ANTIOXIDATIVE PROPERTIES ( TPC , DPPH , FRAP , METAL CHELATING ABILITY , REDUCING POWER AND TAC ) WITHIN SOME CLEOME SPECIES

Applications of antioxidants are increasing due to their multiple roles in removing harmful effects of oxidative stress. for the present study Tpc, dpph, frAp, metal chelating Ability, reducing power and TAc spectrophotometric assays were used for the assessment of antioxidative properties of methanolic extracts of leaves of 5 different Cleome species (Cleome viscosa l., C. simplicifolia (camb.) hook f. & Thoms, C. gynandra l., C. chelidonii l.f. and C. speciosa raf.). The purpose of this study was also to evaluate the comparability of antioxidant capacity measurements in Cleome species by the above assay. This study may provide some supplementary food option by some Cleome species. keywords: ANTioxidANT properTy, chelATiNg AcTiViTy, CLEOME, reduciNg power doi: 10.4462/annbotrm-9958 k3fe(cN)6, shaken well and incubated at 500c for 20 min. After incubation, 1 ml TcA(10%) was added to stop the reaction. it was centrifuged at 3000rpm for 10 min. 1.5 ml supernatant, 1.5 ml d.w. and 0.1 ml fecl3 (0.1%) were mixed and incubated for 10 min and absorbance was read at 700nm on spectrophotometer .The assays were carried out in triplicate and the results are expressed as mean values ± standard deviations. The extract concentration providing 0.5 of absorbance (ec50) was calculated from the graph of absorbance at 700 nm against extract concentration. Ascorbic acid was used as standard. higher absorbance indicates higher reducing power. The total antioxidant capacity of methanol extracts of leaves of Cleome species was evaluated by the phosphomolybdenum method of prieto et al. (1999). An aliquot of 0.5 ml of sample solution (0.2, 0.4, 0.6, 0.8, 1 mg/ml) was combined with 5 ml reagent solution (0.6 m sulfuric acid, 28 mm sodium phosphate and 4 mm ammonium molybdate). The tubes were capped and incubated in a boiling water bath at 950c for 90 min. After the samples had cooled to room temperature, the absorbance of the aqueous solution of each was measured at 695 nm against blank on spectronic 20 visible spectrophotometer. A typical blank solution contained 5 ml of reagent solution and the appropriate volume of the same solvent (methanol) used for the sample and it was incubated under same conditions. for samples, antioxidant capacity is expressed as equivalents of ascorbic acid. RESULTS AND DISCUSSION Total polyphenol contents polyphenols represent the aromatic compounds formed during secondary metabolism in plants. These phenolic compounds, in some instance, affect the fundamental plant processes such as photosynthesis, chlorophyll synthesis, water relations, protein synthesis, respiration, membrane permeability, etc. The phenols and their oxidative products are also known for inhibitory action on various enzyme systems e.g. iAA-oxidase (shekhawat et al., 1980). As polyphenols are known to accumulate during the fungal attack, their role in disease resistance has also been well demonstrated. The importance of polyphenols can be very well recognized at the time of stress. in pharmacological studies, polyphenols are considered as secondary products of plant metabolism without specific physiological function (Adzet, 2002). polyphenols show natural antioxidant properties, therefore impart a tonifying action (Thompson & williams, 1976). some flavonoids act as powerful electron scavengers of free radicals (rice-evans 50 effect of the stable 1, 1-diphenyl-2-picrylhydrazyl (dpph)free radical activity (Braca et al., 2002). The diluted working solutions of the test extracts were prepared in methanol. Ascorbic acid (10mg/ml) was used as standard. 0.002% dpph was prepared in methanol and 3 ml of this solution was mixed with 1 ml of sample solution and standard solution separately. These solution mixtures were kept in dark for 30 min and optical density was measured at 517 nm using spectrophotometer. methanol (1 ml) with dpph solution (0.002%, 3 ml) was used as blank. The optical density was recorded and % inhibition was calculated using the formula given below: percent inhibition absorbance(cont)-absorbance(sam) of dpph activity = ----------------------------------------x100 absorbance(cont) Antioxidant activity assay was also done following the ferric-reducing antioxidant power (frAp) method described by Benzie & strain (1996). The results are expressed as ascorbic acid equivalent antioxidant capacity. The ferrous ion-chelating ability was determined according to the method of decker & welch (1990) with minor modifications. 1.6ml of each extract stock solution (0.2, 0.4, 0.6, 0.8 and 1 mg/ml) were mixed with 2.16 ml of distilled water and 80 μl of 2 mm fecl2 in a test tube. The reaction was initiated by the addition of 160 μl of 5 mm ferrozine. The solutions were well mixed and allowed to stand for 10 min at room temperature. After incubation, the absorbance was measured at 562 nm spectrophotometrically. distilled water (1.6 ml) instead of sample solution was used as a control. distilled water (160 μl) instead of ferrozine solution was used as a blank, which was used for error correction because of unequal colour of the sample solutions. l-ascorbic acid was used as reference standard. All measurements were performed in triplicate. The ferrous ion-chelating ability was calculated as follows: % scavenging activity (Ac – AE/As) (ferrous ion chelating ability) = -------------------------x 100 Ac where, Ac is the Absorbance of control reaction; Ae is the Absorbance of plant extract; As is the Absorbance of standard. The reducing power was determined according to the method (reducing power Assay) of oyaizu (1986). An aliquot of 0.5 ml plant extract (0.2, 0.4, 0.6, 0.8, 1 mg/ml) was mixed with 1 ml phosphate buffer (0.2m, ph 6.6) and 1 ml 1% ApArAdh V. T. / Ann. Bot. (Roma), 2012, 2: 49–56