基于1H NMR导向的大叶桉叶中新颖混源萜成分抗真菌活性及机制研究

The leaf of Eucalyptus robusta is an antifungal traditional Chinese medicine (TCM), however the pharmacodynamic substance foundation of its decoction is still unknown. Previous research revealed that the novel phloroglucinol terpene adducts could significantly inhibit the growth of pathogen, which was the nonvolatile antifungal effective materials of E. robusta. But these adducts with different activities were uneasy to be distinguish and separate from other constituents. Creatively using individual hydroxyl proton singals in its 1H NMR spectrum and the specific antifungal bioassay, and combining with the modern chromatographic method and spectroscopy, we could powerfully identify and separate the terpene adducts and would have a comprehensive view of the adducts constitution. Further investigation on the antifungal bioactivities using fungus corresponded for the clinical diseases, and the mechanism research focused on the inhibition of cell growth and hypha produce, would clarify the function of adducts in the antifungal usage of E. robusta. Thus, this project based on the TCM property theory and the traditional antifungal usage of E. robusta, and aimed on the systemic separation and synergistic action of phloroglucinol terpene adducts. The achievement of the project would provide scientific basis for the development of these adducts and for the antifungal material foundation. In a word, this project is a theoretically and academically valuable fundamental research.

大叶桉叶是传统抗真菌辛味中药，其水煎液的抗真菌物质基础尚不明确。项目前期发现大叶桉叶所含混源萜成分具有新颖的化学结构和显著的多成分多途径协同抗真菌作用，是大叶桉叶主要的非挥发性抗真菌药效物质，但其分离难度大、活性差别明显。创新性的利用该类混源萜成分1H NMR特征羟基氢信号作为分离导向，同时结合特定抗真菌活性导向，利用现代色谱手段和波谱技术，可以快速有效实现该类活性成分全面深入的分离鉴定；在此基础上进一步开展与临床适应症相应的多方面抗真菌活性研究，从对细胞生长、菌丝形成等不同途径抗真菌作用的机制研究，能够系统阐明混源萜成分在中药大叶桉叶抗真菌药效中发挥的作用。基于此，本项目已大叶桉叶传统药性理论与用药习惯为切入点，着眼于其混源萜成分的1H NMR导向系统分离与协同抗真菌作用研究，能够为该类化合物的开发利用，为大叶桉叶的抗真菌物质基础提供科学依据，是一项具有重要理论和学术价值的基础研究项目。

真菌感染是一种常见、多发病，近年来深度感染发病率持续增高，耐药性问题日益严重，反观抗真菌新药研发，进展缓慢，近几十年都未有新抗菌药物分子问世。大叶桉叶是传统抗真菌辛味中药，对其抗菌成分研究长期关注其挥发油成分。项目前期发现其非挥发性间苯三酚混源萜成分具有新颖的化学结构和显著的抗真菌作用。为深入研究该类成分，本项目针对其分离难度大、活性差别明显，创新性的利用该类混源萜成分1H NMR特征羟基氢信号作为分离导向，快速有效的分离鉴定60个该类化合物，30个为新化合物，包括多类新颖骨架的分子。针对该混源萜成分的手性确定问题，本项目首次将Snatzke螺旋规则用于确定其的绝对构型，为类似化合物的后续研究提高重要参考。在此基础上进一步开展的抗真菌活性研究不仅筛选获得了抗真菌活性分子，明确了大叶桉叶中的非挥发性抗菌成分，更深入阐明了该类成分能够通过法尼醇抑制菌丝生长来抑制真菌背膜形成；且该类混源萜类成分是真菌外排泵的底物分子，可以与活性分子或者临床药物竞争外排来发挥协同作用。这些研究成果有力的阐明了大叶桉叶的抗真菌物质基础和多靶点多机制协同抗菌的突出优势，为中药大叶桉叶的进一步现代化新药开发与应用奠定了良好基础。