Chemical Composition Analysis And Metabolomics Study Of Yin-Chen-Hao-Tang Decoction

Yin-Chen-Hao Tang?YCHT?,consists of three traditional Chinese medicinal materials Artemisia capillaris Thunb.,Fructus Gardenia jasminoides Ellis and Rheum officinale Baill.,has been used to relieve liver diseases in China for thousands of years.The traditional decocting method of YCHT,named seperated decocting method?SDM?,is to decoct A.capillaris firstly for period then adding G.jasminoides and R.officinale for further decocting together untill getting the decoction.At present,a simple and easy operating method to prepare YCHT,named combined decocting method?CDM?,is to decoct three medicinal materials together simultaneously.YCHT from these two different decocting method may has some differences in chemical components that may possibly effect the pharmacological activity such as liver protection role.Therefore,it is very important to evaluate the chemical components difference of YCHT prepared from two decocting methods by using modern chromatographic and metabolomic analysis method.Modern pharmacological research reports have shown that YCHT has protection and therapeutical effect on liver injury,but its action mechanism of protecting liver is not clear completely.Usually,the chemical components and its metabolites of traditional Chinese medicine have beneficial effects on human intestinal microbial population that can keep a normal stable equilibrium of intestinal microorganism or inhibit some harmful microorganism.According to our knowledge,the composition and function of human gut microbiota are closely related to the liver injury.As a traditional Chinese medicine formulae for liver protection,YCHT is assumed to has considerable influence on human intestinal microbial population.In this context,the liver protection mechanism of YCHT was explored from the level of"gut-liver"axis that maybe explain how YCHT to effect the gut microorganism and produce liver protection effect or therapeutic action on liver injury.YCHT prepared from two decocting methods was analyzed by high-performance liquid chromatography coupling with quadrupole time-of-flight mass spectrometry for evaluating the chemical consistency difference of the dococtions.YCHT prepared with SDM was infused to stomach of rats induced acute liver injury by carbon tetrachloride?CCl₄?and histological samples,intestinal metabolites and 16s rRNA gene sequences of gut microorganism were analyzed.The Kyoto Encyclopedia of Genes and Genomes?KEGG?data base was used to find the metabolic pathways related to hepatoprotective effect and the correlation between different gut microbiota and different metabolites was evaluated.The main research content of this study includes two parts as following:1.Evaluation of the chemical consistency of YCHT prepared by two methods?SDM and CDM?YCHT prepared by two decoction methods?SDM and CDM?,was analyzed for evaluating the chemical components consistency of YCHT by high-performance liquid chromatography?HPLC?coupling with quadrupole time-of-flight mass spectrometry.The acquired datasets containing sample codes,t_R-m/z pairs and ion intensities were processed with multivariate statistical analyses,such as principal component analysis and orthogonal partial least squared discriminant analysis,to compare the chemical differences completely between the different decoction samples.The chemical components difference of two decoctions from SDM and CDM was screened out by S-plots generated from the orthogonal partial least squared discriminant analysis model and compared with chemical information from an established in-house library.Six components that contributed to the chemical differences mostly were identified as chlorogenic acid,caffeic acid,geniposide,genipin,scopoletin,and 3,5-dicaffeoylquinic acid.The concentrations of genipin and caffeic acid of the decoction from SDM were higher than those from CDM,indicating that YCHT prepared by SDM may present a stronger hepatoprotective effect than YCHT prepared by CDM.These findings indicate that not only a strategy can be established to evaluate chemical consistency of YCHT but also can provide more reasonable scientific interpretation for using decoction by SDM.However,the present result still requires further pharmacological investigations and clinical trials to verify.2.Protective effects of YCHT against acute liver injury induced by CCl₄YCHT prepared with SDM was infused to stomach of rats induced acute liver injury by CCl₄ and an integrated approach based on 16S rRNA gene sequencing combined with high-performance liquid chromatography-mass spectrometry?HPLC-MS?metabolic profiling was performed to assess the effects of YCHT on liver injury.The results showed that YCHT can regulate the relative abundances of gut microbiota and some biomarker candidates were identified.The significant changes of gut microbiota were observed at different classified level of bacterial including phylum,classis,order,familia,genus and species.Compared with the control,nine metabolites of rats were found to return to normal levels after YCHT treatment.The relative abundances of the identified microbiota,including significant elevated amounts of p_Firmicutes,c_Clostridia,o_Clostridiales,f_Ruminococcaceae,g_[Eubacterium]_coprostanoligenes_group,s_uncultured_bacterium_fLachnospiraceae and remarkedly increased amounts of p_Bacteroidetes,c_Bacteroidia,o_Bacteroidales,f_Bacteroidaceae,g_Bacteroides and s_uncultured_bacterium_gBacteroides,were found in model rats compared with controls.Potential biomarkers,including lower levels of LysoPC?16:1?9Z?/0:0?,LysoPC?20:3?5Z,8Z,11Z??,LysoPC?17:0?,LysoPC?20:1?11Z??and 3-hydroxybutyric acid and higher amounts of ornithine,L-kynurenine,hippuric acid and taurocholic acid were supposed involving in several metabolic pathways,such as arginine and proline metabolism,tryptophan metabolism,glycerophospholipid metabolism and primary bile acid biosynthesis.Interestingly,there was a strong correlation between the perturbed gut microbiota in genera Clostridia and Clostridiales and the altered plasma metabolite 3-hydroxybutyric acid.This finding means that the hepatoprotective effects of YCHT may be due to the regulation of the production of the functional metabolite 3-hydroxybutyric acid through changes in the proportions of Clostridia and Clostridiales.These results showed that the hepatoprotective effect of YCHT not only acted some basic metabolic pathways but also depended on the changes in the gut microbiota of liver injury rats.These findings suggest that the 16S rRNA gene sequencing and LC-MS metabolomics analysis can be applied to evaluate the effects of traditional Chinese medicines comprehensively.