基于高通量测序和化学轮廓分析研究黄连在连栀矾溶液发酵炮制中的作用

目的探究黄连Coptidis Rhizoma在连栀矾溶液传统发酵炮制中的作用。方法按照连栀矾溶液传统配方比例,将其拆分为3组配方溶液:连栀矾组(黄连+栀子+白矾)、黄连组(黄连+白矾)和栀子组(栀子+白矾),采用HPLC分析比较3组溶液发酵前后的化学成分变化轮廓,采用Illumina Hiseq高通量测序技术测定并比较发酵溶液中微生物的群落特征。结果传统配方连栀矾经发酵后环烯醚萜类成分发生了较显著的变化(P<0.05),生物碱类成分未发生明显变化,拆分后的黄连组中生物碱类成分和栀子组中环烯醚萜类成分均没有发生明显变化。分别对各组发酵溶液进行测序,根据α多样性分析结果,3组发酵液中真菌多样性为连栀矾组<栀子组<黄连组,真菌的丰富度为栀子组<连栀矾组<黄连组;细菌多样性为连栀矾组<黄连组<栀子组,细菌的丰富度为连栀矾组<栀子组<黄连组。根据β多样性分析结果,3组溶液中的真菌和细菌的群落结构均有明显差异。根据微生物相对丰度分析可知,3组的优势真菌均为子囊菌门、担子菌门和罗兹菌门,不同组间丰度无明显差异;真菌优势属中的孢霉属的丰度在连栀矾组最高,显著高于另外2组(P<0.05),栀子组又显著高于黄连组(P<0.05);韦斯特壳属的丰度在连栀矾组中最低,显著低于黄连组和栀子组(P<0.05),后两者间丰度无明显差异。3组的优势细菌均为变形菌门、厚壁菌门、放线菌门和拟杆菌门,其中连栀矾组中变形菌门的丰度极显著低于黄连组和栀子组(P<0.01),厚壁菌门的丰度在连栀矾组中极显著高于黄连组和栀子组(P<0.01),放线菌门和拟杆菌门的丰度在3组发酵溶液的差异较小;细菌优势菌属中的乳杆菌属在连栀矾组中显著高于黄连组和栀子组(P<0.05),在黄连组中也显著高于栀子组(P<0.05),伯克氏菌属丰度在黄连组和栀子组中无显著差异,都显著高于连栀矾组中的丰度(P<0.05)。结论连栀矾溶液配方中的黄连能提高其中栀子的有效成分的发酵转化,并且是通过对发酵体系中的优势菌属的丰度进行调节而发挥作用的。进一步佐证了传统配方制剂的合理性和科学性。

Based on high-throughput sequencing and chemical profiling to study role of Coptidis Rhizoma in concocting of Lianzhifan solution

Objective To investigate the role of Huanglian(Coptidis Rhizoma)in the traditional fermentation processing of Lianzhifan solution.Methods According to the traditional formula ratio of Lianzhifan solution,it was divided into three groups of formula solutions:Lianzhifan groupHuanglian(Coptidis Rhizoma)+Zhizi(Gardeniae Fructus)+Baifan(Alumen)(LZF)],Coptidis Rhizoma groupCoptidis Rhizoma+Alumen(CR)],and Gardeniae Fructus groupGardeniae Fructus+Alumen(GF)].The chemical composition changes of the three groups of solutions before and after fermentation were analyzed and compared by HPLC.Illumina Hiseq high-throughput sequencing technology was used to determine and compare the community characteristics of microorganisms in the fermentation solution.Results The iridoid composition of the traditional formula LZF group has undergone significant changes after fermentation(P<0.05),and the alkaloid composition has not changed significantly.The alkaloid composition in CR and the iridoids in GF did not change significantly after fermentation.The fermentation solutions of each group were sequenced.According to the results ofαdiversity analysis,the fungal diversity in the three fermentation broths was LZF