Optimized conversion of antiproliferative lignans pinoresinol and epipinoresinol: Their simultaneous isolation and identification by centrifugal partition chromatography and high performance liquid chromatography

Abstract

Abstract High amount of the valuable lignan pinoresinol (PR) was determined in Carduus nutans fruit (7.8 mg/g) for the first time. A preparative separation method using two consecutive, identical steps of centrifugal partition chromatography (CPC) was developed in order (i) to isolate PR and (ii) to subsequently isolate PR and its 7′ epimer epipinoresinol (EPR) simultaneously after an optimized acid treatment which resulted in PR epimerization forming equal amounts of PR and EPR, from C. nutans fruit. As optimal conditions, a two-phase solvent system consisting of methyl tert-butyl ether:acetone:water (4:3:3, v/v/v) for CPC separation, and an acid treatment performed at 50 °C for 30 min for the epimerization were applied. Thus, 33.7 mg and 32.8 mg PR and EPR, in as high as 93.7% and 92.3% purity, were isolated from 10.0 g C. nutans fruit, representing 86.4% and 84.1% efficiency, respectively. Conversion characteristic of PR and EPR in acidic medium, determined as a function of time and temperature of acid treatment provides their unambiguous identification by on-line high performance liquid chromatography (HPLC). Antiproliferative assay of isolated PR and EPR in two different types of colon cancer cell lines (HCT116 and SW480) confirmed that both epimers caused a more significant decrease of viability in HCT116 cells than in SW480 cells, suggesting their similar mechanism of antiproliferative action.