Curcumin as a promising antifungal of clinical interest

Abstract

Objectives

The antifungal activity of curcumin was evaluated against 23 fungi strains and its in vitro inhibitory effect on the adhesion of Candida species to human buccal epithelial cells (BEC) was also investigated.

Methods

The antifungal susceptibility was evaluated by broth microdilution assay following the CLSI (formerly the NCCLS) guidelines. The inhibitory effect of curcumin on the cell adhesion was performed with Candida species and BEC.

Results

Paracoccidioides brasiliensis isolates were the most susceptible to curcumin while the growth of Aspergillus isolates was not affected. Curcumin was much more efficient than fluconazole in inhibiting the adhesion of Candida species to BEC, particularly those strains isolated from the buccal mucosa of AIDS patients.

Conclusions

The lack of antifungal compounds with reduced side effects highlights the importance of studying natural products for this purpose. Curcumin was a more potent antifungal than fluconazole against P. brasiliensis, the causal agent of the neglected disease paracoccidioidomycosis. Curcumin dramatically inhibited the adhesion of Candida species isolated from AIDS patients to BEC, demonstrating that curcumin is a promising lead compound that warrants further investigation into its therapeutical use in immunocompromised patients.

Introduction

Fungal infections have increased significantly, contributing to the cause of morbidity and mortality. The increase in antimicrobial resistance and populations of patients at some risk, in conjunction with the restricted number of commercially available antifungal drugs that still present many side effects, are the cause for this problem.^1,2 These limitations emphasize the need to develop new and more effective antifungal agents. Natural products are attractive prototypes for this purpose due to their broad spectrum of biological activities.³

Curcumin is a yellow–orange polyphenol compound produced by the rhizome of Curcuma longa plants, which is widely used as a spice in Asian cooking. This compound has been shown to possess a wide range of pharmacological activities,⁴ where antifungal activity was assessed by experiments done with crude extracts of C. longa.

This work focused on the evaluation of curcumin antifungal activity against 23 fungi strains of clinical interest as well as its ability to inhibit the adhesion of Candida spp. to human buccal epithelial cells (BEC).

Materials and methods

All chemicals used in this study were obtained from Sigma, unless otherwise stated.

Twenty-three fungi strains, which included Candida spp., Cryptococcus neoformans, Sporothrix schenckii, Paracoccidioides brasiliensis and Aspergillus spp., were the subject of this study (Table 1). Dimorphic fungi (S. schenckii and P. brasiliensis) were employed as the yeast form.

Analysis of in vitro susceptibility was performed by broth microdilution assay following the CLSI (formerly the NCCLS) guidelines for yeasts and filamentous fungi.^5,6 Curcumin was tested in the range of 0.5–256 mg/L and fluconazole (0.06–64 mg/L; Pfizer, São Paulo, Brazil) was included as a positive control. The MIC values correspond to the lowest concentrations that did not allow for the detection of any visual fungal growth.

The adhesion assay was carried out according to Lyon and de Resende.⁷ Briefly, Candida spp. isolates were exposed to curcumin at its MIC value for 1 h, and were then incubated with BEC for another 1 h. Assay with Candida parapsilosis was carried out with 256 mg/L curcumin since its MIC value was not determined. The number of yeast cells that adhered to BEC was quantified by light microscopy (×400 magnification) from 50 randomly chosen BEC.

Results and discussion

The MICs of curcumin that completely abolished the growth of fungi strains are shown in Table 1. P. brasiliensis isolates were the most susceptible to curcumin. Curcumin was 32-fold more potent than fluconazole in the inhibition of P. brasiliensis MG05 growth. Fluconazole was also 4-fold less potent than curcumin in inhibiting the growth of P. brasiliensis Pb18 and B339. The curcumin effect on P. brasiliensis 17 was roughly the same as that of fluconazole, while P. brasiliensis Pb01 and 608 strains were more susceptible to fluconazole (Table 1).

Even though the greatest antifungal activity of curcumin was against P. brasiliensis isolates, promising results were also achieved for this compound against other fungi species. For instance, curcumin was twice as potent as fluconazole in the growth inhibition of the opportunistic yeast S. schenckii (Table 1). S. schenckii promotes infections of hosts with pre-disposing conditions, which includes alcoholics, diabetics, transplant recipients, and patients with haematological malignancies, chronic obstructive pulmonary disease, long-term treatment with corticosteroids and AIDS.⁸

Curcumin (32 mg/L) was able to inhibit the growth of C. neoformans and the clinical isolates of Candida dubliniensis Cd22 and Cd28. Non-albicans Candida species are emerging as colonizers and pathogens causing nosocomial fungal bloodstream infections.⁹Candida albicans was the most susceptible to curcumin among the Candida species studied (Table 1). The growth of the remaining fungi isolates was only affected by curcumin at concentrations ≥256 mg/L.

Curcumin was used to further explore its ability to prevent the adhesion of Candida species to BEC. These experiments were performed with curcumin at its MIC values. Curcumin was able to inhibit the adhesion to BEC of all the Candida species studied, being more potent than the commercial antifungal fluconazole (Figure 1a). C. dubliniensis Cd22 and Cd28 had the most significant reduction in adhesion to BEC (63% and 74%, respectively) in the presence of curcumin. The curcumin effect on all clinical isolates was 2.5–6.0-fold higher than that of fluconazole. Figure 1(b–d) shows the representative images of C. dubliniensis Cd28 adhesion to BEC. These strains were isolated from the oral cavities of AIDS patients at the Santa Maria University Hospital, RS, Brazil. Other C. dubliniensis strains were reported to be recovered from HIV-infected and AIDS patients under fluconazole treatment for oropharyngeal candidiasis, suggesting that this commercial antifungal was either selecting resistant Candida isolates or inducing cell adhesion.¹⁰ The adhesion of microorganisms to host mucosal surfaces is a prerequisite for colonization and infection.⁷ Our results indicate that curcumin is a promising lead compound for the design of new antifungal agents capable of inhibiting the adhesion of C. dubliniensis. The adhesion of Candida tropicalis to BEC was inhibited by 55% in the presence of curcumin while the inhibition caused by fluconazole accounted for only 13%. Curcumin was 2.5-fold more potent than fluconazole at inhibiting the adhesion of C. albicans or C. parapsilosis to BEC (Figure 1a).

To the best of our knowledge, this study reports for the first time the effect of curcumin on the growth and cell adhesion to BEC of fungi of clinical interest. Our in vitro results highlight the potential of curcumin as an effective antifungal against different P. brasiliensis strains, being much more potent than the commercial antifungal fluconazole. This natural product was also much more efficient than fluconazole in inhibiting the adhesion of Candida species to BEC, particularly those strains isolated from the buccal mucosa of AIDS patients.

Synthesis of curcumin analogues and evaluation of their antifungal activities are in progress in our laboratory.

Funding

This work was funded by FAPEMIG, CNPq and CAPES.

Transparency declarations

None to declare.

Acknowledgements

We thank Dr Sydney Alves (Hospital Universitário Santa Maria, RS, Brazil) for kindly providing the isolates of C. dubliniensis.

References