Establishment Of Analytical Methods For Quality Control Of Hirudo And Its Preparation Based On Metabolites Of Gut Microbiota

The evaluation for antithrombin activity of leech,included in the 2015 edition Chinese Pharmacopoeia,plays an important role in the quality assessment of leech.As a kind of animal tradition Chinese medicine with special growth and diet environment,the quality of leech is affected by the intestinal flora and its active metabolites.However,the research about the anaerobic microbial metabolites from leech had not been investigated.This study focused on the intestinal microbial flora of leech and its metabolites.We performed the systematical studies related to the short-chain fatty acid and other metabolites of leech from diverse sources,and evaluated the quality of short-chain fatty acids in Shuxuetong injection.1.With the help of TCMSP,Gene Cards,DAVID and other public databases,the targets of seven compounds,including acetic acid,propionic acid,isobutyric acid,butyric acid,isovaleric acid,valeric acid and hexanoic acid,related to anticoagulant,antithrombotic and neuroprotective functions were searched.For exploring potential pathways on anticoagulant,antithrombotic and neuroprotective functions of short-chain fatty acids,Protein-Protein Interaction(PPI)networks were constructed by using key targets,and Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)analysis was performed.The results showed that there were 37 key targets associated with anticoagulant and antithrombotic functions directly of indirectly related to short-chain fatty acid,covering eight biological pathways,such as cell regulation,arachidonic acid metabolism,folic acid biosynthesis,coagulation system and lipid metabolism.In the investigation about brain neuroprotection,145 key targets were obtained,including biological functions such as cell adhesion,cell growth,cell proliferation,development and regeneration of nerve cells and neuronal signal transmission,which involved in 19 bioinformatics pathways such as amino acid synthesis,glycolipid amino acid metabolism,pentose phosphate and folic acid synthesis.2.The method for determining the contents of seven short-chain fatty acids(including acetic acid,propionic acid,butyric acid,valeric acid,hexanoic acid,isobutyric acid and isovaleric acid)in leech was constructed using gas chromatography(GC).The contents of short-chain fatty acids in 20 batches of leeches from different sources were determined.The results showed that the total contents of short-chain fatty acids were between 1101 and 8571 μg/g,and the contents of acetic acid,propionic acid,butyric acid,valeric acid,isobutyric acid and isovaleric acid varied significantly.The distribution of short-chain fatty acids in different parts of the leech(i.e.body wall and viscera of leech)was further studied.The results showed that the content of short-chain fatty acids per unit weight in the viscera was markedly higher than those in the body wall.During the drying process,the contents of short-chain fatty acids in fresh leech,including acetic acid,propionic acid,isobutyric acid and isovaleric acid,increased significantly due to the metabolism of intestinal microorganisms.However,after the processing of leech with high-temperature talc,the contents of short-chain fatty acids(except acetic acid)decreased and the content of acetic acid increased due to heating.3.The fingerprint of other metabolites in leech was investigated by using ultra-performance liquid chromatography(UPLC),which contained 20 peaks with peak No.11 as the reference peak.By comparison of UPLC fingerprints of leeches from different sources,the results showed that there were significant differences in the fingerprints of leeches from different sources,and the abundance of compounds in Whitmania laevis Baird was obviously higher than that in other species.The distinct difference of fingerprints between processed and raw leeches revealed that abundance and contents of compounds in the leeches significantly changed after processing,which might attribute to the difference of thermal stability of the compounds during processing.4.A rapid quantitative analytical method was established by gas chromatography for determination of short-chain fatty acids in Shuxuetong injection,which was made of leech and earthworm.The contents of acetic acid,propionic acid and butyric acid were 382.2-544.6 μg per ampulla bottle,90.92-217.5 μg per ampulla bottle,39.74-64.27 μg per ampulla bottle in Shuxuetong injection from 36 batches,respectively,showing visible difference on the contents of acetic acid,propionic acid and butyric acid in different batches.The total contents of short-chain fatty acids were 545.79-815.83 per ampulla bottle,relatively stable among different batches of Shuxuetong injection.Through analysis for leeches and earthworm,the raw material of Shuxuetong injection,both leech and earthworm contained short-chain fatty acids.The objective of this study was to establish an analytical method for the anaerobic microbial metabolites of intestinal microorganisms in leech,and apply the analytical method to the quality assessment of Shuxuetong injection.The quality of leech and its Chinese patent medicines was investigated,which is very important for clarifying the differences of multi-source leeches,the influence of processing on the quality of leech,and the distribution rule of components in different parts of the leech.