Studies On The Therapeutic Basis And Effective Mechanisms Of Compound Wurenchun Capsules

Chinese complex prescription（CCP） is the main modality medication of traditionalChinese medicine（TCM）. The therapeutic basis and effective mechanisms is the core ofstudies on CCP and also the key to the modernization of TCM. The relative lag of study onthe therapeutic basis and effective mechanisms of CCP has become the choke point inmodernization of TCM. So, probing into the methodology for the study on therapeutic basisand effective mechanisms of CCP is now the focus in academic circles.There are numerous constituents in CCP, but only the ones or their metabolitesabsorbed into blood have the chances to exert pharmaceutical potency. Nowadays, amajority of studies on therapeutic basis of CCP have been limited to be at in vitro level,such as fractional extraction and tracing of pharmaceutical potency, the effectiveconstituents confirmed by this way are questionable. Taking the human or animal serumafter oral administration of drug as the object, studies by pharmacology（SP） and serumpharmaceutical chemistry（SPC） can simplify the complicated system of CCP. Furthermore,binding SP to SPC is helpful to illuminate the therapeutic basis and effective mechanisms ofCCP actually. But nowadays, the study on SPC fall behind on SP, and combined study iseven less.In view of this, we took Compound Wurenchun Capsules（CWC） as an object to probeinto the methodology of study on therapeutic basis and effective mechanisms of CCP basedon combination SP with SPC. CWC is a Chinese medicine preparation used to treat chronichepatitis and possesses an exactly therapeutic effect. It was composed of FructusSchisandrae Chinensis, Radix et rhizoma notoginseng, Radix bupleuri, and Phyllanthusurinaria, with Fructus Schisandrae Chinensis being main constituent.The research of this subject proceeded with the rat serum obtained after oraladministration of CWC （serum containing CWC）. On the one hand, the constituents inCWC and drug-induced ones in blood were analysed qualitatively and quantitatively by fingerprint technology and by using many detecting means. On the other hand, we acted onmodel of CCL₄-induced injury of primary culture hepatocyte, the pharmacological action ofserum containing drug and its effective mechanisms were evaluated. Finally, the therapeuticbasis of CWC in protecting liver was illuminated roughly by means of "profile-effect"analysis. The result was validated primitively by comparing blank serum to whichcompounds were added in vitro based on concentration in serum with the serum obtainedafter oral administration of CWC for pharmacological effect.There are two parts in this dissertation, literature summarization and experimentalresearch.In part 1,145 references were cited and general thoughts and methods of studies on thetherapeutic basis of CCP, as well as the current status of studies on SP and SPC weresummarized. In addition, the thoughts and methods of this subject were narrated briefly.In part 2, there are 4 aspects of experimental research as follows:1、Qualitative analysis and quantitative determination of the main constituents inCWCBy using fingerprint technology and many detecting instruments, the qualitative andquantitative analysis of the total constituents in CWC was accomplished. Thus foundationwas established for the analysis of drug-induced constituents in rat serum after oraladministration of this preparation. There were 26 common peaks in HPLC fingerprint ofCWC, and these 26 peaks were all affirmed by correlative analysis between fingerprint ofpreparation and its raw medical materials. Peak 1 was due to Fructus Schisandrae Chinensisand Phyllanthus urinaria also, peak 3 and 4 were due to Radix et rhizoma notoginseng.Other 23 ones were all due to Fructus Schisandrae Chinensis. By comparing the retentiontime and spectrograms of chromatographic peaks detected by HPLC-DAD with the ones ofreference substances, 7 constituents out of 26 ones were identified as ginsenoside Rg₁,ginsenoside Rb₁, schisandrin, schisandrol B, schisantherin, deoxyschisandrin, andschisandrin B, respectively. Other 3 ones were deduced tentatively as Gomisin J, Gomisin N,and schisandrin C by comparing the chromagram with the ones in reference literature.Notoginsenoside R₁, ginsenoside Rg₁ and ginsenoside Rb₁ were detected in CWC by usingHPLC-ELSD method, but saponins of Radix bupleuri hadn’t been found yet. Lignans andsaponins were detected by UPLC-MS/MS respectively, and the existence of Gomisin J, Gomisin N, and schisandrin C in CWC was confirmed and saikosaponin d was alsoinspected in this preparation. Methods of HPLC-UV and HPLC-ELSD for simultaneousassaying of 5 lignans and 3 saponins were established on the base of analysis above,respectively, and the content of these constituents in 10 batches CWC were determined.2、Studies on serum pharmacology of CWCFirst of all, model of CCL₄-induced injury of primary culture hepatocyte wasestablished. By single-factor investigation and multiple-factor orthogonal experiment, withhepatocyte increment （assaying of MTT）, alanine aminotransferase （ALT） in cell culturemedium and the content of correlated constituent in serum as pharmacological indices,screenings of the preparative method of serum containing drug was carried out and itssuitable concentration in culture system was confirmed. The impact of form of medication,dosage, using normal animal or model, dose schedule, time of hemospasia, using serum orplasma, and serum containing drug in culture system of different concentration onpharmacological effect and the content of related constituents were investigated, the resultsshowed that normal rats as the donator of serum, using methanolic extract of CWC, 10times equivalent dose for adults every day, intragastric administration twice a day of 8-hourinterval, keeping on 3 days, hemospasia after 2 hours of the last administration was thebetter preparative method of the serum containing drug. Adding 10ï¼…of this serum toculture system could elevate hepatocyte increment and depress ALT in cell culture mediumsignificantly. By measuring the content of malondialdehyde （MDA） and the activity ofsuperoxide dismutase （SOD） in cell culture medium, observing the shape of cell nucleusdyed by DAPI under fluorescence microscope and ultramicrostructure of cells undertransmission electron microscope, and determining the apoptosis rate of hepatocyte by flowcytometer after dyed with Annexin V-FITC/PI, the mechanism of protecting liver of CWCwas approached from antioxidation and repressing apoptosis respectively. The resultshowed that serum containing CWC could resist oxidation and restraint apoptosis ofhepatocyte evidently.3、Studies on serum pharmaceutical chemistry of CWCThe qualitative and quantitative analysis of the main drug-induced constituents inblood was carried out by fingerprint technology and many detecting instruments. Therewere 13 common peaks in HPLC fingerprint of 10 batches serum containing CWC, which represented the drug-induced constituents in blood after oral administration of CWC. Bycomparing the chromagram of serum containing CWC with the one of this preparationdetected in the same condition, it was found that 8 constituents were the original form of thecompounds contained in CWC, and other 5 were metabolites. By comparing the retentiontime and spectrograms detected by HPLC-DAD with the ones of reference substances, 7constituents out of 8 original form ones were identified as schisandrin, schisandrol B,deoxyschisandrin, schisandrin B, Gomisin J, Gomisin N, and schisandrin C, There were 1and 3 spectrograms of metabolites similar to the ones of schisandrin and schisandrol B,respectively, and all the spectrograms of 13 drug-induced constituents possessed the samecharacter as that of dibenzocyclooctadienes. Lignans and saponins were detected byUPLC-MS/MS, schisantherin, ginsenoside Rb₁ and 13 constituents mentioned above werefound out in serum containing drug. The content of schisandrin, schisandrol B,deoxyschisandrin, and schisandrin B in serum containing drug was simultaneousdetermined by HPLC and the content of schisandrin and schisandrin B in serum and plasmawere determined and compared by the same method. The results showed that the content oflignans in each batch of serum was different, but the variance of content of theseconstituents existed group correlation. The content of lignans in plasma was higher than inresum.4、Illumination of the therapeutic basis of CWC by chart-effect relationshipanalysis and the verification of the resultsNine groups of serum were prepared after oral administration of CWC, FructusSchisandrae Chinensis, Radix et rhizoma notoginseng, Radix bupleuri, Phyllanthus urinaria,and CWC without Fructus Schisandrae Chinensis, Radix et rhizoma notoginseng, Radixbupleuri, and Phyllanthus urinaria, respectively. On the one hand, HPLC chromatograms ofthese 9 groups of serum containing drug were detected. On the other hand, the impacts ofthese 9 samples on hepatocyte injury model were evaluated by indices of hepatocyteincrement （MTT method）, and alanine aminotransferase （ALT） in cell culture medium.HPLC chromatograms showed that the 13 drug-induced constituents in serum containingCWC could be all found out in serum after oral administration of Fructus SchisandraeChinensis, as well as CWC without Radix et rhizoma notoginseng, Radix bupleuri, andPhyllanthus urinaria, but none of them could be found out in serum after oral administration of Radix et rhizoma notoginseng, Radix bupleuri, Phyllanthus urinaria, andCWC without Fructus Schisandrae Chinensis. The results of pharmacological testingshowed that the serum containing CWC, Fructus Schisandrae Chinensis, and CWC withoutRadix et rhizoma notoginseng, Radix bupleuri, and Phyllanthus urinaria possessed evidenteffect of protecting liver, but serum containing Radix et rhizoma notoginseng, Radixbupleuri, Phyllanthus urinaria, and CWC without Fructus Schisandrae Chinensis hadn’tthis effect. Furthermore, the effect distinction between Fructus Schisandrae Chinensis andCWC wasn’t significant. It showed significant relativity between chart and effect. Theresults suggested that the material basis of protecting liver in CWC was lignans fromFructus Schisandrae Chinensis, which was the main composition in CWC. In order tovalidate roughly the result, we added schisandrin, schisandrol B, deoxyschisandrin, andschisandrin B to blank serum according to the content of drug in the serum, then comparedthe pharmacological effect of serum containing reference compounds with serum obtaintedafter oral administration of CWC and Fructus Schisandrae Chinensis, respectively. The testresult showed that the serum containing reference compounds possessed the effects ofantioxidation and repressing apoptosis, but the effectiveness was inferior to the serumcontaining CWC generally, and also inferior to the serum containing Fructus SchisandraeChinensis to some extent. The main reason for this result was probably that the sorts ofreference compounds added to blank serum was much less than that of drug-inducedconstituents in blood. This test had a meaning of reference for the study on therapeutic basisof CCP by combined method of SP and SPC.