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|Year : 2008 | Volume
| Issue : 4 | Page : 152--157
The effect of aqueous extract of Embelia ribes Burm on serum homocysteine, lipids and oxidative enzymes in methionine induced hyperhomocysteinemia
Uma Bhandari1, M Nazam Ansari1, F Islam2, CD Tripathi3,
1 Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi, India
2 Department of Toxicology, Faculty of Science, Hamdard University, New Delhi, India
3 Department of Pharmacology, Vardhman Mahavir Medical College, Safdarjung Hospital, New Delhi, India
Department of Pharmacology, Faculty of Pharmacy, Hamdard University, New Delhi
Objective: The present study was designed to evaluate the effect of the aqueous extract of Embelia ribes Burm fruits on methionine-induced hyperhomocysteinemia, hyperlipidemia and oxidative stress in albino rats.
Materials and Methods: Adult male Wistar albino rats were fed with the aqueous extract of Embelia ribes (100 and 200 mg/kg, p.o.) for 30 days. Hyperhomocysteinemia was induced by methionine treatment (1 g/kg, p.o.) for 30 days and folic acid (100 mg/kg, p.o.) was used as a standard drug. The animals were evaluated for various biochemical parameters in serum and brain homogenates, followed by histopathological studies at the end of the study.
Results: Administration of methionine (1 g/kg, p.o.) for 30 days to vehicle control rats produced significant increase (P <0.01) in homocysteine, lactate dehydrogenase (LDH), total cholesterol, triglycerides, low density lipoprotein (LDL-C), very low density lipoprotein (VLDL-C) levels in serum and lipid peroxides (LPO) levels in brain homogenates, with reduction in high density lipoprotein (HDL-C) levels in serum, and glutathione (GSH) content in brain homogenates, as compared to vehicle control rats. Administration of the aqueous extract of Embelia ribes (100 and 200 mg/kg, p.o.) for 30 days, to hyperhomocysteinemic rats, significantly (P <0.01) decreased the levels of homocysteine, LDH, total cholesterol, triglycerides, LDL-C and VLDL-C and increased the HDL-C levels in serum. In addition, a significant (P <0.01) decrease in LPO levels with increase in GSH content was observed in hyperhomocysteinemic rats treated with the aqueous extract of Embelia ribes. The results were comparable to those obtained with folic acid, a standard antihyperhomocysteinemic drug.
Conclusion: The present results provide clear evidence that the aqueous extract of Embelia ribes treatment enhances the antioxidant defense against methionine-induced hyperhomocysteinemia, hyperlipidemia and oxidative stress in brain.
|How to cite this article:|
Bhandari U, Ansari M N, Islam F, Tripathi C D. The effect of aqueous extract of Embelia ribes Burm on serum homocysteine, lipids and oxidative enzymes in methionine induced hyperhomocysteinemia.Indian J Pharmacol 2008;40:152-157
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Bhandari U, Ansari M N, Islam F, Tripathi C D. The effect of aqueous extract of Embelia ribes Burm on serum homocysteine, lipids and oxidative enzymes in methionine induced hyperhomocysteinemia. Indian J Pharmacol [serial online] 2008 [cited 2023 Feb 3 ];40:152-157
Available from: https://www.ijp-online.com/text.asp?2008/40/4/152/43161
Hyperhomocysteinemia is a risk factor for coronary atherosclerotic vascular disease, stroke, and venous thrombosis. It has been associated with Alzheimer's disease and vascular dementia. ,,, Although its pathophysiological mechanisms are complex and not fully understood, much evidence suggests that hyperhomocysteinemia induces vascular and brain damage because of the highly reactive thiol group in homocysteine, which is readily oxidized, leading to the formation of homocysteine, homocysteine mixed disulfides and homocysteine thiolactone. During these oxidative processes, several reactive species are generated.  The methionine cycle is responsible for the formation of homocysteine in the body.
Homocysteine is toxic sulfur containing intermediate amino acid, which results from intracellular conversion of methionine to cysteine, which can induce neuronal dysfunction and cell death. Homocysteine has now been implicated in increased oxidative stress, DNA damage, triggering of apoptosis and excitotoxicity, all important mechanisms in neurodegeneration.  The brain may be particularly vulnerable to high levels of homocysteine in the blood because it lacks two major metabolic pathway for its elimination - betain remethylation and transsulfuration.  Homocysteine is proposed to cause oxidative stress related neurotoxicity  and natural antioxidants play a protective role in hyperhomocysteinemia;  however, there is a paucity of experimental as well as clinical studies which indicates the protective role of medicinal plants in hyperhomocysteinemia.
Embelia ribes Burm, commonly known as 'Vidanga', is a large woody climbing shrub that belongs to the family, Myrsinaceae , which is widely distributed in India, Sri Lanka, Malaysia and South China.  It is highly valued in Ayurveda as a powerful anthelmintic.  In a preliminary study, Tripathi  has demonstrated the blood glucose lowering activity of the decoction of the Embelia ribes fruits in glucose-fed albino rabbits. Bhandari et al . , have reported the diabetic dyslipidemic and antioxidant activity of Embelia ribes Burm in streptozotocin-induced diabetic rats, using gliclazide as a positive control drug. Recently, Bhandari et al .  have reported the cardioprotective activity of the aqueous extract of Embelia ribes, in isoproterenol induced myocardial infarction in albino rats.
However, till date, antihyperhomocysteinemic activity has not been carried out on the aqueous extract of Embelia ribes . Therefore, the present study was undertaken to determine the antihyperhomocysteinemic and antihyperlipidemic activity of the aqueous extract of Embelia ribes in albino rats.
Materials and Methods
Drugs and chemicals
Methionine and folic acid were obtained from Central Drug House, Bombay. All the other chemicals that were used were of analytical grade. Double distilled water was used for all biochemical assays.
Preparation of aqueous extract of Embelia ribes Burm
Dried fruits of Embelia ribes Burm were purchased from the local market, New Delhi, India, in October 2006 and the botanical authentification was carried out by the Department of Botany, Faculty of Science, Hamdard University, New Delhi, India (voucher specimen no. UB 2). The dried and coarsely powdered drug (100 g) was packed in a soxhlet apparatus and was subjected to extraction with water for 72 hours. The filtrate was evaporated under a vacuum drier (Narang Scientific Works Pvt. Ltd., New Delhi, India) and the brown mass residue that was obtained was stored at 4 0 C, for further use. The average yield of the aqueous extract of Embelia ribes was approximately 5.26%. For experimental study, the weighed amount of aqueous extract of Embelia ribes (100 and 200 mg/kg) was dissolved in 1% Tween 80 in distilled water.
Standardization of extract
Preliminary phytochemical screening of the aqueous extract of dried fruits of Embelia ribes was carried out for the detection of phytoconstituents, using standard chemical tests. Alkaloids, carbohydrates, phenolic compounds, flavonoids, proteins and saponins were detected in the extract. HPTLC fingerprints of the aqueous extract was established using CAMAG HPTLC (WinCAT software, version 2.2) and benzene: ethyl acetate (6:4) as the solvent system, which showed the presence of seven spots (R f values: 0.32, 0.34, 0.42, 0.45, 0.52, 0.65 and 0.78) at 520 nm wavelength.
Male adult Wistar albino rats, weighing 200-250 g, procured from the Central Animal House Facility, Hamdard University, New Delhi, were used. The Institutional Animal Ethics Committee (IAEC) approved the present research. Animals were acclimatized under standard laboratory conditions at 25 0 ± 2 0 C, 50 ± 15% RH and normal photoperiod (12 h light : dark cycle) for seven days. The animals were fed with commercial rat pellet diet and water ad libitum .
Methionine-induced hyperhomocysteinemia in rats
The animals were randomly divided into seven groups of ten animals each and treated as follows: Group I served as vehicle control and received only 1% tween 80 in distilled water (2 ml/kg, p.o.) for 30 days. Group II served as pathogenic control and received methionine (1 g/kg, p.o.)  for 30 days. Groups III and IV were administered aqueous extract of Embelia ribes at doses of 100 and 200 mg/kg respectively and co-treated with methionine (1 g/kg, p.o.) for 30 days. Group V served as positive control and received folic acid (100 mg/kg, p.o.)  and co-treated with methionine (1 g/kg, p.o.) for 30 days. Groups VI and VII were administered aqueous extract of Embelia ribes alone at doses of 100 and 200 mg/kg respectively, for 30 days. The blood samples were collected by the retro-orbital plexus using micro-capillary technique from all the groups of rats that were made to fast overnight. Serum was separated for biochemical estimations.
In serum, homocysteine levels were estimated using the Fluorescence Polarization Immunoassay (FPIA) method described by Primus et al. (1988). UV spectrophotometric method of analysis was used for the estimation of LDH (Lum and Gambino, 1974), total cholesterol (Demacher and Hijamaus, 1980), triglycerides (Foster and Dunn, 1973) and HDL-C (Burstein et al ., 1970). Commercial diagnostic kits from SPAN Diagnostics, Udhna, Surat, India were used for cholesterol and triglycerides estimation. Lactate dehydrogenase (LDH) catalyses the oxidation of lactate to pyruvate, accompanied by a simultaneous reduction of NAD to NADH. Lactate dehydrogenase (LDH) activity in serum is proportional to the increase in absorbance, due to the reduction of NAD. Lactate dehydrogenase (LDH) and HDL-C levels were estimated using commercial diagnostic kits from Reckon Diagnostics Pvt. Ltd. Baroda, India. LDL-C and VLDL-C levels were calculated from the formula of Friedward et al . (1972) as given below:
LDL-C = total cholesterol - HDL-C - VLDL-C
VLDL-C = triglycerides / 5
The animals were sacrificed by cervical dislocation and brain tissues were dissected for biochemical estimation and for histopathological studies. Homogenate (10%) of whole brain tissue in ice cold KCl (0.15 M) was used for the assay of the malondialdehyde, according to the method of Ohkawa et al . (1979). In phosphate buffer (0.1 M, pH 7.0), it was used for the assay of glutathione (GSH) content (Sedlak and Lindsay, 1968). Lipid peroxides (LPO) was measured by estimating thiobarbituric acid reactive substances (TBARS) i.e. malondialdehyde (MDA), and glutathione assay was based on the reaction with DTNB i.e. DTNB (5,5'-dithiobis- (2-nitrobenzoic acid)) is reduced by -SH group to form one mole of 2-nitro-5-mercaptobenzoic acid (yellow color).
At the end of the experiment, whole brain tissues from all the groups were subjected to histopathological studies. The tissues were fixed in formalin (10%), routinely processed and embedded in paraffin wax. Paraffin sections (5mm thick) were cut on glass slides and stained with hematoxylin and eosin (H & E), after dewaxing, and examined under a light microscope.
The results are expressed as mean±SEM. Statistical differences between means were determined by one-way analysis of variance (anova), followed by Dunnett t-test. Values of P Embelia ribes was carried out and alkaloids, carbohydrates, phenolic compounds, flavonoids, proteins and saponins were detected in the extract. HPTLC fingerprints of aqueous extract showed the presence of seven spots (R f values: 0.32, 0.34, 0.42, 0.45, 0.52, 0.65 and 0.78) at 520 nm wavelength.
Significant ( P Embelia ribes (group III and IV) significantly ( P P P Embelia ribes (group III and IV) and folic acid (group V) treatment, the above mentioned parameters were regained significantly ( P P Embelia ribes (group III and IV) and folic acid (group V) treatment in methionine-induced hyperhomocysteinemic rats significantly ( P Embelia ribes (100 and 200 mg/kg) treated groups showed normal fibrillary background and absence of degenerative changes or necrosis. The neuronal cell morphology and tissue architecture was retained [Figure 1C] and [Figure 1D]. Photomicrographs of folic acid (100 mg/kg) treated group showed normal fibrillary background, without any pathological symptoms [Figure 1E]. Photomicrographs of the aqueous extract of Embelia ribes (100 and 200 mg/kg) per se groups showed normal fibrillary background and neuronal cell morphology [Figure 1F] and [Figure 1G].
Hyperhomocysteinemia has recently emerged as an independent risk factor for the development of coronary, cerebrovascular and peripheral arterial occlusive disease. Elevated homocysteine promotes atherosclerosis through increased oxidative stress, impaired endothelial function and induction of thrombosis. The role of oxidative stress in neurodegeneration has been intensively studied. Oxidative stress was one important mechanism for homocysteine toxicity in neuronal cells.  Antioxidant treatment restores several toxic effects of homocysteine. 
Bhandari et al., reported the diabetic dyslipidemic and antioxidant activity of Embelia ribes Burm in streptozotocin-induced diabetes in rats, using gliclazide as a positive control drug. In the present study, we examined the homocysteine and lipid lowering potential of the aqueous extract of Embelia ribes (100 and 200 mg/kg, p.o.) in methionine-induced hyperhomocysteinemia and hyperlipidemia in rats.
Several potential mechanisms underlying the deleterious effect of homocysteine in the brain have been proposed, which include oxidative stress, alterations in DNA methylation and activation of the excitotoxic NMDA receptors.  Hyperhomocysteinemia may promote the generation of reactive oxygen species (ROS) such as H 2 O 2 and hydroxyl radicals via the auto-oxidation of sulfhydryl (-SH) group  or by decreasing the intracellular levels of GSH that are involved in the elimination of free radicals. Homocysteine, a thiol containing amino acid derived from demethylation of dietary methionine, may generate partially reduced ROS that are able to stimulate the lipid peroxidation involved in the atherosclerotic process. Thus, an imbalance in dietary methionine may contribute to the development of atherosclerosis by increasing homocysteine levels. 
The data in our present study showed that methionine (1 g/kg, p.o.) treatment for 30 days in pathogenic control group (Group II) rats significantly ( P 
Free radicals generated by hyperhomocysteinemia, initiate lipid peroxidation of the membrane bound polyunsaturated fatty acids, leading to the impairment of the structural and functional integrity of the membrane.  This concurs with the present findings, wherein the levels of LPO were found to be significantly ( P 
In the present study, elevated levels of homocysteine, LDH, total cholesterol, triglycerides, LDL-C and VLDL-C in serum and LPO in brain homogenates were reduced significantly ( P Embelia ribes . Further, there was a decrease in the atherogenic index values, suggesting antihyperhomocysteinemic and antihyperlipidemic potential of Embelia ribes . Further, the levels of HDL-C in serum and GSH in brain homogenates were increased significantly ( P Embelia ribes in both the doses favorably modified various biochemical markers in methionine-induced hyperhomocysteinemic rats significantly ( P Embelia ribes is reported to contain embelin, quercitol (polyphenol), tannins and alkaloids,  which may contribute to its antioxidant activity. In the present study, on standardization of the aqueous extract of Embelia ribes, it was found to contain alkaloids, carbohydrates, flavonoids, phenolic compounds, proteins and saponins. It can thus be concluded that the antioxidant effect of the aqueous extract of Embelia ribes can be due to the content of alkaloids, flavonoids, phenolic compounds and saponins.
Lastly, the light microscopic observations of the brain tissues of the aqueous extract of Embelia ribes treated animals exhibited near normal pattern, thereby further supporting its role as a promising neuroprotective agent.
The present results provide clear evidence that aqueous extract of Embelia ribes treatment enhances the antioxidant defense against methionine-induced hyperhomocysteinemia, hyperlipidemia and oxidative stress in brain. On the basis of above findings, it can be assumed that aqueous extract of Embelia ribes could be a potential source for a novel discovery for central nervous system disorders.
The research project was supported by the project grant to Dr. Uma Bhandari by All India Council of Technical Education, New Delhi, India.
|1||Kang SS, Wong PW, Malinow MR. Hyperhomocysteinemia as a risk factor for occlusive vascular disease. Annu Rev Nutr 1992;12:279-98.|
|2||Loscalzo J. The oxidant stress of hyperhomocysteinemia. J Clin Invest 1996;98:5-7.|
|3||Rodrigo R, Passlacqua W, Araya J, Orellana M, Rivera G. Homocysteine and essential hypertension. J Clin Pharmacol 2003;43:1299-306.|
|4||Faraci FM, Lentz SR. Hyperhomocysteinemia oxidative stress and cerebral vascular dysfunction. Stroke 2004;35:345-7.|
|5||Mattson MP, Shea TB. Folate and homocysteine metabolism in neural plasticity and neurodegenerative disorders. Trends Neurosci 2003;26:137-46.|
|6||Finkelstein JD. The metabolism of homocysteine: Pathways and regulation. Eur J Pediatr 1998;157:S40-4.|
|7||Lipton SA, Kim WK, Choi YB, Kumar S, D'Emilia DM, Rayudu PV, et al. Neurotoxicity associated with dual actions of homocysteine at the N-methyl D-aspartate receptor. Proc Natl Acad Sci U S A 1997;94:5923-8.|
|8||Nappo F, De Rosa N, Marfella R, De Lucia D, Ingrosso D, Perna AF, et al. Impairment of endothelial functions by acute hyperhomocysteinemia and reversal by antioxidant vitamins. JAMA 1999;281:2113-8.|
|9||Guhabakshi DN, Sensarma P, Pal DC. A Lexicon Medicinal Plants of India. Calcutta, India: Naya Prakashan; 2001. p. 135-6.|
|10||Hordegen P, Cabaret J, Hertzberg H, Langhans W, Maurer V. In vitro screening of six anthelmintic plant products against larval Haemonchus contortus with a modified methyl-thiazolyl-tetrazolium reduction assay. J Ethnopharmacol 2006;108:85-9. |
|11||Tripathi SN. Screening of hypoglycemic action in certain indigenous drugs. J Indian Med Yoga Homeopathy 1979;14:159-69.|
|12||Bhandari U, Kanojia R, Pillai KK. Effect of ethanolic extract of Embelia ribes on dyslipidaemia in diabetic rats. Int J Exp Diab Res 2002;3:159-62.|
|13||Bhandari U, Jain N, Pillai KK. Further studies on antioxidant potential and protection of pancreatic β-cells by Embelia ribes in Experimental diabetes. Exp Diab Res 2007;2007:1-6. |
|14||Bhandari U, Ansari MN, Islam F. Cardioprotective effect of aqueous extract of Embelia ribes Burm fruits against isoproterenol- induced myocardial infarction in albino rats. Indian J Exp Biol 2008;46:35-40.|
|15||Yen CH, Lau YT. Vascular responses in male and female hypertensive rats with hyperhomocysteinemia. Hypertension 2002;40:322-8.|
|16||Kapoor P, Bhandari U. To investigate the effect of curcumin on methionine- induced hyperhomocysteinemia in rats. Thesis for M. Pharm (Hamdard University); 2006.|
|17||Omenn GS, Beresford SA, Motulsky AG. Preventing coronary heart disease: B vitamins and homocysteine. Circulation 1998;97:421-4.|
|18||Ho PI, Collins SC, Dhitavat S, Ortiz D, Ashline D, Rogers E, et al. Homocysteine potentiates betaamyloid neurotoxicity: Role of oxidative stress. J Neurochem 2001;78:249-53.|
|19||Jara-Prado A, Ortega-Vazquez A, Martinez-Ruano L, Rios C, Santamaria A. Homocysteine-induced brain lipid peroxidation: Effects of NMDA receptor blockade, antioxidant treatment, and nitric oxide synthase inhibition. Neurotox Res 2003;5:237-43.|
|20||Heinecke JW, Rosen H, Suzuki LA, Chait A. The role of sulfur-containing amino acids in superoxide production and modification of low density lipoprotein by arterial smooth muscle cells. J Biol Chem 1987;262:10098-103.|
|21||Toborek M, Kopieczna-Grzebieniak E, Drózdz M, Wieczorek M. Increased lipid peroxidation as a mechanism of methionine-induced atherosclerosis in rabbits. Atherosclerosis 1995;115:217-24. |
|22||Mehta K, Balaraman R, Amin AH, Bafna PA, Gulati OD. Effect of fruits of Moringa oleifera on the lipid profile of normal and hypercholesterolaemic rabbits. J Ethnopharmacol 2003;86:191-5.|
|23||Ajitha M, Rajnarayana K. Role of oxygen free radicals in Human Disease. Indian Drugs 2001;38:545-54.|
|24||Flohe L. The selenoprotein glutathione peroxidase. In: Dolphin D, Poulson R, Avramsvic O, editors. Glutathione: Chemical, Biochemical and Medical Aspects. John Wiley & Sons, New York 1989.|
|25||Krishna S, Verma KS. Embelia ribes. Indian Forest Bulletin 1941; N.S. No. 102.|