156
International Journal of Essential Oil Therapeutics (2008) 2, 156-157
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International Journal of
Essential Oil Therapeutics
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EORC
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Letter to the Editor In vitro activity of essential oil of Myrtus communis L. against Candida albicans Sir, We recently investigated the antimycotic properties of the essential oil of Myrtus communis L. against different strains of Candida albicans. C. albicans is the most frequently isolated human fungal pathogen. It is known to cause a wide spectrum of mycoses, ranging from deep tissue infections to superficial infections of the skin and mucosa [1]. The fungus typically infects these sites by overgrowth or haematogenous spread from a colonized site within the body. The azoles remain among the most common antifungal drugs, but their intensive clinical use for both therapy and prophylaxis has favoured the emergence of resistant strains. The increase in antifungal drug resistance may be associated with increase of virulence in drug-resistant strains [2]. The threat of increasing resistance to azole drugs, associated with the relative scarcity of antifungal drugs, prompted the use of essential oils. For this reason, we assessed the effect of the essential oil of Myrtus communis on the growth and survival of C. albicans. Twelve strains of C. albicans; four strains originally isolated from AIDS patients with oropharyngeal candidiasis, four isolated from HIV-infected subjects with recurrent vulvovaginitis, two reference strains (ATCC 24433 and 20891) and two laboratory strains (3153 and CA2) were employed in this study. All strains were identified by conventional diagnostic procedures. Susceptibility testing of C. albicans to M. communis and fluconazole was performed according to the NCCLS method M27-A2 for broth dilution antifungal susceptibility testing of yeast [3]. Each antifungal agent was diluted using RPMI1640 medium with L-glutamine, without sodium bicarbonate (Sigma Chemical Co., St Louis, MO, USA) and buffered to pH 7.0 with 0.165 M MOPS buffer (Sigma). Aliquots of 50 μl of two-fold dilutions of drug solutions were dispensed into each well of 96-well microtitre plates. The final Corresponding author. E-mail address:
[email protected] © Essential Oil Resource Consultants. All rights reserved.
concentration of the antifungal agents ranged from 64 to 0.062 mg/l for fluconazole and 2.8 % to 0.0026% for M. communis. Tween 80 (final concentration 0.001% v/v) was included to facilitate oil solubility. At this concentration, no inhibitory effect on yeast was shown by the detergent.The cell density of the suspension by direct cell count using a Thomas camera, and adjusting to a cell density ranging from 0.5 x103 to 2.5 x 103 cfu/ml. 50 μl was added to each well of the microdilution plate, followed by incubation at 30°C for 48 h. To determine MFCs, 10 μl of broth was taken from the well without microbial growth, inoculated onto Sabouraud’s dextrose agar (SDA) and incubated at 30°C for 48h. After 48 h the CFU were counted and MFC was defined as the lowest concentration resulting in the death of 99,9% or more of the initial inoculum. For mycelial development, the cells were grown in RPMI1640 supplemented with 10% fetal murine serum and incubated for 24h at 37°C. The essential oil of M. communis was extracted using a four hour hydrodistillation of the leaves using a Clevenger-type apparatus. The obtained yellowish essential oil chemical composition was then analysed by GC/MS and was mainly found to consist of 1,8-cineole (eucalyptol) (42.25%), α-pinene (34.56%) and limonene (15.94%). It displayed clear activity against all strains of C. albicans (Table 1). All strains had MICs of 0.35% to 1.4%; the majority were resistant to fluconazole. In particular, all resistant strains had MICs of 0.7%. It is very interesting that the MFC were also between 0.7% to 2,8%. Also in this case, 37.5 % of the resistant strains had MFC 0.7%, while 62.5 % had MFC of 1.4%. Besides, we have demonstrated that the essential oil of M. communis is able to inhibit the formation of germ tube at concentrations of 0.0875% v/v as reported in Figure 1. The prevalence of resistant strains of C. albicans is increasing and the identification of new safe and effective antimycotic agents for use in these infections is very important. It is very interesting that low concentrations of essential oil are able to inhibit the hyphae; a major trait of virulence of this fungus. To our knowledge, ours is the first attempt to investigate the anti-Candida effects of the essential oil of myrtle. The results of our study are encouraging, but
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International Journal of Essential Oil Therapeutics (2008) 2, 156-157
additional in vitro testing is needed to characterise the efficacy and safety of this approach in patients with C. albicans infections.
E-mail:
[email protected] References 1.
R. Ragnoa, S. Sivrica, G. Sartorellia, A. Serillib, E. Vavalac, L. Angiolellac*. a Dipartimento Chimica e del farmaco. b Dipartimento di Chimica. c Dipartimento di Scienze di Sanità Pubblica “G.Sanarelli” . Sapienza Università di Roma. *Corresponding author. Mailing address: Department of Public Health Sciences “Sanarelli”, University of Rome “La Sapienza”, Piazzale Aldo Moro, 00161-Rome, Italy.
2.
3.
Bustert J, Schaller M, Korting HC, and Evans EG. Current and future approaches to antimycotic treatment in the era of resistant fungi and immunocompromised hosts. Int J Antimicrob Agents. 2001;7:81-89. Angiolella L, Stringaro A R, De Bernardis F, Posteraro B, Bonito M, Toccacieli L, et al. Increase of virulence and its phenotypic traits in drug-resistant strains of Candida albicans. Antimicrob Agents Chemother. 2008;52:927-36. National Committee for Clinical Laboratory Standards. Reference methods for broth dilution antifungal susceptibility testing of yeast, approved standard. NCCLS document M27-A2. 2002.Waine, PA.
Table 1. In vitro susceptibility of different strains of C. albicans to myrtle essential oil and fluconazole. MIC strains of C. albicans
MFC
myrtle oil (%v/v)
fluconazole (μg/ml)
myrtle oil (% v/v)
fluconazole (μg/ml)
CO23
0.35
0.25
0.7
1
CO23RFK
0.35
0.25
0.7
1
CO23RFLU
0.35
>64
0.7
R
AIDS 6
0.7
>64
0.7
R
AIDS 37
0.7
>64
1.4
R
AIDS 68
1.4
>64
2.8
R
AIDS 126
0.7
0.5
1.4
2
CA2
0.7
>64
1.4
R
3153
0.7
>64
1.4
R
GR5
0.7
>64
1.4
R
ATCC 10231
0.7
>64
0.7
R
ATCC 20891
0.7
4
1.4
16
0 min
90 min
240 min
24h
Control
0,35 % v/v
0,175 % v/v
0,0875 % v/v
Figure 1. Hyphal growth in presence of different concentrations of myrtle oil at different incubation times.
