Determination Of The Contents Of Ingredients And Anti-virulence Effect Of Chebulae Fructus Immaturus And Chebulae Fructus From Different Habitats

Object:Xiqingguo(Chebulae Fructus Immaturus)is the dried immature fruits of Terminalia chebulae,has significant antimicrobial and antivirulence effects,but its chemical composition and main active substances are still unclear.The purpose of this thesis is to identify and analyze the chemical constituents of Xiqingguo,and clarify its antivirulence ingredients.Finally,the chemical constituents and active ingredients of Xiqingguo and Hezi(mature fruits of T.chebulae)from different producing areas are compared,providing a theory basis for the application of Xiqingguo.Methods:1.Using HPLC-ESI-QTOF-MS/MS,the components of Chebulae fructus immaturus.were identified and analyzed.2.With Staphylococcus aureus as the main object of study,using minimum inhibitory concentration(MIC)to identify the antibacterial ability of the extracts from Chebulae fructus immaturus and corilagin by microdilution method.The anti-virulence activity was studied by biofilm inhibition assay,Q-PCR and anti-α-hemolysin assay.3.HPLC is used to establish the fingerprints of 22 batches of T.chebula Retz.fruit samples from different origins.And the content of corilagin was quantified.The proposed method was validated by linearity,intra-day accuracy,inter-day accuracy,LODs and LOQs,repeatability and recovery.Results:1.By HPLC-ESI-QTOF-MS/MS methods,44 substances were identified and most of which were polyphenols.2.The MIC of extracts of Chebulae fructus immaturus and its major component corilagin against S.aureus were 1.25 mg/ml and 25 μg/ml;Both effectively inhibit the growth of biofilm,reduce the expression of genes related to quorum sensing system and inhibitα-hemolysin secretion.3.The results of fingerprint analysis showed that there were about 40 common peaks in the chromatogram of Chebulae fructus immaturus and Chebulae fructus and all samples had the same chromatographic pattern,but the peak abundances were different.The peak at the retention time at 27 minutes was found only in Chebulae fructus.Through the quantitative analysis of corilagin in Chebulae fructus immaturus and Chebulae fructus,the results were as followed: it was relatively high in all batches which ranged from 5.59 mg/g to 13.58 mg/g.The content of corilagin in the Chebulae fructus immaturus from Indonesia was the highest,and the content in Chebulae fructus immaturus was higher than Chebulae fructus.Conclusions:1.Using HPLC-ESI-QTOF-MS/MS technology to determine the composition of the Chebulae fructus immaturus,this method has the advantages of simple and fast,good stability and high sensitivity.2.Chebulae fructus immaturus and corilagin have good antiviral activity against S.aureus,and have low drug resistance,which is potential to be a new antibacterial candidate.3.Fingerprinting is used to compare the chemical constituents of Chebulae fructus immaturus and Chebulae fructus from different producing areas.This method can comprehensively reflect the chemical types and quantities of them,which is scientific and comprehensive.Quantitative analysis by HPLC is used to determine the content of corilagin in Chebulae fructus immaturus and Chebulae fructus.The method has high accuracy and good reproducibility.