Approaches to the mechanism of antifungal activity of Zuccagnia punctata-Larrea nitida bi-herbal combination

Abstract

Background: In our previous study we assessed the synergism of four combinations of Zuccagnia punctata (ZpE) and Larrea nitida (LnE) with the reliable statistical-based MixLow method and found the most anti-C. albicans synergistic ZpE-LnE bi-herbal combination whose composition was quantified by a valid method, according to European Medicines Agency (EMA). Purpose: The purpose of this research was to advance in the study of the mechanisms of action as well as the cytotoxic properties of the ZpE-LnE most synergistic combination found in the previous work. Materials and methods: Minimum Fungicidal Concentration (MFC) of ZpE-LnE was assessed with the microbroth dilution method of the Clinical and Laboratory Standard Institute (CLSI) and the rate of killing was determined with the time-kill assay in the range 0?24h. The morphological alterations were observed with both confocal and fluorescence microscopies on the model yeast Schizosaccharomyces pombe. The ergosterol exogenous assay, the quantification of ergosterol, the sorbitol assay as well as glucan synthase (GS) and chitin synthase (ChS) assays were used to detect the inhibition of the fungal membrane and cell wall respectively. The capacity of ZpE-LnE of inhibiting Candida virulence factors such as adherence to buccal epithelial cells (BECs), germ tube inhibition, and inhibition of phospholipases, proteinases and haemolysins secretion, were assessed with previously reported methods. The effect of ZpE-LnE and each ZpE or LnE alone on cell viability against human hepatoma cell line Huh7 was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT assay. Results: ZpE-LnE was fungicidal rather than fungistatic, killing C. albicans in a shorter time than amphotericin B (AmpB). ZpE-LnE produced malformations in S. pombe cells likely due to a damage in the plasma membrane or the cell wall. ZpE-LnE showed to bind to the membrane ergosterol in the exogenous ergosterol assay, but not to inhibit any step of the ergosterol biosynthesis. ZpE-LnE showed a low or moderate capacity of inhibiting GS and ChS of the fungal cell wall. Regarding the effect on virulence factors, ZpE-LnE significantly decreased the capacity of adhesion to eukaryotic BECs, did not inhibit the germ tube formation and completely 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. Conclusions: 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, makes this combination a good candidate for the development of a new antifungal bi-Herbal Medicinal Product.