Área Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Argentina.
Área Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Argentina; Area Micología y Centro de Referencia en Micología (CEREMIC), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Argentina.
Phytomedicine. 2019 Feb 15;54:291-301. doi: 10.1016/j.phymed.2018.06.045. Epub 2018 Jul 6.
In our previous study the synergism of four combinations of Zuccagnia punctata (ZpE) and Larrea nitida (LnE) exudates with the reliable statistical-based MixLow method was assessed, and the markers of the most anti-C. albicans synergistic ZpE-LnE bi-herbal combination were quantified according to European Medicines Agency (EMA).
To study the mechanisms of action as well as the cytotoxic properties of the ZpE-LnE most synergistic combination found in the previous work.
Minimum Fungicidal Concentration (MFC) and rate of killing of ZpE-LnE were assessed with the microbroth dilution and the time-kill assays respectively. Morphological alterations were observed with both confocal and fluorescence microscopy on the yeast Schizosaccharomyces pombe. The ergosterol exogenous assay, the quantification of ergosterol, the sorbitol as well as glucan synthase (GS) and chitin synthase (ChS) assays were used to detect the effects on the fungal membrane and cell wall respectively. The capacity of ZpE-LnE of inhibiting Candida virulence factors was assessed with previously reported methods. The effect of ZpE-LnE and of ZpE or LnE alone on cell viability was determined on human hepatoma cells line Huh7.
ZpE-Ln E was fungicidal killing C. albicans in a shorter time than amphotericin B and produced malformations in S. pombe cells. ZpE-LnE showed to bind to ergosterol but not to inhibit any step of the ergosterol biosynthesis. ZpE-LnE showed a low or moderate capacity of inhibiting GS and ChS. Regarding inhibition of virulence factors, ZpE-LnE significantly decreased the capacity of adhesion to eukaryotic buccal epithelial cells (BECs), did not inhibit the germ tube formation and inhibited the secretion of phospholipases and proteinases but not of haemolysins. ZpE-LnE demonstrated very low toxicity on Huh7 cells, much lower than that each extract alone.
The fungicidal properties of ZpE-LnE against C. albicans, its dual mechanism of action targeting the fungal membrane's ergosterol as well as the cell wall, its capacity of inhibiting several important virulence factors added to its low toxicity, make ZpE-LnE a good candidate for the development of a new antifungal bi-Herbal Medicinal Product.
在我们之前的研究中，评估了 Zuccagnia punctata（ZpE）和 Larrea nitida（LnE）渗出物的四种组合与可靠的基于统计的 MixLow 方法的协同作用，并根据欧洲药品管理局（EMA）对最抗白色念珠菌协同作用的 ZpE-LnE 双草药组合的标志物进行了定量。
研究我们之前工作中发现的 ZpE-LnE 最协同组合的作用机制和细胞毒性特性。
使用微量肉汤稀释法和时间杀伤法分别评估 ZpE-LnE 的最低杀菌浓度（MFC）和杀菌率。用共聚焦和荧光显微镜观察酵母酿酒酵母的形态变化。用外源性麦角固醇测定法、麦角固醇定量、山梨醇以及葡聚糖合酶（GS）和几丁质合酶（ChS）测定法分别检测真菌膜和细胞壁的影响。用先前报道的方法评估 ZpE-LnE 抑制念珠菌毒力因子的能力。用先前报道的方法评估 ZpE-LnE 对人肝癌细胞系 Huh7 细胞活力的影响。
ZpE-LnE 在比两性霉素 B 更短的时间内杀死白色念珠菌，并在酿酒酵母细胞中产生畸形。ZpE-LnE 显示与麦角固醇结合，但不抑制麦角固醇生物合成的任何步骤。ZpE-LnE 抑制 GS 和 ChS 的能力较低或中等。关于抑制毒力因子，ZpE-LnE 显著降低了与真核口腔上皮细胞（BEC）的粘附能力，不抑制芽管形成，抑制磷脂酶和蛋白酶的分泌，但不抑制溶血素的分泌。ZpE-LnE 在 Huh7 细胞上表现出非常低的毒性，远低于每种提取物单独的毒性。
ZpE-LnE 对白色念珠菌的杀菌特性、其针对真菌膜麦角固醇和细胞壁的双重作用机制、其抑制几种重要毒力因子的能力以及其低毒性，使 ZpE-LnE 成为开发新型抗真菌双草药药用产品的良好候选药物。
