Antifungal activity of tannic acid against Candida spp. and its mechanism of action

Abstract

The increase in fungal resistance is a major public health concern. In this context, Candida spp. is an important genus related to invasive diseases, especially in immunosuppressed patients. The relevance of alternative approaches to increasing fungal resistance stands out, in which products of natural origin demonstrate potential antifungal activity in vitro against Candida spp. In this sense, this work aimed to evaluate the in vitro activity of tannic acid against Candida spp. Minimum inhibitory concentration (MIC) was determined for tannic acid and the antifungals, and the checkerboard assay was performed to analyze the interactions between them. Furthermore, we evaluated the tannic acid antibiofilm activity and its possible mechanism of action. Tannic acid showed MIC ranging to 0.06 to 0.5 µg/ml and showed no loss of effectiveness when combined with antifungals. Also, is safe at the concentrations it exerts its antifungal activity in pre-formed biofilms, as demonstrated by IC50 in murine fibroblasts cells and the hemolytic assay. Additionally, its mechanisms of action can be related with induction of signals that lead to apoptosis in fungal cells.

Introduction

Fungi have emerged as an important cause of disease in humans, especially among immunosuppressed individuals or those hospitalized with serious underlying illnesses. This increase can be attributed to the high number of immunosuppressed patients, including transplant recipients, patients with acquired immunodeficiency syndrome (AIDS), cancer patients undergoing chemotherapy and invasive procedures [1–3].

It is estimated that fungal diseases kill more than 1.5 million and affect over a billion people annually [3] In 2022, the World Health Organization (WHO) published the fungal priority pathogens list to guide research, development and public health action [4]. In this document, the genus Candida spp. was highlighted as an important cause of diseases. Candida spp. constitute a commensal genus that can be detected on the mucosal surfaces of healthy humans [5]. However, in the presence of impaired immune response, Candida spp. can spread and invade the bloodstream, promoting the excessive growth of fungi and leading to complicated opportunistic infections [6]. Globally, Candida spp. have become the third most common cause of bloodstream infections and invasive candidiasis has been attributed to a mortality rate of 40–50% in intensive care units [7].

Candida albicans in the most frequent specie in invasive candidiasis. However, non-albicans infections have increased in the last decades [8]. A change in the epidemiology of Candida infections is occurring, provoking an antifungal susceptibility less predictable [9]. In this context, considering the critical issues of increasing fungal resistance, there is an urgent need for the identification, development, validation and progression of new strategies and approaches that can be easily used to overcome this problem [10].

One of the main factors contributing to the virulence of Candida spp. is the ability to adapt to different habitats for growth and formation of surface-bound microbial communities, known as biofilms [11]. Biofilms are three-dimensional microbial structures that develop on biotic or abiotic surfaces. Biofilm cells are enveloped in an extracellular matrix and are highly tolerant to antimicrobial and immunological insults [12]. When microorganisms persist in this form, they have lower growth rates and greater resistance to antimicrobial treatments, behaving very differently from planktonic cells [13, 14].

Natural products represent an alternative to raise the efficiency of drugs whose effectiveness is weakened by resistance insurgence [15, 16]. In this sense, phenolic compounds such as tannins possess great structural variations and are one of the most diverse groups of secondary metabolites, in which the potential antifungal activity of these compounds has already been reported in the literature [17–21].

Tannic acid (TA) is a tannin typically extracted from oak tree galls and it has been used in many applications, such as development of biomaterials and nanoparticles for drug delivery [22]. It has been widely studied in different fields of science by demonstrating diverse biological properties, such as cardioprotective, tissue regeneration, antidepressant and antitumor [23]. Also, TA presents antibacterial, antiviral and antiparasitic properties in vitro [24–27]. However, literature presents few reports on its antifungal activity and there are no reports about the mechanism of action against Candida spp.

Our study aimed to evaluate the antifungal activity of tannic acid against Candida spp. in planktonic cells, alone and in combination with the antifungals fluconazole (FLC), itraconazole (ITC) and amphotericin B (AMB). Additionally, the study aimed to evaluate the activity of TA in pre-formed biofilms of Candida spp. and elucidate the mechanism of action.

Materials and methods

Drugs and microorganisms

FLC, ITC AMB and TA were obtained from Sigma-Aldrich (MO, USA). ITC and AMB were solubilized in 2.5% dimethyl sulfoxide (DMSO). Tannic acid and fluconazole were solubilized in sterile distilled water.

Thirteen Candida spp. clinical strains were used in this study. Among them, six were sensitive to fluconazole (two Candida albicans, two Candida tropicalis and two Candida parapsilosis) and seven were resistant to fluconazole (two Candida albicans, two Candida tropicalis, two Candida parapsilosis and one Candida glabrata). Candida auris 01256P was also used in this study. Candida parapsilosis ATCC 22,019 and Candida krusei ATCC 6258 were used as controls. The strains were obtained from the culture collection of the Laboratory of Bioprospection in Antimicrobial Molecules of Federal University of Ceará.

Determination of minimum inhibitory concentration (MIC)

Susceptibility testing was performed based on Clinical and Laboratory Standards Institute M27-A3 document [28], using Roswell Park Memorial Institute (RPMI) broth (pH 7.0) buffered with 0.165 M morpholinepropanesulfonic acid (MOPS) (Sigma-Chemical, MO, USA). Minimum inhibitory concentration (MIC) was determined as the lowest concentration of drugs able to inhibiting growth by 50% compared with the growth control. The drugs were tested at concentrations ranging from 0.125 to 64 µg/ml for FLC, 0.03 to 16 µg/ml for TA, ITC and AMB. The susceptibility to FLC was defined according to the Clinical and Laboratory Standards Institute M27-S4 protocol [29]. Candida glabrata was considered resistant to fluconazole if MIC ≥ 64 µg/mL and other Candida spp. when MIC ≥ 8 µg/mL. For Candida auris there is no defined sensitivity cutoff parameter [29].

Combination of TA with the antifungals

TA and the antifungals were combined using the checkerboard technique. The fractional inhibitory concentration index (FICI) was calculated for each combination. The interactions were classified as synergistic (FICI: ≤ 0.5), additive (0.5 < FICI ≤ 1.0), indifferent (1 < FICI ≤ 4.0) or antagonistic (FICI: > 4.0) [30].

Scanning electron microscopy

Planktonic cells treated and not treated of Candida albicans resistant to fluconazole (C. albicans 1) in exponential growth phase were adhered to glass coverslips pretreated with 2.5% silane. Then, fixed with 2.5 % glutaraldehyde (Sigma-Aldrich, MO, USA) in 0.15 M of sodium cacodylate buffer (Electron Microscopy Sciences, PA, USA), with Alcian blue (0.1 %) (Sigma-Aldrich, MO, USA). Cells were incubated overnight at 4 °C, dehydrated with an ascending ethanol concentration, dried with hexamethyldisilazane (Polysciences Europe, Hirschberg an der Bergstraße, Germany) and coated with 10 nm of gold (Emitech Q150T, Quorum Technologies Ltd, Laughton, UK) [31] The analysis was performed using Quanta 450-FEG scanning electron microscope (FEI Company, MA, USA), operating in high-vacuum mode at 20 kV. The concentrations evaluated in this analysis were TA at MIC (0.1 µg/ml), TA 2x MIC (0.2 µg/ml) and TA 4x MIC (0.4 µg/ml).

Cultivation & inhibition of viability of L929 cells

L929 murine fibroblast were cultivated under standard conditions (at 37 °C with 5% CO₂) in minimum essential medium (MEM) containing Earle’s salts. Culture media were supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 µg/ml of penicillin and 100 µg/ml of streptomycin. For evaluation of cytotoxic effects, the cells were cultivated for 2 days under standard conditions and then the medium was replaced with fresh medium containing TA or DMSO solution as negative control. Cell viability was quantified by the ability of living cells to reduce the tetrazolium salt to a purple formazan product. L929 cells were seeded in 96-well plates (0.3 × 10⁶ cells/well) and TA dissolved in sterile distilled water was added. The final concentration of DMSO in the culture medium was kept constant, less than 0.1% (v/v). The effect of the test substances was quantified as the percentage of control absorbance of the reduced dye at 595 nm. [32].

Hemolytic assay

The hemolytic test was performed in 96-well plates using the method described by Cavalcanti et al. 2020 [33]. The wells received 50 µl of 0.85% NaCl solution containing 10 mM CaCl₂. The first well represented the negative control (DMSO, 1%). Tannic acid was tested at concentrations of 100, 500 and 1000 µg/ml. The last well received 50 µL of 0.2% Triton X-100 in saline, to obtain 100% hemolysis (positive control). Each well then received 100 µL of a 2% suspension of mouse erythrocytes in saline containing 10 mM CaCl₂. Subsequently, the plates were incubated for 4 h at room temperature. After this, the plates were centrifuged, the supernatant was transferred to a new plate. The hemoglobin released was measured using a multiplate reader (Spectra Count, Packard, Ontario, Canada) at 540 nm. The experiments were performed in triplicate in three independent trials.

Tests performed by flow cytometry

The following assays were performed with the isolates Candida albicans 1, Candida parapsilosis 1 and Candida tropicalis 1. The strains resistant to fluconazole were selected as representative, due to its clinical relevance. The strains were incubated in yeast nitrogen dextrose medium (YND) (Becton Dickinson, CA, USA) for 24 h at 37 °C. Cell suspensions were prepared from cultures in the exponential growth phase, washed three times with 0.85% saline, then resuspended (∼10⁴ cells/ml) in RPMI. TA was evaluated at concentrations of MIC/2, MIC and 2x MIC for each strain. FLC was evaluated in MIC and AMB at a concentration of 4 µg/mL. FLC and AMB were used as positive control for cell death. Untreated cells were used as negative control [32, 34].

Determination of cell density

This evaluation was performed through the exclusion test with propidium iodide (PI) at a concentration of 2 mg/l. Cells were exposed for 24 h to the drugs and incubated with PI. Cell fluorescence was determined by FACSCalibur (Becton Dickinson, San Jose, CA, USA) flow cytometer. 10.000 events were evaluated in each assay, with cellular debris omitted from the analysis [32, 34].

Analysis of apoptotic markers

The cells were incubated with the drugs and centrifuged (1600× g for 10 min at 4 °C). After, cells were digested with a solution of potassium phosphate buffer pH 6.0, pl 1 M sorbitol and zymolyase 20T 2 mg/ml (Seikagaku Corp., Abingdon, UK) for 2 h at 30 °C for cell wall removal. Following the treatment, protoplasts of Candida spp. were labeled with annexin V and PI, using an apoptosis detection kit (Guava Nexin Kit, Guava Technologies, Inc., CA, USA). The cells were incubated in a binding buffer containing fluorescein isothiocyanate (FITC)-annexin V and then analyzed by flow cytometry. 10,000 events were evaluated in each assay, with the cellular debris omitted from the analysis [32, 34].

Assessment of transmembrane depolarization potential (Δψm)

Cells were washed with phosphate-buffered saline (PBS) and incubated with rhodamine 123 (5 µg/mL) (Sigma, USA) at 37 °C for 15 min away from light. Mitochondrial depolarization was detected by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA). For each experiment, 10.000 events were evaluated. Cellular debris was omitted from the analysis [32, 34].

Detection of ROS

Treated cells were incubated with 20 µM of CM-H₂DCFDA [5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester] (Sigma-Aldrich, MO, USA) during 30 min in the dark at 37 °C. Then, the cells were washed in PBS and immediately analyzed by flow cytometry (FACSCalibur, Becton Dickinson, San Jose, CA, USA) [32, 34].

Assays to determine activity against Candida spp. pre-formed biofilms

Four strains of different Candida species were selected as representatives, C. albicans 1, C. tropicalis 1, C. parapsilosis 1 and the standard strain Candida auris 01256P. Biofilm formation was carried out according to the methodology of Pierce et al. (2008) [35] with modifications. The strains were incubated on Sabouraud dextrose agar for 24 h at 35 ± 2 °C. Then, the yeasts were suspended in 5 mL of YND medium and reincubated under the same conditions. The cells were centrifuged (3000 g, 5 min), washed with PBS and adjusted to standard 0.5 on the McFarland scale in RPMI medium. The concentrations used to evaluate the activity of TA against the pre-formed biofilm by Candida spp. were 16x MIC, 32x MIC, 64x MIC and 128x MIC for each strain. To check cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) (1 mg/mL) (Sigma, USA) was used. After incubation, the dye was then removed and DMSO was added. Reading was performed on a microplate reader Smartreader™ 96 (Accuris instruments, NJ, USA) at 570 nm The growth reduction was compared to the well that corresponds to 100% growth, free of drugs [35].

Data analysis

All assays were performed in triplicate. MIC and FICI values were obtained through arithmetic means. The data from flow cytometry and hemolytic assay were submitted to one-way analysis of variance (ANOVA) followed by the Newman–Keuls test (p < 0.05) [32, 34]. For biofilms, the statistical significance of each assay was analyzed using ANOVA and Tukey’s test (p < 0.05). The GraphPad Prism program (version 6 for Windows, GraphPad Software, La Jolla, CA, USA) was used for the analyzes. The assays were performed in triplicate on different days and the results were averaged [35].

Results

Antifungal activity of tannic acid against Candida Spp

In this study, 54% of the strains were resistant to FLC, 8% susceptible-dose dependent and 38% susceptible. MICs of FLC ranged from 8 to 32 µg/ml for resistant strains and 0.25 to 4 µg/ml for susceptible strains. MICs of ITC ranged from 0.03 to 2 µg/ml and for AMB, MICs values ranged from 0.2 to 2 µg/ml. For TA, MICs ranged from 0.06 to 0.5 µg/ml (Table 1).

Table 1.

– effects of TA and antifungal drugs against strains of Candida spp. sensitive and resistant to fluconazole and their interactions

MIC alone (µg/mL)								MIC associated (µg/mL)
Yeasts	TA	FLC	ITC	AMB	TA/FLC	ICIF	Interaction	TA/ITC	ICIF	Interaction	TA/AMB	ICIF	Interaction
*#Candida. albicans 1	0.1	16	1	0.5	0.2/32	3.5	IND	0.1/0.8	1.6	IND	0.1/0.5	2	IND
Candida albicans 2	0.3	16	0.5	1	0.3/16	2	IND	0.2/0.3	1.2	IND	0.3/1	2	IND
Candida albicans 3	0.5	0.2	0.03	0.5	0.4/0.2	1.8	IND	0.3/0.1	4.3	ANT	0.4/0.4	1.6	IND
Candida albicans 4	0.2	0.2	0.1	0.5	0.4/0.1	2.3	IND	0.1/0.1	1.4	IND	0.2/0.3	1.4	IN
*#Candida parapsilosis 1	0.2	8	0.06	0.5	0.2/6	1.5	IND	0.2/0.1	3	IND	0.2/0.4	1.6	IND
Candida parapsilosis 2	0.2	8	0.2	0.5	0.2/0.8	2	IND	0.2/0.2	2	IND	0.2/0.4	1.6	IND
Candida parapsilosis 3	0.2	4	0.2	0.5	0.2/4	2	IND	0.2/0.2	2	IND	0.2/0.4	1.6	IND
Candida parapsilosis 4	0.3	0.5	0.06	0.5	0.1/0.1	0.5	SYN	0.3/0.06	2	IND	0.3/0.2	1.4	IND
*#Candida tropicalis 1	0.3	8	0.5	0.5	0.2/5	1.3	IND	0.2/0.3	1.1	IND	0.2/0.3	1.2	IND
Candida tropicalis 2	0.2	16	1	1	0.2/16	1.6	IND	0.1/0.7	1.3	IND	0.2/0.8	1.6	IND
Candida tropicalis 3	0.4	0.2	0.1	0.5	0.3/0.2	1.8	IND	0.4/0.1	2	IND	0.4/0.5	1.8	IND
Candida tropicalis 4	0.5	0.2	0.1	0.5	0.2/0.02	0.5	SYN	0.3/0.1	1.3	IND	0.4/0.4	1.5	IND
Candida glabrata	0.06	32	2	2	0.1/53.3	3.2	IND	0.06/2	2	IND	0.05/1.6	1.6	IND
#Candida auris 01256P	0.2	4	0.2	2	0.1/2	1	ADT	0.1/0.2	1.6	IND	0.1/1.3	1.3	IND
Candida parapsilosis ATCC	0.2	2	0.2	0.2	0.1/1	1.1	IND	0.2/0.2	2	IND	0.2/0.2	2	IND
22,019
Candida krusei ATCC 6258	0.1	32	0.2	0.5	0.1/16	1	ADT	0.1/0.2	2	IND	0.1/0.5	2	IND

MIC: minimum inhibitory concentration; ICIF: Fractional inhibitory concentration index; TA: Tannic Acid; FLC: Fluconazole; ITC: Itraconazole; AMB: Amphotericin B; SYN: Synergism; ADT: Additive; IND: Indifferent; ANT: Antagonism

Candida glabrata was considered resistant to fluconazole if MIC ≥ 64 µg/mL and other Candida spp. when MIC ≥ 8 µg/mL. For Candida auris there is no defined sensitivity cutoff parameter

*Strain used in flow cytometry assays

^#Strains used in biofilm assays

The MIC and ICIF values ​​are arithmetic averages of experiments carried out in triplicate

Combination of tannic acid with antifungals

Based in FICI values, indifferent interactions were observed for 100% of the strains when AMB and TA were combined. For ITC, 93.75% of the interactions were indifferent and only one presented antagonist interaction. The combination of FLC and TA presented different results: 75% of the interactions were classified as indifferent, 12.5 synergistic and 12.5% as additive (Table 1).

Scanning cells microscopy

In all concentrations tested, TA caused morphological damage to the external structure of Candida albicans, in relation to the untreated control (Fig. 1a and b). It is possible to observe a decrease in the number of cells, the destruction of cellular architecture and the presence of cellular debris (Fig. 1c and h).

Fig. 1.

Scanning electron microscopy of planktonic cells of Candida albicans. Magnification: 40.000×; bar: 5 μm: a, c, e, g. Magnification: 5.000×; bar: 20 μm: b, d, f, h. Figure parts (b, d, f, h) represent (a, c, e, g) respectively, at smaller magnifications. (a & b) Control (c & d) Treatment with TA at MIC (0,1 µg/ml) (e & f) Treatment with TA at 2x MIC (0,2 µg/ml) (g & h) Treatment with TA at 4x MIC (0,4 µg/ml). Deformations in the cell structure can be seen as well cellular debris (red arrows)

Evaluation of tannic acid cytotoxicity in L929 cells

After TA exposure to murine fibroblasts line L929, it was observed that there was no interference with cell proliferation. The mean inhibitory concentration (IC50) was 65.72 µg/ml, which is a value superior to obtained MIC for all Candida spp. strains.

Evaluation of the hemolysis assay

No hemolytic effects were observed at concentrations in which TA exhibited its antifungal activity. At concentrations to values superior to MICs of 100 µg/ml, 500 µg/ml and 1000 µg/ml hemolysis were 4.67%, 13.9% and 12.45% respectively (Fig. 2).

Fig. 2.

Hemolytic assay. TA was tested at concentrations of 100, 500 and 1000 µg/ml. Triton X-100 in saline was used as positive control. The data from hemolytic assay were submitted to one-way analysis of variance (ANOVA) followed by the Newman–Keuls test (*p < 0.05)

Tannic acid reduces the numerical density of viable cells

After 24 h of exposure, TA induced a reduction in cell viability, observed by PI-negative cells. There were significant effects (p < 0.05) in relation to the control for TA in all concentrations tested. Cells treated with FLC (16 µg/mL) did not show a significant reduction in cell viability compared to the control, while those treated with AMB (4 µg/mL) showed a high reduction in cell density (Fig. 3a).

Fig. 3.

Flow cytometry to identify the mechanism of action of TA after 24 h of exposure in Candida spp. The data from flow cytometry were submitted to one-way analysis of variance (ANOVA) followed by the Newman–Keuls test (*p < 0.05). (a) Cell viability decreased with the increase of concentration of TA. (b) Mitochondrial membrane depolarization increased in higher concentrations of TA. c) ROS production increased in higher concentrations of TA. (d) Higher levels of apoptotic markers were detected with rising concentrations of TA

Mitochondrial depolarization caused by tannic acid in Candida Spp

TA caused transmembrane mitochondrial depolarization at all concentrations tested after 24 h of treatment (p < 0.05) compared to the control. The highest concentration of TA (2x MIC) considerably increased mitochondrial depolarization. FLC did not show this effect on Candida spp. strains (Fig. 3b).

Tannic induces ROS formation in Candida Spp

TA treatment showed high levels of intracellular ROS (p < 0.05), compared to the untreated control at all concentrations tested. For AMB there was a large production of ROS, while FLC did not induce such production. (Fig. 3c).

Exposure to tannic acid leads to cell apoptosis in Candida Spp

TA induced apoptosis of Candida spp. strains. Significant effects were obtained at all concentrations tested and were not detected after treatment with FLC (Fig. 3d).

TA activity against pre-formed biofilms of Candida Spp

TA showed a significant reduction (p < 0.05) in the viability of the pre-formed biofilm in relation to the control at concentrations of 32x MIC (9.6 µg/ml), 64x MIC (19.2 µg/ml) and 128x MIC (38.4 µg/ml) for C. tropicalis. For strains of C. albicans and C. parapsilosis there was a significant reduction in concentrations of 64x MIC (6.4 µg/ml and 12.8 µg/ml) and 128x MIC (12.8 µg/ml and 25.6 µg/ml). For C. auris, a significant reduction was only shown at 128x MIC (25. 6 µg/ml). (Fig. 4)

Fig. 4.

Effect of TA in pre-formed biofilms of Candida spp. (a)Candida auris: 16x MIC (3.2 µg/ml), 32x MIC (6.4 µg/ml), 64x MIC (12.8 µg/ml) and 128x MIC (25.6 µg/ml). (b)Candida albicans: 16x MIC (1.6 µg/ml), 32x MIC (3.2 µg/ml), 64x MIC (6.4 µg/ml) and 128x MIC (12.8 µg/ml). (c)Candida parapsilosis 16x MIC (3.2 µg/ml), 32x MIC (6.4 µg/ml), 64x MIC (12.8 µg/ml) and 128x MIC (25.6 µg/ml). (d)Candida tropicalis: 16x MIC (4.8 µg/ml), 32x MIC (9.6 µg/ml), 64x MIC (19.2 µg/ml) and 128x MIC (38.4 µg/ml). The statistical significance of each assay was analyzed using one-way analysis of variance (ANOVA) and Tukey’s test (*p < 0.05 in relation to the control)

Discussion

The epidemiology of fungal diseases is dynamic and difficult to predict. Furthermore, therapeutic options are restricted to a few classes of toxic and expensive medications [9]. Therefore, there is an urgent need to develop new strategies against fungal resistance, in which natural products could be an alternative. Our results demonstrated that tannic acid stands out as a promising antifungal agent. Scanning electron microscopy showed that TA causes morphological damage to the external structure of Candida albicans cells, and this damage is increased at higher concentrations. Corroborating with these findings, literature presents the compound as an alternative to control the fungal pathogen Penicillium digitatum, considering that TA presented an inhibitory effect on mycelial growth, which was positively correlated with the concentration. It was shown that about 50% of mycelial growth was inhibited by tannic acid at 400 µg/ml [36]. Also, the anticandidal activity of other tannins, such as gallic acid, is already reported in literature at MICs ranging between 12.5 and 72 µg/mL [37, 38].

Hence, this study is relevant to identify the antifungal properties of tannic acid, as well as its possible mechanism of action against Candida spp. In the case of strains resistant to fluconazole, it is possible to observe that the MIC obtained from TA was considerably lower than that from FLC, as demonstrated, for example, for Candida albicans 1 and Candida glabrata, which respectively presented MIC for FLC of 16 µg/ml and 32 µg/ml, while for TA the MICs were 0.1 µg/ml and 0.06 µg/ml.

The obtained results demonstrated that the combinations of tannic acid with antifungals were classified differently, depending on the drug. Regarding the association between TA and the antifungals FLC, ITC and AMB, there is no data available in the literature. Based on the fractional inhibitory concentration index (FICI) values, 93.75% of the interactions with ITC were indifferent and only one strain presented antagonistic interaction. The combination of FLC and TA presented different results: 75% of the interactions were classified as indifferent, 12.5% synergistic and 12.5% as additive. The TA + FLC combination was the most promising, presenting 12.5% synergistic interactions and 12.5% additive interactions, which might indicate a gain in antifungal effect with this association by reducing the concentrations against the microorganism and possibly the toxic effects.

The TA + ITC association mostly demonstrated indifferent interactions. Indeed, only the interaction with one strain was classified as antagonistic. Although FLC and ITC belong to the same class of antifungals, these medications have very different chemical structures. Therefore, different molecular effects can be expected when these drugs are combined with other drugs, since many factors can affect the interactions between different molecules, such as weight, solubility and stereochemical effects between functional groups, among others [39]. Regarding the TA + AMB combination, 100% of the interactions were classified as indifferent, demonstrating that the concomitant use of these active does not cause a reduction in the antifungal effect. In addition to azoles and amphotericin B, it is important that future studies combine tannic acid with other antifungals of first-line therapy of candidemia, such as echinocandins. The increased use of echinocandins has also been associated with the emergence of resistance to these drugs, which affects particularly C. albicans and C. glabrata [40].

Moreover, previous studies already demonstrate synergist interaction of natural products and antifungals used for the treatment of Candida infections [41, 42]. In this sense, it is important that other studies are carried out to understand the possible mechanism of action that occurs in the association of tannic acid and antifungals, for example, in the interference with efflux pumps and transport mechanisms associated with resistance genes [43].

However, it is important to highlight that such available antifungal treatments have narrow therapeutic windows and high toxicity. Among the main toxic effects caused by these antifungals, it is possible to mention hepatotoxicity and nephrotoxicity [44–46]. Therefore, it is extremely important to explore new therapeutic strategies that do not have exacerbated cytotoxic effects. After TA exposure to murine line L929 fibroblasts, we observed no interference in cell proliferation at concentrations observed as effective against the tested Candida spp. Also, no hemolytic effects were observed at the concentrations in which TA exhibited its antifungal activity.

In this work, the antifungal MIC concentrations of TA were considerably lower than the values found to be cytotoxic in L929 cells and those able to cause hemolysis. In this sense, the literature presents well-established values for TA toxicity in animal models [47, 48]. In humans, the cytotoxic effect of TA has not been proven, but a toxic effect has been found at sufficiently high concentrations [22].

With regard to the assays to evaluate the mechanism of action of tannic acid, it is noteworthy that the major concentrations tested (0.2 µg/mL for Candida albicans, 0.4 µg/mL for Candida parapsilosis, and 0.6 µg/mL for Candida tropicalis) substantially increased mitochondrial depolarization and a caused a decrease in cell viability. Also, TA induced apoptosis and had high levels of intracellular ROS compared to the untreated control at all concentrations tested. The action mechanism of TA has not been elucidated yet. In this context, the literature reports that tannins, including TA, may present duality in their activity, being both pro-oxidant and antioxidant, through mechanisms that have not yet been identified [49–51]. Our results demonstrated that TA was able to induce the formation of ROS in Candida, so it exerted a pro-oxidant effect.

This study also demonstrates that TA was able to induce apoptosis in fungal cells. In line with this finding, there are several reports of similar effects generated by TA in cancer cells from different sites, such as breast, prostate, liver and colorectal cancer [52–55]. In this sense, studies have demonstrated that TA interferes with cellular signaling pathways, and consequently in several biological functions, such as cell proliferation and differentiation, in turn resulting in mitochondrial changes that activate an intrinsic apoptosis cascade [56, 57] Probably these effects described against cancer cells also occur in fungal cells, given the structural similarities between fungal and mammalian cells, and since both cell types are eukaryotic. Therefore, our results suggest that the antifungal activity of TA on Candida spp. is exerted through a pro-oxidant activity, in which the production of ROS occurs in fungal cells. These ROS generate oxidative stress, resulting in the induction of signals that mediate apoptosis. As a consequence, the number of viable cells decreases.

Besides planktonic cells, biofilms constitute an important virulence factor in disseminated Candida infections, mainly in patients that use catheters and prosthetic medical devices [10]. Therefore, it is of great importance to develop new antifungal therapies focusing on the prevention and destruction of biofilms. The antibiofilm activity of tannic acid has been already reported against Gram-positive and Gram-negative bacteria [25, 58–60]. Our results demonstrate that tannic acid showed a significant reduction (p < 0.05) in the viability of mature biofilms in relation to the control in different strains Candida spp.

Given the results of the present work, it is possible to envisage applications of the antifungal properties of TA, for example, as topical formulations. In this sense, an antimicrobial application in a hydrogel made from TA was reported. One of the proposed applications for the material is wound protection. Preliminary testing showed that the hydrogel is an efficient barrier against different microorganisms, including Candida albicans [61]. The activity of tannic acid against biofilms reinforces its application in these treatments, since the formation and maturation of biofilms is related to wound infections and improves the resistance of Candida spp. strains.

It is important to highlight that data obtained for cytotoxicity in mammalian cells have demonstrated that TA is safe at the concentrations at which it exerts its antifungal and antibiofilm activity, which supports the hypothesis that it can be used safely by patients. Despite this, more studies must be carried out to elucidate the pharmacokinetic characteristics of TA, in order to determine possible antifungal applications in vivo.

Conclusions

Tannic acid is a potential antifungal and its activity occurs even in low concentrations against Candida spp. It also demonstrated a reduced possibility of toxic effects due to the cytotoxicity and hemolysis tests carried out. Additionally, its mechanisms of action can be related with induction of signals that lead to apoptosis in fungal cells.

Declarations

Competing interest

The authors declare no conflicts of interest concerning this article.