Study On Purification Of Steroidal Saponins And Systematic Separation Of Traditional Chinese Medicine

Steroidal saponins are a class of secondary metabolites with significant pharmacological activity and they are one of the important objects in the study of natural medicines.The separation and purification of steroidal saponins encountered from the difficulties caused by structural diversity and similarity.In this paper,combined with new chromatographic materials and separation technologies,highly efficient preparation methods for steroidal saponins were established and a study on systemic separation of traditional Chinese medicine was performed.Furthermore,quality evaluation and activity screening were carried out.The emphases of the study on separation of steroidal saponins from Paris polyphylla were the enrichment of saponins and the application of two-dimensional(2D)chromatography.According to the difference in the content,solid phase extraction(SPE)was used to enrich a minor saponins fraction and a major saponins fraction.In the light of the difference in the solubility,a 2D-reversed-phase liquid chromatography(RPLC)/hydrophilic interaction liquid chromatography(HILIC)method for the minor saponins fraction and a 2D-HILIC/RPLC method for the major saponins fraction were established.202 and 92 peaks were found in each 2D separation system.Totally,twenty-three compounds including four unreported compounds in Paris polyphylla were purified.The results showed that 2D chromatography method based on RPLC and HILIC was an effective method for the purification of saponins.Quality evaluation was carried out for the major saponins fraction,among which nine compounds were used for accurate qualitative analysis,and five compounds were used for quantitative analysis,accounting for 63.68%of the content.This fraction has the characteristics of clear chemical composition and controllable quality,and can be used as a material resource for new drug development.Study on separation of Anemarrhena asphodeloides focused on systematic separation and class separation of saponins.The extract of Anemarrhena asphodeloides was divided into a strong polar part,a medium polar part and a weak polar part in RPLC mode according to the differences in their polarity.In the medium polar part,there are two kinds of xanthones and saponins.On the basis of their diverse charge characteristics,selective separation was performed using a mixed-mode chromatography stationary phase composed of C18 and quaternary ammonium group.Experiment studied the hydrophobicity of the stationary phase,the effects of pH and salt concentration on the surface charge properties,determination of surface ion exchange capacity and saturated adsorption capacity of xanthones,as well as the effect of acid concentration on the retention behavior of xanthones.Using methanol as mobile phase,the retention of xanthones was controlled by the change of pH value,and a saponins fraction and a xanthones fraction were respectively prepared.Subsequently,twenty-six compounds including fifteen unreported ones in Anemarrhena asphodeloides were purified from the saponins fraction using a separation system based on HILIC and RPLC.A method for the isolation of strong polar part of Anenmarrhena asphodeloides was established,and eleven compounds were prepared from Anemarrhena asphodeloides for the first time.In this method,a hydrophilic reversed-phase material C18HCE was used as the stationary phase and pure water was used as the initial mobile phase.This method could overcome the problem of weak retention in traditional RPLC.Moreover.the effects of the polar groups and the compositions of mobile phase on retention behavior of the purified compounds were investigated,which could provide guidance for the separation of polar compounds.Supercritical fluid chromatography(SFC)was used to isolate the weak polar part of Anemarrhena asphodeloides,and the effects of stationary phase,modifier,column temperature and backpressure on separation performance were investigated.In combination with SFC and RPLC,fifteen compounds were isolated from the weak polar part,and nine of them were first discovered in Anemarrhena asphodeloides.The results proved the complementarity of SFC to RPLC in the separation of the weak polar compounds.Activity screening of the saponins fraction and compounds were carried out.The major saponins fraction of Paris polyphylla had significant inhibitory on the growth of A549 cells and could induce apoptosis through up-regulating promoting caspase-3 expression and down-regulating the expression of Bcl-2a protein.Anti-inflammatory effects and the influences on iNOS and COX-2 proteins expression of twenty-six compounds from Anemarrhena asphodeloides in LPS-stimulated N9 cells were also investigated.Compounds Anemarrhernasaponin ??Plaiycodin D and Platycodin D3 were cytotoxic.Compounds Timosaponin B? and trans-Hinokiresinol could inhibit the production of NO by suppressing the expressions of iNOS and COX-2 in a concentration-dependent manner.In this paper,systematic studies of separation materials,methods and modes on the separation and purification of steroidal saponins were performed.Besides,a systematic separation of traditional Chinese medicine actualized.Moreover,quality evaluation and activity screening were also carried out using insolated compounds.The results have promoting effects on the separation and purification of steroidal saponins,the understanding of chemical compositions of traditional Chinese medicine,the improvement of quality standards and the discovery of active compounds.