Comparison between e cacy of allicin and uconazole against Candida albicans in vitro and in a systemic candidiasis mouse model

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1 RESEARCH LETTER Comparison between e cacy of allicin and uconazole against Candida albicans in vitro and in a systemic candidiasis mouse model Alireza Khodavandi 1, Fahimeh Alizadeh 2, Nabil S. Harmal 1, Shiran M. Sidik 3, Fauziah Othman 4, Zamberi Sekawi 5, Mohammad Ali Farboodniay Jahromi 6, Kee-Peng Ng 7 & Pei Pei Chong 1,8 1 Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia; 2 Islamic Azad University, Yasuj Branch, Yasuj, Iran; 3 Department of Pathology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia; 4 Department of Human Anatomy, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia; 5 Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Selangor, Malaysia; 6 Fars Technological and Environmental Research Center, Division of Medicinal Plants Research, Shiraz, Iran; 7 Department of Medical Microbiology, Faculty of Medicine, University Malaya, Kuala Lumpur, Malaysia; and 8 Institute of Bioscience, Universiti Putra Malaysia, Selangor, Malaysia MICROBIOLOGY LETTERS Correspondence: Pei Pei Chong, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. Tel.: ; fax: ; cpp@medic.upm.edu.my Received 26 August 2010; revised 16 November 2010; accepted 16 November Final version published online January DOI: /j x Editor: Matthias Brock Keywords allicin; fluconazole; in vitro; in vivo. Introduction Recently, the incidence of systemic candidiasis, which is caused by Candida spp., predominantly Candida albicans, has increased (Chowta et al., 2007). This increase over the last two decades has caused a rise in the use of antifungal drugs (Pereira-Cenci et al., 2008). Azoles such as fluconazole or ketoconazole are usually used for treatment of systemic fungal infections. However, one of the biggest problems faced in clinical practice is the emergence of resistance to most of these azole drugs due to mutation (Odds et al., 2003; Looi et al., 2005). Clinically adverse effects are also seen with the use of azoles (Al-Mohsen & Hughes, 1998). Therefore the most urgent challenge in pharmaceutical Abstract The efficacy of allicin compared with fluconazole in alleviating systemic Candida albicans infections was evaluated both in vitro and in vivo through a systemic candidiasis mouse model. Determination of in vitro minimum inhibitory concentrations (MICs) for different C. albicans isolates revealed that both allicin and fluconazole showed different MICs that ranged from 0.05 to 12.5 mgml 1 and 0.25 to 16 mgml 1, respectively. A time kill study showed a significant effect of allicin (P o 0.01) against C. albicans, comparable to that of fluconazole. Scanning electron microscopy observation revealed that, similar to fluconazole, allicin produced structural destruction of C. albicans cell surface at low MIC and lysis or puncture at high MIC concentrations. Treatment of BALB/c mice systemically infected with C. albicans showed that although the allicin treatment (at 5mgkg 1 day 1 ) was slightly less efficacious than fluconazole treatment in terms of the fungal load reduction and host survival time, it was still effective against C. albicans in terms of mean survival time, which increased from 8.4 to 15.8 days. These results demonstrate the efficacy of anticandidal effects of allicin both in vitro and in an animal model of candidiasis and affirm the potential of allicin as an adjuvant therapy to fluconazole. research is the discovery and development of new antifungals from plant and microbial sources. Allicin (diallyl thiosulfinate), one of the sulfur compounds from garlic, has been shown to possess antifungal activity (Yamada & Azuma, 1977). It has been shown that after crushing fresh garlic cloves, allinase rapidly converts the released allin (precursor of allicin) into allicin (Ankri & Mirelman, 1999). Allitridium (diallyl trisulfide), one of the breakdown products from allicin, has also been found to show antifungal activity in vitro (Davis et al., 2003) and in vivo (Davis et al., 1990). Ajoene, one of the other products from allicin, has been shown to have antifungal and antiparasitic properties (Ledezma et al., 2008). Potent antifungal activity of allicin against major species of Candida in vitro

2 88 A. Khodavandi et al. has been reported in our previous work (Khodavandi et al., 2010) but, thus far, there have been very few studies investigating the activity of allicin as an anticandidal agent in vivo. In the present study, the anticandidal activity of pure allicin demonstrated its strong potential activity both in vitro and in vivo. Materials and methods Antifungal agents Allicin was acquired from Alexis Biochemicals Co. (purity Z98%, Batch No. ALX , San Diego, CA) and dissolved at a concentration of 10 mg ml 1 in a mixture of methanol, water and formic acid (60 : 40 : 0.1), and then stored at 20 to 80 1C until use. Fluconazole was purchased from Sigma Chemicals Co. (St. Louis, MO). The stock solution was prepared by dissolving in dimethyl sulfoxide at 5 mg ml 1. The stock solutions were stored frozen at 70 1C until use. For the in vitro study, allicin and fluconazole drug dilutions ranged from 0.05 to 25 mgml 1 and 0.03 to 64 mgml 1, respectively, but for the in vivo work, the two dosages of these drugs were 1 and 5mgkg 1 day 1, respectively. In vitro assays The in vitro efficacy of allicin and fluconazole was measured against C. albicans ATCC and nine clinical isolates obtained from the diagnostic microbiological laboratories of the University of Malaya Medical Center, Kajang Hospital, Selangor and Seremban Hospital, Negeri Sembilan by the broth microdilution method (NCCLS M27 A 2 ). The inhibitory effect of antifungal agents at different time intervals was then studied (C. albicans ATCC 14053). All clinical samples were isolated from patients with systemic candidiasis. The lowest concentrations of antifungals that can inhibit 50% and 90% of Candida growth (minimum inhibitory concentrations, MIC 50 and MIC 90, respectively) compared with untreated growth control were determined as described previously (Khodavandi et al., 2010). In summary, 100 ml of antifungal agents in different concentrations (in standard RPMI 1640 medium with 0.2% glucose buffered to ph 7.0 with M morpholinophosphonyl sulfate) were inoculated with 100 ml of inoculum containing between and yeast cells ml 1 in a 96-well microplate (Brand , Wertheim, Germany). The microplates, including drugs and cells, were incubated at 35 1C and MICs were measured at 530 nm from two independent experiments in three separate technical replicates using an EMax s microplate reader after 24 h. For investigation of the inhibitory effect of allicin and fluconazole on growth of Candida, two different inoculum sizes of C. albicans ATCC ( and cells ml 1 in RPMI 1640 as described before) were grown in the presence of 0.1 mgml 1 of allicin and 2 mgml 1 of fluconazole based on MIC concentration. After 0-, 2-, 4-, 6-, 8-, 12- and 24-h incubation at 35 1C, 100 ml ofthissolutionwascollectedand 10-fold serial dilutions were made and plated on Sabouraud dextrose agar (Difco Laboratories, Detroit, MI). Colonies were counted after 24-h incubation at 37 1C and the number of CFU was calculated. Scanning electron microscopy for morphological observation of Candida after treatment with allicin A suspension of C. albicans ATCC containing cells ml 1 in RPMI 1640 was mixed with different dilutions of allicin and fluconazole (1/2 MIC, 1 MIC and 10 MIC) and incubated at 35 1C for 24 h. Cells were fixed in 2% v/v glutaraldehyde in phosphate-buffered saline (ph 7.2) and washed with sodium cacodylate buffer. For postfixation, samples were rinsed in 1% osmium tetroxide for 2 h at 4 1C, washed again with sodium cacodylate buffer and then dehydrated with ascending ethanol series. After that, samples on coverslips were put into a critical point dryer and then stuck onto the stub. The specimens were coated with gold and observed through a JEOL JSM 6400 scanning electron microscope. In vivo assays For the experiments on the animal model of systemic candidiasis, 4 6-week-old female BALB/c mice were infected intravenously through the tail vein with 200 ml per mouse of C. albicans ATCC ( yeast cells ml 1 ). The mice were divided into five experimental groups of 12 mice each. In the first two groups of mice, allicin (200 ml per mouse) was administered intravenously once daily for 5 days beginning 1 h after Candida injection (postinfection) at 1 and 5 mg kg 1 day 1, respectively (Shadkchan et al., 2004). For the third and fourth groups of mice, fluconazole (200 ml per mouse) was administered via the intraperitoneal route once daily for 5 days starting 1-h postinfection at 1 and 5mgkg 1 day 1, respectively (Rex et al., 1998). For the untreated control group, 200 ml of normal saline was injected into each mouse at 1-h postinfection. The infection was followed up for 28 days and evaluated in terms of mortality and morbidity. For studies of tissue burden, two randomly chosen mice were sacrificed from each experimental group on days 2, 4, 7, 10, 14 and 28 after infection (Shadkchan et al., 2004). Kidneys, liver and spleen from each mouse were aseptically removed and homogenized in 1 ml of sterile normal saline and cultured on Sabouraud dextrose agar plates as explained in the time kill study, and assessed by determination of fungal colonization of viscera.

3 Investigating the effect of allicin on systemic candidiasis 89 Histopathologic analyses were performed for a qualitative confirmation of the result. Tissues were fixed in 10% formalin, then blocked by paraffin wax and cut with a microtome (Leica, model RM2025) in 4-mm thickness. Hematoxylin and eosin and periodic acid-schiff staining were used to observe the tissue and presence of fungal elements. All animal care procedures were supervised and approved by the University of Putra Malaysia Animal Ethics Committee (ACUC NO.: UPM/FPSK/PADS/BRUUH/00278). Statistical analysis For quantitative statistical analysis of inhibitory effects of drugs in vitro and also reduction of fungal load in tissues of mice, data were analyzed in terms of normality and one-way ANOVA was carried out. Moreover, a log rank test was used to determine the survival time. P values of o 0.05 were considered significant. Statistical analysis was performed using SPSS version 17 software (SPSS Inc., Chicago, IL). All experiments were carried out at least in triplicate. 16 mgml 1, respectively, for fluconazole. All samples were sensitive to fluconazole and drug resistance was not seen. The potency of allicin and fluconazole in decreasing the cell number of C. albicans ATCC after 0, 2, 4, 6, 8, 12 and 24 h was significant compared with the control growth (Fig. 1). Figure 1a and b indicate the inhibitory effect of allicin and fluconazole on different inoculum sizes of C. albicans. The significant reduction of Candida treated with allicin and fluconazole started after 4-h incubation (P o 0.01) in comparison to untreated control for both inoculum sizes (Fig. 1). Candida albicans cells grown in RPMI 1640 medium at 35 1C showed typical yeast cells with a smooth surface after 24 h, but cells treated with increasing concentration of allicin or fluconazole displayed changes in surface morphology, with the cell surface becoming rough and irregular. According to Lemar et al. (2005) the main reason for this phenomenon could be a decreased cytoplasmic volume. It was also observed in the present study that higher concentrations of the antifungal agents (such as 10 MIC) destroyed the cell surface, inducing puncture in allicin-treated Results Table 1 shows the MICs of allicin and fluconazole against C. albicans ATCC and some clinical isolates. The results are representative of two independent experiments arranged in triplicate. The MIC 50 and MIC 90 of these isolates ranged from 0.05 to 0.78 mgml 1 and 0.1 to 12.5 mgml 1, respectively for allicin, and from 0.25 to 4 mgml 1 and 2 to Table 1. Interaction of allicin and fluconazole against clinical and standard isolates of Candida albicans by broth microdilution assay after 24-h incubation at 35 1C Allicin w Fluconazole w MIC 50 / Isolates MIC 90 C. albicans ATCC MIC range MIC 50 / MIC 90 MIC range 0.05/ / C. albicans / / C. albicans / / C. albicans / / C. albicans / / C. albicans / / C. albicans / / C. albicans / / C. albicans / / C. albicans / / Strains with numbers only without the ATCC prefix are clinical isolates from local hospitals. w MIC values are mgml 1 from three independent experiments. The MIC values of strains ATCC and 3092 were determined previously and shown in our previous publication (Khodavandi et al., 2010). Fig. 1. Time kill curves of allicin (0.1 mgml 1 ) and fluconazole (2 mgml 1 ) against Candida albicans ATCC at different time intervals: (a) Candida ml 1 ; (b) Candida ml 1. Antifungals: (&) allicin, (m) fluconazole and () untreated control.

4 90 A. Khodavandi et al. (a) (i) (iii) (ii) (iv) (b) (i) (iii) (ii) (iv) Fig. 2. Scanning electron micrograph of Candida albicans ATCC treated with allicin (a) or fluconazole (b) in different concentrations based on MICs: (i) 1/2 MIC; (ii) 1 MIC; (iii) 10 MIC; and (iv) untreated control. samples and causing cell lysis in fluconazole-treated samples (Fig. 2). The results of fungal load determination in the liver, kidney and spleen at different time points indicated a significant reduction of CFUg 1 of the tissue (P o 0.001) starting from the second day postinfection for different dosages of the antifungals. In addition, the reduction of Candida cells CFU in tissues after 28 days postinfection

5 Investigating the effect of allicin on systemic candidiasis 91 Table 2. Tissue fungal loads obtained from mice infected with Candida albicans ATCC and treated with allicin, fluconazole or untreated Day after infection Allicin Allicin w Fluconazole Fluconazole w Untreated control Log 10 CFU g 1 of liver SD a a a a b b b b a c d a c b e d c b a e d c b a e d c b a Log 10 CFU g 1 of spleen SD b a ab ab b c a bc ab d c b b a d d c b a e d c b a e c c b a Log 10 CFU g 1 of kidney SD a a a a b c ab b a d b a c c d a a a a b c b a a d d c b a Results in a row with different superscripts differ significantly (P o 0.05) using Duncan s test. 1mgkg 1 day 1. w 5mgkg 1 day 1. Table 3. The survival time in different groups of treated mice Mice-treated groups MST SE (days) Mortality after 28 days (%) Allicin (1 mg kg 1 day 1 ) (P = 0.163) w Allicin (5 mg kg 1 day 1 ) (P = 0.067) w Fluconazole (1 mg kg 1 day 1 ) (P o 0.05) w Fluconazole (5 mg kg 1 day 1 ) (P o 0.005) w Saline treated The results are representative of three independent experiments for 12 mice in each group. w P value for treated groups compared with untreated control. ranked from 5 mg kg 1 day 1 fluconazole 4 1mgkg 1 day 1 fluconazole 4 5mgkg 1 day 1 allicin 4 1mgkg 1 day 1 allicin (Table 2). As described before, the mortality and morbidity of the treated mice were evaluated for 28 days postinfection. Table 3 also shows the mean survival time (MST) of mice treated with different drugs. Moreover, based on statistical analysis of log rank = in this study, comparison of the mean of survival time between treated and control groups indicated significant differences (P o 0.05) (Fig. 3). Discussion Previous reports have demonstrated the antifungal activity of allicin in vitro against Aspergillus, Trichophyton and Candida spp. (Yamada & Azuma, 1977; Aala et al., 2010). On the other hand, the antifungal potential of allicin against Aspergillus spp. was presented by Shadkchan et al. (2004) using a mouse model of systemic aspergillosis. In this study, we attempted to investigate the potency of allicin against C. albicans, the predominant fungal species isolated from human infections. Allicin alone could exhibit antifungal activity, and when used in synergy with antimicrobial agents, it increased the efficacy of the therapeutic agents (Aala et al., 2010; Khodavandi et al., 2010). For example, combination of allicin with amphotericin B and fluconazole has been demonstrated to have a significant synergistic effect in a mouse model of systemic candidiasis (An et al., 2009; Guo et al., 2010). Garlic and some of its derivatives destroy the Candida cell membrane integrity (Low et al., 2008), inhibit growth (Lemar et al., 2002) and produce oxidative stress (Lemar

6 92 A. Khodavandi et al. Fig. 3. Cumulative mortality of mice (n = 10) infected with Candida albicans ATCC and treated with allicin at 1 and 5 mg kg 1 day 1 (P = 0.163, 0.067, respectively) and fluconazole at 1 and 5 mg kg 1 day 1 (P o 0.01). Mice treated with different dosages of antifungal agents: (m) fluconazole, 5 mg kg 1 day 1 ; (.) fluconazole, 1 mg kg 1 day 1 ; ( ) allicin, 5 mg kg 1 day 1 ; (^) allicin, 1 mg kg 1 day 1 ; () untreated control. et al., 2005) in C. albicans. Most of these abilities are related to an SH-modifying potential, because the activated disulfide bond of allicin has an effect on thiol-containing compounds such as some proteins; however, the main targets of allicin on Candida are not well understood. It has been demonstrated that the antifungal activity of allicin in vivo may be related to some secondary metabolites such as ajoene, diallyl trisulfide and diallyl disulfide, because the chemical structure of allicin is too unstable and converts to these secondary products immediately (Miron et al., 2004). Nonetheless, little is known about the potential in vivo activity of allicin against Candida. In this study, we used fluconazole as the standard anticandidal drug for comparison against allicin. The MICs of allicin and fluconazole against C. albicans fell within the ranges and mgml 1, respectively (Table 1), which is similar to findings from previous reports (Ankri & Mirelman, 1999; Khodavandi et al., 2010). All of the samples were sensitive to fluconazole and drug resistance was not seen. The time kill study demonstrated a significant inhibition of Candida growth comparing untreated controls against those treated with allicin and fluconazole, using inoculum sizes of Candida cells ml 1 (P o 0.05) and Candida cells ml 1 (P o 0.001) after 2- and 4-h incubation, respectively. This demonstrates that allicin decreased the growth of C. albicans almost as efficiently as fluconazole (P ) for both inoculum sizes of Candida, demonstrating a comparable ability to inhibit the growth of the yeast cells (Fig. 1). The presence of pits on the cell surface and cellular collapse with high concentrations of allicin indicates that the cell membrane could be one of the targets of allicin in Candida (Lemar et al., 2002), whereas fluconazole in high concentrations can destroy the Candida cell entirely (Fig. 2). It has been reported that the use of high doses of organosulfur compounds from garlic, such as diallyl sulfide, diallyl trisulfide, allyl methyl sulfide, allyl methyl trisulfide and dipropyl sulfide, predispose to the development of preneoplastic lesions in rat livers (Lee & Park, 2003). It is also important to note that allicin could be toxic for mammalian cells in high concentrations ( 4 60 mgml 1 ), but the lethal dosage for fungus is lower (Rabinkov et al., 1998). In this study, two dosages of antifungal agents, 1 and 5mgkg 1 day 1, were selected. The results showed that allicin could reduce the morbidity and the fungal load in tissues of mice infected with C. albicans. However, these effects cannot be directly attributed to allicin, as it is not stable and converts immediately to other products such as ajoene, which may also have antifungal potential. The fungal load in liver of treated mice showed a significant reduction with increasing time intervals. Although after 1-week postinfection, the fungal load in mice treated with 5 mg kg 1 day 1 of allicin was lower (log 10 mean CFUg 1 = ) compared with the other treated groups, mice treated with 5 mg kg 1 day 1 of fluconazole showed a more significant decrease in fungal load (log 10 mean CFUg 1 = ) thereafter. The results seen in other organs were similar to those seen in the liver (Table 2). Our findings also showed that the fungal load for all concentrations of antifungals during the first week were approximately similar, but after this time the differences between treated groups were significant. This may be due to the intrinsic murine immune responses of BALB/c mice (Ashman & Papadimitriou, 1988) infected at sites surrounding the infection for as long as 5 days postinfection, whereas treated mice were able to suppress Candida infection after at least 1 week. On the other hand, our data suggest that the conditions were approximately constant after 2 weeks postinfection until the last day of the experiments. Data analysis showed a significant reduction in mortality for the two groups treated with fluconazole when compared with untreated control (P o 0.05), whereas no significant difference was observed between the allicin groups treated with 1 and 5 mg kg 1 day 1 dosages and the untreated control group at levels P = and P = 0.067, respectively. However, the survival study suggests that allicin could increase the MST until 16 days, whereas the untreated

7 Investigating the effect of allicin on systemic candidiasis 93 control group showed an MSTof 8.5 days. The percentage of mortality was reduced to 50% by treatment with allicin (Table 3, Fig. 3). The results from the MIC determination seem to suggest a more significant anticandidal potential in vitro of allicin than of fluconazole. However, the time kill curve showed that allicin is comparable to fluconazole in terms of fungal load reduction. The combined results from both the survival studies and fungal load reduction studies in the present work demonstrate that allicin is slightly less efficacious than fluconazole in the treatment of candidiasis. Therefore, it is necessary to discover better treatment modalities or to increase the dosage of allicin, which will require further experiments. Designing a new model of treatment with different dosages of allicin and related compounds as well as a combination of allicin with azoles should be investigated. The molecular mechanisms of the actions of allicin could be investigated further to determine its probable targets in Candida cells. Acknowledgements This project was funded through the Research University Grant Scheme (RUGS) sponsored by the university and a Science Fund sponsored by the Ministry of Science, Technology and Innovation. References Aala F, Yusuf UM, Khodavandi A & Jamal F (2010) In vitro antifungal activity of allicin alone and in combination with two medications against six dermatophytic fungi. Afr J Microbiol Res 4: Al-Mohsen I & Hughes WT (1998) Systemic antifungal therapy: past, present and future. Ann Saudi Med 18: An M, Shen H, Cao Y, Zhang J, Cai Y, Wang R & Jiang Y (2009) Allicin enhances the oxidative damage effect of amphotericin B against Candida albicans. Int J Antimicrob Ag 33: Ankri S & Mirelman D (1999) Antimicrobial properties of allicin from garlic. Microbes Infect 1: Ashman RB & Papadimitriou JM (1988) Murine candidiasis: strain dependence of host responses after immunization. Immunol Cell Biol 66: Chowta MN, Adhikari P, Rajeev A & Shenoy AK (2007) Study of risk factors and prevalence of invasive candidiasis in tertiary care hospital. Indian J Crit Care Med 11: Davis LE, Shen JK & Cai Y (1990) Antifungal activity in human cerebrospinal fluid and plasma after intravenous administration of Allium sativum. Antimicrob Agents Ch 34: Davis SR, Perrie R & Apitz-Castro R (2003) The in vitro susceptibility of Scedosporium prolificans to ajoene, allitridium and a raw extract of garlic (Allium sativum). J Antimicrob Chemoth 51: Guo N, Wu X, Yu L, Liu J, Meng R, Jin J, Lu H, Wang X, Yan S & Deng X (2010) In vitro and in vivo interactions between fluconazole and allicin against clinical isolates of fluconazoleresistant Candida albicans determined by alternative methods. FEMS Immunol Med Microbiol 58: Khodavandi A, Alizadeh F, Aala F, Sekawi Z & Chong PP (2010) In vitro investigation of antifungal activity of allicin alone and in combination with azoles against Candida species. Mycopathologia 169: Ledezma E, Maniscalchi MT & Espinoza DL (2008) Synergy between ajoene and ketoconazole in isolates of Microsporum canis. Rev Iberoam Micol 25: Lee BM & Park KK (2003) Beneficial and adverse effects of chemopreventive agents. Mutat Res : Lemar KM, Turner MP & Lloyd D (2002) Garlic (Allium sativum) as an anti-candida agent: a comparison of the efficacy of fresh garlic and freeze-dried extracts. J Appl Microbiol 93: Lemar KM, Passa O, Aon MA, Cortassa S, Müller CT, Plummer S, O Rourke B & Lloyd D (2005) Allyl alcohol and garlic (Allium sativum) extract produce oxidative stress in Candida albicans. Microbiology 151: Looi CY, D Silva EC, Seow HF, Rosli R, Ng KP & Chong PP (2005) Increased expression and hotspot mutations of the multidrug efflux transporter, CDR1 in azole-resistant Candida albicans isolates from vaginitis patients. FEMS Microbiol Lett 249: Low CF, Chong PP, Yong PVC, Lim CSY, Ahmad Z & Othman F (2008) Inhibition of hyphae formation and SIR2 expression in Candida albicans treated with fresh Allium sativum (garlic) extract. J Appl Microbiol 105: Miron T, Bercovici T, Rabinkov A, Wilchek M & Mirelman D (2004) [3H]Allicin: preparation and applications. Anal Biochem 331: Odds FC, Brown AJP & Gow NAR (2003) Antifungal agents: mechanisms of action. Trends Microbiol 11: Pereira-Cenci T, Del Bel Cury AA, Crielaard W & Ten Cate JM (2008) Development of Candida-associated denture stomatitis: new insights. J Appl Oral Sci 16: Rabinkov A, Miron T, Konstantinovski L, Wilchek M, Mirelman D & Weiner L (1998) The mode of action of allicin: trapping of radicals and interaction with thiol containing proteins. Biochim Biophys Acta 1379: Rex JH, Nelson PW, Paetznick VL, Lozano-Chiu M, Espinel- Ingroff A & Anaissie EJ (1998) Optimizing the correlation between results of testing in vitro and therapeutic outcome in vivo for fluconazole by testing critical isolates in a murine model of invasive candidiasis. Antimicrob Agents Ch 42: Shadkchan Y, Shemesh E, Mirelman D, Miron T, Rabinkov A, Wilchek M & Osherov N (2004) Efficacy of allicin, the reactive molecule of garlic, in inhibiting Aspergillus spp. in vitro, and in a murine model of disseminated aspergillosis. J Antimicrob Chemoth 53: Yamada Y & Azuma K (1977) Evaluation of the in vitro antifungal activity of allicin. Antimicrob Agents Ch 11: