Isolation And Purification Of Bioactive Compounds From Desmodium Styracifolium And Forsythia Suspense Via High-speed Counter-current Chromatography

ABSTRACT:High-speed countercurrent chromatography (HSCCC), a newly liquid-liquid chromatography, was proposed by Ito and has been widely used for separation and purification of chemical compounds from natural products in1982. In this paper, off-line two-dimensional HSCCC was used to separate and purify bioactive compounds from ethyl acetate fraction of the stem bark of Desmodium styracifolium, stepwise elution HSCCC was used to separate and purify bioactive compounds from ethyl acetate of Forsythia suspense, complexation HSCCC was employed for the separation of vanillic acid and β-sitosterol. The optimal conditions for HSCCC separation, identification of isolated compounds were studied.(1) The optimal conditions for HSCCC separation of the ethyl acetate extract of Desmodium styracifolium were investigated. Off-line two-dimensional HSCCC technique in preparative scale has been successfully applied to separate and purify main compounds. The two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water at an optimized volume ratio of1:2:1:2(v/v) was used. A conventional HSCCC was used as the first dimension, and the upper phase of solvent system was used as the stationary phase in the head to tail elution mode at a flow-rate of2.0mL/min and a revolution of900rpm. Recycling HSCCC was served as the second dimension to separate an impure fraction of the first dimensional HSCCC. A total of four well separated substances including vanillic acid (1), β-sitosterol (2), formononetin (3) and aromadendrin (4) were obtained, and their purities and structures were identified by UV, HPLC-MS and1H NMR. The results illustrated that off-line two dimensional HSCCC would be an effective way to isolate compounds in complex samples. By using this method, we could obviously improve the resolution, peak capacity, and resolve problems caused by peak overlap.(2) The optimal conditions for HSCCC separation of the ethyl acetate extract of Forsythia suspense were investigated. HSCCC in preparative scale has been successfully applied to separate and purify main compound from the ethyl acetate extract of Forsythia suspense using stepwise elution with two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at (1:4:1:4,v/v) and (1:4:2:3,v/v). Under the optimized conditions, the preparative HSCCC was performed on350mg of the ethyl acetate of Forsythia suspense yielding phillyrin (12.8mg), isolariciresinol-9’-O-β-D-glucopyranoside (5.3mg), pinoresinol (21.2mg) and phillygenin (8.3mg) in a one-step separation, with purities over90%as determined by HPLC. The structures of the separated compounds were identified by UV, HPLC-MS and1H NMR. The present results of our studies indicate that the new HSCCC technique by varying solvent system is a powerful technique for the separation of natural products with largely partition coefficient.(3) We obtained vanillic acid and β-sitosterol in the separation of Desmodium styracifolium. In this chapter, the optimal conditions for HSCCC separation of vanillic acid and β-sitosterol were investigated. In the separation system, a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water at a volume ratio of1:2:1:2(v/v) with0.1mol/L copper ion as complexation agent in the aqueous phase was employed for the separation of vanillic acid and β-sitosterol. The results showed introduction of chelating metal ion into solvent system are effective ways to implement HSCCC separations by modifing the partition coefficient. There were eighteen graphs, thirteen tables and ninety-seven references in this paper.