An efficient strategy based on liquid-liquid extraction and pH-zone-refining counter-current chromatography for selective enrichment, separation, and purification of alkaloids and organic Acids from natural products.

This study presents an efficient strategy based on liquid-liquid extraction and pH-zone-refining counter-current chromatography for selective enrichment, separation, and purification of alkaloids and organic acids from natural products. First, an acid or base modified two-phase solvent system with maximum or minimum partition coefficient was developed for the liquid-liquid extraction of the crude extract. As a result, alkaloids or organic acids could be selectively enriched in the upper or lower phase. Then pH-zone-refining counter-current chromatography was employed to separate and purify the selectively enriched alkaloids or organic acids efficiently. The selective enrichment and separation of five bufadienolide from toad venom of Bufo marinus was used as an example to show the advantage of this strategy. As a result, 759 mg of selectively enriched bufadienolide was obtained from 2 g of crude extract and the total content of five targets was increased from 14.64 to 83%. A total of 31 mg of marinobufagin-3-adipoyl-l-arginine, 42 mg of telocinobufagin-3-pimeloyl-l-arginine, 51 mg of telocinobufagin-3-suberoyl-l-arginine, 132 mg of marinobufagin-3-suberoyl-l-arginine, and 57 mg of bufalin-3-suberoyl-l-arginine were all simultaneously separated from 500 mg of selectively enriched sample, with the purity of 92.4, 97.5, 90.3, 92.1, and 92.8%, respectively.