In vitro evaluation of antifungal activity of monolaurin against Candida albicans biofilms

Abstract

Monolaurin (also known as glycerol monolaurate) is a natural compound found in coconut oil and is known for its protective biological activities as an antimicrobial agent. The nature of oral candidiasis and the increased antifungal resistance demand the search for novel antifungal therapeutic agents. In this study, we examine the antifungal activity of monolaurin against Candida albicans biofilms (strain ATCC:SC5314/MYA2876) in vitro and investigate whether monolaurin can alter gene expression of host inflammatory cytokines, IL-1α and IL-1β. In a co-culture model, oral fibroblast cells were cultured simultaneously with C. albicans for 24 hrs followed by the exposure to treatments of monolaurin (3.9-2,500 µM), positive control fluconazole (32.2 µM), and vehicle control group (1% ethanol), which was a model used to evaluate the cytotoxicity of monolaurin on fibroblasts as well as to analyze morphological characteristics of biofilms through fluorescence microscopy. In addition, the co-culture model was used for RNA extraction of oral fibroblasts to assess gene expression of host inflammatory cytokines, using quantitative real-time PCR. Our results showed the MIC and MFC of monolaurin were in the range 62.5-125 µM and 125-250 µM, respectively. Biofilm antifungal assay showed significant reduction in Log (CFU/ml) of biofilms treated with 1,250 and 2,500 µM of 1-monolaurin when compared to the control groups . There was also a significant down-regulation of IL-1α and IL-1β in the co-culture treated with monolaurin. It can be concluded that monolaurin has a potential antifungal activity against C. albicans and can modulate the pro-inflammatory response of the host.

Keywords: Antimicrobial agent; Biofilms; Candida albicans; Host inflammatory response; In vitro; MIC/MFC; Monolaurin; Oral candidiasis; Proteolytic enzymes; Virulence factors.

Figure 1. Fungal load of biofilms treated with monolaurin and proteolytic enzymes activity level.

Fungal load of 1-monolaurin treated biofilms. *p < 0.05.

Figure 2. Proteolytic enzymes activity of C. albicans after treatment with monolaurin.

Secreted proteolytic enzymes activity of Candida albicans after treatment with 1-monolaurin; of (A) Proteinase, and (B) Phospholipase enzymes.

Figure 3. Cytotoxic effects of 1-monolaurinon on oral fibroblast cells.

Note that monolaurin is recognized as GRAS (Generally Recognized as Safe), as a food additive by the FDA (Food and Drug Administration), with topical doses of up to 100 mg/ml (Title 21, Code of Federal Regulations, Part 184).

Figure 4. Co-culture fluorescence microscopy of 1-monolaurin.

(A) vehicle control (1% ethanol), (B) positive control (fluconazole), and (C) 1-monolaurin (125 µM); stained with calcofluor white and cytotoxicity assay kit for animal Live/Dead cells (Blue: Candida albicans, Green: live fibroblast cells, and Red: dead fibroblast cells). Scale bar set at 1,000 um at 4×magnification power.

Figure 5. Real-time quantitative gene expression of oral fibroblast cells infected by C. albicans after 1-monolaurin treatments at 62.5 µM and 125 µM concentrations in comparison to vehicle control and positive control fluconazole (32.2 µM).

(A) SAP-1 (B) PLB-1 (C) IL-1α (D) IL-1β (E) IL-8.