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Table of Contents   
ORIGINAL RESEARCH  
Year : 2020  |  Volume : 31  |  Issue : 1  |  Page : 145-148
Variable antifungal activity of curcumin against planktonic and biofilm phase of different candida species


1 Department of Oral Medicine and Radiology, Coorg Institute of Dental Sciences, Virajpet, Karnataka, India
2 Department of Orthodontics, Coorg Institute of Dental Sciences, Virajpet, Karnataka, India
3 Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences, Virajpet, Karnataka, India
4 Department of 3Microbiology, Coorg Institute of Dental Sciences, Virajpet, Karnataka, India
5 Department of School of Dentistry, University of Queensland, Brisbane, Australia

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Date of Web Publication02-Apr-2020
 

   Abstract 


Objective: To evaluate the in vitro antifungal activity of curcumin against 2 strains of Candida albicans (ATCC 90028 and a clinical isolate – JY strain) and 1 isolate each of 3 nonalbicans – Candida species [Candida parapsilosis (ATCC 22019), C. glabrata (ATCC 90030), and C. dublieniensis (MYA 646)]. Materials and Methods: Planktonic MIC of the 4 Candida species was determined using micro broth dilution assay according to CLSI M27-A3 criteria. The biofilm development and sensitivity assay were performed with the 2 C. albicans strains. Results: Curcumin at high concentrations (0.1–2 mg/mL) was effective in inhibiting planktonic organisms of all the 5 tested Candida strains. The planktonic phase and the biofilm phase of C. albicans ATCC 90028 exhibited similar MIC values for curcumin (0.5 mg/mL). Both curcumin and fluconazole were ineffective against the mature biofilms of JY strain. Conclusion: Our results reported here for the first time, in particular for the biofilm state of C. albicans, imply that curcumin a natural product could be used as a therapeutic alternative to conventional antifungals although further investigations are required to evaluate its potential.

Keywords: Antifungal agents, candida, curcumin, fluconazole

How to cite this article:
Narayanan VS, Muddaiah S, Shashidara R, Sudheendra U S, Deepthi N C, Samaranayake L. Variable antifungal activity of curcumin against planktonic and biofilm phase of different candida species. Indian J Dent Res 2020;31:145-8

How to cite this URL:
Narayanan VS, Muddaiah S, Shashidara R, Sudheendra U S, Deepthi N C, Samaranayake L. Variable antifungal activity of curcumin against planktonic and biofilm phase of different candida species. Indian J Dent Res [serial online] 2020 [cited 2021 May 11];31:145-8. Available from: https://www.ijdr.in/text.asp?2020/31/1/145/281817



   Introduction Top


There has been a significant rise in the worldwide incidence of both oral and systemic candidiasis because of a multiplicity of reasons including the high prevalence of immunosuppressed patients on various regimes such as those on cytotoxic therapy, radiation and transplant recipients, and diseases such as HIV. The global emergence of antifungal-resistant Candida strains is a major challenge in this context.[1],[2] The spectrum of Candida species is also shifting with more frequent nonalbicans agents causing infection. For instance, C. glabrata and C. dubliniensis are detected in >20% of isolates of oral candidiasis in both HIV-positive and HIV-negative patients.[3],[4] Such multispecies biofilms of Candida are particularly resistant to conventional antifungals. Hence, antifungal agents with novel mechanisms of action represent an attractive alternative for the management of infections caused by resistant fungi.[5],[6],[7],[8]

Turmeric (Curcuma longa L), a coloring spice, has been used for centuries for the cure of a variety of ailments in Indian and Chinese medicine. Curcumin constitutes the active ingredient of Curcuma longa L. The therapeutic effects of this polyphenolic compound have been extensively researched in the past few decades.[9],[10] Several studies have evaluated the antibacterial, antiviral, antimalarial, and antifungal properties of curcumin and it has been shown to be extremely safe even at a very high dose in various animal and human trials. Some investigators have used curcumin in combination with conventional antifungals.[11] However, the anticandidal activity of curcumin, particularly against nonalbicans species has received relatively little attention.[12],[13]

Therefore, in this study, we investigated the anticandidal activity of curcumin against 2 strains of C.albicans (1 ATCC strain and 1 clinical strain) and 3 ATCC strains of nonalbicans species of Candida, namely C. parapsilosis (ATCC 22019), C. glabrata (ATCC 90030), and C. dubliniensis (MYA 646). We hypothesized that different species of Candida exhibit varying sensitivity to curcumin and the degree of sensitivity depends upon the planktonic or biofilm phenotype.


   Materials and Methods Top


This study was conducted as a collaborative project between the Faculty of Dentistry, Hong Kong, China and Coorg Institute of Dental Sciences, India. All Candida strains used in the study were from the archival collection of Oral Bio-Sciences Laboratories of the Faculty of Dentistry, The University of Hong Kong, China.

Candida strains

A type culture strain of C. albicans (ATCC 90028) and a clinical isolate from a patient with oral candidiasis from Hong Kong (JY strain), and 3 ATCC strains of nonalbicans species: C. parapsilosis (ATCC 22019), C. glabrata (ATCC 90030), and C. dubliniensis (MYA 646) were used in the study. The identity of all the strains was reconfirmed before the studies by using the commercially available APILAB Plus (bioMérieux) system.

All the strains were inoculated onto Sabourauds dextrose agar and incubated for 18 h at 37°C. The resultant yeast cultures were harvested, suspended in yeast nitrogen broth (YNB) with 50 m mol/L glucose and grown for 18 h in a rotary shaker. The overnight growth was harvested by centrifugation, washed with phosphate buffered saline (PBS 0.1 M, pH 7.2), and final yeast suspensions were prepared to a concentration of 1 × 106 cells/mL (0.5 MacFarland Standard Units) through spectroscopy.

Curcumin and fluconazole

Curcumin (C-7727), fluconazole, and other chemicals used in the study were purchased from Sigma Aldrich Chemicals Pvt. Limited, Bangalore, India. Curcumin was diluted in NaOH (0.5M) and PBS (pH 7.2) and fluconazole was diluted using YNB to obtain the test concentrations. The solvent solutions were used as the negative control and fluconazole was used as positive control. These were prepared as per the previously published protocol of ours.[5]

Microbroth dilution assay

Determination of planktonic MIC

Planktonic MIC was determined according to CLSI M27-A3 criteria.[14] 100 μL each of the standard cell suspensions of Candida strains and drugs at different concentrations (in triplicates) were added to 96-well polystyrene microtitre plates (Orange Scientific, Belgium) and incubated at 37°C for 48 h. The optical density of the wells was measured at 492 nm with the microplate reader. The MIC was defined as the lowest concentration of the drug at which there was 90% growth inhibition relative to the controls and the values obtained thereof were statistically analyzed.[11] The optical density of curcumin, a colored solution, was also factored in the calculation.

Determination of biofilm MIC

The biofilm assay was performed on 2 Candida strains – C. albicans (ATCC 90028) and C.albicans (JY strain) – using a previously described method of ours.[15]

The yeast strains were subcultured on SDA for 24 h and the resultant growth harvested, washed in PBS, at 4000 rpm for 10 min. The resultant pellets were resuspended in YNB to yield a 107 cfu/mL yeast suspension. A total 2 mL of the suspension was then transferred into each well of a 24-well plate. The multiwell plates were incubated in a shaker incubator for 1.5 h at 37°C. The YNB and glucose medium were replaced with fresh sterile medium (2 mL/well). Then the plates were incubated at 37°C in a shaker incubator for 4 days for biofilm growth. The spent growth medium in each well was replenished every 24 h.

On day 4, the mature biofilms were gently rinsed with PBS, exposed to different concentrations of 2 mL of either curcumin or fluconazole, prepared as per above, and incubated at 37°C for 24 h. The cells from each well were harvested using an Eppendorf tube, pelleted at 13,000 rpm for 10 min, and washed with PBS twice to remove all curcumin or fluconazole traces attached to the biofilms. The pellets were resuspended in 1 mL sterile PBS, mixed well, and serial dilutions of each biofilm sample were quantified by spiral plating. The seeded plates were incubated for 48 h and cfu/mL were quantified to obtain the MIC[15]

All experiments conducted in triplicate were repeated on 3 different occasions.

Statistical analysis

ANOVA and post hoc analysis were used to compare the control with the drugs. Statistical significance of the data was analyzed using the SPSS software package (SPSS version 17.0). P value <0.05 was considered to indicate statistical significance.


   Results Top


The planktonic MIC of curcumin against C. albicans ATCC 90028was 0.5 mg/mL. However, a 4-fold (2 mg/mL) higher concentration of curcumin was required to inhibit the planktonic phase of the clinical strain (JY strain). Fluconazole inhibited the planktonic ATCC 90028 and the JY strain at 50 and 100 μg/mL, respectively.

The biofilm MIC of curcumin for C. albicans ATCC 90028 was found to be similar to that of the planktonic phase cultures at 0.5 mg/mL. We also noted a dose–response effect of curcumin as the cfu/mL yield decreased as the concentration of curcumin increased. Curcumin was ineffective against mature biofilms of JY strain of C. albicans. Fluconazole did not inhibit biofilms of either of the C. albicans strains even at concentrations 4 times that of planktonic MICs.

Curcumin displayed anti-candidal activity against the planktonic phase of all 3 tested nonalbicans strains, C. parapsilosis (ATCC 22019), C. glabrata (ATCC 90030), and C. dubliniensis (MYA 646) at concentrations of 0.4, 0.5, and 0.1 mg/mL, respectively. These organisms were inhibited by fluconazole at much lower concentrations of 5, 25, and 5 μg/mL, respectively.


   Discussion Top


There is a dire need for the development of newer, safer antifungals but the antifungal activity of curcumin has received little attention to date in the literature. A few early studies have indicated the potential of curcumin as a promising antifungal.[12],[16] Our studies reconfirm the latter findings, but the required dosage to inhibit the yeasts was considerably higher than that of the positive control drug fluconazole. In translational terms, if curcumin were to be used as an antifungal, then the dosage may not be an impediment as it has been found to be tolerable even at high dosages. Children with inflammatory bowel disease tolerated doses up to 2 g twice daily in a dose titration study.[17]

Interestingly, we noted that the MIC of the planktonic phase of the clinical strain (JY strain) for curcumin was 4-fold (2 mg/mL) higher than the ATCC strain. A similar 2-fold, relative difference in MIC values was noted between the clinical and the wild-type (JY) strain for fluconazole. Such relative increased resistance of freshly isolated, wild-type Candida strains is well documented and this is thought to be because of the suppression of the virulent attributes of the yeast because of repeated subculture of the laboratory strains.[18]

Candida strains are more frequently found in their natural habitat as biofilms which are organized communities of organisms that are attached to a surface and encased in a matrix of exopolymeric material. Biofilm formation is associated with increased resistance to conventional antifungal therapy. Among the various fungal species, C. albicans is most commonly associated with biofilms.[19] Hence, in this study the antifungal activity of curcumin against biofilms of 2 strains of C. albicans, ATCC 90028, and a clinical isolate (JY strain which is a good biofilm former) were tested. We were unable to demonstrate a significant difference in the MICs in curcumin between the planktonic and the biofilm phase of the single C. albicans strain (ATCC 90028) we tested. As there are no other reports in the literature comparing the biofilm and planktonic antifungal activity for curcumin further studies with a larger number of strains are warranted to refute or confirm our findings.

We noted in our studies that the growth of nonalbicans Candida species including C. parapsilosis ATCC 22019 and C. glabrata ATCC 90030 was only affected by curcumin at concentrations >0.4 mg/mL. Martin et al. (2009) have reported concentrations of curcumin >256 mg/L that were effective in suppressing the growth of the foregoing Candida species.

Also, they have reported lower concentrations of curcumin (64 mg/L) inhibiting C. albicans. Variations in methodology and strains of Candida could probably account for the disparity in MIC values between these studies.

In a recent study evaluating the antifungal activity, curcumin demonstrated the potent antibiofilm activity of curcumin against clinical and laboratory isolates of C. albicans which are resistant to commonly used antifungals. Curcumin inhibited adhesion of the organism and biofilm growth. Also, curcumin down-regulated the genes involved in adhesion and hyphae formation – adhesin als3 filamentation-associated gene hwp1. However, it did not disrupt the biofilm or directly affect the structure of the yeast cells.[20]

The antifungal activity of curcumin is thought to be a consequence of a multitude of proposed mechanisms. Curcumin has a diverse range of molecular targets related to the transcription factors, growth factors and their receptors,[21] depletion of ergosterol, accumulation of ergosterol biosynthetic precursors, and generation of reactive oxygen species, which eventually results in cell death.[22]

Although curcumin is less potent than conventional antifungals such as fluconazole, its low toxicity even at high doses makes it a possible alternative to the latter. A few recent studies have demonstrated that curcumin when given in combination is synergistic to azoles and polyenes.[10],[11] In phase I trial, no toxicity was observed after daily administration of up to 8000 mg/day of curcumin for 3 months in patients with different types of cancer and oral leukoplakia.[23] In practical terms, it is known that peak serum concentrations of curcumin are reached 20–30 minutes after administration, and the polyphenol totally cleared from the circulation within 2 hours. However, a longer lasting effect of curcumin could possibly be achieved by encapsulation of the polyphenol leading to slow and constant release. This is an avenue worthy of consideration by future workers in order to increase its bioavailability and decrease the dosage and frequency of administration.


   Conclusion Top


To conclude, our data taken together with those of others indicate that curcumin could be an exciting alternate option or an adjunct for the management of candidiasis. Future research with a large number of strains forms different Candida species are needed to confirm our findings, and also to elucidate the mechanisms underlying its antifungal activity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Cheng AL, Hsu CH, Lin JK, Hsu MM, Ho YF, Shen TS, et al. Phase I clinical trial of curcumin, a chemopreventive agent, in patients with high-risk or pre-malignant lesions. Anticancer Res 2001;21:2895-900.  Back to cited text no. 23
    

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Correspondence Address:
Veena S Narayanan
Professor and Head, Department of Oral Medicine and Radiology, Coorg Institute of Dental Sciences, Kanjithanda Kushalappa Campus, Maggula, Virajpet - 571 218, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijdr.IJDR_521_17

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