太子参化感自毒作用物质的识别与筛选研究

Consecutive monoculture problems have become bottlenecks for the sustainable development of natural resources of Chinese medicinal materials. Allelochemical-mediated effects have been considered as one of the major causes of consecutive monoculture problems. To seek a breakthrough for illuminating the structure and interaction of Pseudostellaria heterophylla’s autotoxins, “Zherong Pseudostellariae” and its rhizospheric soil were used as tested materials in this research and the “target constituents knock-out” strategy was applied for autotoxins screening. Hence, contributes were devoted to the identification and screening of the autotoxic allelochemicals of P. heterophylla. ① By comparing thin layer chromatography (TLC) chemical fingerprint of water extraction from rhizospheric soil of Pseudostellaria heterophylla with that of the same fraction from blank soil, the target component fractions were locked and knocked out by preparative thin layer chromatography (PTLC). For the target component fractions, chromatographic profiles were built with high performance liquid chromatography (HPLC). ② Allelopathic effect was carried out with pot experiments to test the autotoxic effects of the target component fractions. ③ In the view of spectrum-effect relationship, key components that lead to consecutive monoculture problems of P. heterophylla can be screened with data analysis, and then their structures were identified as well. Allelopathic effect obtained by pot experiments to simulate the consecutive monoculture problems was used to evaluate the interaction relationships among the target components fractions. ④ Successively, key components were knocked out with the help of gel, preparative chromatography, etc., and those interaction relationships were evaluated by allelopathic effects obtained from tissue culture seeding experiments. Above all, this study was devoted to provide theory basis and technical support for indicating the material basis of consecutive monoculture problems and their action mechanism, along with effectively solving obstacle of Chinese material medical production.

连作障碍已成为中药资源可持续发展的瓶颈,化感物质介导是连作障碍形成的主要原因。为明确太子参化感自毒物质及其互作关系，拟以“柘荣太子参”及其根际土壤为材料，通过构建基于目标成分“敲出”技术体系，开展太子参化感自毒物质的识别与筛选研究。①通过根际与空白土壤水提液薄层指纹图谱比对，锁定并“敲出”目标组分；借助HPLC方法构建各目标组分液相色谱图；②盆栽模拟连作试验对各目标组分、阴性样品及水提液进行连作障碍效应评价；③以目标组分液相色谱图与连作障碍效应“谱效”关系为切入点，筛选出太子参连作障碍中起主导作用的化感成分，并进行结构鉴定；通过盆栽模拟连作障碍效应评价各组分间互作关系；④采用凝胶、制备色谱等技术从目标组分中“敲出”化感成分并通过组培苗的化感效应评价各成分间互作关系。综上为阐明太子参连作障碍物质基础,揭示其连作障碍发生机制及有效解决中药材生产中连作障碍难题提供理论依据和技术支撑。

化感物质及其介导的根际微生态失衡被认为是太子参连作障碍形成的主要原因。研究表明，植物残株及根系分泌物是化感物质（群）的重要来源。目前，引起太子参连作障碍的关键化感物质尚不明确。本项目针对药用植物化感物质筛选鉴定研究中的难点，提出了基于“敲入-敲出”及“谱效相关”理论筛选植物化感物质群。对太子参残株中潜在化感物质群的筛选研究结果表明，以二氯甲烷：甲醇=6: 1及3: 1“敲出”所得组分具有较强化感活性，共包含50种潜在化感物质，涉及到5条代谢通路，且酪氨酸代谢途径与该化合物群的合成积累关系最为密切。此外，采用代谢组学技术及方法对太子参根系分泌物的研究发现，42种化合物可以由太子参释放至根际土壤，该化合物群的形成涉及到33条代谢通路。其中，酪氨酸代谢途径同样是关联代谢物数量最多、贡献最大的途径，提示酪氨酸代谢途径参与了太子参植物体及根系分泌物中化感物质群的积累合成。本项目将“敲入-敲出”及“谱效相关”理论应用于植物化感物质群的筛选研究，并成功实现了太子参化感物质（群）及相关合成代谢途径的筛选，锚定了酪氨酸代谢途径与太子参化感物质群的合成代谢存在密切联系，为进一步研究太子参连作障碍形成机理提供了关键的物质基础。