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In vitro antifungal assay of traditional Argentine medicinal plants
Journal of Ethnopharmacology 102 (2005) 233–238
In vitro antifungal assay of traditional Argentine medicinal plants Liliana Muschietti a,∗ , Marcos Derita b , Valeria S¨ulsen a , Juan de Dios Mu˜noz c , Graciela Ferraro a , Susana Zacchino b , Virginia Martino a a
C´atedra de Farmacognosia, IQUIMEFA (UBA-CONICET), Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, Jun´ın 956 (1113), Buenos Aires, Argentina b C´ atedra de Farmacognosia, Facultad de Ciencias Bioqu´ımicas y Farmac´euticas, Universidad Nacional de Rosario, Suipacha 531 (2000), Rosario, Argentina c C´ atedra de Bot´anica Sistem´atica, Facultad de Ciencias Agropecuarias, Universidad Nacional de Entre R´ıos, CC 24, 3100 Paran´a, Argentina Received 24 January 2005; received in revised form 10 May 2005; accepted 14 June 2005 Available online 1 August 2005
Abstract Methanol extracts from 11 traditionally used Argentine medicinal plants were assayed in vitro for antifungal activity against yeasts, hialohyphomycetes as well as dermatophytes with the microbroth dilution method. Among them, the strongest effect was presented by Eupatorium buniifolium and Terminalia triflora, Trichophyton mentagrophytes and Trichophyton rubrum being the most susceptible species with MICs ranging from 100 to 250 g/ml. Lithrea molleoides showed the broadest spectrum of action inhibiting all of the tested dermatophytes with MIC = 250 g/ml. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Antifungal activity; Argentine medicinal species; Microbroth dilution method
1. Introduction The incidence of opportunistic fungal infections in patients treated with immunosuppressive drugs, intensive chemotherapy, suffering AIDS and neonates is increasing at an alarming rate (Groll et al., 1996; Denning et al., 1997). These mycoses are very difficult to eradicate constituting an enormous challenge for healthcare providers (Meyers, 1990). Although there appear to be an array of drugs for the treatment of systemic and superficial mycoses, none of them is ideal in terms of efficacy, safety or antifungal spectrum (Di Domenico, 1998; Ablordeppey et al., 1999). Many of the drugs have undesirable effects or are very toxic (amphotericin B), produce recurrence, show drug–drug interactions (azoles) or lead to the development of resistance (fluconazole, 5-flucytosine) (White et al., 1998). Although some new
∗
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drugs have emerged as interesting alternatives for the obstinate fungal infections (allylamines or caspofungine) (Vicente et al., 2003), and combination therapy is sometimes used to come these disadvantages, there is a real need for a next generation of safer and more potent antifungal drugs (Bartroli et al., 1998). Numerous useful drugs from higher plants have been discovered by following up ethnomedical uses (Fabricant and Farnsworth, 2001). The diversity of plants growing in Argentina, along with their known ethnopharmacological uses, offer an enormous possibility of finding novel structures with antifungal properties. The selection of the species used in this study was mainly based on their ethnomedical evidence of use for conditions related to microbial infections. These include skin infections, healing of wounds, dandruff, respiratory infections, vaginal mycoses, antiseptic, etc. Some plants without ethnomedical precedents but not previously studied were included too. Data were mainly obtained from the database on Argentine medicinal/toxic plants (Rondina et al., 2003) that covers
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the information of nearly 1800 Argentine medicinal species. Some other literature information was used too (Santos Biloni, 1990). Acacia caven, Bidens subalternans, Eupatorium arnottianum, Eupatorium buniifolium, Jacaranda mimosifolia, Lippia integrifolia, Lithrea molleioides, Morrenia brachystephana, Sebastiania brasiliensis, Sebastiania commersoniana and Terminalia triflora were chosen following an ethnopharmacological approach. Despite no reported ethnomedical uses for Terminalia triflora (Combretaceae), it was included in this study since a closely related species Terminalia australis (traditionally used as an antiseptic agent) shown to possess antifungal properties against Aspergillus and Candida strains (Carparo et al., 2003). Acacia caven (Fabaceae) popularly known as “aromito” or “espinillo” is a tree widely distributed in North and Central Argentina, adjacent Bolivia, Chile, Paraguay, Brasil and Uruguay. The decoction of the bark is traditionally used against bruises, snake bites and skin diseases administered in form of a bath during healing. Against vaginal mycosis, a piece of bark is boiled in 1 l of water, sweetened with honey and drunk several times a day until the sickness disappears (Hilgert, 2001). Additionally, the juice of flowers is applied with cotton as an analgesic for ear-ache and the decoction of flowers used as an antitussive, antihypertensive and antiasthmatic (Martinez Crovetto, 1981). Crude extracts of this plant have demonstrated in vitro antifungal properties against Pycnoporus sanguineus and Fusarium oxysporum (Quiroga et al., 2001). The Asteraceae family is the second largest family of flowering plants with over 13,000 species world-wide. It is the best represented in Argentina with over 200 genera and around 1460 spontaneous species, most of them used for medicinal purposes (Zardini, 1984). Bidens subalternans, Eupatorium arnottianum and Eupatorium buniifolium are species belonging to this family and are commonly found in the North-Eastern and Central regions of Argentina. Bidens subalternans is a herb commonly known as “amor seco”, “amor de viejo” or “espina de erizo”. The decoction of the plant is externally used as an ocular antiseptic and is orally taken in cases of asthma, together with Buddleja madagascariensis and Borago officinalis. It is very accepted to treat aphta and sore throats, being used in gargles or chewing the leaves several times a day. The decoction of the bark is used as a mouthwash for tooth-ache (Martinez Crovetto, 1964, 1981). The dichloromethane extract of this species showed anti-inflammatory activity, maslinic acid and stigmasterol glucoside being responsible for the reported activity (Ortega et al., 1998). Eupatorium arnottianum known as “clavel” or “uou´e” is a subshrub used in traditional medicine against stomachache, as anti-inflammatory and against pain related problems. Aqueous and dichloromethane extracts of this species presented analgesic (Clav´ın et al., 2000) and immunomodulating properties (Fern´andez et al., 2002), respectively. p-
Cymene, ␣-pinene, thymyl acetate and -caryophyllene have been identified in the essential oil as the main compounds (Zygadlo et al., 1995). The decoction of the aerial parts of the shrub Eupatorium buniifolium has been used against rheumatic pains (R´ıos et al., 1993) and as disinfectant (Rojas Acosta, 1905). Twenty grams of aerial parts boiled in 1 l of water is used as a digestive and to treat hepatic affections; it must be drunk four times a day until symptoms disappear (Burgstaller, 1999). Previous phytochemical studies on this species commonly known as “romerillo”, “romerillo colorado” or “chilca” showed the presence of flavonoids, hydroxycinnamic acids derivatives (Muschietti et al., 1990, 1994) and ent-labdanes (Carreras et al., 1998). Pharmacological activities such as antioxidant (Paya et al., 1996), antiviral (Garc´ıa et al., 1990), as well as the identification of three anti-inflammatory compounds have already been reported (Muschietti et al., 2001). Jacaranda mimosifolia (Bignoniaceae) is native to north-west Argentina and Bolivia. Commonly known as “jacaranda”, “caroa” or “tarco”, it is popularly used as an antibiotic (specifically against syphilis) and the bark as a contraceptive (Toursarkissian, 1980). The methanol extract of leaves and stem bark showed antimicrobial activity. Preliminary phytochemical screening of this species revealed the presence of tannins, flavonoids, alkaloids, quinones and traces of saponins (Binutu and Lajubutu, 1994). The genus Lippia (Verbenaceae) includes approximately 200 species of herbs, shrubs and small trees. Nine of them are used in Argentine folk medicine, mainly employed as digestive, stomachic, tonic, nervine, diuretic and emenagogue. Lippia integrifolia is an aromatic shrub, known popularly as “incayuyo”, “pulco”, “poleo”, “t´e del inca” or “manzanillo”, that grows in North-Western and Central Argentina. The decoction of leaves and flowers is traditionally used as an antibiotic (for gonorrhoeal infections), digestive, stomachic, diuretic, emmenagogue, to treat respiratory diseases and sedative (Rondina et al., 2003). It also imparts flavoring to beverages and phytomedicines. Previous chemical research on the essential oil of this species has led to the identification of camphor, limonene, camphene, methyl isoeugenol and germacrene as main compounds (Fricke et al., 1999). The genus Lithrea, from the Anacardiaceae family, is well known for its irritating effects, causing rash and swelling in exposed parts of the body. Lithrea molleoides is a tree which grows in North and Central Argentina, Brazil, Paraguay, Uruguay and Southern Bolivia. A decoction of the twigs of this species, known as “molle de beber”, “chichita” or “´arbol malo” is used in the treatment of respiratory and digestive diseases (Ratera and Ratera, 1980). The infusion of leaves and fruits is said to be diuretic and stomachic, and the resin to treat arthritis (Toursarkissian, 1980). The tincture is used for cough, bronchitis and phlegm (Rondina et al., 2003). Antimicrobial and antiviral activities of this species have already been reported (Kott et al., 1999; Penna et al., 2001). Four new alkylene resorcinols isolated from the MeOH extract showed nematicidal activity (Valcic et al., 2002).
L. Muschietti et al. / Journal of Ethnopharmacology 102 (2005) 233–238
Morrenia brachystephana, as many Asclepiadaceae, is a creeper traditionally known as “taluai”, “tasi” and “isipo’a”. It can be found in North and Central Argentina. The juice obtained from the fruits is employed to treat tetter. The decoction of bark and fruits (30 or 40 g in 1 l of water) is used as a galactagogue and the latex is employed to relieve dental pain (Martinez Crovetto, 1981). The research on this plant has been mainly focused on latex endopeptidases (Cortadi, 2001). Sebastiania brasiliensis and Sebastiania commersoniana (Euphorbiaceae) are two closely related species commonly known as “blanquillo” or “lecher´on”, widely distributed in Entre R´ıos province, Argentina. The decoction of leaves and twigs is useful as an external antiseptic for wounds and the latex is employed for the elimination of warts and as an analgesic for dental caries (Santos Biloni, 1990). The aqueous and alcoholic extracts of Sebastiania brasiliensis and Sebastiania commersoniana have shown antibacterial and antifungal activities. From the hydroalcoholic extract of Sebastiania brasiliensis methylgallate and protocatechuic acid have been identified as the most active compounds on Staphylococcus aureus and Mucor sp., respectively (Penna et al., 2001).
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Terminalia triflora, from the Combretaceae family, popularly known as “lanza amarilla”, “palo amarillo” or “lanza” is a tree widely distributed in North-Western Argentina. Hieronymus (1882) mentioned the use of its bark in the making of posts, furniture and weapons for soldiers and as fuel. Two ellagitannins with anti HIV-RT activity have already been reported from this plant (Martino et al., 2004). Tannins have been demonstrated to have a powerful antifungal action in clinical studies (Latte and Kolodziej, 2000).
2. Materials and methods 2.1. Plant material Plants were collected in their places of origin in Argentina (Fig. 1) between 2002 and 2003, with the exception of Eupatorium arnottianum that was collected in 1995, extracted soon after collection and methanol extract stored at 5 ◦ C. Plant materials were identified by Juan de Dios Mu˜noz, A. Slanis, G. Giberti and A. Cortadi. Voucher specimens are deposited at the following herbariums.
Fig. 1. Provinces of Argentine where species were collected.
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Herbarium of the Universidad Nacional de Entre R´ıos: Acacia caven (ERA-Mu˜noz 5747), Eupatorium arnottianum (ERA-Mu˜noz 3535), Eupatorium buniifolium (ERA-Mu˜noz 5067), Jacaranda mimosifolia (ERA-Mu˜noz 6263), Lithrea molleioides (ERA-Mu˜noz 1714), Sebastiania brasiliensis (ERA-Mu˜noz 1652–1684), Sebastiania commersoniana (ERA-Mu˜noz 1634). Herbarium of the Instituto Miguel Lillo, Tucum´an: Lippia integrifolia (Slanis 178), Terminalia triflora (Slanis 552). Herbarium of the Museo de Farmacobot´anica J. A. Dominguez, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires: Bidens subalternans (BAF 3442). Herbarium of the Universidad Nacional de Rosario: Morrenia brachystephana (UNR 1271). 2.2. Extraction of plant material Aerial parts (10 g) of each species were air-dried, ground to powder and extracted by soaking in methanol (100 ml) at room temperature for 24 h. The extracts were filtered off and the process repeated twice under the same conditions. The filtrates were combined and dried under vacuum. 2.3. Microorganisms and media The microorganisms used for the antifungal evaluation were either acquired from the American Type Culture Collection (ATCC, Rockville, MD) or were clinical isolates from the Centro de Referencia Micol´ogica, Facultad de Ciencias Bioqu´ımicas y Farmac´euticas, Universidad Nacional de Rosario (CEREMIC). These were: Candida albicans (ATCC 10231), Candida tropicalis (C131), Saccharomyces cerevisiae (ATCC 9763), Cryptococcus neoformans (ATCC 32264), Microsporum canis (C112), Microsporum gypseum (C115), Epidermophyton floccosum (C114) Trichophyton mentagrophytes (ATCC 9972), Trichophyton rubrum (C113),
Aspergillus fumigatus (ATCC 26934), Aspergillus flavus (ATCC 9170) and Aspergillus niger (ATCC 9029). The antifungal agents ketoconazole (Janssen Pharmaceutical), amphotericin B (Sigma Chemical Co.) and terbinafine were included in the assays as positive controls. The inocula were prepared according to the method proposed by the National Committee for Clinical Laboratory Standards NCCLS (2002a,b) and were adjusted to 104 spores or cells with colony forming ability/ml (Wright et al., 1983). 2.4. Antifungal assay The antifungal activity was determined by the microbroth dilution assay following the guidelines of the National Committee for Clinical and Laboratory Standards (NCCLS) for yeasts M-27-A2 (NCCLS, 2002a) and filamentous fungi M38-A (NCCLS, 2002b) in 96-well microplates. Each well was inoculated with 100 l of the yeast or filamentous inoculum. The control wells contained only the medium and distilled water. For dermatophyte strains incubations were performed at 28 ◦ C while for yeast and filamentous fungi the plates were incubated at 35–37 ◦ C for 24, 48 or 72 h. MIC was defined as the lowest concentration of extract at which no fungal growth was observed after incubation.
3. Results and discussion The methanol extracts of Argentine medicinal plants were screened for antifungal activity against 12 fungal strains. Results are shown in Table 1. Extracts with MIC values below 1000 g/ml, in any of the microorganisms tested, were considered active since only crude extracts were employed. None of the extracts were active against the yeasts and filamentous fungi (data not shown). In contrast, extracts were
Table 1 Antifungal activity of methanolic extracts of traditional Argentine medicinal plants Species (family)
Antifungal activity MIC (g/ml) M.c.
M.g.
E.f.
T.r.
T.m.
Acacia caven (Molina) Molina (Fabaceae) Bidens subalternans DC. (Asteraceae) Eupatorium arnottianum Griseb. (Asteraceae) Eupatorium buniifolium Hook & Arn. (Asteraceae) Jacaranda mimosifolia D. Don (Bignoniaceae) Lippia integrifolia (Griseb.) Hieron. (Verbenaceae) Lithrea molleoides (Vell.) Engl. (Anacardiaceae) Morrenia brachystephana Griseb. (Asclepiadaceae) Sebastiania brasiliensis Spreng. (Euphorbiaceae) Sebastiania commersoniana (Baill.) L. B. Sm. & B. J. Downs (Euphorbiaceae) Terminalia triflora (Gris.) Lillo (Combretaceae) Ketoconazole Amphotericin B Terbinafine
>1000 >1000 n.t n.t 1000 500 250 n.t 250 250
>1000 >1000 >1000 250 >1000 1000 250 >1000 1000 1000
1000 >1000 n.t n.t 1000 250 250 n.t 250 250
>1000 >1000 >1000 100 >1000 1000 250 >1000 500 500
>1000 >1000 >1000 250 >1000 1000 250 >1000 500 500
n.t 15 50 0.01
250 6.25 12.5 0.006
n.t 25 0.3 0.004
100 6.25 12.5 0.003
100 6.25 12.5 0.006
M.c: Microsporum canis, M.g: Microsporum gypseum, E.f: Epidermophyton floccosum, T.r: Trichophyton rubrum, T.m: Trichophyton mentagrophytes, >1000: not active; n.t: not tested.
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active against dermatophytes with MICs between 100 and 1000 g/ml. Eupatorium buniifolium and Terminalia triflora exhibited the strongest activity against Microsporum gypseum (MIC = 250 g/ml for both extracts), Trichophyton mentagrophytes (MIC values = 250 and 100 g/ml, respectively) and Trichophyton rubrum (MIC values of 100 g/ml for both extracts). Lithrea molleioides inhibited all the dermatophytic strains (MIC = 250 g/ml). Sebastiania brasiliensis, Sebastiania commersoniana and Lippia integrifolia showed a limited spectrum of action and a lower potency against all dermatophytes tested (MICs: between 250 and 1000 g/ml). The fact that the extracts of the above species showed activity against dermatophytes gives support to the ethnopharmacological use of those species and makes them interesting for a further evaluation. It is interesting to note that, among dermatophytes, the most susceptible species was Trichophyton rubrum (MIC = 100 g/ml for Eupatorium buniifolium and Terminalia triflora) which is the etiological agent of 80–93% of all clinical infections produced by dermatophytes (Feresin et al., 2001) suggesting the possibility of using these extracts as starting points for the finding of antifungal agents that selectively inhibit the most prevalent fungus in dermatomycoses. These extracts are being submitted to bioassay-guided fractionations in order to isolate the compound/s responsible for the activity.
Acknowledgments We thank Dr. A. Cortadi and Dr. G. Giberti for collection of plant material. This work was supported by grant PIP CONICET: 02419, B101 UBACYT and is part of the Proyecto Iberoamericano X.7 PIBEAFUN (Search and development of new antifungal agents), within the Programa Iberoamericano de Ciencia y Tecnolog´ıa para el Desarrollo (CYTED). Dr. S. Zacchino thanks to ANPCyT (PICT Redes 00260) and OEA for financial support. M. Derita acknowledges CONICET.
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In vitro antifungal assay of traditional Argentine medicinal plants Liliana Muschietti a,∗ , Marcos Derita b , Valeria S¨ulsen a , Juan de Dios Mu˜noz c , Graciela Ferraro a , Susana Zacchino b , Virginia Martino a a
C´atedra de Farmacognosia, IQUIMEFA (UBA-CONICET), Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires, Jun´ın 956 (1113), Buenos Aires, Argentina b C´ atedra de Farmacognosia, Facultad de Ciencias Bioqu´ımicas y Farmac´euticas, Universidad Nacional de Rosario, Suipacha 531 (2000), Rosario, Argentina c C´ atedra de Bot´anica Sistem´atica, Facultad de Ciencias Agropecuarias, Universidad Nacional de Entre R´ıos, CC 24, 3100 Paran´a, Argentina Received 24 January 2005; received in revised form 10 May 2005; accepted 14 June 2005 Available online 1 August 2005
Abstract Methanol extracts from 11 traditionally used Argentine medicinal plants were assayed in vitro for antifungal activity against yeasts, hialohyphomycetes as well as dermatophytes with the microbroth dilution method. Among them, the strongest effect was presented by Eupatorium buniifolium and Terminalia triflora, Trichophyton mentagrophytes and Trichophyton rubrum being the most susceptible species with MICs ranging from 100 to 250 g/ml. Lithrea molleoides showed the broadest spectrum of action inhibiting all of the tested dermatophytes with MIC = 250 g/ml. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Antifungal activity; Argentine medicinal species; Microbroth dilution method
1. Introduction The incidence of opportunistic fungal infections in patients treated with immunosuppressive drugs, intensive chemotherapy, suffering AIDS and neonates is increasing at an alarming rate (Groll et al., 1996; Denning et al., 1997). These mycoses are very difficult to eradicate constituting an enormous challenge for healthcare providers (Meyers, 1990). Although there appear to be an array of drugs for the treatment of systemic and superficial mycoses, none of them is ideal in terms of efficacy, safety or antifungal spectrum (Di Domenico, 1998; Ablordeppey et al., 1999). Many of the drugs have undesirable effects or are very toxic (amphotericin B), produce recurrence, show drug–drug interactions (azoles) or lead to the development of resistance (fluconazole, 5-flucytosine) (White et al., 1998). Although some new
∗
Corresponding author. Fax: +54 11 4508 3642. E-mail address: [email protected] (L. Muschietti).
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drugs have emerged as interesting alternatives for the obstinate fungal infections (allylamines or caspofungine) (Vicente et al., 2003), and combination therapy is sometimes used to come these disadvantages, there is a real need for a next generation of safer and more potent antifungal drugs (Bartroli et al., 1998). Numerous useful drugs from higher plants have been discovered by following up ethnomedical uses (Fabricant and Farnsworth, 2001). The diversity of plants growing in Argentina, along with their known ethnopharmacological uses, offer an enormous possibility of finding novel structures with antifungal properties. The selection of the species used in this study was mainly based on their ethnomedical evidence of use for conditions related to microbial infections. These include skin infections, healing of wounds, dandruff, respiratory infections, vaginal mycoses, antiseptic, etc. Some plants without ethnomedical precedents but not previously studied were included too. Data were mainly obtained from the database on Argentine medicinal/toxic plants (Rondina et al., 2003) that covers
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the information of nearly 1800 Argentine medicinal species. Some other literature information was used too (Santos Biloni, 1990). Acacia caven, Bidens subalternans, Eupatorium arnottianum, Eupatorium buniifolium, Jacaranda mimosifolia, Lippia integrifolia, Lithrea molleioides, Morrenia brachystephana, Sebastiania brasiliensis, Sebastiania commersoniana and Terminalia triflora were chosen following an ethnopharmacological approach. Despite no reported ethnomedical uses for Terminalia triflora (Combretaceae), it was included in this study since a closely related species Terminalia australis (traditionally used as an antiseptic agent) shown to possess antifungal properties against Aspergillus and Candida strains (Carparo et al., 2003). Acacia caven (Fabaceae) popularly known as “aromito” or “espinillo” is a tree widely distributed in North and Central Argentina, adjacent Bolivia, Chile, Paraguay, Brasil and Uruguay. The decoction of the bark is traditionally used against bruises, snake bites and skin diseases administered in form of a bath during healing. Against vaginal mycosis, a piece of bark is boiled in 1 l of water, sweetened with honey and drunk several times a day until the sickness disappears (Hilgert, 2001). Additionally, the juice of flowers is applied with cotton as an analgesic for ear-ache and the decoction of flowers used as an antitussive, antihypertensive and antiasthmatic (Martinez Crovetto, 1981). Crude extracts of this plant have demonstrated in vitro antifungal properties against Pycnoporus sanguineus and Fusarium oxysporum (Quiroga et al., 2001). The Asteraceae family is the second largest family of flowering plants with over 13,000 species world-wide. It is the best represented in Argentina with over 200 genera and around 1460 spontaneous species, most of them used for medicinal purposes (Zardini, 1984). Bidens subalternans, Eupatorium arnottianum and Eupatorium buniifolium are species belonging to this family and are commonly found in the North-Eastern and Central regions of Argentina. Bidens subalternans is a herb commonly known as “amor seco”, “amor de viejo” or “espina de erizo”. The decoction of the plant is externally used as an ocular antiseptic and is orally taken in cases of asthma, together with Buddleja madagascariensis and Borago officinalis. It is very accepted to treat aphta and sore throats, being used in gargles or chewing the leaves several times a day. The decoction of the bark is used as a mouthwash for tooth-ache (Martinez Crovetto, 1964, 1981). The dichloromethane extract of this species showed anti-inflammatory activity, maslinic acid and stigmasterol glucoside being responsible for the reported activity (Ortega et al., 1998). Eupatorium arnottianum known as “clavel” or “uou´e” is a subshrub used in traditional medicine against stomachache, as anti-inflammatory and against pain related problems. Aqueous and dichloromethane extracts of this species presented analgesic (Clav´ın et al., 2000) and immunomodulating properties (Fern´andez et al., 2002), respectively. p-
Cymene, ␣-pinene, thymyl acetate and -caryophyllene have been identified in the essential oil as the main compounds (Zygadlo et al., 1995). The decoction of the aerial parts of the shrub Eupatorium buniifolium has been used against rheumatic pains (R´ıos et al., 1993) and as disinfectant (Rojas Acosta, 1905). Twenty grams of aerial parts boiled in 1 l of water is used as a digestive and to treat hepatic affections; it must be drunk four times a day until symptoms disappear (Burgstaller, 1999). Previous phytochemical studies on this species commonly known as “romerillo”, “romerillo colorado” or “chilca” showed the presence of flavonoids, hydroxycinnamic acids derivatives (Muschietti et al., 1990, 1994) and ent-labdanes (Carreras et al., 1998). Pharmacological activities such as antioxidant (Paya et al., 1996), antiviral (Garc´ıa et al., 1990), as well as the identification of three anti-inflammatory compounds have already been reported (Muschietti et al., 2001). Jacaranda mimosifolia (Bignoniaceae) is native to north-west Argentina and Bolivia. Commonly known as “jacaranda”, “caroa” or “tarco”, it is popularly used as an antibiotic (specifically against syphilis) and the bark as a contraceptive (Toursarkissian, 1980). The methanol extract of leaves and stem bark showed antimicrobial activity. Preliminary phytochemical screening of this species revealed the presence of tannins, flavonoids, alkaloids, quinones and traces of saponins (Binutu and Lajubutu, 1994). The genus Lippia (Verbenaceae) includes approximately 200 species of herbs, shrubs and small trees. Nine of them are used in Argentine folk medicine, mainly employed as digestive, stomachic, tonic, nervine, diuretic and emenagogue. Lippia integrifolia is an aromatic shrub, known popularly as “incayuyo”, “pulco”, “poleo”, “t´e del inca” or “manzanillo”, that grows in North-Western and Central Argentina. The decoction of leaves and flowers is traditionally used as an antibiotic (for gonorrhoeal infections), digestive, stomachic, diuretic, emmenagogue, to treat respiratory diseases and sedative (Rondina et al., 2003). It also imparts flavoring to beverages and phytomedicines. Previous chemical research on the essential oil of this species has led to the identification of camphor, limonene, camphene, methyl isoeugenol and germacrene as main compounds (Fricke et al., 1999). The genus Lithrea, from the Anacardiaceae family, is well known for its irritating effects, causing rash and swelling in exposed parts of the body. Lithrea molleoides is a tree which grows in North and Central Argentina, Brazil, Paraguay, Uruguay and Southern Bolivia. A decoction of the twigs of this species, known as “molle de beber”, “chichita” or “´arbol malo” is used in the treatment of respiratory and digestive diseases (Ratera and Ratera, 1980). The infusion of leaves and fruits is said to be diuretic and stomachic, and the resin to treat arthritis (Toursarkissian, 1980). The tincture is used for cough, bronchitis and phlegm (Rondina et al., 2003). Antimicrobial and antiviral activities of this species have already been reported (Kott et al., 1999; Penna et al., 2001). Four new alkylene resorcinols isolated from the MeOH extract showed nematicidal activity (Valcic et al., 2002).
L. Muschietti et al. / Journal of Ethnopharmacology 102 (2005) 233–238
Morrenia brachystephana, as many Asclepiadaceae, is a creeper traditionally known as “taluai”, “tasi” and “isipo’a”. It can be found in North and Central Argentina. The juice obtained from the fruits is employed to treat tetter. The decoction of bark and fruits (30 or 40 g in 1 l of water) is used as a galactagogue and the latex is employed to relieve dental pain (Martinez Crovetto, 1981). The research on this plant has been mainly focused on latex endopeptidases (Cortadi, 2001). Sebastiania brasiliensis and Sebastiania commersoniana (Euphorbiaceae) are two closely related species commonly known as “blanquillo” or “lecher´on”, widely distributed in Entre R´ıos province, Argentina. The decoction of leaves and twigs is useful as an external antiseptic for wounds and the latex is employed for the elimination of warts and as an analgesic for dental caries (Santos Biloni, 1990). The aqueous and alcoholic extracts of Sebastiania brasiliensis and Sebastiania commersoniana have shown antibacterial and antifungal activities. From the hydroalcoholic extract of Sebastiania brasiliensis methylgallate and protocatechuic acid have been identified as the most active compounds on Staphylococcus aureus and Mucor sp., respectively (Penna et al., 2001).
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Terminalia triflora, from the Combretaceae family, popularly known as “lanza amarilla”, “palo amarillo” or “lanza” is a tree widely distributed in North-Western Argentina. Hieronymus (1882) mentioned the use of its bark in the making of posts, furniture and weapons for soldiers and as fuel. Two ellagitannins with anti HIV-RT activity have already been reported from this plant (Martino et al., 2004). Tannins have been demonstrated to have a powerful antifungal action in clinical studies (Latte and Kolodziej, 2000).
2. Materials and methods 2.1. Plant material Plants were collected in their places of origin in Argentina (Fig. 1) between 2002 and 2003, with the exception of Eupatorium arnottianum that was collected in 1995, extracted soon after collection and methanol extract stored at 5 ◦ C. Plant materials were identified by Juan de Dios Mu˜noz, A. Slanis, G. Giberti and A. Cortadi. Voucher specimens are deposited at the following herbariums.
Fig. 1. Provinces of Argentine where species were collected.
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Herbarium of the Universidad Nacional de Entre R´ıos: Acacia caven (ERA-Mu˜noz 5747), Eupatorium arnottianum (ERA-Mu˜noz 3535), Eupatorium buniifolium (ERA-Mu˜noz 5067), Jacaranda mimosifolia (ERA-Mu˜noz 6263), Lithrea molleioides (ERA-Mu˜noz 1714), Sebastiania brasiliensis (ERA-Mu˜noz 1652–1684), Sebastiania commersoniana (ERA-Mu˜noz 1634). Herbarium of the Instituto Miguel Lillo, Tucum´an: Lippia integrifolia (Slanis 178), Terminalia triflora (Slanis 552). Herbarium of the Museo de Farmacobot´anica J. A. Dominguez, Facultad de Farmacia y Bioqu´ımica, Universidad de Buenos Aires: Bidens subalternans (BAF 3442). Herbarium of the Universidad Nacional de Rosario: Morrenia brachystephana (UNR 1271). 2.2. Extraction of plant material Aerial parts (10 g) of each species were air-dried, ground to powder and extracted by soaking in methanol (100 ml) at room temperature for 24 h. The extracts were filtered off and the process repeated twice under the same conditions. The filtrates were combined and dried under vacuum. 2.3. Microorganisms and media The microorganisms used for the antifungal evaluation were either acquired from the American Type Culture Collection (ATCC, Rockville, MD) or were clinical isolates from the Centro de Referencia Micol´ogica, Facultad de Ciencias Bioqu´ımicas y Farmac´euticas, Universidad Nacional de Rosario (CEREMIC). These were: Candida albicans (ATCC 10231), Candida tropicalis (C131), Saccharomyces cerevisiae (ATCC 9763), Cryptococcus neoformans (ATCC 32264), Microsporum canis (C112), Microsporum gypseum (C115), Epidermophyton floccosum (C114) Trichophyton mentagrophytes (ATCC 9972), Trichophyton rubrum (C113),
Aspergillus fumigatus (ATCC 26934), Aspergillus flavus (ATCC 9170) and Aspergillus niger (ATCC 9029). The antifungal agents ketoconazole (Janssen Pharmaceutical), amphotericin B (Sigma Chemical Co.) and terbinafine were included in the assays as positive controls. The inocula were prepared according to the method proposed by the National Committee for Clinical Laboratory Standards NCCLS (2002a,b) and were adjusted to 104 spores or cells with colony forming ability/ml (Wright et al., 1983). 2.4. Antifungal assay The antifungal activity was determined by the microbroth dilution assay following the guidelines of the National Committee for Clinical and Laboratory Standards (NCCLS) for yeasts M-27-A2 (NCCLS, 2002a) and filamentous fungi M38-A (NCCLS, 2002b) in 96-well microplates. Each well was inoculated with 100 l of the yeast or filamentous inoculum. The control wells contained only the medium and distilled water. For dermatophyte strains incubations were performed at 28 ◦ C while for yeast and filamentous fungi the plates were incubated at 35–37 ◦ C for 24, 48 or 72 h. MIC was defined as the lowest concentration of extract at which no fungal growth was observed after incubation.
3. Results and discussion The methanol extracts of Argentine medicinal plants were screened for antifungal activity against 12 fungal strains. Results are shown in Table 1. Extracts with MIC values below 1000 g/ml, in any of the microorganisms tested, were considered active since only crude extracts were employed. None of the extracts were active against the yeasts and filamentous fungi (data not shown). In contrast, extracts were
Table 1 Antifungal activity of methanolic extracts of traditional Argentine medicinal plants Species (family)
Antifungal activity MIC (g/ml) M.c.
M.g.
E.f.
T.r.
T.m.
Acacia caven (Molina) Molina (Fabaceae) Bidens subalternans DC. (Asteraceae) Eupatorium arnottianum Griseb. (Asteraceae) Eupatorium buniifolium Hook & Arn. (Asteraceae) Jacaranda mimosifolia D. Don (Bignoniaceae) Lippia integrifolia (Griseb.) Hieron. (Verbenaceae) Lithrea molleoides (Vell.) Engl. (Anacardiaceae) Morrenia brachystephana Griseb. (Asclepiadaceae) Sebastiania brasiliensis Spreng. (Euphorbiaceae) Sebastiania commersoniana (Baill.) L. B. Sm. & B. J. Downs (Euphorbiaceae) Terminalia triflora (Gris.) Lillo (Combretaceae) Ketoconazole Amphotericin B Terbinafine
>1000 >1000 n.t n.t 1000 500 250 n.t 250 250
>1000 >1000 >1000 250 >1000 1000 250 >1000 1000 1000
1000 >1000 n.t n.t 1000 250 250 n.t 250 250
>1000 >1000 >1000 100 >1000 1000 250 >1000 500 500
>1000 >1000 >1000 250 >1000 1000 250 >1000 500 500
n.t 15 50 0.01
250 6.25 12.5 0.006
n.t 25 0.3 0.004
100 6.25 12.5 0.003
100 6.25 12.5 0.006
M.c: Microsporum canis, M.g: Microsporum gypseum, E.f: Epidermophyton floccosum, T.r: Trichophyton rubrum, T.m: Trichophyton mentagrophytes, >1000: not active; n.t: not tested.
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active against dermatophytes with MICs between 100 and 1000 g/ml. Eupatorium buniifolium and Terminalia triflora exhibited the strongest activity against Microsporum gypseum (MIC = 250 g/ml for both extracts), Trichophyton mentagrophytes (MIC values = 250 and 100 g/ml, respectively) and Trichophyton rubrum (MIC values of 100 g/ml for both extracts). Lithrea molleioides inhibited all the dermatophytic strains (MIC = 250 g/ml). Sebastiania brasiliensis, Sebastiania commersoniana and Lippia integrifolia showed a limited spectrum of action and a lower potency against all dermatophytes tested (MICs: between 250 and 1000 g/ml). The fact that the extracts of the above species showed activity against dermatophytes gives support to the ethnopharmacological use of those species and makes them interesting for a further evaluation. It is interesting to note that, among dermatophytes, the most susceptible species was Trichophyton rubrum (MIC = 100 g/ml for Eupatorium buniifolium and Terminalia triflora) which is the etiological agent of 80–93% of all clinical infections produced by dermatophytes (Feresin et al., 2001) suggesting the possibility of using these extracts as starting points for the finding of antifungal agents that selectively inhibit the most prevalent fungus in dermatomycoses. These extracts are being submitted to bioassay-guided fractionations in order to isolate the compound/s responsible for the activity.
Acknowledgments We thank Dr. A. Cortadi and Dr. G. Giberti for collection of plant material. This work was supported by grant PIP CONICET: 02419, B101 UBACYT and is part of the Proyecto Iberoamericano X.7 PIBEAFUN (Search and development of new antifungal agents), within the Programa Iberoamericano de Ciencia y Tecnolog´ıa para el Desarrollo (CYTED). Dr. S. Zacchino thanks to ANPCyT (PICT Redes 00260) and OEA for financial support. M. Derita acknowledges CONICET.
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