Study On The Spectrum-effect Relationship Of Bupluerum Chinense And GC-MS Analysis Of Supercritical-CO₂Fluid Extraction

Objective: Our ongoing effort is consists of four parts. First of all, a quantitativedetermination method of the saikosaponins in Bupluerum chinense usingsaikosaponin-a （SSa） as a standard substance has been developed. The samples whichobtained from different origin and different harvest time were determined. Secondly,the objective is to study the effect of saikosaponins on CCl₄^-induced hepatic injury inthe mice, and to master the efficacy characteristics of saikosaponins, as a result lay thefoundation for exploring the spectrum-effect relationship of Bupluerum chinense.After that, it is to perform the correlation analysis between the chromatographicfingerprints （HPLC-ELSD） that was used to characterize the features of saikosaponinsextracted from Bupluerum chinense and hepatoprotective activity, and to clarify thetherapeutic basis of hepatoprotective of Bupleurum chinense. What is more, to studythe chemical composition and the dynamic change law of supercritical fluid extractionobtained from Bupleurum chinense at Songxian region in Henan province.Methods: The first experiment: the chromogenic reagent was phosphoricacid–dimethylamino benzaldehyde, the standard of saikosaponin-a （SSa） was selectedfor determination. The pharmacodynamics: the experimental models of the acutehepatic injury were induced by intraperitoneal injection of carbon tetrachloride （CCl4）in mice. The different concentration of the saikosaponins was administered by ig for7days. Serum was analyzed for alanine and aspartate aminotransferase （ALT and AST）;liver samples were measured for malonaldehyde （MDA） contents and superoxidedismutase （SOD） activities in normal group, model group and saikosaponins group.The investigation of spectrum-effect relationship: the fingerprint characteristic of then-butanol fraction extracted from Bupleurum chinense was constructed by thetechnology of HPLC with evaporative light scattering detection （ELSD）. The n-butanolfraction effects on acute hepatic injury induced by CCl4in mice were obtained by the research of pharmacology experiments, including the relevant indicators such asthe activities of ALT, AST, SOD and the contents of MDA in serum or liver.Thecorrelation between the chromatographic fingerprint and the hepatoprotective efficacywas carried out with the methods of chemometric and canonical correlation analysis.The last part: the technology of supercritical-CO2fluid extraction （SFE-CO2） was usedto extract the herbal medicinal ingredients, the composition of chemical constituentswas determined by GC-MS spectrometry.Results: The first experiment: the calibration curve of SSa had good linearrelationship in range of0.0306～0.3062mg （r=0.9996）, the relative standarddeviations （RSD） value of methodological study were less than3.30%. Thepharmacodynamics: by compare with the model group, the drug group whichpreventive treatment with the saikosaponins （the dose was20g·kg-1，an equivalent of15.5times clinical dose.）could significantly decreased ALT and AST activities inserum（P<0.05）, at the same time, it could obviously enhance the activities of SOD andreduce the contents of MDA in liver（P<0.05）. The investigation of spectrum-effectrelationship:12co-possessing peaks were defined base on HPLC-ELSD thechromatographic fingerprint, and were divided into two groups that according to theresult of the cluster analysis including “major saikosaponin” cluster and “minorsaikosaponin” cluster. The hepatoprotective efficacy was significant difference amongthe different samples （P<0.05）. The result of cluster analysis and canonical correlationanalysis showed that there are linear correlation between the two variables which wasthe activities of ALT, SOD and the compounds of saikosaponin a, d. The correlationparameter of canonical variables got by two variables was0.842, and the significantlevel was0.039. The last part:36main compounds which accounted for74.32%-90.45%of total extract were identified, the relative content of the fatty acidswhich extracted by SFE-CO2was45.10%-80.95%, the aldehydes was1.61%-5.65%,the ketones was0.65%-1.28%, the esters was4.80%-8.12%.Conclusion: Accoeding to this study, it is suggested that the determination issimple, feasible and reproducible. It can be used to determine the saikosaponins extractfrom Bupluerum chinense. The saikosaponins exists in all samples is obviousdifferences which come from different origin and different harvest time, especiallythere are a highest content when the harvest time is on September28. Thesaikosaponins extract from Bupluerum chinense have protective effects on acutehepatic injury induced by CCl4in mice. The therapeutic basis of hepatoprotective efficacy which can significantly affect the AST activities in serum and the activities ofSOD in liver is mainly decided by SSa and SSd jointly. The research ofspectrum-effect relationship is an effective way for exploring the correlationshipbetween chemical composition and hepatoprotective efficacy.This thinking of dataprocessing which combine with the chemometric and canonical correlation analysis notonly has made up for the deficiencies of the current research method of spectrum-effectrelationship, but also provide a meaningful attempt and an innovative idea.With thedifferent period of harvest, the relative content of fatty acids, aldehydes, ketones andesters are different. This analysis can provide scientific basis for crude drug productionand processing.