Abstract
The 50% ethanol extract from Sebastiania commersoniana. (Baill.) L.B. Sm. & B.J. (Euphorbiaceae), a South American medicinal plant locally used as external antiseptic, was subjected to a phytochemical study and to the determination of its in vitro. antifungal activity. After fractionation of the extract, four flavonoids (quercetin, kaempferol, isorhamnetin, isoquercitrin), four phenolic derivatives (gallicin, gallic, syringic, and caffeic acids), and a coumarin (scopoletin) were identified. The structures of isolated compounds were established by spectroscopic methods and confirmed by comparison with reference samples and literature data. The extract exhibited significant antifungal activity against the dermatophytes Microsporum gypseum., Trichophyton mentagrophytes. and Trichophyton rubrum..
Introduction
Sebastiania commersoniana. (Baill.) L.B. Sm. & B.J. Downs (Euphorbiaceae), commonly known as “blanquillo” or “lecherón,” is a tree of 5 to 10 m high native to subtropical South America and widely distributed in north and northeastern Argentina (Digilio & Legname, Citation1966). “Blanquillo” is a name given to a number of plants of the genus Sebastiania. and refers to the white color of its wood, and “lecherón” refers to its milky latex (Lahitte et al., Citation1998).
Infusions of blanquillo made up with leaves and twigs are used in folk medicine 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, Citation1990).
In a previous article, we reported the antiviral activity of the aqueous extract of S. commersoniana. and the antibacterial and antifungal activities of the aqueous and alcoholic extracts of the same plant, which showed growth inhibitory activity against a panel of Gram-positive and Gram-negative bacteria and fungi (Kott et al., Citation1999; Penna et al., Citation2001).
Although there are many reports on the phytochemistry of the genus Sebastiania. (e.g., Nazaré et al., Citation2005), no references on the chemical constituents of S. commersoniana. are available in the literature.
In this article, we report the phytochemical investigation of the 50% ethanol extract from S. commersoniana. and its in vitro. antifungal activity using several strains of yeasts and filamentous fungi.
Material and Methods
Plant material
S. commersoniana. was collected in Parque Rivadavia, Concordia, Entre Ríos, Argentina and identified by Ing. J. de Dios Muñoz. A voucher specimen (Muñoz 1634) is deposited in the herbarium of the Facultad de Ciencias Agropecuarias de Entre Ríos, Argentina.
Extraction, fractionation, and isolation procedures
Dried and ground aerial parts of S. commersoniana. (650 g) were extracted twice with 50% ethanol for 2 days at room temperature and filtered. The combined filtrates were concentrated under vacuum at 45°C. The extract was suspended in water and then partitioned successively with dichloromethane, ethyl ether, and ethyl acetate. The organic extracts were concentrated to dryness by rotary evaporation at 45°C, yielding 0.20, 0.18, and 3.22%, respectively. The dichloromethane extract (380 mg) was chromatographed on Sephadex LH-20(Sephanex R) and eluted with dichloromethane with increasing amounts of methanol to afford seven fractions. Fraction 5 and 6 were purified by preparative PC in AcOH 15% to obtain quercetin, kaempferol, gallic and syringic acids, gallicin, and scopoletin. Fraction 7 was purified by repeated CC on Sephadex LH-20 eluted with methanol to give five sub-fractions, which were further purified by preparative PC in AcOH 15%, to obtain caffeic acid and isorhamnetin. The ethyl ether extract (350 mg) was chromatographed over Sephadex LH-20, eluted with a gradient of dichloromethane in methanol affording 3 fractions. These fractions were further purified by preparative PC in AcOH 15%, to obtain kaempferol, quercetin, and gallic acid. The ethyl acetate extract (580 mg) was chromatographed on Sephadex LH-20 using dichloromethane-methanol (9:1) with an increasing amount of methanol to give five main fractions. Fractions 3 and 4 were rechromatographed on Sephadex LH-20 and eluted with methanol to give two sub-fractions, which were purified by preparative PC in AcOH 25% to afford quercetin, isoquercitrin, and gallic acid. The structures of all compounds were determined spectroscopically by UV, 1H-NMR, and MS data and confirmed by comparison with data in the literature and with reference samples.
Microorganisms and media
For the antifungal evaluation, the following strains from the American Type Culture Collection (ATCC), Rockville, MD, USA, and from CEREMIC (C) (Centro de Referencia en Micología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531, 2000 Rosario, Argentina) were used. Yeasts: Candida albicans. ATCC 10231, Candida tropicalis. C 131, Saccharomyces cerevisiae. ATCC 9763, and Cryptococcus neoformans. ATCC 32264. Filamentous fungi: Aspergillus fumigatus. ATCC 26934, Aspergillus flavus. ATCC 9170, and Aspergillus niger. ATCC 9029. Dermatophytes: Microsporum gypseum. C 115, Microsporum canis. C 112, Trichophyton mentagrophytes. ATCC 9972, and Trichophyton rubrum. C 110, C 113, C 133-141. Yeasts were grown on Sabouraud- chloramphenicol agar slants for 48 h at 30°C. Cell suspensions were adjusted to 104 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., Citation1983) and adjusted to 104 spores/ml.
Antifungal susceptibility testing
The minimal inhibitory concentration (MIC) of each extract or subextract was determined by using broth microdilution techniques as described by the National Committee for Clinical Laboratory Standards for yeasts (M27-A2) (NCCLS, Citation2002a) as well as for filamentous fungi (M 38 A) (NCCLS, Citation2002b) in microtiters of 96-wells. MIC values were determined in RPMI-1640 (Sigma, St Louis, Mo, USA) buffered to a pH 7.0 with MOPS. The starting inocula were approximately 1 × 104 to 5 × 104 CFU/ml. Microtiter trays were incubated at 35oC for yeasts and hialophytomycetes and at 28–30°C for dermatophyte strains in a moist, dark chamber. MICs were recorded at 48 h for yeasts and at a time according to the control fungus growth for the other fungi. The susceptibility of the standard drugs terbinafine and amphotericin B were defined as the lowest concentration of drug that resulted in total inhibition of fungal growth.
For the assay, extract stock solutions were two-fold diluted with RPMI from 1000–0.98 µg/ml (final volume = 100 µl) and a final DMSO concentration ≤ 1%. A volume of 100 µl of inoculum suspension was added to each well, with the exception of the sterility control where sterile water was added to the well instead. The MIC was defined as the minimum inhibitory concentration of the extract or compound that resulted in total inhibition of the fungal growth. Extracts with MICs ≤ 1000 µg/ml were considered active.
Results and Discussion
The phytochemical investigation of the 50% ethanol extract from S. commersoniana. led to the isolation of quercetin, kaempferol, isorhamnetin, gallicin, scopoletin, syringic, and caffeic acids from the dichloromethane extract; kaempferol and quercetin from the ethyl ether extract; and isoquercitrin and quercetin from the ethyl acetate extract. Some of the described compounds have been previously found in S. brasiliensis., a related species (Penna et al., Citation2001), but this is the first report of these compounds in S. commersoniana..
The crude ethanol extract and the different extracts obtained by L/L partition, were submitted to a screening for antifungal activity against yeasts as well as against hialohyphomycete and dermatophyte fungi. The results are shown in . The tested extracts were active only against dermatophytes with MICs between 100 and 500 µg/ml. These results add new evidence on the antimicrobial activity found by Penna et al. (Citation2001) (who tested other microorganisms) and provide a scientific basis for the traditional use of this medicinal plant as an external antiseptic.
Table 1. In vitro. evaluation of the antifungal activity of different extracts of S. commersoniana. and gallic acid isolated from all active extracts (MIC values are given in µg/ml).
The chromatographic analysis of the active extracts showed the presence of gallic acid as the major common compound for all of them.
In conclusion, this is the first report on the phytochemical study and the antifungal activity of S. commersoniana. extracts. The presence of gallic acid in all active extracts suggests that it could play an important role in antifungal activity. It has been previously demonstrated that gallic acid and its n.-alkyl esters have exhibited antimicrobial activities (Kubo et al., Citation2002).
Further investigations will help to elucidate the role played by each isolated compound in the antifungal activity of the different extracts.
Acknowledgments
The authors wish to thank Ing. J. de Dios Muñoz for collection and identification of the plant material. The financial support of the Universidad de Buenos Aires (Proyectos B 101 and B 046) and ANPCyT PIC R 260 is gratefully acknowledged. This work is a part of Proyecto X-7 PIBEAFUN, Subprograma Iberoamericano de Ciencia y Tecnología para el Desarrollo (CYTED).
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