Studies On The Primary Metabonomics And Pharmacokinetics Of Guizhi-Fuling Capsule

Guizhi-Fuling Capsule（GFC） is a TCM formulation,which is originated from Guizhi-Fuling Wan,first described in the Jin Kui Yao Lue（220 A.D.） written by a famous doctor of Han Dynasty named Zhang Zhongjing.Clinically,GFC is used to treat gynecological malignant tumor.GFC is composed of five kinds of crude herbs,Ramulus Cinnamomi,Poria,Cortex Moutan,Radix Paeoniae Alba,and Semen Persicae.The chemical constituents,quality control methods,the pharmacokinetics and metabolonomics of GFC were investigated in detail.The chemical constituents of Ramulus Cinnamomi,Poria,and Radix Paeoniae Alba were systematically studied and 23 compounds were purified with silica gel,Sephadex LH-20 and ODS column chromatography.Utilizing chemical and spectroscopic methods（UV,NMR, MS）,the structures of 19 compounds were fully characterized.There were seven triterpenes, three monoterpene glycosides,three aromatic acids,and so on.The quality control methods of Poria were studies.A RP-HPLC method was established to determine pachymic acid and dehydro-pachymic acid in poria.A RP-HPLC method with gradient elution was established to determine gallic acid,ethyl gallicate,albiflorin,peoniflorin, benzoylpaeoniflorin and paeonol in GFC.An HPLC-MS method was established to determine dehydrotumulosic acid,tumulosic acid,polyporenic acid C,3-epi-dehydrotumulosic acid, dehydropachymic acid and pachymic acid in poria and GFC.The assay methods are simple, rapid and with good reproducibility and provide a quantitative basis for the quality assessment for herb and GFC. An UPLC-MS method was developed to determine the urine samples.Urine samples were pretreated with protein precipitation using methanol,and the supernatants were separated on a reversed-phase C₁₈ column with gradient elution using a mobile phase of acetonitrile-0.1% formic acid at a flow-rate of 0.25 mL·min^-1.Analysis of urine samples from two groups（the healthy control vs.uterine fibroids model rat） at pre-dose and the 16 th week with established method were illustrated.Visual examination of the LC/MS displayed a clear difference with each other.The analytical data were processed via multivariate analysis such as（Principle component analysis） PCA.PCA scores plot of analytical data describes the general differences between two different groups at the end point of the study whereas little variation is observed at pre-dose.The mechanism of pathological changes may be elucidated with the up- or down-regulated metabolic pathways. The dominant metabolites obtained from loading plot of Masslynex were the differential metabolites between healthy control and pathological rats.A simple HPLC-UV method of determination of cinnamic acid and paeonol were developed in rat plasma,urine and feces.Phenylbutyric acid was selected as internal standard （IS）.Plasma and urine samples were pretreated with protein precipitation using methanol,and the supernatants were separated on a reversed-phase C₁₈ column,using a mobile phase of acetonitrile-0.1%phosphoric acid at a flow-rate of 1.0 mL·min^-1.Feces samples were extracted with methanol.A sensitive and specific method was developed and validated for the determination of albiflorin（AF） and paeoniflorin（PF） in rat plasma,urine and feces by liquid chromatography - mass spectrometry.Plasma and urine samples were extracted using ethyl acetate,and then were separated on a C₁₈ column,using a mobile phase of acetonitrile-0.1%formic acid（25:75, v/v） at a flow-rate of 0.8 mL/min.Geniposide was used as IS.A mass spectrometer equipped with electrospray ionization source was operated in the positive ion mode.A sensitive and specific method was developed and validated for the determination of dehydrotumulosic acid（DTA）,tumulosic acid（TA）,polyporenic acid C（PAC） and 3-epi-dehydrotumulosic acid（3-EDTA） in rat plasma and feces by liquid chromatography - mass spectrometry.Plasma samples were extracted using diethyl ether,and then were separated on a C₁₈ column,using a mobile phase of acetonitrile-0.1%formic acid（75:25,v/v） at a flow-rate of 0.8 mL/min.Luteum was used as the IS.A mass spectrometer equipped with electrospray ionization source was operated in the positive ion mode.These methods were applied to the pharmacokinetic and excretion study of them after intragastic administration of extract of single herbs or formula to rat.The pharmacokinetic and excretion characteristics were significantly different between rats with administration of herbs and rats of GFC or between healthy and pathological rats after administration of GFC because of the effects of co-existing compounds even with the same dosages.Combining the utilization of phytochemistry,pharmaceutical analysis,matabonomics, analytical chemistry,statistics,pharmacology and pharmacokinetics,the quality assessment standards and pharmacokinetics for herbs and GFC were studied,and the metabolomics of uterine fibroids were also investigated.This research provided a beneficial exploration for the modernization of traditional Chinese medicine.