Intestinal Bacterial Transformation And Hepatic Microsomal Metabolism In The Tannin Part Of The Tibetan Medicine Emblica Chinensis

Phyllanthus Emblica is a common Tibetan medicine that belongs to Phyllanthus L.It is referred as one of the " three main fruit" in Tibetan medicine.The other two are myrobalan and myrobalan MAO.The ancient chinese medicine doctors believe that Phyllanthus Emblica have the function of satisfy one's thirst moistening lung and resolving phlegm,heat-clearing and detoxifying,promoting digestion and relieving stasis.The components of Phyllanthus Emblica contain tannins,small molecules phenolic acids,flavonoids,volatile oil,vitamins.They have obvious functions of anti-aging,antitumor,lipid-lowering,decompression,and so on.This paper is on the basis of early research of our team.We established the models of human intestinal bacteria and rat liver microsomal metabolism in vitro to study the metabolism and the composition changes of the tannins parts.It can lay a foundation to further study of the tannins parts' metabolic pathways and efficacy material foundation.In this paper we also established a liver microsomal model to evaluate 6 kinds of CYP450 enzyme activity at the same time which is called "Cocktail" probe.With the method,we can study the impact of tannins parts and its major constituents to CYP450 enzyme activities and provide the basis for the clinical medication.The concrete research content is as follows:Part ? The extraction and content determination of tannins parts of Phyllanthus EmblicaTotal tannins parts of Phyllanthus Emblica were extracted and purificated with the established methods.The contents of total tannins was 54.17%.The mass fraction of gallic acid,corilagin and ellagic acid were 4.54%,1.61%,3.67%respectively.Part ? The metabolism study of total tannins parts of Phyllanthus Emblica with intestinal bacteria.In the study,total tannins was mixed culture with human intestinal bacteria in vitro in the conditions of 37?,anaerobic.Metabolites were detected with high performance liquid chromatography(HPLC)and high performance liquid chromatography-mass spectrometry(HPLC-MS).Gallic acid corilagin and ellagic acid,the main ingredients of total tannins in the phyllanthus emblica increased at the beginning of cultivation and decreased later.We find 9 new metabolites after the total tannins were transformed by human intestinal bacteria metabolism in vitro.4 of them were confirmed to be pyrogallic acid methyl-derivative of gallic urolithinB,urolithinA.The result tell us that total tannins in phyllanthus emblica can be transformed to be phenolic acids which have smaller molecular weight by human intestinal bacteria in vitro.This study can provide datas for metabolism of tannins part in phyllanthus emblica in human intestinal bacteria in vitro and lay a foundation for dynamics study of tannins part in phyllanthus emblica in vivo.Part ? The microsomal metabolism research of total tannins parts of Phyllanthus Emblica.In this part,the models of rat liver microsomal metabolism was established.We use it to study the metabolism of total tannins parts,gallic acid and corilagin in vitro.After hatched 30 minutes in vitro temperature,samples were centrifuged with rotate speed of 12000r.min-1.HPLC-MSn method was used to detective the metabolites.We did not find new metabolites from the samples of gallic acid and total tannins parts in liver microsomal metabolism after 30 minutes.However,two metabolites were detective in the corilagin sample,and they were confirmed as gallic acid ellagic acid.Part ? The effects of total tannins parts of Phyllanthus Emblica to liver enzyme activity.In this chapter,we established a liver microsomal model to evaluate 6 kinds of CYP450 enzyme activity at the same time.HPLC was used to obtain each peak area of probe and peak area of the internal standard compound,then calculate the ratio of peak area.Using Graphpad prism 5.0 software to do nonlinear regression analysis and compute the half inhibitory concentration IC50 of the positive inhibitors and tannins parts to CYP450 enzyme.The result shows that the positive inhibitors behaved strong inhibitory effect with IC50 are all less than 1 ?mol.L-1 however,the total tannins parts of Phyllanthus Emblica showed little effect to liver enzyme activity with IC50 are all more than 50?g.mL-1.