| Yin Chen Hao Tang(YCHT)is one of the most famous herbal formulas recorded in Shanghanlun of Zhongjing Zhang in Han dynasty.YCHT has long been used in clinical practice for treating jaundice.It contains three component herbs: Artemisia capillaris Thunb.(Yinchen),Gardenia jasminoides Ellis(Zhizi)and Rheum Palmatum L.(Dahuang).YCHT can be used to treat liver injury and liver fibrosis.However,present researches about the effective substances and protective mechanism of YCHT against liver injury are not comprehensive.Thus,in this study,we firstly conducted chemome analysis of YCHT,and network pharmacology was further applied to investigate its protection mechanism against liver injury based on above results.In vitro and in vivo quantitative studies of some relevant active components in YCHT were also conducted.The content of this study could be divided into four parts:(1)The rapid identification of ingredients of Yin Chen Hao TangUHPLC-Q-TOF/MS is a highly sensitive and qualitative liquid chromatograph mass spectrometer technology,which can quickly separate and identify complex systems in a short period of time.In this study,the chemical constituents in YCHT were quickly identified with this analysis method.The ACQUITY UPLC HSS T3 column(2.1 mm × 100 mm,1.8 μm)was used for separation.The mobile phase was 0.1% formic acid aqueous solution-0.1% formic acid acetonitrile with gradient elution.The mass spectra were collected in positive and negative ion modes,respectively.After analyzing the data based on the established database,140 compounds have been identified.This method laid the foundation for the network pharmacology research.(2)Network pharmacology-based study of compound-target interaction of Yin Chen Hao Tang.In this study,18 representative hepatoprotective active ingredients were screened from YCHT by text mining based on above chemical substance group results.The potential targets of these compounds were then predicted using the HTDocking Anti-Liver Fibrosis platform.At last,a compound-target network of YCHT was constructed by Cytoscape to illustrate inhibition of hepatic stellate cell proliferation and activation and antioxidant stress might be its action mechanism against liver injury.(3)In vitro quantitative analysis of 14 components in Yin Chen Hao Tang.Scoparone,catechin,chlorogenic acid,scopoletin,hyperosid,esculetin,geniposide,quercetin,genipin,rhein,aloe-emodin,chrysophanol,emodin and physcion are active ingredients in YCHT against liver injury.In this study,we developed a novel HPLC-DAD based analytical method for simultaneous determination of 14 constituents in YCHT.An Agilent Eclipse Plus C18(4.6 mm × 250 mm,5 μm)column was chose for the separation,and the mobile phase is acetonitrile-0.1% phosphoric acid solution with gradient elution.The contents in 3 samples were in the ranges of 0.136-0.168 mg·g-1 for scoparone,0.080-0.104 mg·g-1 for catechin,0.403-0.470 mg·g-1 for chlorogenic acid,0.008-0.009 mg·g-1 for scopoletin,0.061-0.068 mg·g-1 for hyperoside,0.020-0.027 mg·g-1 for esculetin,0.269-0.306 mg·g-1 for geniposide,0.036-0.048 mg·g-1 for quercetin,0.005-0.006 mg·g-1 for genipin,0.122-0.164 mg·g-1 for rhein,0.037-0.044 mg·g-1 for aloe-emodin,0.030-0.037 mg·g-1 for chrysophanol,0.011-0.014 mg·g-1 for emodin and 0.007-0.010 mg·g-1 for physcion.Our established method can be used for quantitative analysis and quality control of different batches of YCHT.(4)Comparative pharmacokinetic of eight compounds in Yin Chen Hao Tang after oral administration in normal and acute liver injury rats.After oral administration of traditional Chinese medicine,the content of active ingredients in plasma is directly related to the therapeutic effect,which is affected by disease state.In this study,we developed and applied an UHPLC-MS/MS method for comparative pharmacokinetic study of eight major bioactive components(scoparone,scopoletin,geniposide,rhein,aloe-emodin,chrysophanol,emodin and physcion)from YCHT in normal and acute liver injured rats.Results reveal that there are significant differences in the pharmacokinetics of scoparone,geniposide,aloe-emodin,rhein,chrysophanol and physcion in hepatic injured rats as compared to in control except for scopoletin and emodin.The results provided some reference for the clinical safety and rational administration of YCHT.In conclusion,the chemical substances of YCHT were clarified,and we constructed a "Component-Target" network based on this chemome results by network pharmacology to explain the action mechanism of YCHT against liver injury.We developed a HPLC quantitative analysis method to determine of 14 compounds of potential liver-protect activity in vitro,and the in vivo quantitative method of 8 components.After the construction of rats with acute liver injury,the pharmacokinetic behavior of YCHT in normal and diseased rats was studied,which indicated that the efficacy of the drug was affected by the physiological state.This study provides a reference for the quality control and the rational use of YCHT,and also provides a new idea for the modernization of traditional Chinese medicine formulas. |
