Indian Journal of Experimental Biology Vol. 48, April 2010, pp. 378-382
Neuroprotective effect of hydroalcoholic extract of dried fruits of Trapa bispinosa Roxb on lipofuscinogenesis and fluorescence product in brain of D-galactose induced ageing accelerated mice D B Ambikar, U N Harle, R A Khandare, V V Bore & N S Vyawahare* Department of Pharmacology, AISSMS College of Pharmacy, Kennedy Road, Pune 411 001, India Received 14 January 2009; revised 16 December 2009 Effect of hydroalcoholic extract T. bispinosa (TB) was studied on fluorescence product and biochemical parameter like lipid peroxidation, catalase activity and glutathione peroxidase activity in the brain of female albino mice. Ageing was accelerated by the treatment of 0.5 ml 5% D-galactose for 15 days. This resulted in increased fluorescence product, increase lipid peroxidation and decrease antioxidant enzyme like glutathione peroxides and catalase in cerebral cortex. After cotreatment with hydroalcoholic extract of TB (500 mg/kg, po) there was decrease in fluorescence product in cerebral cortex. Moreover, TB inhibited increase lipid peroxidation and restores glutathione peroxidase and catalase activity in cerebral cortex as compare to ageing accelerated control group. To conclude TB found to be effective antioxidative agent which could to some extent reverse D-galactose induced ageing changes resulted due to oxidative damage. Keywords: Fluorescence product, Lipid peroxidation, Lipofuscin granules, Trapa bispinosa
Medicinal herbs that are relatively safe, cheap and easily available constitute the cornerstone of traditional medicinal practice worldwide. These medicinal plants represent a great deal of untapped reservoir of drugs and the structural diversity of their component molecules makes a valuable source of novel lead compounds1. Trapa bispinosa Roxb (TB) a floating aquatic herb, is one of the medicinal plants that have been used as nerve tonic from time immemorial2. The acrid juice is used for diarrhea and dysentery. The fruits are used as intestinal astringent, aphrodisiac, antiinflammatory, antileprotic, in urinary discharges, fractures, sore throat, bronchitis, and anemia2. TB reported to have hepatoprotective activity3, free radical scavenging activity4, antimicrobial activity5 antitumor activity and antioxidant activity6,7. TB possesses significant facilitatory effect on aversively motivated learning and memory in mice8. TB fruit is also claimed as nerve tonic and useful in nervous debility2, however these properties have not been significantly documented so far. Stress is one of the primary reasons for referrals to psychiatrists and root cause of a number of brain disorders. Modern day diseases are largely due to this ________________ *Correspondent author Telephone: +91-020-26058204 Fax: +91-020-26058204 E-mail:
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hideous stress9. Oxidative stress and reactive oxygen species (ROS) are proposed to be major contributors to the aging process and many age linked neurodegenerative diseases such as Alzheimer’s and Parkinson’s diseases10. Accumulated evidence highlights that oxidative damage is an essential source of aging11. During aging, a general decline in various biochemical and physiologic functions is noted in most organs, resulting in increased susceptibility to age associated diseases12. The brain is particularly vulnerable to oxidative stress because of its high metabolic rate and low antioxidant defenses13. The accumulation of free radicals progressively damages brain structure and function14. D-galactose is a normal reducing sugar in the body. At its normal level, it is usually converted into glucose by galactose-1-phosphate uridyltransferase and galactokinase. However, at high levels, it can be oxidised into aldehydes and H2O2 in presence of galactose oxidase15. Administration of D-galactose induces symptoms similar to those in normal aging; many investigations using rodents have demonstrated that the injection of D-galactose can lead to excessive formation of ROS, neuronal damage and a significant decline in learning and memory capacity16,17. Decline in learning and memory processes is commonly observed in aged patient11. Moreover it is also well reported that the susceptibility to neuronal cell
AMBIKAR et. al: NEUROPROTECTIVE EFFECT OF TRAPA BISPINOSA FRUIT EXTRACT
damage due to increased oxidative stress is linked with advancing age10. Wherein enzymatic and nonenzymatic antioxidants scavenge these free radicals and prevent cell mediated neuronal deterioration18. Science D-galactose treated mouse are believed to serve as an induced ageing model and has been recommended for related investigation19. The present study has been undertaken to investigate the potential role of TB in reducing oxidative stress in the brain of D-galactose induced mice by quantifying lipid peroxidation and various antioxidant enzyme activities. In addition, the effect of TB on fluorescence product formed in cerebral cortex has also been studied. Materials and Methods Plant material─The plant material (dry fruits of Trapa bispinosa) collected from Maihar region of Madhya Pradesh, India was authenticated by Botanical Survey of India (voucher specimen numberBSI/WC/Tech/2008-976). The hydroalcoholic extract of dried fruit was prepared at approved laboratory of Green Chem, Bangalore, India using following procedure. Preparation of extract⎯Dried fruits were extracted with 50% mixture of hydroalcohol and concentrated. The concentrated mass was washed several times with petroleum ether to remove the resinous matter. Then the mass was diluted with mixture of hydroalcohol (75 + 25), filtered and concentrated, dried to get the powdered form of the extract20 (yield: 0.25 %). Chemicals and drugs⎯D-galactose, 5, 5-dithiobisnitrobenzoic acid (Loba Chemicals), thiobarbituric acid, trichloroacetic acid, ascorbate (SD Fine Chemical), glutathione (Ozone India) were used. Animals─Six months old female Swiss albino mice (18-22 g) certified as healthy by a veterinary physician were used. The female mice were reported to be ideal for D-galactose model and are more susceptible towards D-galactose induced changes in brain as compared to male mice21. These mice were maintained at 25° ± 2°C and 45-55% RH and under standard environmental conditions (12:12 h L:D cycle). These mice had free access to food and water. Institutional Animal Ethics Committee (IAEC) approved the protocol (CPCSEA/IAEC/PC-04/092K7) and entire study has carried out as per standard guideline of IAEC. Acute toxicity test⎯Mice were subjected to acute oral toxicity study as per guidelines (AOT 425)
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suggested by the Organization for Economic Co-operation and Development (OECD-2001). The mice were observed continuously for 2 h for behavioral and autonomic profiles and for any sign of toxicity or mortality up to 7 days22. D-galactose treatment⎯18 female albino mice were divided in to three groups of 6 each. First group served as control and received 0.5 ml saline per day for 15 days. Second group mice were injected with 0.5 ml 5% D-galactose (sc) per day for 15 days as served as ageing accelerated group. The rats of third group were injected with a daily dose of 0.5 ml 5% Dgalactose (sc) plus TB 500 mg/kg (po). On 15th day, 60 min after the dose of TB and D-galactose mice were sacrificed. Brain of each mouse was dissected out and subjected to biochemical estimation of lipofuscin content, lipid peroxidation, glutathione peroxides and catalase activity 21. Biochemical investigations
Measurement of fluorescence⎯Lipofuscin contents from whole cerebral cortex, were extracted in chloroform: methanol mixture (2:1 v/v) and fluorescence was measured on photofluorometer using 1µg of quinine sulfate/ml of 0.1N sulfuric acid as standard and 0.1N sulfuric acid as blank21. Assay of lipid peroxidation─As a measure of lipid peroxidation, malonaldialdehyde (MDA) levels was estimated according to method of Kakkar et al.23 by measuring thiobarbituric acid reactive substances (TBARS). Initially the colour developed was red at 532 nm using spectrophotometer and expressed as nM/mg of protein. Activities of glutathione peroxidase (GPx) was estimated24-26; the colour developed was read at 420 nm using spectrophotometer and expressed as U/mg of protein. The activity of catalase (CAT) was measured according to the method of Claiborne27 and the protein content of brain tissue was estimated by following the method of Lowry et al28. Statistical analysis⎯The data were expressed as mean ± SE and analysed statistically using one way ANOVA followed by Tukey’s post hoc test. Results Biochemical investigations
Fluorescence content⎯Significant (P
