Abstract
Context: Subcutaneous mycoses are chronic infections caused by slow growing environmental fungi. Latin American plants are used in folk medicine to treat these afflictions. Moreover, the potential of the rich Latin American biodiversity for this purpose has not been fully explored.
Objectives: The aim of the study was to screen Latin American plant extracts against two species of subcutaneous fungi: Sporothrix schenckii and Fonsecaea pedrosoi.
Materials and methods: One hundred ninety-five organic extracts from 151 Latin American plants were screened against two subcutaneous fungi by the agar dilution method at a concentration of 100 µg/mL, and minimum inhibitory concentrations (MICs) of active extracts were determined. Positive (amphothericin B) and negative (50% ethanol) controls were used.
Results and discussion: Twenty eight extracts showed activity at ≤100 µg/mL. Of these, four extracts from Gnaphalium gaudichaudianum DC (Asteraceae), Plumeria rubra L (Apocynaceae), Tecoma stans (L.) Juss. ex Kunth. (Bignoniaceae), and Trichostigma octandum (L.), H. Walter showed activity against F. pedrosoi at MIC 12.5 µg/mL; and, four extracts from Bourreria huanita (Lex.) Hemsl. (Boraginaceae), Phytolacca bogotensis Kunth (Phytolaccaceae), Monnina xalapensis Kunth (Polygalaceae) and Crataegus pubescens (C. Presl) C. Presl (Rosaceae) against S. schenckii. This is the first report on antifungal activity of the Latin American plants against these two subcutaneous fungi.
Conclusion: S. schenkii and F. pedrosoi were inhibited by B. huanita (MIC: 12.5 and 25 µg/mL), G. gaudichaudianum (MIC: 50 and 12.5 µg/mL) and T. triflora (MIC: 25 µg/mL).
Introduction
Sporotrichosis is a subcutaneous fungal disease, usually associated with trauma. It is caused by the dimorphic fungus Sporothrix schenckii. It occurs globally in tropical and subtropical regions and is characterized by nodular lesions, associated with lymphadenopathy (CitationMorris-Jones, 2002; CitationRamos-e-Silva et al., 2007), which become disseminated in AIDS patients (CitationWare et al., 1999). The available therapy in the majority of patients consists of treatment with terbinafine (CitationHull & Vismer, 1992), potassium iodide (CitationSandhu & Gupta, 2003), amphotericin B (CitationKohler et al., 2007), itraconazole and ketoconazole (CitationAlvarado-Ramírez & Torres-Rodríguez, 2007).
Chromoblastomycosis is a traumatically induced chronic subcutaneous mycotic infection, which is caused by several species of pigmented saprophytic molds. Among them, Fonsecaea pedrosoi is one of the most common pathogens associated with this disease, which progresses slowly over many years. The currently available drugs 5-flucytosine, fluconazole, ketoconazole, amphotericin B (CitationLupi et al., 2005; CitationLópez Martínez & Méndez Tovar, 2007), itraconazole and terbinafine (CitationGupta et al., 2002) show toxicity, limited efficacy, and require longer duration of treatments. This results in the development of resistance as reported for itraconazole (Andrade et al., 2003). Therefore, there is an urgent need to discover newer antifungal drugs for subcutaneous mycoses.
Plants constitute an invaluable source of antifungal compounds because of their unmatched availability of chemical diversity, and many efforts have been made worldwide to identify medicinal plants with antifungal properties (CitationRai & Mares, 2003; CitationCos et al., 2006; CitationMaregesi et al., 2008), some of them in Latin America (CitationAgüero et al., 2007; CitationCruz et al., 2007; CitationDanelutte et al., 2003; CitationEscalante et al., 2002; CitationMalheiros et al., 2005, CitationSvetaz et al., 2010).
There have been a few studies reported on antifungal screening of medicinal plants against subcutaneous fungi. For example, against S. schenckii, CitationRojas et al. (2003) reported results of antifungal screening of 24 Peruvian plants; the essential oil of Origanum vulgare L. (Lamiaceae) showed activity against seven isolates with a MIC of 250 µL/mL (CitationCleff et al., 2008); the ethanol extract of Agapanthus africanus (L.) Hoffmanns (Agapanthaceae) rhizomes showed activity at a MIC of 250 µg/mL, and a saponin was isolated and active at MIC: 15.6 µg/mL (CitationSingh et al., 2008); and, pseudodillapiol, isolated from Piper abutiloides Kunth (Piperaceae), showed activity at 12.5 µg/mL (CitationJohann et al., 2008).
On the other hand, four studies on plants acting against F. pedrosoi showed moderate activity (MIC 200–625 µg/mL) in Brazil, on essential oil of Lippia origanoides Kunth. (Verbenaceae) (CitationOliveira et al., 2007), aqueous extract of Caesalpinia pyramidalis Tul. (Caesalpinaceae) and Ziziphus joazeiro Mart. (Rhamnaceae) (CitationCruz et al., 2007), seven Artemisia species (CitationLopes-Lutz et al., 2008), and methanol extract from Pterocaulon alopecuroides Lam (Asteraceae) (Daboit et al., 2009).
The only study that investigated both fungi is one on the defatted ethanol extract of Curcuma longa L. (Zingiberaceae) conducted in Thailand, which showed inhibition of S. schenckii (MIC 114 µg/mL) and F. pedrosoi (459 µg/mL), although the isolated curcumin did not present activity against neither (CitationApisariyakul et al., 1995).
Within the framework of a multinational collaborative Organization of American States (OAS) project coordinated by one of the authors (MPG) during 2001–2004 with the participation of research centers from Argentina, Bolivia, Brazil, Colombia, Costa Rica, Guatemala and Panama, the potential of 327 plant species (92 families and 251 genera) from Latin America were screened for antifungal properties against a panel of human opportunistic fungi (CitationSvetaz et al., 2010).
We now report here the antifungal activity of 151 species from Latin America used either ethnomedically for skin or dermatomucosal diseases or collected at random in 0.1 h biodiversity plots in protected reserves against S. schenckii and F. pedrosoi, the two most prevalent agents of subcutaneous infections.
Materials and methods
Plant material
One hundred fifty-one plants from 64 families from seven countries involved in the OAS project were chosen and collected (). These included 112 species used in ethnomedicine for the following conditions of traditional therapeutic indications: antibiotic, antifungal, antiseptic, infections, itching, respiratory diseases, skin ailments, ulcerations, vaginitis, venereal diseases, vulnerary, and wound healing, and 39 species collected at random from 0.1 ha biodiversity plants in protected reserves.
Table 1. Antifungal activity against Sporothrix schenckii and Fonsecaea pedrosoi of extracts from Latin American plant species (MIC µg/mL).
The taxonomic identity of each plant was established by taxonomists of participating countries from Argentina, Elisa Petenatti and Martha Gattuso; from Bolivia, Rosy de Michel, Genevieve Bourdy and Andrés Roca; from Colombia, Ricardo Callejas, Edgar Linares, Zaleth Cordero, and Santiago Díaz; from Costa Rica, Luis Guillermo Acosta, Alexander Rodríguez and Diego Vargas; from Guatemala, Mario Véliz and Juan José Castillo; and, from Panama, Mireya Correa.
Voucher specimens were deposited in each country’s Herbarium. Argentina: National University of San Luis (UNSL), San Luis: National University of Rosario (UNR), Rosario: National University of Entre Ríos (ERA), Paraná: and the Herbarium of the Botanical Institute of North East (IBONE). Bolivia: National Herbarium of Bolivia (LPB), La Paz, Brazil: Herbário Barbosa Rodrigues (HBR), Itajaí, SC. Colombia: National Colombian Herbarium (COL), Bogotá. Costa Rica: National Institute for Biodiversity, INBio (INB), San José. Guatemala: CEMAT-FARMAYA Ethnobotany Herbarium (CFEH), Guatemala. Panamá: FLORPAN at the Herbarium of the University of Panamá (PMA), Panamá ().
Preparation of extracts
From 151 species, 195 extracts were prepared by percolation with different solvents; percolates were concentrated in vacuo in a rotary evaporator at a temperature <40°C and dried in a vacuum dryer.
Antifungal assay
Activity was evaluated by the procedure described by CitationBrancato and Golding (1983) for filamentous fungi, modified by Mac Rae et al. (1988) and previously used to determine activity against dermatophytes (CitationCáceres et al., 1993). Due to its marked survival in the environment, only the saprophytic phase was studied. Screw cap tubes containing Takashio agar slant for sporulation (CitationVanbreuseghem et al., 1970) were inoculated with the fungi and incubated at 27°C for 21 days; the spores were removed with sterile saline solution, counted, and a suspension containing 100 spores/µL was prepared. For screening, tubes containing 13.5 mL of Sabouraud agar were autoclaved, cooled to 50°C, 1.5 mL of an extract containing 1000 µg/mL were added, poured into Petri dishes, cooled, and incubated at 35°C to check for sterility (final concentration 100 µg/mL). Four equidistant holes were made in each agar-extract dish with sterile Durham tubes; 30 µL of the spore suspension were placed in each hole, incubated al 27°C for 21 days for S. schenckii and 28 days for F. pedrosoi; as controls, dishes containing 35% ethanol (growth 100%) and 100 µg/mL amphotericin B (growth 0%) were prepared. Amphotericin B was chosen as positive control since it is used as drug for the treatment of both fungi (CitationKohler et al., 2007; López Martínez & Mendez Tovar, 2007). The colony diameter was measured in millimeter using a vernier and an extract was considered active when it showed 75% inhibition of the colony diameter in comparison with the control. For minimum inhibitory concentration (MIC), extract-agar dishes were challenged with fungal spores in the same manner as above, but using serial dilutions of extracts in the agar dishes (CitationGaitán et al., 2007).
Results
Different parts of the plants were collected either from biodiversity plots or based on ethnomedical use. One hundred ninety five extracts from 151 plant species were evaluated by the agar dilution method at a fixed screening concentration of 100 µg/mL. At this concentration, 28 extracts showed activity (MIC ≤100 µg/mL), 13 against both fungi, 11 against only S. schenckii, and 4 against F. pedrosoi. Then the MIC of each of the active extract was assessed with the agar dilution assay according to the method of CitationGaitán et al. (2007). Of these, four extracts from Gnaphalium gaudichaudianum DC (Asteraceae), Plumeria rubra L (Apocynaceae), Tecoma stans (L.) Juss. ex Kunth. (Bignoniaceae) and Trichostigma octandum (L.), H. Walter showed activity against F. pedrosoi at MIC 12.5 µg/mL, and four extracts from Bourreria huanita (Lex.) Hemsl. (Boraginaceae), Phytolacca bogotensis Kunth (Phytolaccaceae), Monnina xalapensis Kunth (Polygalaceae) and Crataegus pubescens (C. Presl) C. Presl (Rosaceae) showed activity against S. schenckii; and the activity of 10 extracts could not be confirmed due to insufficient material (). No significant difference was found among plants chosen on the basis of ethnomedical use (41/112, 36.6%) or collected from biodiversity plots (13/39, 33.3%) (Z test of two independent populations, P = 0.823).
For interpretation of results, we considered that an extract was inactive when MIC was >100 μg/mL, moderately active when MIC was 25–50 μg/mL and strongly active when MIC was 12.5 μg/mL. Although several extracts (167, 85.6%) did not show any activity against the studied fungi, the eight species mentioned above showed strong activity and others showed moderate activity, such as Terminalia triflora (Griseb.) Lillo (Combretaceae), which showed an MIC of 25 µg/mL against both fungi, and Aristolochia gibertii Hook. (Aristolochiaceae), Piper scabrum Lam (Piperaceae), and Lippia graveolens Kunth. (Verbenaceae) were active against S. schenckii at MIC of 25 µg/mL.
Discussion
Only 18 (9.2%) extracts showed inhibitory activity against one or both of the subcutaneous fungi, S. schenkii and F. pedrosoi studied (MIC ≤50 µg/mL). Eight (4.1%) extracts were active at MIC of 12.5 µg/mL. Only one extract of the flower of B. huanita inhibited both fungi. This is a rare tree native to Mesoamerica, and its flowers are used popularly for skin ailments and ulcers (CitationOrellana, 1987), as well as for several nervous ailments (CitationTorres, 2007). Previous information on this plant is negligible; one study showed lack of antifungal activity in the ethanol extract of the flowers against Candida, Aspergillus and dermatophytes (CitationSvetaz et al., 2010).
G. gaudichaudianum is a herb native to Argentina, where it is used in folk medicine for the treatment of respiratory diseases and as a vulnerary, but no activity has been demonstrated previously against a panel of pathogenic fungi (CitationSvetaz et al., 2010).
T. triflora is a tree native to South America used in folk medicine as antiseptic, but results of its antifungal activity are controversial, since in one study the methanol extract showed activity against Trichophyton mentagrophytes, T. rubrum (100 µg/mL) and Microsporum gypseum (250 µg/mL) (CitationMuschietti et al., 2005), while another study showed no antifungal activity (CitationSvetaz et al., 2010). Other important activities have been demonstrated by the aqueous extract of leaves, such as pro-oxidant activity (CitationDesmarchelier et al., 1997), and inhibition of polymerase and ribonuclease activities of HIV reverse transcriptase at IC50 1.6–1.8 µg/mL (CitationMartino et al., 2002); by bioguided fractionation, active principles were shown to be punicalin and 2-O-galloylpunicalin (CitationMartino et al., 2004). Other Terminalia spp. have shown activity against S. schenckii at 20–40 µg/mL such as extracts of seven South African species (CitationMasoko et al., 2005), as well as the acetone extract of T. sambesiaca Engl. & Diels, and hexane and acetone extracts of T. phanerophlebia Engl. & Diels (30 µg/mL) (CitationShai et al., 2008); from stem bark of T. barachystemma was isolated punicalagin with important activity against Candida spp. (CitationLiu et al., 2009).
The other five species showed activity, but only to one of the fungi. In the case of P. bogotensis no specific information was found, but phytolaccosides isolated from Phytolacca tetramera Hauman showed activity against a panel of opportunistic fungi (CitationEscalante et al., 2002); from P. rubra, plumericin and isoplumericin were isolated, showing molluscicidal and antibiotic activity (CitationHamburger et al., 1991); and, the chloroform extract of T. stans gave the best inhibition zone against fungal activity (CitationGandhi Ramesh, 2010), but no activity was shown against pathogenic fungi (CitationSvetaz et al., 2010). From C. pubescens, M. xalapensis, and T. octandrum, no reference of biocidal activity was detected in the literature.
We describe here for the first time the inhibitory activity of extracts from G. gaudichaudianum, P. rubra, T. stans and T. octandrum against F. pedrosoi, and from B. huanita, C. pubescen, M. xalapensis and P. bogotensis, against S. schenckii.
Conclusion
It is noteworthy that three species showed good activity, not described before, against two subcutaneous fungi, S. schenkii and F. pedrosoi, which are usually resistant to most of the available antibiotics, B. huanita (MIC: 12.5 and 25 µg/mL, respectively), G. gaudichaudianum (MIC: 50 and 12.5 µg/mL, respectively), and T. triflora (MIC 25 µg/mL to both fungi). The antifungal activity of extracts from P. rubra, T. stans and T. octandrum against F. pedrosoi and from C. pubescen, M. xalapensis and P. bogotensis, against S. schenckii, is reported for the first time. Only in 36% of plants screened the found activity in this study may explain their ethnomedical use.
Declaration of interest
The authors acknowledge the financial support from Organization of American States (OAS grant /AICD/106), Secretaría Nacional de Ciencia, Tecnología e Innovación (Panama), ANPCyT (Argentina), DIGI-USAC (Guatemala), and Ibero-American Program on Science and Technology for Development (CYTED, RIBIOFAR Red 0284). Technical assistance from Nancy del Cid, Ana Beatriz Suarez, Ana Margarita García, Diego Vargas and Alex Espinoza are kindly appreciated. We are indebted to all the taxonomists who participated in this study in identifications of plants studied and to the National authorities of the respective countries for granting collection permissions.
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