Isolation of hyperoside and isoquercitrin from Camellia sasanqua as antioxidant agents

Two antioxidant active compounds were isolated from the methanol extract of Camellia sasanqua using various in vitro assays: 1,1-diphenyl-2-picrylhydrazyl (DPPH), β-carotene bleaching and reducing power assays. The ethyl acetate (EtOAc) fraction of the methanol extract had the highest DPPH radical-scavenging activity with an Inhibition Concentration (IC50) value of 18.3 ± 1.63 μg mL(-1). Sephadex LH-20 column chromatography was used to separate the EtOAc fraction into eight fractions (F1-F8). Antioxidant activity was significantly higher in fraction 5 with an IC50 value 14.61 ± 0.02 μg mL(-1). Fraction 5 was further separated by HPLC preparative with Capcellpak C18 MG followed by the Cosmosil 5C18-AR-II column, using a guided DPPH radical-scavenging assay. The compounds isolated were identified as: Hyperoside (1) and isoquercitrin (2) after recrystallization from ethanol, based on Mass Spectrum (MS) and Nuclear Magnetic Resonance (NMR) analyses. Their DPPH radical-scavenging activities based on the 50% scavenging concentration decreased in the following order: Isoquercitrin (21.6 mM) > hyperoside (27.5 mM). The antioxidant activities of hyperoside and isoquercitrin were 67.52 ± 0.64 and 64.33 ± 0.51%, respectively, in the β-carotene bleaching assay. These compounds were found to have good reducing powers (OD value: 2.5-3.8) at concentrations of 50-140 μg mL(-1), using the potassium ferricyanide reduction method. Although, these compounds are well-known, hyperoside (1) was isolated from this herb for the first time.