|Year : 2012 | Volume
| Issue : 3 | Page : 236-243
Antihyperlipidemic and antioxidant activities of Bruguiera cylindrinca (L)
Vijai Lakshmi1, Ravi Sonkar2, Ashok K Khanna2
1 Department of Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow, Uttar Pradesh, India
2 Department of Biochemistry Division, Central Drug Research Institute, Lucknow, Uttar Pradesh, India
|Date of Web Publication||10-Aug-2012|
Department of Biochemistry, CSM Medical University, Central Drug Research Institute, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Atheroclerosis is the major cause of morbidity and mortality world wide. The drugs used these days have some or other side effects. Aim: Therefore it is an urgent need to develop a safe and cheaper drugs for the society. We have planned this study to investigate lipid lowering and antioxidant activities of Bruguiera cylindrinca in triton and cholesterol fed rats. Materials and Methods: Different extract and fractions were collected from B. cylindrica. Two major compounds were isolated from there and have been tested for their lipid lowering activity along with increase in high density lipoprotein,cholesterol, compared with the cholesterol fed animals. Results: On chronic feeding of active fraction and pure compounds (at 100 mg / kg b.w.) for 30 days. The pure compounds isolated from the active fraction were found showing lipid lowering activity similar to the fraction. Conclusion: Structre modification of the two pure compounds isolated from the active fraction is required for further enhacement of biological activity.
Keywords: B. cylindrica, hypolipidemic activity, lipoprptein metabolism, triton model
|How to cite this article:|
Lakshmi V, Sonkar R, Khanna AK. Antihyperlipidemic and antioxidant activities of Bruguiera cylindrinca (L). Chron Young Sci 2012;3:236-43
|How to cite this URL:|
Lakshmi V, Sonkar R, Khanna AK. Antihyperlipidemic and antioxidant activities of Bruguiera cylindrinca (L). Chron Young Sci [serial online] 2012 [cited 2019 Dec 13];3:236-43. Available from: http://www.cysonline.org/text.asp?2012/3/3/236/99596
| Introduction|| |
Atherosclerosis is the major cause of myocardial morbidity and mortality world wide. Lipoproteins are responsible for the initiation of atherosclerosis and associated disorders. Elevated level of plasma concentration of cholesterol, lipoproteins (LDL) and triglycerides are recognized as a major cause in the development of atherosclerosis and coronary heart disease , Oxidative stress starts in low density lipoproteins of the plasma by hydroxyl radicals generated by the metal ions prsent in the serum, due to alteration in their oxidation states. Currently several drugs are being used in the treatment of the dyslipidemia.  Statins are used to lower serum level, which are known or side effects, such as myositis, athralgias, gastrointestinal upset etc. Statins such as atarovastatin, atarovastatin, fluvastatin, simvastatuin and and provastatin act as a inhibitors of HMGCoA reductase enzyme.
Reductase is an enzyme involved in denovo synthesis of cholesterol and upgradation of LDL. Therefore it is required to develop drugs for the treatment of hyperlipidemia reducing their Side effects. The involvement of hydroxyl free radicals have been found to be a major causative factor for the peroxidative damage and progression of atherosclerosis in hypolipidemic subjects. 
The antioxidant compounds in a diet are mostly derived from the plant sources.  Little is known about the lipid lowering and anti oxidant potential of the mangroves. Oxygen via its transformation produces Reactive oxygen species (ROS) such as superoxide, hydroxy radicals and hydrogen peroxide.
Free radicals attack and induce oxidative damage to various biomolecules including proteins, lipids, lipoproteins and DNA. , The body possesses several defense systems comprising enzymesand radical scavengers.  Antioxidants act as a inhibitors in the oxidation process and are found to inhibit oxidative chain reactions at a small concentrations and eliminate the threat of pathological processes.  Phenolic compounds present in medicinal plants have shown powerful antioxidant property.  The antioxidant compounds in a typical diet are mostly derived from plant sources and polyphenolic components of higher plants which act anti-carcinogenic action.  Apart from antioxidant studies, the present study also involves evalutation of antihyperlipidemic activity.
| Materials and Methods|| |
Collection of plant material
Stems with leaves of B. cylindrica were collected from the South Andaman Coast of India in the month of march 2007 by the botany division of the Institute. The voucher specimen has been preserved in the botanydivision with a voucher specimen number 484.
Extraction and fractionation procedure
The air dried powdered plant material (1.0 kg) was extracted with 95% ethanol four times (4×4.0l) and the combined ethanol extact was filtered, concentrated under reduced pressure below 50°C to get agreen viscous mass,which was finally dried under high vacum to get the ethanol extract (30.0 g). The plant material left after the extraction with 95% ethanol, was further extracted with 50% aqueous ethanol, three times (3×4.0Lit.) and the combined extract was concentrated under reduced pressure below 50°to get viscous residual which was dried under high vacum for 2 hours toget the 50% aqueous ethanol extract (12.0 g,). Both of these extracts showed promising lipid lowering activity,therefore ethanol extract (20.0 g) was fractionated into four fractions by maceration with hexane, chloroform and n-butanol successively and each of these were concentrated under reduced pressure below 50° to get each of the residual masses. Hexane soluble (1.5 g), chloroform soluble (2.6 g), n-butanol soluble (5.6 g) respectively and the n-butanol butanol insoluble fraction (10.3 g). On chromatography of the hexane and chloroform fractions over the plates of silicagel, these two showed similar tlc pattern, therefore were mixed together and chromatographed over a column of silicagel. Only two pure compounds could be isolated in large amounts, which were bio-evaluated. These compounds were identified as lupeol (1) and β-amyrin (2) by comparision of physicochemical data with those reported in the literature , and co-tlc with the authentic samples.
Spectral data of compound (1)
White crystalline powder; mp 212°C, IR (KBr) cm- 1 3324, 2930, 1630, 1452, 1038, 874; EIMSm/z 425 [M+ - H], 409 [M+ - OH] 218, 207-and 189, 1 HNMR(CDCl3, 300 MHz) δvalues, 0.79, 0.89, 0.90, 0.95, 0.99, 1.07 and 1.70, (s, for six methyls) 2.42 (1H,dt, J= 3.5 and 9.6 Hz, H-19), 3.19 (1H, dd, J=4.6 and 11.6 Hz,H-3), 4.57 (1H, brs, H-29), 4.68 (1H, brs, H-29); 13 C NMR(CDCl3, 75 MHz) δ values, 39.4, 27.6, 79.5, 39.6, 55.3, 18.4, 34.7, 41.2, 50.7, 37.5, 21.3, 25.5,38.4, 43.2, 27.8, 35.9, 43.4, 48.3, 48.6, 151.1, 30.2, 40.4, 28.4, 15.4,16.6, 16.9, 14.9, 18.4, 109.5, 19.6.
Spectral data of compound (2)
EI-MSm/z 426 [M] + ; 411[M + -CH3], 408 [M + -H2O] , 393 [M + -H2O- CH3], 1 HNMR(300 MHz, CDCl3) δ values: 0.79 (6H,s,Me-28, 30) , 0.84 (3H,s, Me-29), 0.86,(3H,s Me-25), 1.0, (3H,s, Me- 23)1.02, (3H,s, Me-24) 1.05, (3H s,Me-26), 1.17, (3H s,Me-27) of eight tertiary methyls 3.24 (1H,dd, J=5,11 Hz geminal to secondary alcoholic group). 13 C- δ values; 15.8, 15.9, 16.8, 18.6, 23.2,23.6, 26.2, 26.3, 26.8, 27.2, 28.4, 28.6, 31.0, 32.5, 32.8, 33.4, 34.6, 36.7, 37.2, 38.5, 38.8, 39.6, 41.4, 46.4, 47.4, 47.6, 55.3, 79.0, 121.9, 145.4.
Male adult rats of charles foster strain (150-200 g) bred in the animal house of the institute and were kept in a room with controlled temperature at 25-26°C , humidity 60 -80% and 12/12 hourslight/darkcycle, light from 8.00-20.00 underhygenic conditions. Animals were acclimatized for one week before startingthe experiment. The animal had free access to the normal diet and water adlibitum.
Lipid lowering activityina triton induced hyperlipidemic rat model
Rats were divided into 9 groups control, triton treated, triton plus extracts, fractions and compounds and a standard drug Gemfibrozil (100mg/kg) treated groups with six animals ineach group. In the 18 h acute experiment, hyperlipidemia was developed by administration of triton WR-1339 (Sigma Chemical Company, st Louis, M.O, U.S.A) at a dose of 400mg/kg b.w. intraperitoneally to animals in all groups except the control. The extracts and the fractions of B. cylindrica and Gemfibrozil were macerated with 0.2% w/w aqueousgumacacia and fed orally at a dose of 200mg/ kg simultaneously with triton plusdrug treated groups, and diet was withdrawn. Animals in the control and triton group without treatment with extracts and fractions were given the same amount of gumacacia suspension (vehicle). After 18 h of treatment the animals were anaesthetized with thiopentone solution (50 mg/kg) prepared in normal saline and 1.0 ml blood was withdrawn from retro orbital pluxus using glass capillaryin EDTA - coated eppendorf tubes (3.0 mg/ml blood). The blood was centrifuged at 25000 psi for 10 min at 4°C and the plasma was separated. The plasma was diluted with normal saline at a ratio of 1:3 and used for the analysis of total cholesterol (TC) phospholipids (PL) and triglycerides (TG) by standard enzymatic methods ,, using an auto analyzer (Beckmann Coulter Synchron- CX 5 clinical system, U.S.A.) Kits were purchased from Beckmann Coulter International, U.S.A.) and post-heparin lypolytic activity (PHLA) were assayed  using spectrophotometer.
Antioxidant activity (Generation of free radicals)
Superoxide anions were generated enzymatically  by Xanthine (160 mM), xanthine oxidase (0.04 μM) and nitroblue tetrazolium (320 μM) in the absence or presence of compounds (1,2) of compounds (100μg and 200μg/ml) in 100 mM phosphate buffer. The reaction mixtures were incubated at 37°C and after 30 minutes the reaction was stopped by adding 0.5ml glacial acetic acid. The amount of formazone was measured at 560 nm on a spectrophotometer. Percentage inhibition was calculated taking absorption coefficient of formazone as 7.2×10 3 M/cm. in another set of experiment, an effect of compounds on generation of hydroxyl radicals (OH•) was also studied by nonenzymic reactants. Briefly OH• was also studied by nonenzymic system comprised of deoxy- ribose (2.0 mM) FeSO4. 7H2O (2.8 mM) sodiumcarbonate (2.0 mM) and H2 O 2 (2.8mM) in 50 mM KH2PO4 buffer, pH 7.4 toa final volume of 2.5 ml. The above reaction mixtures in the absence or presence of compounds (100 μg/ml and 200 μg/ml) were incubated at 37°C for 90 minutes. Reference samples and reagent blanks were also run simultaneously. Malondialdehyde (MDA) content in both experimental and reference samples were estimated spectrophotometrically by thiobarbituric acid method. 
Cholestrol induced hyperlipidemia
Hyperlipidemia was induced similarly in animals as before.Extracts and fractions were fed orally (100 mg/kg,b.w.) simultaneously with cholesterol in the drug treated groups. Control animals received the same amount of normal saline or ground nut oil. At the end of the experiments ratswere fasted overnight and blood was withdrawn. The animals were killed and their liver were promptlyexcised.
Biochemical analysis of plasma/serum
Serum from the second set of experiments with cholesterol induced hyperlypemia were analyzed for their TC, PL, TG and protein bystandard procedure.
Biochemical analysis of tissue
Liver was homogenised (10% w/v) in cold 10 mM phosphate buffer, pH 7.2 and used for the analysis of total lipolytic activity,  the lipid homogenate was used for the estimation of TC, PL, TG and protein.
Faecal bile acid
The rats faeces were collected from all groups over 30 days and processed for the estimation of cholicacid and de- oxycholic acid. 
Data was analyzed using t-test. Hypolipidemic groups were compared with control and extracts/fractions treated hyperlipidemic. (P<0.05) was considered a significant difference.
| Results and Discussion|| |
Effect of extracts/fractions in triton induced hyperlipidemia
Administration of triton WR-1339 caused a marked increase in the levels of serum lipids of TC (3.32 fold), PL (2.74 fold), TG (2.91 fold) and protein (2.07 fold). After treatment with the extracts/fractions of B. cylindrica, the ethanol extract and its five fractions as well as pure compounds showed a significant reversal in TC (15-31%), PL (18-31%) and TG (14-32%) and protein (12-34%) respectively against triton [Table 1]. It is obvious from this study some fractions did inhibit cholesterol biosynthesis and potentiates the activity of lypolitic enzymes toan early clearance of lipids from the circulation in triton induced hyperlipidemia. We have successfully used this model for the evaluation of the lipid lowering activity of natural products. ,,,
|Table 1: Lipid lowering activity of crude extract/fractions/pure compounds in triton treated hyperlipemic rats|
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Effect of extract/fractions/pure compounds in cholesterol induced hyperlipidemia
Cholesterol feedingto rats suppressed PHLA (33%) and plasma LCAT activity (51%) as well as increased their serum levels of TC (1.62 F), PL (1.56F), TG (1.48F) and protein (1.44F); a significant lowering in B. cylindrica crude extract and its five fractions showed reversal of TC (16-28), PL (26- 39), TG (13-24%) and protein (12-19%). These lipid and protein level followed by reactivation of plasma enzymes was observed after treatment with crude extract, fractions and pure compounds [Table 2] analysis of hyperlipaemic serum showed a marked increased in the levels of lipids and apoproteins constituting β - lipoproteins and these effects were pronounced for VLDL-TC(4.10F), VLDL-PL(2.0 F) and VLDL -TG(2.23 F). Treatment with the crude extract, fractions and purecompounds of the B. cylindrica significantly reduced these levels of VLDL lipids TC, PL, TGby (5-33%, 11-36%, 21-32%) respectively as well as LDL-TC (15-29%), PL (12-28%), TG (19-34%) and apo- LDL (9- 35%) respectively. In hyperlipidemic animals, lipid and apoprotein constituents of HDL were shown to be decreased (23%) and were partially recovered after marine fractions 9-26%) treatment [Table 2]a-c. The data in [Table 2]a-c. Show that cholesterol induced hyperlipidemia in rats caused inhibition of hepatic lipolytic activity (-47%). Treatment with B. cylindrica crude extract, its five fractions and its pure compounds significantly restored the enzyme activity (8-22%). The increased levels of TC, PL and TG in liver (1.62, 1.56, 1.48 fold) respectively of cholesterol fed rats were lowered by their treatment with B. cylindrica crude extract, its five fractions and pure compounds. These marine fractions enhances marine fractions enhances the synthesis of LDL as well as receptor protein and inhibit the oxidative modification of LDL to accelerate the turnover of LDL- in liver.
Marine fractions may also inhibit the HMG-CoA reductase, the rate limiting enzyme in the hepatic cholesterol biosynthesis. Feeding with cholesterol results in the expansion of cellular cholesterol pools within hepatocytes. 
The abnormalities in the synthesis and the catabolism of the body lipids are closely related to LCAT deficiency and hepatic dysfunction in hypoglycemia.
LCAT, a key enzyme involved in body lipid metabolism,solely synthesized in liver and therefore hepatoprotective action of marine fractions may contribute to improve liver function, reactivation of LCAT and thus the regulation of lipid in experimental animals. Enhancement of faecal excretion of holicacid (7-25%) and deoxycholic acidby (13- 36%) by treatment with B. cylindrica crude extract and its fractions [Table 3] indicate that this drug interfered with the absorption of dietary cholesterol in the small intestine.
|Table 3: Effect of crude extract/fractions/pure compounds on hepaticlipid, lipoprotein lipase and faecal bile acid activity in hyperlipemic rats|
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Effect of fractions on oxygen free radical generation in vitro
The Scavenging potential of B. cylindrica crude extract and its fractions and pure compounds (100 and 200 μg/ml) against formation of O -2 and OH• in non- enzymic systems was studied [Table 4]. The significant decrease in superoxide anions by (18-77%) inhibition and hydroxylradical by (29-43%) inhibition and in microsomallipid peroxidation showed more antioxidant activity (20-39%) inhibition and in above test system the involvement of hydroxyl free radicals (OH• ) hasbeen found to be a major causative factors for peroxidative damage to lipoproteins.  To overcome these ailments, adrughaving multifold properties such as lipid lowering, antidiabetic and antioxidant activities together is in great demand.
|Table 4: Effect of crude extract/fractions/pure compounds on superoxideion, hydroxyl radical an lipid|
peroxidation in microsomes
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| Conclusion|| |
The pure compounds isolated from the active fraction were found showing lipid lowering activity similar to the fraction. Further work is required to synthesize the analogs of pure compounds to enhance the activity.
| Acknowledgments|| |
We are thankful to the Director CSIR-CDRI, Lucknow for providing necessary research facilities as well as his keen interest in marine natural products. We are grateful to the Ministry of Earth Sciences, Government of India, New Delhi for providing financial assistance.Weare also thankful to HRDG, Council of Scientific and Industrial Research,Government of India, New Delhi for providing to VL emeritus scientistship which helped in compiling the work. We acknowledge to Dr. M. N. Srivastava for the collection of marine material to carry out the present studies.
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[Table 1], [Table 2], [Table 3], [Table 4]