Antihyperlipidemic activity of Sapindus emarginatus in Triton WR-1339 induced albino rats

 

Srikanth Jeyabalan and Muralidharan Palayan

Department of Pharmacology and Toxicology, C.L.Baid Metha College of Pharmacy Jyothi Nager, Thoraippakkam,

Chennai-600 097.Tamilnadu, India

*Corresponding Author E-Mail: srikanthcologist@rediffmail.com

 

ABSTRACT:                         

Hyperlipidemia is the greatest risk factor of coronary heart disease.  Currently available hypolipidemic drugs have been associated with number of side effects.  Herbal treatment  for  hyperlipidemia  has  no  side  effects  and  is relatively cheap  and  locally available.  Literature claims that Saponins are able to reduce hyperlipidemia.  Based on high saponin content in herbal plants, Sapindus emarginatus (SE) was selected and the present study focus on the anti-hyperlipidemic  activity  of  methanol  extract  of  pericarps of  SE  against  Triton  induced  hyperlipidemia  in  rats.  SE was  administered  at  a dose  of  100 and 200mg/kg  (p.o)  to  Triton  induced  hyperlipidemic  rats.  Fenofibrate was used as reference standard. The statistical analyses were carried out using one way ANOVA followed by Dunnet’s multiple comparisons test.SE shows a significant decrease in the levels of serum cholesterol, phospholipid, triglyceride, LDL, VLDL and significant increase in the level of serum HDL at the dose of 100 and 200mg/kg  (p.o) against Triton induced hyperlipidemic in rats.  Methanol extracts decreased serum level of total cholesterol by 69.72%. On the other hand aqueous extract of SE increased the serum HDL cholesterol level by 24.11%.  The reduction of LDL cholesterol level by extract was 30.31%.

 

KEY WORDS: Sapindus emarginatus, Hyperlipidemia, Triglycerides, Lipoproteins.

 


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.

 


 

Table 1: Effect of Methanolic extract of Sapindus emarginatus on HDL, LDL, VLDL in Serum of Control and Experimental Rats

Groups                                                                                                             Parameters

                                                                           HDL                                     LDL                       VLDL

Group-I control                                                   23.22 ± 2.31                         24.67 ± 1.78          14.66 ± 2.51

Group-II Triton treated                                       17.71 ± 6.10*                       154.49 ± 8.51*      23 ± 2.01*


Group-III                 Triton + CS (100 mg/kg)                    19.62 + 4.56**                     42.56 + 3.56**      17.84 + 2.68**

Group-IV Triton + CS (200 mg/kg)                     24.11 ± 3.11**                     30.31 ± 3.51**      15.3 ± 2.11**

Group-V Triton + Fenofibrate                             24.30 ± 3.10**                     25.71 ± 3.34**      14.4 ± 2.10**

Values are in mean ± SE; Number of animals in each group = 5; *p < 0.05 Vs Group I; ** p <0.05 Vs Group II

 

Table 2: Effect of Methanolic extract of Sapindus emarginatus on Cholesterol, Triglycerides, Phospholipids in Serum of Control and Experimental Rats

Groups                                                                                                                            Parameters            

                                                                           Cholesterol                           Triglyceride                          Phospholipids

Group-I control                                                   62.55 ± 5.52                         73.32 ± 5.57                         156.27 ± 9.32

Group-II Triton treated                                       195.20 ± 10.58*                   115 ± 5.57*                          207.22 ± 10.81*

Group-III               Triton + CS (100 mg/kg)                     78.56 + 6.38 **                    85.24 + 5.38**                     196.24 + 7.56**


Group-III Triton + CS (200 mg/kg)                     69.72 ± 5.53**                     76.5 ± 5.96**                        177.71 ± 6.23** Group-IV Triton + Fenofibrate                              65.43 ± 2.51**                     72.0 ± 11.01**                      59.54 ± 7.53**

 Values are in mean ± SD; Number of animals in each group = 5; * p < 0.05 Vs Group I; ** p < 0.05 Vs Group II

 


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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Received on   .  .2008       Modified on   .  .2008

Accepted on   .  .2008       © RJPT All right reserved

Research J. Pharm. and Tech.2 (2): April.-June.2009,319-323