Study On The Chemical Basis Of Yinchenhao Decoction And Qushi Huayu Fang In The Treatment Of Liver Disease Based On The In Vivo Disposition Process

Yinchenhao decoction?YCHD?is a commonly used prescription for protecting liver and gallbladder in modern clinic,composed of Artemisia capillaris Thunb.,Gardenia jasminoides Ellis.,and Rheum officinale Baill.YCHD was first discribed by Zhongjing Zhang in“Treatise on Febrile Diseases”in the Eastern Han Dynasty,mainly curing for hot and humid jaundice.Qushi Huayu Fang?QHF?is a clinic-empirical TCM prescription for NAFLD treatment,which was developed by Shuguang Hospital affiliated to Shanghai University of Traditional Chinese Medicine,and composed of five TCMs comprising Artemisia capillaris Thunb.,Gardenia jasminoides Ellis.,Polygonum cuspidatum Sieb.Et Zucc.,Curcuma Longa L.,and Hypericum japonicum Thunb.ex Murray.Even though the effacacy of YCHD and QHF has been proved both in the bench and bedside,the unclear chemical basis for their effacacy restrict the in-depth study of action mechanism,as well as difficult to establish scientific and reasonable quality control methods and standards.In this work,a stragety binding the in vivo and in vitro research was adopted to investigate the chemical basis of YCHD and QHF.The ingredients of two TCM prescriptions and their in vivo disposition,including of the metabolism,pharmacokinetics,and distributions in plasma and target organs of the major constituents,were first illuminated,which contributed to pinpoint the constituents and active metabolites of YCHD and QHF with high exposure in vivo as the possible attributers for the effacacy.Then,the activities of those screened compounds were explored and validated in cell and animal models,and the main chemical basis of hepatoprotective YCHD and anti-NAFLD QHF were clarified finally.Besides,a network pharmacological analysis was conducted based on the compounds of YCHD and QHF detected in vivo to investigate their targets and pathways,and the networks of components-targets-disease were established preliminarily,which could provide scientific hints for further intensive research on the mechanism of YCHD and QHF.1.The research on the metabolism of YCHD in normal and liver-injury ratsThe qualitative analysis using HPLC-Q-TOF-MS/MS was performed on the portal vein plasma,the liver,and the systemic plasma of normal and CCl₄-induced liver-injury rats after orally administration of YCHD,to investigate the influence of liver injury on the in vivo metabolism of YCHD.There were 10 ingredients of YCHD identified in the biosamples of YCHD-treated normal rats,constiting of two organic acids?chlorogenic acid and 3,4-dicaffeoylquinic acid?,one lignin phenol?4-hydroxyphenylacetone?,one coumarin?scopoletin?,two iridoids?genipin gentiobioside and geniposide?,two monoterpenes?jasminodiol and 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxylic acid?,and two anthraquinones?rhein and emodin?.In vitrue of the regular mass spectrometric fragmentation pattern of above compounds,a total of 26 metabolites were identified in the biosamples,consisting of 25 metabolites in the portal vein plasma,9 metabolites in the liver,and 22metabolites in the systemic plasma.The major metabolic pathways included of hydrolysis,decarboxylation,dehydroxylation,oxidation,methylation,sulfation,and glucuroniadation,when the glucuronidated or sulfated conjugates accounted for 69%of all metabolites.The ingredients identified in the liver-injury rats were same as those identified in the normal rats,while the metabolites in two groups were slightly different.Compared with those in normal rats,two more metabolites?aloe emodin glucuronide and chrysophanol sulfate?were identified in the portal vein plasma of liver-injury rats.Seven metabolites were identified in the liver of model rats,without the genipin glucuronide and chrysophanol glucoside glucuronide.There were 22 metabolites detected in the systemic plasma,lacking two dehydroxy metabolites of 3,4-dicaffeyl quininic acid and one genipin sulfate,as well as plus two phase II metabolites of anthraquinone?aloe emodin glucuronic acid conjugates and the sulfate of emodin?and a methyl ester of caffeic acid.The qualitative analysis showed that the liver injury induced by CCl₄ only caused tiny changes in the amount and kinds of the metabolites in rats,but no influence on the prototypes.2.The research on the pharmacokinetics and liver distribution of YCHD in normal and liver-injury rats and the hepatoprotective effacacy of possible active constituentsOn the basis of the above systematic qualitative analysis,the prototypes and one metabolite?genipin?identified in vivo were quantified in the extraction and biosamples of normal and liver-injury rats by using an HPLC-QQQ-MS/MS method.Five prototypes including of geniposide,chlorogenic acid,rhein,4-hydroxyphenylacetone,and emodin,as well as the metabolite genipin,were analyzed accurately in vivo.In the YCHD extraction,geniposide was of highest concentration?1.00,relative ration to geniposide?,and then chlorogenic acid?0.47?,rhein?0.01?,4-hydroxyphenylacetone?0.01?,and emodin?0.001?.when rats were orally dosed of YCHD,geniposide also displayed the highest AUC levels in vivo.However,inconformity was also observed between the sequence of AUCs in vivo and the concentration in extraction of some compounds.For example,chlorogenic acid,who was the second most abundant compound in YCHD,had low AUCs in the portal vein and systemic plasma,which were lower than 10%of the AUC of geniposide,and no exposure in the liver.The AUC levels of rhein in the plasma and the liver were all in second place,but its amount in extraction was only accountig for 1%of that of geniposide.And the AUC of rhein in the systemic plasma was higher than that in the portal vein plasma,which suggested the biotransformation from other anthraquinones to rhein in the liver of rats.Comparing the AUCs of compounds in normal and liver-injury rats,the exposure levels of rhein,4-hydroxyphenylacetone,and emodin decreased about 50%?P<0.001?in the liver of model rats,manifesting that the liver injury may influence the exposure of compounds in vivo significantly.The qualitative analysis suggested that only small difference existed in the prototypes and metabolites of YCHD between the normal and liver-injury rats,while the quantative analysis showed the AUC levels of active compounds decreased significantly in the liver-injury rats,especially the target organ.This result figured out that qualitative and quantative analysis were both needed in the study on the constituents of traditional Chinese medicines,and the quantative analysis was often more important.The activity study was performed on the six compounds of YCHD who displayed certain exposure levels in vivo.results showed that six compounds all could raise the vitality?P<0.01?and SOD levels?P<0.05?of injured cells,and decrease the LDH levels?P<0.01?of injured cells.Except emodin,other five compounds could prevent the rise of AST and ALT levels?P<0.05?of cells induced by CCl₄.Besides,geniposide and rhein,the two compounds displayed high AUCs in vivo,could also cut down the AST and ALT levels of liver-injury rats.The above results proved that the two compounds screened by in vivo disposition process research?especially the quantative analysis of compounds in multiple body parts?,including of geniposide and rhein,could prevent the liver injury induced by CCl₄significantly,which were preliminarily clarified as the major chemical basis of the hepatoprotective YCHD.3.The investigation of the hepatoprotective mechanism of YCHD based on the network pharmacologyThe network pharmacology analysis was performed on the six prototypes detected in the liver and chlorogenic acid,a active compound detected in the plasma and the quality control of the mornach medicine Artemisia capillaris Thunb.A total of 194proteins were selected as the candidate targets of YCHD,while 65 targets with node amounts?10 were considered as the key targets according to the PPI network.The enrichment analysis indicated that the most correlated pathways to YCHD included pathways in cancers,PI3K-Akt signal pathway,EGFR tyrosine kinase inhibitor resistance,Hepatitis B,AGE-RAGE signaling pathway in diabetic complications,and so on.The PI3K-Akt signal pathway has been proved to be closely related to the oxidative stress,inflammatory response,insulin resistance,and liver fibrosis.Moreover,five constituents including of geniposide,emodin,rhein,scopoletin,and chlorogenic acid had regulatory effects on the PI3K-Akt signal pathway.Above results indicated that the PI3K-Akt signaling pathway may play an important role in the hepatoprotective activity of YCHD,derserving the further research.4.The research on the ingredients of QHF and its metabolism in rats and in vitro incubation system of intestinal bacteriaA total of 66 ingrendients were identified in QHF extraction relevant to six kinds of structure types,covering organic acids,iridoids,flavones,stilbenes,anthraquinones,and naphthols.There were 14 prototypes detected in the portal vein plasma,the liver,and the systemic plasma of rats after oral administration of QHF extraction,which were three organic acids?chlorogenic acid,4-hydroxyacetophenone,and hydroxybenzoic acid?from Artemisia capillaris Thunb.,two iridoids?genipin gentiobioside and geniposide?form Gardenia jasminoides Ellis.,two naphthols?torachryson sulfate?,two stilbenes?polydatin and resveratrol?,and four anthraquinones?emodin-8-O-glucoside,emodin,et al?from Polygonum cuspidatum Sieb.Et Zucc.,and a flavone?quercetin?from Hypericum japonicum Thunb.ex Murray.In addition,there were 34 compounds identified as the metabolites of QHF in rats.Phase II metabolism was the major metabolic pathway of QHF,while the hydroxylation and hydrogenation were the main phase I metabolic reaction of emodin and resveratrol in vivo,respectively.The results of in vitro metabolism by intestinal bacteria showed that chlorogenic acid,iridoid glycosides,anthraquinone glycosides,stilbene glycosides,and flavone glycosides could be hydrolyzed rapidly by the intestinal bacteria,and the corresponding metabolites such as caffeic acid,emodin,resveratrol,and quercetin could also be further metabolized in the incubation system,which demonstrated the intestinal metabolism played an important role in the in vivo metabolism of QHF.The study in this part profiled the ingredients of QHF extraction and its metabolic characteristics in rats,as well as confirmed the 14 prototypes and major metabolites of QHF detected in vivo,which provided the chemical foundations for the following quantitative analysis.5.The research on the pharmacokinetics and liver distribution of QHF in mice after single and continuous oral administration and the anti-NAFLD effacacy of possible active compoundsThe six most abundant components in QHF extraction were geniposide?1.00,relative ration to geniposide?,polydatin?0.29?,genipin gentiobioside?0.17?,emodin-8-O-glucoside?0.15?,chlorogenic acid?0.11?,and quercitrin?0.06?.The concentrations of other compounds were all lower than 5%of the concentration of geniposide.Considering that mice were usually used to establish the NAFLD model,and successive administration was adopted in the pharmacological experiment generally,we first investigated the pharmacokinetics and liver distribution of QHF in mice after single and continuous oral administration.In the systemic plasma,the liver,and the kidney of mice,geniposide was always of highest exposure levels whatever the mice were dosed once or continuously.In the single dosage group,the total exposure level of all compounds except geniposide was only about 5%of the AUC of geniposide in the systemic plasma.The five most abundant compounds in the liver were geniposide?1.00,relative ration to geniposide?,resveratrol?0.24?,emodin?0.11?,quercetin?0.07?and polydatin?0.04?,while they were geniposide?1.00,relative ration to geniposide?,genipin gentiobioside?0.03?,resveratrol?0.02?,emodin?0.02?,and 4-hydroxyphenylacetone?0.01?in the kidney.Compared with those of single-dosed rats,the AUCs of resveratrol and quercetin decreased 83%?P<0.01?and 36%?P<0.05?in the liver of rats with continuous administration,respectively.In the kidney of rats dosed continuously,the exposure level of emodin increased about 75%?P<0.01?,while the AUC of resveratrol reduced 58%?P<0.01?,and the AUCs of genipin gentiobioside,geniposide,and genipin also decreased a little bit?P<0.05?.Moreover,continuous dosage also influenced the concentration-time curces of resveratrol in the systemic plasma and the liver of mice,reflected by the disappearance of double-peak phenomenon when mice were dosed of QHF continuously.The above results suggested that the major compounds with high exposure levels in mice included geniposide,resveratrol,and emodin,whether the mice were administered of QHF singlely or repeatedly.Geniposide itself was of the highest content,and the genipin gentiobioside with the third high content in QHF could also be transformed to geniposide in vivo.However,the the contents of emodin and resveratrol in the QHF were both very low.According to the metabolic study,polydatin whose content was at second place,could be metabolized to resveratrol,and emodin-8-O-glucoside with content at forth place could be metabolized to emodin,with the hydrolysis of intestinal bacteria,which may be the major reason for the high AUC levels of resveratrol and emodin.After the comprehensive consideration of the qualitative and quantitative results in vivo and in vitro,geniposide,genipin gentiobioside,polydatin,emodin-8-O-glucoside,and chlorogenic acid were chosen as the main possible active compound composition,and the anti-NAFLD activity of the composition was investigated in the NAFLD mice.The activity research showed that the composition of five compounds could decrease the amounts of TG?P<0.01?,inhibit the fat deposition and degeneration in the liver of NAFLD mice,and display the hepatoprotection effect to a certain degree.6.The investigation of the anti-NAFLD mechanism of QHF based on the network pharmacologyAccording to the network pharmacological analysis of 14 prototypes and one active metabolites of QHF with in vivo distribution,an systematic network was established between the major compounds of QHF with in vivo distribution,the targets,and the NAFLD.The key target in the network included PTGS2,CASP3,MMP1,EGFR,MAOB,TNF,MMP2,and PTPN1.The results of pathway enrichment analysis showed that the pathways of anti-NAFLD QHF mainly consisted of metabolic disease pathways,cancer pathways,viral infectious liver disease,and systemic immunity and signal transduction pathways.These results primarily explored the potential targets nad pathways of anti-NAFLD QHF,which provided some clues for the further in-depth investigation of the anti-NAFLD mechanism of QHF.