Studies On Chemical Constituents Of Scutellaria Baicalensis And Metabolism Of Its Extract In Rats

Scutellaria baicalensis Georgi(S.baicalensis),a perennial herb of the Labiatae family,has long been used as a medicinal herb in East Asian countries such as China.Korea and Japan under the name "Huang Qin" in Chinese.Traditionally,S.baicalensis has been applied to treat diarrhea,jaundice,painful micturition,high fever,suppurative infection,hematemesis,epistaxis,hematuria?metrorrhagia,and bleeding during pregnancy.It has been used for more than two thousand years,and now still play important role in clinic.Systematic investigation on the chemical constituents of S.baicalensis was carried out,which resulted in the isolation of thirty one compounds,their structures were determined by chemical and spectroscopic methods,including twenty four flavonoids:skullcapflavone II(1),dihydrooroxylin A(2),wogonin(3)?oroxylin A(4),5,2'-dihydroxy-7,8-dimethoxyflavone(5),2'-hydroxy-6,7,8-trimethoxyflavone(6),5,7,2',5'-tetra hydroxy-8?6'-dimethoxyflavone(7),5,7-dihydroxy-6,8-dimethoxyflavone(8),5,7,2'-trihydroxy-6-methoxyflavone(9),5,7,2'-trihydroxy-6,8-dimethoxyflavone(10),scutellarein(12),baicalein(13),chrysin(14),norwogonin(15),5,7,2'-trihydroxyflavone(16)?5,7,2',5'-tetrahydroxyflavonol(19),5,6,7-trihydroxy-4'-methoxyflavone(20),baicalin(21),baicalein 7-O-?-D-glucopyanoside(22),wogonoside(23),chrysin 7-O-?-D-glucopyranuronoside(24),norwogonin 7-O-?-D-gluco pyranuronoside(26),norwogonin 7-O-?-D-glucopyranuronoside methyl ester(27),oroxylin A 7-O-?-D-glucopyranuronoside(28).Three phenylpropanoids:(E)-methyl-3-(4-hydroxy-3-methoxy phenyl)acrylate(11),trans-caffeic acid methyl ester(17),trans-caffeic acid(18);one phenylethanoid glycoside:martynoside(25),and three phenolic acids:benzoic acid(29),p-hydroxybenzoic acid(30),protocatechuic acid(31).Among them,compound 27 was a new compound,compound 11 and 17 were isolated for the first time from genus Scutellaria,and compound 18 was isolated from this plant for the first time.Resin adsorption as a means to separate and purify baicalin and wogonoside from extracts of Scutellaria baicalensis was investigated.Among the ten tested resins,the non-polar resin HPD-100 offered the best adsorption and desorption properties.Langmuir and Freundlich isotherms were used to describe the interactions between solutes and resin at different temperatures,and the equilibrium experimental data were well fitted to the two isotherms.Column packed with HPD-100 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process.After one round treatment with HPD-100 resin,the contents of baicalin and wogonoside were 3.6-fold and 12.0-fold increased with recovery yields of 85.7%and 65.6%,respectively.In addition,a laboratory preparative-scale separation was carried out under the final conditions.The results showed that the preparative separation of baicalin and wogonoside can be easily and efficiently achieved via adsorption and desorption on HPD-100 resin.The developed method is a promising basis for large-scale preparation of baicalin and wogonoside from Scutellaria baicalensis extracts.Recrystallizations and preparative HPLC were used for further purification of baicalin and wogonoside from the high contents fractions,respectively.The purities of the final products were around 98%and 99%for baicalin and wogonoside,respectively.Metabolism of S.baicalensis in rats was investigated.First,14 metabolites were isolated from the urine samples of rats after oral administration of Scutellaria baicalensis extract.Their structures were elucidated by UV,1H and 13C-NMR,MS,or CD techniques as baicalein(M1),wogonin(M2),chrysin(M3),norwogonin(M4),baicalin(M5),baicalein 6,7-di-O-?-D-glucopyranuronoside(M6),baicalein 6-O-?-D-glucopyranosyl-7-O-?-D-glucopyranuronoside(M7),baicalein 6-O-?-D-gluco pyranuronosyl-7-O-?-D-glucopyranoside(M8),oroxylin A 7-O-?-D-glucopyranuronoside(M9),wogonoside(M10),wogonin 7-O-sulfate(M11),chrysin 7-O-?-D-glucopyranuronoside(M12),norwogonin 8-O-?-D-glucopyranuronoside(M13),pinocembrin 7-O-?-D-glucopyranuronoside(M14).Among them,M11 was a new compound;M3,M8,M13 and M14 were obtained for the first time as metabolites of S.baicalensis.The identified metabolites were assigned into three groups,including 5,6,7-trihydroxyflavone derivatives(M1,M5,M6,M7,M8 and M9),5,7,8-trihydroxyflavone derivatives(M2,M4,M10,M11 and M13)and 5,7-dihydroxyflavone derivatives(M3,M12 and M14).In order to clarify the real existing form of S.baicalensis in vivo,UPLC/Q-TOF-MS/MS was used for the analysis of metabolites in rat urine,plasma,and bile samples.Baicalin,wogonoside and S.baicalensis extract were orally given to three groups of rats,respectively.After oral administration,their metabolites were characterized by UPLC/Q-TOF-MS/MS based on the authentic standards obtained above.Under the established chromatographic and MS conditions,a total of 22 metabolites were identified as metabolites of S.baicalensis in rats.Finally,the metabolic pathways of S.baicalensis in rats were fully profiled.