHCl₃ CH₃OH (2% v/v). This lipid extract was used for the estimation of lipid parameters ²¹.

Biochemical analysis:

The serum and liver extract were assayed for total cholesterol, triglycerides, phospholipids, high-density lipoprotein (HDL), low-density lipoprotein (LDL), very low-density lipoprotein (VLDL) using standard protocol methods²².

Statistical analysis:

The results were expressed as mean + S.E.M. Statistical analysis was carried out by using ANOVA followed by Dunnet’s multiple comparison tests using Graph pad PRISM software version 4.03 (2005).P values < 0.05 were considered as statistically significant

RESULTS:

The methanol extract of S.emarginatus was found to be non–toxic up to the dose of 2 g/kg and did not cause any death of the tested animals. The Phytochemical tests with the methanol extract of S.emarginatus indicated the presence of carbohydrates, flavanoids, glycosides, terpenes, saponins and gums and mucilage. Hyperlipidemia is associated with heart disease, which is the leading cause of death in the world. The lowering of the levels of harmful lipids to satisfactory values have been confirmed by several experimental animal and interventional studies indicating lowered morbidity and mortality in coronary heart diseases. The results are discussed under the lipid profile in serum and the lipid profile in liver. Lipid profile in serum and liver indicates that increased phospholipids (PL), triglyceride (TG) and cholesterol levels were significantly reduced by treatment of 100 and 200 mg/kg of SE.LDL and VLDL levels were significantly increased in triton-injected animals to control rats. The results are shown in Tables 1, 2, 3, and 4. The SE markedly lowers the levels of serum cholesterol and VLDL. The decrease in cholesterol may indicate increased oxidation of mobilized fatty acids of inhibition or lipolysis. The present investigation shows that all triton induced rats displayed hyperlipidemia as shown by their elevated levels of serum and liver cholesterol, triglyceride, PL, VLDL, LDL and the reduction in the HDL level. It can be concluded that SE 100 and 200 mg/kg treatment was effective in cholesterol, PL, TG, VLDL, LDL and HDL in a dose dependant manner..

DISCUSSION:

Triton Wr-1339 has been widely used to block clearance of triglyceride-rich lipoproteins to induce acute hyperlipidemia in several animals²³.This model is widely used for a number of different aims²⁴ particularly, in rats it has been used for screening natural or chemical hypolipidemic drugs²⁵. Interestingly, the results of the present study show that extract of Sapindus emarginatus produced a significant reduction in cholesterol level and also it reversed Triton induced hyperlipidemia in rats.

Schurr et al demonstrated that a parenteral administration of a dose of Triton Wr-1339 to adult rats induced hyperlipidemia. Our present studies clearly show that Methanol Sapindus emarginatus extract at a dose of 100 and 200 mg/kg significantly lowered both plasma triglycerides and cholesterol levels. The large increase in plasma cholesterol and triglycerides due to Triton Wr-1339 injection results mostly from an increase of VLDL secretion by the liver accompanied by a strong reduction of VLDL and LDL catabolism ²⁶. The reduction of total cholesterol by the Sapindus emarginatus extract was associated with a decrease of its LDL fraction, which is the target of several hypolipidemic drugs. This result suggests that cholesterol-lowering activity of the herb extract can be result from the rapid catabolism of LDL cholesterol through its hepatic receptors for final elimination in the form of bile acids as demonstrated ²⁷.

It is well known that HDL-Cholesterol levels have a protective role in Coronary artery disease²⁸. Similarly increased level of serum LDL-cholesterol results in increased risk for the development of atherosclerosis ²⁹.The increased level of HDL- cholesterol and decreased cholesterol level along with its LDL fraction which is evident from the results could be due to an increased cholesterol excretion and decreased cholesterol absorption through gastro intestinal tract. Thus the decreasing cholesterol levels in the body under the influence pf S.emarginatus could have enhanced the enzymatic by a positive feed back mechanism.

It is found that some saponins increase the permeability of intestinal mucosal cells in vitro, inhibit active mucosal transport and facilitate uptake of substances that are normally not absorbed ³⁰. Saponins also lower transmural potential difference across the small intestine of the rat³¹.Saponins have long been known to have a lytic action on erythrocyte membranes and this property has been used for their detection. The haemolytic action of saponins is believed to be the result of the affinity of the aglycone moiety for membrane sterols, particularly cholesterol, with which they form insoluble complexes³².and in fact, flavonoids have exhibited a variety of pharmacological activites, including the anti-atherogenesis and antioxidant effect ³³.

The result strongly suggests that the hypolipidemic activity of this medicinal plant could be attributed to the presence of the valuable saponins and flavonoids in the extract. This extract supplementation also resulted in significant attenuation in the level of LDL and HDL in serum towards the control level, which again strengthens the hypolipidemic effect of this extract. The antihyperlipidemic activity of Sapindus emarginatus (100 and 200 mg/kg) against Triton Wr-1339 showed significant activity when compared to fenofibrate treated groups in a dose dependant manner.

Thus, our study showed that administration of Methanolic extract of 100 and 200 mg of Sapindus emarginatus was more effective to manage hyperlipidemia.The active ingredients present here may recover the disorders in lipid metabolism noted in hyperlipidemic state.

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