Potential of plant oils as inhibitors of Candida albicans growth

Abstract

Minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) were determined for 38 oils of plant origin against Candida albicans. Four strains including one standard strain were used in this study. The antifungal agents, Fluconazole and Amphotericin B were used as positive controls. The standard strain (ATCC10231) used in this study was found to be highly resistant to Fluconazole: 3000 μg ml⁻¹ of Fluconazole was required to inhibit the growth of this strain partially, and complete inhibition could not be achieved. Other Candida strains were sensitive to 5 μg ml⁻¹ of Fluconazole. All the strains used were sensitive to Amphotericin B. Of the 38 oils tested, 23 were found effective and fifteen were ineffective. Based on their MFCs, effective oils were categorized into three categories. Seven oils, which exerted fungicidal effect at less than 0.15% concentration of oils, were grouped into the most effective class. The oils exhibiting MFCs in the range of 0.16–1.5% concentration were considered moderately effective. Nine oils, which required more than 1.5% concentration, were regarded as less effective. The Fluconazole-resistant strain (MTCC 227) was sensitive to at least 23 of the plant oils. Results of this study indicate that oils of plant origin may find use as potential anti-Candida agents.

1 Introduction

Amphotericn B and Fluconazole are two important agents used in antifungal chemotherapy against human pathogenic fungi [1–6]. Amphotericin B is used as the drug of choice in terminally ill patients [3,7]. The azole drug, Fluconazole is widely prescribed against various Candida albicans infections [2,3,8–10]. Even though very widely acclaimed for their efficacy, these drugs are known to have side effects [1,3,6,11]. Fluconazole is fungistatic in nature and there are reports of emergence of Fluconazole resistance among clinical isolates of C. albicans[5–7,12–15]. Thus there is a need for better, novel antifungal agents against infections caused by C. albicans which are effective and have lesser side effects.

Many natural agents like plant products including extracts, oils, etc. are traditionally used against various ailments [16–18]. Some of the plant products are effective against fungal and bacterial infections [17,19–23]. Essential oils like Tea tree oil, Lemon grass oil, Citronella oil, Palmarosha oil and some of their constituents have been tested against the in vitro growth of C. albicans[23,26,27].

We have tested the in vitro efficacy of 38 plant oils against four isolates of C. albicans. Minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) of the effective oils are reported. Based on their efficacy (MFC), the oils are classified into three categories.

2 Materials and methods

2.1 Chemicals and strains

A standard strain, Type strain ATCC10231 (MTCC 227/CA IV), a reference strain suggested for Amphotericin B assay, and three clinical isolates of C. albicans were used in this study. The three clinical isolates of C. albicans, viz. SRTCC I (CA I), SRTCC II (CA II), SRTCC III (CA III), were obtained from the SRTCC Culture Collection Center, School of Life Sciences, SRTM University, Nanded (MS). India. The standard strain was purchased from The Institute of Microbial Technology (IMTECH), Chandigarh, India.

Thirty-eight plant oils were used in this study. Some of the oils were kindly provided by the Central Institute of Medicinal and Aromatic Plants (CIMAP), Lucknow, India, and the remaining oils and the antifungal drugs, Amphotericin B and Fluconazole were purchased from the local market.

2.2 Inocula preparation

All the strains of C. albicans used in this study were grown at 30 °C for 24 h in a shaking incubator using Yeast Extract Peptone medium and Sabouraud Dextrose medium. Cells were harvested by centrifugation, suspended in sterile distilled water and used immediately. The cell number was determined by using a hemocytometer and adjusted to 5 × 10⁶ cfu ml⁻¹.

2.3 Screening of plant oils for anti-Candida activity by disc diffusion assay

The anti-Candida activity of 38 plant oils was tested by standard disc diffusion assay [23]. The Sabouraud Dextrose Agar plates were prepared and seeded with 5 × 10⁶ cfu ml⁻¹ using the inocula of Candida strains prepared as above. Aseptically, 5 μl of plant oils were spotted onto 5-mm filter paper discs (Whatman No. 1). These discs then were transferred to the centre of the seeded plates. Three plates were used for each concentration and incubated at 30 ± 2 °C. The diameter of the zone of inhibition was measured after 48-h incubation.

2.4 Determination of minimum inhibitory concentration (MIC) of plant oils by agar dilution method

Minimum Inhibitory Concentrations (MICs) of plant oils were determined by agar dilution assay (23) The agar plates were prepared by adding YPD agar containing various concentrations of plant oils (i.e., 0.03–3% v/v). Tween 20 0.5% (v/v) was added to enhance the oil solubility. These plates were inoculated with 10³ cfu, using the inocula of Candida strains prepared as above. Plates were kept in triplicates for each concentration. Plates with Tween 20 but without any plant oil were used as control. All these plates were incubated at 30 ± 2 °C. Plates were observed and numbers of colonies were counted after 48 h of incubation. The MIC values were determined as the lowest concentration of oil preventing visible growth of Candida strains [23].

2.5 Determination of minimum fungicidal concentration (MFC) of plant oils using Broth macro dilution method

Minimum fungicidal concentration (MFC) of plant oils was determined by Broth macro dilution assay (NCCLS M27-A, reference method recommended for the antifungal susceptibility testing of yeast) [24]. A range of concentrations (0.03–3% v/v) of plant oil was prepared in YPD broth medium in flasks. Tween 80 was included at a final concentration of 0.001% (v/v) to enhance oil solubility. Each flask was inoculated with 5 × 10⁵ cfu ml⁻¹ of the Candida strains. Flasks containing Tween 20 but lacking any plant oil were used as control. The flasks were incubated at 30 ± 2 °C, in an orbital shaking incubator (100 rpm) for 48 h. From each flask 5 μl of culture was inoculated on YPD plates and incubated at 30 ± 2 °C for 48 h. The plates were observed and MFCs were determined as the lowest concentration of the plant oil completely inhibiting growth of Candida strains.

3 Results

3.1 Screening of plant oils for anti-Candida activity

The isolates of C. albicans tested showed different sensitivity to the oils. However, the majority of the oils were found effective and showed considerable activity at very low concentrations. Twenty-five among the 38 oils tested caused a 1–30 mm zone of inhibition (ZOI). Seventeen oils caused a 10–20 mm ZOI. Six oils showed a 1–9 mm ZOI against all four strains tested. Jasmine and lavender oil failed to inhibit the growth of the Candida strains except CA IV. (Table 1). (13 oils failed to inhibit growth of the C. albicans strains at 5-μl doses.)

3.2 Determination of MIC of plant oils against C. albicans

MICs of the 23 effective oils and two widely prescribed drugs, Fluconazole and Amphotericin B, were determined against the four isolates of C. albicans. The oils exhibited concentration-dependent inhibition of growth. In a few cases, the strains tested showed different sensitivity but there was no considerable difference in MIC (Table 2).

All four isolates were found sensitive to Amphotericin B. A 2.5 μg ml⁻¹ concentration of Amphotericin B was sufficient to inhibit growth of strain CAI and strain CA IV (ATCC10231) completely. Strain CA II and CA III required 3 μg ml⁻¹ Amphotericin B for total growth inhibition (Table 2).

The response to Fluconazole was found to be divergent. Strain Ca III was the most sensitive and only 2 μg ml⁻¹ concentration of Fluconazole was enough for complete growth inhibition. Strains CA I and CA II required 5 μg ml⁻¹ of Fluconazole () for complete growth inhibition (Table 2). Strain CA IV was highly resistant to Fluconazole: 3000 μg ml⁻¹, i.e., 1500-fold the MIC of strain CA III, was required for a partial growth inhibition. Complete growth inhibition could not be achieved in this strain even at a very high concentration.

A 0.01% concentration of Cinnamon oil was enough to completely inhibit growth of all four isolates. Jasmine and Lavender oils caused only slight inhibition of growth and failed to inhibit growth completely. Seven oils inhibited growth of the C. albicans strains completely, at the concentration range of 0.03–0.15%. For 11 oils MIC ranged from 0.16% to 1.0% Five oils required a concentration of more than 1.0% (Tables 2 and 3).

3.3 Determination of MFC of plant oils against C. albicans

Minimum fungicidal concentration (MFC) is defined as the lowest concentration of oil resulting in the death of 99.9% of the inoculum. All the oils inhibiting growth showed fungicidal activity except Jasmine and Lavender oils. In general, it was observed that the fungicidal concentration was higher than MIC, except in a few cases like Peppermint, Geranium, Ocimum, Clove and Gingergrass oils, which caused the death of 99.9% of the inoculum at MIC. According to their MFC against ATCC10231, effective oils could be classified into three classes. The oils that caused fungicidal effect at 0.03–0.15% concentration are considered as the most effective (ME) ones. Oils requiring 0.15–1.0% concentration are included in the moderately effective group (MoE). Those found fungicidal at more than 1.0% concentration are considered as less effective (LE).

Seven oils were found to be most effective (ME) (Tables 2 and 3). Cinnamon oil was the best, having fungicidal effect at 0.03% concentration in all four isolates of C. albicans. Clove oil was fungicidal at 0.12% concentration and there was no difference between MIC and MFC. In the majority of the ME oils, a 2–4-fold of MIC, was required for fungicidal effect. The response of Candida isolates to some members of this group was divergent, i.e., of strain CA II in the case of Japanese mint oil and Gingergrass oil, CA III in the case of Lemongrass oil and CA IV in the case of Ginger grass oil (Table 2).

Peppermint and Tea tree oil were the best among the group of seven MoE members, causing fungicidal effect at 0.25% concentration (Table 3). Tulsi oil was fungicidal at 0.5% concentration, 1% was the MFC of Camphor, Lemon, Ocimum and Ylang-ylang oils. Sensitivity difference for a few members of this group was also found. Two-fold of MIC was required for these oils except for Peppermint and Ocimum oil (Table 2).

Nine oils were found to be less effective (Table 3). These oils required more than 1.0% to exert fungicidal effect. MFC of these oils ranged from 1.0% to 3% except in the case of Jasmine, Ginger and Juniper oil, which were not fungicidal up to the 3% concentration. CA II and CA III showed different sensitivity (Tables 2 and 3).

4 Discussion

Anti-Candida activity of some of the essential oils like Tea tree oil is well known [23,26,27]. Many of the preparations used against vaginal candidiasis caused by C. albicans contain Tea tree oil as an active component [23]. Several oils of plant origin have been suggested in alternative medicines against microbial infections including candidiasis and gingivitis [17,19–21,23,25,26,28,29]. However, azole antifungal agents and derivatives continue to dominate as drugs of choice against Candida infections, as topical applications or as oral drugs [1–3,10,15]. Except for Tea tree oil, which has a history of more than 80 years of use in Australia, not many herbal products have entered into the market [23,30]. Some products containing Rosemary oil are used against fungi causing dandruff [31,32]. In vitro anti-Candida activity of essential oils like Tea tree, Lemongrass, Citronella, Peppermint and Palmarosa have been reported [22,23,26,27]. We have examined the efficacy of 38 oils of plant origin for the in vitro anti-Candida activity against four isolates of C. albicans (Tables 1 and 2). At least 23 of the oils tested showed promise as effective agents. These oils could be classified according to their efficacy (Table 3). One of the isolates which was found to be resistant to Fluconazole was inhibited at very low concentration of all the effective oils of plant origin tested in this study (Table 2).

Disc diffusion assay is a standard method widely used for quick screening of natural products for antifungal activity [23]. We have screened plant oils using this very convenient assay method. This study indicates that caution is needed since different oils may have different diffusion rates on agar plates and this may contribute to variation in the inhibitory zones, leading to erroneous conclusions regarding their antifungal activity. For example, some of the oils which exhibited smaller inhibition zones compared to others were very effective against Candida strains in the NCCLS broth dilution assay which does not involve diffusion. For example, Cinnamon oil exhibited a ZOI of 24 mm in the disc diffusion assay, where Lemongrass oil showed 30 mm. But in the MFC assay Cinnamon oil (0.01%) was found better than Lemongrass oil (0.06%). Fungicidal activity is considered as a desirable quality for antifungal agents, since it could totally eliminate the fungus from tissues. Amphotericin B is fungicidal, is known to be very effective against human pathogenic fungi and is used as the drug of choice in systemic fungal infections, in spite of its severe side effects [2,3,5,7]. Fluconazole, the widely-prescribed antifungal agent against Candida infections, is fungistatic in nature and may require prolonged use [1,2,7].

It is encouraging to note that the majority of the oils used in this study were fungicidal at low concentrations. Not much information is available on the mode of action of the natural products inhibiting Candida growth. More Fluconazole/azole-resistant strains need to be included in future studies. The plant oils could find use as anti-Candida agents against azole-resistant strains. Most of the oils used in this study have a long history of use in food, confectionery and as components of perfume [18,32]. However, if they are to be considered in topical preparations a careful exploration of their probable irritating and other undesirable effects in humans need to be undertaken.

Acknowledgements

Authors are thankful to Dr. S.A. Suryawanshi, Hon'ble Vice Chancellor, SRTM University, Nanded (MS) India, for encouragement.

References