Chromatographic Behavior And Regularity Of Separation Of Steroidal Saponins From Traditional Chinese Medicine By Supercritical Fluid Chromatography

Supercritical fluid chromatography(SFC) is an efficient chromatographic technology, and has the characteristics of gas chromatography and liquid chromatography. SFC is an important supplement to HPLC and GC. SFC has the advantages of high efficiency, short analysis time, low organic solvent consumption and environmental friendliness. SFC has been utilized for the analysis and separation of natural products by the development of the technique in recent years.Steroidal saponin is an important kind of constituents in traditional Chinese medicine. There are a lot of pharmacological activites, especially for cardiovascular and cerebrovascular diseases. Some steroidal saponins also show potential value for new drug development, such as Timosaponin BII, which should significatnly up-regulate the cholinergic receptors, relieve cerebral ischemia and ischemic injury, and enhance the abilitiy of learning and memory with good safety. Column chromatography on silica gel and C18, and preparative HPLC with C18 are used for separation and purification of steroidal saponins. HPLC and UPLC have been used for analysis of steroidal saponins. But, there are some challenges in separating some steroidal saponins, such as isomers.Chromatographic conditions were studied and optimized for separations of steroidal saponins by SFC. The chromatographic behaviors of steroidal saponins in SFC were summarized for further studies.Steroidal saponins chemically consist of a steroidal aglycone and the linked oligosaccharide moieties. Among them, furostanol saponin contains two sugar chains at positions C-3 and C-26 generally, and spirostanol saponin has one sugar chain at position C-3 and a closed F ring. Spirostanol saponins are known to be formed from furostanol saponins by hydrolysis of glucosyl at C-26 and a subsequent dehydration condensation reaction between the newly formed hydroxyl group at position C-26 and the hydroxyl group at position C-22. There are many different properties between furostanol saponin and spirostanol saponin because of their different structures. The hydroxyl group at position C-22 in furostanol saponin is activated and easy to react with methanol, ethanol and so on. So, chromatographic conditions of SFC for the separation of furostanol and spirostanol saponins were optimized respectively.Firstly, we systematically studied the effects of chromatographic conditions, such as column, co-solvent, additive, back pressure and temperature, on the separation of the mixtural sample. The sample contains 18 spirostanol saponins and the mixture of two isomeric spirostanol saponins. We learned the sequence of the influence of the chromatographic conditions: column > co-solvent > additive, back pressure and temperature. The Diol and DEA columns packed with polar materials were more suitable for the separation of the spirostanol saponins. The eluting ability of methanol was the strongest. When the acid, basic and salt were added in methanol, the separation of spirostanol saponins was not improved. The retention of spirostanol saponins was less influenced by temperature and back pressure.According to the structures and polarities, the 20 spirostanol saponins were divideded into 4 groups. The conditions of SFC were optimized for the best separations of each group. The retentions of spirostanol saponins with the different sugar chains and aglycones in SFC were studied. The spirostanol saponins were better separated on the Diol column, but the sapogenins were co-eluted on the Diol column. The HSS C18 SB column was appropriate for the differentiation of sapogenins and weak polar spirostanol saponins. Sapogenins and weak polar spirostanol saponins were eluted before the polar spirostanol saponins. The saponins in each group were also separated by HPLC, respectively. We compared the separations of spirostanol saponins by SFC and by HPLC. SFC is time-saving and quite effective in separating the spirostanol saponins which share the same aglycone and vary in sugar chains. SFC is also sensitive to the number and the position of hydroxyl groups in the aglycones. HPLC(C18) is good at separating the spirostanol saponins with the same sugar moiety and various aglycones, especially different double bonds in aglycones. Therefore, HPLC(C18) and SFC are complementary in separating spirostanol saponins.Then, we systematically studied the effects of chromatographic conditions on the separation of the mixture of 3 furostanol saponins. The conditions of SFC included column, co-solvent, additive and temperature. When we separated the sample on 1-AA and Diol columns, the hydroxyl group at position C-22 in furostanol saponin reacted with methanol. The extent of the reaction on the Diol column was stronger than that on the 1-AA column. The resolution and peak shape of furostanol saponins on the Diol column were better than those on the 1-AA column. So, the Diol column was used as the stationary phase. When methanol and ethanol were used, the reactions of C22-OH occurred in the elution. The reaction of the hydroxyl with methanol was stronger than that with ethanol. But the resolution and retention time of furostanol saponins by using methanol were better than those by using ethanol. Methanol was considered as the co-solvent. When acid was added into methanol, the separation of furostanol saponins was not improved. However, the addition of aqueous ammonia obviously suppressed the reaction of the hydroxyl at C-22. The more aqueous ammonia was added, the stronger the retention of furostanol saponins was. When aqueous ammonia was added more, tailing of the peak shapes were more seriously. The elution ability of mobile phase was improved and the peak shapes were better, while 3% water was added into methanol with 0.2% NH3·H2O. When the temperature increased, the reaction of the hydroxyl at C-22 in furostanol saponins was stronger. When the temperature decreased, the elution time was longer. The optimized SFC conditions were as follows: stationary phase was the Diol column, mobile phase was CO2-MeOH/H2O(97/3, v/v)(0.2% NH3·H2O), temperature was 40 °C, back pressure was 1600 psi. Therefore, SFC was not only used for hydrophobic spirostanol saponins, and also used for the separation of hydrophilic furostanol saponins.At last, we separated the mixture of 10 furostanol saponins and profiled the extract of Dioscorea zingiberensis C. H. Wright by SFC-Q-TOF/MS. According to mass spectrometry data, the structures of 19 steroidal saponins were characterized in the extract.This thesis systematically studied the effects of conditions for the separation of steroidal saponins and summarized the chromatographic behaviors of SFC for spirostanol and furostanol saponins. The extract of Dioscorea zingiberensis C. H. Wright was well profiled and characterized by SFC. SFC has advantages on separating hydrophobic spirostanol saponins. SFC and HPLC(C18) are complementary in separating spirostanol saponins. Hydrophilic furostanol saponins can also be separated by SFC. In a word, SFC can be used for the separation of polar natural products.