Antifungal effects of herbal essential oils alone and in combination with ketoconazole against Trichophyton spp.

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1 Journal of Applied Microbiology 2004, 97, doi: /j x Antifungal effects of herbal essential oils alone and in combination with ketoconazole against Trichophyton spp. S. Shin and S. Lim College of Pharmacy, Duksung Women s University, Dobongku, Seoul, South Korea 2003/0470: received 4 June 2003, revised 6 July 2004 and accepted 7 July 2004 ABSTRACT S. S H I N A N D S. L IM Aims: To determine the effects of herbal essential oils on Trichophyton spp. growth and to evaluate the effects of Pelargonium graveolens oil and its main components citronellol and geraniol combined with ketoconazole against Trichophyton spp. Methods and Results: Growth inhibition of six Trichophyton spp. by herbal essential oils was accessed and the combined effects of P. graveolens oil and its main components citronellol and geraniol were evaluated using a checkerboard microtitre assay against T. schoenleinii, T. erinacei and T. soudanense. The essential oil fraction of P. graveolens and its main components, geraniol and citronellol, exhibited strong synergism with ketoconazole against T. schoenleinii and T. soudanense, with fractional inhibitory concentration (FIC) indices in the range of 0Æ18 0Æ38. Conclusions: The antifungal effects of ketoconazole against Trichophyton spp. are enhanced significantly by administering it in combination with the essential oil fraction of P. graveolens or its main components, because of strong synergism, especially against T. soudanense and T. schoenleinii. Significance and Impact of the Study: The combination of ketoconazole and the essential oil fraction from P. graveolens or its main components for treatment of infections caused by Trichophyton species may reduce the minimum effective dose of ketoconazole, and thus minimize the side-effects of ketoconazole. Keywords: citronellol, essential oil, geraniol, ketoconazole, Pelargonium graveolens, synergism, Trichophyton. INTRODUCTION Natural antifungal agents are in great needed because currently used therapeutics have toxic side-effects, may interact with other drugs, and become ineffective as a result of the rapid development of fungal resistance (Gundidza 1993; Hammer et al. 1998, 1999; Shahi et al. 1999). Trichophyton is a fungal species that causes superficial mycoses commonly known as tinea infections in various areas in humans and other animals. Ketoconazole is one of the commonly used antifungal drugs administered orally for the treatment of both superficial and deep infections caused by Trichophyton. However, the unpleasant side-effects of Correspondence to: Seungwon Shin, College of Pharmacy, Duksung Women s University, Ssangmoondong 419, Dobongku, Seoul, , South Korea ( swshin@duksung.ac.kr). this drug include nausea, abdominal pain and itching, and its toxicity limits its therapeutic use in many cases. Moreover, the therapeutic response may be slow, and thus inappropriate for treatment of patients with severe or rapidly progressive mycoses. In addition, the efficacy of ketoconazole is poor in immunosuppressed patients and in the treatment of meningitis. Essential oils are one of the most promising groups of natural compounds for the development of safer antifungal agents. However, their poor absorption from the human intestine and relatively mild antifungal activity compared with commercial, synthetic antifungal drugs may ultimately limit their clinical application in the treatment of systemic fungal infections (Garg and Siddiqui 1992; Adam et al. 1998; Bidlack et al. 2000; Shin and Kang 2001). ª 2004 The Society for Applied Microbiology

2 1290 S. SHIN AND S. LIM Many essential oils are useful for treatment of tinea and their in vitro and in vivo antifungal effects have been evaluated (Hammer et al. 2000; Inouye et al. 2001; Harris 2002). In general, their antifungal activity is mild compared with commonly used antibiotics. Many essential oils are only fungistatic and high concentrations are needed for fungicidal activity (Cassella et al. 2002). To enhance the efficacy of essential oils, the combined use of different oils has been evaluated recently for potential synergistic effects. The combination of essential oils with synthetic antifungals will probably result in a more effective therapy though (Yoon et al. 1994; Suresh et al. 1997; Giordani et al. 2001; Shin and Kang 2003). However, synergistic effects between essential oils and common antifungal drugs against Trichophyton have not yet been reported until now. In order to develop a broad-spectrum natural anti- Trichophyton agent, we evaluated the activities of essential oils, used for the treatment of fungal infections in aromatherapy and complementary medicine, against six species of Trichophyton fungi by broth dilution and disc diffusion tests. In addition, we evaluated the synergistic effects of the essential oils from P. graveolens, a potent oil containing the highly active and stable primary alcohols citronellol and geraniol, and ketoconazole, a commonly used therapeutic for Trichophyton infections, against Trichophyton spp. MATERIALS AND METHODS Preparation of samples for testing anti-trichophyton activities The essential oils from Cedrus atlantica (wood), Citrus bergamia (fruit), Cymbopogon citratus (leaf), Eukalyptus golbulus (leaf), Juniperus communis (fruit), Lavandula angustifolia (flower), Melaleuca alternifolia (leaf), Pelargonium graveolens (leaf), Pogestemon patchouli (leaf), Rosmarinus officinalis (herb), Styrax tonkinensis (wood) and Thymus vulgaris (herb) were extracted by steam distillation. The compositions of the essential oils were analysed by gas chromatography-mass spectrometry (GC-MS) on a Hewlett-Packard 6890 GC and Hewlett-Packard 5973 MSD apparatus using an HP-5 capillary column (Shin 2003). Citronellol, geraniol, benzoic acid, thymol, 1,8-cineol and ketoconazole were purchased from Sigma Chemical Co. (St Louis, MO, USA). Fungal strains Six Trichophyton spp. were obtained from the Korean Culture Center of Microorganisms (KCCM). Trichophyton erinacei KCCM 60411, T. mentagrophytes KCCM 11950, T. rubrum ATCC 6345, T. tonsurans KCCM 11866, T. schoenleinii KCCM and T. soudanense KCCM were cultured in yeast and malt extract broth (YM; Difco 0712) for 48 h at 25 C. The turbidity of the cell suspension was measured at 600 nm and adjusted with medium to match the 0Æ5 McFarland standard [ colony-forming units (CFU) ml )1 ]. Determination of MIC Essential oil samples were serially diluted with YM broth to obtain solutions with 0Æ25 64 mg ml )1 essential oil; 10 ll Tween 80 was added to each solution. After shaking, 100-ll aliquots of the essential oil solutions were added to wells of 96- well microtitre plates. A 100-ll suspension of each of the six Trichophyton species was adjusted to CFU ml )1, and then added to individual wells and cultivated at 25 C. The MIC was defined as the lowest concentration that completely inhibited visible fungal growth after 72 h. Each organism was also cultured with a blank solution containing Tween 80 at concentrations equivalent to those in the test solutions to certify that these vehicles did not affect fungal growth. Disc diffusion assay Fungal broth culture aliquots were added to SabouraudÕs dextrose agar medium and uniformly distributed. Sterile paper discs (8 mm; Advantec, Dublin, CA, USA) were impregnated with 50 ll of2æ5 50% (1Æ25 25 mg per disc) ethanol solutions of each agent, and after alcohol evaporation the discs were placed on the culture plates. The width of the zone of inhibition (mm) was measured from the boundary of the disc after cultivation at 25 C for 72 h. Values are given as mean and S.D. of tests performed in triplicate. Checkerboard microtitre test Ten serial twofold dilutions of geraniol, citronellol, the essential oil fraction from P. graveolens, and ketoconazole were prepared using the same solvents as in the MIC tests. Fifty-micolitre aliquots of each P. graveolens oil dilution were added to the wells of a 96-well plate in a vertical orientation and 10-ll aliquots of each ketoconazole dilution were added in a horizontal orientation so that the plate contained various concentration combinations of the two compounds. Following this, each well was inoculated with 100 ll (ca CFU per well) of one of three Trichophyton (T. erinacei, T. soudanense or T. schoenleinii) fungal suspensions and cultivated at 25 C. Fractional inhibitory concentration (FIC) was calculated by dividing the MIC of the combination of geraniol and ketoconazole by the MIC of geraniol or ketoconazole alone. The FIC index, obtained by adding both FIC, was interpreted as indicating a synergistic effect when it was 0Æ5, as additive or indifferent when it was >0Æ5 and 2Æ0, and as antagonistic when it was >2Æ0

3 HERB OILS AGAINST TRICHOPHYTON SPP (White et al. 1996). Similar checkerboard experiments were also performed to test the combined effect of the essential oil fraction from P. graveolens or citronellol and ketoconazole. An isobologram was constructed from the checkerboard data to depict the synergism between P. graveolens oil and ketoconazole against Trichophyton spp. A checkerboard experiment was also performed to determine the effect of combining citronellol or geraniol with ketoconazole. Statistical treatment of the results The analysis of data was performed with the SPSS program version 11Æ0 (SPSS Inc., Chicago, IL, USA). Analysis of variance tests were conducted using the general one-way ANOVA with post hoc comparison of mean values by LSD. RESULTS MIC of the herbal oils and their main components We used the main components (benzoic acid, 1,8-cineol, thymol, citronellol and geraniol) of the essential oils in S. tonkinensis, E. golbulus, T. vulgaris and P. graveolens, respectively, as determined by GC-MS analysis of the oils (Shin 2003). MIC assay results of the tested oils are shown in Table 1. Most of the oils evaluated exhibited significant inhibitory activity against all six of the Trichophyton species tested. The fungal sensitivity did not differ remarkably in the broth dilution and agar methods. However, the MIC and MFC of the essential oils tested varied significantly. Among the oil fractions tested, C. citratus and E. golbulus were the most potent inhibitors of all fungi tested, with MIC of <0Æ125 0Æ25 mg ml )1 and MFC of <0Æ125 1 mg ml )1. As expected, T. vulgaris and its main component thymol had an especially high MIC value ranging between 0Æ25 and 1mgml )1. Thymol was more active than the total oil fraction of T. vulgaris. The oil of P. graveolens as well as its main components, citronellol and geraniol, also strongly inhibited these fungi, with MIC of 0Æ25 2 mg ml )1. The oils of P. patchouli, which are widely used as therapeutics for various microbial infections, exhibited unexpectedly moderate or relatively poor activity in this study. Growth inhibition of Trichophyton spp. on Sabouraud s agar plates The results of the disc diffusion tests (Table 2) also indicate that these oils are significantly potent against Trichophyton spp. Consistent with the MIC assay results, the oils of C. citrates, L. angustifolia, P. graveolens, R. officinalis and T. vulagris exhibited strong inhibition of >39 mm in most of the experimental concentrations of 25 and 12Æ5 mg per disc. The results from tests with T. vulagris and its main component thymol, and P. graveolens and its main components citronellol and geraniol were similar, at 1Æ25 mg per disc. However, not all the fungal inhibition results on Table 1 Minimum inhibitory concentration (MIC)* and minimum fungicidal concentration (MFC) of herbal oils against Trichophyton spp. Fungi T. erinacei T. mentagrophytes T. rubrum T. schoenleinii T. soudanense T. tonsurans Essential oil MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC MIC MFC C. atlantica Æ25 0Æ5 0Æ5 1 C. bergamia Æ C. citratus 0Æ25 0Æ5 <0Æ125 <0Æ125 <0Æ125 0Æ125 <0Æ125 0Æ125 <0Æ125 <0Æ125 <0Æ125 0Æ125 E. globulus 0Æ25 1 0Æ25 0Æ5 <0Æ125 0Æ5 0Æ25 0Æ5 0Æ25 0Æ5 <0Æ125 0Æ125 J. communis 0Æ Æ L. angustifolia 0Æ Æ Æ25 0Æ5 1 2 M. alternifolia 0Æ5 3,12 1 >32 1 >32 2 > P. graveolens 0Æ5 2 0Æ5 1 0Æ5 0Æ5 0Æ5 0Æ5 0Æ25 0Æ5 0Æ5 1 P. patchouli 8 >32 >32 > Æ5 >32 8 >32 R. officinalis Æ S. tonkinensis Æ T. vulgaris 0Æ Æ5 1 0Æ Benzoic acid <0Æ125 0Æ125 0Æ25 0Æ5 0Æ25 <0Æ25 <0Æ25 <0Æ25 0Æ25 0Æ5 0Æ25 0Æ5 1,8-Cineol 0Æ25 2 <0Æ125 0Æ25 0Æ5 0Æ5 0Æ25 0Æ5 0Æ25 0Æ5 0Æ5 1 Citronellol 0Æ Æ Geraniol 0Æ5 2 0Æ Æ5 1 0Æ25 0Æ5 0Æ5 1 Thymol 0Æ25 0Æ5 0Æ5 1 0Æ5 1 0Æ5 1 0Æ5 1 0Æ5 1 *Values are given as mean values (mg ml )1 ) from the triplicate experiments.

4 1292 S. SHIN AND S. LIM Table 2 Growth inhibition (mm)* of Trichophyton spp. on SabouraudÕs agar plates Fungi Essential oil T. erinacei T. mentagrophytes T. rubrum T. schoenleinii T. soudanense T. tonsurans C. atlantica I 7Æ3 ±0Æ76 f 9Æ2 ±0Æ29 e 1Æ0 ±0Æ00 a 7Æ3 ±0Æ58 e 8Æ7 ±0Æ58 d 7Æ2 ±0Æ76 d II 3Æ3 ±0Æ76 c 5Æ3 ±0Æ29 c 0Æ7 ±0Æ29 a 4Æ3 ±1Æ15 c 5Æ8 ±0Æ76 c 3Æ8 ±0Æ29 b C. bergamia I 8Æ0 ±0Æ50 f 6Æ0 ±1Æ00 c 8Æ7 ±0Æ76 e 3Æ3 ±0Æ58 b 14Æ3 ±1Æ53 3Æ3 ±0Æ58 II 5Æ0 ±2Æ00 e 2Æ8 ±0Æ29 b 6Æ0 ±1Æ00 d 2Æ5 ±1Æ50 a 7Æ8 ±0Æ29 2Æ3 ±0Æ58 a C. citratus I >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 II >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 E. globules I 25Æ0 ±1Æ53 21Æ0 ±2Æ08 15Æ0 ±2Æ00 9Æ8 ±0Æ58 >39Æ0 10Æ0 ±0Æ29 f II 18Æ0 ±0Æ00 13Æ0 ±1Æ53 9Æ0 ±2Æ00 e 6Æ2 ±1Æ15 d >39Æ0 5Æ8 ±0Æ76 c III 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a J. communis I 2Æ8 ±0Æ29 b 5Æ8 ±0Æ29 c 3Æ8 ±0Æ29 c 6Æ5 ±1Æ32 5Æ2 ±0Æ29 b 4Æ7 ±0Æ58 II 1Æ7 ±0Æ29 b 2Æ7 ±0Æ58 b 11Æ0 ±0Æ58 3Æ3 ±0Æ29 b 2Æ8 ±0Æ29 2Æ2 ±0Æ29 a L. angustifolia I >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 II >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 M. alternifolia I 13Æ0 ±0Æ58 8Æ7 ±0Æ58 e 9Æ3 ±0Æ58 e 16Æ0 ±1Æ15 f 23Æ3 ±1Æ53 5Æ2 ±0Æ29 c II 4Æ8 ±0Æ29 e 5Æ3 ±0Æ29 c 6Æ7 ±1Æ15 e 8Æ3 ±0Æ58 11Æ7 ±0Æ29 e 3Æ5 ±0Æ87 b P. graveolens I >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 II >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 III 4Æ0 ±0Æ50 6Æ8 ±1Æ04 d 4Æ3 ±1Æ04 d 10Æ5 ±1Æ32 8Æ7 ±1Æ26 9Æ5 ±0Æ50 f P. patchouli I 5Æ3 ±1Æ04 e 3Æ0 ±0Æ00 b 3Æ3 ±1Æ53 c 7Æ0 ±0Æ50 e 4Æ7 ±0Æ58 b 7Æ3 ±0Æ58 d II 3Æ3 ±0Æ58 c 2Æ7 ±0Æ29 b 2Æ3 ±0Æ58 b 6Æ0 ±0Æ00 d 4Æ5 ±0Æ78 b 5Æ7 ±0Æ58 c R. officinalis I >39Æ0 >39Æ0 33Æ0 ±1Æ15 >39Æ0 >39Æ0 31Æ0 ±0Æ58 II >39Æ0 >39Æ0 20Æ0 ±0Æ29 >39Æ0 >39Æ0 17Æ0 ±0Æ00 S. tonkinensis I 15Æ0 ±1Æ53 16Æ0 ±0Æ50 17Æ0 ±0Æ29 17Æ0 ±0Æ29 f 19Æ0 ±2Æ00 29Æ0 ±1Æ53 II 8Æ7 ±0Æ58 f 11Æ0 ±0Æ58 13Æ0 ±0Æ50 11Æ0 ±0Æ29 16Æ2 ±0Æ76 19Æ0 ±1Æ00 III 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a T. vulgaris I >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 II >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 >39Æ0 III 4Æ5 ±0Æ87 7Æ8 ±1Æ04 4Æ7 ±0Æ58 d 10Æ8 ±1Æ26 11Æ3 ±0Æ64 e 8Æ2 ±1Æ04 e Benzoic acid III 1Æ3 ±0Æ29 a 2Æ5 ±0Æ50 b 5Æ7 ±0Æ58 d 2Æ8 ±0Æ29 b 6Æ3 ±0Æ58 c 5Æ8 ±0Æ29 c 1,8-cineol I 9Æ5 ±0Æ50 2Æ0 ±0Æ00 a 2Æ7 ±0Æ58 b 7Æ7 ±1Æ53 e 1Æ2 ±0Æ29 a 3Æ0 ±0Æ00 II 1Æ5 ±0Æ50 a 1Æ2 ±0Æ76 a 1Æ8 ±0Æ29 a 3Æ7 ±0Æ58 b 0Æ0 ±0Æ00 a 1Æ8 ±0Æ29 a III 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0Æ0 ±0Æ00 a 0±0Æ00 a Citronellol III 4Æ2 ±0Æ29 d 5Æ7 ±0Æ58 c 3Æ8 ±0Æ29 c 4Æ5 ±0Æ50 c 6Æ5 ±0Æ87 8Æ5 ±0Æ50 e Geraniol III 4Æ0 ±0Æ00 d 6Æ2 ±0Æ76 c 3Æ7 ±0Æ29 c 4Æ7 ±0Æ58 c 8Æ8 ±0Æ29 d 9Æ3 ±0Æ58 f

5 HERB OILS AGAINST TRICHOPHYTON SPP Table 2 (Contd) Fungi Essential oil T. erinacei T. mentagrophytes T. rubrum T. schoenleinii T. soudanense T. tonsurans Thymol III 8Æ8 ±0Æ76 f 7Æ3 ±0Æ58 d 9Æ3 ±0Æ58 e 9Æ3 ±0Æ58 6Æ3 ±1Æ53 c 5Æ8 ±0Æ29 c I, 25 mg per disc; II, 12Æ5 mg per disc; III, 1Æ25 mg per disc. *The width (mm) of growth inhibition of fungi was measured from the boundary of discs. The values are the mean and S.D. from the triplicate experiments. Data in each column followed by the same letters are not significantly different at the 5% level. Sabouraud s agar plates were consistent with results from the MIC assays. For example, the radius of inhibition by E. globulus oil and its main component 1,8-cineol was smaller than expected based on its MIC. J. communis and P. patchouli oils were relatively ineffective against all the fungi tested, with inhibition zones between 2Æ8 and 7Æ3 mm even at 25 mg per disc on Sabouraud s dextrose agar plate. The widths of the fungal inhibition zones were generally dose dependent. Combined effects of P. graveolens oil and its main components and ketoconazole To identify synergism of the essential oils with and ketoconazole to enhance the efficacy of antifungal drugs, checkerboard assays were performed and used to construct an isobologram. Among the 12 essential oil fractions used in the MIC and disc diffusion tests, we chose P. graveolens oil, which was highly active against all five Trichophyton spp. and contained, relatively stable primary monoterpene alcohols (citronellol and geraniol) as main components. As shown in Table 3, the MIC of ketoconazole combined with P. graveolens oil against T. erinacei, T. schoenleinii and T. soudanense were remarkably decreased, with FIC indices ranging between 0Æ18 and 0Æ56. Moreover, fungal susceptibility to ketoconazole was enormously improved by combination with the oil or its main components. In an experiment with T. erinacei and T. soudanense, the FIC of ketoconazole when combined with citronellol or geraniol was 0Æ06 and 0Æ13. FIC indices indicate the strongest synergism between P. graveolens oil and ketoconazole against T. soundanense, with an FIC index of 0Æ18. Similar results were obtained by the combination of ketoconazole with geraniol or citronellol, with an FIC index of 0Æ18. The isobologram based on the checkerboard titre assay results against T. schoenleinii Table 3 Fractional inhibitory concentration (FIC) and FIC indices T. erinacei T. schoneleinii T. soudanense Sample MIC o MIC c FIC FICI MIC o MIC c FIC FICI MIC o MIC c FIC FICI P. graveolens oil ketoconazole P. graveolens oil (mg ml )1 ) 0Æ5 0Æ25 0Æ50 0Æ56 0Æ5 0Æ125 0Æ25 0Æ31 0Æ25 0Æ031 0Æ12 0Æ18 Ketoconazole (lg ml )1 ) Æ06 8 0Æ5 0Æ Æ06 Citronellol Ketoconazole Citronellol (mg ml )1 ) 0Æ5 0Æ25 0Æ50 0Æ56 1 0Æ25 0Æ25 0Æ38 0Æ5 0Æ062 0Æ12 0Æ18 Ketoconazole (lg ml )1 ) Æ Æ Æ06 Geraniol Ketoconazole Geraniol (mg ml )1 ) 0Æ5 0Æ25 0Æ50 0Æ56 0Æ5 0Æ125 0Æ25 0Æ38 0Æ25 0Æ031 0Æ12 0Æ18 Ketoconazole (lg ml )1 ) Æ Æ Æ06 MIC o, MIC of one sample alone; MIC c, MIC of one sample of the most effective combination. MIC of oil in combination with ketoconazole FIC of oil ¼ MIC of oil alone MIC of ketoconazole in combination with oil FIC of ketoconazole ¼ MIC of ketoconazole alone FICI ¼ FIC of oil þ FIC of ketoconazole

6 1294 S. SHIN AND S. LIM (a) (b) (c) Ketoconazole (mg ml 1 ) P. graveolens oil (mg ml 1 ) Citronellol (mg ml 1 ) Geraniol (mg ml 1 ) Fig. 1 Isobolograms revealing the synergistic and additive effect of Pelargonium graveolens oil (a), citronellol (b) and geraniol (c) with ketoconazole on Trichophyton schoenleinii growth inhibition (a) (b) (c) Ketoconazole (mg ml 1 ) P. graveolens oil (mg ml 1 ) Citronellol (mg ml 1 ) Geraniol (mg ml 1 ) Fig. 2 Isobolograms revealing the synergistic action of Pelargonium graveolens oil (a), citronellol (b) and geraniol (c) in combination with ketoconazole on Trichophyton soudanense growth inhibition (Fig. 1) also indicates synergism between the combined compounds by the obviously deviating curves to the left. The patterns of the curves for P. graveolens oil and geraniol activity were highly similar. The isobologram against T. soudanense confirms these results. The curves depicted in Fig. 2 also show strong synergism from the combination of ketoconazole with P. graveolens oil, citronellol or geraniol by the distinctly left-deviating curves (Davidson and Parish 1989). Thus, geraniol is responsible for a large part of the activity of the P. graveolens oil fraction (Nidiry 1998). DISCUSSION With the goal of developing a new highly active antifungal therapeutic combination of herbal essential oils and synthetic antifungal agents, we began by comparing the antifungal properties of 12 essential oils and some of their main components against six species of Trichophyton that commonly cause tinea infections. These filamentous fungi act as dermatophytes in humans and cattle by digesting keratin. Tinea is typically treated by topical or systemic administration of polyene or azole antibiotics, including ketoconazole, which are efficacious but problematic, as previously mentioned (Sugar et al. 1987; Von Moltke et al. 1996; Giordani et al. 2001; McMichael 2001). The in vivo and in vitro anti-trichophyton activity of herbal essential oils has focused mainly on T. rubrum and T. mentagrophytes (Kishore et al. 1993; Wannissorn et al. 1996; Adam et al. 1998; Shahi et al. 1999; Inouye et al. 2001; Cassella et al. 2002; Cimanga et al. 2002; Giamperi et al. 2002; Harris 2002; Patra et al. 2002). However, the efficacy of herbal oils and their main components against the broad-spectrum Trichophyton spp., and especially the combined efficacy of these oils with ketoconazole, have not previously been

7 HERB OILS AGAINST TRICHOPHYTON SPP evaluated extensively. We did not compare our MIC and disc diffusion test data with data of previous reports because essential oil composition is variable and dependent on many factors such as plant chemotype, and culture, harvesting and storage conditions. Therefore, results from unrelated studies may differ because of these uncontrolled variables rather than anti-fungal activity (Lis-Balchin et al. 1996; Pandey et al. 1996; Kaul et al. 1997, 1998; Cosentino et al. 1999; Rao et al. 2001; Faleiro et al. 2003). Based on our MIC and disc diffusion test results, we selected P. graveolens oil and its main components, the relatively stable monoterpene primary alcohols, citronellol (17Æ20%) and geraniol (5Æ89%), to investigate in combination with ketoconazole. The oils from C. citrates, L. angustifolia, R. officinalis and T. vulgaris also exhibited strong inhibition against all the fungi tested in this study. Alcohols could be clinically practical antifungal agents because of their stability, especially in different ph conditions, while the clinical utility of phenolic compounds such as eugenol or thymol may be restricted in higher ph conditions. The synergistic effects of P. graveolens oil in combination with ketoconazole against Trichophyton spp. had not previously been investigated. Of the Trichophyton species tested, little variation in susceptibility to the oils alone was observed. We selected three relatively rare pathogenic Trichophyton species to investigate the synergistic effects of the oil and the main oil components and ketoconazole. However, the FIC results and the FIC indices differed. As shown in Table 3, the combination of P. graveolens oil and its components with ketoconazole exhibited especially strong synergistic inhibition against T. soudanense and T. schoenleinii. In the same experiment, inhibition by the combination was additive against T. erinacei. We cannot currently explain the mechanistic reason for these differences, as the antifungal mechanisms of essential oils have not yet been clarified. However, the inhibitory mechanism may be associated with the fungal cell wall composition or construction, upon consideration of the active mechanism of ketoconazole (Godwin and Michniak 1999; Scholar and Pratt 2000). In conclusion, we suggest the combination of P. graveolens oil and ketoconazole for the treatment of Trichophyton species, especially T. soudanense and T. schoenleinii, which originated in Africa and have spread to other parts of the world in recent decades (Nzelibe and Gugnani 1984; Kalter and Hay 1988; Ruebben and Krause 1996; Lamb and Rademaker 2001) may reduce the efficacious dose of ketoconazole and thus minimize the side-effects of ketoconazole. 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