Antifungal Activity of Some Medicinal Plant Extracts Against Candida albicans and Cryptococcus neoformans

The ethanol extracts of clove (Eugenia caryophyllus Bullock & Harrison) and sweet flag (Acorus calamus Linn.) were investigated for their antifungal activity in comparison with eugenol and amphotericin B (AmB) by using the National Committee for Clinical Laboratory Standards (NCCLs) M27-P broth microdilution method. Two medicinal plant extracts, eugenol and amphotericin B were used to determine their minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) against 28 clinical isolates of Candida albicans and 25 clinical isolates of Cryptococcus neoformans. The MICs of clove, sweet flag, eugenol and AmB against C. albicans were 17.41+8.64 mg/ml, 28.8+16.32 mg/ml, 12.16+4.53 mg/ml and 0.23+0.1 μg/ml respectively. The MFCs were 67.5+15.39 mg/ml, >75 mg/ml, 15.4+6.47 mg/ml and 0.47+0.21 μg/ml respectively. The same extracts and antifungal drugs which were tested against C. albicans were also tested against C. neoformans. The MICs were 2.43+0.95 mg/ml, 3.02+1.97 mg/ml, 6.28+3.4 mg/ml and 0.28+0.15 μg/ml, respectively. The MFCs were 22.22+12.71 mg/ml, 30.82+27.11 mg/ml, 10.06+4.9 mg/ml and 0.51+0.25 μg/ml respectively. The results showed that C. albicans was significantly (p<0.01) more susceptible to the extract of clove than sweet flag, whereas C. neoformans was significantly susceptible to the clove extract (p>0.05). Moreover, the extract of clove showed significantly (p<0.01) more potent inhibitory activity against C. neoformans than eugenol, while it showed significantly (p<0.01) less inhibitory activity against C. albicans than eugenol. AmB, the drug of choice for invasive infection treatment, remains as one of the most effective antifungal drugs. These data indicate that the extracts of clove and sweet flag were potential fungistatic agents against yeasts, whereas AmB and eugenol showed fungicidal effects. INTRODUCTION The incidence of fungal infections has increased significantly in the last 20 years (Poeta et al.,1999). In immunocompromised patients, the emergence of candida infections with both primary drug and azole-resistance have been described (Willocks et al., 1991; Cameron et al., 1993; Pfaller et al., 1994). Amphotericin B has been provided for the standard treatment of the most systemic fungal infections (Medoff and Kobayashi, 1980). Unfortunately, treatment with amphotericin B, especially for long-term periods, can lead to adverse effects in patients, or to the development of resistant organisms during the course of therapy (Kovacicova et al., 2001). In the quest for new antifungal agents, low toxicity and broad spectrum fungicidal activities are needed for effective management of the infections. Eugenia caryophyllus Bullock & Harrison (clove) and Acorus calamus Linn. (sweet flag) have eugenol as a major constituent. These medicinal plants have been used in traditional medicine in Thailand and certain medical applications. Both plants have been reported to possess inhibitory properties to filamentous fungi in vitro (Hitokoto et al., 1980; Tragoolpua, 1996). Proc. Int. Conf. on MAP Eds. J. Bernáth et al. Acta Hort. 597, ISHS 2003 218 Only limited knowledge is available regarding the antifungal activities of the plant, which is also used for other technological purposes. Therefore, the aim of this study was to determine the antifungal activities of some medicinal plant extracts against clinical isolates of C. neoformans and C. albicans by using the broth microdilution method. MATERIALS AND METHODS Plant Material Flowers of Eugenia caryophyllus Bullock & Harrison (clove) and rhizomes of Acorus calamus Linn. (sweet flag) were selected for study. Plant extracts were prepared as follows. Air dried plant materials (200 g) were finely ground before being infused in 95% ethanol and sonicated in an ultrasonic bath (Bandelin Sonorex super RF 510H) for 30 min. The extracts were then filtered through Whatman filter paper No.1. The filtrate was evaporated and concentrated using a rotary vacuum evaporator (Fabry et al., 1996; Tragoolpua, 1996). The concentrated plant material was then soaked in 10 ml 95% ethanol. Finally, the ethanolic extracts were dried, weighed and kept at -20°C in sterile bottles. Fungal Isolation Fifty-three clinical isolates (28 of C. albicans and 25 of C. neoformans) were isolated from oral, vaginal, urine and cerebrospinal fluid of human immunodeficiency virus (HIV) – positive or – negative patients from Maharaj Nakorn Chiang Mai and Chiang Rai regional hospital, Thailand. The reference strain, C. albicans ATCC 90028, was included in all susceptibility tests as a control. The isolates were identified according to a standard procedure (Mahon and Manuselis, 1995) and cultured on Sabouraud dextrose agar (SDA) plates (BBL, Cockeysville, Md) at 35 °C for 24-48 h to ensure optimal growth before testing. Assay Medium (Cormican and Pfaller, 1996; National Committee for Clinical Laboratory Standards,1997.) RPMI 1640 powder (with L-glutamine, without bicarbonate; BIOCHOM KG, Leonorenstr.2-6. D-12247 Berlin) was prepared in distilled water and adjusted to pH 7.0 with 0.165 M morpholinopropanesulfonic acid (MOPS, Sigma). This assay medium was filter sterilized by using 0.2 μm millipore size filters (Acrodisc 32, Gelman Sciences), aliquoted and stored at 4 °C until use. Drug and Medicinal Plant Extracts Preparation Ten serial twofold dilutions in RPMI 1640 of AmB, eugenol and two medicinal plant extracts were prepared from stock solutions and arranged in rows, as well as a growth control well (without drug) and a purity control well, which contained yeast-free medium (Cormican and Pfaller, 1996). Stock solutions of AmB deoxycholate (Fungizone, Squibb Industria Farmaceutica S.A., Esplugues-Barcelona, Spain) and eugenol were prepared at 16,000 μg/ml and 500 mg/ml in dimethyl sulfoxide (Sigma), respectively. The final concentration ranges used were 1.6 to 0.003 μg/ml for AmB, 50 to 0.98 mg/ml for eugenol and 75.0 to 0.15 mg/ml for both ethanol plant extracts. Inocula Preparation Yeast inocula were prepared as previously described (Archiesi et al., 1994; Anaissie et al., 1996; Cormican and Pfaller, 1996). Briefly, yeast was grown on Sabouraud dextrose agar plates for 24 h (C. albicans) or 48 h (C. neoformans). For each isolate, five colonies were grown until their diameters were at least 1 mm. Then, the colonies were picked off and suspended in 0.85% saline solution. The suspension was adjusted to the turbidity of a 0.5 McFarland standard at a wavelength of 530 nm. Quantitative colony plate counts were determined on SDA to verify the inoculum size. Testing of antifungal activity was performed in 96-well round-bottomed microtitration