JAC Advance Access originally published online on April 14, 2006
Journal of Antimicrobial Chemotherapy 2006 57(6):1231-1234; doi:10.1093/jac/dkl132
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In vitro anticandidal activity of xanthorrhizol isolated from Curcuma xanthorrhiza Roxb
1 Department of Biotechnology & Bioproducts Research Center (BRC), Yonsei University 134 Sinchon-dong, Seodaemun-gu, Seoul 120-749, Korea 2 Research Center for Bioresources and Biotechnology, Bogor Agricultural University Bogor 16880, Indonesia 3 Department of Laboratory Medicine & Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Yonsei University Seoul 120-749, Korea
*Corresponding author. Tel: +82-2-2123-5881; Fax: +82-2-362-7265; E-mail: jkhwang{at}yonsei.ac.kr
Received 30 July 2005; returned 19 September 2005; revised 15 March 2006; accepted 20 March 2006
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Abstract |
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Objectives: Xanthorrhizol, isolated from the methanol extract of Curcuma xanthorrhiza Roxb., was investigated for its anticandidal activity using six Candida species.
Methods: The in vitro susceptibility tests for xanthorrhizol were carried out in terms of MIC and minimal fungicidal concentration (MFC) using the NCCLS M27-A2 broth microdilution method. Time–kill curves were determined to assess the correlation between MIC and fungicidal activity of xanthorrhizol at concentrations ranging from 0 MIC to 4x MIC.
Results: All Candida species showed susceptibility to xanthorrhizol in the MIC range 1.0–15.0 mg/L for Candida albicans, 1.0–10 mg/L for Candida glabrata, 2.0–8.0 mg/L for Candida guilliermondii, 2.5–7.5 mg/L for Candida krusei, 2.5–25 mg/L for Candida parapsilosis and 2.0–8.0 mg/L for Candida tropicalis. Time–kill curves demonstrated that xanthorrhizol was able to kill the Candida strains with MFCs of 20 mg/mL, 15 mg/mL, 12.5 mg/mL, 10 mg/L, 30 mg/mL and 10 mg/L for C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis, respectively.
Conclusions: The potent anticandidal activity of xanthorrhizol may support the use of C. xanthorriza for the treatment of candidiasis.
Keywords: C. xanthorriza , MIC , MFC , antifungals
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Introduction |
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Candida species are now recognized as a significant cause of hospital-acquired infection. Candida albicans is the organism most often associated with serious fungal infection and it is showing increased resistance to traditional antifungal agents.1 Recently, non-C. albicans species, such as Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis and Candida tropicalis, have also shown dramatic increases in fungal infections and antifungal resistance.2
The development of resistance in known fungal pathogens and emergence of new fungal pathogens intrinsically resistant to the currently available antibiotics demonstrate the urgent importance of identifying novel antifungal agents. The antifungal effect of essential oils of many plants and their specific anticandidal activity are well established.3 Curcuma xanthorrhiza Roxb., commonly known as Javanese turmeric, has been traditionally used in South-East Asian countries for food and medicinal purposes. Xanthorrhizol, isolated from C. xanthorrhiza, has been reported to possess anticariogenic activity against Streptococcus mutans.4 In the present research, we found that xanthorrhizol exhibited antifungal activity against C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis. To our knowledge, this is the first report showing that xanthorrhizol possesses anticandidal activity against pathogenic non-C. albicans species.
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Materials and methods |
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Candida isolates, growth conditions and inoculum
The reference Candida species (C. albicans, C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis and C. tropicalis) used in this study were all obtained from the American Type Culture Collection (Rockville, MD, USA). Clinical Candida isolates were obtained from The Research Institute of Bacterial Resistance, College of Medicine, Yonsei University, Korea. The clinical isolates were from body fluid, blood, urine, sputum and genitals of the patients. The species were identified by conventional methods or using Vitex YBC cards (bioMérieux, Marcy l'Étoile, France), according to the manufacturer's instructions. All clinical Candida isolates were susceptible to fluconazole and voriconazole. The strains were cultured in Sabouraud dextrose broth (SDB) or on Sabouraud dextrose agar (SDA) (Difco) for 48 h at 35°C. A standardized inoculum (a McFarland standard) for each isolate was 5 x 106 cfu/mL.5
Anticandidal agents
Xanthorrhizol was isolated as the pure form from the ethylacetate fraction of the methanol extract of C. xanthorrhiza according to the method of Hwang et al.4 Xanthorrhizol was dissolved in 10% dimethylsulfoxide (DMSO) to obtain 1 g/L stock solutions. DMSO at 10% was found not to kill the Candida. Amphotericin B, purchased from Sigma Chemical Co. (St Louis, MO, USA), was dissolved in sterile distilled water to obtain 1 g/L stock solution. The concentrations of individual antifungal agents ranged as follows: xanthorrhizol, 10–1000 mg/L; amphotericin B, 1–75 mg/L.
In vitro susceptibility tests
In vitro susceptibility tests were performed to evaluate MICs and minimal fungicidal concentrations (MFCs) using the method described in the guidelines of NCCLS M27-A2.5 Briefly, MICs were determined for all species with the adjusted inoculum suspension of 5 x 106 cfu/mL by diluting the suspension 1:100 with MOPS-buffered RPMI 1640 medium to give a final inoculum concentration of 5 x 104 cfu/mL. Individual antifungal agents were diluted 1:10 in MOPS-buffered RPMI 1640 medium containing 5 x 104 cfu/mL inoculum, yielding an initial inoculum of 4.5 x 103 cfu/mL. The final concentration of individual antifungal agents ranged as follows: xanthorrhizol, 1–100 mg/L; amphotericin B, 0.1–7.5 mg/L. A 200 µL aliquot of each suspension was placed in 96-well round-bottomed microtitration plates. The plates were incubated at 35°C, and endpoints were read visually after 48 h. MFCs were determined for each anticandidal agent/Candida species/medium combination as outlined for MICs by removing the medium from each well showing no visible growth and subculturing onto SDA plates. The plates were incubated at 35°C until growth was seen in the growth control plates.
Time–kill curves
Time–kill assays were performed in standard MOPS-buffered RPMI 1640.5 Before the tests were performed, the Candida species were subcultured at least twice and grown for 24 h at 35°C on SDA plates. The adjusted inoculum suspension of 5 x 106 cfu/mL was diluted 1:10 in MOPS-buffered RPMI 1640 medium to give a final inoculum concentration of 5 x 105 cfu/mL. Each concentration of xanthorrhizol was diluted 1:10 in MOPS-buffered RPMI 1640 medium containing 5 x 105 cfu/mL. This procedure yielded an initial inoculum of 4.5 x 105 cfu/mL. Final concentrations of xanthorrhizol were 0x MIC, 0.5x MIC, MIC, 2x MIC and 4x MIC for each Candida species. Cultures (5 mL final volume) were incubated at 35°C with agitation (200 rpm). At pre-determined time points (0, 2, 4, 8, 12, 24 and 48 h) 100 µL aliquots were removed and transferred to Eppendorf tubes, centrifuged (3900 g at 4°C for 1 min) and rinsed twice with 0.9 mL of sterile distilled water to obtain xanthorrhizol-free cells. Pellets were suspended in 100 µL of sterile distilled water and serially diluted. An appropriate volume (100, 40 or 20 µL, depending on the dilution and the concentration of xanthorrhizol) was spread onto SDA plates and incubated at 35°C for 48 h or more (until the colonies were seen on the plates) to determine the numbers of cfu/mL.
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MICs and MFCs of xanthorrhizol compared with those of amphotericin B are summarized in Table 1. All species were susceptible to xanthorrhizol. Interestingly, there were three strains of clinical C. albicans and one strain each of clinical C. glabrata and clinical C. parapsilosis that were not killed by more than 20 mg/L amphotericin B, but the strains were killed by xanthorrhizol, at 5.0–10.0 mg/L for clinical C. albicans, 5.0 mg/L for clinical C. glabrata and 7.5 mg/L for clinical C. parapsilosis.
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The killing activity of xanthorrhizol for each species is represented in Figure 1. The fungicidal activity of xanthorrhizol was fast acting against all Candida tested; the reduction in the number of cfu/mL was >3 log units (99.9%). The fungicidal endpoints for C. albicans and C. parapsilosis were reached after 48 h of incubation at 4x MIC, while those for C. guilliermondii, C. krusei and C. tropicalis were reached after 12 h, and that for C. glabrata was reached after 24 h, at 4x MIC. The strongest fungicidal activity of xanthorrhizol was observed for C. guilliermondi, C. krusei and C. tropicalis. The fungicidal endpoint of C. guilliermondii, C. krusei and C. tropicalis was reached after 12 h at 4x MIC and after 24 h at 2x MIC. These data demonstrated that the ability to kill Candida species is dependent on the concentration of xanthorrhizol and the species.
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Discussion |
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In this research, we used MOPS-buffered RPMI 1640 medium for measuring the MICs of amphotericin B and xanthorrhizol. The results showed that MICs of amphotericin are high, particularly for C. parapsilosis. A broth microdilution technique using RPMI 1640 is suitable for most antifungals except for amphotericin B, as apparent resistance to this drug is not always detected in RPMI 1640. MOPS-buffered RPMI 1640 is not an ideal medium for measuring the MICs of amphotericin.5
Generally, MICs and MFCs of amphotericin B against Candida species are in good agreement with previous reports.2,6 The isolates with reduced susceptibility to amphotericin B may also exhibit reduced susceptibility to xanthorrhizol. There was no previous report for xanthorrhizol against pathogenic non-C. albicans species. In the present study xanthorrhizol has been shown to possess powerful in vitro activity against a broad range of distantly related Candida species. An anticandidal which has broad-spectrum activity against non-C. albicans species may have clinical applications. Snydman7 reported that the spectrum of invasive fungal infections is changing and rising with the frequencies of infections due to non-C. albicans species.
Among the six species of Candida used in this study, C. guilliermondii, C. krusei and C. tropicalis were the most susceptible to xanthorrhizol followed by C. glabrata, C. albicans and C. parapsilosis. Knowledge of the in vitro pharmacodynamic characteristics of C. guilliermondii is still poor and limited to amphotericin B.8 C. glabrata infection is second or third in frequency after C. albicans and is associated with a high mortality rate in at-risk hospitalized patients. However, its epidemiology, pathogenesis, treatment and antifungal resistance are poorly understood.7 Moreover, compared with other Candida species, especially with C. albicans, higher levels of all azole antifungals are required for comparable activity against C. glabrata isolates.9 Our study showed that C. glabrata was more susceptible to xanthorrhizol compared with C. albicans. This result suggested that xanthorrhizol may be effective in treating C. glabrata infection. C. parapsilosis is the most frequently isolated yeast species from hospital patients and the most common non-C. albicans species causing infection in cancer patients.10 Our results also demonstrated that xanthorrhizol has the ability to inhibit and to kill C. parapsilosis in vitro.
Overall, xanthorrhizol may be potentially valuable as a natural compound for fungal infections, even though its anticandidal activity is weaker than that of amphotericin B against six species of Candida. However, the clinical applications of xanthorrhizol are challenging to interpret in this study due to a lack of pharmacokinetic and safety studies. Unanswered concerns exist about the in vivo efficacy and toxicity of xanthorrhixol. Future research towards these objectives, based on animal models, may resolve these issues.
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None to declare.
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Acknowledgements |
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We thank Dr Jim Kaput for critical suggestions to improve the manuscript.
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References |
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1 Jarvis WR. (1995) Epidemiology of nosocomial fungal infections, with emphasis on Candida species. Clin Infect Dis 20:1526–30.[Web of Science][Medline]
2 Nguyen MH, Peacock JE, Morris AJ, et al. (1996) The changing face of candidemia: emergence of non-Candida albicans species and antifungal resistance. Am J Med 100:617–23.[CrossRef][Web of Science][Medline]
3 Knoblock K, Pauli A, Iberl N. (1989) Antibacterial and antifungal properties of essential oil components. J Ess Oil Res 1:119–28.
4 Hwang JK, Shim JS, Baek NI, et al. (2000) Xanthorrhizol: a potential antibacterial agent from Curcuma xanthorrhiza against Streptococcus mutans. Planta Med 66:196–7.[Medline]
5 National Committee for Clinical Laboratory Standards. (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeasts—Second Edition: Approved Standard M27-A2 (NCCLS, Wayne, PA, USA).
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Canton E, Peman J, Gobernado M, et al. (2004) Patterns of amphotericin B killing kinetics against seven Candida species. Antimicrob Agents Chemother 48:2477–82.
7 Snydman DR. (2003) Shifting patterns in the epidemiology of nosocomial Candida infections. Chest 123:500–3S.
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Bonaventura GD, Spedicato I, Picciani C, et al. (2004) In vitro pharmacodynamic characteristics of amphotericin B, caspofungin, fluconazole, and voriconazole against bloodstream isolates of infrequent Candida species from patients with hematologic malignancies. Antimicrob Agents Chemother 48:4453–6.
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Sanguinetti M, Posteraro B, Fiori B, et al. (2005) Mechanisms of azole resistance in clinical isolates of Candida glabrata collected during a hospital survey of antifungal resistance. Antimicrob Agents Chemother 49:668–79.
10 Krcmary V and Barnes AJ. (2002) Non-albicans Candida spp. causing fungaemia: pathogenicity and antifungal resistance. J Hosp Infect 50:243–60.[CrossRef][Web of Science][Medline]
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