PREPARATIVE ISOLATION AND PURIFICATION OF PHENOLIC ACIDS FROM THE DRIED BUDS OF LONICERA JAPONICA THUNB BY HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY IN GRADIENT ELUTION MODE

A new high speed counter-current chromatography (HSCCC) method for preparative separation and purification of three phenolic acids from the dried buds of Lonicera japonica Thunb was developed by using gradient elution mode. Two pairs of two-phase solvent system composed of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid at volume ratios of 1:5:1:5 and 1:5:2:5 were used in HSCCC separation. The upper phase of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid (1:5:1:5, v/v) was used as the stationary phase. The lower phase of n-hexane-ethyl acetate-methanol-0.01 M hydrochloric acid (1:5:1:5, v/v) and (1:5:2:5, v/v) were used as the mobile phase in gradient elution mode. From 200 mg of the crude extract 51.6 mg of 3-caffeoylquinic acid, 7.5 mg of caffeic acid, and 42.4 mg of 3,5-O-dicaffeoylquinic acid were obtained in one-step HSCCC separation. The purities of the obtained compounds were all above 95% as determined by HPLC area normalization method, and their chemical structures were identified by 1H-NMR and 13C-NMR.