Separation of Six Lupane-Type Triterpenoid Saponins from Leaves of Acanthopanax gracilistylus by HSCCC with Preparative-HPLC

High efficiency and less solvent consumption are the essential requirements of high-speed counter current chromatography (HSCCC), especially for the preparation and purification of natural products. In this manuscript, an efficient HSCCC strategy with preparative high performance liquid chromatography (preparative-HPLC) was successfully developed to rapidly separate and purify six lupane-type triterpenoids (including acankoreoside C (1), acangraciliside S (2), acankoreoside B (3), acankoreoside D (4), acantrifoside A (5) and acankoreoside A (6)) from leaves of the Acanthopanax gracilistylus. The effective separation was achieved using ethyl acetate–nbutanol–methanol–water (3:0.3:0.8:4, v/v/v/v) as the two-phase solvent system, in which the mobile phase was eluted at an optimized flow rate of 2.0 mL/min and a revolution speed of 900 rpm. HSCCC preparation was performed on 400 mg of crude sample yielding 5.3 mg of compound 3, 6.4 mg of compound 4, 10.6 mg of compound 5, 35.8 mg of compound 6 with purities of 95.6%, 96.3%, 96.1%, 97.2%, respectively, 17.2 mg of a mixture of compounds 1 and 2, which was further separated by preparative-HPLC yielding 5.9 mg of compound 1, and 4.5 mg of compound 2 with purities of 96.8% and 94.6%, respectively, as determined by HPLC at 210 nm. Their chemical structures were identified by nuclear magnetic resonance (NMR) technology. All compounds were evaluated for their anti-inflammatory activity with lipopolysaccharide (LPS)-induced RAW264.7 cell. The compounds 3 and 4 showed weakly inhibitory effect of nitric oxide (NO) production with low cytotoxicity.