Rapid Isolating Of Active Compounds From Salvia Miltiorrhiza Bunge And Andrographis Paniculata By Novel Counter Current Chromatography Methods

Natural products represent a rich source of biologically active compounds and are an example of molecular diversity, with recognized potential in drug discovery and development. Clearly, how to quickly and effectively generate and characterize natural product compound libraries, either in pure compound or in a small number of compound mixtures, is a major challenge for natural product chemistry .This paper, some novel CCC methods will be used to separate natural products from Salvia miltiorrhiza Bunge and Andrographis paniculata. Those new methods will make CCC be applied in isolation of natural produce more largely and conveniently.At first, the choice of solvent systems for CCC separations is absolutely crucial. However, for a long time, the accurate solvent compositions in solvent system have just been researched. In this paper, we used GC method to analyze the components of solvent mixture and established a new CCC method without presaturation of the two-phase solvent mixture to isolate tanshinones from Salvia miltiorrhiza Bunge directly. The CCC method combines the results of CCC solvent system selection and components analyses of solvent mixture by GC, and thus it is possible to add accurately each individual solvent to prepare single saturated solvent phase without presaturation. The optimum CCC solvent system is a system of hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v), which has been determined by usual solvent system selection and CCC runs. As a result, over 98% pure tanshinone IIA and over 94% pure tanshinone I have been obtained by using less solvent volume.In addition, a linear gradient CCC method was applied to purify more tanshinones from Salvia miltiorrhiza Bunge. The system of hexane-ethyl acetate-ethanol-water (8:2:7:3, v/v) and (8:2:5:5, v/v) were selected as linear gradient solvent systems. As a result, with the best CCC separation conditions, four tanshinones including dihydrotanshinone I, cryptotanshinone, tanshinone I and tanshinone IIA were successfully isolated with the purity 95%, 95%, above 95% and above 98% respectively. At last, continuous elution-extrusion counter-current chromatography was developed for the efficient large-scale isolation and purification of a cytotoxic natural diterpenoid combination of 14-deoxy-andrographolide and 14-deoxy-11,12-didehydroandrographolide with mole ratio of 1:2 as well as pure andrographolide by using an optimum solvent system composed of hexane-ethyl acetate-ethanol-water (5:5:4:6, v/v). A new operation of continuous elution-extrusion CCC can save more solvent and elution time and been done more conveniently. As results, about 9 mg of pure andrographolide and 17 mg of component combination of 14-deoxy-andrographolide and 14-deoxy-11,12-didehydroandrographolide with mole ratio of 1:2 have been obtained by one injection.The structures of all obtained compounds have been identified by electrospray ionization mass spectrometry (ESI-MS), or high resolution ESI-MS, one- and two-dimensional nuclear magnetic resonance (1D- and 2D-NMR) spectra NMR spectra.