Isolation of antioxidant response element inducing compounds from Yongdamsagan-Tang by the gradient elution mode of centrifugal partition chromatography

Abstract

Bioactivity guided fractionation by centrifugal partition chromatography (CPC) with gradient elution mode and antioxidant response element (ARE)-luciferase assay was applied to determine bioactive compounds from Yongdamsagan-Tang [1] which is traditionally used for the treatment of liver disease. Yongdamsagan-tang is composed of ten ingredints: Gentianae Radix, Scutellariae Radix, Gardeniae Fructus, Alismatis Rhizoma, Akebiae Caulis, Plataginis semen, Angelicae Gigantis Radix, Rehmanniae Radix, Bupleuri Radix and Glycyrrhizae Radix.

Crude extract of Yongdamsagan-Tang was fractionated by linear gradient elution mode of CPC that was designed as a two-phase solvent system composed of n-hexane-acetonitrile-water (10:2:8, v/v), ethyl acetate-acetonitrile-water (10:2:8, v/v) and water saturated n-butanol-acetonitrile-water (10:2:8, v/v). Firstly, lower phase consisted of n-hexane-acetonitrile-water (10:2:8, v/v) was used as stationary phase and upper phase of n-hexane-acetonitrile-water (10:2:8, v/v) was eluted as mobile phase. Then, mobile phase changed to upper phase of ethyl acetate-acetonitrile-water (10:2:8) and finally upper phase of water saturated n-butanol-acetonitrile-water (10:2:8, v/v) was pumped to generate a linear gradient elution, increasing the mobile phase polarity. Gradient elution CPC method allowed separation and isolation of compounds with diverse polarities in Yongdamsagan-Tang. After gradient CPC, 13 fractions were obtained and ARE-luciferase induction effects were evaluated. Among active fractions, compounds were further purified by HPLC and the chemical structure of purified compounds were elucidated by 1H NMR, 13C NMR and ESI-MS. As a result, baicalein and wogonin were considered as ARE-luciferase inducing compounds in Yongdamsagan-Tang.

Acknowledgements: This study was supported by a grant from the Korean HealthTechnology R&D Project, Ministry of Health and Welfare, Republic of Korea (HI14C0018).