Rahimifard N, Shoeibi SH, Pakzad SR, Sabzevari O, Ajdary S, Haghighi S, Bagheri F, Sabzikaran M. Antifungal Activity of the Native Essential Oil of Zataria Multiflora on Candida Albicans, Aspergillus Niger and Aspergillus Flavus from Iran. Biomed. Pharmacol. J.2008;1(2)

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Manuscript received on :June 10, 2008
Manuscript accepted on :August 19, 2008
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N. Rahimifard^1-2*, SH. Shoeibi^1,2, SR. Pakzad^1,2, O. Sabzevari^³,  S. Ajdary⁴, S. Haghighi⁵, F. Bagheri⁵ and M. Sabzikaran⁵

^¹Food and Drug Laboratory Research center (FDLRC), Tehran (Iran).

^²Food and Drug Control Laboratories (FDCLs), Ministry of Health (MOH), Tehran (Iran).

³Pharmacy faculty, Tehran University of Medical Sciences(TUMS), Tehran (Iran).

⁴Immunology Department, Pasteur Institute, Tehran (Iran).

⁵Microbiology Department, Pharmaceutical sciences branch, Islamic Azad University, Tehran (Iran).

Corresponding Author E-mail:, rahimifn@yahoo.com

Abstract

The composition of the native essential oil of Zataria multiflora and its antifungal activity on Candida albicans, Aspergillus niger and Aspergillus flavus fungal strains were studied in Iran. Essential oil from the aerial parts of the plant was obtained by hydrodistillation and analysed by GC and GC-MS. The oil showed high contents of p-Cymene, carvacrol and thymol. The MIC was used to evaluate the antifungal activity against Candida albicans ATCC 10231, Aspergillus niger ATCC 9642 and Aspergillus flavus ATCC 9643. Antifungal activity was evaluated for the essential oil and simultaneously for Amphotricin B. Results showed that Zataria multiflora essential oil exhibited a significant activity against fungi, and its MIC on Candida albicans, Aspergillus niger and Aspergillus flavus were respectively 0.125, 0.125 and 0.125 µg ml-1 (ppm).The present study indicates that Zataria multiflora essential oil has considerable antifungal activity, deserving further investigation for clinical applications.

Keywords

Zataria multiflora;  MIC(minimal inhibitory concentration); Antifungal

Introduction

Fungal infections have been increasing in recent years due to a growing number of high-risk patients, particularly immunocompromised hosts. Candida is the third- or fourth-most-common isolate innosocomial bloodstream infections in the USA. In addition, The mortalityrate due to invasive aspergillosis increased by 357 % between1980 and 1997 in the USA .In spite of the introduction of new antifungal drugs, they arelimited in number. The increase of fungal resistance to classicaldrugs, the treatment costs, and the fact that most availableantifungal drugs have only fungistatic activity, justify thesearch for new strategies (Eugenia pinto, 2006).

Aromatic plants have been widely used in folk medicine. It isknown that most of their properties are due to their volatileoils. Essential oils from many plants are known to possess antifungalactivity, but only limited informationexists about activity toward human fungal pathogens. They havebeen empirically used as antimicrobial agents, but the mechanismsof action are still unknown.

Some essential oils show an important antifungal activity against yeasts, dermatophyte fungi and Aspergillus strains, which could predict therapeutic benefits, mainly for diseases with mucosal, cutaneous and respiratory tract involvement.  (Pina-Vaz et al., 2004; Salgueiro et al., 2003, 2004),

The objective of our present research was to evaluate the antifungal activity and investigate the mechanism of action of Zataria multiflora  essential oil from Iran.

Method

Essential oil analysis: Essential oil from the aerial parts of the plant was obtained by hydrodistillation and analysed by GC and GC-MS. Gas chromatography (GC) and GC–mass spectrometry (MS) analysis of essential oil of thyme were performed. The oil showed high contents of p-cymene, carvacrol and thymol.

Plant material and chemicals

Aerial parts of the plants were collected at the flowering stage from Tokestan, 11^th Km. Gorgan-Mashhad Road,Gorgan, Iran.

Thymol (99.5 %) was purchased from DBH, and carvacrol, -terpinene and p-cymene (all 99.5 %) from Fluka.

Essential oil analysis

Essential oil was isolated by water distillation for 3 h from air-dried material, using a Clevenger-type apparatus, according to the procedure described in the European Pharmacopoeia (Council of Europe, 1997).

Gas chromatography (GC–FID)

Gas chromatography analysis was performed on a Hewlett-Packard Model 5890 Series II gas chromatograph equipped with flame ionization detector and capillary column HP-101 (Methyl silicone fluid), 25 m × 0·2 mm i.d., coating thickness 0·2 μm. Chromatographic conditions were as follows: helium as carrier gas at 1·0 ml min⁻¹; injector and detector temperatures, 250°C and 300°C. Oven temperature was isothermal at 70°C for 2 min, then increased to 200°C, at a rate of 3°C min⁻¹ and held isothermal for 15 min. Volume injected 1 μl. Split ratio 1 : 50.

Gas chromatography–mass spectrometry

Essential oil was also analysed by Hewlett Packard GC–MS (model 5890 series II) with mass selective detector (model 5971A). Two columns of different polarity were used: an HP-101 column (Methyl silicone fluid, Hewlett Packard; 25 m × 0·2 mm i.d., film thickness 0·2 μm) and an HP-20M column (Carbowax 20M, Hewlett Packard; 50 m × 0·2 mm i.d., film thickness 0·2 μm). Oven temperature was programmed as follows: isothermal at 70°C for 4 min, then increased to 180°C, at a rate of 4°C min⁻¹ and subsequently held isothermal for 15 min (for HP-20M column); isothermal at 70°C for 2 min, then increased to 200°C, at a rate of 3°C min⁻¹ and held isothermal for 15 min (for HP-101 column). Carrier gas was helium, flow rate: 1 ml min⁻¹; injector temperature: 250°C; volume injected: 1 μl; split ratio: 1 : 50. MS conditions: ionization voltage: 70 eV; ion source temperature: 280°C; mass range: 30–300 mass units.

Qualitative and quantitative determination

The individual peaks were identified by comparison of their retention indices to those of authentic samples, as well as by comparing their mass spectra with the Wiley 6·0 library (Wiley, New York, NY, USA) and NIST98 (National Institute of Standards and Technology, Gaithersburg, MD, USA) mass spectral database and literature (Adams 1995).

The percentage composition of the samples was computed from the GC peak areas by using the normalization method (without correction factors). Quantitative results are mean of data derived from duplicate GC-FID analyses.

Isolation and detection of Fungi

The antifungal activity of Zataria multiflora  essential oil was evaluated against Candid albicans ATCC 10231 , Aspergillus niger ATCC 9642 , and Aspergillus flavus ATCC 9643.The fungal isolates were identified by standardmicrobiology methods and stored in Sabouraud dextrose brothwith glycerol at –70 °C.

Fungi were plated on Sabouraud 2% (w/v) glucose agar (SGA),  and incubated at 25 ± 2°C for the 5–7 days.

For the antifungal activity testing, Zataria multiflora  essential oil was dissolved in 96% (v/v) ethanol and then diluted with 30% (v/v) ethanol in distilled water with 0·1% (w/v) Tween 80. Final concentrations of the essential oil was 2% (w/v) ..

Antifungal activity testing by dilution method

Minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) were determined by using the serial broth dilution method described by Pepeljnjak et al. (2003). Serial twofold dilutions in DMSO, ranging from 0.02 to 20 µl ml^–1, were tested for essential oil. In addition, the reference antifungal compounds, fluconazole (Pfizer) for Candida albicans or amphotericin B (Sigma) for Aspergillus, were used as standard antifungal drugs. Twofold serial dilutions ranging from 0.25 to 128 µg ml^–1 for fluconazole and 0.016 to 16 µg ml^–1 for amphotericin B were used. Quality control determinations of the MICs of fluconazole and amphotericin B were performed by testing C. parapsilosis ATCC 90018 and C. krusei ATCC 6258. The results obtained were withinthe recommended limits.

MIC is defined as the lowest concentration of extract or essential oil that allows no more than 20% growth of the fungus, visualized as a reduced number of colonies after removing the loop with approx. 10 μl of each dilution, and then inoculated on SGA and incubated at 25 ± 2°C for 7 days. MFC is defined as the lowest concentration of essential oil that completely inhibited the growth of fungi. These experiments performed in duplicate were repeated independentlythree times and yielded essentially the same results. A rangeof values is presented where different results were obtained.Two growth controls, RPMI medium and RPMI with 2.0 % (v/v) DMSO,were included for each strain.

Statistics: The data obtained as MIC and MFC of  essential oil, expressed in μg ml⁻¹, were statistically analysed by using the Wilcoxon matched pairs test. The level of P < 0·05 was considered statistically significant.

Results and discussion

The oil was obtained from air-dried plant material in a yield of 1.8 % (v/w).

Evaluation of MIC showed that the oil was active against all the tested strains (Table 1). Zataria multiflora  essential oil exhibited significant antifungal activity. MIC value 0.125 µl ml^–1 against Aspergillus strains and Candida. It is difficult to attribute the activity of a complex mixture to particular constituents. Nevertheless, it is reasonable to speculate that the activity of this oil can be related to the presence of p-cymene, carvacrol and thymol. The importance of the phenolic hydroxyl groups for the antimicrobial activity of the monoterpenoids has previously been reported (Adam et al., 1998; Aligiannis et al., 2001; Dorman & Deans, 2000; Nostro et al., 2004; Sivropoulou et al., 1996).

Table 1: Antimicrobial activity (MIC) and (MFC) of the essential oil of the Zataria multiflora  for Candida albicans, Aspergillus niger and Aspergillus flavus.

Results were obtained from three independent experiments performed in duplicate.

In conclusion, the findings of the present study indicate that Zataria multiflora  essential oil has potential as a topical antifungal agent against fungi that are pathogenic to humans. This essential oil is a broad-spectrum agent that inhibites Aspergillus and Candida species,

Given the results described above, particularly the possiblemechanisms of action, which might induce side-effects in humans,these antifungals require further investigation.

The results presented should stimulate studies on toxicity,improved formulations and the determination of optimal concentrationsfor clinical applications, as well as comparative studies alongsidecurrently used drugs of the therapeutic efficacy of essentialoils to control infections.

References

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   Aligiannis, N., Kalpoutzakis, E., Mitaku, S. & Chinou, I. B. (2001). Composition and antimicrobial activity of the essential oils of two Origanum species. J Agric Food Chem 38, 4168–4170.[CrossRef].
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