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Antifungal effects of the volatile oils from Allium plants against Trichophyton species and synergism of the oils with ketoconazole.

Abstract

In an attempt to develop stable and safe antifungal agents from natural products (daily foodstuffs in particular), the activities of essential oils from Allium sativum for. pekinense, A. cepa, and A. fistulosum against three Trichophyton species responsible for severe mycoses in humans were investigated and compared with activity of allicin in this study. The fungistatic activities of Allium oils were evaluated by the broth dilution method and disk diffusion assay. The combined effects of Allium oils with ketoconazole were tested by the checkerboard titer test. Among the tested oils, A. sativum for. pekinense oil exhibited the strongest inhibition of growth of T. rubrum, T. erinacei, and T. soudanense with MICs (minimum inhibiting concentrations) of 64 [micro]g/ml, while the activities of A. cepa and A. fistulosum were relatively mild. The inhibiting activities of the oils on Sabouraud agar plates were dose dependent against Trichophyton species. Additionally, these oils showed significant synergistic antifungal activity when combined with ketoconazole in the checkerboard titer test and disk diffusion test.

[c] 2005 Elsevier GmbH. All rights reserved.

Keywords: Allium spp.; Antifungal activity; Trichophyton; Volatile oils; Synergism; Ketoconazole; Allicin

Introduction

Allicin, a well-known compound of Allium spp., is so unstable that upon generation it readily changes into other compounds (Xinjian et al., 1992; Emiko and Takao, 1997; Talia et al., 1998; Serge and David, 1999; Larry et al., 2001; Bocchini et al., 2001). Thus, allicin has not been conclusively proven to be responsible for the known health benefits of garlic. Moreover, recent characterization of the pharmacokinetics and metabolism of organosulfur compounds in garlic has revealed that allicin is not biologically active in the body (Hughes and Lawson, 1991; Yasuo and Keizo, 1997). Therefore, compounds in Allium spp. other than allicin may very well be responsible for the beneficial activities.

Essential oils are one of the most promising groups of natural compounds from which a new prototype of antifungal agents may be developed (Dikshit et al., 1986; Garg and Siddiqui, 1992; Gundidza, 1993; Ayer et al., 1996; Adam et al., 1998; Bhaskara-Reddy et al., 1998; Garg and Dengre, 1998; Bidlack et al., 2000; Inouye et al., 2001), even though they appear to have relatively mild activities against human pathogenic fungi compared to commercial synthetic antifungal drugs. Consequently, the antifungal activities of plant essential oils have been evaluated after administration in combination with synthetic drugs to evaluate for synergism (Rhee and Kim, 1999; Hammer et al., 2000; Giordani et al., 2001; Shin and Kang, 2003).

Trichophyton is a fungal species that causes superficial mycoses commonly known as tinea infections in humans and other animals (Harris, 2002; Patra et al., 2002; Shin, 2004). 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 this drug include nausea, abdominal pain, and itching, and its toxicity limits its therapeutic use in many cases (Sugar et al., 1987). 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 (Craven and Graybill, 1984).

In order to develop stable and safe antifungal agents from natural products (daily foodstuffs in particular), the composition and antifungal activities of the essential oils from A. sativum for. pekinense, A. cepa, and A. fistulosum against Trichophyton spp. were investigated and compared in this study. In addition, in an attempt to achieve a more powerful and safer therapy (Shin, 2003), the combined effects of Allium oils and their main components with ketoconazole were determined by checkerboard titer tests and isobolograms constructed with combined minimum inhibiting concentrations (MICs).

Materials and methods

Sample preparation

The essential oils were obtained by steam distillation for 5 h in a simultaneous steam distillation--extraction apparatus from A. sativum for. pekinense Makino (bulbs), A. cepa L.(bulbs), and A. fistulosum L. (whole plant), which were purchased from the National Agriculture Cooperative Federation in Seoul. Allyl sulfide (A35801), allyl disulfide (A317691), and ketoconazole (K1003) were purchased from Sigma-Aldrich Korea, LTD. Allicin was extracted from the bulbs of A. sativum for. pekinense with ethanol, according to the method of Cavallito et al. (Cavallito and Bailey, 1994). The concentration of allicin was adjusted by comparing the activity with the synthesized allicin using allyl disulfide as starting compound. Allicin synthesis was carried out by the method of Lawson et al. (Larry et al., 2001; Lawson et al., 1991). The synthesized allicin was analyzed by high-pressure liquid chromatography, infrared spectroscopy, mass spectrometry, and nuclear resonance spectrometry. All data are in accordance with the literature.

Essential oil analysis

The essential oil fractions of the three Allium species were analyzed by the Hewlett-Packard 6890 GC and the Hewlett-Packard 5973 MSD apparatus (Agilent 5973 network mass selective detector, 300 [degrees]C with a fused silica capillary column (HP-5MS, 30 m x 0.25 mm x 0.25 [micro]m)). The injector was adjusted to 250 [degrees]C and the oven temperature was as follows: initial temperature: 40 [degrees]C for 3 min, 2[degrees]C/min up to 150 [degrees]C, and then 20 [degrees]C/min up to 220 [degrees]C. The final temperature was 220 [degrees]C with a hold time of 5 min.

Fungal strains

The tested Trichophyton species were obtained from the Korean Culture Center of Microorganisms (KCCM). T. erinacei KCCM 60411, T. rubrum ATCC 6345, and T. soudanense KCCM 60448 were cultured in yeast and malt extract broth (YM, Difco 0712) for 48 h at 25 [degrees]C. The turbidity of the cell suspensions was measured at 600 nm and adjusted with medium to match the 0.5 McFarland standard ([10.sup.5]-[10.sup.6] colony forming units (CFU))/ml.

Minimal inhibitory concentration (MIC)

The MICs of the antifungal agents against the various fungi were determined by the broth micro dilution method. The Allium oil fractions, allicin, allyl sulfide, and ally disulfide were serially diluted with 10% dimethyl sulfoxide (DMSO) and added with 10 [micro]l of Tween 80 to prepare the solutions, which contained from 160 to 0.625 mg/ml of oils, respectively. Ketoconazole was similarly diluted in DMSO to generate a series of concentrations ranging from 100 to 0.78 [micro]g/ml per testing well. After shaking, 100 [micro]l of the antifungal agent solutions were added to the wells of 96-well plates. The suspension of each organism was adjusted to [10.sup.4]-[10.sup.5] CFU/ml, added to the individual wells at 100 [micro]l/well ([10.sup.3]-[10.sup.4] CFU/well), and cultivated at 24-28 [degrees]C. The MIC was defined as the lowest concentration that completely inhibited visible fungal growth in the wells after 72 h of incubation. MIC values were determined in duplicate and were retested if the values differed. Each organism was also cultured with a control solution containing Tween 80 and DMSO at levels equivalent to those in the test compound solutions to certify that they did not affect fungal growth. The tests were performed in triplicate to confirm the values.

Disk diffusion assay

Fungal broth culture aliquots adjusted to [10.sup.4]-[10.sup.5] CFU/ml were added to Sabouraud dextrose agar medium and distributed uniformly. Sterile paper discs (8 mm, Advantec, Toyo Roxhi Kaisha) were impregnated with 50 [micro]l of 25% (v/v, 12.5 mg) or 50% (v/v, 25.0 mg) ethanol and antifungal solution and placed on the culture plates after removing the ethanol by evaporation. The diameter of the zone of inhibition (mm) around the disk was measured after cultivation at 24-28 [degrees]C for 2 days. The values shown (Table 2) are the means [+ or -] SD of tests performed in triplicate.

Checkerboard titer test and isobologram construction

Eight serial two-fold dilutions of A. sativum for. pekinense oil and ketoconazole were prepared with the same solvents used in the MIC tests. Fifty microliter aliquots of each oil dilution were added to the wells of 96-well plates in a vertical orientation, and 10 [micro]l aliquots of each ketoconazole dilution were added in a horizontal orientation so that the plate contained various concentration combinations of the two compounds. A 100 [micro]l suspension of each Trichophyton fungi ([10.sup.4] CFU/well) was added to each well and cultured for 3 days. Fractional inhibitory concentrations (FICs) were calculated as the MIC of the combination of A. sativum for. pekinense oil and ketoconazole divided by the MIC of A. sativum for. pekinense oil or ketoconazole alone. The FIC index was calculated by adding both FICs and was interpreted as a synergistic effect when it was [less than or equal to]0.5, as additive or indifferent when it was >0.5 to 2.0, and as antagonistic when it was >2.0. A checkerboard experiment was also performed to determine the effect of combining allicin with ketoconazole (White et al., 1996).

Results and discussion

Analysis and comparison of volatile oils from Allium species

To identify the composition of the tested oils, we analyzed the oils derived from steam distillation of the three Allium species by GC-MS, even though oils of Allium species, especially of A. sativum for. pekinense, have been evaluated extensively. The composition of these oils differs greatly among species, and in many cases also among the same species, possibly due to analytical techniques, chemotypes, culture climate, and other culture conditions, which may affect biological activities (Coley-Smith, 1986; Calvey et al., 1998; Schulz and Kruger, 2002). The Allium species used in the experiments for this study are commonly cultivated for production of nutritional supplements in Korea.

As shown in Table 1, the three oils differed widely in composition as well as in fragrance. Di-2-propenyl trisulfide (32.82%) and di-2-propenyl disulfide (29.12%) represent the volatile fraction of A. sativum for. pekinense, while a relatively large amount of dipropyl disulfide (16.76%) was identified in A. cepa oil. The composition of A. cepa identified in this study differed greatly from other previous reports (Jarvenpaa et al., 1998). The main components of A. fistulosum were dipropyl disulfide (46.32%), trans-propenyl propyl disulfide (11.32%), and dimethyl trisulfide (5.86%). Most importantly, not even a trace of allicin was detected in any of the three oils. There were significant differences in composition of the oil fraction from A. fistulosum from that reported by Pino et al. (2000). Differences in cultivars of the plants might be one of the possible reasons for this.

MIC results

MIC assay results of the tested oils against the three Trichophyton fungi are shown in Table 2. We compared the activities of the volatile oils of Allium species with allicin and with diallyl sulfide and allyl sulfide, which are the expected decomposition products of allicin. Ketoconazole, one of the commonly used antifungal drugs for tinea infections, was the positive control in this experiment. As expected, among the oil fractions tested, A. sativum for. pekinense oil was the most potent inhibitor of all three Trichophyton spp., with MICs of 64 [micro]g/ml, equivalent to 25-50% of the activity of allicin (16-32 [micro]g/ml). Due to the relatively weak activity of the oil fraction compared to allicin, we repeated the same experiments after heating the samples for 3 h connected with cooling apparatus. The MICs of the oil were virtually unchanged, while the MIC of the allicin solution was dramatically lowered by heating. In fact, allicin was not detected in the solution after heating. The activity of A. sativum oil was 12.5-25% of the activity of ketoconazole. This is a remarkable level of activity for a natural product, especially after considering the side effects and toxicity associated with ketoconazole. The oils from A. cepa and A. fistulosum exhibited much weaker activity, with MICs of over 128 [micro]g/ml. The MICs did not differ significantly with regard to tested fungal species. In contrast, MICs differed in previous similar experiments conducted with Aspergillus species by Yin and Chao (Yin and Tsao, 1999). In their report, the sensitivities of Aspergillus species (MIC of A. sativum oil: 35-104 [micro]g/ml) varied significantly.

Combined effects with ketoconazole

To explore the possibility of developing a more powerful combination therapy of A. sativum oil with ketoconazole, the disk diffusion tests and the checkerboard micro-titer tests were performed with combined samples. Table 3 demonstrates a significant synergism of ketoconazole in combination with A. sativum volatile oil fraction, and also with allicin. Moreover, the greater than four-fold difference in width of the inhibited zone between A. sativum volatile oil fraction and allicin following separate administration was decreased significantly by combination of these samples with ketoconazole. The FIC and FICI results calculated from the checkerboard micro-titer tests are listed in Table 4. The startling combination effects of ketoconazole and A. sativum oil were confirmed in this test again with FICIs ranging from 0.09 to 0.12 against the same species of Trichophyton. Ketoconazole combined with allicin resulted in additive effects, with FICIs from 0.53 to 0.75. The isobologram depicted in Fig. I was constructed using the well concentrations which resulted in the greatest fungal inhibition in the checkerboard titer tests. The curve patterns deviating to the left indicate that the effective concentrations were significantly lowered by combination more extensively than by simple additive reduction.

[FIGURE 1 OMITTED]

Acknowledgments

This study was supported by a grant from the Korea Science and Engineering Foundation (KOSEF: R06-2002-004-01007-0).

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M.-S. Pyun, S. Shin*

College of Pharmacy, Duksung Women's University, Seoul 132-714, Republic of Korea

Received 29 October 2004; accepted 22 March 2005

*Corresponding author. Tel.: +82 2 901 8384; fax: +82 2 901 8386.

E-mail address: swshin@duksung.ac.kr (S. Shin).
Table 1. Main components of volatile oils from Allium species analyzed
by GC-MS

 A.
Compounds (peak area %) RI A. sativum A. cepa fistulosum

Methyl thiirane 606 -- 1.76 0.30
Bis 1-methyl ethyl disulfide 610 -- 0.34 0.08
2-Methyl pentanal 791 -- 0.08 0.04
3,3-Thio bis-l-propene 831 0.87 -- --
2,4-Dimethyl thiophene 863 0.03 0.04 0.16
2,5-Dimethyl thiophene 905 0.06 0.63 3.94
Methyl propyl disulfide 923 -- 1.37 0.62
Methyl cis-propenyl disulfide 923 0.13 -- 0.48
Methyl trans-propenyl disulfide 930 0.24 1.94 1.04
1-Ethyl thio methyl propene 942 -- 0.13 --
N,N'-Dimethyl thiourea 945 1.46 -- --
Dimethyl trisulfide 955 0.51 2.18 5.86
2-Pentyl furan 972 -- 0.05 0.16
Octyl aldehyde 984 -- 0.08 0.18
Limonene 1006 -- 0.04 --
Phenyl acetaldehyde 1025 -- 1.58 1.06
[gamma]-Terpinene 1036 -- 0.04 --
3-Ethyl methyl phenol 1067 -- 0.15 0.02
Di-2-propenyl disulfide 1071 32.82 -- 2.60
Linalool 1083 -- -- 0.42
Di-2-propenyl propyl disulfide 1072 -- 0.15 --
Dipropyl disulfide 1088 0.11 16.75 46.32
Trans-propenyl propyl disulfide 1095 0.30 4.96 11.32
Methyl 2-propenyl trisulfide 1130 7.40 0.26 0.40
1-Methoxy 2-benzene 1128 -- 1.41 --
Methyl propyl trisulfide 1137 0.15 8.51 6.22
1,3,5-Trithiane 1138 0.26 -- --
3-Methyl thio 1-propene 1144 0.03 4.55 2.24
4,5-Dimethyl thiazole 1158 0.02 1.35 --
1-Methyl thio-1-propene 1153 0.12 -- --
L-Borneol 1168 -- -- 0.50
Isobuthyl isothiocyanate 1174 -- 0.05 0.00
3-Vinyl-4H-1,2-dithiin 1175 1.99 -- --
Camphene 1178 -- -- 0.16
[alpha]-Terpineol 1187 -- -- 0.20
Dodecane 1190 -- 0.04 0.10
2-Vinyl-4H-1,3-dithiin 1195 5.87 -- --
1,4-Dimethyl tetrasulfide 1203 Trace 1.68 1.28
Benzene thiazole 1216 -- 1.03 0.72
Geraniol 1254 -- -- 0.14
2,5-Dimethylthiazole 1289 0.01 -- --
1-Hexadecanol 1304 -- -- 1.92
2-Octadecene 1306 -- -- 2.24
Di-2-propenyl trisulfide 1312 29.12 3.22 --
Di-1-propenyl sulfide 1313 -- -- --
Tridecanol 1315 -- -- 0.22
4-Vinyl 2-methoxy phenol 1319 -- -- 2.18
2-Tridecanone 1321 -- 0.16 1.18
Dipropyl trisulfide 1327 -- 12.46 --
Trans-propenyl propyl trisulfide 1333 -- 8.05 --
Cis-propenyl propyl trisulfide 1333 -- 2.43 --
3,5-Dimethyl 1,2,4-thiolane 1335 -- 4.59 --
[alpha]-Copaene 1374 -- 0.08 --
Tridecanal 1412 -- -- 0.14
[beta]-Caryophyllene 1419 -- 0.24 0.12
2-Methyl-3-isothiozolone 1431 -- -- 0.30
[alpha]-Humulene 1453 -- 0.07 --
[beta]-Ionone 1490 -- -- 0.18
2-Undecanone 1500 -- 0.70 0.16
Tridecanal 1518 -- 0.00 0.60
[delta]-Cadinene 1524 -- 0.15 --
[alpha]-Calacorene 1543 -- 0.04 --
Diallyl tetrasulfide 1555 6.35 6.01 --
Nerulidol 1577 -- 2.03 0.00
Hexadecanal 1622 -- 0.01 1.38
T-muurolol 1662 -- 0.74 --
Farnesol 1747 -- 0.53 --
1,4-Dimethyl tetrasulfide 1755 0.28 -- --
1,2-Dithiane-4-one 1758 0.05 -- --
In Total 88.18 92.66 97.18

Note: GC retention indices (RI) calculated against [C.sub.9]-[C.sub.24]
n-alkanes on a HP-5MS column.

Table 2. MICs against Trichophyton spp.

 MIC ([micro]g/ml)
Samples T. rubrum T. erinacei T. soudanense

Allium sativum 64 64 64
A. cepa >128 >128 >128
A. fistulosum >128 >128 >128
Allicin 32 16 16
Allyl sulfide >128 128 128
Allyl disulfide >128 128 128
Ketoconazole 16 8 16

Table 3. Comparison of inhibitory activities against Trichophyton spp.
by disk diffusion test

 Growth inhibition zone (mm) (a)
Sample T. rubrum T. erinacei T. soudanense

1. A. sativum oil 3.3[+ or -]0.76 3.3[+ or -]0.58 4.7[+ or -]0.58
2. A. sativum 17.0[+ or -]0.29 14.3[+ or -]1.53 15.0[+ or -]1.53
 oil--KE
3. Allicin 16.0[+ or -]0.50 17.0[+ or -]0.29 23.0[+ or -]1.15
4. Allicin--KE 19.5[+ or -]0.87 21.5[+ or -]1.50 25.5[+ or -]1.32
5. KE 7.3[+ or -]0.58 9.5[+ or -]1.32 8.0[+ or -]1.00
6. DMSO 0.0[+ or -]0.00 0.0[+ or -]0.00 0.0[+ or -]0.00
7. Ethanol 0.0[+ or -]0.00 0.0[+ or -]0.00 0.0[+ or -]0.00

1. A. sativum oil (200 [micro]g/disk) alone, 2. A. sativum oil plus
ketoconazole (200 [micro]g/disk + 100 [micro]g/disk), 3. allicin
(50 [micro]g/disk) alone, 4. allicin plus ketoconazole (50 [micro]g/disk
+ 100 [micro]g/disk), 5. ketoconazole (100 [micro]g/disk) alone, and, 6
and 7. negative controls.
(a) Values are mean [+ or -] SD (mm) from the experiments in triplicate.
The diameter of the disk (8 mm) is not included.

Table 4. Fractional inhibiting concentrations (FICs) and FIC indices
(FICI)

 T. rubrum T. erinacei T. soudanense
Combinations FIC FICI FIC FICI FIC FICI

1. A. sativum -- ketoconazole 0.06 0.12 0.06 0.09 0.06 0.09
 0.06 0.03 0.03
2. Allicin -- ketoconazole 0.50 0.53 0.50 0.75 0.06 0.56
 0.03 0.25 0.05

FIC of A. sativum oil or allicin =
[MIC in combination with ketoconazole]/
[MIC of A. sativum oil or allicin alone].
FIC of ketoconazole =
[MIC of ketoconazole in combination with the oil or Allicin]/
[MIC of ketoconazole alone].
FICI = FIC of the oil or allicin + FIC of ketoconazole.
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Date:Jun 1, 2006
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