| Massa Medicata Fermentata(MMF),also known as Liushenqu and Liuqu,was first published in Yaoxinglun.It is a traditional compound fermented drug made by Semen Armeniacae Amarum,Semen Vignae,flour,wheat bran,Herba Artemisiae Annuae,Herba Polygoni and Herba Xanthii.It is commonly used in clinic to treat spleen and stomach weakness,diet stagnation,chest and abdominal distension,infantile food accumulation and so on.As a commonly used drug,MMF has not been included in the Pharmacopoeia of the People’s Republic of China.There are great differences in fermentation methods across the country.Besides,there is a lack of unified and quantifiable indicators for quality evaluation,and it is unable to focus on the key factors affecting its quality because of the complexity and uncontrollability of the fermentation process,which limits the in-depth research and clinical application of MMF.In this study,the changes of chemical components before and after fermentation were comprehensively studied.The effects of fermentation mode and fermentation season on MMF fermentation were also observed and the changes of amino acids during processing were analyzed.In addition,the pharmacological mechanism of amino acids in the treatment of gastrointestinal diseases was predicted based on network pharmacology.Based on these results,the key factors and the quality markers affecting MMF fermentation would be revealed,so as to lay a foundation for the construction of scientific and reasonable quality control methods.The main chemical components of unfermented and fermented MMF were identified by UHPLC-Q-Orbitrap HRMS.The relative peak area of each compound was used as a variable for statistical analysis,and the differences of chemical components before and after fermentation were compared.A total of 54 chemical components were characterized from unfermented MMF,while 44 compounds were identified from fermented MMF,mainly including organic acids,flavonoids and amino acids.Through statistical analysis on the differences of various chemical components before and after fermentation,proline,tyrosine,leucine and other amino acids increased significantly in fermented MMF,while eleven components such as amygdalin,prunasin,chlorogenic acid and cryptochlorogenic acid were not detected after fermentation.According to the preparation method of the current ministry standard of the People’s Republic of China,the fermentation of MMF was carried out.The fermentation characteristics of MMF fermented at different season and different fermentation process were observed,which showed that both fermentation season and the fermentation method could have significant impact on the fermentation degree and the quality of MMF.Taking the mycelium length,color,and distribution uniformity as indexes,the method of plastic film covered fermentation with fresh-keeping film(9 holes)was finally optimized.Based on precolumn derivatization combined with ultra-performance liquid chromatography with photo-diode array(UPLC-PDA)technology,phenyl isothiocyanate(PITC)acetonitrile solution was used as derivatization reagent to detect the content of aspartic acid,glutamic acid,serine,glycine,glutamine,histidine,alanine,proline,tyrosine,valine,isoleucine,leucine,phenylalanine and lysine in MMF.The results of methodological test showed that the linear relationship was good for 14 amino acid components(r~2>0.999).The RSD value of intra-day precision was not more than 1.5%and the RSD value of inter-day precision was not more than 2.9%.The RSD value of the repeatability and the stability test results were not more than 1.6%.The results of mean recovery ranged from 93.8%to 108.4%with the RSD value not more than 2.5%.The method was applied to the determination of 21batches of MMF and the results showed that there were great differences in the content of amino acids in different batches of MMF.The content of tested amino acids in MMF in different fermentation days was detected by the established UPLC-PDA method.And the content of amino acids in MMF before and after drying and different processed MMF samples were detected by the established detection method.The results showed that the content of amino acids increased during fermentation.There was little difference in the content of most amino acids before and after drying,and the content of some amino acids such as glycine,valine,isoleucine,leucine and phenylalanine increased significantly after drying.In addition,the results showed that the content of amino acids decreased after stir frying with bran,for instance,aspartic acid decreased from 1.262 mg/g to 1.073 mg/g,glutamine decreased from 4.377 mg/g to 3.124 mg/g,and phenylalanine decreased from 1.315 mg/g to 1.185 mg/g.And the content of amino acid decreased significantly after charring,and the content of glutamate,histidine,tyrosine,valine,leucine,phenylalanine,lysine was lower than the quantitative limit,which was difficult to detect.The pharmacological mechanism of amino acids in the treatment of gastrointestinal diseases was predicted based on network pharmacology.Through Gene Cards database,Swiss Target prediction database and others,the key targets of amino acids related to diseases and activity were obtained.Then protein interaction network analysis and KEGG pathway analysis were carried out by using the key targets,and the“Amino acids in MMF-targets-disease and biological activity”network was constructed by Cytoscape.The results showed that in the role of anti-inflammatory,antioxidant and bacteriostatic activities,170 key targets were obtained,involving 34 metabolic pathways.In the treatment of vomiting,dyspepsia and constipation,151key targets were obtained,involving 24 metabolic pathways.The amino acids in MMF mainly exert anti-inflammatory and antioxidant activities and treat symptoms such as vomiting and dyspepsia through targets such as ADORA3,NOS1 and PEPD,so as to achieve the purpose of treating diseases. |
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