Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 38, Num. 2, 2006, pp. 131-132

Indian Journal of Pharmacology, Vol. 38, No. 2, March-April, 2006, pp. 131-132

Research Letter

Antioxidant potential of methanolic extract of Dolichos biflorus Linn in high fat diet fed rabbits

A. Kottai Muthu, S. Sethupathy*, R. Manavalan, P.K. Karar

Department of Pharmacy, Annamalai University, Annamalai Nagar. *Division of Biochemistry,Rajah Muthiah Medical College, Annamalai University, Annamalai Nagar-608002. India E-mail: arthik03@yahoo.com
Correspondence Address:Department of Pharmacy, Annamalai University, Annamalai Nagar, arthik03@yahoo.com

Code Number: ph06032

Dolichos biflorus Linn (Fabaceae), is commonly known as Kollu in Tamil and horse gram in English. It has been reported to lower lipids in rats.[1] A high fat diet induces oxidative stress in the cells by producing reactive oxygen species. Therefore, in this study, the influence of the Dolichos biflorus extract on high fat diet (HFD) induced oxidative stress in rabbits, has been investigated.

Whole plants of D. biflorus were collected from Sankaran Koil, Tirunelveli district of Tamilnadu, India. Taxonomic identification was made by the Botanical Survey of Medicinal Plant Unit, Siddha, Government of India, Palayamkottai, Tamilnadu. Four month-old whole plants were dried in the shade, segregated, and pulverized by a mechanical grinder and passed through a 40 mesh sieve. The powder was extracted by methanol in Soxhlet apparatus by continuous hot percolation method. After filtration through Whatmann filter paper No 40, the filtrate was vacuum dried at 35 to 40oC. The extracts were stored in screw cap vials at 4oC until further use. The extractive value of the methanolic extract was 8.13% w/w. The methanolic extract of D.biflorus was subjected to preliminary phytochemical screening to find out the presence of active principles. The extracts were suspended in 2% tween 80.

New Zealand white rabbits, weighing 900-1050 g were procured from the central animal house, Rajah Muthiah Medical College, Annamalai University. The animals were kept in cages, 2 per cage, with 12:12 h light/dark cycle at 25 + 2oC. The animals were maintained on their respective diets and water ad libitum . Animal ethical committee′s clearance was obtained for the study.

Rabbits were divided into the following five groups, with six rabbits in each group:

Group I : (Control): Standard chow diet.
Group II : High fat diet (HFD)
Group III : High fat diet plus methanolic extract of D.biflorus (dose I -200 mg/kg body weight)
Group IV : High fat diet plus methanolic extract of D.biflorus (dose II -400 mg/kg body weight)
Group V : High fat diet plus standard drug atorvastatin (1.2 mg/kg body weight).

The compositions of the two diets were as follows:

Control diet
Wheat flour 22.5%, roasted Bengal gram powder 60%, skimmed milk powder 5%, casein 4%, refined oil 4%, salt mixture with starch 4%, and vitamin and choline mixture 0.5%.

High fat diet
Wheat flour 20.5%, roasted Bengal gram 52.6%, skimmed milk powder 5%, casein 4%, refined oil 4%, coconut oil 9%, salt mixture with starch 4%, vitamin and choline mixture 0.5%, and cholesterol 0.4%.

Rabbits in groups III and IV were orally fed the methanolic extracts of D. biflorus dose I and dose II, respectively, and rabbits in group V were fed standard drug atorvastatin. The D. biflorus extracts and atorvastatin were suspended in 2% tween 80 separately and fed to the respective groups of rabbits by oral intubation. At the end of 11 weeks, all the animals were sacrificed by cervical decapitation after overnight fasting.

Portions of the tissues from liver, heart, and aorta were blotted, weighed, and homogenized with methanol (3 volumes). The lipid extract obtained by the method of Folch et al .[2] was used for the estimation of thiobarbituric acid reactive substances[3] (TBARS). Another portion of the tissues was homogenized with phosphate buffer saline and used for the estimation of reduced glutathione[4] (GSH), catalase[5] (CAT), and superoxide dismutase[6](SOD).

Results were expressed as mean + SE of 6 rabbits in each group. One-way analysis of variance (ANOVA) with Scheffe′s multiple comparisons test, were used to determine the statistical significance. P< 0.05 was considered significant.

The average body weight was found to have increased in high fat diet fed rabbits compared with those in control group. After administration of two doses of D.biflorus extract it was found to have decreased. [Table - 1] TBARS had significantly increased and GSH had significantly decreased in liver, heart, and aorta of rabbits fed HFD ([Table - 1]; Group II) compared with those in control group I. Administration of D.biflorus extract significantly lowered the level of TBARS and enhanced the level of GSH. Higher dose of the plant extract was found to be more effective and showed comparable results with standard drug atorvastatin on these two parameters. Activities of antioxidant enzymes, that is, SOD and CAT in different groups are given in [Table - 2]. These two enzymes showed a marked reduction in activity in liver, heart, and aorta of rabbits in group II which had been fed HFD. Supplementation of D.biflorus extract with HFD significantly improved the activities of SOD and CAT in the above tissues of rabbits in groups III and IV as compared with group II. Preliminary phytochemical study shows that methanolic extract of the experimental plant contains phytoconstituents, such as, alkaloids, steroids, flavonoids, and isoflavone.

Elevated levels of TBARS in liver, heart, and aorta in group II rabbits are a clear manifestation of excessive formation of free radical and activation of lipid peroxidation. The significant decline in the level of TBARS, in rabbits administered with D.biflorus (Groups III and IV), unveils the antioxidant potential of the D.biflorus extract. Both doses of the D.biflorus extract (groups III and IV) help to restore the GSH levels near normal. The higher dose has more effect, which was comparable to atorvastatin. Increase in GSH concentration in animals treated with D.biflorus extract may be due to increased activity of the enzyme, glutathione reductase, which catalyses the conversion of oxidized glutathione to reduced glutathione in liver. It may also be due to enhanced synthesis/transport of GSH.

Restoration of the activities of SOD and CAT to near normal, observed in tissues of rabbits supplemented with D.biflorous may be due to the removal of toxic intermediates by the plant extract in HFD fed rabbits. The components of the plant extract may also directly activate the antioxidant enzymes.[7] It is concluded that administration of D.biflorus manifests a protective action against HFD induced oxidative stress in different tissues in rabbits. However, further studies are needed to isolate the active principles, elucidate their structures, and determine their pharmacological activities.

References

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