Research Journal of Chemical Sciences ______________________________________________ ISSN 2231-606X Vol. 2(2), 54-60, Feb. (2012) Res.J.Chem.Sci. International Science Congress Association 54 Hypolipidaemic effect of leucodelphinidin derivative from Ficus bengalensisLinn on cholesterol fed rats Mathew B.C.*, Yoseph B.A., Dessale T., Daniel R.S., Alemayehu A., Campbell I.W. and Augusti K.T.1, 2, 3Department of Medical Biochemistry, Faculty of Medicine, University of Gondar, ETHIOPIA 4,5Department of Biotechnology, Faculty of Natural and Computational Sciences, University of Gondar, ETHIOPIA Victoria Hospital, Bute Medical School, University of St Andrews, Scotland, UNITED KINGDOM Department of Biochemistry, University of Kerala, Trivandrum, INDIA Available online at: www.isca.in (Received 7th December 2011, revised 17th December 2011, accepted 12th January 2012)Abstract Administration of leucodelphinidin derivative isolated from the bark of Ficus bengalensis and another flavonoid quercetin (100mg/kg/day) in hypercholesterolemic rats provoked significant reduction in serum total cholesterol, LDL-cholesterol and an increase in the HDL-cholesterol levels. Significant decrease in atherogenic index was noted in these rats treated with the different flavonoids . There was an increased concentration of total bile acids in the liver and also increase in the fecal excretion of bile acids and neutral sterols in the rats fed cholesterol containing diet as compared to those fed normal diet. Feeding the flavonoids further significantly increased the concentrations of hepatic bile acids and the fecal excretion of bile acids and neutral sterols as compared to the control cholesterol diet fed group. The activity of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase was not inhibited by the flavonoids, instead there was an increased cholesterogenesis as was evident by an increased incorporation of labeled acetate into both free and esterfied cholesterol on treatment with leucodelphinidin and quercetin. These results demonstrated that the flavonoids exerted their hypocholesterolemic effects by increasing fecal bile acids and cholesterol excretion. Acute toxicity studies with the leucodelphinidin derivative showed no toxic reactions up to a dose of 4g/kg dose level. Keywords: Ficus bengalensis, leucodelphinidin, quercetin, hypocholesterolemic effect.Introduction Phytochemicals have drawn a considerable amount of attention as they play an important role in healthcare. Drug discovery using natural products is pivotal in current herbal medical research. Among the different phytochemicals, flavonoids, which are polyphenolic compounds, have attracted considerable attention for their wide variety of biological activities. Ficus bengalensis Linn (Banyan tree) is a large evergreen tree with aerial roots. Medicinal properties of this plant have been described in the literature of traditional systems of medicine like ayurveda, siddha, unani and homeopathy. Bioactive compounds isolated from different parts of the plant are effective in the treatment of various ailments including dysentery, diarrhea, diabetes, piles, rheumatism, leucorrhoea and menorrhagia. The infusion of the bark of this tree is used for the treatment of diabetes mellitus . We have studied the hypoglycemic effects of flavonoids isolated from extracts of the bark in diabetic rats , rabbits and dogs 4,5,6.Two flavonoids namely 5,7-dimethyl ether of leucopelargonidin-3-O--L rhamnoside and 5,3’-dimethyl ether of leucocyanidin 3-O--D galactosyl cellobioside were reported to possess insulinogenic action from our laboratory7,8. The antioxidant and related properties of these flavonoids were also demonstrated by us 9,10,11.The present study aimed to evaluate the hypolipidaemic effects of a flavonoid namely 5,7,3’ trimethylether of leucodelphinidin 3-O--L rhamnoside which was isolated from the bark of F. Bengalensis. Studies were carried out in 2% cholesterol fed rats and the results compared with those of a structurally similar synthetic flavonoid quercetin with known hypolipidaemic action. The structures of both flavonoids used in this study are shown in figure 1A and 1B. Material and Methods Chemicals: Quercetin dehydrate was purchased from Sigma (St Louis, MO USA).[14 C] acetate (specific activity 1.5466 GBq/mmol) was purchased from BRIT (Mumbai, India). All other chemicals used were of high purity and analytical grade and were purchased from Sisco Laboratories Kochi, Kerala. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(2), 54-60, Feb. (2012) Res.J.Chem.SciInternational Science Congress Association 55 CO O C OH OCH OHOCHOH H O H H OH O H H OHCHOH(A) HO OH O O OH OH OH (B) Figure-1 Structure of flavonoids used in this study A:Leucodelphinidin derivative (5, 7, 3’ trimethylether of leucodelphinidin 3-0--L rhamnoside, B: QuercetinPlant material and extraction: 5, 7, 3’ trimethylether of leucodelphinidin 3-0--L rhamnoside was isolated from the alcoholic extract of the defatted bark of F. bengalensis Linn according to the method of Subramanium and Mishra with slight modifications12. Fresh bark of banyan tree (after authentication) was collected locally and the middle saffron colored part of it was separated and dried under the sun. It was then cut into small pieces, powdered and defatted by extraction with petroleum ether (B.P.40-60C) and solvent ether exhaustively (24 h each) in a soxhlet apparatus. These extracts were discarded. The bark powder was taken out of the soxhlet and dried again to remove the solvents. It was put into the soxhlet and extracted exhaustively with double distilled alcohol (8h). The alcoholic extract was collected and the solvent removed under reduced pressure. The tarry residue left behind was stirred with enough water to dissolve the entire water soluble fraction. The mixture was allowed to stand overnight when a red precipitate and a red solution were separated. The precipitate was filtered out and allowed to dry in the funnel. This water insoluble residue was dissolved in methanol:chloroform (30:2.5V/V) mixture and chromatographed over silica gel-G of 60-120 mesh. Elution of column with methanol-chloroform (1:11 V/V) gave the red coloured compound 5, 7, 3’ trimethylether of leucodelphinidin 3-0--L rhamnoside whose structure is shown in figure1A. Yield obtained was 200 mg/100 gm bark powder. The compound with a melting point of 171C is soluble in ethyl alcohol, methyl alcohol and ethyl acetate. With alcoholic hydrochloric acid, it developed a purple colour, which deepened on warming. With FeCl it gave a blue colour which is characteristic of flavonoids. Animals and diet: Male albino rats Sprague-Dawley Strain, (weight 100-120 g) were randomly divided into four groups of 12 rats each. The grouping and treatment were as follows. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(2), 54-60, Feb. (2012) Res.J.Chem.SciInternational Science Congress Association 56 Group 1- Rats fed control diet (normal control). Group 2- Rats fed 2% cholesterol diet (cholesterol control). Group 3- Rats fed 2% cholesterol diet + leucodelphinidin derivative (100 mg/kg body wt/day). Group 4- Rats fed 2% cholesterol diet + quercetin (100 mg/kg body wt/day). The rats were housed in plastic cages individually in a controlled environment conditions (22-28C, 60-70% relative humidity-and 12-h and 12-h dark/light cycle), fed ad libitumon standard laboratory feed from Hindustan Lever Ltd, Bangalore, India (approximate composition protein,221 g/kg; fat, 40 g/kg; fibre, 36g/kg; minerals, 50 g/kg; energy, 15.16kJ/g) and water. The flavonoid derivatives were administered as suspensions in normal saline through a gastric tube at a dose of 100mg/kg body weight/day. Duration of the experiment was 90 days. The experiments were carried out with the approval of the Institutional Animal Ethics Committee. Sampling procedures: At the end of the experimental period their body weights were determined and fecal samples were collected from one half of each group and stored at -20C for bile acid estimation. The rats of these subgroups i.e., six rats from each group were deprived of food for 16 h and then anesthetized with ether inhalation and killed by decapitation. Blood samples were collected into tubes without an anticoagulant, kept at room temperature for 1h, and serum separated by centrifugation at 4C for 20 min at 1500 g. Serum was stored at -20C until further analyzed. Their gain in body and liver weights were also assessed. Livers were removed to ice-cold containers, sliced and portions equivalent to about 500 mg were taken for analysis. Analytical procedures: Serum total cholesterol was determined by the cholesterol oxidase method 13. Triacylglycerols in the serum were estimated by the glycerol phosphate oxidase method14. Serum VLDL+LDL was precipitated using heparin-MnCl solution and the lipids in the VLDL+LDL fraction were extracted by and cholesterol estimated13,15. LDL cholesterol was separately assessed by subtraction of VLDL cholesterol equivalent to 1/5 TAG. Fecal samples from the rats of all groups were homogenized with an equal volume of water and lyophilized to a fine powder. From this powder fecal neutral sterols and bile acids were extracted and estimated16. Activity of 3-hydroxy-3-methylglutaryl- coenzyme A (HMG-CoA) reductase in liver was determined by estimating the ratio of HMG-CoA/Mevalonate17. The rats left behind in the other half of each group i.e., six rats each in all groups were used for studying the incorporation of 1,2-14C-acetate into hepatic cholesterol as described below. The rats were deprived of food overnight for 16hr and they were injected (ip) with 0.5 ml solution of 1,2-14C sodium acetate (10µ Ci/100gm) at 9hr. After 3 hr, the rats were sacrificed by decapitation. The liver was quickly removed to ice-cold containers and gently blotted and weighed. The liver was extracted with chloroform: methanol and individual lipids were separated by TLC on silica gel G plates with a solvent mixture of hexane-diethyl ether- acetic acid (80:20:1, by vol). Authentic lipid standards were run concurrently and the TLC plates were developed using iodine vapor. Individual lipids were located as spots corresponding to standards. Silica gel corresponding to the lipids was scraped and extracted with chloroform and the radioactivity associated with each lipid was measured separately by liquid scintillation counter. Acute toxicity study: Acute toxicity of the leucodelphinidin derivative was carried out in rats according to standard protocol18. Different doses of the derivative dissolved in distilled water was administered orally at doses up to 4000 mg/kg body weight and animals were observed closely for a period of 72 hr for behavioral changes, toxic reactions and mortality. Statistical analysis: All values are expressed as means and standard deviations. Data were analyzed by one-way ANOVA and the results significant at 1-5% level are considered in this study. The statistical analysis were done using SPSS Statistical Package for Windows version 10.0 (SPSS, Chicago, IL, USA). Results and Discussion Animal growth: The diet consumption was found to be more or less the same for all the groups (11.2±1.1 g/rat). Feeding of high cholesterol diet significantly increased the body and liver weights over the normal control by 21% and 13% (p0.05). Treatment with the flavonoids for 90 days significantly decreased the same (7-9%) in groups 3 and 4 over the cholesterol control (p0.05). Serum lipids: To compare the effect of the two flavonoids on serum lipids, serum collected from rats of all four groups were analyzed for total cholesterol as well as LDL-cholesterol and HDL-cholesterol. These results and the calculated atherogenic index (Total cholesterol/HDL-C) are given in table-1. The concentration of total cholesterol and LDL cholesterol in the serum of cholesterol fed rats were significantly higher as compared to that of the normal diet fed rats ( p0.01). There was significant reduction in total cholesterol in the serum of rats treated with the flavonoids (p0.01). Serum HDL cholesterol was slightly lowered in the cholesterol fed rats, but treatment with both the flavonoids resulted in a significant increase in the levels of serum HDL-C as compared to the cholesterol diet fed rats (p0.01).and. Similarly the atherogenic index (Total cholesterol/ HDL-C) in the treated group of rats was also significantly lowered as compared to that of the control (p0.01). The effect of both the flavonoids on these parameters were more or less the same. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(2), 54-60, Feb. (2012) Res.J.Chem.SciInternational Science Congress Association 57 Hepatic bile acids and excretion of fecal bile acids and neutral sterols: The concentration of hepatic bile acids and the excretion of fecal bile acids and neutral sterols were estimated and the results are given in table-2. There was a significant increase in the concentration of total bile acids in the liver and also increase in the fecal excretion of bile acids and neutral sterols in the rats fed cholesterol containing diet as compared to those fed normal diet (p0.01). Feeding the flavonoids further significantly increased (p0.01) the concentrations of hepatic bile acids and the fecal excretion of bile acids and neutral sterols as compared to the control cholesterol diet fed group. The leucodelphinidin derivative showed a slightly better effect than quercetin. Synthesis of hepatic cholesterol: The synthesis of hepatic cholesterol was estimated by assaying the activity of HMG-CoA reductase (Ratio of HMG CoA/Mevalonate) and the concomitant in vivo incorporation of labeled acetate into hepatic cholesterol. These results are given in table-3. The ratio of HMG CoA/Mevalonate indicates the activity of HMG CoA reductase in a reverse order i.e., lower ratio indicating higher activity and higher ratio lower activity. Feeding of high cholesterol diet significantly decreased HMG CoA reductase activity in liver (p0.01). This was reflected in the significant decrease (p0.01) in the incorporation of labeled acetate into free and esterified cholesterol in the liver of rats fed cholesterol rich diet. The decrease in activity was restored to near normal levels on treatment with the flavonoids (p0.01) as was shown by a significant increase in the incorporation of labeled acetate into both free and esterified cholesterol in the flavonoid treated groups (p0.01).The effects of both the flavonoids on hepatic cholesterol synthesis were more or less the same. Table-1 Concentration of cholesterol in serum and lipoproteins (Mean values ± standard deviations for six rats per group) TC, total cholesterol.Second group values except HDL-C are significantly higher than the normal (p0.01) and 3 and 4group valuesare significantly lower than that of the 2nd. 3 and 4 HDL-C values are significantly higher than 2nd (p0.01) Groups TC (mg/100ml serum) Serum lipoproteins ( mg/100ml serum) HDLc LDLc Atherogenic index TC/HDLc Normal diet (Normal control) 68.2±1.21 43.2±0.50 16.6±0.23 1.57±0.006 2% Cholesterol diet (Cholesterol control) 159.2±1.91a 41.8±0.56 93.8±1.5a 3.80±0.038 a 2% Cholesterol diet+ Leucodelphinidin (100mg/kg body wt) 108.2±1.81 b 63.9±0.53 c 36.6±0.63 b 1.69±0.008 b 2% Cholesterol diet+ Quercetin (100mg/kg body wt) 106.2±1.81 b 64.9±0.53 c 34.6±0.63 b 1.63±0.009 b Table-2 Concentration of hepatic bile acids and fecal excretion of bile acids and neutral sterols. (Mean values ± standard deviations for six rats per group).Second group values are significantly higher than the normal (p0.01) and 3 and 4group values are significantly higher than that of 2nd (p0.01) Groups Hepatic bile acids ( mg/100gm wet tissue) Fecal excretion (mg/rat/day) Bile acids Neutral sterols Normal diet (Normal control) 29.2±0.12 23.6±0.16 17.6±0.11 2% Cholesterol diet (Cholesterol control) 39.4±0.21a 31.8±0.52a 43.2±0.53a 2% Cholesterol diet + Leucodelphinidin (100mg/kg body wt) 60.8±0.51 b 55.9±0.5386.1±0.73 b 2% Cholesterol diet+Quercetin (100mg/kg body wt) 57.1.2±0.42 b 52.5±0.53 c79.6±0.62 b Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(2), 54-60, Feb. (2012) Res.J.Chem.SciInternational Science Congress Association 58 Table-3 Rate of cholesterol synthesis and in vivo incorporation of (1,2-14C) acetate into hepatic cholesterol.(Mean values ± standard deviations for six rats per group).*Ratio of HMGCoA/Mevalonate is inversely proportional to HMG CoA reductase activity. Secondgroup values show significantly lower activities of HMG CoA reductase and also decreased incorporation of (1,2-14C) acetate into hepatic cholesterol. 3 and 4group values show significantly higher activities of HMG CoA reductase and also increased incorporation of (1,2-14C) acetate into hepatic cholesterol. (p0.01) Groups Ratio of HMG CoA/ Mevalonate* Free cholesterol Ester cholesterol ( count/min/g tissue) Normal diet (Normal control) 2.28±0.025 1480±17.12 390.1±3.42 2% Cholesterol diet (Cholesterol control) 3.42±0.038943.5±6.52216.2±2.50 2% Cholesterol diet + Leucodelphinidin (100mg/kg body wt) 2.33±0.029 b1325.0±10.25376.1±3.34 b 2% Cholesterol diet + Quercetin (100mg/kg body wt) 2.31±0.022 b1309.5±10.39382.6±3.69 b The results obtained in this study clearly indicate that the flavonoids studied have beneficial effects to a significant level on metabolism of rats fed cholesterol, containing diet. It is noteworthy to observe that the hypolipidaemic effect of leucodelphinidin is more or less the same as quercetin. The significant decrease observed in serum cholesterol in the rats treated with leucodelphinidin derivative and quercetin further extended an influence to decrease serum LDL-C also. Furthermore, their HDL-C showed significant increase on treatment as compared to the cholesterol diet fed rats. Studies in our laboratory and elsewhere with other ficus bioflavonoids have shown to decrease both total and LDL-C in hyperlipaedemic rats and rabbits10,19. In Germany a study in overweight subjects with a high cardiovascular risk phenotype have provided strong evidence supporting the cardioprotective properties of quercetin. Treatment with quercetin at a dose of 150 mg/d for six weeks was found to reduce systolic pressure and plasma oxidized low-density lipoprotein concentrations20. Significant decreases in serum total and LDL-C were attained in female chickens treated with quercetin21. The protective effect of flavonoids as observed in our study on HDL-C is in agreement with similar results obtained by other workers using flavonoids from green tea , and fenugreek seeds22,23. Brutieridin and melitidin, flavonoids of bergamot juice are structural analogues of statins and were shown to inhibit HMG-CoA reductase the enzyme that catalyzes conversion of HMG-CoA to mevalonate, which is the rate limiting step in the cholesterol synthesis pathway24. However, in the present study such an inhibitory effect on HMG-CoA reductase was not observed on treatment with leucodelphinidin or quercetin. Instead the increased incorporation of labeled acetate into both free and esterified cholesterol in the treated groups indicate increased hepatic cholesterogenesis. The fact that the concentration of total bile acids was significantly higher in rats treated with the ficus flavonoid and quercetin indicates an increased hepatic degradation of cholesterol to bile acids. Increased excretion of neutral sterols and bile acids was also observed indicating that more of the cholesterol is degraded in the liver and removed via bile acids in hyperlipidaemic rats treated with the flavonoids. Gallic acid, catechin and epicatechin, the major polyphenolic compounds in grape seed show cholesterol lowering activity by inhibiting pancreatic cholesterol esterase, binding of bile acids and reducing solubility of cholesterol in micelles which may result in delayed cholesterol absorption25. The hypocholesterolemic effects of Chinese green tea, silimarin and glycyrrhiza glabra root powder also have shown to be mediated via accelerated bile acid and neutral sterol elimination through feces with an increased hepatic bile acid production22,26,27. Atherosclerosis is a progressive inflammatory disease affecting large and medium-sized arteries that may manifest as coronary heart disease, cerebrovascular disease or peripheral vascular disease. Coronary artery obstruction and myocardial infarction are the number one killers in the world. Epidemiological data demonstrate that the risk for coronary heart disease and other forms of atherosclerotic vascular disease rises with plasma cholesterol possible mode of action of flavonoids in regulating hypercholesterolemia can be attributed to blocking the entero-hepatic circulation of bile acids due to increased binding to flavonoids with bile acids conjugates and their subsequent excretion. Liver cells respond to this situation by increasing the number of LDL receptors and also by increasing the rate of cholesterol synthesis. But as the inhibition of bile acid recycling may far outweigh cholesterol synthesis, the decrease in both plasma total and LDL-C is noted in hypercholesterolemic rats treated with the flavonoids. The molecular structure of flavonoids plays a decisive role in its potential as a lipid lowering agent. Studies in HepG2 cells with two polymethoxylated citrus flavonoids (PMFs), tangertin and nobiletin have shown that flavonoids with a fully methoxylated A-ring lower blood cholesterol and triacylglycerol concentrations by suppressing hepatic apoB secretion28. It is pertinent to note here that the leucodelphinidin derivative from ficus has two methoxy groups in its A-ring and further studies are warranted to relate its molecular structure with lipid lowering properties. Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(2), 54-60, Feb. (2012) Res.J.Chem.SciInternational Science Congress Association 59 The leucodelphinidin derivative did not show any toxic effects even at a high dose of 4000 mg/kg in experimental animals. The methylation of many of the hydroxyl groups and absence of toxophore group (-CO-CH=CH-O-) in the structure of leucodelphinidin might have conferred on them non-toxicity. In contrast although quercetin also showed hypocholesterolemic and related effects its consumption for humans is debatable as it belongs to a class of toxic flavonoids29. Acute toxicity studies revealed no mortality up to a dose of 4.0 g/kg body weight. Hence the LD50 may be more than 4.0 g/kg body weight. The drug may be used at ordinary doses of 50-500 mg/ kg dose level. Conclusion Our observations suggest that leocodelphinidin may be useful as a naturally occurring non-toxic hypocholesterolemic agent. Further investigations are required to evaluate the mechanisms of action of this polymethoxylated ficus flavonoid in lowering the risk of cardiovascular diseases. Acknowledgement The authors acknowledge with thanks DGHS, New Delhi for financial assistance in the form of DGHS scholarship to the first author (BCM) and ICMR, New Delhi, for Senior Research Fellowship to the second author (RSD) and also Prof P.R. Sudhakaran, Head of Department, Department of Biochemistry, University of Kerala, Trivandrum, India for offering all the facilities to carry out this work. The authors are also pleased to acknowledge Professor Sisay Yifru, Dean, Faculty of Medicine, Gondar College of Medical Sciences Ethiopia for all his help and support offered during the preparation of this article. References 1. Babalola O.O., Ojo O.E. and Oloyeda F.A., Hepatoprotective activity of aqueous extract of the leaves of Hyptis suaveolens (l) Poit on acetaminophen induced hepatotoxicity in rabbits. Research Journal of Chemical Sciences,1(7), 85-88 (2011) 2. Mathew B.C. and Daniel R.S., Effect of isoflavones on cardiovascular health: low but not out either. J. Clin Biochem.,43, 129-130 (2008) 3. Mumanocha N., Chandra S.K., Sharma V., Sangameswaran B. and Saluja M., Anti-rheumatic and antioxidant activity of extract of stem bark of Ficus bengalensis,Research Journal of Chemical Sciences, 1(2), 2-8 (2011) 4. Geetha B.S., Mathew B.C. and Augusti K.T., Hypoglycemic effects of leucodelphidin derivative isolated from Ficus bengalensis (Linn), Indian J Physiol Pharmacol., 38(3), 220-222 (1994) 5. Augusti K.T., Hypoglycemic action of bengalenoside: Aglucoside isolated from Ficus bengalensis Linn, in normal and alloxan diabetic rabbits, Indian J Physiol Pharmacol., 19, 218-220 (1975) 6. Augusti K.T., Daniel R.S., Cherian S., Sheela C.G. and Sudhakaran Nair C.R., Effect of leucope-lergonidin derivative from Ficus bengalensis Linn on diabetic dogs. Indian J Med Res., 99, 82-86 (1994) 7. Cherian S., Sheela C.G., Augusti K.T., Insulin sparing action of leucopelergonidin derivative isolated from Ficus bengalensis Linn, Indian J Expl Biol., 33, 608-611 (1995) 8. Kumar R.V., Augusti K.T., Insulin sparing action of a leucocyanidin derivative isolated from ficus bengalensis Linn, Indian J Biochem Biophys., 31(1), 73-76 (1994) 9. Daniel R.S., Mathew B.C., Devi K.S., Augusti K.T., Antioxidant effect of two flavonoids from the bark of Ficus bengalensis Linn in hyperlipidemic rats, Indian J. Exp Biol., 36, 902-906 (1998) 10. Daniel R.S., Devi K.S., Augusti K.T. and Sudhakaran Nair C.R., Mechanism of action of antiatherogenic and related effects of Ficus bengalensis Linn. Flavonoids in experimental animals, Indian J Exp Biol.,41, 296-303 (2003) 11. Augusti K.T., Anuradha S. and Prabha S.P. et al. Nutraceutical effects of garlic oil, its nonpolar fraction and a Ficus flavonoid as compared to vitamin E in CCl4 induced liver damage in rats, Indian J Exp Biol.,43(5), 437-444 (2005) 12. Prema M.S. and Misra G.S., Chemical constituents of Ficus bengalensis, Indian J Chem., 15B, 762-763 (1977) 13. Zlatkis A., Zak B. and Boyle A.J., A new method for the direct determination of serum cholesterol, J. Lab Clin Med.,41, 486-492 (1953) 14. Fossati P. and Prencipe L., Serum triglycerides determined colorimetrically with enzyme that produces hydrogen peroxide, Clin Chem.,28, 2077-2080 (1982) 15. Gidez L.I., Miller G.J., Burstein M., Slagle S. and Eder H.A., Seperation and quantitation of subclasess of human plasma high density lipoproteins by a simple precipitation procedure. J. Lipid Res.,23, 1206-1223 (1982) 16.. Menon V.P. and Kurup P.A., Effect of fiber rich polysaccharide from blackgram on cholesterol metabolism in rats fed normal and atherogenic diet. Biomedicine, 24, 248-253 (1976) 17. Rao A.V. and Ramakrishnan S., Indirect assessment of hydroxyl methyl glutaryl CoA reductase (NADPH) Research Journal of Chemical Sciences __________________________________________________________ ISSN 2231-606X Vol. 2(2), 54-60, Feb. (2012) Res.J.Chem.SciInternational Science Congress Association 60 activity in liver tissue, Clin Chem., 21, 1523-1525 (1975) 18. Zimmerman M., Ethical guidelines for investigation of experimental pain in conscious animals, Pain., 16, 109-110 (1983) 19. Shukla R., Anand K., Prabhu K.M. and Murthy P.S., Hypocholesterolemic effect of water extract of the bark of banyan tree Ficus bengalensis, Indian J Clin Biochem., 10, 14-18 (1995) 20. Egert S., Bosy-Westphal A. and Seiberi J., Quercetin reduces systolic blood pressure and plasma oxidized low-density lipoprotein concentrations in overweight subjects with high-cardiovascular disease risk phenotype: a double blinded, placebo-controlled cross-over study, Br J Nutr.,102(7), 1065-1074 (2009) 21. Qureshi A.A., Reis J.C., Qureshi N., Papasian C.J., Morrison D.C. and Schaefer D.M., -Tocotrienol and quercetin reduce serum levels of nitric oxide and lipid parameters in female chickens. Lipids in Health and Disease, 10, 39, (2011) 22. Yang T.T. and Koo M.W., Chinese tea lowers cholesterol level through an increase in fecal lipid excretion, Life Sci., 66(5), 411-423 (2000) 23. Belquith-Hadriche O., Bouaziz M., Jamussi K., El Feki A., Sayadi S. and Makni-Ayedi F., Lipid lowering and antioxidant effects of an ethyl acetate extract of fenugreek seeds on high-cholesterol-fed rats, J Agric Food Chem.,58(4), 2116-2122 (2010) 24. Leopoldini M., Malaj N., Toscano M., Sindona G. and Russo N., On the inhibitor effects of bergamot juice flavonoids binding to the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme, J. Agric Food Chem.,58(19), 10768-10773 (2010) 25. Ngamukote S., Makyen K., Thialawech T. and Adisakwattana S., Cholesterol-lowering activity of the major polyphenols in grape seed, Molecules., 16(6)5054-5061 (2011) 26. Crocenzi F.A., Pellegrino J.M., Sanchez Pozzi E.J., Mottino A.D., Garay E.A., Roma M.G., Effect of silymarin on biliary bile salt secretion in the rat, Biochem Pharmacol., 59(8), 1015-1022 (2000) 27. Visavadiya N.P. and Narasimhacharaya A.V., Hypocholesterolemic and antioxidant effects of glycyrrhiza glabra (Linn) in rats, Mol Nutr Food Res., 50(11), 1080-1086 (2006) 28. Lin Y., Vermeer M.A. and Bos W. et al., Molecular structures of citrus flavonoids determine their effects on lipid metabolism in HepG2 cells by primarily suppressing apoB secretion., J Agric Food Chem.,59(9), 4496-4503 (2011) 29. Chen X.W., Serag E.S., Sneed K.B., Zhou S.F., Herbal bioactivation, molecular targets and toxicity relevance, Chem. Biol. Interact., 192(3), 161-176 (2011)