Studies On The Chemical Constituents Of Ilex Rotunda Thunb.and Their Metabolic Transformation By Intestinal Bacteria In Vitro

Ilex rotunda Thunb.(Holly family)is an evergreen arbor with a wide distribution in Eastern Asia(China,Japan and Indonesia).The barks of I.rotunda have been used as a traditional Chinese medicine,called "Jiu Bi Ying".Phytochemical investigations on I.rotunda reported occurrences of triterpenoid saponins,phenolic glycosides,etc,in which triterpenoid saponins are the main chemical constituents and also the main active ingredients.Modern pharmacological studies have shown that it has anti-inflammatory,antibacterial pharmacological actions and cardiovascular pharmacological effects.It's commonly used in the treatment of various infectious diseases,cardiovascular diseases,other inflammatory diseases such as colds,tonsillitis,pharyngitis,acute gastroenteritis,diarrhea,bleeding wound,scalds,burns and bone pain.On the basis of preliminary studies,this thesis further studied on the chemical constituents of I.rotunda and choosed typical chemical components to discuss their metabolic transformation by intestinal bacterial in vitro,in order to furtherclarify the chemical composition types of I.rotunda and preliminary probe into their metabolic characteristics and law.This research will provide useful data for the deeper digging of the medicinal value of I.rotunda.In this study,the n-BuOH layer from the MeOH extract of the stem barks of this plant was subjected to column chromatography over macroporous resin,silica gel,RP C18 and semi-preparative HPLC procedures,to afford four new compounds(1,2,4,10)and seven known compounds(3,5-9,11).Their structures were elucidated on the basis of extensive spectroscopic analysis,including 1H-NMR,13C-NMR,HSQC,HMBC,1H-1H COSY and NOESY,and chemical evidences,and also by the comparison of their NMR data with those of related compounds.The ten compounds were 3-O-[?-L-rhamnopyranosyl-(1?2)-?-D-glucopyranosyl-(1?2)-?-D-arabinopyranosyl]-3?,19?-dihydroxyurs-12-en-28-oic-O-?-D-glucopyranosyl ester(1),3-O-[a-L-rhamnopy-ranosyl-(1 ?2)-?-D-glucopyranosy 1-(1 ?2)-?-D-arabinopyranosyl]-3?,19a-dihydroxyurs-12-en-28-oic-O-?-D-glucopyranosyl ester(2),ilexoside K(3),3-O-?-D-glucopyranos-y1-(1?2)-?-D-xylopyranosyl siaresinolic acid 28-O-?-D-glucopyranoside(4),ilexoside O(5),ilexpublesnin E(6),3?-0-?-D-glucopyranosyI-(1?2)-a-L-arabinopyranosyl-19-h-ydroxyl-20a-urs-12-en-28-oic acid 28-O-?-D-glucopyranosyl ester(7),19a,23-dihydroxyurs-12-en-28-oic acid 3?-O-[?-D-glucuronopyranoside-6-O-methylester]-28-O-?-D-glucopyranosyl ester(8),oblonganoside I(9),3-O-[?-D-glucopyranosyl-(1?2)-?-D-xylopyranosyl]-3?,19?-dihydroxyurs-12-en-28-oic-O-?-D-glucopranosyl ester(10),Chikusetsusaponin V methyl ester(11).They are all pentacyclic triterpenoid saponins and compounds 3,5 to 9,11 were found from this plant for the first time.We investigated the metabolism of five representative components isolated from this plant by rat intestinal bacteria in vitro.The study on the metabolism of peduncloside found two metablites could be detected,one of them was prototype(peduncloside),and another one was its aglycone(rotundic acid).From the investigation on metabolism of syringin,there were three metablites to be detected,and one of them was prototype,one could be its aglycone(synapyl alcohol)on analysis by LC-MSn,and the other was(+)-syringaresinol,which was separated by means of RP C,8 column chromatography,and analyzed by 1H-NMR,13C-NMR and LC-MSn.Concerning the metabolism of ilekudinoside H,three ingredients were detected,one of them was prototype,and its metabolic rate was very slowly.About the metabolism of 3?-O-?-D-glucopyranosyl-(1-2)-a-L-arabinopyranosyl-20(R)-19a-hydroxyurs-12-en-28-oic acid 28-O-?-D-glucopyranosyl ester,four ingredients were detected,and after 48 h,the whole of 3?-O-?-D-glucopyranosyl-(1-2)-a-L-arabinopyranosyl-20(R)-19a-hydroxy-urs-12-en-28-oic acid 28-O-?-D-glucopyranosyl ester was almost completely metabolized to the other metabolites.From the metabolism of 3-O-[a-L-rhamnopyranosyl-(1?2)-?-D-glucopyranosyl-(1?2)-?-D-arabinopyranosyl]-3?,19a-dihydroxyurs-12-en-28-oic-O-?-D-glucopyranosyl ester,four ingredients were detected,and the metabolic rate was very slowly within 12 h,but during 12 to 24 h the ilexoside ? was rapidly metabolized.These results indicated that the intestinal bacteria played an important role in the metabolism of the triterpenoid saponins of Ilex rotunda Thunb.