Abstract
The current study was undertaken to evaluate the in vitro. antimicrobial and cytotoxic activity of six crude extracts obtained from the leaves and flowers of Distictis buccinatoria. (DC.) A.H. Gentry (Bignoniaceae). Antimicrobial activity was tested against the Gram-positive bacteria Staphylococcus aureus., Streptococcus pyogenes., and Streptococcus faecalis.; the Gram-negative bacteria Escherichia coli., Klebsiella pneumoniae., and Salmonella typhi.; and the fungi Candida albicans., Trichophyton mentagrophytes., Trichophyton rubrum., and Aspergillus niger.. The cytotoxic activity of each extract was determined using two human tumor cell lines in culture, nasopharyngeal carcinoma (KB) and colon carcinoma (HCT-15). The results showed that extracts from D. buccinatoria. possess antimicrobial activity against the Gram-positive bacteria and against both dermatophyte fungal species. The strongest antibacterial activity observed was that of the dichloromethane extract prepared from flowers, and the best antifungal activity was demonstrated by the dichloromethane extract from the leaves. The hexane and dichloromethane extracts from the flowers exhibited cytotoxicity against KB cells. These results support the traditional folk medicinal uses of this plant.
Introduction
Infectious and parasitic diseases, such as acute lower respiratory infections, tuberculosis, diarrhea, HIV/AIDS, and malaria, are major causes of morbidity and mortality worldwide (WHO, Citation2004). In addition, in recent years, there has been a growing incidence of opportunistic fungal infections due to the increasing immunocompromised population, including organ transplant recipients, neonates, cancer, and HIV/AIDS patients. Moreover, antibiotic resistance is a growing problem. These facts have triggered an extended search for new drugs to treat infectious diseases (Fostel & Lartey, Citation2000). Cancer treatment is poorly defined in terms of traditional folk medicine; nevertheless, some medicinal plants used for the treatment of infectious diseases, fever, and inflammation have shown cytotoxic in vitro. activity against human tumor cell lines and are an important source of potential anticancer agents (Cragg & Newman, Citation2005; Kim et al., Citation2005).
According to World Health Organization estimates, herbal remedies serve the health needs of about 80% of the world's population, especially in developing countries (WHO, Citation2001). Data on prescription drugs dispensed from community pharmacies in the United States from 1959 to 1980 indicate that about 25% contained plant extracts or active compounds derived from plants (Newman et al., Citation2000). Thus, because natural products are rich sources of biologically active compounds, there has been an increasing interest in the development of new antimicrobial agents from plants in recent years (Raskin et al., Citation2002).
In Mexico, medicinal plants have been used in rural areas for thousands of years to treat gastrointestinal, respiratory, hepatic, genital, urinary, and skin illness, as well as conditions consistent with cancer symptomatology (Martínez, Citation1944; Díaz, Citation1977; Lozoya et al., Citation1987; Fuentes & Aviles, Citation1997). However, scientific investigation to determine the therapeutic potential of these plants is scarce. The research presented below is part of a series of studies performed in our laboratory as part of a permanent program to investigate the antimicrobial and cytotoxic potential of local medicinal plants (Popoca et al., Citation1998; Navarro et al., Citation1998Citation2003; Rojas et al., Citation2001).
Distictis buccinatoria. (DC.) A.H. Gentry (Bignoniaceae), locally known as “tonacaxochitl,” is a native Mexican plant that has been used since pre-Hispanic times for medical purposes (De la Cruz, Citation1964; Navarro, Citation1992). Nowadays, empirical midwives and herbalists in the state of Morelos in Mexico use the decoction of its flowers to treat cough, angina, inflammation, pharyngitis, and coughs with blood. To date, no biological activity or chemical information regarding this plant has been reported. In this study, we report on the in vitro. antibacterial, antifungal, and cytotoxic activities of the hexane, dichloromethane, and methanol extracts from the flowers and leaves of D. buccinatoria..
Materials and Methods
Collection of plant material
Aerial parts of D. buccinatoria. were collected during spring 2004 from plants growing in Tetela del Volcan, Morelos State, Mexico. Voucher specimens were prepared and authenticated by Margarita Aviles and Macrina Fuentes at the Herbarium of the Instituto Nacional de Antropología e Historia Morelos (INHAM) Medicinal Botanical Garden in the city of Cuernavaca, in the state of Morelos, Mexico; the classified reference voucher was deposited at this institution under the code number INHAM-2007.
Preparation of extracts
Plant materials (leaves and flowers) were shade-dried at room temperature, powdered, and individually extracted sequentially with n.-hexane, dichloromethane, and methanol (100 g per 1500 mL) at room temperature. Each solvent was replaced three-times with fresh solvent, remaining in contact with the plant material for 48 h on each occasion. After filtration, the extracts were concentrated under low pressure at 40°C. Finally, the percentage yield (w/w) for each type of extract was determined. The resulting crude extracts were stored at − 20°C until they were tested.
Microorganisms and media
The microorganisms used for the antimicrobial evaluation were purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). The following Gram-positive bacteria were used: Staphylococcus aureus. (ATCC 29213), Streptococcus pyogenes. (ATCC 08668), and Streptococcus faecalis. (ATCC 29212). The following Gram-negative strains were used: Escherichia coli. (ATCC 25922), Klebsiella pneumoniae. (ATCC 10031), and Salmonella typhi. (ATCC 06539). The following fungi were tested: Candida albicans. (ATCC 10231), Trichophyton mentagrophytes. (ATCC 28185), Trichophyton rubrum. (ATCC 28188), and Aspergillus niger. (ATCC 10335). All bacteria were maintained on tryptic soy agar (TSA; Merck, Darmstadt, Germany) and assayed on Mueller-Hinton agar (MH; Merck). Defibrinated sheep blood (5%) was added to the medium for Streptococcus pyogenes.. The filamentous fungi were maintained on potato dextrose agar (PDA; Merck). Sabouraud 4% glucose-agar (SGA; Merck) was used to maintain the yeast and as the assay medium.
Antimicrobial activity assays
The antibacterial and antifungal activities of the crude extracts were determined using the agar dilution method (Jorgensen et al., Citation1999). The hexane, dichloromethane, and methanol extracts were individually dissolved in 2% dimethylsulfoxide (DMSO; Merck). Further two-fold dilutions of each extract were performed using sterile distilled water and were added to melted agar culture medium in 100 mm × 15 mm Petri dishes (Falcon Franklin Lakes, New Jersey, USA) at the following final concentrations: 0.25, 0.50, 1, 2, 4, and 8 mg/mL. Agar culture medium and DMSO 2% were used as negative controls. The procedures followed for the antibacterial and antifungal assays are described below.
Antibacterial activity assay
Bacterial suspensions for each bacterium were prepared by transferring five or six colonies, chosen after overnight growth on tryptic soy agar, to 5 mL of Mueller-Hinton broth (MHB; Merck). Cultures were incubated at 36°C until they were visibly turbid, and the suspensions were then diluted until the turbidity matched the 0.5 McFarland standard turbidity equivalents [108 colony-forming units (CFU)/mL]. Microbial suspensions were further diluted 1:10 to obtain a concentration of 107 CFU/mL. The diluted inoculum of each bacterium was applied with a loop calibrated to deliver 0.002 mL, resulting in a spot covering a circle of 5 to 8 mm diameter and containing 104 CFU/mL. The plates were incubated for 24 h at 36°C. Gentamicin (Sigma, St. Louis, Missouri, USA) was used as reference standard. Observations were performed in duplicate, and results are expressed as the lowest concentration of plant extract that produced a complete suppression of colony growth, the minimal inhibitory concentration (MIC) (Ríos et al., Citation1988).
Antifungal activity assay
C. albicans. inoculum was prepared by diluting the yeast suspension (adjusted to McFarland 0.5 scale) in 0.85% NaCl solution to a final concentration of 105 cell/mL. The filamentous fungi inoculums were prepared by diluting the scraped cells mass in 0.85% NaCl to a final concentration of 106 spores/mL. The diluted inoculums were delivered on the top of the solidified agar with a loop calibrated to deliver 0.005 mL. The plates were incubated at 29°C. Fungal growth was checked both in control plates prepared without any test sample and in experimental plates; the time at which fungal growth was checked was either 24, 48, or 72 h, depending on the incubation time required for a visible growth; 24 h for C. albicans., 48 h for A. niger., and 72 h for the dermatophytes. Experiments were duplicated, and results were expressed as MIC. Positive controls were prepared with miconazole (Sigma) and nystatin (Merck).
Cytotoxic assay
In accordance with international methods and following the regulations of the National Cancer Institute (NCI) described in the literature (Suffness & Pezzuto, Citation1991; Villarreal et al., Citation1992), HCT-15 (colon) and KB (nasopharynx) human tumor cell lines (donated by Dr. J. Pezzuto of the University of Illinois at Chicago) were maintained in RPMI 1640 (in vitro.) culture medium supplemented with 10% heat-inactivated fetal bovine serum (in vitro.) and were cultured at 37°C in an atmosphere of 5% CO2 in air (100% humidity). During the log phase of their growth cycle, the cells were treated with different concentrations of the extracts (1, 10, and 100 µg/mL), each concentration being repeated three-times, and were incubated for 72 h at 37°C in a humidified atmosphere of 5% CO2. The cell concentration was determined by protein analysis (Oyama & Eagle, Citation1956). The results, the mean of three distinct experiments, were expressed as the concentration that inhibited 50% of growth with respect to control growth after the incubation period (ED50). The values were estimated from a semi-log plot of the extract concentration (µg/mL) against the percent of viable cells. Ellipticine (Sigma) was used as a positive control.
Results and Discussion
In , the plant parts employed, the percentage yield, and the obtained MIC values from the antibacterial and antifungal assays of the crude extracts are summarized. The solid dilution method used in this investigation has been employed frequently and recommended as a good method for determining the relative potency of complex extracts and for establishing their antimicrobial spectrum (Ríos & Recio, Citation2005). Although there is no agreement on the level of acceptance for plant extracts when compared with standard antibiotics and some authors consider only activity comparable with standard antibiotics, in our experience and according to previous antimicrobial studies carried out in our laboratory, crude plant extracts with MIC values between 2.5 and 15 mg/mL (Lozoya et al., Citation1992; Navarro et al., Citation1996) have led to the isolation of strong antimicrobial compounds (Navarro & Delgado Citation1999; Alvarez et al., Citation2001; Zamilpa et al., Citation2002) and to the production of standardized phytodrugs with high rates of clinical and mycological effectiveness with no side effects (Herrera et al., Citation2003Citation2004). Under this criterion, and considering that in this study only crude extracts were employed, extracts with MIC values of 8 mg/mL or below against any of the microorganisms tested were considered active.
Table 1.. Antibacterial and antifungal activity of crude extracts from D. buccinatoria. (MIC, mg/mL)
From all extracts assayed, five of them (83%) showed antimicrobial activity against at least two of the microorganisms tested. All extracts were active against the Gram-positive bacteria Staphylococcus aureus. and Streptococcus pyogenes., the most active being the dichloromethane extract from flowers with MIC values of 0.5 mg/mL for both bacteria, whereas only the dichloromethane extract from flowers was active against the Gram-positive bacterium Streptococcus faecalis. with a MIC value of 8 mg/mL. None of the crude extracts were active against the Gram-negative bacteria. The MIC values obtained in the antifungal evaluation showed that the hexane and dichloromethane extracts from flowers and leaves and the methanolic extract from leaves were active against the dermatophytes (T. mentagrophytes. and T. rubrum.) with MIC values of 4–8 mg/mL; the dichloromethane extract from leaves was the most active with MIC values of 4 mg/mL. No antifungal activity was observed against Candida albicans. and Aspergillus niger..
shows the cytotoxic data of the tested organic extracts against the two cultured human tumor cell lines. According to the NCI guidelines (Geran et al., Citation1972), extracts with ED50 values of 20 µg/mL or below are considered active. As shown, the hexane and dichloromethane extracts from the flowers displayed significant cytotoxicity against KB cells, with ED50 values of 15.8 and 8.3 µg/mL, respectively, whereas none of the organic extracts were active against HCT-15 cells. The cytotoxic activity exhibited by the hexane and dichloromethane extracts against the KB cells coupled with the non-cytotoxicity against the HCT-15 cells is a significant observation that suggests that D. buccinatoria. is a good candidate for future antitumoral testing.
Table 2.. Cytotoxicity of crude extracts from D. buccinatoria. (ED50, µg/mL)
No previous reports on the pharmacological activity or chemical nature of Distictis. could be found in the literature. Experimental studies carried out on some Bignoniaceae species used in traditional folk medicine for the treatment of infectious diseases have displayed antimicrobial and antitumoral activities: Catalapa ovata. G. Don has been shown to possess antimicrobial, antitumoral, and anti-inflammatory properties (Moon et al., Citation2003); Clystoma ramentaceum. Bur. & K. Schum and Mansoa hirsute. DC. inhibited the growth of the fungi Aspergillus niger. and Fusarium oxysporum. (Rocha et al., Citation2004); a mixture of ellagitannins isolated from Punica granatum. L. and two naphthoquinones isolated from Tabebuia avellanedae. Lorenz ex Grisebach demonstrated antibacterial activity against multiresistant Staphylococcus aureus. strains (Machado et al., Citation2003); the alcoholic extracts of the pods and flowers of Tecoma sambucifolia. H.B.K. displayed cytotoxicity against a human hepatoma cell line (Alguacil et al., Citation2000); the biological screening of some fractions derived from the leaves and liana of Macfadyena unguiscati. (L.) revealed antitumoral and antitrypanosomal activities (Duarte et al., Citation2000); and an aqueous ethanol extract of Jacaranda caucana. Pittier showed in vivo. antitumoral and in vitro. cytotoxic activity against the P-388 lymphocytic leukemia system (Ogura et al., Citation1977).
The results obtained in the current investigation indicate that D. buccinatoria. possesses antibacterial, antifungal, and cytotoxic properties, showing a strong correlation between the reported uses of this plant in Mexican folk medicine and the obtained experimental data. It also supports the importance of ethnopharmacology as a guide in the selection of plants for the discovery of bioactive compounds.
Phytochemical bioguided studies are currently under way in order to isolate and characterize the compounds responsible for the antimicrobial and cytotoxic activities of D. buccinatoria..
Acknowledgments
The authors would like to thank empirical midwife Sofia Barranco and traditional healer Fladiano Saavedra for ethnobotanical information, as well as M.C. Margarita Aviles and Macrina Fuentes for assistance in the collection and identification of plant specimens. We also wish to acknowledge the help of Mrs. Katharine M. Roman and Mr. Yuriy Roman from the University of Wisconsin-Madison for their collaboration in the revision of the English writing of this paper.
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