基于药用植物化学型研究策略探索平栽黄芪化学和药效变异规律

In order to solve the shortage of wild Astragali Radix (AR) supply, a new flat-cultivated AR emerged as required. The previous study showed that the cultivation pattern may have greater impacts on the chemical constituents than the species or origins. However, whether or not the chemical variation of the flat-cultivated AR could define the Chemotype? Are there significant differences in the efficacy of the two ARs? What is the biological mechanism of chemical variation? Answering these scientific questions is of great importance for clinical application of AR. This project is guided by the Chemotype research strategy of medicinal plants and pharmacodynamic evaluation thought of compound formula. Chemical analysis combined with bioinformatics was applied to verify the degree and pattern of chemical variation of ARs, and the molecular marker technique coupled with transcriptome sequencing technology was used to obtain the genetic background of the two kinds of ARs to illuminate the variation mechanism at the molecular level. In addition, a quality evaluation method was developed based on the efficacy advantage of the two kinds of ARs by comparing the blood-tonifing effect in Danggui Buxue Decoction and the stasis-dispelling effect in paired-herb Astragali Radix and Salviae Miltiorrhizae Radix. Meanwhile, the metabolomic approach was used to improve the sensitivities of traditional pharmacodynamic indicators. Finally, the chemistry and efficacy were linked up by integrating a variety of data analysis techniques to elucidate the scientific implications of chemical variation leading to the differences in efficacy. In conclusion, the results are of great significance for the scientific evaluation of the quality of AR, explication of clinical dosage and the oriented application and development of different AR resources.

平栽黄芪是为解决仿野生黄芪供应紧缺而发展的新技术种植药材，前期研究表明该种植方式对黄芪化学成分的影响远大于物种和产地因素。然而，平栽黄芪化学变异幅度是否达到界定化学型的程度？是否引起显著的药效差异？化学变异的生物学机制是什么等科学问题的解决对临床应用至关重要。本项目以药用植物化学型研究策略和复方药效评价思路为指导，采用化学分析结合生物信息学手段探明平栽蒙古黄芪化学变异的程度和规律；应用分子标记与转录组测序技术获得两类黄芪的遗传背景，并阐释平栽方式下化学变异的形成机制。构建在当归补血汤复方中比较两类黄芪补血功效、在黄芪丹参药对中比较它们的祛瘀功效的药材质量评价方法，通过代谢组学辅助提高传统药效学指标灵敏性，明确各类黄芪的药效优势，采用多种数据分析关联化学与药效，阐释化学变异导致药效差异的科学内涵。研究结果对科学评价黄芪药材质量、明确临床用量、实现不同黄芪资源的定向应用与发展具有重要意义。

项目以药用植物化学型研究策略和复方药效评价思路为指导，首先构建了基于诊断离子技术的黄芪化学成分的分析策略，对山西恒山山脉周边相近气候下的蒙古黄芪（1-6年仿野生芪，1-3年平栽芪）各类化学成分的积累规律进行了描述和比较，二者在化学组成上存在显著差异。进一步基于DNA分子标记技术，发现蒙古黄芪具有丰富的遗传多样性且仿野生芪的遗传多样性水平较高，并找到了分别与生长年限（855-3*，827-4*）及生长方式（840-2*，866-3*）相关的ISSR条带；基于Illumina HiSeq与PacBio高通量转录组测序、qRT-PCR、HPLC含测、UHPLC-ESI-Q-TOF-MS/MS代谢组学等技术，发现CHI、CHS、PAL基因是异黄酮类成分累积的关键酶基因，而MVK、CMK、PMK、MVD、SS基因，则是黄芪甲苷形成的关键酶基因。其次，成功构建了血虚小鼠和局部脑缺血大鼠用于两类黄芪的药效评价，通过代谢组学辅助提高传统药效学指标灵敏性，进一步比较两类黄芪在当归补血汤复方配伍环境中的的补血功效、在黄芪丹参药对配伍环境中的祛瘀功效。研究发现两种栽培方式黄芪在“补血”“活血行滞”均有效，而仿野生黄芪配伍的当归补血汤对血虚模型小鼠的药效更佳，反之，平栽黄芪配伍的黄芪-丹参药对对局部脑缺血大鼠的治疗作用尤佳。同时，二者发挥不同药效的化学成分也是不同的，其中黄酮类成分对补气功能的药理贡献较大，而皂苷类成分对活血行滞功能的贡献较大。本项目共发表学术论文15篇，其中研究性论文11篇，包括 SCI 论文4篇，综述性文章8篇；授权专利1件；获得山西省科技进步二等奖1项，制定中华中医药学会行业标准一项。培养硕士研究生6名；参加会议6人次。总之，本项目明确了不同种植方式黄芪化学变异的形成机制及其导致药效差异的科学内涵。研究结果对科学评价黄芪药材质量、明确临床用量、实现不同黄芪资源的定向应用与发展具有重要意义。