In vitro Antioxidant Potentiality of Purified Anthocyanin from Floral Petals of Wild Balsam Species

activities of different polarity extracts of plants grown widely under different climatic conditions. Synthetic AOXs were used enormously in food industries to prevent oxidative reactions in living organisms. However, because of potential health hazards associated with dietary intake of these chemical AOXs, the focus of biologists is to explore plant‐based compounds as natural AOXs. The AOX research an area in the pharmaceutical field as well as in the food industry. Herbals, particularly medicinal ones, have been largely studied for their AOX power for the past several years. AOXs phytochemicals Rajendranpillai Arathy et al. Int. J. Pharm. Sci. Drug Res. July-August, 2020, Vol 12, Issue 4, 313-319 314 from aromatic, spicy, medicinal, and other herbals were screened to isolate natural AOX formulations for food, cosmetic, and other applications. [3] It is also documented that higher intake of natural AOXs rich food is connected with lower risks of degenerative disorders, particularly lifestyle disorders. Anthocyanins are accepted as the largest group of water‐soluble pigments in nature with diversified colors in petals like blue, purple, red, and orange. Anthocyanin refers to anthos mean flowers, and kyanos, dark blue. [4] Common sources of anthocyanins were blueberries, cherries, raspberries, strawberries, black currants, purple grapes, and red wine. Anthocyanin belongs to flavonoids, but they are distinguished due to their capacity to form flavylium cations. In the present study, AOX potentiality of purified anthocyanin from wild balsam species is analyzed by different in vitro protocols. Materials and Methods Plant Materials For the whole study, the fresh, healthy f lowers of Impatiens balsamina (wild) collected from the natural habitats of hilly regions of Trivandrum, Kerala. Estimation of Anthocyanin Content 1‐gram flower was homogenized with acidified methanol (1% HCl), and the anthocyanin content was quantified by the standard protocol of Sutharut and Sudarat. [5] The absorbance of each dilution was read at 510 and 700 nm against distilled water as blank. Purification and Characterization of Anthocyanin from Wild Balsam Anthocyanin from fresh f lowers of wild balsam was extracted by acidified methanol (100% methanol:1% HCl, 99:1, v/v), i.e., the petals were immersed in acidified methanol for overnight in dark at low temperature (4°C). Subsequently, the crude solution obtained was filtered through cheesecloth and concentrated in rotary vacuum evaporator under vacuum at temperature not exceeding 40°C. Then, the aqueous extract was partitioned with ethyl acetate, and the remaining water extract was concentrated in rotary vacuum evaporator, temperature not exceeding 40°C. It was further fractionated by using Sephadex LH‐20, followed by C18 as separation matrices. The aqueous extract was loaded on to a Sephadex LH‐20 using 100% methanol. Based on the TLC profile, the fractions were subjected to purification by C‐18 open‐column chromatography using acidified water (1% HCl), and the column was eluted with a gradient of acidified methanol (1% HCl) and water (0:100 to 50:50). From reverse phased (RP) C‐18 open‐column chromatography, based on the TLC profile, the pure fractions were concentrated and kept at low temperature (‐20°C). Purified anthocyanin from the wild balsam was subjected to NMR analysis for their structural identification. AOX Activity Hydrogen Peroxide Scavenging Hydrogen peroxide scavenging activity was determined according to a previously described method of Ruch et al.[6] DPPH Assay AOX activity was also determined by DPPH assay using the standard protocol of Saeed et al.[7] Metal Chelating Activity The chelation of ferrous ions was estimated by the method of Dinis et al.[8] The absorbance of the solution was, thereafter, measured at 562 nm. ABTS Radical Scavenging Activity ABTS (2,2’‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic) acid) assay was performed according to the protocol of Delgado‐Andrade et al.[9] FRAP Assay The FRAP assay was carried as per the proven protocol of Benzie and Strain. [10] The principle of this method was based on the reduction of ferric‐tripyridyl triazine complex to ferrous colored form in the presence of AOXs. β‐Carotene Bleaching (BCB) Test AOX activity of anthocyanin was determined according to a slightly modified version of the β‐carotene bleaching (BCB) method. [11] Statistical Analysis SPSS 18.0 stat ist ical sof t ware was used for data analysis. Data from different assay treatments were analyzed by one‐way analysis of variance (ANOVA). The Student‐Neuman‐Keuls post‐hoc test was used to determine significance for individual experimental conditions. Differences with p < 0.01 or p < 0.05 were regarded as significant.