Development of kinetic model for testing antifungal effect of Thymus vulgaris L. and Cinnamomum cassia L. essential oils on Aspergillus flavus spores and application for optimization of synergistic effect

Published Date
15 July 2015, Vol.99:131–137, doi:10.1016/j.bej.2015.03.024

Author 

M. Pekmezovic ^a

K. Rajkovic ^b

A. Barac ^a

L. Senerović ^c

V. Arsic Arsenijevic ^a,,

aNational Reference Medical Mycology Laboratory, Institute of Microbiology and Immunology, Faculty of Medicine, University of Belgrade, dr Subotića 1, 11 000 Belgrade, Serbia

bHigh Chemical and Technological School for Professional Studies, Kosančićeva 36, 37 000 Kruševac, Serbia

cInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010 Belgrade, Serbia

Received 7 November 2014. Revised 4 March 2015. Accepted 25 March 2015. Available online 27 March 2015.

Highlights

• Evaluation of antifungal effect of T. vulgaris and C. cassia against A. flavus.
• •
  Application of logistic kinetic model to the experimental data for optimal ratio calculation.
• •
  Optimal mixture has the synergistic effect with lower MIC and MFC values.
• •
  Modeling of results can advance synergistic studies by predicting optimal mixture.

Abstract

The antifungal effect of essential oils (EOs) of Thymus vulgaris L. (EO_{T. vulgaris}) and Cinnamomum cassia L. (EO_{C. cassia}) against Aspergillus flavus spores was evaluated by determining minimum inhibitory concentration, minimum fungicidal concentrations and fungicidal kinetics. Kinetic model of fungicidal activity of individual EO_{T. vulgaris}and EO_{C. cassia} was developed and its parameters were used to make EO mixture with optimal ratio of individual EOs. Synergism and speed of fungicidal effect of EO mixtures against A. flavus spores were evaluated.

Kinetic model revealed optimal ratio EO_{T. vulgaris}: EO_{C. cassia} as 14:1 in mixture. Observed result was validated by comparing fungicidal effect of this mixture to commonly used ratio 1:1 and intermediate 7:1 EO mixtures. All three mixtures showed partial fungicidal synergism, but the time point of fungicidal effect was different. The fastest fungicidal effect was observed in 14:1 EO mixture (after 90 min), followed by 7:1 (110 min) and 1:1 (180 min). The difference in the speed of fungicidal effect could not be detected using standard microdilution susceptibility tests, which demonstrated the importance and usefulness of developed kinetic model for further investigations.

Abbreviations

ATCC, American Type Culture Collection

EO_C.cassia, Cinnamommum cassia essential oil

EOs, essential oils

EO_T.vulgaris, Thymus vulgaris essential oil

DMSO, dimethyl sulfoxide

FBS, fetal bovine serum

FICI, fraction inhibitory concentration index

IA, invasive aspergillosis

k, death constant

MFC, minimum fungicidal concentration

MIC, minimum inhibitory concentration

MRC5, human lung fibroblasts cell line

MRPD, mean relative percentage deviation

MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

p values, the level of significance

R, ²coeficient of determination

SDA, Sabouraud dextrose agar

t, time

y₀, initial percent of spores

y_m, maximum possible percent of reduced spores

y, _rpercent of reduced spores

y_v, percent of viable spores

F-values, the Fisher statistic

Keywords

• Antifungal effect
• Aspergillus flavus
• Cinnamomum cassia
• Kinetic model
• Synergism

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