Separation of chemical constituents from three plant medicines by counter-current chromatography using a three-phase solvent system at a novel ratio

• A three-phase solvent system composed of n-hexane–methyl acetate–acetonitrile–water at a novel volume ratio of 4:3:4:4 is proposed.
• Three separate two-phases, i.e. upper/lower, upper/middle, and middle/lower from this system are successfully used for CCC separations of three plant medicines.
• This system offers a novel CCC methodology and demonstrates better selectivity for a selection of compounds that are difficult to separate using HPLC.
• Five compounds are separated by CCC for the first time.

A solvent system of n-hexane, methyl acetate, acetonitrile, and water at a novel volume ratio of 4:3:4:4 forms three layers, i.e. upper phase (UP), middle phase (MP), and lower phase (LP), with a volume ratio of 1:1.20:1.42 at room temperature (25 °C). All three two-phases from this three-phase solvent system were successfully used to separate some chemical constituents from three plant medicines with counter-current chromatography (CCC). Eight coumarins (B1–B8) were obtained from petroleum ether extract of fresh roots of Angelica dahurica (Baizhi) with a stationary phase of UP and a mobile phase of LP. Six diarylheptanoids (L1–L6) were obtained from petroleum ether extract of dried rhizomes of Alpinia officinarum (Liangjiang) with a stationary phase of UP and a mobile phase of MP. Three chemical constituents (Z1–Z3) were obtained from ethyl acetate extract of fresh rhizomes of Anemarrhena asphodeloides (Zhimu) with a stationary phase of MP and a mobile phase of LP. Preparative HPLC was used for further purification if necessary. Seventeen chemical constituents were identified as oxypeucedanin hydrate (B1), byakangelicin (B2), byakangelicol (B3), bergapten (B4), oxypeucedanin (B5), imperatorin (B6), phellopterin (B7), isoimperatorin (B8), 5-hydroxy-7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-3-heptanone (L1), 7-(4-hydroxy-3-methoxyphenyl)-1-phenyl-4E-en-3-heptanone (L2), 5-hydroxy-1,7-diphenyl-3-heptanone (L3), 1,7-diphenyl-4E-en-3-heptanone (L4), 5-hydroxy-1,7-diphenyl-4E,6E-dien-3-heptanone (L5), isomers of 1,7-diphenyl-3,5-heptandione and 5-hydroxy-1,7-diphenyl-4E-en-3-heptanone (L6), mangiferin (Z1), timosaponin A-III (Z2), and 2,6,4′-trihydroxy-4-methoxy-benzophenone (Z3) by means of MS, 1H and 13C NMR studies. Five compounds of B3, L3, L5, L6, and Z3 were isolated by CCC for the first time.