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The antifungal activity and mechanisms of action of quantified extracts from berries, leaves and roots of Phytolacca tetramera.
Phytomedicine 60 (2019) 152884
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Original Article
The antifungal activity and mechanisms of action of quantified extracts from berries, leaves and roots of Phytolacca tetramera.☆
T
Estefanía Butassia, Laura A. Svetaza, Shuaizhen Zhoub,c, Jean-Luc Wolfenderc, Juan C.G. Cortésd, ⁎ Juan C. Ribasd, Caridad Díaze, José Pérez-del Palacioe, Francisca Vicentee, Susana A. Zacchinoa, a
Área Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Suipacha 531, 2000 Rosario, Argentina Natural Products Chemistry Department, & State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China c School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU-Rue Michel-Servet 1, CH-1206 Geneva 4, Switzerland d Instituto de Biología Funcional y Genómica y Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas (CSIC)/Universidad de Salamanca, 37007 Salamanca, Spain e Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento 34, E-18016 Granada, Spain b
ARTICLE INFO
ABSTRACT
Keywords: Phytolacca tetramera Quantified extracts Candida albicans Candida glabrata Phytolaccagenin Phytolaccoside B
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 nonstandardized 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
Abbreviations: AmphB, amphotericin B; ChS, chitin synthase; CLSI, Clinical and Laboratory Standards Institute; CV, coefficient of variation; CW, Calcofluor White; EMA, European Medicines Agency; GlcNAc, N-acetylglucosamine; GS, (1,3)β-glucan synthase; ICH, International Council for Harmonization; MIC, minimum inhibitory concentration; NikZ, nikkomycin Z; PapB, papulacandin B; PBS, phosphate buffered saline; PhytB, phytolaccoside B; PhytE, phytolaccoside E; PhytG, phytolaccagenin; RSD, relative standard deviation; TCA, trichloroacetic acid; UHPLC-ESI-MS/MS, ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry; UPLC-ESI-MS, ultra performance liquid chromatography-electrospray ionization-mass spectrometry ☆ In honor of Prof. Hildebert Wagner, on the occasion of his 90th birthday. ⁎ Corresponding author. E-mail address: [email protected] (S.A. Zacchino). https://doi.org/10.1016/j.phymed.2019.152884 Received 23 January 2019; Received in revised form 25 February 2019; Accepted 9 March 2019 0944-7113/ © 2019 Elsevier GmbH. All rights reserved.
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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.
Introduction
Materials and methods
Amongst plants of the Phytolaccaceae family, Phytolacca tetramera Hauman (common name ‘ombusillo’) is an endemic species from Argentina that grows in an area of a strong anthropic impact. Due to this, P. tetramera has been included in the category of species in critical risk (Hernández et al., 1998) and rare species (Delucchi and Correa, 1992). In previous studies carried out in our laboratory (Escalante et al., 2002) the antifungal activity of methanol (MeOH), dichloromethane (DCM), butanol (BuOH) and aqueous berry extracts of P. tetramera was assessed against a panel of human pathogenic opportunistic fungi using the non-standardized agar dilution method. By bioassay-guided fractionation, three monodesmosidic triterpenoid saponins - phytolaccosides (Phyt) B, E and F - were isolated from BuOH extract, PhytB being the most active one against both yeasts and filamentous fungi (Escalante et al., 2002). Mainly based on our previous studies, an agreement for the creation of an area for research and development for the conservation of P. tetramera was signed between the Faculty of Agrarian and Forestry Sciences of the National University of La Plata and the Roads Department of the Province of Buenos Aires (Petri et al., 2010). In addition, several attempts have been made to achieve propagation techniques that allow for maintaining or increasing the population of P. tetramera. The objectives were to reforest natural areas, to conserve the ecosystem's biodiversity and to maintain their germplasm in vivo (Basiglio Cordal et al., 2014; Hernández et al., 1998; 2009). Besides protecting the population of P. tetramera plants, the final aim of all these efforts was to obtain enough material of the plant to gain knowledge on this species and its medicinal potentialities. Here, we deepen in the study of the antifungal activity of extracts derived from P. tetramera with the following aims: (i) to study not only extracts from berries as in the previous work, but also extracts from leaves and roots, this time using quantified extracts following the European Medicine Agency (EMA) guidelines (EMA, 2010); (ii) to use the standardized method of the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2017) for antifungal assessment of the extracts against Candida albicans and Candida glabrata; and (iii) to study the mechanism of antifungal action of the most potent extracts. Regarding aim (i), EMA defines quantified extracts as those for which the content of selected markers is determined. To follow these guidelines, it is necessary to select the appropriate markers. Once they are established, they must be quantified in the corresponding extracts. With respect to aim (ii), the use of the most recent guidelines of the CLSI (2017) for yeasts assures more reproducible and more reliable results. In this work, C. albicans and C. glabrata were selected for antifungal assessment due to C. albicans is the most common cause of opportunistic fungal infections in immunocompromised hosts worldwide (Pfaller and Diekema, 2007; Pfaller et al., 2010) while C. glabrata is the second leading cause of candidemia, particularly in adult patients with hematologic malignancies (Malani et al., 2005; Pfaller and Diekema, 2007). Concerning aim (iii), the most active extract was subjected to the study of the mechanism of anti-Candida action. Morphological studies were carried out by using phase contrast and fluorescence microscopies on the yeast models Schizosaccharomyces pombe and C. albicans. Finally, we performed cellular and enzymatic assays targeting either the fungal membrane or the fungal cell wall to know the mechanism of action of the most active extract.
Plant material P. tetramera berries, leaves and roots were collected in the town of Arditi, Magdalena, Buenos Aires province (Argentina) (35° 04´ lat. S −57° 34´ long. W) in March 2013. The plant was identified by Prof. Martha Gattuso and a voucher specimen (MG 134/2) was deposited in the herbarium of the Plant Biology area, School of Pharmaceutical and Biochemical Sciences, National University of Rosario (UNR), Rosario, Argentina. Preparation of P. tetramera extracts Dried berries, leaves and roots (500 g each) were extracted by maceration with MeOH (24 h, 3 × 800 ml) at room temperature by using mechanical agitation. Solutions were filtered and evaporated in vacuum, resulting in MeOH extracts from berries (PtMEb) (236.98 g, 47.40% W/W), from leaves (PtMEl) (89.96 g, 17.99% W/W) and from roots (PtMEr) (39.29 g, 7.86% W/W). Then, 100.9 g, 47.2 g and 16.1 g of PtMEb, PtMEl and PtMEr, respectively, were dissolved in H2O and successively extracted with DCM (3 × 300 ml). The solutions were concentrated to dryness, resulting in DCM extracts of berries (PtDEb, 30.92 g, 30.65% W/W), leaves (PtDEl, 4.87 g, 10.32% W/W) and roots (PtDEr, 2.57 g, 15.94% W/W). The remaining water solutions were extracted with BuOH (3 × 300 ml). The solutions were concentrated to dryness resulting in BuOH extracts of berries (PtBEb, 50.54 g, 50.09% W/W), leaves (PtBEl, 27.13 g, 57.48% W/W) and roots (PtBEr, 2.73 g, 16.98% W/W). The water solutions were lyophilized, resulting in aqueous extracts (AqE) of berries (PtAqEb, 9.28 g, 9.20% W/W), leaves (PtAqEl, 7.45 g, 15.78% W/W) and roots (PtAqEr, 1.96 g, 12.18% W/ W). Source of standard compounds PhytB was isolated from PtBEb, as previously described (Escalante et al., 2002). Its purity was determined by HPLC-ESI-MS (≥ 95%) and 13C NMR spectrum (Supplementary material). PhytE and PhytG were purchased from PhytoLab GmbH & Co. (Phyproof® Reference Substances) (Vestenbergsgreuth, Germany). Qualitative and quantitative chemical analysis of P. tetramera extracts TLC development was carried out using a CAMAG equipment (Muttenz, Switzerland) with an automatic injector (model ATS4 22003), a development tank (model ADC2 220221), a visualizer (model 220145) and a scanner (Scanner4 211421). For the analysis, 20 μl of the different extracts (5 mg/ml) and 10 μl of each marker (1 mg/ml) were injected, and developed with a DCM: MeOH: H2O 50:10:1 mobile phase. The plate was revealed at 254 nm, 365 nm and with p-anisaldehyde-sulfuric acid. An integrated software Win-CATS V.4.06 was used for the analysis. The retention factor (Rf) was calculated for PhytB, PhytE and PhytG. UHPLC chromatographies were performed on extracts from berries in a Thermo Dionex Ultimate 3000 UHPLC interfaced to a Thermo QExactive Plus mass spectrometer with a heated electrospray ionization (HESI) source. Separation was performed on a Waters Acquity UPLC® BEH column (1.7 μm, 2.1 × 100 mm i.d.). An elution gradient was used with UPLC quality H2O and acetonitrile, both supplemented with 0.10% formic acid (solutions A and B, respectively). The elution 2
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gradient started with 5% B for 1 min, followed by a linear increase in B up to 100% for 8 min. The gradient was maintained at 100% B for 3 min. Before each injection, the column was equilibrated for 4 min with 5% B. The flow rate was 0.6 ml/min at 40 °C. Each sample was dissolved in 85% MeOH at a concentration of 1 mg/ml for injection. UPLC chromatography of extracts from leaves and roots was carried out on an Agilent 1290 UPLC interfaced to a triple quadrupole mass spectrometer Api4000 (Sciex), equipped with an ESI ion source. An XBridge Amide (Waters) column (3.5 μm, 2.1 × 100 mm i.d.) was used. The elution gradient was prepared with ultrapure H2O and HPLC quality ACN, both supplemented with 0.10% NH3 20% (solutions A and B respectively). The elution gradient started with 90% B for 0.5 min, followed by a linear increase in B up to 30% for 5.2 min, and then an increase up to 90% in B for 1.3 min. The flow rate was 275 μl/min. Each sample was dissolved in MeOH/H2O 85:15 and injected in triplicate. Calibration curves of pure compounds PhytB and PhytG were constructed by using five different concentrations (in triplicate) of each compound, prepared from stock solutions. The data were acquired using the Selected Ion Monitoring mode. The analytical methods were validated for linearity, limit of detection and quantification (LOD and LOQ) and intra- and inter-day variability, following the International Council for Harmonization (ICH) guidelines of Technical Requirements for Pharmaceuticals for Human Use (ICH, 1996). The percentage (%) of recovery was used to evaluate the accuracy of the method. The results of this validation are shown in Supplementary material.
glabrata CCC 115-2000 from the Centro de Referencia en Micología (CCC, CEREMIC, School of Pharmaceutical and Biochemical Sciences, UNR, Rosario, Argentina) were used. Strains were grown on Sabouraudchloramphenicol agar slants for 48 h at 30 °C. They were then maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and subcultured every 15 days to prevent pleomorphic transformations. The inocula were obtained according to reported procedures (CLSI, 2017) and adjusted to 1–5 × 103 cells with colony-forming units (CFU)/ml. Determination of fungal growth inhibition percentage Broth microdilution technique M27 4th ed for yeasts (CLSI, 2017) was performed in 96-well microplates. For the assay, extract or compound test-wells (ETWs or CTWs) were prepared with stock solutions of each extract or compound in DMSO (maximum concentration ≤ 2%), diluted with RPMI-1640 to final concentrations of 1000 μg/ml for the extracts and 250 μg/ml for the compounds. An inoculum suspension (100 μl) was added to each well (final volume in the well = 200 μl). A growth control well (GCW) (containing medium, inoculum, and the same amount of DMSO used in an ETW or CTW, but extract- or compound-free) and a sterility control well (SCW) (sample, medium, and sterile water instead of inoculum) were included for each fungus tested. Microtiter trays were incubated in a moist dark chamber at 30 °C for 48 h for both C. albicans and C. glabrata. Microplates were read in a VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA, USA). Itraconazole (Itra) (Sigma-Aldrich, St. Louis, MO, USA) was used as positive control. Tests were performed in triplicate. Reduction of growth for each extract or compound concentration was calculated as follows:% of inhibition = 100 - (OD405 ETW (or CTW) − OD405 SCW) x100/(OD405 GCW−OD405 SCW). The means ± SD (standard deviations) were the data used for the dose-response curves that represent ‘% inhibition’ vs. ‘concentration’ of each extract or compound. Dose-response curves were constructed with SigmaPlot 11.0 software. MIC50 of each extract or compound was defined as the minimum concentration that inhibits 50% of the fungal growth. Extracts with MIC50 < 100 µg/ ml were considered to have high activity and were subjected to further studies. Extracts with 100 ≤ MIC50 ≤ 1000 µg/ml were considered to have low activity, while those with MIC50 > 1000 µg/ml were considered inactive.
UHPLC-ESI-MS validation The linearities of pure PhytB and PhytG calibration curves were established by calculating the slope, intercepts and R2 coefficient (Table S2, Supplementary material). The regression equation and R2 showed good linearity response. Linear range, LOD and LOQ were calculated for each marker. Intra- and inter-day variability was determined at three different concentrations five times within on the same day and on five different days, respectively. Variations were expressed by the coefficient of variation (CV), which allows evaluating the statistical quality of the estimates. The resulting CV values (3.68–12.79) (Table S3) confirm the precision of the method. The recoveries of analytes were determined at three concentrations of pre-analyzed sample solutions which were spiked with known quantities of the standards and injected in triplicate to perform recovery studies. The percentage of recovery for both PhytB and PhytG was between 90.8 and 105.4% (RSD <4%, n = 3), confirming the accuracy of the proposed method.
Studies of mechanisms of antifungal action Morphological studies by using phase contrast and fluorescence microscopies Fission yeast S. pombe 972 h− (wild type) provided by one of the authors (J.C. Ribas) and C. albicans CCC 125-2000 were selected for morphological studies. Early logarithmic phase cells of the model S. pombe and C. albicans were grown in YES (Yeast Extract with Supplements) and Sabourauddextrose (SD) broth at 28 °C, respectively and then treated with PtDEb at MIC50 and compared to untreated cells (control). Then, treated and untreated cells were concentrated by centrifugation (1000 g, 1 min) and examined by phase contrast and fluorescence microscopies. For fluorescence microscopy, the cells were resuspended in a solution of the fluorochrome Calcofluor White (CW, Fluorescent Brightener 28 or CW F3543, Sigma-Aldrich) at a final concentration of 50 µg/ml (from a stock solution of 10 mg/ml in water or PBS). Then the cells were imaged using a Leica DM RXA fluorescence microscope (Leica, Wetzlar, Germany) with the appropriate UV filter (Leica filter cube type A, excitation filter BP 340–380, dichromatic mirror 400, and suppression filter LP 425), a PL APO 63 × /1.32 oil PH3 objective and a digital camera (DFC350FX; Leica). The images were captured through the CW4000 cytoFISH software (Leica) (Muñoz et al., 2013). The images were processed with Adobe Photoshop CS2 software. All the analyses were repeated from three to four independent experiments, and representative images of the analyzed phenotype were selected.
UPLC-ESI-MS validation Pure PhytB showed a quadratic calibration curve, while PhytG presented a linear calibration curve in the ranges detailed in Table S4. For both compounds, the R2 coefficient showed a good response. Intra-day variability was determined at three different concentrations three times on the same day. The variations were expressed by the CV. The obtained CV values (0.2–17) (Table S5) confirm the precision of the method. The recoveries of analytes were determined at three concentrations of pre-analyzed sample solutions which were spiked with known quantities of the standard solutions and injected in duplicate to perform recovery studies. The % of recovery for both PhytB and PhytG was between 87.5 and 115% (CV <17%, n = 2), confirming the accuracy of the proposed method. Antifungal activity Microorganisms and media For the antifungal evaluation, C. albicans CCC 125-2000 and C. 3
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Fungal cell wall as the target
following the guidelines of CLSI (2017). MIC50 values were read at day 7 (Frost et al., 1995). The drug papulacandin B (PapB) (Novartis, Basel, Switzerland), known inhibitor of fungal (1,3)β-D-glucan synthase, was used as standard positive drug.
Sorbitol cellular assay The MIC50 of PtDEb against C. albicans CCC 125-2000 and C. glabrata CCC 115-2000 were determined either in the absence or in the presence of 0.8 M sorbitol (Sigma-Aldrich) added to the assay medium,
Fig. 1. Curves of concentration (µg/ml) vs. inhibition percentage (%) of P. tetramera methanol, DCM, BuOH and Aq extracts from: (A) berries (PtMEb, PtDEb, PtBEb, PtAqEb); (B) leaves (PtMEl, PtDEl, PtBEl, PtAqEl); (C) roots (PtMEr, PtDEr, PtBEr, PtAqEr), against Candida albicans and Candida glabrata. The dotted line shows 50% inhibition. 4
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Enzymatic assays related to the fungal cell wall
Results and discussion
Enzyme extract preparation Extracts of S. pombe 972 h− and C. albicans CCC 125-2000 cells were obtained as described by Martins et al. (2011). Early logarithmic phase cells grown in 100 ml of culture medium were collected, washed once with buffer A (50 mM Tris‐HCl pH 7.5, 1 mM EDTA and 1 mM β-mercaptoethanol), suspended in 100 µl of the same buffer per g of cells (wet weight) containing 50 µM GTPγS to preserve enzyme activity, and finally, broken with glass beads in a FastPrep FP120 apparatus (BIO 101, Thermo Savant Inc., New York, NY, USA) (one 15 s pulse at speed = 6). The broken material was collected and the cell debris was removed by low speed centrifugation (5000 g, 5 min at 4 °C). The supernatant was centrifuged at 48,000 g for 30 min at 4 °C and the pellet was re-suspended in buffer A containing 33% glycerol and 50 µM GTPγS (at a concentration of about 2–5 mg protein/ml) and stored at −80 °C.
Antifungal activity of P. tetramera extracts The antifungal activity of twelve P. tetramera extracts obtained from berries, leaves and roots (PtMEb, PtMEl, PtMEr, PtDEb, PtDEl, PtDEr, PtBEb, PtBEl, PtBEr, PtAqEb, PtAqEl, PtAqEr) was assessed with the standardized CLSI microbroth dilution method for yeasts (CLSI, 2017) against C. albicans and C. glabrata as explained in Materials and methods. To determine the MIC50 for each extract, the percentage of growth inhibition was plotted against the corresponding concentration of the extract for each yeast (Fig. 1). The MIC50 values for the different extracts against C. albicans and C. glabrata are summarized in Table 1. PtDEb induced a 50% of C. albicans and C. glabrata growth inhibition at 62.5 µg/ml and thus being a high active extract according to the score (below 100 µg/ml) defined in Materials and methods (Fig. 1A). Differently, PtMEb and PtBEb showed a lower activity with 275 µg/ml ≤ MIC50 ≤ 750 µg/ml and AqE did not show any activity below 1000 µg/ml. PtMEl, PtDEl, PtBEl and PtMEr, PtDEr, PtBEr showed no activity (MIC50 > 1000 µg/ml) except for PtDEl, that showed a % MIC50 = 750 µg/ml against C. glabrata. The fact that PtMeb and PtDEl behave differently against the different species of Candida genus (C. glabrata and C. albicans) has already been observed in previous works. For example, the echinocandins papulacandins A-D showed to inhibit C. albicans and other Candida spp. but not Candida guilliermondii (Traxler et al., 1977); and ascosteroside, an acidic terpenoid glucoside isolated from the ascomycetous fungus Ascotricha amphitricha, is a strong inhibitor of C. glabrata but not of C. albicans (Onishi et al., 2000). One possible reason for this dictinct behavior could be the differences in the fungal wall composition between the two spp. that makes the compound/s to reach the target of the plasma membrane more easily in one sp than in the other (Cortés et al., 2019).
(1,3)β-D-glucan synthase (GS) assay The standard GS assay mixture contained 5 mM UDP-D-[14C]glucose (4 × 104 cpm/200 nmol), 150 µM GTPγS, 0.75% bovine serum albumin (BSA), 2.1 mM EDTA, 75 mM (pH 8.0), 7.5% (v/v) glycerol, and 5 µl enzyme extract (approximately 10–15 µg of protein in 5 µl of enzyme extract) in a total volume of 40 µl. Two µl of DMSO or PtDEb (kept in a stock solution of 50 mg/ml in DMSO at −20 °C), were added to each reaction. The reaction mixture was incubated for 90 min at 30 °C and the reaction was stopped by the addition of 1 ml of 10% trichloroacetic acid (TCA). All reactions were carried out in duplicate. PapB was used as the standard positive drug. Chitin synthase (ChS) assay ChS activity was measured as previously described (Choi and Cabib, 1994) with a slight modification as follows: for the proteolytic activation step of ChS, reaction mixtures were prepared with 0.5 M 2[N-morpholino]ethanesulfonic acid (MES) at pH 6.5, 40 mM MgCl2, 5 mM UDP-[U-14C]GlcNAc (2 × 104 cpm/50 nmol), 2 µl of trypsin at the optimal concentration for enzyme activation (0.005 µg/µl) and 10 µl of enzyme extract (20–30 µg protein) in a total volume of 46 µl. Two µl of DMSO or PtDEb (kept in a stock solution of 50 mg/ml in DMSO at −20 °C) were added to each reaction. The reaction mixture was incubated for 15 min at 30 °C. Proteolysis was stopped by adding 2 µl of trypsin inhibitor solution at a concentration 1.5 times higher than the trypsin solution used and by cooling the tubes on ice. GlcNAc was added to a final concentration of 32 mM, followed by incubation for 90 min at 30 °C and stopped by the addition of 1 ml of 10% TCA. All reactions were carried out in duplicate. Nikkomicin Z (NikZ) was used as standard positive drug.
Qualitative chemical analysis of P. tetramera extracts The qualitative analysis of the PtDEb extract by UHPLC-ESI-MS/MS allowed the detection of PhytB, PhytE and PhytG. The phytolaccosides PhytB and PhytE were previously described to have antifungal activity (Escalante et al., 2002). Additionally, the phytolaccagenin PhytG, which is the genin of PhytB, was detected as an important peak in the chromatographic analysis (Fig. 2). The presence of these compounds in the PtDEb extract was also confirmed by comparing the retention times (Rt), the mass value and the fragments obtained by MS/MS with those of the reference compounds (Table 2). The analysis of berry, leaf and root extracts along with pure PhytB, PhytE and PhytG through thin layer chromatography showed that PhytB, PhytE and PhytG are not present in the aqueous extracts. Although PhytB appears in all the remaining extracts, it seems more abundant in the PtDEb extract. PhytG is exclusively observed in PtMEb and PtDEb extracts (Fig. 3).
Fungal plasma membrane as the target Exogenous ergosterol effect assay The MIC50 values of PtDEb, either in the absence or in the presence of different concentrations (50, 100 and 200 µg/ml) of ergosterol (Sigma-Aldrich) added to the assay medium, were determined by following the guidelines of CLSI (2017) against C. albicans CCC 125-2000 and C. glabrata CCC 115-2000. AmphB, which is known for its capacity for binding to ergosterol, was used as the positive drug. MIC50 was determined after 48 h of incubation (Escalante and Zacchino, 2007).
Antifungal activity of pure components The antifungal activity of PhytB, PhytE and PhytG was evaluated against C. albicans and C. glabrata (Table 3). PhytB and PhytG were active against both pathogens with a MIC50 of 62.5 and 50 µg/ml, respectively. Differently, PhytE did not show activity (MIC50 > 250 µg/
Table 1 Concentrations (µg/ml) to achieve 50% inhibition (MIC50) of the different extracts of Phytolacca tetramera against C. albicans (Ca) CCC 125-2000 and C. glabrata (Cg) CCC 115-2000 determined with the microbroth dilution method recommended by CLSI (2017). Itra = Itraconazole
Ca Cg
PtMEb
PtDEb
PtBEb
PtAqEb
PtMEl
PtDEl
PtBEl
PtAqEl
PtMEr
PtDEr
PtBEr
PtAqEr
Itra
375 275
62.5 62.5
750 750
i i
i i
i 750
i i
i i
i i
i i
i i
i i
0.25 0.70
5
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Fig. 2. UHPLC-ESI-MS chromatogram of P. tetramera berries DCM extract (PtDEb). Retention time (Rt, min) are: PhytE (3.95 min), PhytB (4.26 min) and PhytG (4.75 min).
ml). These results are in agreement with those described for PhytB and PhytG in previous reports (Escalante et al., 2002; Di Liberto et al., 2010). Based on all these results, PhytB and PhytG were selected as active markers (EMA, 2010) and were quantified in all the extracts where they are present (Fig. 3).
is a very good model for detecting changes in the cellular morphology. Thus, an important alteration of the cell wall results in clear and strong changes in the S. pombe morphology, inducing round morphology and/ or cell lysis (Cortés et al., 2016). Phase contrast microscopy images (Fig. 4, left) showed that S. pombe rod-shape cells treated with PtDEb at MIC50 are smaller than the control and they appear wrinkled and brighter. It could be also observed that some cells were deformed and swollen when compared to the control. The wrinkled and brighter appearance of S. pombe cells indicates a phenotype of dead cells, which was more abundant during the process of cell separation. This phenotype of cell death after septum completion and the start of cell separation was similar to that described for cells lacking the essential and most abundant (1,3)β-D-glucan synthase Bgs4 or the essential (1,3)α-Dglucan synthase Ags1/Mok1 (Muñoz et al., 2013; Cortés et al., 2012), suggesting that PtDEb could affect the synthesis of the (1,3)β-D-glucan and/or (1,3)α-D-glucan of S. pombe cell wall. The cell wall was analyzed by staining the cells with the fluorochrome CW that binds to fibrillar (1,4)β-polysaccharides, such as chitin or cellulose. In the case of S. pombe, which does not have chitin in the cell wall, CW specifically binds with high affinity to the linear (1,3)β-D-glucan of the primary septum and with much less intensity to the growing poles (Cortés et al., 2007). Disturbance of either the cell wall or the plasma membrane might cause that CW penetrates in the cell cytoplasm, and this event can be observed by fluorescence microscopy. Thus, the observation of cells displaying a CW-stained cytoplasm suggests that either the cell wall or the plasma membrane might be altered in the presence of PtDEb. CW-staining of S. pombe control cells shows an intense fluorescence in the division septum, a less intense fluorescence in the growing ends and a complete absence of fluorescence in the cell cytoplasm, indicating that the fluorochrome did not penetrate in the cell cytoplasm (Fig. 4, right). Differently, S. pombe cells growing in the presence of the extract appeared dead and showing a bright CW-staining fluorescence in the cell cytoplasm. This observation indicates that this extract induced either cell wall and/or plasma membrane disturbances, which would facilitate the entry of the dye into the cell.
Quantitation of active markers in P. tetramera extracts The results of quantitative analyses of active markers in MeOH, DCM and BuOH extracts using the UHPLC-ESI-MS and UPLC-ESI-MS validated methods are shown in Table 4. The most potent extract of P. tetramera (PtDEb) showed the highest amount of active markers, followed by PtMEb and PtBEb. The extracts from leaves and roots contained a low level of both PhytB and PhytG. Mechanisms of antifungal action studies The mechanism of action of the most active extract PtDEb was studied. To know the mode of action of a specific compound is an important next step in the development of Herbal Medicinal Products. The main modes of action of the antifungal agents that are currently in clinical use are the binding to the ergosterol of fungal membrane (AmphB and other polyenes), the inhibition of some steps of ergosterol biosynthesis (allylamines, azoles), and the disruption of the fungal cell wall by inhibiting the synthesis of (1,3)β-D-glucan that is the main fungal cell wall polymer (echinocandins) (Mathew and Nath, 2009). Morphological studies using phase contrast and fluorescence microscopies The morphological alterations induced by antifungal compounds have often provided insight into their mechanism of action (Gunji et al., 1983; Fukushima et al., 1993). Thus, the morphology of the yeasts S. pombe and C. albicans in the presence of inhibitory concentrations of PtDEb was analyzed by phase contrast (Figs. 4 and 5, left) and fluorescence microscopies (Figs. 4 and 5, right). Because S. pombe exhibit rod shape and divides by medial fission it
Table 2 Identification data of standard compounds PhytE, PhytB, and PhytG: retention time (Rt), molecular formula (MF), molecular weight (MW), molecular ion (Mol. ion) and MS/MS fragments in P. tetramera berries extracts using UHPLC-ESI-MS/MS. The detected compounds had the greatest responses under the negative mode and so, the [M−H]− was used as the precursor ion. Compound
Rt (min)
MF
MW
Mol. Ion [M-H]−
PhytE PhytB PhytG
3.95 4.26 4.75
C42H66O16 C36H56O11 C31H48O7
826.96 664.38 532.71
825.4289 663.3760 531.3333
6
Fragments 531.3333; 663.3764; 483.3124; 589.9089 531.3325; 499.3069; 640.0847 517.3145; 453.3023
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Fig. 3. TLC plate treated with p-anisaldehyde sulfuric acid of P. tetramera berries, leaves and roots MeOH extracts (PtMEb, PtMEl and PtMEr), DCM extracts (PtDEb, PtDEl and PtDEr), BuOH extracts (PtBEb, PtBEl and PtBEr) and aqueous extracts (PtAqEb, PtAqEl and PtAqEr). PhytB (Rf= 3.7), PhytE (Rf=1.4) and PhytG (Rf= 4.9) were used as standard compounds. Table 3 MIC50 (µg/ml) values of PhytB, PhytG and PhytE against C. albicans (Ca) CCC 125-2000 and C. glabrata (Cg) CCC 115-2000 determined with the microbroth dilution method recommended by CLSI (2008).
Ca Cg
PhytB
PhytE
PhytG
Itra
62.5 62.5
i i
50 50
0.25 0.70
PhytB: phytolaccoside B; PhytE: phytolaccoside E; PhytG: phytolaccagenin; Itra: Itraconazole; (i): > 250 µg/ml. Table 4 Content (mg of compound/g of plant) of active markers PhytB and PhytG in the nine P. tetramera extracts analyzed by UHPLC-ESI-MS (Pt berries extracts) and UPLC-ESI-MS (Pt leaves and roots extracts). Values are the mean ± Standard.
Berries Leaves Roots
Extracts
PhytB (mg/g)
PhytG (mg/g)
PtMEb PtDEb PtBEb PtMEl PtDEl PtBEl PtMEr PtDEr PtBEr
66.12 ± 0.33 155.24 ± 5.27 36.27 ± 0.30 2.11 ± 0.28 0.61 ± 0.11 3.13 ± 0.66 0.09 ± 0.01 0.56 ± 0.10 0.55 ± 0.21
24.92 ± 0.09 121.48 ± 1.36 0.68 ± 0.03 0.01 ± 0.005 0.06 ± 0.0002 nd 0.004 ± 0.0003 0.017 ± 0.005 nd
Fig. 4. Left: phase contrast microscopy. Right: fluorescence microscopy obtained by staining S. pombe cells with CW. Control: untreated cells; Treated: S. pombe treated with PtDEb (at IC50). Same lens (63x) and exposure times were used for both images. The arrows indicate S. pombe cells with a smaller size, appearing wrinkled and lighter or deformed and swollen. It is observed that CW penetrated into the cell.
phase contrast, also indicative of cell death or sickness.
(n = 3). nd: not detected.
Assays related to the interaction of PtDEb with the fungal cell wall
Regarding C. albicans, phase contrast microscopy images (Fig. 5, left) showed that cells treated with the extract become swollen or elongated and refringent, which suggest sick or dead cells due to an altered plasma membrane. There was also an enrichment of chained cells, indicating a defect in the final process of cell separation. Control cells showed a slight CW-staining fluorescence that appeared evenly distributed along the cell wall due to the presence of chitin. However, cells treated with the extract displayed a more intense CW-staining fluorescence of both cell wall and septum, and they also exhibited CWstaining in the cell cytoplasm, suggesting that the extract also causes alterations in the cell wall and/or the plasma membrane of C. albicans (Fig. 5, right). As in S. pombe, the cells displaying cytoplasmic CWstaining fluorescence appeared clearer or brighter when observed by
Fungal cells are encased in a rigid cell wall that provides osmotic integrity, maintains mechanical strength and defines the cell shape (Cortés et al., 2016), and thus this structure is vital for the growth and survival of the fungal cells. The cell wall is also a differential structure that is found in the fungal cells, but it is absent in mammalian cells. Therefore, cell wall represents an ideal target for developing new antifungal agents, since agents acting by this mechanism of action would not be toxic for human host cells. To determine whether PtDEb could act through the inhibition of the synthesis or assembly of the main polymers of the fungal cell wall, both cellular and enzymatic assays were performed.
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Table 5 IC50 values (µg/ml) of S. pombe (Sp) β(1,3)-D-glucan synthase, C. albicans (Ca) (1,3)-D-glucan synthase and Ca chitin synthase, in the presence of DCM extract of berries (PtDEb) and standard drugs papulacandin B (PapB) and nikkomycin B (NikZ).
PtDEb PapB NikZ
Sp β(1,3)-D-glucan synthase
IC50 (µg/ml) Ca β(1,3)-D-glucan synthase
Ca chitin synthase
> 312.5 0.019 –
> 312.5 0.01 –
> 2500 – 0.20
Table 6 Exogenous ergosterol (0–200 (μg/ml) effect on PtDEb MIC50 against C. albicans (Ca) and C. glabrata (Cg). AmphB was used as standard positive drug.
Fig. 5. Images obtained by phase contrast microscopy (left) and fluorescence by staining with CW (right) of C. albicans cells untreated (control) and treated with PtDEb (at IC50). Same lens (63x) and exposure time were used for both images. The arrows indicate C. albicans cells that are swollen or show an elongated shape. It is observed that CW penetrates into the cell.
Ergosterol concentration (μg/ml)
Ca
Cellular sorbitol assay Cg
The presence of the osmotic stabilizer sorbitol in the culture medium can stabilize those cells displaying a weakened cell wall (Ribas et al., 1991; Muñoz et al., 2013). Consequently, it is frequent that the effects caused by cell wall antifungals can be reversed by the presence of sorbitol in the growing medium, and thus antifungal compounds might exhibit a higher MIC in the presence of sorbitol (Frost et al., 1995). MIC50 determination for PtDEb was conducted both with and without sorbitol. Results showed that when C. albicans was treated with PtDEb in the presence of sorbitol, the MIC50 values did not shift to a higher value than those without sorbitol (results not shown), thus suggesting that PtDEb does not exclusively act through the inhibition of the cell wall synthesis.
PtDEb PhytG PhytB AmphB PtDEb PhytG PhytB AmphB
0 50 MIC50 (µg/ml) 62.5 125 50 125 62.5 62.5 0.20 >16 62.5 125 50 125 62.5 62.5 0.20 >16
100
200
500 125 62.5 >16 500 125 62.5 >16
500 250 62.5 >16 500 250 62.5 >16
endocytosis, vacuole fusion, cell division and cell signaling (Anderson et al., 2014). Therefore, the binding of PtDEb to this sterol could affect multiple cellular functions, leading to the death of fungal cells. In fact, the treatment of S. pombe cells with theonellamide (TNM), an antifungal compound that binds to the ergosterol, causes an activation of the (1,3)β-D-glucan synthase catalytic subunit Bgs1 and cell death (Nishimura et al., 2010). To determine whether PtDEb binds to fungal ergosterol, its MIC50 against C. albicans and C. glabrata was determined both with and without the addition of exogenous ergosterol to the culture medium. If the mode of action of the PtDEb is the binding to ergosterol, it will bind preferably to the most attainable exogenous ergosterol rather than to the plasma membrane ergosterol. Consequently, if PtDEb binds to ergosterol, a higher MIC50 will be obtained (Escalante et al., 2008). We found that the MIC50 of PtDEb against C. albicans and C. glabrata is enhanced in a dose-dependent way in the presence of increasing concentrations (50, 100 and 200 μg/ml) of exogenous ergosterol (Table 6). These results suggest that PtDEb binds to the ergosterol of the plasma membrane of Candida cells. The active markers PhytG and PhytB were also tested for their capacity to bind to ergosterol, finding that PhytG (but not PhytB) binds to ergosterol (Table 6). The inability of PhytB to bind to ergosterol is consistent with our previous report (Escalante et al., 2008). The antifungal AmphB, whose mode of action is binding to ergosterol, was used as a positive control drug. As expected, the presence of exogenous ergosterol leads to an enhanced MIC50 value for this antifungal drug (Table 6).
Enzymatic GS and ChS assays To analyze whether PtDEb inhibits the activity of the cell wall synthases, we tested S. pombe and C. albicans GS and ChS activities in the presence of PtDEb. GS and ChS are the enzymes that catalyze the synthesis of the main polymers of the fungal cell wall, (1,3)β-D-glucan and chitin, respectively. Thus, the activity of both GS and ChS was assayed by measuring the incorporation of either soluble UDP[14C]glucose into insoluble (1,3)β-D-glucan or UDP-[U-14C]GlcNAc into insoluble chitin in the presence of PtDEb (Ribas et al., 1991; Choi and Cabib, 1994). The GS and ChS in vitro assays showed that PtDEb did not inhibit either S. pombe and C. albicans GS or ChS activities (Table 5). In all the cases the enzymatic IC50 was higher than 312.5 µg/ml, which is a concentration five times higher than the MIC50 for PtDEb. The standard drugs PapB (an inhibitor of the GS) and NikZ (an inhibitor of the ChS) showed IC50 values of 0.02 and 0.01 µg/ml, respectively (Table 5). These results suggest that the defects of the cell wall observed in S. pombe and C. albicans cells are not caused by an inhibition of the cell wall synthases. One possibility is that those cell wall defects could be due to an indirect compensatory mechanism that alters the composition the cell wall polysaccharides.
Conclusion The antifungal activity of twelve P. tetramera extracts coming from berries, leaves and roots has been assessed in this work. PtDEb showed the highest antifungal activity against C. albicans and C. glabrata. Besides, PtDEb displayed the highest amount of the selected active markers PhytG and PhytB. Interestingly, PtDEb seems to disrupt the fungal plasma membrane by binding to ergosterol, which seems to cause cell wall damage and cell death. Interestingly, PhytG (but not PhytB) also binds to ergosterol, proving that it acts through the same
Assays related to the interaction with the fungal membrane Binding of PtDEb to ergosterol Plasma membrane ergosterol plays many essential roles in fungal cell physiology, including functional regulation of membrane proteins, 8
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mechanism of action of PtDEb extract. Globally, our data have shown that the antifungal PtDEb, that acts against the fungal plasma membrane, is the most interesting extract to be developed as an Herbal Medicinal Antifungal Product. These results have also confirmed the medicinal properties of the threatened species P. tetramera and they give a strong reason for the definitive protection of this species and its natural environment.
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Conflict of interest Authors declare that they do not have any conflict of interest. Acknowledgments Susana A. Zacchino and Laura Svetaz (L.S.) acknowledge Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), PICT20141170 and PICT2016-1833 and National University of Rosario, Argentina (UNR) for funds. J.C.R. acknowledges MINECO (BIO201569958-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 postdoctoral 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. We would like to thank the staff from the English Department of the School of Biochemical and Pharmaceutical Sciences (UNR) for the English language correction of the manuscript. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2019.152884. References Anderson, T., Clay, M., Cioffi, A., Diaz, K., Hisao, G., Tuttle, M., Uno, B., 2014. Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat. Chem. Biol. 10, 400–406. Basiglio Cordal, M.A., Adema, M., Briones, V., Villarreal, B., Panarisi, M.H., W., Abedini, Sharry, S., 2014. Induction of somatic embryogenesis in Phytolacca tetramera, medicinal species of Argentina. Emir. J. Food Agric 26, 552–557. Choi, W., Cabib, E., 1994. The use of divalent cations and pH for the determination of specific yeast chitin synthetases. Anal. Biochem. 219, 368–372. CLSI (Clinical and Laboratory Standards Institute), 2017. Reference Method For Broth Dilution Antifungal Susceptibility Testing of yeasts. Approved Standard-Document M27, 4 ed. Wayne, Pennsylvania, USA. Cortés, J.C.G., Curto, M.A., Carvalho, V.S.D., Pérez, P., Ribas, J.C., 2019. The fungal cell wall as a target for the development of new antifungal therapies. Biotechnol. Adv. https://doi.org/10.1016/j.biotechadv.2019.02.008. Cortés, J.C.G., Konomi, M., Martins, I., Muñoz, J., Moreno, M., Osumi, M., Ribas, J.C., 2007. The (1,3)β‐D‐glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation. Mol. Microbiol. 65, 201–217. Cortés, J.C.G., Ramos, M., Osumi, M., Pérez, P., Ribas, J.C., 2016. The cell biology of fission yeast septation. Microbiol. Mol. Biol. Rev. 80, 779–791. Cortés, J.C.G., Sato, M., Muñoz, J., Moreno, M.B., Clemente-Ramos, J.A., Ramos, M., Okada, H., Osumi, M., Durán, A., Ribas, J.C., 2012. Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission. J. Cell Biol. 198, 637–656.
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Original Article
The antifungal activity and mechanisms of action of quantified extracts from berries, leaves and roots of Phytolacca tetramera.☆
T
Estefanía Butassia, Laura A. Svetaza, Shuaizhen Zhoub,c, Jean-Luc Wolfenderc, Juan C.G. Cortésd, ⁎ Juan C. Ribasd, Caridad Díaze, José Pérez-del Palacioe, Francisca Vicentee, Susana A. Zacchinoa, a
Área Farmacognosia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario (UNR), Suipacha 531, 2000 Rosario, Argentina Natural Products Chemistry Department, & State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, China c School of Pharmaceutical Sciences, EPGL, University of Geneva, University of Lausanne, CMU-Rue Michel-Servet 1, CH-1206 Geneva 4, Switzerland d Instituto de Biología Funcional y Genómica y Departamento de Microbiología y Genética, Consejo Superior de Investigaciones Científicas (CSIC)/Universidad de Salamanca, 37007 Salamanca, Spain e Fundación MEDINA, Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía, Parque Tecnológico de Ciencias de la Salud, Avda. del Conocimiento 34, E-18016 Granada, Spain b
ARTICLE INFO
ABSTRACT
Keywords: Phytolacca tetramera Quantified extracts Candida albicans Candida glabrata Phytolaccagenin Phytolaccoside B
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 nonstandardized 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
Abbreviations: AmphB, amphotericin B; ChS, chitin synthase; CLSI, Clinical and Laboratory Standards Institute; CV, coefficient of variation; CW, Calcofluor White; EMA, European Medicines Agency; GlcNAc, N-acetylglucosamine; GS, (1,3)β-glucan synthase; ICH, International Council for Harmonization; MIC, minimum inhibitory concentration; NikZ, nikkomycin Z; PapB, papulacandin B; PBS, phosphate buffered saline; PhytB, phytolaccoside B; PhytE, phytolaccoside E; PhytG, phytolaccagenin; RSD, relative standard deviation; TCA, trichloroacetic acid; UHPLC-ESI-MS/MS, ultra high performance liquid chromatography-electrospray ionization-tandem mass spectrometry; UPLC-ESI-MS, ultra performance liquid chromatography-electrospray ionization-mass spectrometry ☆ In honor of Prof. Hildebert Wagner, on the occasion of his 90th birthday. ⁎ Corresponding author. E-mail address: [email protected] (S.A. Zacchino). https://doi.org/10.1016/j.phymed.2019.152884 Received 23 January 2019; Received in revised form 25 February 2019; Accepted 9 March 2019 0944-7113/ © 2019 Elsevier GmbH. All rights reserved.
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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.
Introduction
Materials and methods
Amongst plants of the Phytolaccaceae family, Phytolacca tetramera Hauman (common name ‘ombusillo’) is an endemic species from Argentina that grows in an area of a strong anthropic impact. Due to this, P. tetramera has been included in the category of species in critical risk (Hernández et al., 1998) and rare species (Delucchi and Correa, 1992). In previous studies carried out in our laboratory (Escalante et al., 2002) the antifungal activity of methanol (MeOH), dichloromethane (DCM), butanol (BuOH) and aqueous berry extracts of P. tetramera was assessed against a panel of human pathogenic opportunistic fungi using the non-standardized agar dilution method. By bioassay-guided fractionation, three monodesmosidic triterpenoid saponins - phytolaccosides (Phyt) B, E and F - were isolated from BuOH extract, PhytB being the most active one against both yeasts and filamentous fungi (Escalante et al., 2002). Mainly based on our previous studies, an agreement for the creation of an area for research and development for the conservation of P. tetramera was signed between the Faculty of Agrarian and Forestry Sciences of the National University of La Plata and the Roads Department of the Province of Buenos Aires (Petri et al., 2010). In addition, several attempts have been made to achieve propagation techniques that allow for maintaining or increasing the population of P. tetramera. The objectives were to reforest natural areas, to conserve the ecosystem's biodiversity and to maintain their germplasm in vivo (Basiglio Cordal et al., 2014; Hernández et al., 1998; 2009). Besides protecting the population of P. tetramera plants, the final aim of all these efforts was to obtain enough material of the plant to gain knowledge on this species and its medicinal potentialities. Here, we deepen in the study of the antifungal activity of extracts derived from P. tetramera with the following aims: (i) to study not only extracts from berries as in the previous work, but also extracts from leaves and roots, this time using quantified extracts following the European Medicine Agency (EMA) guidelines (EMA, 2010); (ii) to use the standardized method of the Clinical and Laboratory Standards Institute (CLSI) (CLSI, 2017) for antifungal assessment of the extracts against Candida albicans and Candida glabrata; and (iii) to study the mechanism of antifungal action of the most potent extracts. Regarding aim (i), EMA defines quantified extracts as those for which the content of selected markers is determined. To follow these guidelines, it is necessary to select the appropriate markers. Once they are established, they must be quantified in the corresponding extracts. With respect to aim (ii), the use of the most recent guidelines of the CLSI (2017) for yeasts assures more reproducible and more reliable results. In this work, C. albicans and C. glabrata were selected for antifungal assessment due to C. albicans is the most common cause of opportunistic fungal infections in immunocompromised hosts worldwide (Pfaller and Diekema, 2007; Pfaller et al., 2010) while C. glabrata is the second leading cause of candidemia, particularly in adult patients with hematologic malignancies (Malani et al., 2005; Pfaller and Diekema, 2007). Concerning aim (iii), the most active extract was subjected to the study of the mechanism of anti-Candida action. Morphological studies were carried out by using phase contrast and fluorescence microscopies on the yeast models Schizosaccharomyces pombe and C. albicans. Finally, we performed cellular and enzymatic assays targeting either the fungal membrane or the fungal cell wall to know the mechanism of action of the most active extract.
Plant material P. tetramera berries, leaves and roots were collected in the town of Arditi, Magdalena, Buenos Aires province (Argentina) (35° 04´ lat. S −57° 34´ long. W) in March 2013. The plant was identified by Prof. Martha Gattuso and a voucher specimen (MG 134/2) was deposited in the herbarium of the Plant Biology area, School of Pharmaceutical and Biochemical Sciences, National University of Rosario (UNR), Rosario, Argentina. Preparation of P. tetramera extracts Dried berries, leaves and roots (500 g each) were extracted by maceration with MeOH (24 h, 3 × 800 ml) at room temperature by using mechanical agitation. Solutions were filtered and evaporated in vacuum, resulting in MeOH extracts from berries (PtMEb) (236.98 g, 47.40% W/W), from leaves (PtMEl) (89.96 g, 17.99% W/W) and from roots (PtMEr) (39.29 g, 7.86% W/W). Then, 100.9 g, 47.2 g and 16.1 g of PtMEb, PtMEl and PtMEr, respectively, were dissolved in H2O and successively extracted with DCM (3 × 300 ml). The solutions were concentrated to dryness, resulting in DCM extracts of berries (PtDEb, 30.92 g, 30.65% W/W), leaves (PtDEl, 4.87 g, 10.32% W/W) and roots (PtDEr, 2.57 g, 15.94% W/W). The remaining water solutions were extracted with BuOH (3 × 300 ml). The solutions were concentrated to dryness resulting in BuOH extracts of berries (PtBEb, 50.54 g, 50.09% W/W), leaves (PtBEl, 27.13 g, 57.48% W/W) and roots (PtBEr, 2.73 g, 16.98% W/W). The water solutions were lyophilized, resulting in aqueous extracts (AqE) of berries (PtAqEb, 9.28 g, 9.20% W/W), leaves (PtAqEl, 7.45 g, 15.78% W/W) and roots (PtAqEr, 1.96 g, 12.18% W/ W). Source of standard compounds PhytB was isolated from PtBEb, as previously described (Escalante et al., 2002). Its purity was determined by HPLC-ESI-MS (≥ 95%) and 13C NMR spectrum (Supplementary material). PhytE and PhytG were purchased from PhytoLab GmbH & Co. (Phyproof® Reference Substances) (Vestenbergsgreuth, Germany). Qualitative and quantitative chemical analysis of P. tetramera extracts TLC development was carried out using a CAMAG equipment (Muttenz, Switzerland) with an automatic injector (model ATS4 22003), a development tank (model ADC2 220221), a visualizer (model 220145) and a scanner (Scanner4 211421). For the analysis, 20 μl of the different extracts (5 mg/ml) and 10 μl of each marker (1 mg/ml) were injected, and developed with a DCM: MeOH: H2O 50:10:1 mobile phase. The plate was revealed at 254 nm, 365 nm and with p-anisaldehyde-sulfuric acid. An integrated software Win-CATS V.4.06 was used for the analysis. The retention factor (Rf) was calculated for PhytB, PhytE and PhytG. UHPLC chromatographies were performed on extracts from berries in a Thermo Dionex Ultimate 3000 UHPLC interfaced to a Thermo QExactive Plus mass spectrometer with a heated electrospray ionization (HESI) source. Separation was performed on a Waters Acquity UPLC® BEH column (1.7 μm, 2.1 × 100 mm i.d.). An elution gradient was used with UPLC quality H2O and acetonitrile, both supplemented with 0.10% formic acid (solutions A and B, respectively). The elution 2
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gradient started with 5% B for 1 min, followed by a linear increase in B up to 100% for 8 min. The gradient was maintained at 100% B for 3 min. Before each injection, the column was equilibrated for 4 min with 5% B. The flow rate was 0.6 ml/min at 40 °C. Each sample was dissolved in 85% MeOH at a concentration of 1 mg/ml for injection. UPLC chromatography of extracts from leaves and roots was carried out on an Agilent 1290 UPLC interfaced to a triple quadrupole mass spectrometer Api4000 (Sciex), equipped with an ESI ion source. An XBridge Amide (Waters) column (3.5 μm, 2.1 × 100 mm i.d.) was used. The elution gradient was prepared with ultrapure H2O and HPLC quality ACN, both supplemented with 0.10% NH3 20% (solutions A and B respectively). The elution gradient started with 90% B for 0.5 min, followed by a linear increase in B up to 30% for 5.2 min, and then an increase up to 90% in B for 1.3 min. The flow rate was 275 μl/min. Each sample was dissolved in MeOH/H2O 85:15 and injected in triplicate. Calibration curves of pure compounds PhytB and PhytG were constructed by using five different concentrations (in triplicate) of each compound, prepared from stock solutions. The data were acquired using the Selected Ion Monitoring mode. The analytical methods were validated for linearity, limit of detection and quantification (LOD and LOQ) and intra- and inter-day variability, following the International Council for Harmonization (ICH) guidelines of Technical Requirements for Pharmaceuticals for Human Use (ICH, 1996). The percentage (%) of recovery was used to evaluate the accuracy of the method. The results of this validation are shown in Supplementary material.
glabrata CCC 115-2000 from the Centro de Referencia en Micología (CCC, CEREMIC, School of Pharmaceutical and Biochemical Sciences, UNR, Rosario, Argentina) were used. Strains were grown on Sabouraudchloramphenicol agar slants for 48 h at 30 °C. They were then maintained on slopes of Sabouraud-dextrose agar (SDA, Oxoid) and subcultured every 15 days to prevent pleomorphic transformations. The inocula were obtained according to reported procedures (CLSI, 2017) and adjusted to 1–5 × 103 cells with colony-forming units (CFU)/ml. Determination of fungal growth inhibition percentage Broth microdilution technique M27 4th ed for yeasts (CLSI, 2017) was performed in 96-well microplates. For the assay, extract or compound test-wells (ETWs or CTWs) were prepared with stock solutions of each extract or compound in DMSO (maximum concentration ≤ 2%), diluted with RPMI-1640 to final concentrations of 1000 μg/ml for the extracts and 250 μg/ml for the compounds. An inoculum suspension (100 μl) was added to each well (final volume in the well = 200 μl). A growth control well (GCW) (containing medium, inoculum, and the same amount of DMSO used in an ETW or CTW, but extract- or compound-free) and a sterility control well (SCW) (sample, medium, and sterile water instead of inoculum) were included for each fungus tested. Microtiter trays were incubated in a moist dark chamber at 30 °C for 48 h for both C. albicans and C. glabrata. Microplates were read in a VERSA Max microplate reader (Molecular Devices, Sunnyvale, CA, USA). Itraconazole (Itra) (Sigma-Aldrich, St. Louis, MO, USA) was used as positive control. Tests were performed in triplicate. Reduction of growth for each extract or compound concentration was calculated as follows:% of inhibition = 100 - (OD405 ETW (or CTW) − OD405 SCW) x100/(OD405 GCW−OD405 SCW). The means ± SD (standard deviations) were the data used for the dose-response curves that represent ‘% inhibition’ vs. ‘concentration’ of each extract or compound. Dose-response curves were constructed with SigmaPlot 11.0 software. MIC50 of each extract or compound was defined as the minimum concentration that inhibits 50% of the fungal growth. Extracts with MIC50 < 100 µg/ ml were considered to have high activity and were subjected to further studies. Extracts with 100 ≤ MIC50 ≤ 1000 µg/ml were considered to have low activity, while those with MIC50 > 1000 µg/ml were considered inactive.
UHPLC-ESI-MS validation The linearities of pure PhytB and PhytG calibration curves were established by calculating the slope, intercepts and R2 coefficient (Table S2, Supplementary material). The regression equation and R2 showed good linearity response. Linear range, LOD and LOQ were calculated for each marker. Intra- and inter-day variability was determined at three different concentrations five times within on the same day and on five different days, respectively. Variations were expressed by the coefficient of variation (CV), which allows evaluating the statistical quality of the estimates. The resulting CV values (3.68–12.79) (Table S3) confirm the precision of the method. The recoveries of analytes were determined at three concentrations of pre-analyzed sample solutions which were spiked with known quantities of the standards and injected in triplicate to perform recovery studies. The percentage of recovery for both PhytB and PhytG was between 90.8 and 105.4% (RSD <4%, n = 3), confirming the accuracy of the proposed method.
Studies of mechanisms of antifungal action Morphological studies by using phase contrast and fluorescence microscopies Fission yeast S. pombe 972 h− (wild type) provided by one of the authors (J.C. Ribas) and C. albicans CCC 125-2000 were selected for morphological studies. Early logarithmic phase cells of the model S. pombe and C. albicans were grown in YES (Yeast Extract with Supplements) and Sabourauddextrose (SD) broth at 28 °C, respectively and then treated with PtDEb at MIC50 and compared to untreated cells (control). Then, treated and untreated cells were concentrated by centrifugation (1000 g, 1 min) and examined by phase contrast and fluorescence microscopies. For fluorescence microscopy, the cells were resuspended in a solution of the fluorochrome Calcofluor White (CW, Fluorescent Brightener 28 or CW F3543, Sigma-Aldrich) at a final concentration of 50 µg/ml (from a stock solution of 10 mg/ml in water or PBS). Then the cells were imaged using a Leica DM RXA fluorescence microscope (Leica, Wetzlar, Germany) with the appropriate UV filter (Leica filter cube type A, excitation filter BP 340–380, dichromatic mirror 400, and suppression filter LP 425), a PL APO 63 × /1.32 oil PH3 objective and a digital camera (DFC350FX; Leica). The images were captured through the CW4000 cytoFISH software (Leica) (Muñoz et al., 2013). The images were processed with Adobe Photoshop CS2 software. All the analyses were repeated from three to four independent experiments, and representative images of the analyzed phenotype were selected.
UPLC-ESI-MS validation Pure PhytB showed a quadratic calibration curve, while PhytG presented a linear calibration curve in the ranges detailed in Table S4. For both compounds, the R2 coefficient showed a good response. Intra-day variability was determined at three different concentrations three times on the same day. The variations were expressed by the CV. The obtained CV values (0.2–17) (Table S5) confirm the precision of the method. The recoveries of analytes were determined at three concentrations of pre-analyzed sample solutions which were spiked with known quantities of the standard solutions and injected in duplicate to perform recovery studies. The % of recovery for both PhytB and PhytG was between 87.5 and 115% (CV <17%, n = 2), confirming the accuracy of the proposed method. Antifungal activity Microorganisms and media For the antifungal evaluation, C. albicans CCC 125-2000 and C. 3
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Fungal cell wall as the target
following the guidelines of CLSI (2017). MIC50 values were read at day 7 (Frost et al., 1995). The drug papulacandin B (PapB) (Novartis, Basel, Switzerland), known inhibitor of fungal (1,3)β-D-glucan synthase, was used as standard positive drug.
Sorbitol cellular assay The MIC50 of PtDEb against C. albicans CCC 125-2000 and C. glabrata CCC 115-2000 were determined either in the absence or in the presence of 0.8 M sorbitol (Sigma-Aldrich) added to the assay medium,
Fig. 1. Curves of concentration (µg/ml) vs. inhibition percentage (%) of P. tetramera methanol, DCM, BuOH and Aq extracts from: (A) berries (PtMEb, PtDEb, PtBEb, PtAqEb); (B) leaves (PtMEl, PtDEl, PtBEl, PtAqEl); (C) roots (PtMEr, PtDEr, PtBEr, PtAqEr), against Candida albicans and Candida glabrata. The dotted line shows 50% inhibition. 4
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Enzymatic assays related to the fungal cell wall
Results and discussion
Enzyme extract preparation Extracts of S. pombe 972 h− and C. albicans CCC 125-2000 cells were obtained as described by Martins et al. (2011). Early logarithmic phase cells grown in 100 ml of culture medium were collected, washed once with buffer A (50 mM Tris‐HCl pH 7.5, 1 mM EDTA and 1 mM β-mercaptoethanol), suspended in 100 µl of the same buffer per g of cells (wet weight) containing 50 µM GTPγS to preserve enzyme activity, and finally, broken with glass beads in a FastPrep FP120 apparatus (BIO 101, Thermo Savant Inc., New York, NY, USA) (one 15 s pulse at speed = 6). The broken material was collected and the cell debris was removed by low speed centrifugation (5000 g, 5 min at 4 °C). The supernatant was centrifuged at 48,000 g for 30 min at 4 °C and the pellet was re-suspended in buffer A containing 33% glycerol and 50 µM GTPγS (at a concentration of about 2–5 mg protein/ml) and stored at −80 °C.
Antifungal activity of P. tetramera extracts The antifungal activity of twelve P. tetramera extracts obtained from berries, leaves and roots (PtMEb, PtMEl, PtMEr, PtDEb, PtDEl, PtDEr, PtBEb, PtBEl, PtBEr, PtAqEb, PtAqEl, PtAqEr) was assessed with the standardized CLSI microbroth dilution method for yeasts (CLSI, 2017) against C. albicans and C. glabrata as explained in Materials and methods. To determine the MIC50 for each extract, the percentage of growth inhibition was plotted against the corresponding concentration of the extract for each yeast (Fig. 1). The MIC50 values for the different extracts against C. albicans and C. glabrata are summarized in Table 1. PtDEb induced a 50% of C. albicans and C. glabrata growth inhibition at 62.5 µg/ml and thus being a high active extract according to the score (below 100 µg/ml) defined in Materials and methods (Fig. 1A). Differently, PtMEb and PtBEb showed a lower activity with 275 µg/ml ≤ MIC50 ≤ 750 µg/ml and AqE did not show any activity below 1000 µg/ml. PtMEl, PtDEl, PtBEl and PtMEr, PtDEr, PtBEr showed no activity (MIC50 > 1000 µg/ml) except for PtDEl, that showed a % MIC50 = 750 µg/ml against C. glabrata. The fact that PtMeb and PtDEl behave differently against the different species of Candida genus (C. glabrata and C. albicans) has already been observed in previous works. For example, the echinocandins papulacandins A-D showed to inhibit C. albicans and other Candida spp. but not Candida guilliermondii (Traxler et al., 1977); and ascosteroside, an acidic terpenoid glucoside isolated from the ascomycetous fungus Ascotricha amphitricha, is a strong inhibitor of C. glabrata but not of C. albicans (Onishi et al., 2000). One possible reason for this dictinct behavior could be the differences in the fungal wall composition between the two spp. that makes the compound/s to reach the target of the plasma membrane more easily in one sp than in the other (Cortés et al., 2019).
(1,3)β-D-glucan synthase (GS) assay The standard GS assay mixture contained 5 mM UDP-D-[14C]glucose (4 × 104 cpm/200 nmol), 150 µM GTPγS, 0.75% bovine serum albumin (BSA), 2.1 mM EDTA, 75 mM (pH 8.0), 7.5% (v/v) glycerol, and 5 µl enzyme extract (approximately 10–15 µg of protein in 5 µl of enzyme extract) in a total volume of 40 µl. Two µl of DMSO or PtDEb (kept in a stock solution of 50 mg/ml in DMSO at −20 °C), were added to each reaction. The reaction mixture was incubated for 90 min at 30 °C and the reaction was stopped by the addition of 1 ml of 10% trichloroacetic acid (TCA). All reactions were carried out in duplicate. PapB was used as the standard positive drug. Chitin synthase (ChS) assay ChS activity was measured as previously described (Choi and Cabib, 1994) with a slight modification as follows: for the proteolytic activation step of ChS, reaction mixtures were prepared with 0.5 M 2[N-morpholino]ethanesulfonic acid (MES) at pH 6.5, 40 mM MgCl2, 5 mM UDP-[U-14C]GlcNAc (2 × 104 cpm/50 nmol), 2 µl of trypsin at the optimal concentration for enzyme activation (0.005 µg/µl) and 10 µl of enzyme extract (20–30 µg protein) in a total volume of 46 µl. Two µl of DMSO or PtDEb (kept in a stock solution of 50 mg/ml in DMSO at −20 °C) were added to each reaction. The reaction mixture was incubated for 15 min at 30 °C. Proteolysis was stopped by adding 2 µl of trypsin inhibitor solution at a concentration 1.5 times higher than the trypsin solution used and by cooling the tubes on ice. GlcNAc was added to a final concentration of 32 mM, followed by incubation for 90 min at 30 °C and stopped by the addition of 1 ml of 10% TCA. All reactions were carried out in duplicate. Nikkomicin Z (NikZ) was used as standard positive drug.
Qualitative chemical analysis of P. tetramera extracts The qualitative analysis of the PtDEb extract by UHPLC-ESI-MS/MS allowed the detection of PhytB, PhytE and PhytG. The phytolaccosides PhytB and PhytE were previously described to have antifungal activity (Escalante et al., 2002). Additionally, the phytolaccagenin PhytG, which is the genin of PhytB, was detected as an important peak in the chromatographic analysis (Fig. 2). The presence of these compounds in the PtDEb extract was also confirmed by comparing the retention times (Rt), the mass value and the fragments obtained by MS/MS with those of the reference compounds (Table 2). The analysis of berry, leaf and root extracts along with pure PhytB, PhytE and PhytG through thin layer chromatography showed that PhytB, PhytE and PhytG are not present in the aqueous extracts. Although PhytB appears in all the remaining extracts, it seems more abundant in the PtDEb extract. PhytG is exclusively observed in PtMEb and PtDEb extracts (Fig. 3).
Fungal plasma membrane as the target Exogenous ergosterol effect assay The MIC50 values of PtDEb, either in the absence or in the presence of different concentrations (50, 100 and 200 µg/ml) of ergosterol (Sigma-Aldrich) added to the assay medium, were determined by following the guidelines of CLSI (2017) against C. albicans CCC 125-2000 and C. glabrata CCC 115-2000. AmphB, which is known for its capacity for binding to ergosterol, was used as the positive drug. MIC50 was determined after 48 h of incubation (Escalante and Zacchino, 2007).
Antifungal activity of pure components The antifungal activity of PhytB, PhytE and PhytG was evaluated against C. albicans and C. glabrata (Table 3). PhytB and PhytG were active against both pathogens with a MIC50 of 62.5 and 50 µg/ml, respectively. Differently, PhytE did not show activity (MIC50 > 250 µg/
Table 1 Concentrations (µg/ml) to achieve 50% inhibition (MIC50) of the different extracts of Phytolacca tetramera against C. albicans (Ca) CCC 125-2000 and C. glabrata (Cg) CCC 115-2000 determined with the microbroth dilution method recommended by CLSI (2017). Itra = Itraconazole
Ca Cg
PtMEb
PtDEb
PtBEb
PtAqEb
PtMEl
PtDEl
PtBEl
PtAqEl
PtMEr
PtDEr
PtBEr
PtAqEr
Itra
375 275
62.5 62.5
750 750
i i
i i
i 750
i i
i i
i i
i i
i i
i i
0.25 0.70
5
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Fig. 2. UHPLC-ESI-MS chromatogram of P. tetramera berries DCM extract (PtDEb). Retention time (Rt, min) are: PhytE (3.95 min), PhytB (4.26 min) and PhytG (4.75 min).
ml). These results are in agreement with those described for PhytB and PhytG in previous reports (Escalante et al., 2002; Di Liberto et al., 2010). Based on all these results, PhytB and PhytG were selected as active markers (EMA, 2010) and were quantified in all the extracts where they are present (Fig. 3).
is a very good model for detecting changes in the cellular morphology. Thus, an important alteration of the cell wall results in clear and strong changes in the S. pombe morphology, inducing round morphology and/ or cell lysis (Cortés et al., 2016). Phase contrast microscopy images (Fig. 4, left) showed that S. pombe rod-shape cells treated with PtDEb at MIC50 are smaller than the control and they appear wrinkled and brighter. It could be also observed that some cells were deformed and swollen when compared to the control. The wrinkled and brighter appearance of S. pombe cells indicates a phenotype of dead cells, which was more abundant during the process of cell separation. This phenotype of cell death after septum completion and the start of cell separation was similar to that described for cells lacking the essential and most abundant (1,3)β-D-glucan synthase Bgs4 or the essential (1,3)α-Dglucan synthase Ags1/Mok1 (Muñoz et al., 2013; Cortés et al., 2012), suggesting that PtDEb could affect the synthesis of the (1,3)β-D-glucan and/or (1,3)α-D-glucan of S. pombe cell wall. The cell wall was analyzed by staining the cells with the fluorochrome CW that binds to fibrillar (1,4)β-polysaccharides, such as chitin or cellulose. In the case of S. pombe, which does not have chitin in the cell wall, CW specifically binds with high affinity to the linear (1,3)β-D-glucan of the primary septum and with much less intensity to the growing poles (Cortés et al., 2007). Disturbance of either the cell wall or the plasma membrane might cause that CW penetrates in the cell cytoplasm, and this event can be observed by fluorescence microscopy. Thus, the observation of cells displaying a CW-stained cytoplasm suggests that either the cell wall or the plasma membrane might be altered in the presence of PtDEb. CW-staining of S. pombe control cells shows an intense fluorescence in the division septum, a less intense fluorescence in the growing ends and a complete absence of fluorescence in the cell cytoplasm, indicating that the fluorochrome did not penetrate in the cell cytoplasm (Fig. 4, right). Differently, S. pombe cells growing in the presence of the extract appeared dead and showing a bright CW-staining fluorescence in the cell cytoplasm. This observation indicates that this extract induced either cell wall and/or plasma membrane disturbances, which would facilitate the entry of the dye into the cell.
Quantitation of active markers in P. tetramera extracts The results of quantitative analyses of active markers in MeOH, DCM and BuOH extracts using the UHPLC-ESI-MS and UPLC-ESI-MS validated methods are shown in Table 4. The most potent extract of P. tetramera (PtDEb) showed the highest amount of active markers, followed by PtMEb and PtBEb. The extracts from leaves and roots contained a low level of both PhytB and PhytG. Mechanisms of antifungal action studies The mechanism of action of the most active extract PtDEb was studied. To know the mode of action of a specific compound is an important next step in the development of Herbal Medicinal Products. The main modes of action of the antifungal agents that are currently in clinical use are the binding to the ergosterol of fungal membrane (AmphB and other polyenes), the inhibition of some steps of ergosterol biosynthesis (allylamines, azoles), and the disruption of the fungal cell wall by inhibiting the synthesis of (1,3)β-D-glucan that is the main fungal cell wall polymer (echinocandins) (Mathew and Nath, 2009). Morphological studies using phase contrast and fluorescence microscopies The morphological alterations induced by antifungal compounds have often provided insight into their mechanism of action (Gunji et al., 1983; Fukushima et al., 1993). Thus, the morphology of the yeasts S. pombe and C. albicans in the presence of inhibitory concentrations of PtDEb was analyzed by phase contrast (Figs. 4 and 5, left) and fluorescence microscopies (Figs. 4 and 5, right). Because S. pombe exhibit rod shape and divides by medial fission it
Table 2 Identification data of standard compounds PhytE, PhytB, and PhytG: retention time (Rt), molecular formula (MF), molecular weight (MW), molecular ion (Mol. ion) and MS/MS fragments in P. tetramera berries extracts using UHPLC-ESI-MS/MS. The detected compounds had the greatest responses under the negative mode and so, the [M−H]− was used as the precursor ion. Compound
Rt (min)
MF
MW
Mol. Ion [M-H]−
PhytE PhytB PhytG
3.95 4.26 4.75
C42H66O16 C36H56O11 C31H48O7
826.96 664.38 532.71
825.4289 663.3760 531.3333
6
Fragments 531.3333; 663.3764; 483.3124; 589.9089 531.3325; 499.3069; 640.0847 517.3145; 453.3023
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Fig. 3. TLC plate treated with p-anisaldehyde sulfuric acid of P. tetramera berries, leaves and roots MeOH extracts (PtMEb, PtMEl and PtMEr), DCM extracts (PtDEb, PtDEl and PtDEr), BuOH extracts (PtBEb, PtBEl and PtBEr) and aqueous extracts (PtAqEb, PtAqEl and PtAqEr). PhytB (Rf= 3.7), PhytE (Rf=1.4) and PhytG (Rf= 4.9) were used as standard compounds. Table 3 MIC50 (µg/ml) values of PhytB, PhytG and PhytE against C. albicans (Ca) CCC 125-2000 and C. glabrata (Cg) CCC 115-2000 determined with the microbroth dilution method recommended by CLSI (2008).
Ca Cg
PhytB
PhytE
PhytG
Itra
62.5 62.5
i i
50 50
0.25 0.70
PhytB: phytolaccoside B; PhytE: phytolaccoside E; PhytG: phytolaccagenin; Itra: Itraconazole; (i): > 250 µg/ml. Table 4 Content (mg of compound/g of plant) of active markers PhytB and PhytG in the nine P. tetramera extracts analyzed by UHPLC-ESI-MS (Pt berries extracts) and UPLC-ESI-MS (Pt leaves and roots extracts). Values are the mean ± Standard.
Berries Leaves Roots
Extracts
PhytB (mg/g)
PhytG (mg/g)
PtMEb PtDEb PtBEb PtMEl PtDEl PtBEl PtMEr PtDEr PtBEr
66.12 ± 0.33 155.24 ± 5.27 36.27 ± 0.30 2.11 ± 0.28 0.61 ± 0.11 3.13 ± 0.66 0.09 ± 0.01 0.56 ± 0.10 0.55 ± 0.21
24.92 ± 0.09 121.48 ± 1.36 0.68 ± 0.03 0.01 ± 0.005 0.06 ± 0.0002 nd 0.004 ± 0.0003 0.017 ± 0.005 nd
Fig. 4. Left: phase contrast microscopy. Right: fluorescence microscopy obtained by staining S. pombe cells with CW. Control: untreated cells; Treated: S. pombe treated with PtDEb (at IC50). Same lens (63x) and exposure times were used for both images. The arrows indicate S. pombe cells with a smaller size, appearing wrinkled and lighter or deformed and swollen. It is observed that CW penetrated into the cell.
phase contrast, also indicative of cell death or sickness.
(n = 3). nd: not detected.
Assays related to the interaction of PtDEb with the fungal cell wall
Regarding C. albicans, phase contrast microscopy images (Fig. 5, left) showed that cells treated with the extract become swollen or elongated and refringent, which suggest sick or dead cells due to an altered plasma membrane. There was also an enrichment of chained cells, indicating a defect in the final process of cell separation. Control cells showed a slight CW-staining fluorescence that appeared evenly distributed along the cell wall due to the presence of chitin. However, cells treated with the extract displayed a more intense CW-staining fluorescence of both cell wall and septum, and they also exhibited CWstaining in the cell cytoplasm, suggesting that the extract also causes alterations in the cell wall and/or the plasma membrane of C. albicans (Fig. 5, right). As in S. pombe, the cells displaying cytoplasmic CWstaining fluorescence appeared clearer or brighter when observed by
Fungal cells are encased in a rigid cell wall that provides osmotic integrity, maintains mechanical strength and defines the cell shape (Cortés et al., 2016), and thus this structure is vital for the growth and survival of the fungal cells. The cell wall is also a differential structure that is found in the fungal cells, but it is absent in mammalian cells. Therefore, cell wall represents an ideal target for developing new antifungal agents, since agents acting by this mechanism of action would not be toxic for human host cells. To determine whether PtDEb could act through the inhibition of the synthesis or assembly of the main polymers of the fungal cell wall, both cellular and enzymatic assays were performed.
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Table 5 IC50 values (µg/ml) of S. pombe (Sp) β(1,3)-D-glucan synthase, C. albicans (Ca) (1,3)-D-glucan synthase and Ca chitin synthase, in the presence of DCM extract of berries (PtDEb) and standard drugs papulacandin B (PapB) and nikkomycin B (NikZ).
PtDEb PapB NikZ
Sp β(1,3)-D-glucan synthase
IC50 (µg/ml) Ca β(1,3)-D-glucan synthase
Ca chitin synthase
> 312.5 0.019 –
> 312.5 0.01 –
> 2500 – 0.20
Table 6 Exogenous ergosterol (0–200 (μg/ml) effect on PtDEb MIC50 against C. albicans (Ca) and C. glabrata (Cg). AmphB was used as standard positive drug.
Fig. 5. Images obtained by phase contrast microscopy (left) and fluorescence by staining with CW (right) of C. albicans cells untreated (control) and treated with PtDEb (at IC50). Same lens (63x) and exposure time were used for both images. The arrows indicate C. albicans cells that are swollen or show an elongated shape. It is observed that CW penetrates into the cell.
Ergosterol concentration (μg/ml)
Ca
Cellular sorbitol assay Cg
The presence of the osmotic stabilizer sorbitol in the culture medium can stabilize those cells displaying a weakened cell wall (Ribas et al., 1991; Muñoz et al., 2013). Consequently, it is frequent that the effects caused by cell wall antifungals can be reversed by the presence of sorbitol in the growing medium, and thus antifungal compounds might exhibit a higher MIC in the presence of sorbitol (Frost et al., 1995). MIC50 determination for PtDEb was conducted both with and without sorbitol. Results showed that when C. albicans was treated with PtDEb in the presence of sorbitol, the MIC50 values did not shift to a higher value than those without sorbitol (results not shown), thus suggesting that PtDEb does not exclusively act through the inhibition of the cell wall synthesis.
PtDEb PhytG PhytB AmphB PtDEb PhytG PhytB AmphB
0 50 MIC50 (µg/ml) 62.5 125 50 125 62.5 62.5 0.20 >16 62.5 125 50 125 62.5 62.5 0.20 >16
100
200
500 125 62.5 >16 500 125 62.5 >16
500 250 62.5 >16 500 250 62.5 >16
endocytosis, vacuole fusion, cell division and cell signaling (Anderson et al., 2014). Therefore, the binding of PtDEb to this sterol could affect multiple cellular functions, leading to the death of fungal cells. In fact, the treatment of S. pombe cells with theonellamide (TNM), an antifungal compound that binds to the ergosterol, causes an activation of the (1,3)β-D-glucan synthase catalytic subunit Bgs1 and cell death (Nishimura et al., 2010). To determine whether PtDEb binds to fungal ergosterol, its MIC50 against C. albicans and C. glabrata was determined both with and without the addition of exogenous ergosterol to the culture medium. If the mode of action of the PtDEb is the binding to ergosterol, it will bind preferably to the most attainable exogenous ergosterol rather than to the plasma membrane ergosterol. Consequently, if PtDEb binds to ergosterol, a higher MIC50 will be obtained (Escalante et al., 2008). We found that the MIC50 of PtDEb against C. albicans and C. glabrata is enhanced in a dose-dependent way in the presence of increasing concentrations (50, 100 and 200 μg/ml) of exogenous ergosterol (Table 6). These results suggest that PtDEb binds to the ergosterol of the plasma membrane of Candida cells. The active markers PhytG and PhytB were also tested for their capacity to bind to ergosterol, finding that PhytG (but not PhytB) binds to ergosterol (Table 6). The inability of PhytB to bind to ergosterol is consistent with our previous report (Escalante et al., 2008). The antifungal AmphB, whose mode of action is binding to ergosterol, was used as a positive control drug. As expected, the presence of exogenous ergosterol leads to an enhanced MIC50 value for this antifungal drug (Table 6).
Enzymatic GS and ChS assays To analyze whether PtDEb inhibits the activity of the cell wall synthases, we tested S. pombe and C. albicans GS and ChS activities in the presence of PtDEb. GS and ChS are the enzymes that catalyze the synthesis of the main polymers of the fungal cell wall, (1,3)β-D-glucan and chitin, respectively. Thus, the activity of both GS and ChS was assayed by measuring the incorporation of either soluble UDP[14C]glucose into insoluble (1,3)β-D-glucan or UDP-[U-14C]GlcNAc into insoluble chitin in the presence of PtDEb (Ribas et al., 1991; Choi and Cabib, 1994). The GS and ChS in vitro assays showed that PtDEb did not inhibit either S. pombe and C. albicans GS or ChS activities (Table 5). In all the cases the enzymatic IC50 was higher than 312.5 µg/ml, which is a concentration five times higher than the MIC50 for PtDEb. The standard drugs PapB (an inhibitor of the GS) and NikZ (an inhibitor of the ChS) showed IC50 values of 0.02 and 0.01 µg/ml, respectively (Table 5). These results suggest that the defects of the cell wall observed in S. pombe and C. albicans cells are not caused by an inhibition of the cell wall synthases. One possibility is that those cell wall defects could be due to an indirect compensatory mechanism that alters the composition the cell wall polysaccharides.
Conclusion The antifungal activity of twelve P. tetramera extracts coming from berries, leaves and roots has been assessed in this work. PtDEb showed the highest antifungal activity against C. albicans and C. glabrata. Besides, PtDEb displayed the highest amount of the selected active markers PhytG and PhytB. Interestingly, PtDEb seems to disrupt the fungal plasma membrane by binding to ergosterol, which seems to cause cell wall damage and cell death. Interestingly, PhytG (but not PhytB) also binds to ergosterol, proving that it acts through the same
Assays related to the interaction with the fungal membrane Binding of PtDEb to ergosterol Plasma membrane ergosterol plays many essential roles in fungal cell physiology, including functional regulation of membrane proteins, 8
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mechanism of action of PtDEb extract. Globally, our data have shown that the antifungal PtDEb, that acts against the fungal plasma membrane, is the most interesting extract to be developed as an Herbal Medicinal Antifungal Product. These results have also confirmed the medicinal properties of the threatened species P. tetramera and they give a strong reason for the definitive protection of this species and its natural environment.
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Conflict of interest Authors declare that they do not have any conflict of interest. Acknowledgments Susana A. Zacchino and Laura Svetaz (L.S.) acknowledge Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT), PICT20141170 and PICT2016-1833 and National University of Rosario, Argentina (UNR) for funds. J.C.R. acknowledges MINECO (BIO201569958-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 postdoctoral 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. We would like to thank the staff from the English Department of the School of Biochemical and Pharmaceutical Sciences (UNR) for the English language correction of the manuscript. Supplementary materials Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2019.152884. References Anderson, T., Clay, M., Cioffi, A., Diaz, K., Hisao, G., Tuttle, M., Uno, B., 2014. Amphotericin forms an extramembranous and fungicidal sterol sponge. Nat. Chem. Biol. 10, 400–406. Basiglio Cordal, M.A., Adema, M., Briones, V., Villarreal, B., Panarisi, M.H., W., Abedini, Sharry, S., 2014. Induction of somatic embryogenesis in Phytolacca tetramera, medicinal species of Argentina. Emir. J. Food Agric 26, 552–557. Choi, W., Cabib, E., 1994. The use of divalent cations and pH for the determination of specific yeast chitin synthetases. Anal. Biochem. 219, 368–372. CLSI (Clinical and Laboratory Standards Institute), 2017. Reference Method For Broth Dilution Antifungal Susceptibility Testing of yeasts. Approved Standard-Document M27, 4 ed. Wayne, Pennsylvania, USA. Cortés, J.C.G., Curto, M.A., Carvalho, V.S.D., Pérez, P., Ribas, J.C., 2019. The fungal cell wall as a target for the development of new antifungal therapies. Biotechnol. Adv. https://doi.org/10.1016/j.biotechadv.2019.02.008. Cortés, J.C.G., Konomi, M., Martins, I., Muñoz, J., Moreno, M., Osumi, M., Ribas, J.C., 2007. The (1,3)β‐D‐glucan synthase subunit Bgs1p is responsible for the fission yeast primary septum formation. Mol. Microbiol. 65, 201–217. Cortés, J.C.G., Ramos, M., Osumi, M., Pérez, P., Ribas, J.C., 2016. The cell biology of fission yeast septation. Microbiol. Mol. Biol. Rev. 80, 779–791. Cortés, J.C.G., Sato, M., Muñoz, J., Moreno, M.B., Clemente-Ramos, J.A., Ramos, M., Okada, H., Osumi, M., Durán, A., Ribas, J.C., 2012. Fission yeast Ags1 confers the essential septum strength needed for safe gradual cell abscission. J. Cell Biol. 198, 637–656.
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