Antifungal activity and cytotoxicity of extracts and triterpenoid saponins obtained from the aerial parts of Anagallis arvensis L.
Graphical abstract
Introduction
C. albicans is an opportunistic fungal pathogen, and a common cause of invasive fungal infections in humans, producing infections that can involve any organ (Soberón et al., 2015). There is a limited number of therapeutic antifungal agents, and a decrease in the activity, even for new drugs (e.g. echinocandins), due to an increase of resistance mechanisms, enhanced by the ability of some strains to form biofilms (Favre-Godal et al., 2015). Biofilms are structured microbial communities with a complex three-dimensional architecture characterized by a network of adherent cells connected by water channels and encapsulated within an extracellular matrix (Bachmann et al., 2002). Most candidiasis are related with the formation of biofilms, which show resistance to antifungal compounds, thus increasing the concentration of antifungals, which may become toxic (Bachmann et al., 2002). Fluconazole (FLU) is the main therapeutic antifungal drug employed in developing countries (Flynn et al., 2009). The fungistatic action of azoles is an aspect that triggers the development of drug resistance (Sanglard et al., 2003), an effect that could be avoided through association of drugs. Drug associations could be described as indifferent (i.e. no interaction), antagonistic or synergistic (Soberón et al., 2015). An advantage of using drugs combinations is the possibility to obtain a synergistic effect, which could lead to a fungicidal mix, highly desirable to increase efficacy and reduce resistance development (Fiori and Van Dijck, 2012). These facts indicate the need for the discovery of compounds with antifungal activities against both planktonic and biofilm cells (Denning and Perlin, 2011), or compounds that could be combined with commercial fungistatic drugs to yield a fungicidal association (Soberón et al., 2015).
Higher plants are interesting sources of antimicrobial agents (Soberón et al., 2014). Anagallis arvensis L. (Primulaceae) is a small annual weed spread all over the world, used to treat fungal infections in Argentinean northwestern traditional medicine (Rondina et al., 2010), and also in other counties, such as India (Mitscher, 1975) or Palestine (Ali-Shtayeh et al., 1998). Aerial parts are used to prepare an ointment for the treatment of external infections (López et al., 2011). The leaves are consumed raw by humans and other mammals, as sheeps (Middleditch, 2012). There are few reports on the antifungal activity of A. arvensis extracts: Al-Abed et al. (1993) evaluated the antifungal activity against phytopathogenic fungi, Ali-Shtayeh and Abu Ghdeib (1999) proved the antifungal activity against dermatophytes, and López et al. (2011) proved the antifungal activity of an ethanolic extract against C. albicans. All these reports deal with raw extracts. This article describes the antifungal activity study of A. arvensis ethanolic extracts, activity guided purification, structural elucidation and the antifungal activity analysis of compounds obtained from A. arvensis ethanolic extract, alone and combined with FLU against planktonic C. albicans cells. The isolated compounds were also evaluated on their haemolytic and genotoxic effects. The most active compound isolated was also evaluated on its ability to inhibit C. albicans sessile cells.
Section snippets
Chemicals
Analytical and HPLC grade solvents were from Sintorgan Labs (Buenos Aires, Argentina). FLU, menadione, and 2H-tetrazolium-2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] hydroxide salt (XTT) were from Sigma-Aldrich (MO, USA). Sodium phosphate, KCl and NaCl were from Biopack (Buenos Aires, Argentina). Sabouraud dextrose (SD) medium and agar were from Britania Labs (Buenos Aires, Argentina). RPMI 1640 medium was from Microvet Labs (Buenos Aires, Argentina). CHROMagar® Candida
Activity-guided fractionation of A. arvensis ethanolic extract
The extraction yield obtained from EE was 30.3±3.2 g of EM per 100 g of dry plant material. A detailed flow chart of the purification procedure is shown in Fig. 1. The EM obtained in HX, CH, nBu and Aq was 0.94 mg, 2.70 g, 5.93 g and 0.30 g, respectively. Neither of HX, CH and Aq exhibited growth inhibition against the C. albicans ATCC10231 strain (i.e. no inhibition halo observed), while nBu showed inhibitory activity against this strain, with inhibition halo diameters of 8.0±0.2 cm (experiments
Conclusion
The bioassay-guided fractionation from aerial parts of A. arvensis allowed the detection of AnC as the compound with the highest antifungal activity against C. albicans FLU sensitive and FLU resistant strains. AnC combinated with FLU yielded a synergistic mix with fungicidal activity against both strains. This monodesmosidic triterpenoid saponin exerted inhibitory activity against C. albicans sessile cells, and the toxicity on red blood cells was almost 60 times higher than MICs obtained on
Acknowledgements
This work was supported by grants from Secretaría de Ciencia, Arte e Innovación Tecnológica of the Universidad Nacional de Tucumán [grant number 26D535], the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET; Buenos Aires, Argentina) [grant number PIP840], and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT; Buenos Aires, Argentina) [grant number PICT 2013 N°1064].
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