The Study On The Spectrum-effect Relationship Of Wulingzhi's Anticoagulant Effect And Quality Standards

Objective:Faeces Trogopterori,the dry excrement of Trogopterus xanthipes Milne-Edwars,is a kind of special traditional Chinese medicine commonly used in the clinical practice of traditional Chinese medicine.Its quality standard was stipulated in the 1977 and 1990 editions of Chinese Pharmacopoeia,with only characteristics and identification items.The 2015 edition of Shaanxi provincial standard for medicinal materials added microscopic identification and thin-layer chromatography.The lack of research on quality standards leads to the uneven quality of Faeces Trogopterori on the market,and the phenomenon of fake and inferior products emerge in endlessly.In this research,the method of spectrum-effect relationship is used to investigate the main effective components on anticoagulant effect of Faeces Trogopterori.At the same time,the fingerprint for quality control of Faeces Trogopterori and the content of total bile acids in Faeces Trogopterori is analyzed.This study can provide the basis for clarifying the anticoagulant substance of Faeces Trogopterori and improving its quality standard.Methods:1.Ultra High Performance Liquid Chromatography-Ultraviolet Visible(UPLC-UV)technology was used to establish the fingerprint of Faeces Trogopterori water extracting-alcohol precipitation solution,and Ultra High Performance Liquid Chromatography-Ultraviolet Visible-Linear Ion Trap-Orbitrap Mass Spectrum(UPLC-UV-LTQ-Orbitrap MS)was used to identify the chemical components to identify the common peaks of the fingerprint in spectrum-effect relationship.High Performance Liquid Chromatography-Ultraviolet Visible(HPLC-UV)was used to establish fingerprint of Faeces Trogopterori for its quatily control.UPLC-UV-LTQ-Orbitrap MS was used to identify the chemical components in Faeces Trogopterori after transforming the HPLC-UV fingerprint into UPLC-UV fingerprint.Mass spectrometry information combined with UV fingerprints to identify the common peaks of HPLC fingerprint.2.Using Thrombin Time(TT),Prothrombin Time(PT),Activated Partial Thromboplastin Time(APTT)as indicators,the collected Faeces Trogopterori was tested for anticoagulant activity in vitro.3.Based on the above-mentioned fingerprint and pharmacodynamics,the correlation between the common peaks of fingerprint and anticoagulant activity of each sample was analyzed by using the method of gray relational analysis.By comparing the correlation degree between the common peaks of fingerprint and anticoagulant effect,the material basis of anticoagulant effect of Faeces Trogopterori was preliminarily explored.4.Total Bile Acid Kit was used to determine the content of total bile acid in Faeces Trogopterori,so as to preliminarily improve the quality standard of Faeces Trogopterori.Results:1.The water extracting-alcohol precipitation solution fingerprint of Faeces Trogopterori were established and 40 common peaks were marked by UPLC-UV fingerprint.The similarity of fingerprint was 0.893 to 0.994.38 components were identified by UPLC-UV-LTQ-Orbitrap MS,and 9 common peaks of 40 common peaks were preliminarily identified.Besides,14 common peaks were marked in HPLC-UV fingerprint for quality control,the relative retention time and relative peak area of the common peaks were all in line with the requirements.The similarity of the fingerprint of 11 batches of samples was 0.825 to 0.999.55 components were identified by UPLC-UV-LTQ-Orbitrap MS,and 6 common peaks of the 14 common peaks were preliminarily identified.2.The anticoagulant effect of Faeces Trogopterori in vitro showed that 11 batches of samples all showed significant prolongation of thrombin time(TT).Among them,the anticoagulant effect of S4 sample was the best.The PT,TT,APTT of S4 sample all showed higher clotting time prolongation rate than other samples,the other samples had different effects on the three indicators.3.The spectrum-effect relationship results showed that the chemical components represented by all 40 common peaks in the fingerprint of Faeces Trogopterori were related to the anticoagulant effect,among which 13 common peaks were more related to the anticoagulant effect.4.The content of total bile acid in 11 batches of samples was determined.The content of bile acid in S8 samples was most in all sample.The content of bile acid in S3,S4,S6 and S11 samples all were 16.33?mol·L-1.Conclusion:In this study,the spectrum-effect relationship of anticoagulant effect and quality standard of Faeces Trogopterori were preliminary explored and improved.Firstly,the UPLC-UV fingerprint of Faeces Trogopterori spectrum effect relationship sample was established and its chemical compositions were identified.Besides,the anticoagulant effect of Faeces Trogopterori was determined.Then the anticoagulant spectrum-effect relationship was established to screen the material basis of the anticoagulant effect of Faeces Trogopterori.Secondly,the HPLC-UV and UPLC-UV fingerprints of Faeces Trogopterori quality control sample were established,its chemical composition were identified and the content of bile acids in Faeces Trogopterori was determined to further improve the quality standards of Faeces Trogopterori.