Antifungal activity of some Brazilian Hypericum species.
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.
[c] 2004 Elsevier GmbH. All rights reserved.
Keywords: Hypericum; Antifungal activity; Agar dilution method
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, 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 medicine in different parts of the world, from which several antifungal (Decosterd 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 (Decosterd 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 (Correa 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
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 Paraiso do Sul and Cacapava do Sul, in July and August, 1998, respectively. H. connatum Lam. and H. piriai Arechav, were collected in Capao do Leao in January, 1999. H. carinatum Griseb. was collected in Glorinha, RS. in January, 1999 and H. ternum A. St.-Hil. in Sao 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 X 24 h), yielding 11%, 15% and 15%, respectively, of total methanol crude extracts (TMCE). H. caprifoliatum, H. polyanthemum, H. ternum and H. carinatum (ca. 80g) were extracted successively in a Soxhlet apparatus with petroleum ether, chloroform, and methanol for 12h. These extracts were evaporated to dryness in vacuo at 45[degrees]C, 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 Micologica, Facultad de Ciencias Bioquimicas y Farmaceuticas, 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, C140, 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 Sabouraud-chloramphenicol agar slants, for 48h at 30[degrees]C. Cell suspensions were adjusted to [10.sup.6] 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 [10.sup.6] spores/ml.
The fungistatic activities of different extracts were evaluated via the agar dilution method by using 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 [micro]g/ml. The final concentrations of DMSO in the assay did not exceed 2%. Using a micropipette, an inoculum of 5 [micro]l of the yeast, cell or spore suspensions was added to each Sabouraud-chloramphenicol 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 72h at 30[degrees]C (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. MI[C.sub.50] and MI[C.sub.90] values are the lowest extract concentration at which 50% and 90% of the clinical isolates were inhibited (Marco et al. 1998).
Results and discussion
To carry out the antifungal evaluation with agar dilution assays, extracts in concentrations of up to 1000 [micro]g/ml were incorporated into the growth media according to Material and methods. Extracts with MIC values [less than or equal to]1000 [micro]g/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 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 [micro]g/ml and, interestingly enough, inhibited 5/7 species with MIC values [less than or equal to]500 [micro]g/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 [micro]g/ml (MI[C.sub.90] and MI[C.sub.50] values = 1000 and 500 [micro]g/ml, respectively). The same extract displayed strong activities against all the clinical strains of T. mentagrophytes and T. rubrum tested, with MI[C.sub.50] and MI[C.sub.90] values of 250 and 500 [micro]g/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, 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.
Table 1. In vitro evaluation of the antifungal activity of different extracts of Hypericum species (Hypericaceae) Plant species Extract (a) Voucher specimen C.a. (c) C.n. (d) S.c. (e) A.fu. (f) H. caprifoliatum PET n.a n.a n.a n.a Bordignon, 1400 CLF n.a n.a n.a n.a MET n.a n.a n.a n.a H. myrianthum TMCE n.a n.a n.a n.a Bordignon, 1402 H. piriai TMCE n.a n.a n.a n.a Bordignon, 1528 H. ternum MET n.a n.a n.a n.a Bordignon et al., 1715 PET n.a 250 100 500 CLF 250 250 100 250 H. carinatum MET n.a n.a n.a n.a Bordignon et al., 1520 PET n.a n.a n.a n.a CLF n.a n.a n.a n.a H. connatum TMCE n.a n.a n.a n.a Bordignon and Salazar, 1527 H. polyanthemum MET n.a n.a n.a n.a Bordignon et al., 1429 PET n.a n.a n.a n.a CLF n.a n.a n.a n.a Amphotericin B 8 2 5 20 Ketoconazole 0.7 0.4 6.25 3 Plant species Extract (a) MIC value ([micro]g/ml) (b) Voucher specimen A.fl. (g) A.n. (h) M.c. (i) M.g. (j) H. caprifoliatum PET n.a n.a n.a n.a Bordignon, 1400 CLF n.a n.a n.a n.a MET n.a n.a n.a n.a H. myrianthum TMCE n.a n.a n.a n.a Bordignon, 1402 H. piriai TMCE n.a n.a n.a n.a Bordignon, 1528 H. ternum MET n.a n.a n.a n.a Bordignon et al., 1715 PET 500 500 n.a. 250 CLF 1000 250 250 250 H. carinatum MET n.a n.a n.a n.a Bordignon et al., 1520 PET n.a n.a n.a n.a CLF n.a n.a n.a n.a H. connatum TMCE n.a n.a n.a n.a Bordignon and Salazar, 1527 H. polyanthemum MET n.a n.a n.a n.a Bordignon et al., 1429 PET n.a n.a n.a n.a CLF n.a n.a n.a n.a Amphotericin B 30 12.5 15 6.25 Ketoconazole 3 0.4 30 6.25 Plant species Extract (a) MIC value ([micro]g/ml) (b) Voucher specimen E.f. (k) T.r. (l) T.m (m) H. caprifoliatum PET n.a n.a n.a Bordignon, 1400 CLF n.a n.a n.a MET n.a n.a n.a H. myrianthum TMCE n.a n.a n.a Bordignon, 1402 H. piriai TMCE n.a n.a n.a Bordignon, 1528 H. ternum MET n.a n.a n.a Bordignon et al., 1715 PET 100 500 100 CLF 250 500 100 H. carinatum MET n.a n.a n.a Bordignon et al., 1520 PET n.a n.a n.a CLF 500 n.a n.a H. connatum TMCE n.a n.a n.a Bordignon and Salazar, 1527 H. polyanthemum MET n.a n.a n.a Bordignon et al., 1429 PET 500 n.a n.a CLF n.a n.a n.a Amphotericin B 15 15 12.5 Ketoconazole 0.3 25 6.25 (a) MET -- methanolic extract; PET -- petroleum ether extract; CLF -- chloroform extract; TMCE -- total methanol crude extract. (b) 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. Table 2. Antifungal activity of the chloroform extract of Hypericum ternum against Candida spp Hypericum ternum (chloroform) Amphotericin B Ketoconazole Candida tropicalis 250 15 3 (CEREMIC 131-2000) Candida parapsilosis 1000 30 25 (CEREMIC 118-2000) Candida glabrata 500 30 5 (CEREMIC 115-2000) Candida lusitaniae 1000 10 10 (CEREMIC 124-2000) Candida krusei 500 0.7 1 (CEREMIC 117-2000) Candida kefyr 500 0.7 5 (CEREMIC 123-2000) Candida colliculosa 500 1.5 0.7 (CEREMIC 122-2000) Table 3. In vitro susceptibilities of clinical fungi isolates to the chloroform extract of Hypericum ternum MIC value ([micro]g/ml) Organism (No. of isolates) Range 50% 90% C. albicans (7) 250-1000 500 1000 T. rubrum (10) 250-500 250 500 T. mentagrophytes (10) 125-500 250 500
This work was supported by grants to SAZ (Agencia de Promociones Cientificas y Tecnologicas 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).
Received 23 July 2003; accepted 19 November 2003
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R. Fenner (a), M. Sortino (b), S.M. Kuze Rates (a), R. Dall'Agnol (a), A. Ferraz (a), A.P. Bernardi (a), D. Albring (a), C. Nor (a), G. von Poser (a,*), E. Schapoval (a), S. Zacchino (b)
(a) Programa de Pos Graduacao em Ciencias Farmaceuticas, UFRGS, Universidade Federal do Rio Grande do Sul, Porto Alegre, Av. Ipiranga, 2752, RS, Porto Alegre 90610-000, Brazil
(b) Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario- Suipacha Rosario, Argentina
*Corresponding author. Tel.: +55-51-33165258; fax: +55-51-33305610.
E-mail address: firstname.lastname@example.org (G. von Poser).
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|Author:||Fenner, R.; Sortino, M.; Rates, S.M. Kuze; Dall'Agnol, R.; Ferraz, A.; Bernardi, A.P.; Albring, D.;|
|Publication:||Phytomedicine: International Journal of Phytotherapy & Phytopharmacology|
|Date:||Mar 1, 2005|
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