Study On Chemical Constituents And Biological Activity Of Allium Chinense G.Don

Allium chinense G.Donis is a plant of genus Allium in Liliaceae Allium family.Its main functions are treating chest pain,heart problems,abdominal swelling and pain,headache and tumors.Its dried bulbs are one of the sources of the traditional Chinese medicine Allii macrostemonis bulbus.At present,scholars at home and abroad have done more research on the chemical components of Allii macrostemonis Bulbus and Allii macrostemon Bge.（another source of Chinese medicinal material Allium chinense G.Don）,but there are few reports on the chemical composition of Allium chinense G.Don.In order to further clarify the medicinal material basis of Allium chinense G.Don and improve its quality standards,this paper conducted an in-depth study on the chemical composition,contents determination and bioactivity of Allium chinense G.Don.In the paper,several separation methods such as silica gel column chromatography,ODS column chromatography,preparative high performance liquid chromatography and others have been used to separate steroidal saponins from the 98%ethanol extracts of fresh Allium chinense G.Don..Eighteen steroidal saponin monitors were purified,and by using spectral data and physical and chemical properties analysis,they were identified as Gitogenin（X-1）,Laxogenin（X-2）,6-one-5α-cholano-22,16-lactone-3-hydroxyl-3-O-β-D-xylopyranosyl-（1→4）-[α-L-arabinopyranosyl-（1→6）]-β-D-glucop yranoside（X-3）,（25R）-5α-furostane-2β,3β,22α,26-tetraol-26-O-β-D-glucopyranoside（X-4）,（25R,S）-26-O-β-D-glucopyranosyl-5α-spirotane-3β-ol-3-O-β-D-glucopyra nosyl-（1→2）-[β-D-glucopyranosyl-（1→3）]-(6-acetyl-β-D-glucopyranosyl-（1→4）-β-D-galacopyranoside（X-5）,Macrostemonoside B（X-6）,（25R）-6-one-26-O-β-D-glucopyranosyl-5α-furostane-3β,22α,26-triol-3-O-β-D-xylopyranosyl-（1→4）-β-D-glu copyranoside（X-7）、（25R）-6-one-5α-furostane-3β,22α,24β,26-tetraol-3-O-β-D-xylopyranosyl-（1→4）-[α-L-arabinopyranosyl-（1→6）]-β-D-glucopyranoside（X-8）,（25R）-6-one-26-O-β-D-glucopyranosyl-5α-furostane-3β,22α,26-triol-3-O-β-D-glucop yranoside（X-9）,（25R）-6-one-26-O-β-D-glucopyranosyl-5α-furostane-3β,22,26-triol-3-O-α-L-arabinopyranosyl-（1→6）-β-Dglucopyranoside（X-10）,（25R）-26-O-β-D-glucopyranosyl-5α-furostane-3β,26-diol-3-O-β-D-glucopyranosyl-（1→2）-[β-D-glucop yranosyl-（1→3）]-β-D-glucopyranosyl-（1→4）-β-D-galacopyranoside（X-11）,（25R）-26-O-β-D-glucopyranosyl-5α-furostane-2α,3β,22,26-tetraol-3-O-β-D-glucopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→3）]-β-D-glucopyranosyl-（1→4）-β-D-galacopyranos ide（X-12）,（25R）-6-one-5α-furostane-3β,22α,26-triol-26-O-β-D-glucopyranoside（X-13）,5α-cholano-22,16-lactone-3-hydroxyl-3-O-β-D-glucopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→3）]-β-D-glucopyranosyl-（1→4）-β-D-galacopyranoside（X-14）,（25R）-26-O-β-D-glucopyranosyl-5α-spirotane-2α,3β-diol-3-O-β-D-glucopyranosyl-（1→2）-[β-D-glucopyranosyl-（1→3）]-β-D-glucopyranosyl-（1→4）-β-D-galacopyranosi de（X-15）,（25R）-6-one-26-O-β-D-glucopyranosyl-5α-furostane-3β,22,26-triol-3-O-β-D-xylopyranosyl-（1→4）-[α-L-arabinopyranosyl-（1→6）]-β-D-glucopyranoside（X-16）,（25R）-26-O-β-D-glucopyranosyl-5α-furostane-3β,12β,22,26-tetraol-3-O-β-D-glucop yranosyl-（1→2）-[β-D-glucopyranosyl-（1→3）]-β-D-glucopyranosyl-（1→4）-β-D-galac opyranoside（X-17）,Macrostemonoside A（X-18）.Among them,X-10 was isolated from Allium chinense G.Don for the first time.X-14,X-3,X-11,X-7,X-8,X-4 isolated from Allium chinense G.Don are new compounds.Among them,compound X-10 was isolated from Allium chinense G.Don for the first time,compound X-14,X-3,X-11,X-7,X-8,X-4 are new compounds,especially,the sapogenin structures of compound X-14 and X-3 were proved to be the new structure framework in natural products.On the basis of the above chemical composition research in this paper,solid-phase extraction-high performance liquid chromatography-evaporative light scattering detector（SPE-HPLC-ELSD）were used to determine three furostanol saponins in Allium chinense G.Don.These three furostanol saponins were isolated in this paper and they are compound X-6,X-10 and X-16.Through single factor investigation,the optimal experimental conditions of this method were determined and described as follow:pure water is the extraction solvent,the liquid-to-material ratio is 15 times（V/W,m L/g）;Heating and refluxing extraction 3 times,the extraction time is 1.5 h,1.0 h,0.5h,respectively;D-101 type macroporous adsorption resin is the stationary phase,the mass was 3 g;15%ethanol-water solution（V/V,m L/m L）was used as eluent,and the volume is 12 m L;40%ethanol-water solution（V/V,m L/m L）as the eluent,and the volume is 21 m L.Then,the chromatographic conditions for HPLC contents determination were determined and the methodological investigation was established.Under these experimental conditions,the peak area y（lg S average）and the sample mass x（lgm）of compound X-6,X-10 and X-16 showed a good linear relationship in the range of 0.40～19.72μg,0.20～10.32μg and 0.40～19.96μg,respectively.The relative standard deviation（RSDs）value of precision,repeatability,stability and sample recovery were all less than 2%（n=6）.The experimental results showed that the established determination method has the advantages of high accuracy and good precision.Assay results showed that the contents of the three compounds in different batches of fresh Allium chinense G.Donare different,and the contents in the fresh Allium chinense G.Don A medicinal materials are higher.The average contents of the three compound X-6,X-10 and X-16 were 0.144 mg·g^-1,0.166 mg·g^-1 and 0.532 mg·g^-1,respectively.Finally,In this paper,CCK-8 method was used to study the inhibitory effects of six new furostanol saponins（compound X-3,X-4,X-7,X-8,X-11 and X-14）and two known furostanol saponins（compound X-6 and X-16）on the proliferation of four kinds of tumor cells.Also,Through H₂O₂ induced PC12 neuronal cell oxidative damage model,the antioxidant activities of eight furostanol saponinswere investigated.The experimental results showed that all the compounds from Allium chinense G.Don have no obvious inhibitory effect on tumor cell growth at a concentration of 80μg·m L^-1,however,compound X-11 gave a significant protective effect on the damage of PC12neuronal cell induced by H₂O₂ and presented dose-dependent relationship.In summary,this paper conducted an in-depth study on the chemical components of Allium chinense G.Don,from which 6 new furosteroid compounds,1 compound obtained for the first time and 11 known compounds were separated and identified.A SPE-HPLC-ELSD method was established for the determination of three furosteroid saponins in fresh Allium chinense G.Don.The inhibitory effects of eight furostanol saponins on the proliferation of four types of tumor cells and antioxidant activity were studied.The research results not only enriched the types and quantities of steroidal saponins in Allium chinense G.Don,but also provided new scientific basis for the in-depth study of medicinal substances in Allium chinense G.Don.and for the development and utilization of steroidal saponins.