百药煎不同菌种发酵品特征性成分差异比较及潜在分子作用机制探讨

Comparison of characteristic components of Baiyaojian fermented by different strains and its potential molecular mechanism

目的分析探讨黑曲霉Aspergillus niger(BYJ.H-1)、米根霉Rhizopus oryzae(BYJ.G-1)及蜡样芽孢杆菌Bacillus cereus(HMB2)、巨大芽孢杆菌Bacillus megaterium(HMB5)、伯顿丝孢毕赤酵母菌Burton Pichia pastoris(HMY1)、克鲁维酵母菌Kluyveromyces(HMY2)4菌混合菌种3种优势菌种/群发酵的百药煎特征性成分差异及百药煎发挥“润肺化痰、止血止泻、解热生津”功效的潜在分子生物机制,为百药煎饮片的生产和临床应用提供参考。方法以百药煎中没食子酸(gallic acid,GA)、表没食子儿茶素(epigallocatechin,EGC)、2,4,6-三-O-没食子酰-β-D-葡萄糖(2,4,6-tri-O-galloyl-β-D-glucose,TGβG)、2,4,6-三-O-没食子酰-α-D-葡萄糖(2,4,6-tri-O-galloyl-α-D-glucose,TGαG)4种特征性成分的HPLC峰面积为指标,通过主成分降维(principal component analysis,PCA)结合聚类分析法(cluster analysis,CA)对比不同菌种发酵品的差异;在中医药理论基础上采用网络药理学预测百药煎治疗呼吸道疾病(respiratory disease,RD)、水液代谢失调(water metabolism imbalance,WI)和消化道出血(alimentary tract hemorrhage,AH)的分子生物学机制。结果3种优势菌种/群发酵品在聚类结果中存在交叉现象,群集的聚类趋势无规律且与菌种类别无关联;百药煎中的没食子酸、EGC、没食子酸甲酯(methyl gallate,MG)、没食子酸乙酯(ethyl gallate,EG)和表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)5种活性成分与RD/WI/AH间存在99个共有靶点,经过蛋白互作和网络拓扑分析后得到98个核心靶点,主要包括蛋白激酶B1(protein kinase B 1,AKT1)、信号传导及转录激活蛋白3(signal transducer and activator of transcription 3,STAT3)、表皮生长因子(epidermal growth factor,EGF)、丝裂原活化蛋白激酶1/3(mitogen-activated protein kinase 1/3,MAPK1/3)等,基因本体(gene ontology,GO)分析结果显示这些靶点主要涉及激酶活性、连接酶结合和受体蛋白结合等分子生物功能,京都基因和基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)分析结果显示有154条信号通路与百药煎治疗RD/WI/AH有关,主要涉及到病毒感染、癌变、细胞因子调节等信号通路。结论3种优势菌种/群发酵百药煎外在性状及内在成分无显著差异,百药煎的功效应用是多成分、多靶点、多途径协同作用的结果,研究为百药煎饮片的生产、质量控制和临床应用提供了参考。

Objective To compare the differences of characteristic components of Baiyaojian fermented by Aspergillus niger(BYJ.H-1),Rhizopus oryzae(BYJ.G-1),and mixed strains of Bacillus cereus(HMB2),Bacillus megaterium(HMB5),Burton Pichia pastoris(HMY1),and Kluyveromyces(HMY2),analyze the potential molecular biological mechanism of Baiyaojian to exert the effects of“moistening lungs and eliminating phlegm,stopping bleeding and diarrhea,relieving fever and promoting fluid production”,so as to provide reference for the production and clinical application of the decoction pieces.Methods Four characteristic components including gallic acid,epigallocatechin,2,4,6-tri-O-galloyl-β-D-glucose(TGβG)and 2,4,6-tri-O-galloyl-α-D-glucose(TGαG)were selected,and then the HPLC characteristic peak areas of the four components were used as indicators to compare the differences of three different strains fermented products through principal component analysis(PCA)combined with cluster analysis(CA).Simultaneously,on the basis of traditional Chinese medicine theory,network pharmacology was used to predict the molecular biological mechanism of Baiyaojian for the treatment of respiratory disease(RD),water metabolism imbalance(WI)and alimentary tract hemorrhage(AH).Results There was a cross phenomenon in the clustering results of different strains fermented products,the clustering trend of clusters was irregular and not related to the type of strain.There were 99 common targets between five active ingredients including gallic acid,epigallocatechin,methyl gallate,ethyl gallate and epigallocatechin gallate and RD/WI/AH,then 98 core targets were obtained after protein interaction and network topology analysis that mainly including AKT1,STAT3,EGF,MAPK1/3 and so on.GO analysis results showed that these targets mainly involved molecular biological functions such as kinase activity,ligase binding,and receptor protein binding,etc.KEGG analysis results showed that there were 154 signal pathways related to Baiyaojian to treat RD/WI/AH,such as viral infection,canceration and cytokine regulation.Conclusion There was no significant difference in the external characters and internal components of three dominant strains/groups of fermented Baiyaojian.The efficacy of Baiyaojian was the result of multi-component,multi-target and multi-channel synergy.This study provides a reference for the production,quality control and clinical application of Baiyaojian pieces.