Effect of Cinnamomum verum leaf essential oil on virulence factors of Candida species and determination of the in-vivo toxicity with Galleria mellonella model

Abstract

BACKGROUND Essential oils (EO) extracted from Cinnamomum verum has been used as an antimicrobial agents for centuries. The effects of C. verum leaf oil against virulence of microorganisms is not well studied yet. OBJECTIVES This study evaluates the effect of C. verum leaf oil against three virulence factors of Candida albicans, C. tropicalis and C. dubliniensis and its in-vivo toxicity. METHODS Chemical composition of EO was determined using gas chromatography-mass spectrometry (GC-MS). Minimum inhibitory concentration (MIC) was determined using clinical and laboratory standards institute (CLSI) M27-A3 broth microdilution. Effect of EO on initial adhesion was quantified using XTT assay after allowing Candida cells to adhere to the polystyrene surface for 2 h. Biofilm formation of Candida in the presence of EO was quantified using XTT viability assay. Efficacy on reduction of germ tube formation was evaluated using standard protocol. Visualisation of biofilm formation and progression under the EO treatment were done using scanning electron microscope (SEM) and Time lapses microscope respectively. In-vivo toxicity of EO was determined using Galleria mellonella larvae. Chlorhexidine digluconate: positive control. RESULTS Eugenol was the main compound of EO. MIC was 1.0 mg/mL. 50% reduction in initial adhesion was achieved by C. albicans, C. tropicalis and C. dubliniensis with 1.0, > 2.0 and 0.34 mg/mL respectively. 0.5 and 1.0 mg/mL significantly inhibit the germ tube formation. MBIC 50 for forming biofilms were ≤ 0.35 mg/mL. 1.0 mg/mL prevent biofilm progression of Candida. SEM images exhibited cell wall damages, cellular shrinkages and decreased hyphal formation. No lethal effect was noted with in-vivo experiment model at any concentration tested. CONCLUSION C. verum leaf oil acts against virulence factors of Candida and does not show any toxicity.

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