Study On The Diversity Of C₂₁ Steroids In DaiBaijie Based On HPLC-ESI-MS/MSⁿ

Daibaijie is the root of Dregea sinensis Hemsl,belongs to genus Dregea E.Mey in the family Asclepiadaceae.It has been used for blood detoxification,dehumidification,swelling and rheumatism treatment etc.By the guide of HPLC-ESI-MS/MS~n,this artical isolated C21 steroidal glycosides from ethyl acetate fraction in 95%ethanol extraction of Daibaijie(Dregea sinensis Hemsl).Finally,12 compounds were obtained via separation including 8 new and 4 known compounds.Their structures were identified by combining NMR with MS~n technology(NMR-MS~n)and the hydrolysis experiment of glycoside.The 8 new structures of steroidal glycosides were established as:3-O-[β-D-gluc opyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranoside]-11α-O-tigloyl-12β-O-cinnamoyl-tenacigenin C(1),3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyran osyl-(1→4)-β-D-oleandropyranoside]-11α-O-tigloyl-12β-O-cinnamoyl-deacylcondurangog enin A(2),3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-m ethyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranoside]-11α-O-tigloyl-12β-O-cinnamoyl-drevogenin P(3),3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranoside]-11α-O-cinnamoyl-12β-O-benzoyl-deacylcondurangogenin A(4),3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucopyran osyl-(1→4)-6–deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranoside]-11α,12β-O-benzoyl-deacylcondurangogenin A(5),3-O-[β-D-glucopyranosyl-(1→4)-β-D-gluc opyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyranoside]-11α-O-cinnamoyl-12β-O-benzoyl-tenacigenin C(6),3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandropyr anoside]-11α-O-cinnamoyl-12β-O-benzoyl-marsdenin(7),3-O-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-6-deoxy-3-O-methyl-β-D-allopyranosyl-(1→4)-β-D-oleandr opyranoside]-11α-O-tigloyl-12β-O-cinnamoyl-tenacigenin B(8).The 69 C21 steroidal structures of the minor components in Daibaijie were elucidated based on the fragment ions and accurate molecular weight provided by HPLC-ESI-MS/MS~nand HR-MS.As a result,59 compounds unreported previous and 10 known compounds were deducted.All C21 steroidal glycosides in this work can be classified into the following types based on the steroidal structure:C-8-R4,C-14-OH,C-17-R5,C-5/C-6 double bond(Type E);C-8-R4,C-14-OH,(Type F);C-8/C-14 epoxy ring(Type G);C-8/C-14 epoxy ring,C-5/C-6 double bond(Type H).For the activity study of the minor components,191 C21 steroidal glycosides database was built including the all reported isolated structures and predicted structures in Daibaijie.By using computer assisted drug screening technique,191 C21 steroids docked with 80proteins’structures associated with tumor and immune.By molecular docking,proteins and potential compounds with activity were selected out.The results showed that most of the 81compounds in this paper had the binding trends with IL-2Rα,TNF-αand TLR4 proteins,but no bound with tumor proteins.The most active compounds were selected,they were compound 5,6,18,23,30,78,79 and 80.Moreover,SPR experiment in vitro verified the reliability of computer virtual screening and 5 compounds were chosen selectively.They were compound 6,18,30,78 and 80.Among them,compound 6 was only bounded with TNF-α;compound 30 and 78 were both bounded with IL-2Rαand TLR4;compound 18and 80 were only bounded with IL-2Rα.The ADMET predictions in Discovery Studio showed that compound 30 was the potential candidate of pills.The CDOCKER in Discovery Studio gave us the binding mode of the 5 compounds with proteins,that is,hydrogen bond interaction and Pi-Pi interaction were the main interactional founctions,also,it was related with the types of the substitutes of the compounds.The 12 isolated compounds were evaluated for their anti-tumor activity and inhibiting experiments on mouse spleen cells in vitro.And compounds 2,5,6,8 and 34 showed moderate cytotoxicity against HL-60.Besides,only compounds 32 and 33 had the activity of inhibiting the proliferation of mouse spleen cells in the cell immunity assay.And compound 31 showed the antagonism with LPS on the mouse spleen cell proliferation.However,the crude extraction of Daibaijie can inhibited the proliferation significantly and antagonized the function of Con A or LPS for the T or B spleen cells proliferation in vitro significantly.