Journal of Ethnopharmacology 72 (2000) 111 – 117 www.elsevier.com/locate/jethpharm Antifungal activity of volatile constituents of Eugenia dysenterica leaf oil Théo R. Costa a, Orionalda F.L. Fernandes a, Suzana C. Santos b, Cecı́lia M.A. Oliveira b, Luciano M. Lião b, Pedro H. Ferri b,*, José R. Paula c, Heleno D. Ferreira d, Beatriz H.N. Sales e, Maria do Rosário R. Silva a a Departamento de Microbiologia, Instituto de Patologia Tropical e Saúde Pública, Uni6ersidade Federal de Goiás, 74605 -050 Goiânia, GO, Brazil b Laboratório de Bioati6idade Molecular, Instituto de Quı́mica, C.P. 131, Uni6ersidade Federal de Goiás, 74001 -970 Goiânia, GO, Brazil c Faculdade de Farmácia, Uni6ersidade Federal de Goiás, 74605 -050 Goiânia, GO, Brazil d Departamento de Biologia Geral, Instituto de Ciências Biológicas, Uni6ersidade Federal de Goiás, 74001 -970 Goiânia, GO, Brazil e Departamento de Quı́mica, Uni6ersidade Federal de São Carlos, C.P. 676, 13565 -905 São Carlos, SP, Brazil Received 4 February 2000; received in revised form 6 March 2000; accepted 16 March 2000 Abstract The essential oil from the hydrodistillation of Eugenia dysenterica leaves consisted mainly of b-caryophyllene and a-humulene as the major sesquiterpene, while limonene and a-thujene were the major monoterpene hydrocarbons. The main oxygenated mono and sesquiterpene constituents were a-terpineol and b-caryophyllene oxide, respectively. The oil was investigated against eight strains of Candida albicans, 35 strains of Cryptococcus neoformans var. neoformans, and two C. neoformans var. gattii isolated from HIV-infected individuals with candidosis or cryptococcal meningitis using the agar dilution method. Based on the minimal inhibitory concentration (MIC) values, the most significant results were obtained against Cryptococcus strains. It was observed that 22 strains were inhibited at a concentration of 250 mg/ml, whereas four exhibited potent inhibition with MIC values below 125 mg/ml against 106 UFC/ml organisms. We found MICs ]3.12 mg/ml for 91.6, 50 and 30% of all Cryptococcus strains in relation of amphotericin B, fluconazole and itraconazole, respectively. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Antifungal activity; Candida albicans; Cryptococcus neoformans; Essential oil; Eugenia dysenterica; Immunocompromised patients 1. Introduction * Corresponding author. Within the framework of our research on natural antiinfectious agents, investigations were con- 0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 8 7 4 1 ( 0 0 ) 0 0 2 1 4 - 2 112 T.R. Costa et al. / Journal of Ethnopharmacology 72 (2000) 111–117 ducted on Eugenia dysenterica DC. (synon. Stenocalyx dysentericus Berg., Myrtus dysenterica M.), a shrubby tree of Southern Brazil with edible cherry-like fruits, in Brazil called ‘cagaiteira’ (Corrêa, 1984). The plant is well known in Brazilian Cerrado medicine (Septı́mio, 1994) and the leaves are part of preparations used for medical diarrhoeic care and dysentery. The fruits are considered to have a possible increase in their economic importance through cultivation, which is already occurring with E. uniflora L. (‘pitangueira’). Antimicrobial activities have been reported for essential oils and expressed juice of the Eugenia genus, including dermatophyte strains isolated from patients with dermatophytosis (Lima et al., 1993). While the dermatophyte group of fungi is of common occurrence in the tropics (Caceres et al., 1991), the incidence of infections due to Candida and Cryptococcus species, particularly Candida albicans and Cryptococcus neoformans, associated with acquired immune deficiency syndrome (AIDS) have increased dramatically in the last two decades in all countries (Greene, 1990). Some antifungal drugs, such as polyene macrolides and azoles, are currently used in antifungal therapies with certain limitations due to side effects (Kullberg, 1997). Therefore, the development of more effective and less toxic antifungal agents is required for the treatment of patients with common and rare fungal infectious diseases (Janssen and Cauwenbergh, 1990). In our effort to screen antifungal extracts of native plants from Brazilian Cerrado, we evaluated the antifungal activity of plants used in traditional medicine for several purposes including antimicrobial effects. The aim of this study was to test the antifungal activity of E. dysenterica and the determination of the composition of its essential oil which, to our knowledge, have not yet been performed. In the present paper, we analysed the composition of hydrodistillate from leaves and their antimicrobial activity against eight strains of Candida albicans, 35 strains of Cryptococcus neoformans var. neoformans, and two C. neoformans var. gattii isolated from human immunodeficiency virus-infected individuals with oral candidosis or cryptococcal meningitis. 2. Materials and methods 2.1. Plant material Eugenia dysenterica DC. (Myrtaceae) was collected in Catalão city, Goiás State, Brazil, in April 1997, authenticated by Professor Heleno D. Ferreira, Departamento de Botânica, Universidade Federal de Goiás. Voucher specimens are deposited at the herbarium of the Universidade Federal de Goiás. 2.2. Extraction The essential oil of E. dysenterica leaves was isolated by hydrodistillation in a Clevenger-type apparatus of aqueous homogenates of mature and immature leaves dried at room temperature (32°C) until no more condensing oil could be seen (6 h). The aqueous phase was saturated with sodium chloride and extracted with diethyl ether. The ether was dried over anhydrous sodium sulfate and concentrated at room temperature. 2.3. Analysis Oil sample analysis was performed on a Shimadzu QP5000 gas chomatograph interfaced to a mass spectrometer (GC – MS) instrument employing the following conditions: Column: DB-5 (J & W) fused silica capillary column (30 m long ×0.25 mm i.d.×0.25 mm film thickness composed of 5% phenylmethylpolysiloxane) connected to an ion trap detector operating in Electronic Impact mode at 70 eV; carrier gas: Helium (1 ml/min); injector and ion-source temperatures were 225 and 250°C, respectively. The oven temperature was programmed from 60°C (isothermal for 2 min), with an increase of 3°C/min, to 240°C, then 10°C/min to 280°C, ending with a 10 min isothermal at 280°C. The calculation of the retention indexes was made through co-injection with a n-alkanes series (Van Den Dool and Kratz, 1963). Identification of the oil constituents were made based on the retention indexes (Adams, 1995) and by comparison of mass spectra with the library, and by co-injection of authentic sample (b-caryophyllene oxide) (Sigma, St. Louis, MO). Compound con- T.R. Costa et al. / Journal of Ethnopharmacology 72 (2000) 111–117 centrations were calculated from GC peak areas and they were arranged in order of GC (DB-5) elution. 2.4. Microorganisms All the eight strains of Candida albicans, two strains of Cryptococcus neoformans var. gattii and 35 of C. neoformans var. neoformans used in this study were clinical isolates from human immunodeficiency virus-infected individuals with oral candidosis or patients with cryptococcal meningitis in the Hospital of Tropical Diseases, Goiás State, Brazil. The strains were identified according to standard procedure (Van Rij, 1984) by Dr Orionalda F. L. Fernandes, Departamento de Microbiologia, Universidade Federal de Goiás. Cryptococcus neoformans varieties were determined in Canavanine-Glycine-Bromothymol Blue agar (Difco, USA) following the procedure described by Kwonchung et al. (1982). Isolates were kept by repeated transfer to Sabouraud Dextrose agar medium (BBL, Beckton Dickinson Microbiology Systems, USA). C. neoformans var. gattii (serotype B) and C. neoformans var. neoformans (serotypes A and D) (Public Health Service, MA) were kindly provided by Dr Claudete R. de Paula, Departamento de Microbiologia, Universidade de São Paulo and used as reference strains from previous knowledge of their sensitivity towards antifungal agents. 2.5. Drugs Fluconazole (Pfizer, Belgium), amphotericin B (Bristol-Myers Squibb, USA) and itraconazole (Johnson and Johnson, USA) were suspended in sterile physiological Tris buffer (pH 7.4, 0.05 M) and included in assay as positive controls. All other chemicals were purchased from Sigma Chemical (USA). 2.6. Antifungal assay Antifungal activity was measured using a dilution in agar technique (Alves and Cury, 1992). The essential oil (100 mg) was solubilized in 1 ml of dimethyl sulfoxide (DMSO) and serially two- 113 fold diluted in Yeast Nitrogen Base Phosphate (YNBP) agar (Merck, Germany) to obtain a concentration range of 15.6– 1000 ml/ml. YNBP agar plates containing only DMSO diluted in the same way, which did not influence fungal growth, were included as controls. All fungal strains were suspended in sterile physiological Tris buffer (pH 7.4, 0.05 M), homogenised and adjusted to an OD (530 nm) of 0.05 (equivalent to 1×106 UFC/ml). This suspension was used as the inoculum for the test in the agar plates. Fungal suspensions (3 ml) were inoculated using a automatic micropippete (Brand), and plates (diameter: 25 cm) were incubated at 37°C for 48 h. The minimal inhibitory concentration (MIC) was defined as the minimal concentration of the essential oil which completely inhibited the visible growth of the fungus. The sensitivity of all cryptococcal strains to antifungal agents amphotericin B, fluconazole, and itraconazole, included as positive controls, was tested using the same technique. All antifungal assays were tested in duplicate. 3. Results and discussion Simple hydrodistillation of E. dysenterica leaves produced a clear, colourless to pale yellow oil, with a yield of 0.15%. The essential oil was a mixture with more than 50 compounds, 42 of which were identified, corresponding to 87.8% of the total oil. The identified components with their percentages and relative retention times on a DB5 column are listed in Table 1. GC– MS analysis of the essential oil indicated that it consists of ca. 60% sesquiterpenoids (17.6% oxygenated sesquiterpenes and 42.4% sesquiterpenes hydrocarbons) and ca. 28% monoterpenoids (9.6% oxygenated monoterpenes and 18.2% monoterpenes hydrocarbons). b-Caryophyllene oxide (5.4%) was the major oxygenated sesquiterpene and bcaryophyllene (14.8%), together a-humulene (10.9%), were the major sesquiterpenes hydrocarbons. a-Terpineol (6.1%) and limonene (5.5%), together a-thujene (5.6%) and sabinene (3.9%), were the major oxygenated monoterpene and monoterpenes hydrocarbons, respectively. Similar relative amounts of these major constituents are 114 T.R. Costa et al. / Journal of Ethnopharmacology 72 (2000) 111–117 Table 1 Constituents of E. dysenterica DC. leaf oil Compound no. Name RRTb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 a-Thujene a-Pinene Sabinene b-Myrcene a-Phellandrene o-Cymene Limonene 1,8-Cineole b-Phellandrene b-Z-Ocimene b-E-Ocimene Isoterpinolene b-Linalool a-Fenchol Canphene hydrate Isoborneol 4-Terpineol a-Terpineol a-Copaene b-Caryophyllene Cis-thujopsene Unknown Unknown a-Humulene 9-Epi-(E)-caryophyllene g-Muurolene Germacrene D b-Selinene a-Selinene a-Muurolene a-Bulnesene g-Cadinene Unknown d-Cadinene a-Calacorene Unknown Caryophyllene alcohol b-Caryophyllene oxidea Globulol Unknown Unknown Humulene epoxide II Unknown Unknown Unknown 1-Epi-cubenol Cedr-8(15)-en-9-a-ol Cubenol a-Muurolol Selin-11-en-4-a-ol Unknown Unknown Khusinol 5.47 5.85 6.69 7.13 7.57 8.27 8.47 8.52 8.79 9.18 9.58 10.76 11.22 11.75 13.20 13.98 14.50 15.14 23.41 25.36 26.11 26.30 26.54 26.76 27.01 27.69 27.85 28.07 28.45 28.69 28.92 29.24 29.56 29.68 30.37 30.74 31.44 32.00 32.17 32.62 32.73 33.03 33.21 33.34 33.82 33.96 34.10 34.32 34.48 34.79 34.92 35.20 35.42 Total volatile fraction (%) 100.0 a Co-injection with authentic sample. Relative retention time (min). c Kovat’s index determined on DB-5 column; for conditions see Section 2.3 b KIc 932 945 974 989 1003 1020 1025 1026 1033 1042 1052 1081 1092 1105 1140 1159 1172 1187 1374 1419 1438 1442 1448 1454 1460 1476 1480 1485 1494 1500 1506 1514 1521 1524 1541 1550 1567 1580 1585 1595 1598 1606 1611 1614 1627 1631 1635 1641 1645 1653 1657 1664 1670 % 4.6 0.1 3.9 1.1 0.2 0.1 5.5 0.3 1.3 0.6 0.2 0.4 1.1 0.4 0.3 0.8 0.6 6.1 3.8 14.8 1.2 0.5 0.5 11.0 0.3 0.8 0.2 0.4 0.5 0.7 1.9 0.8 0.8 5.8 0.2 0.8 1.1 5.4 0.4 0.7 1.9 3.2 0.2 1.8 4.2 2.5 2.3 1.0 0.3 0.9 0.8 0.2 0.5 T.R. Costa et al. / Journal of Ethnopharmacology 72 (2000) 111–117 115 Table 2 In vitro antifungal activity of essential oil from E. dysenterica leaves against different Cryptococcus neoformans clinical isolates and reference strains MICa (mg/ml) Cryptococcus neoformans Variety neoformans 500 250 125 62.5 31.25 15.6 Total 11(31.3)d 21(60) 1(2.9) 1(2.9) 1(2.9) 35(100) Reference strains gattii Serotype Ab 1(50) 1(50) Serotype Db Serotype Bc 1(100) 1(100) 1(100) 1(100) 1(100) 2(100) 1(100) a Minimal inhibitory concentration. C. neoformans var. neoformans. c C. neoformans var. gattii. d Number of strains (%). b reported for clove terpenes from the E. caryophyllata (Zheng et al., 1992). Amounts of bcaryophyllene oxide and b-caryophyllene higher than 20% were reported also for essential oils of leaves of E. in6olucrata, E. plicato-costata, and E. schuechiana (Henriques et al., 1993). On the other hand, smaller relative amounts of these compounds were reported in essential oils from leaves of E. tinguyensis, E. rostrifolia (Henriques et al., 1993), E. caryophyllata (Sangwan et al., 1990), and for different chemotypes of E. uniflora L. (synon. E. michelii Lam., Stenocalyx michelii Berg., S. brasiliensis Berg.). The oxygenated sesquiterpenes, selina-1,3,5(11)-trien-8-one, oxidoselina-1,3,7(11)-trien-8-one and furanodiene, which represents more than 50% of the essential oil of E. uniflora leaves (Nigerian chemotype) (Weyerstahl et al., 1988), and more than 65% of those of Northeastern Brazilian chemotype (Morais et al., 1996) were not detected in the leaves of E. dysenterica. The preliminary antifungal screening of essential oil afforded the Cryptococcus species as the only sensitive strains in randomised eight yeast isolates from patients with cryptococcal meningitis. Despite the fact that the volatile constituents from leaves, clove buds and fruits of Eugenia may act as effective antifungal defences against various plant pathogens (Wilson et al., 1997), dermatophytes (Lima et al., 1993) and opportunistic fungi (Adebajo et al., 1989), the essential oil from leaves of Eugenia dysenterica at concentrations up to 1000 mg/ml did not show antifungal activity against any of the Candida strains tested. In contrast, the oil was active against all eight Cryptococcus strains. At concentrations below 500 mg/ml, the extract suppressed the yeast growth. The above results prompted us to investigate the MIC against all 37 Cryptococcus clinical isolates (Table 2). It is important to note that 70.3% of Cryptococcus isolates were inhibited by the essential oil at concentrations below 250 mg/ml. The sensitivity of the same fungal isolates to commonly used antifungal agents is described in Table 3. 5.4% of the strains were resistant to amphotericin B, with 12.5 mg/ml needed for inhibition. Likewise, fluconazole and itraconazole showed MICs value at 25 mg/ml and ]12.5 mg/ml for 8.1% and 10.8% of the all strains, respectively. These MIC values were similar to that of the essential oil against 2.7% of strains isolated. Other than these, the essential oil was less potent than antifungal agents against the fungal strains tested. Bioassay-directed fractionation of the essential oil is in progress to isolate and identify the compounds responsible for the antifungal activity. 116 Antifungals C. neoformans Minimal inhibitory concentration (mg/ml) 25 Amphotericin B Fluconazole Itraconazole a Var. neoformans Var. gattii Serotype Aa Serotype Bb 6.25 3.12 1.56 1(2.9)c 1(50) 3(8.8) 27(79.4) 1(50) 3(8.8) 0.78 0.19 0.05 0.025 B0.025 1(100) 1(100) Var. neoformans Var. gattii Serotype A Serotype B 3(11.5) Var. neoformans Var. gattii Serotype A Serotype B 3(7.9) C. neoformans var. neoformans. C. neoformans var. gattii. c Number of strains (%). b 12.5 2(7.6) 9(34.6) 1(50) 12(46.2) 1(50) 1(100) 1(100) 1(2.6) 3(7.9) 5(13.1) 11(28.9) 1(50) 1(2.6) 14(36.8) 1(50) 1(100) 1(100) T.R. Costa et al. / Journal of Ethnopharmacology 72 (2000) 111–117 Table 3 In vitro activity of antifungal agents against Cryptococcus neoformans clinical isolates and reference strains T.R. Costa et al. / Journal of Ethnopharmacology 72 (2000) 111–117 Acknowledgements We thank Dr A.J. Marsaioli (Universidade Estadual de Campinas-IQ) for his encouragement, critical comments and for supplying the authentic sample. Thanks are also due to CNPq/PIBIC for fellowships to T.R.C. and Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (CNPq), PADCT III/QEQ (Grant No. 620166/975), and Fundação de Apoio à Pesquisa (FUNAPE/UFG) for their financial support. References Adams, R.P., 1995. Identification of Essential Oil Components by Gas Chromatography/Mass Spectroscopy. Allured, Illinois. Adebajo, A.C., Oloke, K.J., Aladesanmi, A.J., 1989. Antimicrobial activities and microbial transformation of volatile oils of Eugenia uniflora. Fitoterapia 60, 451 – 455. Alves, S.H., Cury, A., 1992. Estudo comparativo entre as técnicas de diluição em caldo para Candida. Revista de Patologia Tropical 34, 259 – 262. Caceres, A., Lopez, B.R., Giron, M.A., Logemann, H., 1991. 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