Evaluation of synergistic anticandidal and apoptotic effects of ferulic acid and caspofungin against Candida albicans.

This study aimed to investigate the synergy between anticandidal and apoptotic effects of ferulic acid and caspofungin against Candida albicans and Candida glabrata, with the help of a quantitative checkerboard microdilution assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) as a viability dye. Apoptotic effects of caspofungin and ferulic acid concentrations (alone and combined) were analyzed for C. albicans and C. glabrata based on annexin V-propidium iodide binding capacities using flow cytometric analysis. C. albicans showed a synergistic effect, represented by a fractional inhibitory concentration index of < 0.5, but C. glabrata showed no synergistic effect (fractional inhibitory concentration index > 0.5). Early and late apoptotic effects of caspofungin and ferulic acid concentrations (1 μg/mL and 1000 μg/mL) were calculated as 55.7% and 18.3%, respectively, while their necrotic effects were determined as 5.8% and 51.6%, respectively, using flow cytometric analyses. The apoptotic effects of the combination of caspofungin and ferulic acid at concentrations of 1 μg/mL and 1000 μg/mL on C. albicans and C. glabrata were 73.0% and 48.7%, respectively. Ferulic acid also demonstrated a synergistic effect in combination with caspofungin against C. albicans. Another possibility is to combine the existing anticandidal drug with phytochemicals to enhance the efficacy of anticandidal drug.

1. Introduction

A considerable increase in the incidence of deep fungal infections has been observed in the last two decades in hospital environments due to the increase in organ transplantations, rise in the incidence of AIDS, and use of invasive devices (such as catheters, artificial joints, and valves), and also in immu-nocompromised patients [1,2].

Candida albicans and Candida glabrata have been highly associated with several opportunistic fungal infections [3]. Candida species are the basis for the development of new antifungal drugs. However, increasing levels of Candida species resistant to the current antifungal drugs have been observed, making these agents ineffective [4]. Therefore, other therapies, which are more effective and safer than the current ones, are being explored; namely the use of plant extracts enriched in phenolic compounds [5,6].

Although the advances in medical and chemical fields have increased life expectancy, an increasing resistance of pathogenic microorganisms to conventional drugs has been observed due to the random use of chemicals and drugs, leading to the development of other complications [7].

Ferulic acid is a phenolic acid widely distributed in the plant kingdom [8]. It is one of the most abundant phenolic acids. It may be found in high concentrations in foods such as navy bean, corn bran, wheat bran, eggplant, artichokes, and beets [9]. It presents a wide variety of potential therapeutic effects helpful in the treatment of cancer, diabetes, and lung and cardiovascular diseases; hepatoprotective, neuro-protective, and photoprotective effects; and antimicrobial and anti-inflammatory properties [10,11].

Caspofungin and other agents in the echinocandin class of antifungals have assumed an increasingly important role in the therapy of invasive candidiasis [12]. These agents are nontoxic and show potent fungicidal activity against C. albicans and other Candida species [13]. Although the mechanism of action of echinocandins is known, the physiological mechanisms by which they cause cell death are not defined [14].

The minimum inhibitory concentration (MIC) values of caspofungin and ferulic acid using the microbroth dilution assay, fractional inhibitory concentration (FIC) index using the checkerboard microdilution assay, and mechanisms of C. albicans and C. glabrata cell death caused by caspofungin and ferulic acid were determined in this study. The flow cytometric analysis showed that caspofungin caused both apoptosis and necrosis of C. albicans and C. glabrata cells.

2. Materials and Methods

2.1. Fungal strains and chemicals

C. albicans (ATCC 90028) and C. glabrata (ATCC 90030) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Caspofungin diacetate (SML0425) and ferulic acid (1270311) were purchased from Sigma (St. Louis, MO, USA).

2.2. Antimicrobial assay

MIC is defined as the lowest concentration of the antimicrobial agent that inhibits the bacterial growth, as detected by the lack of visual turbidity. The microbiological assay was performed according to the Clinical and Laboratory Standards Institute M7-A7 broth microdilution method [15].

2.3. Checkerboard microdilution assay for ferulic acid and caspofungin

Synergy was tested by the checkerboard method; a two-dimensional array of serial concentrations of test compounds, which has been used most frequently to assess antimicrobial combinations in vitro. The tested dilutions were based on the MIC of the two compounds. The checkerboard test was used as the basis to calculate the FIC index [16]. The effects of the combination of caspofungin with ferulic acid were investigated by the checkerboard broth microdilution method. Drug interaction was classified as synergistic, additive, or less-than-additive based on the FIC index, which is the sum of FIC indexes for each drug. The FIC index of each drug was calculated as the MIC of that drug in combined treatment divided by the MIC of the drug used alone. Drug–drug interactions were considered synergistic if the FIC index was < 0.5; additive if the FIC index was = 1.0; and less-than-additive if the FIC index was > 0.5.

2.4. Analysis of apoptosis caused by Candida species using flow cytometry

C. albicans and C. glabrata cells (2 × 106/mL) were incubated in Sabouraud Dextrose Broth with 1 μg/mL caspofungin and 1000 μg/mL ferulic acid for 24 hours at 30°C. C. albicans and C. glabrata cells were harvested by centrifugation and washed in 0.1M potassium phosphate buffer. Annexin V/propidium iodide (PI) assays were performed according to the staining kit protocol, using 5 μg annexin V and 5 μg PI at 37°C for 20 minutes. The cells were analyzed using a BD Accuri C6 flow cytometer (Becton–Dickinson, Mansfield, MA, USA) [17].

3. Results

The primary aim of this study was to determine the MIC and FIC index of caspofungin and ferulic acid, which induced both apoptosis and necrosis, using flow cytometry. The checkerboard microdilution assay showed that ferulic acid and caspofungin exhibited antifungal activity against C. albicans with an MIC value of 40 μg/mL and 2 μg/mL, respectively, and against C. glabrata with an MIC value of 20 μg/mL and 4 μg/mL, respectively. The FIC index of ferulic acid and caspofungin was 0.0375 (Table 1). The apoptotic effects of caspofungin and ferulic acid concentrations (alone and combined) after the 24-hour incubation period, which were analyzed for C. albicans and C. glabrata based on annexin V–PI binding capacities using flow cytometry, are depicted in Figure 1. Flow cytometric analyses revealed early and late apoptotic effects of 1 μg/mL caspofungin on C. albicans as 2.1% and 53.6%, respectively, while their necrotic effects were determined as 5.8%. Especially late apoptotic effects on C. albicans were found to be increased (Figure 1). The late apoptotic effects of the combination of caspofungin and ferulic acid (1 μg/mL and 1000 μg/mL) on C. albicans and C. glabrata were 63.4% and 44.9%, respectively (Table 2 and Figure 1). These findings indicated that the anticandidal effect of the combination of caspofungin and ferulic acid on C. albicans than on C. glabrata increased depending on the concentration and prolonged incubation period. The results of flow cytometric analysis were used to determine the apoptotic effects of caspofungin and ferulic acid alone and combined against C. albicans and C. glabrata. Caspofungin exerted apoptotic activity against C. albicans by directly killing the cells (resulting in necrosis) and causing others to undergo programmed cell death (apoptosis). The synergistic activity of the combination of caspofungin and ferulic acid caused a pronounced reduction in the MIC of C. albicans and C. glabrata.

Table 1

Results of ferulic acid and caspofungin combination treatment on Candida albicans and Candida glabrata.

	Ferulic acid (μg/mL)(A)	Caspofungin (μg/mL)(B)	Combination (μg/mL)		FIC> 0.5–< 0.5
			(A + B)	(B + A)
C. albicans	40	2	10	0.25	0.0375
C. glabrata	20	4	2.5	0.25	0.1875

C. albicans showed an synergistic effect (FIC < 0.5). C. glabrata showed a nonsynergistic effect (FIC > 0.5). FIC = fractional inhibitory concentration.

Figure 1

Candida albicans and Candida glabrata apoptosis assay stained with annexin V-propidium iodide using flow cytometry.

Table 2

Comparison of percentages of viable, early apoptotic, late apoptotic, and necrotic cells on Candida albicans and Candida glabrata stained with annexin V–propidium iodide using flow cytometry

Microorganism	C. albicans	C. glabrata
Control
Viable	98.0	94.8
Early apoptotic	0.5	0.2
Late apoptotic	1.0	1.4
Necrotic	0.5	3.5
Caspofungin (1 μg/mL)
Viable	38.5	7.8
Early apoptotic	2.1	1.8
Late apoptotic	53.6	62.3
Necrotic	5.8	28.1
Ferulic acid (1000 μg/mL)
Viable	30.2	47.9
Early apoptotic	0.0	0.1
Late apoptotic	18.3	4.3
Necrotic	51.6	47.7
Caspofungin (1 μg/mL) + ferulic acid (1000 μg/mL)
Viable	26.1	14.7
Early apoptotic	7.6	3.8
Late apoptotic	63.4	44.9
Necrotic	3.0	36.6

Results are shown as percentages.

4. Discussion

Only a few classes of antifungals such as polyenes, azoles, echinocandins, allylamines, and flucytosine are available for the treatment of Candida infections [18]. However, studies on their mechanism of action alone or in synergism with known antifungals are still scarce [19]. This study aimed to explore the potential use, proposed mechanisms of action, and limitations of the phenolic acids in anticandidal therapy [20]. No study to date has evaluated the synergistic effect of caspofungin and ferulic acid on Candida species.

However, caspofungin has been reported to mediate apoptosis in C. albicans when used alone. Hao and colleagues discovered that caspofungin induced apoptosis in C. albicans [13]. The results of their study correlated with the present findings: 0.5 μg caspofungin induced apoptosis in C. albicans (early and late apoptosis rate 25%). Another study on the resistance of C. albicans to fluconazole using a combination of fluconazole and Rubus chingii extract reported a significant effect of the combination on the resistance [21]. Also, a synergistic effect of caffeic acid and fluconazole was found on the resistance of C. albicans to fluconazole [22]. A study on the effects of fluconazole and nystatine on C. albicans reported a 64-fold decrease in the MIC value [23]. Stringaro et al [24] reported the antifungal activity of the essential oil of Mentha suaveolens, alone or in combination with other antifungal drugs, against C. albicans.

Regarding the studies on the antifungal activities of ferulic acid, the most significant effect was found against C. albicans. In spite of various biological activities of ferulic acid, no real clinical usage of this compound has been reported. Clinical trials exploring the effect of ferulic acid on different ailments and diseases, namely, colon and pancreatic cancers, multiple myeloma, myelodysplastic syndromes, Alzheimer’s disease, and psoriasis, are still in progress [25].

Therefore, more comprehensive researches are required for a definite conclusion on the suitability of phenolic acids as successful antifungal agents in the future.

The present study highlighted the low toxic potential of phytochemicals as antifungal compounds and suggested the possible use of synergistic drug–herb combinations for the treatment of fungal contaminations. This study also highlighted various systemic interaction patterns. The findings may help identify new drug combinations with a novel mechanism of action.

In conclusion, this study demonstrated the inhibitory effect of different concentrations of ferulic acid and caspofungin on C. albicans and C. glabrata. It can be hypothesized that these effects are probably not produced by a single compound, but are a synergistic effect of different compounds. Substantial evidence is available regarding the synergistic effect of ferulic acid and existing antifungal agents, which may become a promising anticandidal treatment. However, more comprehensive research is needed to validate these fungal treatment strategies. This study evaluated the synergetic effect of two compounds on Candida species. It provided a different perspective in managing opportunistic fungal infections, especially against Candida species. The combination of ferulic acid and caspofungin exhibited a stronger fungicidal activity compared with ferulic acid or caspofungin alone.