| Pheretima was a common animal-derived traditional Chinese medicine.Pheretima was habitual divided into Guang Pheretima and Hu Pheretima.Guang Pheretima was the dry body of Pheretima aspergillum(E.Perrier),and the Hu Pheretima was the dry body of Pheretima vulgaris Chen or Pheretima guillelmi(Michaelsen)or Pheretima pectinifera Michaelsen.Pheretima nature and flavor were cold and salt,respectively.It has clearing heat,calming shock,dredging collaterals,relieving asthma and diuresis properties.Pheretima was usually treated in arthralgia,hemiplegia,lung heat,cough and asthma,and so on.Clinically,Pheretima was usually used to cure asthma,scald,stroke and its sequelae,etc.As the modern pharmacological researches shown,Pheretima extract possessed good effects on anti-inflammatory,anti-fibrosis,anti-tumor and neuroprotective.Pheretima origin was intermingle.Pheretima,Eisenia foetida and Amynthas were the common varieties of China.However,there was not a convenient and quick method to recognize the four species documented in Chinese Pharmacopoeia.The mean of Pheretima quality control was relative single.In the latest pharmacopoeia,microscopic observation and thin layer chromatogram was set as identification items.Water soluble extract and inspection items,including impurity and humidity and so on,were set as quality control markers.At present,it was urgent for seeking a handy and efficient method on Pheretima quality control.What’s more,the chemical components of Pheretima were complexincluding amide acid,nucleoside,purine,base,peptide and protein.It made the anti-coagulation material basis be ambiguous.Based on the above problems,the fact that Pheretima could effectively treat ischemic stroke was seemed as research basis in this study.DNA barcode,intelligent combined with artificial annotation analysis,stoichiometry as well as chemical fingerprint were integrated for establishing a standard quality analysis system of Pheretima.The specific experiments were as follow:1.Based on DNA barcode technology,the decoction pieces family was detected by analyzing mitochondrion COⅠ gene of forty commercial batches Pheretima.Finally,in forty batches of goods,there were 5 batches of Pheretima aspergillum,11 batches of Pheretima vulgaris,4 batches of Amynthas fuscatus and 6 batches of Metaphire magna,etc.2.Based on ultra-high performance liquid chromatogram-quadruple time of flight-mass spectrum(UPLC-Q-TOF-MS),the MS information of Pheretima water extract was obtained.By integrating artificial and intelligent annotation tools,such as GNPS,Met DNA and HMDB,the qualitative analysis of Pheretima was performed.In order to carrying out accurate annotation,the MS fragment pathways were concluded and the relative literature was referenced.In conclusion,there were 128 compositions which were successfully identified,including 33 amide acids,22 dipeptides,20 alkaloids,12 nucleosides,11 organic acids,5phospholipids and 25 other ingredients.3.By using UHPLC-Q-To F-MS,the MS information which contained 15 batches Pheretima aspergillum and 27 batches Pheretima vulgaris was obtained.After analyzing with multivariate statistical analysis,there were 179 and 155 variables which was recognized under positive and negative acquisition modes.Among them,31 compounds were identified.Some compounds such as instance adenosine and citrulline had higher contents in Pheretima aspergillum,while others such as histidine and sucrose had higher contents in Pheretima vulgaris.Based on network pharmacology analysis of variable compounds,there were 65 potential targets and 83 signal pathways were predicted.What’s more,it became clearly that the effect on LPA,LCN2,SOD2 and AKT1 were different between Pheretima aspergillum and Pheretima vulgaris.Their main mechanisms against stroke might disaccord.4.The extraction process(40 ℃,220 W,40 min)was optimized by orthogonal experimental design.The chemical fingerprint of 40 batches Pheretima was successfully established.Six common peaks were identified,they were hypoxanthine,urine,phenylalanine,inosine,guanosine and tryptophan.The result of network pharmacology showed that 6 common ingredients could relate to 51 potential targets against ischemic stroke.It suggested that the six compounds could be served as efficiency Q-Marker of Pheretima.The multiple compounds HPLC-DAD quantitative method was established.At the same time,anti-coagulate activity was determined by fibrinogen-thrombin titration.Then,the gray correlation analysis and “contentanticoagulation activity” heatmap was performed.It verified that urine,phenylalanine,guanosine and tryptophan could be served as anti-coagulation Q-markers of Pheretima.Ultimately,the total content of efficiency Q-Markers in Pheretima should no less than 0.3%according the 95% confidence interval. |
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