Letters in Applied Microbiology 2003, 37, 185–187
Antimycotic activity of Melaleuca alternifolia essential oil and its major components B. Oliva1, E. Piccirilli1, T. Ceddia1, E. Pontieri1, P. Aureli2 and A.M. Ferrini2 1
Department of Experimental Medicine, Section of Microbiology, University of L’Aquila, L’Aquila, and 2Istituto Superiore di Sanita`, Laboratorio Alimenti, V.le Regina Elena, 299-Rome, Italy
2002/357: received 21 November 2002, revised 19 February 2003 and accepted 15 May 2003
ABSTRACT B . O L I V A , E . P I C C I R I L L I , T . C E D D I A , E . P O N T I E R I , P . A U R E L I A N D A . M . F E R R I N I . 2003.
Aims: The aim of this study was to analyse the antimycotic properties of Melaleuca alternifolia essential oil (tea tree oil, TTO) and its principal components and to compare them with the activity of 5-fluorocytosine and amphotericin B. Methods and Results: The screening for the antimycotic activity was performed by serial twofold dilutions in Roswell Park Memorial Institute medium with the inclusion of Tween-80 (0Æ5%). TTO and terpinen-4-olo were the most active compounds. Conclusions: The majority of the organisms were sensitive to the essential oil, with TTO and terpinen-4-olo being the most active oils showing antifungal activity at minimum inhibitory concentration values lower than other drugs. Significance and Impact of the study: This study provides a sample large enough to determine the antifungal properties of TTO and terpinen-4-olo and suggests further studies for a possible therapeutic use. Keywords: antifungal activity, essential oil, Melaleuca alternifolia, tea tree.
INTRODUCTION The essential oil of Melaleuca alternifolia (Australian tea tree oil, TTO) has been successfully used as a topical agent to control wound infections (Altman 1989; Syed et al. 1999). However, new interest in natural oils as a possible alternative to antibiotics requires a better definition of the antimicrobial properties of these agents. As fungal pathogens are important causes for topical infections it is important to evaluate the antifungal activity of the essential oil of M. alternifolia. So far, the antimycotic properties of this oil have only been investigated on a limited number of fungal species (BanesMarshall et al. 2001). This paper presents the first detailed investigation of the antimycotic properties of this oil using a large collection of strains, belonging to different genera and Correspondence to: Brunello Oliva, Department of Experimental Medicine, Section of Microbiology, University of L’Aquila, Coppito II, 67100 L’Aquila, Italy (e-mail:
[email protected]).
ª 2003 The Society for Applied Microbiology
species including numerous ATCC strains and clinical isolates. MATERIALS AND METHODS Essential oil and its major components M. alternifolia of known composition (a-tuiene 0Æ9%, a-pinene 2Æ7%, a-terpinene 10Æ5%, 1,8-cineolo 3%, c-terpinene 23%, terpinolene 3Æ8%; terpinolen-4-olo 48Æ9% and a-terpineolo 2Æ7%) was obtained from Variati, Milano. Principal components of the essential oil, i.e. terpinen4-olo, cineolo, terpinolene, a-terpinene and c-terpinene were also purchased separately from Sigma-Aldrich (Milan, Italy). Fungal strains A total of 115 strains were used: Candida lipolytica ATCC 9773, C. parapsilosis ATCC 22019, 96144, 96140, Blastoschizomyces capitatus ATCC 200923, 200924, 200925,
2 1 1 1 1 1 1–2 1 1–2 1–2 1 1–2 1 4 1 M, Melaleuca alternifolia; T, terpinen-4-olo; C, cineolo; 5-Flu, 5-fluorocytosine; AMB, amphotericine B; t, terpinolene; a-t, a-terpinene; c-t, c-terpinene.
8 1 8 0Æ25 1 0Æ25 2 to >128 0Æ25 to >128 0Æ25 to >128 0Æ25–1 0Æ25–1 0Æ25–64 0Æ5 2 4 4 4 4 2 2 >4 4 1–4 4 to >4 1–2 1–2 >4 2 2 1 >4 >4 4 4 2 >4 4 to >4 4–4 4 2–4 2 4 to >4 4 2 2 4 4 >4 2 4 4 2–4 1–4 1 to >4 1–2 1–4 2–4 4 4 1 1 1 1 1 1 1 0Æ5–1 0Æ25–1 1 0Æ25–1 1 0Æ5–1 1 0Æ25 1 0Æ06 0Æ125 0Æ125 0Æ125 0Æ125 0Æ125 0Æ06 0Æ06–0Æ125 0Æ06–0Æ125 0Æ06–0Æ125 0Æ06–0Æ125 0Æ06–0Æ125 0Æ06 0Æ06 0Æ125 0Æ125 0Æ125 0Æ125 0Æ125 0Æ125 0Æ25 0Æ06–0Æ125 0Æ06–0Æ125 0Æ125 0Æ03–0Æ125 0Æ125 0Æ125–0Æ25 0Æ125 0Æ03 0Æ125
t C T M
Minimum inhibitory concentration (MIC) (lg ml)1)
The MIC values of M. alternifolia essential oil and its major components together with the MIC values of 5-fluorocytosine and amphotericin B are listed in Table 1. For most species MIC ranges and MIC90 values are reported. When one member of a tested species was used, only the MIC value is reported. Previous tests proved that the inclusion of 0Æ5% v/v of Tween-80 did not affect fungal growth. The essential oil from M. alternifolia displayed potent antifungal
Species (no. of isolates)
RESULTS
Table 1 Susceptibility of various fungal isolates to Melaleuca alternifolia essential oil
The fungal strains were grown in Sabouraud dextrose agar, five colonies were picked and suspended in sterile saline. They were diluted appropriately in Roswell Park Memorial Institute medium (RPMI) 1640 medium (NCCLS 1997) to give an inoculum of 5 · 103 cells ml)1. The RPMI medium was supplemented with Tween-80 (0Æ5%) (May et al. 2000) to dissolve the oils. Tween-80 did not affect fungal growth. MICs were determined after 24 or 48 h incubation at 30C. The MIC of 5-fluorocytosine and amphotericine B (Sigma-Aldrich) were carried out according to NCCLS (1997) recommendations supplementing the broth with 0Æ5% Tween-80 to standardize the conditions; MICs were determined by twofold serial dilutions of the compounds on RPMI medium. The MIC was defined as the lowest concentration of compound that completely inhibited visible growth after 24–48 h of incubation at 30C.
a-t
Minimum inhibitory concentration (MIC) of essential oil, its components and antimycotic drugs
lipolytica (13) parapsilosis (57) albicans (9) glabrata (5) lusitanie (3) capitatus (25) guilliermondii (1) incospiqua (1) krusei (1)
c-t
Strains were identified according to morphological–biochemical procedures and by the commercial kit ID 32C (Biomerieux, Rome, Italy). The strains were cultured in Sabouraud broth or agar (Oxoid), and stored in glycerol (33%) at )80C.
C. C. C. C. C. B. C. C. C.
Strains identification and growth conditions
MIC range MIC90 MIC range MIC90 MIC range MIC90 MIC range MIC90 MIC range MIC90 MIC range MIC90 MIC range
5-Flu
AMB
200926, 200927, 200928, 200929, 62963, 62964 (13 strains); environmental origin: three C. parapsilosis and one C. lipolytica; clinical isolates from vaginal swabs, blood, urine, catheters, onycomycosis, and sputum: C. lipolytica (13 strains), C. parapsilosis (57 strains), C. albicans (nine strains), C. glabrata (five strains), C. lusitaniae (three strains), C. guillermondi (one strain), C. incospicua (one strain), and C. krusei (one strain). The Blastoschizomyces were isolated mostly from immuno-compromised patients from Italy (Pescara, L’Aquila, Roma, Reggio Calabria, Pisa), Germany, Holland and UK.
MIC90 MIC range MIC90
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ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 185–187
ANTIFUNGAL ACTIVITY OF TEA TREE OIL
activity. Among its components, terpinen-4-olo and cineole also possessed strong antifungal activity. Comparison of MIC data demonstrate that the essential oil and some of its components are more potent than 5-fluorocytosine and amphotericin B. DISCUSSION Standardizing the method for determining MIC is important when evaluating the antimicrobial properties of oils, in order to permit comparison of the data generated by different laboratories. For this purpose we chose the NCCLS reference method for antifungal susceptibility tests, which is now internationally recognized. The results reported in Table 1 were obtained from analysis of 115 different strains belonging to different genera. This study provides a sample large enough to judge the in vitro antimycotic activity of the oils tested. It appears that M. alternifolia (TTO) essential oil and one of its components terpinen4-olo are the most active agents. Cineole shows some activity, but it has been reported to cause skin irritation, therefore it is not of primary interest for in vivo application. The MIC values obtained for TTO and terpinen-4-olo are similar to each other and lower than those obtained for 5-fluorocytosine and amphotericin B. The fact that these oils act on Gram-negative and Grampositive bacteria and fungi indicates a common mode of action. This probably results from membrane damage, loss of K+ and small molecules (Carson et al. 2002 ) which is followed by inhibition of respiration (Cox et al. 2000).
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The results reported here suggest that further studies on the possible therapeutic use of essential oils for fungal infections would be of interest. REFERENCES Altman, P.M. (1989) Australian tea tree oil – a natural antiseptic. Australian Journal of Biotechnology 3, 247–248. Banes-Marshall, L., Cawley, P. and Phillip, C. (2001) In vitro activity of Melaleuca alternifolia (tea tree) oil against bacterial and Candida spp. isolates from clinical specimens. British Journal of Biomedical Science 58, 139–145. Carson, C.F., Mee, B.J. and Riley, T.V. (2002) Mechanism of action of Melaleuca alternifolia (tea tree) oil on Staphylococcus aureus determined by time-kill, lysis, leakage and salt tolerance assays and electron microscopy. Antimicrobial Agents Chemotherapy 46, 1914– 1920. Cox, S.D., Mann, C.M., Markham, J.L., Bell, H.C., Gustavson, J.E., Warmington, J.R. and Wyllie, S.G. (2000) The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil). Journal of Applied Microbiology 88, 170–175. May, J., Chan, C.H., King, A., Williams, L. and French, G.L. (2000) Time-kill studies of tea tree oils on clinical isolates. Journal of Antimicrobial Chemotherapy 45, 639–643. NCCLS (1997) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts; Approved Standard M27-A. Wayne, PA: National Committee for Clinical Laboratory Standard. Syed, T.A., Qureshi, Z.A., Ali, S.M., Ahmad, S. and Ahmad, S.A. (1999) Treatment of toenail onychomycosis with 2% butenafine and 5% Melaleuca alternifolia (tea tree) oil in cream. Tropical Medicine and International Health 4, 284–287.
ª 2003 The Society for Applied Microbiology, Letters in Applied Microbiology, 37, 185–187
