Centrifugal Partition Chromatography의 기울기 용리 시스템을 이용한 용담사간탕의 항산화 효소 유도 성분 추적 분리 연구

Abstract

Yongdamsagan-Tang is one of the traditional oriental medicine (TOM) prescriptions used for liver disease and the prescription was described as the clinical effects of damp-heat for liver. Two methods of in vitro assay were used to evaluate the scientific efficacy of the Yongdamsagan-Tang. One of them, the antioxidant activity in the cell, is the antioxidant response element (ARE)-luciferase assay which can generate the antioxidant enzyme through the mechanism of nuclear factor E2-related factor 2 (Nrf2)-ARE. And the other antioxidant activity was carried out using the high performance liquid chromatography (HPLC) radical scavenging online assay to confirm the immediate response of antioxidant molecules. The centrifugal partition chromatography (CPC) method based on the principle of solvent fractionation was applied to the antioxidant enzyme inducer from Yongdamsagan-Tang to isolate an active compounds. Various natural compounds such as flavonoids, saponins, coumarins, anthraquinones, phenolic acids and naphthoquinones were efficiently separated by the conventional CPC method. The effective separation from active compounds was compared between the conventional method and new designed method, which is the activity-guided isolation method using n-hexane, ethyl acetate, water-saturated n-butanol, acetonitrile and water (HEB-AW) solvent with gradient elution system. 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 elution 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 and 13C NMR. As a result, baicalein and wogonin were considered as ARE-luciferase inducing compounds and the free radical scavenging compounds were baicalin and baicalein in Yongdamsagan-Tang. These results suggest that the conventional activity-guided isolation method can be complemented by applying the HEB-AW solvent system with gradient elution CPC technology, and that the new method is more efficient.