Study On Isolation,Identification Of C₂₁ Steroidal Compounds From Cynanchum Bungei And Their Inhibitory Activities Of Hedgehog Signaling Pathway

Cynanchum bungei Decne.is a famous traditional Chinese medicine as a tonic medicine or health food for centuries.The extract from Cynanchum bungei has anti-tumor activity,the C₂₁ steroidal compounds were determined as main ingredients of this plant.Since activation of the Hedgehog signaling pathway is implicated in several types of human cancers,the inhibitors targeting at hedgehog signaling pathway have become a new hotspot for anti-cancer drugs.This paper systematically studied the chemistry of C₂₁ steroidal compounds from Cynanchum bungei Decne.and their activities of inhibition against Hedgehog signaling pathway.By using modern chromatographic separation techniques,twenty-two C₂₁ steroidal compounds were obtained,including fourteen C₂₁ steroidal glycosides?1-14?and eight C₂₁ steroidal aglycones?21-28?.Meanwhile,their chemical structures were identified by modern spectroscopic techniques and six C₂₁ steroidal glycosides?3-8?and two C₂₁ steroidal aglycones?25 and 27?were new compounds.The activities of inhibition against Hedgehog signaling pathway of these C₂₁ steroidal aglycones were measured.Fourteen C₂₁ steroidal glycosides?1-14?were isolated from alcohol extract from the roots of Cynanchum bungei.Their structures were determined on the basis of extensive spectroscopic methods including 1H NMR,13 C NMR,DEPT,HMQC,HMBC and HRESI-MS.Compounds 1-14 were determined as Kidjoranine 3-O-?-D-glucopyranosyl?1?4?-?-L-cymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?1?,Caudatin 3-O-?-D-glucopyranosyl?1?4?-?-L-cymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?2?,Kidjoranine 3-O-?-D-glucopyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-Dcymaropyra-noside?3?,12-O-nicotinoyl-20-O-cinnamonacyl-sarcostin-O-?-D-glucopyranosyl?1?4?-?-L-cymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosy l?1?4?-?-D-digitoxopyranoside?4?,Penupogenin 3-O-?-D-glucopyranosyl?1?4?-?-Lcymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-digit oxopyranoside?5?,Kidjoranine 3-O-?-D-glucopyranosyl?1?4?-?-L-cymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-digitoxopyranoside?6?,Caudatin 3-O-?-D-glucopyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?7?,12-O-benzoyl-deacetylmetaplexigenin 3-O-?-D-glucopyranosyl?1?4?-?-Lcymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?8?,Caudatin 3-O-?-D-glucopyranosyl?1?4?-?-L-cymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-cymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?9?,Caudatin 3-O-?-L-cymaropyranosyl?1?4?-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?10?,Caudatin 3-O-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?11?,Kidjoranine3-O-?-D-cymaropyranosyl?1?4?-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?12?,Kidjoranine 3-O-?-L-diginopyranosyl?1?4?-?-D-cymaropyranoside?13?,Caudatin 3-O-?-D-cymaropyranosyl?1?4?-?-L-diginopy-ranosyl?1?4?-?-D-cymaropyranoside?14?,respectively.Compounds 3-8 were new compounds.Eight C₂₁ steroidal aglycones?21-28?and baishouwu benzophenone?29?were isolated from the acidic hydrolysis products of the crude steroidal glycosides from the roots of Cynanchum bungei.Their structures were determined on the basis of extensive spectroscopic methods including 1H NMR,13 C NMR,DEPT,HMQC,HMBC and HRESI-MS.Compounds 21-28 were determined as 12-O-benzoyl-deacetylmetaplexigenin?21?,12-O-acetyl-20-O-?2-methylbutanoyl?-sarcostin?22?,20-O-Acetyl-penupogenin?23?,Gracigenin?24?,8,14-seco-caudatin?25?,Penupogenin?26?,?20S?-12b-cinnamoyloxy-20-acetyloxy-3b,5,17-trihydroxy-8,14-seco-5b,17a-pregn-6-ene-8,14-dione?27?,Isoikemagenin?28?,respectively.Among them,25 and 27 were new compounds.The inhibitory activities against the hedgehog signaling pathway of compounds 25-28 and compounds 15-20,30,31 isolated in the early stages were determined by Dual luciferase reporter gene assay based on Gli activity,GANT-58 as the positive control.The results showed that compounds 18,19,20,26,30 and 31 displayed inhibitory activities on the hedgehog signaling pathway in vitro,with IC50 value being 6.4±0.9 ?M,7.2±0.7 ?M,9.7±2.3 ?M,14.1±6.6 ?M,2.9±0.3 ?M and 3.9±0.2 ?M,respectively.The absolute configuration of compounds 30 and 31 isolated from the root of Cynanchum bungei was identificated.The absolute structures of methylbutyric acyl from30 and 31 were determined by comparing the data of 1H NMR,13 C NMR,Optical rotations,MS and retention time on UPLC with those of methylbutyric acyl-?S?-?-?-1-ethyl benzene,a pair of epimer,obtained by chemical synthHRESIs,respectively.Then compounds 30 and31 were determined as 12-O-cinnamoyl-20-O-?S?-?+?-?-methylbutyryl–sarcostin and 12-Ocinnamoyl-20-O-?R?-?-?-?-methylbutyrylsarcostin,respectively.