Antioxidant activity of Trikatu megaExt - International Journal of ...

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701promote health, immunity, and longevity.According to Ayurveda, they strengthen all tissueof the body, prevent aging, promote intellect, andprevent disease.Research envisaged (Justification- Aim-Objectives): In the literature we found somereports on the antioxidant properties of one or theother individual herb constituents of Trikatu.However, there is any report on the mixture ofthese 3 herbs for pharmacological screening ofsuch therapeutic activity to the antioxidant .Asthere is synergistic effect or mutual potential oftherapeutic action when such herbs of similarnature are mixed together. In the light of this, itwas thought worthy to evaluate antioxidant activityof Trikatu and their correlation with antioxidantactivity. Such studies are important to substantiatethe claims documented with regard to Trikatu inancient Ayurvedic texts.Materials and MethodsPlant material and extraction procedures: Theherbs of Trikatu (Piper nigrum, Piper longum, andZingiber officinale) were collected from localvender of 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 of Trikatu megaHerb.Trikatu megaHerb was subjected to successiveextraction in soxhlet apparatus, using non polar topolar solvent (Pet.ether, Benzene, Choloroform,Ethyl acetate, 70% ethanol and water). Each of allsix extracts was concentrated by distilling thesolvent and air dried. All six extracts were mixedtogether to prepare Trikatu megaExtract(megaExt).The megaExt was subjected toqualitative phytochemical analysis for presence ofvarious constituents like Alkaloids, Carbohydrate,Glycosides, Terpanoids, Protein and Amino acids,Phenolic and Tannins, Flavanoids, Oils and Fats,Saponins.Experimental / methodDPPH radical scavenging activity: The method isbased on the reduction of colored solution of DPPH(1, 1-diphenyl-2picryl hydrazyl) in presence of testdrug measured at 517nm. The free radicalscavenging capacity of theTrikatu megaExtractwas determined using DPPH method of DPPHsolution (0.004% w/v) was prepared in 95%methanol. Ascorbic acid solution preparation:1000µg/ml stock solution was prepared bydissolving 10mg of ascorbic acid in 10 ml ofmethanol. 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 of this megaExtract solution was 10mg /100 mL. Stock solution 2ml, 4ml, 6ml, 8ml &10ml of this solution were taken in five test tubes& by serial dilution with same solvent were madethe final volume of 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 of these test tubes containingmegaExtract (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 of 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 of the control(blank, without extract) and At was theabsorbance in the presence of the extract.Superoxide radical scavenging activity:Thisactivity was measured using NBT (Nitro bluetetrazolium reagent). The method is based ongeneration of superoxide radical (O - 2 ) by autooxidation of hydroxylamine hydrochloride inpresence of NBT, which gets reduced to nitrite.Nitrite in presence of EDTA gives a color that canbe measured at 560 nm. Various concentrations(20, 40, 60, 80, 100 µg/ml) of test solutions weretaken in test tube. To this, reaction mixtureconsisting of 1 ml of 50 mM sodium carbonate, 0.4ml of 24 mM NBT 0.2 ml of 0.1 mM EDTAsolution were added to the test tube and zerominute reading was taken at 560 nm. The reactionwas initiated by the addition of 0.4 ml of 1 mMhydroxylamine hydrochloride to the abovesolution. Reaction mixture was incubated at 25 0 Cfor 15 minute; the reduction of 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 of the control(blank, without extract) and At was the absorbancein the presence of the extract.Reducing power: The reducing power of themegaExt was determined according to the method.Various concentrations of the mega Ext (20, 40,60, 80, 100 µg/ml) in 1.0 ml of 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 of trichloroacetic acid (2.5 ml, 10%) wereadded to the mixture, which was then centrifuged at3000 rpm for 10 min. The upper layer of solution(2.5 ml) was mixed with distilled water (2.5 ml)Vol. 2 (2) Apr – Jun 2011 www.ijrpbsonline.com 625

International Journal of 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 of 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 of difference betweenindividual groups.RESULTS & DISCUSSIONPhytochemical screening reveals that the majorconstituents of Trikatu megaExtract are phenoliccompound, glycosides alkaloid, and flavanoidwere, phenolic compounds which may beresponsible for the activities of antioxidant.1. DPPH radical scavenging activity:Trikatu megaExt had significant scavenging effecton the DPPH free radical which increased withincreasing concentration from 20-100 µg/ml. Thescavenging effect of sample was lower than that ofAscorbic acid. The sample posseses statisticallysignificance DPPH free radical scavenging activity(P

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-3701Table 3. Effect of megaExt of Trikatu in reducing powerAbsorbanceS.No. Conc.µg/mlSampleMean ± SEMAscorbic acid1. 20 0.375±0.001 0.460±0.00032. 40 0.494±0.0009 0.6212±0.00023. 60 0.658±0.0005 0.948±0.00024. 80 0.929±0.0009 1.142±0.00045. 100 1.161±0.0004 1.3091±0.0006DPPH Assay6050% Inhibition403020sampleascorbic acid1000 20 40 60 80 100 120Concentration (µg/ml)Graph: 1. Comparative effect of megaExt of Trikatu (sample) and Ascorbic acid on DPPH assaySuperoxide Scavenging120100% Inhibition806040sampleascorbic acid2000 20 40 60 80 100 120Concentration (µg/ml)Graph: 2. Comparative effect of megaExt of Trikatu (sample) and Ascorbic acid on Superoxidescavenging activityVol. 2 (2) Apr – Jun 2011 www.ijrpbsonline.com 627

International Journal of Research in Pharmaceutical and Biomedical Sciences ISSN: 2229-37011.4Reducing Power1.21Absorbance0.80.60.4sampleascorbic acid0.200 20 40 60 80 100 120concentration (µg/ml)Graph: 3 Comparative effect of megaExt of Trikatu (sample) and Ascorbic acid on reducing power.References1. Bano G, Amla V, Raina RK, Zutshi U,Chopra CL, The effect of piperine onpharmacokinetics of phenytoin in healthyvolunteers, Planta Med., 1987; 53: 568–569.2. Cadenas E. Davies KJ. Mitochondrial freeradical generation, oxidative stress, andaging. Free Radic. Biol. Med.2000;29:222–230.3. Dash B, Junius M. (Eds.), the drugs. In: AHandbook of Ayurveda. ConceptPublishing Company, New Delhi, 1987.,97.4. Finkel T.; Holbrook, N. J. Oxidants,oxidative stress and thebiology of ageing.Nature 2000; 408:239–247.5. Gulcin Ilhami, 2005. The antioxidantand radical scavenging activities ofblack pepper (Piper nigrum) seeds.International Journal of Food Sciencesand Nutrition 2005;56(7):491-499.6. Johri RK. and Zutshi U, Ayurvedicformulation 'Trikatu' and its constituents,Journal of Ethnopharmacology, 1992; 37:85-91.7. Mandal S, Yadav S, and Nema RK.Antioxidants: A Review. Journal ofChemical and Pharmaceutical Research2009; 1 (1): 102-104.8. Marnett LJ. Oxyradicals and DNAdamage. Carcinogenesis 2000;21:361–370.9. Nirmala NR, Urmila MT. and SharadiniAD, Adaptogenic Properties of SixRasayana Herbs Used in AyurvedicMedicine, Phytother.Res., 1999;13:275-291.10. Sabu MC, Kuttan R, Antidiebiticactivity of medicinal plants and itsrelationship with their antioxidantactivity. J.Ethanopharmacology 2002;81: 155-160.11. Stadtman ER, Levine RL. Proteinoxidation. Ann. N. Y. Acad. Sci.2000;899:191–208.12. Vani T, Rajani M, Sarkar S, Shishoo CJ.Antioxidant properties of the ayurvedicformulation Triphala and itsconstituents. Inter. J. Pharmacognosy,1997;35: 313-317.13. Yla¨-Herttuala S. Oxidized LDL andatherogenesis. Ann. N. Y. Acad. Sci.1999;874:134–137.Email:Vol. 2 (2) Apr – Jun 2011 www.ijrpbsonline.com 628