Studies On The Material Basis Of Hepatoprotective Effect Of Yinchenhao Decoction

Yinchenghao Decoction (YCHD), originated from Treatise on Febrile Disease written by Zhang Zhongjing in Han Dynasty, consists of Artemisiae Capillaris (AC), Fructus Gardeniae (FG) and Rhei radix et rhizome (RRR). It has significant effects on choleretic and jaundice-relieving, anti-hepatic injury, treating of fatty liver and resisting liver fibrosis, and has a broad application in the field of liver disease treatment. Studying and investigating the material basis for efficacy in liver disease is of great significance to develop innovative Chinese medicine which has clear composition, quality control and stable curative effect, and also of great significance to tap contemporary clinical application value of this classical prescription from a deeper level.There are numerous constituents in Chinese complex prescription, but only the ones or their metabolites absorbed into blood (collectively referred to as the drug-induced constituents by this research group) have the chances to exert pharmaceutical potency, and the effective substances must take blood as the medium to the target organ or tissue to produce efficacy. Therefore, some scholars have put forward a research strategy based on "serum containing drug" to study on pharmacodynamic material basis of Chinese complex prescription. The strategy includes "serum pharmacology" and "serum pharmacochemistry". The so-called "serum containing drug" means to the serum or plasma collected from animals or people on medication; the "serum pharmacology" refers to the pharmacodynamic evaluation on the cell model treated on medicated serum in vitro; the "serum pharmacochemistry" refers to the qualitative and quantitative analysis on medicated serum by modern analytic methods. Correlation analysis of the two methods can illustrate the pharmacodynamic material basis of Chinese complex prescription.On that account, this study established serum pharmacology and serum pharmacochemistry research method of YCHD, respectively. Taken serum containing drugs collected at 10 time-points after ig administration of YCHD and at the setting time after ig administration of 7 decomposed recipes of YCHD as object, on the one hand, fingerprints and contents of the main composition of these 17 serum containing drug were determined, on the other hand, the hepatoprotective effect of these 17 serum containing drug was evaluated, finally, the pharmacodynamic material basis of hepatoprotective effect of YCHD was clarified preliminarily by the analysis of profile-effect relationship and dose-effect relationship between the results of serum pharmacochemistry and serum pharmacology by using the comparison of drug-time curve and time-activity curve and data processing technique.The dissertation has two parts:the review and the experimental research.Part one, Literature reviewIt cited 213 articles, which illustrated the progress in the research of pharmacodyamic material basis of YCHD on hepatoprotective effect, and in the studies of pharmacodynamic material basis by combining results of serum pharmacochemistry with results of serum pharmacology. Based on this, the research thought and project design of this subject was introduced.Part Two, Experiment research1. Studies on serum pharmacology of YCHD(1) The preparative method of serum containing drug of YCHDModel of CCl4-induced injury of hepatocyte was established and rats were used as the donor to prepare serum containing drug of YCHD. Taking effect of serum containing drug on hepatocyte proliferation as an index, orthogonal experiment was carried out to screen the preparative method of serum containing drug. Results indicated that choosing the normal rats as the donator of serum,10 times equivalent dose of the extract of YCHD for adults, orally administration twice every day for 3 days, and plasma collection after 1 h of the last administration were comparatively rational. Serum containing drug of YCHD in this preparative method had significant protective effect on injured hepatocytes.(2) The establishment of the model of liver in jury in HL-7702 cells by H2O2This experiment investigated the time and concentration of H2O2 administrated to establish the model by using cell proliferation and AST infiltrated as indices. Results displayed that the appropriate time of H2O2 administration was 4h, and the appropriate concentration of injury model were 0.5mmol/L for cell proliferation test and 2.5mmol/L for AST infiltrated detection, respectively.(3) Investigation of the addition of serum containing drug in the systemTaking the cell proliferation and AST infiltrated as indices,5%,10%,20% and 40% of serum containing drug without protein were investigated to choose the appropriate addition in the system. The results showed that 5% of serum containing drug obviously promoted the cell proliferation of hepatocytes injury and reduced the AST infiltrated.2. Studies on serum pharmacochemistry of YCHD(1) HPLC-UV fingerprints of serum containing drugFingerprints of 10 batches serum containing drug of YCHD were determined and common modes of fingerprints were established at 238nm and 440nm. Compared with the retention time and UV absorption spectra of chromatographic peaks of blank serum, fingerprint in vitro and reference substance, drug-induced constituents of serum containing drug were analyzed. The results showed that similarities of fingerprints of 10 batches serum containing drug of YCHD at 238nm and 440nm were good.9 drug-induced constituents were found in fingerprint of serum containing drug at 238nm.15 drug-induced constituents were found in fingerprint of serum containing drug at 440nm; 4 of them were the common constituents with ones at 238nm.6 out of the all 20 common peaks were the original compounds contained in YCHD, other 14 were metabolites.3 constituents out of 6 original form ones were identified as geniposidic acid and geniposide (iridoids due to FG), and rhein (anthraquinone due to RRR), others 3 were identified as a anthraquinone due to RRR and 2 crocetin derivatives due to FG. Out of 14 metabolites,1 was identified as the metabolite of iridoid,4 were identified as the metabolites of anthraquinone, and 9 were identified as the metabolites of crocetin derivatives.(2) The content determination of geniposidic acid, geniposide and rhein in serum containing drug of YCHDA method for simultaneous determination of 3 drug-induced constituents (including geniposidic acid, geniposide and rhein) in serum containing drug of YCHD by HPLC was established. The results showed that the linear range of geniposidic acid, geniposide and rhein were 0.0019～0.57μg (r=0.9998),0.0018～0.54μg (r=0.9999) and 0.0016～0.48μg (r=0.9999), respectively. This method was simple, accurate and specific, and could be used for the determination of 3 constituents in serum containing drug of YCHD.3. Analysis of the profile-effect relationship and dose-effect relationship of pharmacodyamic material basis of hepatoprotective effect of YCHD(1) Analysis of the profile-effect relationship and dose-effect relationship of serum containing drug of YCHD at various time-points for hepatoprotective effectSerum containing drug of YCHD were prepared after oral administration of YCHD at 10 different time. Fingerprints, the content of geniposidic acid, geniposide and rhein in serum containing drug of YCHD, and their hepatoprotective effect were determined, profile-effect relationship and dose-effect relationship were analyzed by using the comparison of drug-time curve and time-activity curve and data processing technique. The results showed that the constituents in serum containing YCHD had significant correlation with hepatoprotective effect included original form anthraquinones due to RRR and their metabolites, original form iridoids due to FG and their metabolites, original form crocetin derivatives due to FG and their metabolites. Out of them,2 metabolites of anthraquinones, a original form anthraquinone, and a metabolite of crocetin derivatives had better therapeutic effects. The results also showed that anthraquinones including rhein and iridoids including geniposide and geniposidic acid had protection and toxigenicity on hepatocytes, that was to say the low dose had a protective effect and high dose produced toxicity.(2) Analysis of the profile-effect relationship and dose-effect relationship of serum containing drug of decomposed recipes of YCHD for hepatoprotective effectSerum containing drug of decomposed recipes of YCHD were prepared after oral administration of decomposed recipes of YCHD at 1h. Fingerprints, the content of geniposidic acid, geniposide and rhein in serum containing drug of decomposed recipes of YCHD, and their hepatoprotective effect were determined using the same method of analysis of serum containing drug of YCHD at various time-points, and the profile-effect relationship and dose-effect relationship of serum containing drug of decomposed recipes of YCHD were also analyzed using data processing technique. The results showed that the constituents in serum containing YCHD had significant correlation with hepatoprotective effect included original form anthraquinones due to RRR and their metabolites, original form iridoids due to FG and their metabolites, metabolites of crocetin derivatives due to FG. Out of them, a metabolites of anthraquinones had better therapeutic effects.The results of study on profile-effect relationship and dose-effect relationship of the serum containing drug showed that the pharmacodynamic material basis of hepatoprotective effect of YCHD included original form anthraquinones due to RRR and their metabolites, original form iridoids due to FG and their metabolites, metabolites of crocetin derivatives due to FG. Out of them, a metabolite of anthraquinones had better therapeutic effects.