侧柏叶抗红色毛癣菌主要活性成分α-蒎烯以ERG-3为靶点发挥抗真菌作用

doi:10.11843/j.issn.0366-6964.2023.04.032

畜牧兽医学报 ›› 2023, Vol. 54 ›› Issue (4): 1690-1702.doi: 10.11843/j.issn.0366-6964.2023.04.032

侧柏叶抗红色毛癣菌主要活性成分α-蒎烯以ERG-3为靶点发挥抗真菌作用

张潇¹, 李丹丹¹, 田红利¹, 欧春敏¹, 杨龙², 欧阳五庆^2*, 郑寅^1*

1. 贵州大学动物科学学院, 贵阳 550025;
2. 西北农林科技大学动物医学院, 杨凌 712100

收稿日期:2022-08-08 出版日期:2023-04-23 发布日期:2023-04-27
通讯作者: 郑寅,主要从事中药活性成分分析、纳米新制剂和皮肤癣菌病防治研究,E-mail:yzheng@gzu.edu.cn;欧阳五庆,主要从事新兽药研发、纳米生物技术和细胞生物学研究,E-mail:oywq506@sina.com
作者简介:张潇(1997-),男,贵州遵义人,硕士生,主要从事中草药抗皮肤癣研究,E-mail:836970241@qq.com
基金资助:
贵州大学人才引进项目（贵大人基合字2018[13]号）；贵州大学培育项目（贵大培育[2020]82号）；贵州省科技厅基础研究计划项目（黔科合基础-ZK[2021]-170）

Alpha-pinene Is the Main Active Ingredient of Cacumen Platycladi against Trichophyton rubrum with ERG3 as Its Target

ZHANG Xiao¹, LI Dandan¹, TIAN Hongli¹, OU Chunmin¹, YANG Long², OUYANG Wuqing^2*, ZHENG Yin^1*

1. College of Animal Science, Guizhou University, Guiyang 550025, China;
2. College of Veterinary Medicine, Northwest A&F University, Yangling 712100, China

Received:2022-08-08 Online:2023-04-23 Published:2023-04-27

摘要/Abstract

摘要： 红色毛癣菌（Trichophyton rubrum，T.rubrum）是皮肤癣最主要病原；侧柏叶（Cacumen Platycladi）是侧柏[Platycladus orientalis （L.） Franco]的干燥嫩枝梢及叶，被《苗族医学》收录用于治疗皮肤癣。本研究旨在探讨侧柏叶抗T.rubrum的主要活性物质及其抗真菌机制，为抗皮肤癣新药研发提供帮助。本研究通过测定最小抑菌浓度和孢子萌发率分析了侧柏叶活性物质对T.rubrum的抑菌活性，使用电镜观察T.rubrum菌丝形态和孢子超微结构，并检测侧柏叶活性物质对T.rubrum细胞膜通透性、完整性和麦角甾醇含量的影响，从而阐述侧柏叶活性物质对T.rubrum抑菌机理，最后采用GC-MS和qRT-PCR方法找到其抗T.rubrum的作用靶点。结果显示，侧柏叶石油醚部位的α-蒎烯为主要抗菌活性成分，MIC为32 μg·mL^-1，极显著抑制孢子萌发（P<0.01）；α-蒎烯可致细胞膜发生明显损伤和内容物流出、通透性极显著增加（P< 0.01）以及24 h内细胞膜中麦角甾醇和24（28）脱氢麦角甾醇含量显著下降（P<0.05）；引起脂质色谱图中ERG3活性抑制相关的“麦角甾烷-7，24-二烯-3β-醇”色谱峰出现；可极显著降低ERG3的相对表达量（P<0.01）。结果表明，α-蒎烯是侧柏叶中发挥抗T.rubrum活性最主要成分且以ERG3为其抗真菌靶点。

关键词: 红色毛癣菌, 侧柏叶, α-蒎烯, 细胞膜, 麦角甾醇, ERG3

Abstract: Trichophyton rubrum (T. rubrum) is an important pathogen causing dermatophytosis. The dry twig and leaf of Platycladus orientalis (L.) Franco is called Cacumen Platycladi and recorded in the "Miao Medicine" for the treatment of tineas. The study aimed to investigate the main anti-T. rubrum components and mechanisms of Cacumen Platycladi, and to provide potential candidate anti-T. rubrum agents. In this study, the antifungal activity of Cacumen Platycladi main ingredient against T. rubrum was determined by minimum inhibitory concentration and spore germination rates. The mycelial morphology and spores ultrastructure of T. rubrum were observed by using electron microscopy; and the effects of the active substances of Cacumen Platycladi on the permeability, integrity, and ergosterol content of T. rubrum cell membrane were further examined, so as to elucidate the mechanism of T. rubrum inhibition by the active substances of Cacumen Platycladi. Finally, we used GC-MS and qRT-PCR to find its target of action against T. rubrum. The results showed that the petroleum ether fraction of Cacumen Platycladi exhibited the strongest anti-T. rubrum activity and in which α-pinene was proved to be the main ingredient with a MIC 32 μg·mL^-1. Alpha-pinene could induce membrane damage, higher significant cellular leakage (P<0.01) and ergosterol and 24(28)dehydroergosterol amount significant decreasing within 24 h (P<0.05); Cause highly significant reduction (P<0.01) of the relative expression of ERG3 gene and the "ERG3 inhibition" related mark sterol Ergosta-7,22-dien-3β-ol occured in sterols pattern. The results showed that α-pinene is the most potent anti-T. rubrum component in Cacumen Platycladi and ERG3 was its target.

Key words: Trichophyton rubrum, Cacumen Platycladi, α-pinene, cell membrane, ergosterol, ERG3

中图分类号:

S859.7

张潇, 李丹丹, 田红利, 欧春敏, 杨龙, 欧阳五庆, 郑寅. 侧柏叶抗红色毛癣菌主要活性成分α-蒎烯以ERG-3为靶点发挥抗真菌作用[J]. 畜牧兽医学报, 2023, 54(4): 1690-1702.

ZHANG Xiao, LI Dandan, TIAN Hongli, OU Chunmin, YANG Long, OUYANG Wuqing, ZHENG Yin. Alpha-pinene Is the Main Active Ingredient of Cacumen Platycladi against Trichophyton rubrum with ERG3 as Its Target[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(4): 1690-1702.

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侧柏叶抗红色毛癣菌主要活性成分α-蒎烯以ERG-3为靶点发挥抗真菌作用

Alpha-pinene Is the Main Active Ingredient of Cacumen Platycladi against Trichophyton rubrum with ERG3 as Its Target

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