Total Phenolic Content and Antioxidant Activity of Flavonoids Isolated From Leaves of Selected Citrus Species

Water and 80% methanol crude extracts from the leaves of Citrus aurantifolia, C. hystrix, C. grandis, and Citrofortunella microcarpa were investigated for their total phenolic content and its relationship with total antioxidant activity. The total phenolic content was determined using a Folin-Ciocalteu reagent whereas determination of total antioxidant activity was carried out using a β-carotene bleaching method. Among the citrus leaves extracts, the highest total phenolic content was found from the water extract of Citrofortunella microcarpa whereas the water extract of C. maxima and Citrofortunella microcarpa gave the highest total antioxidant activity and was comparable with the antioxidant activity of marker BHT. However, no clear correlation was found between the total phenolic content and the total antioxidant activity of citrus leaves in both crude extract. Eleven flavonoid components were isolated and identified from the 80% methanolic extract of the leaves of Citrus aurantifolia, C. hystrix, C. grandis and Citrofortunella microcarpa. The isolation and purification of all flavonoid components were carried out using standard paper chromatography technique. The isolated components were identified basis of their spectral analysis with shift reagents, Rf values, colour reactions under UV and UV+NH3, hydrolysis with acid and co-chromatogram with standard markers. The successfully isolated flavonoids were consisted from the group of flavone, flavone C-glycoside and flavonol. The methylated and glycosylated flavonoids were also isolated. The antioxidant capacity of isolated flavonoids was measured by two assays; a β-carotene bleaching method and a DPPH scavenging method. Statistical analysis showed that the total antioxidant and free radical scavenging activity of the isolated flavonoids was significantly low compared to the markers. Among the isolated flavonoids, results showed that at the 60th minute, quercetin 4’-O-glucoside exhibited the highest total antioxidant activity. Quercetin 4’-O-glucoside also showed a significantly higher free radical scavenging activity in DPPH scavenging method than the other isolated flavonoids. The lowest activities in both assays were exhibited by vitexin 7-O-arabinoside and isovitexin.

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