Study On The Steroidal Saponins And Bioactivity In Allium Chinense G.don

Xie is a perennial herb of Allium genus in Liliaceae and its Latin scientific name is Allium chinense G.Don（abbreviated to A.chinense）.Its dry bulb is one of the main sources of Allii macrostemonis bulbus.A.chinense contains a variety of biologically active substances,such as steroidal saponins,flavonoids,sulfur compounds,volatile oil and polysaccharides,etc.Among these substances,steroidal saponins are the main biologically active ingredients,which have pharmacological effects such as lowering blood lipids,anti-inflammatory,antioxidant,anti-tumor,antibacterial and antiviral,etc.The research on the extraction,separation and structure identification of the chemical components of medicinal plants has always been the most important research part in the process of excavating new bioactive components from natural products.In this paper,the chemical structure,content determination method and biological activity of the steroidal saponins in A.chinense have been studied in depth,which can not only provide a strong basis for the establishment of the quality standard of A.chinense and Allii macrostemonis bulbus,but also provide a certain reference for the new medical application of the steroidal saponins in A.chinense.In this paper,a variety of separation methods were used,including D-101macroporous adsorption resin,silica gel column chromatography,ODS column chromatography and preparative HPLC,and a total of 17 kinds of steroidal saponins were isolated from fresh A.chinense bulb using deionized water as extraction solvent.By using various spectral techniques,such as nuclear magnetic resonance（NMR）,infrared spectroscopy（IR）,mass spectrometry（MS）,etc,the structures of 17 steroidal saponins were identified,of which 5 were new and 3 were isolated from A.chinense for the first time.Among the new compounds,FA-6,FA-8,FA-9 and FA-15 are four new furostanol saponins.Their common structural characteristics are as follow:Both C-3 and C-26 are connected with sugar groups,among which C-26 is glucose,but the difference is that C-3 has different sugar chain composition.The aglycone of FA-6 is a simple and unsubstituted parent nucleus.FA-8 and FA-9 have a carbonyl substitution at the C-6 position of the parent nucleus,while FA-15 has a hydroxyl substitution at the C-2 position.FA-14 is a new spirostanol saponin whose structure in its parent nucleus is characterized by the C-6 position being replaced by a carbonyl group and the C-3 position being connected with sugar groups.A method for the determination of spirostanol saponins（FA-3,FA-4 and FA-11）in fresh A.chinense by high performance liquid chromatography（HPLC-ELSD）was established,and matrix solid phase dispersion（MSPD）was used as the sample pretreatment method.Firstly,the HPLC chromatographic conditions were optimized,and then the best experimental conditions were determined through single factor investigation,the results are as follow:silica gel was used as dispersant,and the mass ratio of silica gel to fresh A.chinense lyophilized powder was 4:1;Rinse with 30 m L cyclohexane,and then elute with 25 m L volume ratio of 70%ethanol.Secondly,a methodological study was carried out,and the results show that FA-3,FA-4,and FA-11 can exhibit good linear relationships between peak area and injection volume in the ranges of 0.32～10.24μg,0.34～10.56μg,and 0.32～10.24μg,respectively.At the same time,the method has good precision,repeatability,stability and sample recovery,and the relative standard deviation（RSD）of the method was less than 2%（n=6）.The results show that the analytical method established in this paper has the advantages of high accuracy and good precision.The results of content determination showed that the average contents of compounds FA-3,FA-4 and FA-11 in fresh A.chinense from different areas were 0.341 mg·g^-1,0.112 mg·g^-1and 0.128 mg·g^-1,respectively.In this paper,the lipase inhibitory activity of 13 steroidal saponins isolated from fresh A.chinense was studied by using in vitro model method.The results showed that some monomer compounds in A.chinense had good lipase inhibition.The results showed that compounds FA-4,FA-7 and FA-10 had strong inhibitory effect on lipase in the concentration range of 0 to 120μM,with IC₅₀values of 12.51±0.91,17.51±0.90 and 15.12±0.46μM,respectively.Compounds FA-3,FA-11 and FA-14have certain inhibitory effects on lipase in the concentration range of 0～500μM,and their IC₅₀values are 153.77±3.73,207.45±9.55 and 244.75±1.63μM,respectively.To sum up,five new steroidal saponins were isolated from fresh A.chinense,and a method for the determination of spirostanol saponins in A.chinense was established.The inhibitory effect of steroidal saponins on lipase was investigated through in vitro experiments and the structure-activity relationship was analyzed.In short,the research results of this paper are not only of great significance to the formulation of quality standards for A.chinense and Allii macrostemonis bulbus,but also provide new research ideas and methods for the development of green weight-loss products.