Quality Evaluation Of Ampelopsis Grossedentata And Metabolic-related Study Of Its Bioactive Ingredient Dihydromyricetin

Vine tea is one of the most widely studied traditional Chinese medicinal and edible herb in recent years,which consists of the tender stem and leaves of Ampelopsis grossendentata(Hand.-Mazz)W.T.Wang.(family: Vitaceae;genus: Ampelopsis Michx.).And it was reported that vine tea could be used in the treatment of sore throat,skin disease,cold and fever,diabetes and alcohol.Ampelopsis grossendentata is widely distributed in the south of the Yangtze River,such as Hubei,Hunan,Chongqing,Guizhou,Guangxi and Fujian Province.Due to the differences of the climate,habitat and other conditions in different regions,the quality of Ampelopsis grossendentata from different regions is various.However,the quality of Ampelopsis grossendentata from various regions is still not systematically guaranteed and monitored until now.Dihydromyricetin(DMY)is found as the most abundant(with content of more than 30%)and bioactive flavanonol compound in Ampelopsis grossedentata.The numerous biological activities of DMY have long been demonstrated such as anti-oxidant,anti-inflammation,anti-bacterial,anti-cancer,hepatoprotective and cardio protective effects,and improvement of insulin resistance.According to the previous studies,DMY showed poor oral bioavailability,and it was predominantly distributed in the gastrointestinal tract and then eliminated in the feces.However,there are few reports about the metabolism and related study of DMY.Therefore,the thesis is focused on the Ampelopsis grossedentata and its bioactive compound DMY,and carried out from three parts including the quality evaluation of Ampelopsis grossedentata,the metabolism in vivo of DMY and the interaction of DMY with gut microbiota,which could provide important information for drug development and clinical application of DMY.Thus,the thesis is divided into three parts.1.Application of fingerprint combined with quantitative analysis of multi-component by single marker(QAMS)in quality evaluation of Ampelopsis grossedentataThe HPLC fingerprint of Amepelosis grossedentata from different regions was established.Twenty-four common peaks were found.Then the fingerprint results were further analyzed by similarity and pattern recognition analysis.The similarity result showed that the chemical compositions of Amepelosis grossedentata from different regions were similar but existed some differences.The results of cluster analysis and principal component analysis showed that the samples of Amepelosis grossedentata could be classified into three groups.Meanwhile,three important common components were identified as dihydromyricetin,myricitrin and myricetin,and QAMS method of the contents was established.The determination results showed that the contents of three components varied greatly in different regions,especially the content of DMY.In summary,the method of HPLC fingerprint combined with QAMS method can be used for the quality control of Amepelosis grossedentata,which provides scientific basis for the improvement of the quality standard of Amepelosis grossedentata.2.The metabolism study of DMY in ratsThe study was first conducted to investigate the metabolites profile of DMY in rats using UPLC-QTOF-MS/MS.In the part,the mass fragmentation of DMY standard in the negative ionization mode was performed as the first step.Meanwhile,the UPLC-Q-TOF-MS/MS method for detection and identification of DMY metabolites was established,and used for the metabolism study of DMY in rats.Based on the method,a total of eight metabolites were detected and identified in urine and feces.Three metabolites(M4,M7 and M8)were reported for the first time.Besides,it was found that the predicted metabolic pathways of DMY including reduction,dehydroxylation,methylation,and sulfation glucuronidation were proposed.The main metabolites including six phase I and II metabolites formed by reduction(M2),dehydroxylation(M3 and M5),reduction and dihydroxylation(M6)and methylation(M7 and M8)were mainly detected in the feces samples,while the few metabolites including four phase II metabolites derived from glucuronidation(M1),sulfation(M4)and methylation(M7 and M8)could be detected in the urine samples.However,the metabolites of DMY were hardly found in the plasma samples.The present findings may provide the theoretical basis for evaluating the intracorporal process of DMY,and will be helpful for its future development and application,which could also provide important scientific basis for the development of our next part study.3.Interactions of DMY with gut microbiotaThe interactions of DMY with gut microbiota were first studied in the part.Through the metabolism study of DMY by fecal microflora in vitro,it was found that DMY could be metabolized into three metabolites by fecal microflora via reduction and dehydroxylation pathways,and the dehydroxylation metabolite was the dominant one.Meanwhile,in order to consider the influence of gut microbiota metabolism on the pharmacokinetics of DMY,the pharmacokinetics of DMY in control and pseudo-germ-free rats were compared.It was shown that AUC could only slightly increase,however,Cmax could significantly increase in the pseudo-germ-free rats compared with the control rats,which indicated the gut microbiota metabolism played an important role in the pharmacokinetics of DMY.In addition,the long-term influence of DMY on gut microbiota composition by using 16 S r RNA pyrosequencing was further investigated.And it was found that DMY could markedly alter the richness and diversity of the gut microbiota and modulate the gut microbiota composition.The present findings will provide new ideas and theoretical support for the research on the pharmacological mechanism of DMY,contribute to the elucidation of the reason of its low bioavailability but high bioactivity and provide the theoretical and technical support for the study of other similar ingredients from TCM.