In vitro antifungal activity of eugenol and vanillin ...

In vitro antifungal activity of eugenol and vanillin against Candida albicans and Cryptococcus neoformans CHUENCHIT BOONCHIRD A N D T. W. FLEGEL

Can. J. Microbiol. Downloaded from www.nrcresearchpress.com by Peking University on 06/03/13 For personal use only.

Department of Microbiology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok, Thailand Accepted July 22, 1981

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BOONCHIRD, C., and T. W. FLEGEL.1982. In vitro antifungal activity of eugenol and vanillin against Candida albicans and Cryptococcus neoformans. Can. J. Microbiol. 28: 1235- 1241. Eugenol and vanillin were examined for in vitro antifungal activity against the medically important yeasts, Candida albicans and Cryptococcus nzoformans. Minimal inhibitory concentrations (MIC) and minimal fungicidal concentrations (MFC) were determined for each compound against 3 1 strains of C . albicans and 33 strains of C. neoformans. With eugenol, the mean MIC's for C. albicans and C . neoformans were 625 and 293 pg/mL, respectively, while the mean MFC's were 1209 and 521 pg/mL, respectively. With vanillin, the mean MIC's for C . albicans and C . neoformans were 1250 and 738 pg/mL, respectively, while the mean MFC's were 5000 and 1761 pg/rnL, respectively. With C . albicans, inhibition and retardation of growth were similar for yeast and mycelial forms, but germ tube formation was inhibited at concentrations lower than those which inhibited growth. Short-term toxicity tests with mice using the intraperitoneal route gave maximum tolerated doses of 62.5 mg/kg for eugenol and 125 mg/kg for vanillin and excluded their use as therapeutic agents for systemic mycoses.

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BOONCHIRD, C., et T. W. FLEGEL.1982. In vitro antifungal activity of eugenol and vanillin against Candida albicans and Cryptococcus neoformans. Can. J. Microbiol. 28: 1235- 1241. On a CtudiC l'activitt antifongique de l'eugtnol et de la vanilline contre des levures d'importance mCdicale, Candida albicans et Cryptococcus neoformans. Pour chacun de ces composts, on a mesurt les concentrations minimales inhibitrices (CMI) et les I concentrations minimales fongicides (CMF) vis-a-vis de 31 souches de C , albicans et de 33 souches de C. neoformans. Pour l'eugCno1, les CMI moyennes sont de 625 pg/mLpour C. albicans et de 293 pg/mLpour C . neoformans alors que les CMF sont de 1209 et 521 pg/mL respectivement. Avec la vanilline, les CMI moyennes pour C . albicans et C . neoformans sont de 1250 et 738 pg/mL, alors que les CMF sont de 5000 et 1761 pg/mL respectivement. Chez C . albicans, l'inhibition et le retard de la / croissance sont semblables pour la forme levure et pour la forme mycklienne, mais la formation des tubes germinatifs est inhibte i a des concentrations inftrieures B celles qui inhibent la croissance. Des essais de toxicit6 court terme faits chez la souris par voie j intrapCritonCale ont dCmontrC que les doses maximales tolCrtes Ctaient de 62,s mg/kg pour I'eugCnol et de 125 mg/kg pour la 1 vanilline, ce qui rend ces agents inutilisables dans le traitement des mycoses systCmiques. [Traduit par le journal]

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Introduction Until recently, the general development of chemoherapy for fungal infections has not been extensive, and present only a few antifungal agents are available for e treatment of systemic yeast infections. The most commonly employed are amphotericin B and 5-fluoro. -::: ,.. cytosine (Bennett 1974). Unfortunately, treatment with . these drugs, especially for extended periods, can lead to problems with toxicity to the patients (amphotericin B) or with the development of resistant organisms during the course of therapy (5-fluorocytosine). Since the incidence of systemic yeast infections is increasing, ts are being made to develop new chemotheraagents or combinations of agents to treat them. ile working with the yeast form of the jelly fungus irobasidium magnum, one of the authors (T. W. unpublished) observed marked inhibition of in culture medium containing eugenol and (Fig. 1). Since it was known that this fungus is robably related to the pathogenic yeast Cryptococcus neoformans (Flegel 1976), a preliminary test was

carried out to determine whether these chemicals also inhibited growth of the latter. Affirmative results led to a similar test and similar results with Candida albicans, the most frequently encountered fungal opportunist in systemic infections. Eugenol is a major constituent of oil of clove (Eugenia caryophyllata) and vanillin is a major constituent of alcoholic extracts of the vanilla bean (Vanilla planijiolia, V. tahitensis, and V. pompona). Both have long been used in certain medical applications and in flavoring, both are approved by the Food and Drug Administration of the United States (Anonymous 1978; Opdyke 1975), and both have been reported to possess inhibitory properties (H. Hitokoto, S. Morozumi, T. Wauke, and S. Sakai. 1979. Abstr. Annu. Meet. Am. Soc. Microbiol. 79: 204 (cited in Food Sci. Technol. Abstr. 12: 4T204); Ivanova et al. 1965; Maruzzella and Liguori 1958; Rudman 1963) to filamentous fungi in vitro. The purpose of this study was to quantify the inhibition of several strains of C. neoformans and C.

0008-4166/82/111235-07$01 .OO/O 01982 National Research Council of Canada/Conseil national de recherches du Canada . . . .

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CAN. J. MICROBIOL. VOL. 28, 1982

autosterilization. They were stored at 4OC when not in use. Working solutions were prepared on the day of use by first


&OCH~

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making a1100 further dilutions 1:10 dilution of the stocks in theinsame YNBmedium. and thenThe by maximum concentration of DMSO was 1% at working 4 \ 4\ concentrations of eugenol and vanillin. Inocula were prepared from 24- and 48-h cultures of C. CH;CH=CH2 CHO albicans and C . neoformans, respectively, at 37OC on SDA. Cell suspensions were prepared in YNB and adjusted to an optical density (OD) of 0.02 at 530nm (Bausch & Lomb Spectronic 20 colorimeter). The inoculum consisted of approximately 5 X lo5 cells/mL. The precise inoculum was determined by 10-fold serial dilution in0.85% NaCl and plate Eugenol Vanillin counts on SDA. The experiments for determination of MIC and MFC were FIG. 1. Structural formulae of eugenol and vanillin. ~erformedin du~licatefor each isolate. One millilitre of broth h 12 x 75 m& tubes was inoculated with 0.05 mL of a albicans by eugenol and vanillin, and via toxicity tests, standardized suspension of the test organism, For growth to the possibility using them treat controls, broth free of antimicrobial agent was also inoculated. mycoses caused by these fungi. Since C . albicans is After incubation at 37Oc for 18-24 h with C. albicans and for dimorphic, it would be further necessary to determine 48 h with C . neoformans the tubes were exarninedand the MIC whether inhibitory activity was selective for only the was recorded. The MIC was defined as the lowest concentration of antimicrobial agent which inhibited macroscopic yeast or mycelial phase. growth. The MFC was determined by subculturing on SDA Materials and methods approximately 0.01 mL from tubes showing no macroscopic growth and from control tubes containing-no antimicrobial Chemicals and culture media Eugenol (USP XVIII) was purchased from E. Merck agent. After subsequent incubation at 37OC for 24 or 48 h, the (Darmstadt, Germany). Vanillin was supplied by BDH MFC (defined as the lowest concentration of antimicrobial Chemicals Ltd. (Poole, England). Dimethylsulfoxide agent from which subcultures were negative or yielded fewer (DMSO) and propylene glycol were acquired from Fisher than three colonies) was recorded (Shadomy and EspinelScientific Co. All chemicals used were of analytical grade. All Ingroff 1980). of the culture media and constituents in this study were obtained from Difco Laboratories (Detroit, MI). These were Kinetics of inhibition of growth and killing of organisms by eugenol and vanillin Sabouraud dextrose agar (SDA), Sabouraud dextrose broth The following description is given for C . albicans but it (SDB), Bacto-yeast nitrogen base (YNB), Bacto-dextrose, Bacto-peptone, and Bacto-sucrose. L-Asparagine was sup- applies also to C . neoformans with variations concerning it included in parentheses. Candida albicans CBS 5763 (or C. plied by Sigma Chemical Co., (St. Louis, MO). neoformans IF0 1319) from stock cultures were transferredon Organisms SDA and maintained at 37OC to verify purity. Then, the culture Candida alblcans CBS 5763 and Cryptococcus neoformans was used to inoculate 50 mL of SDB in a 150-mL Erlenmeyer IF0 1319 were used as reference strains. Thirty strains of C . flask, incubated on a gyratory shaker (New Brunswick albicans were isolated from clinical sources. Thirty-two Scientific Co., New Brunswick, NJ) at 37OC and 200 rpm for strains of C. neoformans were obtained from human origin and 18-24h (48 h). This culture was transferred into fresh SDB avian excreta (pigeon, dove, budgengar). The identity of all and adjusted to an OD of 0.05 at 530 nm. The cell suspension strains was confirmed on the basis of mcroscopic morphology corresponded to 1.2 X lo6 cells/mL. Five rnillilitres of this and biochemical reactions (Silva-Hutner and Cooper 1980). standard suspension was added to 45mL of SDB in flasks All strains were maintained at 4OC on SDA slants. containing 156, 313, 625, 1250, and 2500 kg eugenol/mL and 313,6257 1250,2500, and 5000 ygvanillin/*. The cultures Susceptibility testing were incubated at 37OC and 200rpm. Samples of 3 mL each The broth dilution technique was employed (Shadomy and were taken at 4-h intervals to determine the OD and the number Espinel-Ingroff 1980) to determine susceptibility of test of viable cells. The OD was measured using uninoculated SDB organisms to eugenol and vanillin. Susceptibility was excontaining matching concentrations of eugenol and vanillin as pressed as the minimal inhibitory concentration (MIC) and the blanks. minimal fungicidal concentration (MFC). The steps involved in determining MIC and MFC are described below. Yeast and mycelial forms of C. albicans Each experiment was performed in YNB supplementedwith In order to evaluate the effect of eugenol and vanillin on the 1% dextrose and 0.15% L-asparagine. The medium was yeast and mycelial phases of C . albicans, a two-part experiadjusted to pH 7 before filter sterilization (Beggs et al. 1976). ment was performed. In the first, the effect of the two agents on Stock solutions of eugenol and vanillin were prepared by jlament initiation was tested. In the second, the effect of the dissolving each in DMSO. These solutions were protected two agents on jlament growth (extension) was tested. The from light and allowed to stand for 30 min before use to permit experiment was performed in sucrose peptone medium (SP)

BOONCHIRD AND FLEGEL

TABLE1. In vitro antifungal activity of eugenol and vanillina


MIC, kg/mL

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Organism

Eugenol

Vanillin

Eugenol

Vanillin

C . albicans C. neoformans Degree of significanceb

31 33 -

625kO 293k52 p