Introduction

Drug-resistant pathogens and serious side effects are major problems associated with antifungal chemotherapy [1]. Candida species are the fourth leading cause of nosocomial blood stream infections in the USA, with treatment costs estimated to be more than two to four billion US dollars annually [2] and with attributable mortality rates estimated to be 38–49 % [3]. The epidemiology of invasive fungal infections is changing, although Candida albicans remains the most important fungal agent. However, a notable increase in infections caused by non-albicans species (Candida tropicalis, Candida glabrata, Candida parapsilosis, and Candida krusei) has been reported, and infections by these species account for 36–63 % of all cases [4].

Notwithstanding the increasing need for effective therapy, the range of antifungal agents available is limited, and some of the most effective agents are also toxic. In addition, while the azoles have been used successfully for the treatment of Candida infections, numerous reports of treatment failures are now appearing in the literature [5]. Amphotericin B, a polyene fungicidal agent, has been the standard treatment for Candida infections for decades, but the toxicity of its conventional form and the costs of its lipid forms limit its use [6]. More recently, azole antifungal compounds, which have excellent efficacy–toxicity profiles, have emerged as the principal drugs used in the treatment of Candida infections in non-neutropenic patients [7]. As already emphasized, the administration of azoles for treating fungal infections has encountered certain limitations, such as their low water solubility, low bioavailability, and frequent side effects consequent on the requirement of high doses and/or long-term administration [8]. However, with the increasing clinical use of azole, resistance is emerging in clinical isolates from immunocompromised patients. In addition, azole is only fungistatic; this characteristic probably contributes to the development of resistance. Therefore, novel fungal therapies for effective management of Candida infections are urgently required to meet this dangerous situation.

Acorus calamus Linn., very popularly known as “sweet flag,” is native to Central Asia, North America, and Eastern Europe [9]. Rhizome of this plant is widely used in the Indian systems of medicine, such as Ayurveda, Siddha, and Unani [10]. Moreover, the rhizomes of this plant are widely used in the number of diseases like epilepsy, mental ailments, chronic diarrhea, and dysentery. A. calamus is also used in the conditions of stomatopathy, hoarseness, flatulence, dyspepsia, helminthiasis, amenorrhea, dysmenorrheal, nephropathy, calculi, and stragury [11]. Studies on chemical composition of Acorus spp. have revealed that α- and β-asarones are the major bioactive compounds [12, 13]. A number of bioactivities are reported to α- and β-asarone, like antibacterial, antihelmintic, antifungal properties [14], and anticancer activity [15]. However, little is known about its synergistic interaction with clinically used antibiotics against pathogenic fungi.

The present study was undertaken to evaluate in vitro synergistic effect of α- and β-asarones isolated from A. calamus in combination with two azole and amphotericin B drugs against pathogenic Candida species. The present work highlights a promising role of asarones in combination with the three most widely used drugs for anticandidal drug therapy.

Materials and Methods

Chemical and Antifungal Agents

The microbiological media were purchased from HiMedia, Mumbai, India. The fluconazole and clotrimazole and polyene (amphotericin B) (Fig. 1) were purchased from Sigma-Aldrich, USA. Antifungal stock solutions were prepared in distilled water and sterilized by syringe filtration (Millipore). Three stock solutions were maintained at −20 °C.

Fig. 1
figure 1

Structure of azoles and amphotericin B drugs purchased from Sigma-Aldrich are used in the present study. a Fluconazole, b clotrimazole, and c amphotericin B

Full size image

Isolation of α- and β-Asarones

The A. calamus samples were purchased from the local market of Trivandrum, India. A voucher specimen has been deposited at the Agroprocessing and Natural Product Division, NIIST, Trivandrum. The rhizomes (250 mg) of A. calamus were shade-dried, and the powdered A. calamus sample was extracted with 90 % aqueous methanol (MeOH) on a shaker for 4 days. The MeOH extract was filtered through Whatman no. 1 filter paper and concentrated in vacuum using a rotavapor (BUCHI). The remaining residue was dissolved in distilled water (pH 5.0) and extracted twice with an equal volume of EtOAc at room temperature. The ethyl acetate (EtOAc) layers were pooled and extracted with distilled water to remove the hydrophilic compounds. The resulting organic solvent layers were evaporated under reduced pressure. The concentrated EtOAc extract was subjected to flash column chromatography over silica gel (60F254, 100–200 μm, Merck) with successive elutions with CHCl3/MeOH in increasing order of polarity (90:10, 80:20, 70:30, 50:50, 30:70, 10:90, v/v). Each of the CHCl3/MeOH elutes was concentrated and evaluated for antifungal activity. The active CHCl3/MeOH (80:20) and CHCl3/MeOH (30:70) elutes that exhibited antifungal activity against Candida spp. were further purified on a silica gel column (15 × 200 mm) with a stepwise gradient of n-hexane and Me2CO (100:0, 90:10, 80:20, 70:30, 50:50, 30:70, 10:90, 100:0 v/v). The concentrates of 90 % hexane/10 % acetone and 100 % acetone elutes were separated by the gel filtration of the C26/100 column packed with Sephadex LH-20 resin using MeOH as a mobile phase at a 0.5 ml/min rate. The 2 ml fractions were collected using a fraction collector (Bio-Rad). Each of all the fractions was bioassayed for antifungal activity against Candida spp. The active fractions 47–75 and 103–155 were subjected to preparative TLC on glass plates (20 × 20 × 0.2 mm) coated with silica gel (60GF254, Merck). The TLC plates developed in the n-hexane/EtOAc (8:2) solvent system were visualized under UV light. The activity of bands on TLC plates was examined by bioautographic technique. Scraped active bands were collected and extracted with 100 % MeOH at 28 °C in a shaking incubator for 24 h. The pooled extract was bioassayed against Candida spp. The concentrated extracts from antifungal-active TLC bands were further purified on Sephadex LH-20 column (10 × 950 mm) chromatography. The Sephadex LH-20 column was eluted with MeOH at a flow of 0.2 ml/min. The isolation of the antifungal compounds ∞ and β-asarone was performed by HPLC system on a Prep C18 column (7 μm, 7.8 × 300 mm, Waters) using a linear gradient solvent system from 50 % CH3CN in H2O to 70 % CH3CN in H2O at a flow rate of 2 ml/min. The separation was monitored at an absorbance of 270 nm by a UV detector (Schimadzu). Isolated pure α- and β-asarone were confirmed by comparing the retention time of isolated asarones with authentic α- and β-asarone from Sigma-Aldrich.

Test Candida spp.

The Candida strains used in the study were C. albicans MTCC 277 and C. tropicalis MTCC 184, which were procured from the Microbial Type Culture Collection and Gene Bank (MTCC) Division, CSIR-Institute of Microbial Technology (IMTECH), Chandigarh, India. The two Candida species was subcultured in potato dextrose agar (PDA) and broth (PDB) (HiMedia Laboratories Limited, Mumbai, India) at 37 °C for 48 h to ensure viability and purity prior to activity testing.

Inoculum Preparation

Stock inoculum of the Candida species were prepared by picking freshly growing colonies from 24-h-old cultures grown on PDA plates and suspended in 5 ml of sterile saline (0.85 %). Cell density was adjusted using a spectrophotometric method at 600 nm wavelength to achieve the turbidity equivalent 0.5 McFarland standard. The dilution of the Candida species stock suspension was adjusted from 1–5 × 106 cells/ml.

Antifungal Susceptibility Testing by Determining the MIC

The in vitro antifungal activities of the azoles, amphotericin B, and α- and β-asarones were determined using a broth microdilution method, which was performed in accordance with Clinical and Laboratory Standards Institute guidelines [16]. Minimal inhibitory concentration (MIC) values were determined in 96-well flat-bottomed microtiter plates by measuring the optical density (OD) of the Candida cultures. In all experiments, the test medium was Roswell Park Memorial Institute (RPMI) 1640 (Sigma-Aldrich) containing L-glutamine but lacking sodium bicarbonate, buffered to pH 7.0 with 0.165 M MOPS (Sigma-Aldrich). Candida cultures were prepared from 1-day-old cultures grown on PDA slants. Candida suspensions were diluted in RPMI 1640 to give a final inoculum of 5 × 103 CFU/ml. Series of twofold dilutions were prepared in RPMI 1640 and were mixed with equal amounts of cell. The final concentrations for azoles and amphotericin B in the wells were 0.5–128 μg/ml, and for asarones, it was 2–1000 μg/ml.

The microplates were incubated for 48 h at 37 °C, and the OD was measured at 600 nm with a microtiter plate reader (Bio-Rad). Uninoculated medium was used as the background for the spectrophotometric calibration; the growth control wells contained inoculum suspension in the drug-free medium. The solvent control wells contained inoculum suspension in the drug-free solvent containing (1 %) medium to prove that solvent had no inhibitory effect on the investigated fungi at the applied concentration. For calculation of the extent of inhibition, the OD600 nm of the drug-free control cultures was set at 100 % growth. The MICs for asarones were the lowest concentration of drugs that produced an optically clear well, while the MICs for azoles and amphotericin B were the lowest concentration of drugs that produced a prominent decrease in turbidity. The quality control strains were included every time an isolate was tested. All experiments were repeated at least three times.

Synergistic Activity Between Asarones and Azoles and Amphotericin B

To evaluate the combined effects of azoles and amphotericin B with asarones, checkerboard experiments were carried out [17, 18]. Briefly, cells were grown overnight in PDA medium at 37 °C with constant agitation. After incubation, the cells were washed in PBS and suspended as 4 × 103 cells/ml solution. A 100 μl aliquot of microtiter plate containing RPMI medium in the absence and presence of different concentrations of asarones, azoles, and amphotericin B, alone or in combination. In brief, serial double dilutions of the test compounds were prepared (in μg/ml): 0.015–2 (azoles and amphotericin B) and 0.12–32 (asarones). Serial dilutions of the combination of asarones with azoles and amphotericin B were mixed in PDB. Plates were incubated at 37 °C for 24 h, and the MIC was determined by measuring the optical density at 600 nm. The MIC was defined as the lowest concentration of drug, alone or in combination with other agents, inhibiting the growth of yeast (100 % inhibition of growth compared to the control).

Drug interaction was based on the fractional inhibitory concentration index (FICI) (Odds 2003), a non-parametric model defined by the equation:

$$ \mathrm{FICI}=\mathrm{F}\mathrm{I}\mathrm{C}\ \mathrm{of}\ \mathrm{asarones}+\mathrm{F}\mathrm{I}\mathrm{C}\ \mathrm{of}\ \mathrm{azoles}/\mathrm{amphotericin}\ \mathrm{B} $$

where FIC of asarones is defined as MIC of asarones in combination with FLC divided by the MIC of asarones alone and FIC of azoles/amphotericin B as MIC of azoles/amphotericin B in combination with asarones divided by the MIC of azoles/amphotericin B alone. The values calculated by the FICI equation will determine the interaction relations as follows: values ≤0.5 represent synergistic interactions, ˃4.0 antagonistic effect, and values in between these two represent no interaction [19].

Time-Kill Curve Analysis

A time-kill curve (CFU as a function of time) is evaluated to study the rate and extent of reduction in Candida spp. burden when treated with asarones and azoles/amphotericin B individually and in combinations. The experiments were conducted in PDB for 48 h. The concentrations used are the MIC values of corresponding asarones plus azole drugs in alone (see Table 1). An initial inoculum of approximately 1 × 106 CFU/ml was taken for all the experiments. Samples (0.1 ml) were collected at 0, 2, 4, 6, 8, 12, 24, and 48 h and serially diluted in normal saline and aliquoted in PDA. These plates were then incubated at 37 °C for 48 h. The colonies were counted after 48 h incubation. The broth without any agent was used as the control. The data were plotted as log CFU/ml versus time (h) for each time point. Tests were performed three times. Synergism was defined as a decrease of ≥1 log10 CFU/ml in antifungal activity produced by the combination compared with the agent alone after 48 h. Lowest limit of quantification of time-kill assay was 0.5 log10 CFU/ml (99.99 % reduction).

Table 1 Synergistic activity of the ∞- and β-asarone with azoles and amphotericin B against Candida species
Full size table

Inhibition of Biofilm Formation Assay

Candida biofilms were developed on polystyrene surfaces of 96-well plates as per standard methodologies [20]. One hundred microliters of a cell suspension (1 × 106 cells/ml) in PBS was inoculated, and plates were incubated at 37 °C for 90 min to allow attachment of cells onto the surface. Non-adhered cells were removed by washing the wells with sterile PBS, two to three times. RPMI 1640 medium (200 μl) was added to each well, and the plates were incubated at 37 °C for 24 h to allow biofilm formation. RPMI 1640 medium with 1:1 concentrations of test compound was added immediately after the adhesion phase to observe any effects on the development of biofilms. After incubation, wells were washed to remove any released cells, and biofilm growth was analyzed by MTT-metabolic assay.

Statistical Analysis

All statistical analyses were performed with SPSS (Version 17.0; SPSS, Inc., Chicago, IL, USA). In MIC and FICI determinations, when the results were different in both experiments, we made another test for final result. One-way ANOVA-Duncan’s multiple range test was used in time-kill assay. Data for time-kill analysis was presented as means ± standard deviations. Statistical significance was defined as p < 0.05.

Results

Purification of α- and β-Asarone from A. calamus

A. calamus was extracted with 90 % MeOH. The concentrated crude extracts were then partitioned with EtOAc. Antifungal activity against Candida spp. was detected in the EtOAc layer. The active organic layers were pooled, concentrated, and further chromatographed on a silica gel (100–200, Merck) column. The silica gel column was eluted with stepwise gradients of CHCl3 and MeOH. The fractions eluted with CHCl3 and MeOH (80:20 and 30:70 v/v) showed a high level of antifungal activity against Candida spp. and was rechromatographed on a silica gel column using a stepwise gradient elution system of n-hexane and Me2CO. The 90 % hexane/10 % acetone and 100 % acetone fractions that were highly active against Candida spp. were further purified by Sephadex LH-20 column (26 × 950 mm) chromatography. Antifungal activity against Candida spp. was detected in fractions 47–75 and 103–155. The antifungal compounds of Sephadex LH-20 fractions active against Candida spp. were further purified by TLC. Silica gel plates loaded with the antifungal-active fractions were developed by a solvent system of n-hexane/EtOAc (8:2, v/v). MeOH elutes of the active band scraped from preparative TLC plates were bioassayed against Candida spp. using a paper disc method and then purified by gel filtration chromatography of Sephadex LH-20 (10 × 950 mm column). The antifungal compound active against 47–75 and 103–155 was detected by bioautography on a silica TLC plate. The clear inhibition zone occurred at the position of Rf 0.40 and 52 on the silica gel TLC plates developed by a solvent system of n-hexane/EtOAc (8:2, v/v). The HPLC profile of the antifungal compound α- and β-asarone showed an antifungal active peak at the retention time of 12.34 and 15.86 min, respectively at 270 nm, which matched with the authentic α- and β-asarone from Sigma-Aldrich. The yield of α- and β-asarone was 21.1 and 43.56 mg, respectively. The chemical structure of α- and β-asarone is shown in Fig. 2.

Fig. 2
figure 2

Structure of asarones. a α-asarone and b β-asarone

Full size image

Antifungal Activity

The MIC values of α- and β-asarone, azoles, and amphoteric B alone are shown in Table 1. It is evident from the table that the highest activity was recorded for β-asarone in the range of 64–125 μg/ml. While α-asarone exhibited activity at higher concentration (250–500 μg/ml), for azole drugs, the activity ranges from 1 to 4 μg/ml and for amphotericin B, the activity ranges from 1 to 2 μg/ml.

Checkerboard Assay

The combined activities of α- and β-asarone with azoles and amphotericin B from the in vitro checkerboard interactions against the Candida species are summarized in Table 1. FIC, FIC index, and interpretations for the activities of α- and β-asarones with azoles and amphotericin B against the test Candida species recorded significant synergistic interaction. Significant synergistic interaction was recorded by β-asarone with azoles and amphotericin B. Antagonism and indifference was not recorded for any combinations. In the combination of ∞ and β-asarone with azoles and amphotericin B, the MIC values have reduced to more than eight times.

Time-Kill Assay

Time-kill assays for the synergistic combinations (α- and β-asarone with azoles and amphotericin B) on Candida species are shown in Fig. 3. The maximum reduction of Candida species growth by α-asarone with azoles and amphoteric B was recorded between 2 and 4 h (p < 0.05) (Fig. 3a). At 48 h, this combination reduced 99.99 % of Candida species. Significant reduction was recorded by the combination α-asarone plus clotrimazole and amphotericin B, and at 48 h, this combination resulted in 100 % inhibition. Regrowth of the Candida species was recorded by α-asarone alone after 12 h.

Fig. 3
figure 3

Time-kill curve of asarones, azoles, and amphotericin B alone and in combination against Candida species. The strains at a starting inoculum density of 106 CFU/ml were used. At 0, 2, 4, 6, 8, 12, 24, and 48 h, aliquots were removed from each tube to examine the cell viability. The experiments were performed three times. Data are expressed as mean ± standard deviation. a α-asarone and b β-asarone. The x-axis represents time, and the y-axis represents logarithmic Candidal survival. CFU colony-forming unit

Full size image

For β-asarone with azoles and amphoteric B, maximum reduction in the Candida albicans was recorded by β-asarone and fluconazole and the maximum reduction was recorded between 0 and 2 h. This combination reduced 99.99 % of C. albicans at 12 h (p < 0.05) (Fig. 3b). Whereas against C. tropicalis, maximum reduction was recorded by β-asarone plus fluconazole and amphotericin B and the maximum reduction was recorded between 0 and 2 h and reduced 99.99 % of C. tropicalis at 12 h (p < 0.05) (Fig. 3b). The combination of asarone with azoles and amphoteric B never recorded regrowth even after 48 h.

Biofilm Inhibition

α- and β-asarone alone recorded the inhibition of biofilms of Candida sp. at MIC concentration of 69–80 % (Fig. 4). Whereas, the combination of α- and β-asarone with azoles and amphotericin B recorded significant inhibition of biofilms than the individual compounds. Significant inhibition was recorded by β-asarone plus fluconazole against C. tropicalis (12 %) (Fig. 4). This clearly indicated that combination of asarones and antibiotics has better inhibition of biofilm formation.

Fig. 4
figure 4

Effect of the combination of asarones and drugs on the inhibition of biofilm formation on Candida species. a α-asarone and b β-asarone

Full size image

Discussion

In recent years, candidiasis has reemerged with higher prevalence and mortality rates that are nearly 45 % among immunocompromised population groups [21]. Combination therapy is one approach that can be used to improve the efficacy of antimicrobial therapy for difficult-to-treat infections [22]. Attempts have been made to cope with treatment failures either by combining different antifungals or by combining antifungals with non-antifungals [23]. However, assessing the nature and intensity of drug interactions is still a debated issue. In the present study, we investigated the combined effects of asarones plus azoles and amphotericin B against Candida species by the checkerboard microdilution method and the time-killing test.

Phongpaichit et al. [24] reports the significant antifungal activity of A. calamus extracts against various pathogenic fungi like Trichophyton rubrum, Microsporum gypseum, and Penicillium marneffei with IC50 values of 0.2, 0.2, and 0.4 mg/ml, respectively. However, this extract recorded moderate activity against yeasts: C. albicans, Cryptococcus neoformans, and Saccharomyces cerevisiae (MIC 0.1–1 mg/ml). In addition, authentic α- and β-asarones were also tested for their antimicrobial potential. Both α- and β-asarones exhibited good antimicrobial activities against the fungi and yeasts than those of rhizome and leaf extracts. The study clearly suggested that A. calamus rhizomes and leaves must possess active principle α- and β-asarones which is believed to be responsible for their antimicrobial activities; further, it was established that β-asarone has high antimicrobial activity as compared to the α-asarone [14]. Similar in our study, also α- and β-asarones are the major compounds present in A. calamus and β-asarone exhibited high anticandidal activity than the α-asarone.

Rajput and Karuppayil [25] reported the anticandidal properties of A. calamus rhizome (ethyl acetate) extract and its active principle, β-asarone. β-asarone exhibited promising growth inhibitory activity at 0.5 mg/ml. Similarly, in the present study, also β-asarone recorded more activity. Minimum fungicidal concentration (MFC) of β-asarone was highly toxic to C. albicans, killing 99.9 % inoculum within 120 min of exposure. β-asarone inhibited C. albicans morphogenesis and biofilm development at subinhibitory concentrations [25].

In this study, we have investigated the in vitro anticandidal activity of α- and β-asarone, azoles, and amphotericin B alone and in combination by checkerboard assay to determine whether synergism, indifference, or antagonism would be the principal response against two Candida species. Very interestingly, the results of our study clearly indicated that asarones in combination with azoles and amphotericin B predominantly recorded synergistic effect (Table 1). Significant synergistic activity was recorded by β-asarone and amphotericin B against C. tropicalis. Synergy will gave significantly greater activity provided by two agents combined than that provided by the sum of each agent alone [26]. Currently, no information regarding the anticandidal activity of asarones in combination with azoles and amphotericin B drugs is available in literature. The in vitro synergistic activity of asarones plus azoles and amphotericin B against Candida species is reported for the first time. Further we have checked the inhibition of asarones and combination of asarones and antibiotics against Candida species, and the results clearly indicated that the combination of asarones and antibiotics inhibited the biofilm formation significantly than the asarones alone. C. albicans biofilms on implanted medical devices are a major source of life-threatening blood stream infections. Fluconazole, a frontline drug for the treatment of C. albicans infections, was relatively ineffective against the yeast in such biofilms, while amphotericin B was active against them [27]. Similarly, in the present study, also asarones in combination with amphotericin B recorded significant inhibition in biofilm formation.

In our study, significant synergy was recorded by asarones in combination with antibiotics. Positive interactions by checkerboard microdilution were confirmed by time-killing test in our study. Time-kill curves can provide growth kinetic information overtime. This method is often used to detect the differences in the rate and extent of antifungal activity over time. In our experiment, there was great agreement between the FICI method and the time-kill curves. The previous literatures also demonstrated the accordance between the two methods [28, 29].

Improving the efficiency of antifungal compounds can be done by using combination therapy, i.e., by combining agents with different antifungal mechanisms [30] or combining antifungal and non-antifungal compounds [29, 31]. In the present study, clinically used antifungal agents were combined with the α- and β-asarone, and the numerous synergistic interactions noted during the study demonstrated that asarones might be of use in natural compound and antifungal combination therapy. In the present study, the significant enhancement in the activity of asarones and azoles and amphotericin B may be due to the inhibition of ergosterol. Ergosterol is an essential component of the fungal plasma membranes, and the inhibition of its synthesis negatively influences the membrane fluidity [32]. The antifungal effect of asarones are due, in part, to their role on ergosterol levels, as well as to their indirect effect on cell signaling, proliferation, and differentiation through inhibition of the synthesis of important terpenoids. The precise mechanism of action of asarones and azoles and amphotericin B on yeast cells still need to be elucidated deeply.

Conclusions

In conclusion, to the best of our knowledge, this study reports for the first time the ability of asarones to sensitize in vitro Candida species to azoles and amphotericin B. This study suggests that asarones is not only a natural compound capable of inhibiting Candida species growth but is also able to act synergistically with azoles and amphotericin B in very low doses. Further studies are warranted to explore the mechanism behind the antifungal activity of asarones plus antibiotics (azoles and amphotericin B). Our findings are very much encouraging in view of the growing treatment failures and antibiotic resistance in Candida species and suggest a way of treating resistant Candida infections through an asarones plus azoles and amphotericin B combination approach.