基于“微生物群-化学成分-毒性”整体关联性的化风丹药母发酵减毒作用研究

Hua feng dan which is a Guizhou province traditional famous drug, mainly used in the clinical treatment of wind-phlegm stagnation, apoplectic hemiplegia, epilepsy etc. and has a good effect. The hua feng dan preparation is includ in the national non-material cultural heritage list, its preparation is divide into medicine preparation and formulations preparation. The medicine preparation is the specialties of the preparation. The medicine contains toxic medicinal materials, previous studies have confirmed that the process of fermentation can reduce the drug toxicity, but it is worth to note that detoxification scientific connotation was not clear. Based on this finding, we hypothesize that the hua feng dan medicine detoxification is microbial degradation or transformation toxic chemical constituent. We applied the advanced technology including PCR-DGGE, LC-MS and high-throughput sequencing to study the the overall correlation of microbial, alkaloids chemical constituent change and toxicity change, identify the microbial flora convert chemical constituent. Through the research of the project, it can not only further clarify the scientific connotation of hua feng dan medicine fermentation, but also provide new ideas and new methods for the modernization fermentation of toxic traditional Chinese medicine and ethnic medicine. It has important theoretical significance and application value.

化风丹是贵州省传统名药，临床治疗风痰闭阻、中风偏瘫、癫痫等症，疗效显著。化风丹的制作技艺入选国家级非物质文化遗产名录中医传统制剂方法，其制备分为药母和成药制备，独特之处在于药母的发酵。药母中含有毒药材，前期研究发现发酵可降低药母毒性，但减毒作用科学内涵不清楚。基于此发现，本项目提出“化风丹药母发酵减毒是微生物使药母中毒性成分降解或转化为低毒性物质”假说。本项目拟采用PCR-DGGE、高通量测序、液质联用等技术，研究药母发酵过程中微生物菌群、化学成分、毒性三者变化的整体关联性，并明确对毒性成分进行转化的微生物菌群。通过项目研究，既能更深入系统阐明化风丹药母发酵减毒的科学内涵，又为有毒中药（民族药）现代化发酵减毒提供新思路和新方法，具有重要的理论意义和应用价值。

有毒中药是中药的重要组成部分,在中药复方制剂中应用广泛，阐明中药复方炮制减毒的科学性，是确保临床用药安全的前题。化风丹药母是由川乌、半夏、天南星、白附子、郁金打粉，加入神曲和牛胆汁混匀，自然条件下发酵2-3月后入药。药母中含有毒性较大的药物川乌，前期研究表明发酵可以降低川乌中双酯型生物碱的含量使药母毒性降低，但药母发酵减毒的科学内涵并不清楚。本项目综合运用高效液相色谱法、高通量测序技术、微生物平板培养等方法和技术，对不同批次不同发酵时间化风丹药母中生物碱含量进行测定；开展了不同发酵时间药母中微生物菌群多样性和动态变化的检测；比较了不同发酵时间药母的毒性情况；对药母中微生物进行分离纯化鉴定，并进行传统发酵与单菌发酵药母生物碱变化的对比。通过项目研究发现化风丹药母发酵其中乌头碱、新乌头碱、次乌头碱含量降低，苯甲酰乌头碱、苯甲酰新乌头碱、苯甲酰次乌头碱含量升高；药母发酵后小鼠最大耐受剂量增加且对PC12细胞增殖抑制率降低；药母发酵过程中有16个细菌属和7个真菌属与生物碱有显著相关性，芽孢杆菌属、片球菌属、酵母属、毕赤酵母属等属于优势微生物，其中阿氏芽孢杆菌、汉逊德巴利酵母菌、米曲霉均能降低双酯型生物碱含量。本课题的研究揭示药母发酵减毒科学内涵，为有毒中药发酵炮制研究提供思路和借鉴。