Abstract
The antifungal and antibacterial activity of 10 crude extracts from four different species, all of them used in Mexican folk medicine for the treatment of infectious diseases, were tested in vitro. for antimicrobial activity against Staphylococcus aureus., Streptococcus faecalis., Escherichia coli, Klebsiella pneumoniae., Salmonella typhi., Candida albicans., Trichophyton mentagrophytes., and Trichophyton rubrum.. All extracts from the above plants showed some degree of antimicrobial activity against at least two microorganisms tested. The strongest antibacterial activity was found in the water extract of Hibiscus sabdariffa. and the methanol extract of Lysiloma acapulcensis., whereas the methanol extract from Loeselia mexicana. showed the best antifungal activity against dermatophytes.
Introduction
Infectious diseases represent an important problem to health and represent one of the main causes of morbidity and mortality worldwide (World Health Organization, Citation2004). In the past years, there has been an increasing incidence of infectious diseases due to a growth in immunocompromised population, such as cancer and HIV/AIDS patients. This fact coupled with the resistance and the toxicity of several antibiotics support the search for new drugs (Fostel & Lartey, Citation2000). In this sense, medicinal plants offer many possibilities for discovering new molecules with antimicrobial activity (Navarro and Delgado, Citation1999; Ríos et al., Citation2003; González et al., Citation2004).
Mexico is a country where folk medicine still plays a very import role in the health care system (Argueta, Citation1994). We are currently investigating medicinal plants with potential biomedical uses in the treatment of infectious diseases (Lozoya et al., Citation1992; Navarro et al., Citation1996; Rojas et al., Citation2001). In order to extend our studies in this field, we decided to explore the antibacterial and antifungal activities of Hibiscus sabdariffa., Loeselia mexicana, Lysiloma acapulcensis., and Miconia mexicana., which were collected on the basis of ethnobotanical surveys realized from the states of Morelos and Chiapas, Central and Southern México. To our knowledge, there are no reports describing the antimicrobial properties and chemical constituents of these plants. Therefore, our objective was to investigate the in vitro. antimicrobial activity of 10 extracts from the above species against two Gram-positive bacteria, two Gram-negative bacteria, one yeast, and two mycelia fungi that are common causes of several infectious diseases. The current work will contribute to enhance our knowledge of the antimicrobial properties of four Mexican medicinal plants, and these results may lead to the development of new therapeutic alternatives in phytomedicine.
Materials and Methods
Collection of plant material
Aerial parts of Loeselia mexicana. and Lysiloma acapulcensis. were collected in March 2004 in their natural habitat from different regions in Morelos State, México. Miconia mexicana. was collected in January 2001 in Oxchiuc, Chiapas State, México. Voucher specimens were prepared and authenticated by M. en C. Margarita Aviles and M. en C. Macrina Fuentes and were deposited at the herbarium of Instituto Nacional de Antropología e Historia de Morelos (INAHM) in the city of Cuernavaca, Morelos State. Hibiscus sabdariffa. was collected in February 2004 from cultivate grown controlled at Centro de Investigación Biomédica del Sur, Xochitepec, in Morelos. This plant was botanically authenticated by M. en C. Abigail Aguilar at the herbarium of the Instituto Mexicano del Seguro Social (IMSSM). The voucher specimen numbers are given in . Traditional practices by herbalists in Morelos State provided the basis for selecting the part of the plant to be tested.
Table 1 List of medical plant used to evaluate their antimicrobial properties
Preparation of extracts
Loeselia mexicana, Lysiloma acapulcensis., and Miconia mexicana. were dried at room temperature for a period of 2 weeks and then milled into fine powder, which was successively extracted with n.-hexane, dichloromethane, and methanol (100 g/1500 ml) at room temperature. Each solvent was replaced three-times with fresh solvent, remaining in contact with the plant material 24 h each time. After filtration, the extracts were concentrated to residue by removing the solvents in a rotavapor at 40°C. Roughly powdered calyces (100 g) from Hibiscus sabdariffa. were macerated in 1000 ml of distilled water by shaking at room temperatures for 24 h twice. The extract was filtered and freeze-dried. The yield of the extracts was quantified, and the obtained material was protected from direct light and stored at less than 4°C until its use.
Antibacterial activity
The bacteria used for the antibacterial assays were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). The following strains were used: Staphylococcus aureus. ATCC 6358; Streptococcus faecalis. ATCC 10231; Escherichia coli. ATCC 8937; Klebsiella pneumoniae. ATCC 13883, and Salmonela typhi. ATCC 06539.
The antibacterial activity was measured by determining the minimal inhibitory concentration (MIC) and was carried out by employing the agar dilution method (Ríos et al., Citation1988). The crude extracts (n.-hexane, dichloromethane, and methanol) were dissolved in dimethyl sulfoxide (DMSO; 2% v/v); to obtain a concentration of 40 mg/ml. Dilutions were prepared from these solutions to obtain final concentrations of 8.0 to 0.5 mg/ml. The inoculum for each organism was prepared from cultures containing 108 colony-forming units (CFU)/ml.The diluted (1:20) inoculum was applied as a spot by means of a calibrated loop to deliver 0.002 ml, resulting in a spot inoculum covering a circle of 5–8 mm diameter and containing 104 CFU. The plates were incubated for 24 h at 37°C. Gentamicin (2.5–120 µg/ml) (Sigma) was used as reference standard. Observations were performed by duplicate, and results are expressed as the lowest concentration of plant extract able to produce a complete suppression of colony growth on agar (minimum inhibitory concentration; MIC).
Antifungal activity
The following three strains were used as test fungi: Trychophyton mentagrophytes. ATCC 28185, Trychophyton rubrum. ATCC 28188, and Candida albicans. ATCC 10231. The filamentous fungi were maintained on potato dextrose agar (PDA; Merck, Germany) at 27°C. Sabouraud glucose agar (SGA; Merck, Germany) was used to keep up the yeast and as assay medium.
The antifungal assay was performed by the agar dilution method using Petri dishes (Falcon, USA) (Rahalison et al., Citation1994; Gadhi et al., Citation2001). The stock solution of extracts and reference compounds, nystatin (Merck, Germany) and miconazole (Sigma), were two-fold serial diluted yielding concentrations in the range from 8.0 to 0.125 mg/ml and from 128 to 1 µg/ml, respectively. Final concentrations of DMSO in the test were less than 2% (v/v). Final inoculum of 105 cell/ml for Candida albicans. and 106 spore/ml for filamentous fungi was placed on top of the solidified agar with a loop calibrated to deliver 0.005 ml. Experiments were carried out in duplicate and incubation at 29°C. The fungal growth was checked, first in control plates prepared without any test sample, after 24, 48, and 72 h, depending on the incubation period required for a visible growth: 24 h for Candida albicans., 24 h for Aspergillus niger., and 72 h for dermatophytes.
Results and Discussion
shows the botanical name, voucher specimen numbers, local name, and popular uses of the studied plants. In , the plant part used, the percentage yield, and the obtained MIC values of the corresponding extracts are summarized. Considering that in this screening only crude extracts were tested, extracts with MIC values of 8.0 mg/ml or below were considered active.
Table 2 Minimal inhibitory concentration (MIC, mg/ml) of the organic crude extract
The obtained results show that all the tested plants were active against at least two of the assayed fungi. As antifungal, the most interesting were the methanol extracts of Lysiloma acapulcensis. (C. albicans. MIC = 2.0, T. mentagrophytes. MIC = 1.0, and T. rubrum. MIC = 1.0 mg/ml) and Loeselia mexicana. (C. albicans. MIC = 4.0, T. mentagrophytes. MIC = 0.250, and T. rubrum. MIC = 0.250 mg/ml). Also, the dichloromethanol and hexane extracts of L. mexicana. showed an important activity against dermatophytes ().
Concerning the antibacterial properties, the most interesting results were obtained from methanol extracts of Lysiloma acapulcensis., Miconia mexicana., and Hibiscus sabdariffa. (water extract), with the last extract being the most active against the Gram-positive and Gram-negative bacteria. Thus, MIC of 0.5 mg/ml was found against S. aureus. and S. faecalis., while a MIC of 1.0 mg/ml was found against E. coli, K. pneumoniae, and S. typhi..
These screenings showed that all tested plant extracts possess interesting antimicrobial properties. This explains the use of these plants in folk medicine for the treatment of various illnesses whose symptoms might involve infectious diseases. Consequently, in order to elucidate the responsible active compound, we have currently started a bioguided fractionation of the above extracts.
Acknowledgment
We want to thank Myrna and Francois Mages for technical assistance. This investigation was partially supported by project IMSS-FOFOI: 2005/1/I/066.
References
- Aguilar A, Camacho JR, Chino S, Jacquez P, Lopez ME (1994): Herbario Medicinal del Instituto Mexicano del Seguro Social. Información Etnobotánica.. México, IMSS, p. 156.
- Argueta A, Cano L, Rodarte M (1994): Atlas de las Plantas de la Medicina Tradicional Mexicana, Tomo 1-3. México, D.F., Instituto Nacional Indigenista, p. 1786.
- Avíles M (2001): Informe del Programa de Parteras Tradicionales en el Estado de Morelos. Cuernavaca, Morelos, México, Centro INAH, p. 65.
- Avíles M, Suaréz G (1994): Catálogo de Plantas Medicinales Jardín Etnobotánico Centro. Cuernavaca, Morelos, México, INAH, p. 47.
- Berlin B, Berlin EA, Breedlove DE, Duncan T, Jara VM, Laughlin RM, Velasco T (1990): La herbolaria médica Tzeltal-Tzotzil en los altos de Chiapas.. Gobierno del Estado, Chiapas, México, p. 154.
- Fostel J, Lartey P (2000): Emerging novel antifungal agents. Drug Discov Today 5: 25–32. [INFOTRIEVE], [CSA]
- Gadhi C, Benharref A, Jana M, Basile A, Contet-Audonneau N, Fortier B (2001): Antidermatophic properties of extracts from the leaves of Aristolochia paucinervis. pomel. Phytother Res 15: 79–81. [INFOTRIEVE], [CROSSREF], [CSA]
- González M, Zalimpa A, Marquina S, Navarro V, Alvarez L (2004): Antimycotic spirostanol saponins from Solanum Hispidum. leaves and their structure-activity relationships. J Nat Prod 67: 938–941. [CROSSREF], [CSA]
- Lozoya X, Navarro V, Zurita M (1992): Solanum chrysotrichum. (Schldl.), a plant used in Mexico for the treatment of skin mycosis. J Ethnopharmacol 36: 127–132. [INFOTRIEVE], [CROSSREF], [CSA]
- Monroy OC, Castillo P (2000): Plantas Medicinales Utilizadas en el Estado de Morelos. Centro de Investigaciones Biológicas, Universidad Autónoma del Morelos, México, p. 400.
- Navarro V, Villareal M, Rojas G, Lozoya X (1996): Antimicrobial evaluation of some plants used in Mexican traditional medicine for the treatment of infectious diseases. J Ethnopharmacol 53: 143–147. [INFOTRIEVE], [CROSSREF], [CSA]
- Navarro V, Delgado G (1999): Two antimicrobial alkaloids from Bocconia arborea.. J Ethnopharmacol 66: 223–226. [INFOTRIEVE], [CROSSREF], [CSA]
- Rahalison L, Hamburger M, Monod M, Frenk E, Hostettmann (1994): Antifungal test in phytochemical investigations: Comparison of bioautographic methods using phytophatogenic and human pathogenic fungi. Planta Med 60: 41–44. [INFOTRIEVE], [CSA]
- Ríos J, Recio M, Villar A (1988): Screening methods for natural products with antimicrobial activity: A review of the literature. J Ethnopharmacol 23: 127–149. [CROSSREF], [CSA]
- Ríos M, Aguilar B, Navarro V (2003): Two New Benzofuranes from Eupatorium aschenbornianum. and their Antimicrobial Activity. Planta Med 69: 967–970. [CROSSREF], [CSA]
- Rojas G, Lévaro J, Tortoriello J, Navarro V (2001): Antimicrobial evaluation of certain plants used in Mexican traditional medicine for the treatment of respiratory diseases. J Ethnopharmacol 74: 97–101. [INFOTRIEVE], [CROSSREF], [CSA]
- World Health Organization (2004): The World Health Report 2004. Changing History. Statistical Annex. Deaths by Cause, Sex and Mortality Stratum in WHO Regions, Estimates for 2002. Geneva, Switzerland, WHO, pp. 120–122.