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Antifungal activity of some Brazilian Hypericum species
ARTICLE IN PRESS
Phytomedicine 12 (2005) 236–240 www.elsevier.de/phymed
Antifungal activity of some Brazilian Hypericum species R. Fennera, M. Sortinob, S.M. Kuze Ratesa, R. Dall’Agnola, A. Ferraza, A.P. Bernardia, D. Albringa, C. No¨ra, G. von Posera,, E. Schapovala, S. Zacchinob a
Programa de Po´s Graduac¸a˜o em Cieˆncias Farmaceˆuticas, UFRGS, Universidade Federal do Rio Grande do Sul, Porto Alegre, Av. Ipiranga, 2752, RS, Porto Alegre 90610-000, Brazil b Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario- Suipacha Rosario, Argentina Received 23 July 2003; accepted 19 November 2003
Abstract Crude methanolic extracts and fractions from the aerial parts of seven species of Hypericum (H. caprifoliatum Cham. and Schltdl., H. carinatum Griseb., H. connatum Lam., H. ternum A. St.-Hil., H. myrianthum Cham. and Schltdl., H. piriai Arechav. and H. polyanthemum Klotzsch ex Reichardt) growing in southern Brazil were analyzed for their in vitro antifungal activity against a panel of standardized and clinical opportunistic pathogenic yeasts and filamentous fungi, including dermatophytes, by the agar dilution method. Chloroform and hexane extracts of H. ternum showed the greatest activity among extracts tested. r 2004 Elsevier GmbH. All rights reserved. Keywords: Hypericum; Antifungal activity; Agar dilution method
Introduction Human infections, particularly those involving the skin and mucous membranes, are increasing at an alarming rate, especially in tropical and subtropical developing countries, with dermatophytes and Candida sp. being the most common pathogens. This increase is directly related to the growing population of immunocompromised individuals, resulting from changes in medical practice such as the use of intensive chemotherapy and immunosuppressive drugs. HIV and other diseases causing immunosuppression have also contributed to this problem (Groll et al. 1996; Denning et al. 1997; Portillo et al. 2001). Human mycoses are not always successfully treated, since the available antifungal drugs are ineffective, Corresponding author. Tel.: +55-51-33165258; fax: +55-5133305610. E-mail address: [email protected] (G. von Poser).
0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2003.11.004
produce many adverse effects, show recurrence, or lead to the development of resistance. It is therefore essential to research for more effective and less toxic new antifungal agents (Zacchino et al. 1999). Although most antibiotics in clinical use have been obtained from microorganisms, a renewed interest in plant antimicrobials has emerged during the last 20 years. Because only a very small fraction of the known plant species of the world have been evaluated for the presence of antifungal compounds, and there is a rapid rate of plant species extinction, many efforts are necessary to collect and to screen plants in order to avoid the lost of valuable sources of potential leads for the development of novel and environmentally safe antifungal agents. In the course of our ongoing project for the detection of antifungal compounds in native Southern Brazilian plants, we selected species of the genus Hypericum to begin our screening for antifungal compounds. The genus includes various species used in traditional
ARTICLE IN PRESS R. Fenner et al. / Phytomedicine 12 (2005) 236–240
medicine in different parts of the world, from which several antifungal (De´costerd et al. 1986), antibiotic (Ishiguro et al. 1986), antiviral (Jacobson et al. 2001) and anticancer (Jayasuriya et al. 1989) compounds have been isolated. Most active compounds are of a phenolic nature, such as flavonoids, xanthones and phloroglucinol derivatives. Among the latter some are related to the well-known hyperforin, isolated from H. perforatum (Trifunovic et al. 1998), while others possess a phloroglucinol unit conjugated with a filicinic acid moiety (Ishiguro et al. 1986; Rocha et al. 1995). Phloroglucinol derivatives, found frequently in the lipophilic fractions of several Hypericum species, have demonstrated antibacterial activities against microorganisms such as Staphylococcus aureus, Bacillus cereus, B. subtilis and Nocardia gardenen. Their presence could justify the popular use of some Hypericum species as wound healing agents and in the treatment of some infectious diseases (Ishiguro et al. 1986; Jayasuriya et al. 1991; Yamaki and Ishiguro 1994; Rocha et al. 1995). Other substances, such as benzopyrans (De´costerd et al. 1986), xanthones (Ishiguro et al. 1999) and flavonoids (Ishiguro et al. 1993) are reported to be responsible for the antimicrobial activity against various bacteria and fungi in some species of Hypericum. In this paper, we report the antifungal evaluation of 17 extracts from 7 species of Hypericum, some of them reported in the Brazilian traditional medicine as useful in treating infectious diseases (Correˆa 1984; Mentz et al. 1997). Extracts of H. caprifoliatum Cham. and Schltdl., H. carinatum Griseb., H. connatum Lam., H. ternum A. St.-Hil., H. myrianthum Cham. and Schltdl., H. piriai Arechav. and H. polyanthemum Klotzsch ex Reichardt, were tested against a panel of human opportunistic pathogenic fungi by the agar dilution method. The most active extracts were then evaluated against panels of different clinical isolates of Candida species as well as different strains of Candida albicans, Trichophyton rubrum and Trichophyton. mentagrophytes, in order to gain insight into the actual antifungal potentiality of these extracts for the development of new antifungal agents.
Material and methods Plant material Aerial parts of Hypericum caprifoliatum Cham. and Schltdl. were collected in the ‘‘Morro Santana’’, Porto Alegre in May, 1998. H. myrianthum Cham. and Schltdl, and H. polyanthemum Klotzsch ex Reichardt were collected in Paraı´ so do Sul and Cac¸apava do Sul, in July and August, 1998, respectively. H. connatum Lam. and H. piriai Arechav. were collected in Capa˜o do Lea˜o in
237
January, 1999. H. carinatum Griseb. was collected in Glorinha, RS, in January, 1999 and H. ternum A. St.-Hil. in Sa˜o Francisco de Paula, RS, in October, 1999. Voucher specimens have been deposited in the herbarium of the Federal University of Rio Grande do Sul (ICN).
Preparation of plant extracts Dried and powdered plant material was submitted to two distinct extraction processes depending on the species employed. H. myrianthum, H. piriai and H. connatum (100 g) were extracted with methanol (drug/ solvent ratio=1:10 w/v) by maceration (3 24 h), yielding 11%, 15% and 15%, respectively, of total methanol crude extracts (TMCE). H. caprifoliatum, H. polyanthemum, H. ternum and H. carinatum (ca. 80 g) were extracted successively in a Soxhlet apparatus with petroleum ether, chloroform, and methanol for 12 h. These extracts were evaporated to dryness in vacuo at 45 1C, affording 3.0–4.0% PET, 2.5–3.5% CLF, and 6.0–7.0% MET, respectively.
Microorganisms and media For the antifungal evaluation, strains from the American Type Culture Collection (ATCC), Rockville, MD, USA and CEREMIC (C), Centro de Referencia Micolo´gica, Facultad de Ciencias Bioquı´ micas y Farmace´uticas, Suipacha 531-(2000)-Rosario, Argentina were used. C. albicans ATCC10231, C. tropicalis C131, C. parapsilosis C118, C. glabrata C115, C. lusitaniae C124, C. krusei C117, C. kefyr C123, C. colliculosa C122; strains of C. albicans C125, C126, C127, C128, C129, C130, C131, Saccharomyces cerevisiae ATCC9763, Cryptococcus neoformans ATCC32264, Aspergillus flavus ATCC9170, A. fumigatus ATTC26934, A. niger ATCC9029, Epidermophyton floccosum C114, strains of T. rubrum: C110, C113, C133, C134, C135, C136, C137, C138, C139, C 140, C141, strains of T. mentagrophytes ATCC9972, C199, C201, C202, C206, C126, C127, C128, C129, C130, C131, Microsporum canis C112, and M. gypseum C115. Yeasts were grown on Sabouraudchloramphenicol agar slants, for 48 h at 30 1C. Cell suspensions were adjusted to 106 viable yeast cells/ml, in sterile distilled water. Filamentous fungi were maintained on Sabouraud-dextrose agar (SDA, Oxoid) and subcultured every 15 days to prevent pleomorphic transformations. Spore suspensions were obtained according to reported procedures (Wright et al. 1983) and adjusted to 106 spores/ml.
Antifungal assays The fungistatic activities of different extracts were evaluated via the agar dilution method by using
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Sabouraud-chloramphenicol agar for yeasts and filamentous fungi according to reported procedures (Mitscher et al. 1972; Zacchino et al. 1998, 1999; Feresin et al. 2001). Stock solutions of extracts were diluted in DMSO to produce serial decreasing dilutions ranging from 0.25 to 1000 mg/ml. The final concentrations of DMSO in the assay did not exceed 2%. Using a micropipette, an inoculum of 5 ml of the yeast, cell or spore suspensions was added to each Sabouraudchloramphenicol agar tube. The antifungal agents ketoconazole (Janssen Pharmaceutical) and amphotericin B (Sigma Chemical Co.) were included in the assay as positive controls. Drug-free solution was also used as a blank control. The tubes were incubated for 24, 48 or 72 h at 30 1C (according to the control fungus growth) up to 15 days for dermatophyte strains. The minimal inhibitory concentration (MIC) value was defined as the lowest extract concentration, showing no visible fungal growth after incubation time. MIC50
Table 1.
Results and discussion To carry out the antifungal evaluation with agar dilution assays, extracts in concentrations of up to 1000 mg/ml were incorporated into the growth media according to Material and methods. Extracts with MIC values p1000 mg/ml were considered active. Among the Hypericum extracts tested, only the PET and CLF extracts of H. ternum showed significant antifungal activity. Results are shown in Table 1. These extracts present a broad spectrum of action inhibiting all the fungi tested, including C. albicans, the causative fungus of many superficial infections and over 90% of
In vitro evaluation of the antifungal activity of different extracts of Hypericum species (Hypericaceae)
Plant species Voucher specimen H. caprifoliatum Bordignon, 1400 H. myrianthum Bordignon, 1402 H. piriai Bordignon, 1528 H. ternum Bordignon et al., 1715 H. carinatum Bordignon et al., 1520 H. connatum Bordignon and Salazar, 1527 H. polyanthemum Bordignon et al., 1429 Amphotericin B Ketoconazole a
and MIC90 values are the lowest extract concentration at which 50% and 90% of the clinical isolates were inhibited (Marco et al. 1998).
Extracta
MIC value (mg/ml)b C.a.c
C.n.d
S.c.e
A.fu.f
A.fl.g
A.n.h
M.c.i
M.g.j
E.f.k
T.r.l
T.mm
PET CLF MET TMCE
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
TMCE
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
MET PET CLF MET PET CLF TMCE
n.a n.a. 250 n.a n.a n.a n.a
n.a 250 250 n.a n.a n.a n.a
n.a 100 100 n.a n.a n.a n.a
n.a 500 250 n.a n.a n.a n.a
n.a 500 1000 n.a n.a n.a n.a
n.a 500 250 n.a n.a n.a n.a
n.a n.a. 250 n.a n.a n.a n.a
n.a 250 250 n.a n.a n.a n.a
n.a 100 250 n.a n.a 500 n.a
n.a 500 500 n.a n.a n.a n.a
n.a 100 100 n.a n.a n.a n.a
MET PET CLF
n.a n.a n.a 8 0.7
n.a n.a n.a 2 0.4
n.a n.a n.a 5 6.25
n.a n.a n.a 20 3
n.a n.a n.a 30 3
n.a n.a n.a 12.5 0.4
n.a n.a n.a 15 30
n.a n.a n.a 6.25 6.25
n.a 500 n.a 15 0.3
n.a n.a n.a 15 25
n.a n.a n.a 12.5 6.25
MET—methanolic extract; PET—petroleum ether extract; CLF—chloroform extract; TMCE—total methanol crude extract. n.a.—not active. c Candida albicans ATCC 10231. d Cryptococcus neoformans ATCC 32264. e Saccharomyces cerevisiae ATCC 9763. f Aspergillus fumigatus ATCC 26934. g Aspergillus flavus ATCC 9170. h Aspergillus niger ATCC 9029. i Microsporum canis C 112. j Microsporum gypseum C 115. k Epidermophyton floccosum C 114. l Trichophyton rubrum C113. m Trichophyton mentagrophytes ATCC 9972. b
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Table 2.
Candida Candida Candida Candida Candida Candida Candida
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Antifungal activity of the chloroform extract of Hypericum ternum against Candida spp
tropicalis (CEREMIC 131-2000) parapsilosis (CEREMIC 118-2000) glabrata (CEREMIC 115-2000) lusitaniae (CEREMIC 124-2000) krusei (CEREMIC 117-2000) kefyr (CEREMIC 123-2000) colliculosa (CEREMIC 122-2000)
Hypericum ternum (chloroform)
Amphotericin B
Ketoconazole
250 1000 500 1000 500 500 500
15 30 30 10 0.7 0.7 1.5
3 25 5 10 1 5 0.7
the systemic or deep yeast infections, particularly in immunocompromised patients. It colonizes the wound site in burn patients, causing mortality in 73% of bone marrow recipients (Meyers 1990) and there is evidence of invasive candidal infections in patients with hematological malignances due to intensive myelosuppressive chemotherapy (Meunier-Carpentier 1984; Bodey et al. 1992). In addition, a number of non-albicans Candida strains are currently emerging (Powderly et al. 1999). Considering the activity shown by the chloroform extract of H. ternum against a standardized C. albicans strain, it was evaluated against different species of the Candida genus (Table 2) and against several strains of C. albicans isolates (Table 3). In addition, because the chloroform extract of H. ternum was strongly active against dermatophytes, we tested it against several strains of T. rubrum and T. mentagrophytes, which are the main cause of athlete’s foot and onichomycoses in human beings. Athlete’s foot is the most prevalent superficial infection in the developed world (Evans 1997) and onichomycoses affects 2–13% of the population worldwide and up to 30% of groups at high risk such, as elderly and diabetic people (Levy 1997; Gupta et al. 1998). Results showed (Table 2) that the chloroform extract of H. ternum inhibited all the species of Candida genus tested, with MIC values between 250 and 1000 mg/ml and, interestingly enough, inhibited 5/7 species with MIC values p500 mg/ml. As for the activity of this extract against different isolates of C. albicans, our results showed that it inhibited all strains tested, with MIC values between 250 and 1000 mg/ml (MIC90 and MIC50 values=1000 and 500 mg/ml, respectively). The same extract displayed strong activities against all the clinical strains of T. mentagrophytes and T. rubrum tested, with MIC50 and MIC90 values of 250 and 500 mg/ ml, respectively, for both fungi (Table 3). These results showed that the chloroform extract of H. ternum possesses antifungal properties not only against standardized strains of clinically important fungi, but against their clinical isolates, making this extract promising for further studies. The antifungal activity of H. ternum could not be attributed to polyphenol compounds such as tannins,
Table 3. In vitro susceptibilities of clinical fungi isolates to the chloroform extract of Hypericum ternum Organism (No. of isolates)
C. albicans (7) T. rubrum (10) T. mentagrophytes (10)
MIC value (mg/ml) Range
50%
90%
250–1000 250–500 125–500
500 250 250
1000 500 500
which are responsible for the antimicrobial activity of several plants (Kolodzeij et al. 1999; Hwang et al. 2001; Panizzi et al. 2002), as these compounds are found in the most polar non-active extracts. The fact that the best antifungal properties of H. ternum were found in its chloroform extract suggests that the activity could be due to phloroglucinol derivatives, which are present in the lipophilic extract and are a group of natural products with recognized antimicrobial activity. In conclusion, the chloroform extract of H. ternum possesses a broad spectrum of activity against a panel of opportunistic fungi. It also showed activity when tested against different species of Candida spp. and against clinical isolates of C. albicans, T. mentagrophytes and T. rubrum, responsible for most fungal infections in immunocompromised patients. This promising extract opens the possibility of finding new clinically effective antifungal compounds.
Acknowledgements This work was supported by grants to SAZ (Agencia de Promociones Cientı´ ficas y Tecnolo´gicas de la Argentina PICT99 # 06-06454) and is part of the collaborative research within the ‘‘Bioactive Natural Products and their Applications’’ nucleus of the Association of Universities of the Montevideo Group (AUGM). Collaboration from the Iberoamerican Program of Science and Technology for Development (CYTED) (Project X.7) is gratefully acknowledged. SAZ is grateful to the OEA (Project: Profit of the Regional Flora).
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Phytomedicine 12 (2005) 236–240 www.elsevier.de/phymed
Antifungal activity of some Brazilian Hypericum species R. Fennera, M. Sortinob, S.M. Kuze Ratesa, R. Dall’Agnola, A. Ferraza, A.P. Bernardia, D. Albringa, C. No¨ra, G. von Posera,, E. Schapovala, S. Zacchinob a
Programa de Po´s Graduac¸a˜o em Cieˆncias Farmaceˆuticas, UFRGS, Universidade Federal do Rio Grande do Sul, Porto Alegre, Av. Ipiranga, 2752, RS, Porto Alegre 90610-000, Brazil b Facultad de Ciencias Bioquı´micas y Farmace´uticas, Universidad Nacional de Rosario- Suipacha Rosario, Argentina Received 23 July 2003; accepted 19 November 2003
Abstract Crude methanolic extracts and fractions from the aerial parts of seven species of Hypericum (H. caprifoliatum Cham. and Schltdl., H. carinatum Griseb., H. connatum Lam., H. ternum A. St.-Hil., H. myrianthum Cham. and Schltdl., H. piriai Arechav. and H. polyanthemum Klotzsch ex Reichardt) growing in southern Brazil were analyzed for their in vitro antifungal activity against a panel of standardized and clinical opportunistic pathogenic yeasts and filamentous fungi, including dermatophytes, by the agar dilution method. Chloroform and hexane extracts of H. ternum showed the greatest activity among extracts tested. r 2004 Elsevier GmbH. All rights reserved. Keywords: Hypericum; Antifungal activity; Agar dilution method
Introduction Human infections, particularly those involving the skin and mucous membranes, are increasing at an alarming rate, especially in tropical and subtropical developing countries, with dermatophytes and Candida sp. being the most common pathogens. This increase is directly related to the growing population of immunocompromised individuals, resulting from changes in medical practice such as the use of intensive chemotherapy and immunosuppressive drugs. HIV and other diseases causing immunosuppression have also contributed to this problem (Groll et al. 1996; Denning et al. 1997; Portillo et al. 2001). Human mycoses are not always successfully treated, since the available antifungal drugs are ineffective, Corresponding author. Tel.: +55-51-33165258; fax: +55-5133305610. E-mail address: [email protected] (G. von Poser).
0944-7113/$ - see front matter r 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2003.11.004
produce many adverse effects, show recurrence, or lead to the development of resistance. It is therefore essential to research for more effective and less toxic new antifungal agents (Zacchino et al. 1999). Although most antibiotics in clinical use have been obtained from microorganisms, a renewed interest in plant antimicrobials has emerged during the last 20 years. Because only a very small fraction of the known plant species of the world have been evaluated for the presence of antifungal compounds, and there is a rapid rate of plant species extinction, many efforts are necessary to collect and to screen plants in order to avoid the lost of valuable sources of potential leads for the development of novel and environmentally safe antifungal agents. In the course of our ongoing project for the detection of antifungal compounds in native Southern Brazilian plants, we selected species of the genus Hypericum to begin our screening for antifungal compounds. The genus includes various species used in traditional
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medicine in different parts of the world, from which several antifungal (De´costerd et al. 1986), antibiotic (Ishiguro et al. 1986), antiviral (Jacobson et al. 2001) and anticancer (Jayasuriya et al. 1989) compounds have been isolated. Most active compounds are of a phenolic nature, such as flavonoids, xanthones and phloroglucinol derivatives. Among the latter some are related to the well-known hyperforin, isolated from H. perforatum (Trifunovic et al. 1998), while others possess a phloroglucinol unit conjugated with a filicinic acid moiety (Ishiguro et al. 1986; Rocha et al. 1995). Phloroglucinol derivatives, found frequently in the lipophilic fractions of several Hypericum species, have demonstrated antibacterial activities against microorganisms such as Staphylococcus aureus, Bacillus cereus, B. subtilis and Nocardia gardenen. Their presence could justify the popular use of some Hypericum species as wound healing agents and in the treatment of some infectious diseases (Ishiguro et al. 1986; Jayasuriya et al. 1991; Yamaki and Ishiguro 1994; Rocha et al. 1995). Other substances, such as benzopyrans (De´costerd et al. 1986), xanthones (Ishiguro et al. 1999) and flavonoids (Ishiguro et al. 1993) are reported to be responsible for the antimicrobial activity against various bacteria and fungi in some species of Hypericum. In this paper, we report the antifungal evaluation of 17 extracts from 7 species of Hypericum, some of them reported in the Brazilian traditional medicine as useful in treating infectious diseases (Correˆa 1984; Mentz et al. 1997). Extracts of H. caprifoliatum Cham. and Schltdl., H. carinatum Griseb., H. connatum Lam., H. ternum A. St.-Hil., H. myrianthum Cham. and Schltdl., H. piriai Arechav. and H. polyanthemum Klotzsch ex Reichardt, were tested against a panel of human opportunistic pathogenic fungi by the agar dilution method. The most active extracts were then evaluated against panels of different clinical isolates of Candida species as well as different strains of Candida albicans, Trichophyton rubrum and Trichophyton. mentagrophytes, in order to gain insight into the actual antifungal potentiality of these extracts for the development of new antifungal agents.
Material and methods Plant material Aerial parts of Hypericum caprifoliatum Cham. and Schltdl. were collected in the ‘‘Morro Santana’’, Porto Alegre in May, 1998. H. myrianthum Cham. and Schltdl, and H. polyanthemum Klotzsch ex Reichardt were collected in Paraı´ so do Sul and Cac¸apava do Sul, in July and August, 1998, respectively. H. connatum Lam. and H. piriai Arechav. were collected in Capa˜o do Lea˜o in
237
January, 1999. H. carinatum Griseb. was collected in Glorinha, RS, in January, 1999 and H. ternum A. St.-Hil. in Sa˜o Francisco de Paula, RS, in October, 1999. Voucher specimens have been deposited in the herbarium of the Federal University of Rio Grande do Sul (ICN).
Preparation of plant extracts Dried and powdered plant material was submitted to two distinct extraction processes depending on the species employed. H. myrianthum, H. piriai and H. connatum (100 g) were extracted with methanol (drug/ solvent ratio=1:10 w/v) by maceration (3 24 h), yielding 11%, 15% and 15%, respectively, of total methanol crude extracts (TMCE). H. caprifoliatum, H. polyanthemum, H. ternum and H. carinatum (ca. 80 g) were extracted successively in a Soxhlet apparatus with petroleum ether, chloroform, and methanol for 12 h. These extracts were evaporated to dryness in vacuo at 45 1C, affording 3.0–4.0% PET, 2.5–3.5% CLF, and 6.0–7.0% MET, respectively.
Microorganisms and media For the antifungal evaluation, strains from the American Type Culture Collection (ATCC), Rockville, MD, USA and CEREMIC (C), Centro de Referencia Micolo´gica, Facultad de Ciencias Bioquı´ micas y Farmace´uticas, Suipacha 531-(2000)-Rosario, Argentina were used. C. albicans ATCC10231, C. tropicalis C131, C. parapsilosis C118, C. glabrata C115, C. lusitaniae C124, C. krusei C117, C. kefyr C123, C. colliculosa C122; strains of C. albicans C125, C126, C127, C128, C129, C130, C131, Saccharomyces cerevisiae ATCC9763, Cryptococcus neoformans ATCC32264, Aspergillus flavus ATCC9170, A. fumigatus ATTC26934, A. niger ATCC9029, Epidermophyton floccosum C114, strains of T. rubrum: C110, C113, C133, C134, C135, C136, C137, C138, C139, C 140, C141, strains of T. mentagrophytes ATCC9972, C199, C201, C202, C206, C126, C127, C128, C129, C130, C131, Microsporum canis C112, and M. gypseum C115. Yeasts were grown on Sabouraudchloramphenicol agar slants, for 48 h at 30 1C. Cell suspensions were adjusted to 106 viable yeast cells/ml, in sterile distilled water. Filamentous fungi were maintained on Sabouraud-dextrose agar (SDA, Oxoid) and subcultured every 15 days to prevent pleomorphic transformations. Spore suspensions were obtained according to reported procedures (Wright et al. 1983) and adjusted to 106 spores/ml.
Antifungal assays The fungistatic activities of different extracts were evaluated via the agar dilution method by using
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Sabouraud-chloramphenicol agar for yeasts and filamentous fungi according to reported procedures (Mitscher et al. 1972; Zacchino et al. 1998, 1999; Feresin et al. 2001). Stock solutions of extracts were diluted in DMSO to produce serial decreasing dilutions ranging from 0.25 to 1000 mg/ml. The final concentrations of DMSO in the assay did not exceed 2%. Using a micropipette, an inoculum of 5 ml of the yeast, cell or spore suspensions was added to each Sabouraudchloramphenicol agar tube. The antifungal agents ketoconazole (Janssen Pharmaceutical) and amphotericin B (Sigma Chemical Co.) were included in the assay as positive controls. Drug-free solution was also used as a blank control. The tubes were incubated for 24, 48 or 72 h at 30 1C (according to the control fungus growth) up to 15 days for dermatophyte strains. The minimal inhibitory concentration (MIC) value was defined as the lowest extract concentration, showing no visible fungal growth after incubation time. MIC50
Table 1.
Results and discussion To carry out the antifungal evaluation with agar dilution assays, extracts in concentrations of up to 1000 mg/ml were incorporated into the growth media according to Material and methods. Extracts with MIC values p1000 mg/ml were considered active. Among the Hypericum extracts tested, only the PET and CLF extracts of H. ternum showed significant antifungal activity. Results are shown in Table 1. These extracts present a broad spectrum of action inhibiting all the fungi tested, including C. albicans, the causative fungus of many superficial infections and over 90% of
In vitro evaluation of the antifungal activity of different extracts of Hypericum species (Hypericaceae)
Plant species Voucher specimen H. caprifoliatum Bordignon, 1400 H. myrianthum Bordignon, 1402 H. piriai Bordignon, 1528 H. ternum Bordignon et al., 1715 H. carinatum Bordignon et al., 1520 H. connatum Bordignon and Salazar, 1527 H. polyanthemum Bordignon et al., 1429 Amphotericin B Ketoconazole a
and MIC90 values are the lowest extract concentration at which 50% and 90% of the clinical isolates were inhibited (Marco et al. 1998).
Extracta
MIC value (mg/ml)b C.a.c
C.n.d
S.c.e
A.fu.f
A.fl.g
A.n.h
M.c.i
M.g.j
E.f.k
T.r.l
T.mm
PET CLF MET TMCE
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
n.a n.a n.a n.a
TMCE
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
n.a
MET PET CLF MET PET CLF TMCE
n.a n.a. 250 n.a n.a n.a n.a
n.a 250 250 n.a n.a n.a n.a
n.a 100 100 n.a n.a n.a n.a
n.a 500 250 n.a n.a n.a n.a
n.a 500 1000 n.a n.a n.a n.a
n.a 500 250 n.a n.a n.a n.a
n.a n.a. 250 n.a n.a n.a n.a
n.a 250 250 n.a n.a n.a n.a
n.a 100 250 n.a n.a 500 n.a
n.a 500 500 n.a n.a n.a n.a
n.a 100 100 n.a n.a n.a n.a
MET PET CLF
n.a n.a n.a 8 0.7
n.a n.a n.a 2 0.4
n.a n.a n.a 5 6.25
n.a n.a n.a 20 3
n.a n.a n.a 30 3
n.a n.a n.a 12.5 0.4
n.a n.a n.a 15 30
n.a n.a n.a 6.25 6.25
n.a 500 n.a 15 0.3
n.a n.a n.a 15 25
n.a n.a n.a 12.5 6.25
MET—methanolic extract; PET—petroleum ether extract; CLF—chloroform extract; TMCE—total methanol crude extract. n.a.—not active. c Candida albicans ATCC 10231. d Cryptococcus neoformans ATCC 32264. e Saccharomyces cerevisiae ATCC 9763. f Aspergillus fumigatus ATCC 26934. g Aspergillus flavus ATCC 9170. h Aspergillus niger ATCC 9029. i Microsporum canis C 112. j Microsporum gypseum C 115. k Epidermophyton floccosum C 114. l Trichophyton rubrum C113. m Trichophyton mentagrophytes ATCC 9972. b
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Table 2.
Candida Candida Candida Candida Candida Candida Candida
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Antifungal activity of the chloroform extract of Hypericum ternum against Candida spp
tropicalis (CEREMIC 131-2000) parapsilosis (CEREMIC 118-2000) glabrata (CEREMIC 115-2000) lusitaniae (CEREMIC 124-2000) krusei (CEREMIC 117-2000) kefyr (CEREMIC 123-2000) colliculosa (CEREMIC 122-2000)
Hypericum ternum (chloroform)
Amphotericin B
Ketoconazole
250 1000 500 1000 500 500 500
15 30 30 10 0.7 0.7 1.5
3 25 5 10 1 5 0.7
the systemic or deep yeast infections, particularly in immunocompromised patients. It colonizes the wound site in burn patients, causing mortality in 73% of bone marrow recipients (Meyers 1990) and there is evidence of invasive candidal infections in patients with hematological malignances due to intensive myelosuppressive chemotherapy (Meunier-Carpentier 1984; Bodey et al. 1992). In addition, a number of non-albicans Candida strains are currently emerging (Powderly et al. 1999). Considering the activity shown by the chloroform extract of H. ternum against a standardized C. albicans strain, it was evaluated against different species of the Candida genus (Table 2) and against several strains of C. albicans isolates (Table 3). In addition, because the chloroform extract of H. ternum was strongly active against dermatophytes, we tested it against several strains of T. rubrum and T. mentagrophytes, which are the main cause of athlete’s foot and onichomycoses in human beings. Athlete’s foot is the most prevalent superficial infection in the developed world (Evans 1997) and onichomycoses affects 2–13% of the population worldwide and up to 30% of groups at high risk such, as elderly and diabetic people (Levy 1997; Gupta et al. 1998). Results showed (Table 2) that the chloroform extract of H. ternum inhibited all the species of Candida genus tested, with MIC values between 250 and 1000 mg/ml and, interestingly enough, inhibited 5/7 species with MIC values p500 mg/ml. As for the activity of this extract against different isolates of C. albicans, our results showed that it inhibited all strains tested, with MIC values between 250 and 1000 mg/ml (MIC90 and MIC50 values=1000 and 500 mg/ml, respectively). The same extract displayed strong activities against all the clinical strains of T. mentagrophytes and T. rubrum tested, with MIC50 and MIC90 values of 250 and 500 mg/ ml, respectively, for both fungi (Table 3). These results showed that the chloroform extract of H. ternum possesses antifungal properties not only against standardized strains of clinically important fungi, but against their clinical isolates, making this extract promising for further studies. The antifungal activity of H. ternum could not be attributed to polyphenol compounds such as tannins,
Table 3. In vitro susceptibilities of clinical fungi isolates to the chloroform extract of Hypericum ternum Organism (No. of isolates)
C. albicans (7) T. rubrum (10) T. mentagrophytes (10)
MIC value (mg/ml) Range
50%
90%
250–1000 250–500 125–500
500 250 250
1000 500 500
which are responsible for the antimicrobial activity of several plants (Kolodzeij et al. 1999; Hwang et al. 2001; Panizzi et al. 2002), as these compounds are found in the most polar non-active extracts. The fact that the best antifungal properties of H. ternum were found in its chloroform extract suggests that the activity could be due to phloroglucinol derivatives, which are present in the lipophilic extract and are a group of natural products with recognized antimicrobial activity. In conclusion, the chloroform extract of H. ternum possesses a broad spectrum of activity against a panel of opportunistic fungi. It also showed activity when tested against different species of Candida spp. and against clinical isolates of C. albicans, T. mentagrophytes and T. rubrum, responsible for most fungal infections in immunocompromised patients. This promising extract opens the possibility of finding new clinically effective antifungal compounds.
Acknowledgements This work was supported by grants to SAZ (Agencia de Promociones Cientı´ ficas y Tecnolo´gicas de la Argentina PICT99 # 06-06454) and is part of the collaborative research within the ‘‘Bioactive Natural Products and their Applications’’ nucleus of the Association of Universities of the Montevideo Group (AUGM). Collaboration from the Iberoamerican Program of Science and Technology for Development (CYTED) (Project X.7) is gratefully acknowledged. SAZ is grateful to the OEA (Project: Profit of the Regional Flora).
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