Separation Of Alkaloids From Cephalotaxus Fortunine By High-Speed Countercurrent Chromatography

Cephalotaxine-type alkaloids are the antitumor components in the twigs,leaves,roots and seeds of Cephalotaxus fortunine.It is very important to find a way to increase the use of the limited resource by finding an efficient purification technology of the alkaloids.The pH-zone-refining countercurrent chromatography was developed on the basis of high-speed countercurrent chromatography(HSCCC), which is the important branch of the countercurrent chromatography (CCC).Separation of cephalotaxine-type alkaloids in Cephalotaxus fortunine by pH-zone-refining countercurrent chromatography (pH-zone HSCCC) was studied in this paper.Traditional high-speed countercurrent chromatography was used for the separation of cephalotaxine-type alkaloids.But it did not work by changing the polarity of solvent systems. Based on pH-zone countercurrent chromatographic theory,a technology of separating cephalotaxine-type alkaloids from crude extract was established.The excellent separations were obtained by pH-zone HSCCC using solvent system ethyl acetate-petroleum ether-water or solvent system tert-butyl methyl ether(TBME)-water. For each separation,800 mg of crude extract could be separated with a HSCCC equipped a 400 ml-column to yield 3 substances epi-wilsonine, cephalotaxine and acetylcephalotaxine with a purity of more than 95%. This method can be scaled up to a pharmaceutical grade separation which is very promising for practical application.Six alkaloid monomers were obtained by pH-zone HSCCC and a assisted use of preparative HPLC.The six alkaloid monomers was drupacine(â… ),wilsonine(â…¡),cephalotaxine(â…¢),epi-wilsonine(â…£), fortunine(â…¤) and acetylcephalotaxine(â…¥).Their chemical structures were identified with the spectra of ESI-MS,¹H-NMR and ¹³C-NMR. The activities of inhibiting cancer cells A549,HL-60,MCF-7 and HO8910 of chiral isomers wilsonine and epi-wilsonine were tested. The results show that the activity of wilsonine is obviously better than epi-wilsonine.