The antifungal activity and mechanisms of action of quantified extracts from berries, leaves and roots of Phytolacca tetramera

Background: Phytolacca tetramera is an endemic plant from Argentina that is currently at serious risk because its environment is subjected to a high anthropic impact. A previous study has shown that berry extracts obtained from this plant display antifungal activity against multiple human-pathogenic fungi when tested with a non-standardized method. Further evidences of the antifungal properties of other parts of the plant and studies of mechanism of antifungal action of the antifungal chemically characterized extracts are required. Purpose: This study aimed to gain further evidence of the antifungal activity of P. tetramera berry, leaf and root extracts in order to find the most active extract to be developed as an Herbal Medicinal Antifungal Product. The medicinal usefulness of P. tetramera extracts as antifungal agents will serve as an important support to create concience and carry out actions tending to the preservation of this threatened species and its environment. Materials and methods: Chemical analysis of all P. tetramera extracts, including quantitation of selected markers, was performed through UHPLC-ESI-MS/MS and UPLC-ESI-MS techniques according to the European Medicines Agency (EMA). The antifungal activity of the quantified extracts was tested with the standardized CLSI microbroth dilution method against Candida spp. Antifungal mechanisms of the most active extract were studied by examination of morphological changes by phase-contrast and fluorescence microscopies and both, cellular and enzymatic assays targeting either the fungal membrane or the cell wall. Results: The antifungal activity of twelve P. tetramera extracts was tested against Candida albicans and Candida glabrata. The dichloromethane extract from berries (PtDEb) showed the best activity. Phytolaccagenin (PhytG) and phytolaccoside B (PhytB) were selected as the main active markers for the antifungal P. tetramera extracts. The quantitation of these active markers in all extracts showed that PtDEb possessed the highest amount of PhytG and PhytB. Finally, studies on the mechanism of antifungal action showed that the most active PtDEb extract produces morphological changes compatible with a damage of the cell wall and/or the plasma membrane. Cellular and enzymatic assays showed that PtDEb would not damage the fungal cell wall by itself, but would alter the plasma membrane. In agreement, PtDEb was found to bind to ergosterol, the main sterol of the fungal plasma membrane. Conclusion: Studies of the anti-Candida activity of P. tetramera extracts led to the selection of PtDEb as the most suitable extract, confirming the antifungal properties of the threatened species P. tetramera. The new data give a valuable reason for the definitive protection of this sp. and its natural environment thus allowing further studies for the future development of an Herbal Medicinal Antifungal Product.Susana A. Zacchino and Laura Svetaz (L.S.) acknowledge Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), PICT2014-1170 and PICT2016-1833 and National University of Rosario, Argentina (UNR) for funds. J.C.R. acknowledges MINECO (BIO2015-69958-P), Junta de Castilla y León/FEDER (CSI068P17 and >Escalera de Excelencia> CLU-2017-03), Spain, for funds. Estefanía Butassi (E.B.) acknowledges Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) for doctoral and post-doctoral fellowships. L.S. is a member of the CONICET Researcher career; L.S. and E.B. belong to the teaching staff of Pharmacognosy area. Jean-Luc Wolfender and Shuaizhen Zhou (S.Z.) acknowledge Sino Swiss Science and Technology Cooperation Program (SSSTC) IP18-092011 for the funding of the post-doctoral stay of S.Z. The MEDINA authors Francisca Vicente, Caridad Díaz and José Pérez-del Palacio disclosed the receipt of financial support from Fundación MEDINA, a public-private partnership of Merck Sharp & Dohme de España S.A./Universidad de Granada/Junta de Andalucía, Spain