<strong>International</strong> <strong>Journal</strong> <strong>of</strong> Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701promote health, immunity, and longevity.According to Ayurveda, they strengthen all tissue<strong>of</strong> the body, prevent aging, promote intellect, andprevent disease.Research envisaged (Justification- Aim-Objectives): In the literature we found somereports on the antioxidant properties <strong>of</strong> one or theother individual herb constituents <strong>of</strong> <strong>Trikatu</strong>.However, there is any report on the mixture <strong>of</strong>these 3 herbs for pharmacological screening <strong>of</strong>such therapeutic <strong>activity</strong> to the antioxidant .Asthere is synergistic effect or mutual potential <strong>of</strong>therapeutic action when such herbs <strong>of</strong> similarnature are mixed together. In the light <strong>of</strong> this, itwas thought worthy to evaluate antioxidant <strong>activity</strong><strong>of</strong> <strong>Trikatu</strong> and their correlation with antioxidant<strong>activity</strong>. Such studies are important to substantiatethe claims documented with regard to <strong>Trikatu</strong> inancient Ayurvedic texts.Materials and MethodsPlant material and extraction procedures: Theherbs <strong>of</strong> <strong>Trikatu</strong> (Piper nigrum, Piper longum, andZingiber <strong>of</strong>ficinale) were collected from localvender <strong>of</strong> sagar, an expert in the field authenticatedby the competent expert. Dried in shade, coarselypowdered and all three powdered drugs were mixedin 1:1:1 w/w, to preparation <strong>of</strong> <strong>Trikatu</strong> megaHerb.<strong>Trikatu</strong> megaHerb was subjected to successiveextraction in soxhlet apparatus, using non polar topolar solvent (Pet.ether, Benzene, Cholor<strong>of</strong>orm,Ethyl acetate, 70% ethanol and water). Each <strong>of</strong> allsix extracts was concentrated by distilling thesolvent and air dried. All six extracts were mixedtogether to prepare <strong>Trikatu</strong> <strong>megaExt</strong>ract(<strong>megaExt</strong>).The <strong>megaExt</strong> was subjected toqualitative phytochemical analysis for presence <strong>of</strong>various constituents like Alkaloids, Carbohydrate,Glycosides, Terpanoids, Protein and Amino acids,Phenolic and Tannins, Flavanoids, Oils and Fats,Saponins.Experimental / methodDPPH radical scavenging <strong>activity</strong>: The method isbased on the reduction <strong>of</strong> colored solution <strong>of</strong> DPPH(1, 1-diphenyl-2picryl hydrazyl) in presence <strong>of</strong> testdrug measured at 517nm. The free radicalscavenging capacity <strong>of</strong> the<strong>Trikatu</strong> <strong>megaExt</strong>ractwas determined using DPPH method <strong>of</strong> DPPHsolution (0.004% w/v) was prepared in 95%methanol. Ascorbic acid solution preparation:1000µg/ml stock solution was prepared bydissolving 10mg <strong>of</strong> ascorbic acid in 10 ml <strong>of</strong>methanol. From this 20, 40, 60, and 80,100 µg/mlascorbic acid solutions were prepared. Themegaextracts were mixed with 95% methanol toprepare the stock solution (10 mg/100mL). Theconcentration <strong>of</strong> this <strong>megaExt</strong>ract solution was 10mg /100 mL. Stock solution 2ml, 4ml, 6ml, 8ml &10ml <strong>of</strong> this solution were taken in five test tubes& by serial dilution with same solvent were madethe final volume <strong>of</strong> each test tube up to 10 mlwhose concentration was then 20µg/ml, 40µg/ml,60µg/ml, 80µg/ml & 100µg/ml respectively.Freshly prepared DPPH solution (0.004% w/v) wasadded in each <strong>of</strong> these test tubes containing<strong>megaExt</strong>ract (20 µg/mL., 40µg/mL., 60µg/mL.,80µg/mL., and 100µg/mL.) and after 10 min, theabsorbance was taken at 517 nm using aspectrophotometer. Control sample was preparedcontaining the same volume without any extractwas used as blank. % scavenging <strong>of</strong> the DPPH freeradical was measured using the following equation.Results are shown in table and graphically% inhibition = [(Ao-At) / Ao x 100]Where Ao was the absorbance <strong>of</strong> the control(blank, without extract) and At was theabsorbance in the presence <strong>of</strong> the extract.Superoxide radical scavenging <strong>activity</strong>:This<strong>activity</strong> was measured using NBT (Nitro bluetetrazolium reagent). The method is based ongeneration <strong>of</strong> superoxide radical (O - 2 ) by autooxidation <strong>of</strong> hydroxylamine hydrochloride inpresence <strong>of</strong> NBT, which gets reduced to nitrite.Nitrite in presence <strong>of</strong> EDTA gives a color that canbe measured at 560 nm. Various concentrations(20, 40, 60, 80, 100 µg/ml) <strong>of</strong> test solutions weretaken in test tube. To this, reaction mixtureconsisting <strong>of</strong> 1 ml <strong>of</strong> 50 mM sodium carbonate, 0.4ml <strong>of</strong> 24 mM NBT 0.2 ml <strong>of</strong> 0.1 mM EDTAsolution were added to the test tube and zerominute reading was taken at 560 nm. The reactionwas initiated by the addition <strong>of</strong> 0.4 ml <strong>of</strong> 1 mMhydroxylamine hydrochloride to the abovesolution. Reaction mixture was incubated at 25 0 Cfor 15 minute; the reduction <strong>of</strong> NBT was measuredat 560 nm. Absorbance was recorded and %inhibition was calculated according to thefollowing equation.% inhibition = [(Ao-At) / Ao x 100]Where Ao was the absorbance <strong>of</strong> the control(blank, without extract) and At was the absorbancein the presence <strong>of</strong> the extract.Reducing power: The reducing power <strong>of</strong> the<strong>megaExt</strong> was determined according to the method.Various concentrations <strong>of</strong> the mega Ext (20, 40,60, 80, 100 µg/ml) in 1.0 ml <strong>of</strong> demonized waterwere mixed with phosphate buffer (2.5 ml, 0.2M,pH 6.6) and 1% potassium ferricynide (2.5 ml).The mixture was incubated at 50 0 C for 20 min.aliquots <strong>of</strong> trichloroacetic acid (2.5 ml, 10%) wereadded to the mixture, which was then centrifuged at3000 rpm for 10 min. The upper layer <strong>of</strong> solution(2.5 ml) was mixed with distilled water (2.5 ml)Vol. 2 (2) Apr – Jun 2011 www.ijrpbsonline.com 625
<strong>International</strong> <strong>Journal</strong> <strong>of</strong> Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701and a freshly prepared fecl 3 solution (0.5 ml, 1%).The absorbance was measured at 700 nm.Increased absorbance <strong>of</strong> the reaction mixtureindicated increased reducing power.STATICAL ANALYSISAll the values are expresses as mean ± SD anddata was analyzed by one-way ANOVA, usingGraphpad INSTAT. The post-hock analysis wascarried out by Dunnet’s multiple comparison teststo estimate the significance <strong>of</strong> difference betweenindividual groups.RESULTS & DISCUSSIONPhytochemical screening reveals that the majorconstituents <strong>of</strong> <strong>Trikatu</strong> <strong>megaExt</strong>ract are phenoliccompound, glycosides alkaloid, and flavanoidwere, phenolic compounds which may beresponsible for the activities <strong>of</strong> antioxidant.1. DPPH radical scavenging <strong>activity</strong>:<strong>Trikatu</strong> <strong>megaExt</strong> had significant scavenging effecton the DPPH free radical which increased withincreasing concentration from 20-100 µg/ml. Thescavenging effect <strong>of</strong> sample was lower than that <strong>of</strong>Ascorbic acid. The sample posseses statisticallysignificance DPPH free radical scavenging <strong>activity</strong>(P
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