Elsevier

Journal of Ethnopharmacology

Volume 203, 5 May 2017, Pages 233-240
Journal of Ethnopharmacology

Antifungal activity and cytotoxicity of extracts and triterpenoid saponins obtained from the aerial parts of Anagallis arvensis L.

https://doi.org/10.1016/j.jep.2017.03.056Get rights and content

Abstract

Ethnopharmacological relevance

Anagallis arvensis L. (Primulaceae) is used in argentinean northwestern traditional medicine to treat fungal infections. We are reporting the isolation and identification of compounds with antifungal activity against human pathogenic yeast Candida albicans, and toxicity evaluation.

Aim of the study

to study the antifungal activity of extracts and purified compounds obtained form A. arvensis aerial parts, alone and in combinations with fluconazole (FLU), and to study the toxicity of the active compounds.

Materials and methods

Disk diffusion assays were used to perform an activity-guided isolation of antifungal compounds from the aerial parts of A. arvensis. Broth dilution checkerboard and viable cell count assays were employed to determine the effects of samples and combinations of FLU + samples against Candida albicans. The chemical structures of active compounds were elucidated by spectroscopic analysis. Genotoxic and haemolytic effects of the isolated compounds were determined.

Results

Four triterpenoid saponins (1–4) were identified. Anagallisin C (AnC), exerted the highest inhibitory activity among the assayed compounds against C. albicans reference strain (ATCC 10231), with MIC-0 =1 µg/mL. The Fractional Inhibitory Concentration Index (FICI=0.129) indicated a synergistic effect between AnC (0.125 µg/mL) and FLU (0.031 µg/mL) against C. albicans ATCC 10231. AnC inhibited C. albicans 12–99 FLU resistant strain (MIC-0 =1 µg/mL), and the FICI=0.188 indicated a synergistic effect between AnC (0.125 µg/mL) and fluconazole (16 µg/mL). The combination AnC+ FLU exerted fungicidal activity against both C. albicans strains. AnC exerted inhibitory activity against C. albicans ATCC 10231 sessile cells (MIC50=0.5 µg/mL and MIC80=1 µg/mL) and against C. albicans 12–99 sessile cells (MIC50=0.75 µg/mL and MIC80=1.25 µg/mL). AnC exerted haemolytic effect against human red blood cells at 15 µg/mL and did not exerted genotoxic effect on Bacillus subtilis rec strains.

Conclusions

The antifungal activity and lack of genotoxic effects of AnC give support to the traditional use of A. arvensis as antifungal and makes AnC a compound of interest to expand the available antifungal drugs.

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].

References (46)

  • N. Shoji et al.

    Triterpenoid glycosides from Anagallis arvensis

    Phytochemistry

    (1994)
  • J.R. Soberón et al.

    Free radical scavenging activities and inhibition of inflammatory enzymes of phenolics isolated from Tripodanthus acutifolius

    J. Ethnopharmacol.

    (2010)
  • J.R. Soberón et al.

    Antibacterial activities of Ligaria cuneifolia and Jodina rhombifolia leaf extracts against phytopathogenic and clinical bacteria

    J. Biosci. Bioeng.

    (2014)
  • S. Sparg et al.

    Biological activities and distribution of plant saponins

    J. Ethnopharmacol.

    (2004)
  • X. Xia et al.

    Triterpenoid saponins from Lysimachia candida Lindl

    Pharmacogn. J.

    (2013)
  • H. Abe et al.

    The effects of saikosaponins on biological membranes

    Planta Med.

    (1978)
  • A.S. Al-Abed et al.

    Antifungal effects of some common wild plant species on certain plant pathogenic fungi

    Dirasat. Ser. B Pure Appl. Sci.

    (1993)
  • M.S. Ali‐Shtayeh et al.

    Antifungal activity of plant extracts against dermatophytes

    Mycoses

    (1999)
  • S.P. Bachmann et al.

    In vitro activity of caspofungin against Candida albicans biofilms

    Antimicrob. Agents Chemother.

    (2002)
  • Clinical Laboratory Standards Institute (CLSI), 2008. protocols. Reference method for broth dilution antifungal...
  • J.J. Coleman et al.

    Characterization of plant-derived saponin natural products against Candida albicans

    ACS Chem. Biol.

    (2010)
  • M. Cuenca-Estrella et al.

    Combined activity in vitro of caspofungin, amphotericin B, and azole agents against itraconazole-resistant clinical isolates of Aspergillus fumigatus

    Antimicrob. Agents Chemother.

    (2005)
  • D.W. Denning et al.

    Azole resistance in Aspergillus: a growing public health menace

    Future Microbiol.

    (2011)
  • Cited by (25)

    • A comprehensive review on the botany, traditional uses, phytochemistry, pharmacology and toxicity of Anagallis arvensis (L).: A wild edible medicinal food plant

      2023, Food Bioscience
      Citation Excerpt :

      Sheep intoxicated with A. arvensis showed difficulty in breathing, gait stiffness, depression, weakness of the leg, recumbence and ultimately, sudden decrease in temperature followed by coma. Post-mortal lacerations included lungs and liver redundancy and also haemorrhage of the heart, kidneys, and intestines (Schneider, 1978; Stephens, 2002, pp. 75–134, Al-Sultan, Hussein, & Hegazy, 2003). Similarly, Al-Mujalli (2008) conducted a toxicity study of A. arvensis for one month in sheep and reported its toxicity in the daily dose of 5 g/kg BW.

    • Antitumor and radiosensitizing effects of Anagallis arvensis hydromethanolic extract on breast cancer cells through upregulating FOXO3, Let-7, and mir-421 Expression

      2022, Pharmacological Research - Modern Chinese Medicine
      Citation Excerpt :

      Most importantly, five major triterpenoid saponins (oleanane) were indicated through high-featured LC-HR-ESI-MS relying on the peak intensity rank, predominantly triterpenoidal saponins (oleanane), such as anagallisin (A and C), and anagallo saponin (II, IX, and VI) that sheds light on them as novel anticancer and radiosensitizers saponins with potential efficacy against BCCs according to metabolome-based dereplication of AAE. Previous studies revealed that triterpenoid saponins identified from AAE arial parts exerted antimicrobial and spermatogenesis suppressive effects, which supports their medicinal uses in traditional and modern Chinese medicine as antimicrobial and in male contraception [31,15]. Moreover, Saponins, like most compounds isolated from Chinese medicines, affect multiple targets, and current data has yet to provide a comprehensive vision of the mechanisms that are involved [32].

    • Triterpenoid saponins from Anagallis monelli ssp. linifolia (L.) Maire and their chemotaxonomic significance

      2022, Phytochemistry
      Citation Excerpt :

      This chain was found in saponins of Anagallis (Glombitza and Kurth, 1987b; Shoji et al., 1994a and b), Lysimachia (Kohda et al., 1989, Podolak et al., 2013), Cyclamen (Altunkeyik et al., 2012; Bencharif-Betina et al., 2012; Dall’Acqua et al., 2010; El Hosry et al., 2014), Ardisia (Jia et al., 1994), Myrsine (Bloor and Qi, 1994), and Androsace genera (Waltho et al., 1986) (Table 7). This tetrasaccharide sequence S2 can be substituted with glucose at C-4 of glcII or glcI in the case of Anagallis saponins (Shoji et al., 1994a and b; Soberón et al., 2017), at C-3 or C-6 of glcII in the case of Cyclamen saponins (Çalis et al., 1997a, 1997b), or at C-4 of the terminal glucose (glcI) in the case of Androsace saxifragaefolia saponins (Waltho et al., 1986); however, it was the only study found for the genus Androsace. In addition, this common chain was substituted with xylose at C-4 of glcII in the case of Lysimachia saponins (Podolak et al., 2013), or with rhamnose at C-3 of glcII in saponins of Ardisia gigantifolia (Mu et al., 2010).

    View all citing articles on Scopus
    View full text