Abstract
Ethanol extracts from the leaves of Cayaponia podantha. Cogn. (Cucurbitaceae), Nectandra falcifolia. (Nees) Castiglioni (Lauraceae), and Paullinia elegans. Cambess. (Sapindaceae), as well as from the aerial parts of Helicteres gardneriana. St. Hil. & Naud. (Sterculiaceae) and Melochia arenosa. Benth. (Sterculiaceae), all naturally occurring species in the Brazilian part of the Upper Paraná River and all belonging to genera used in folk medicine, were screened for anti-inflammatory activity using the carrageenan-induced pleurisy model in rats, and for antimicrobial activity using a broth microdilution assay against Staphylococcus aureus., Bacillus subtilis., Escherichia coli., Pseudomonas aeruginosa., Candida albicans., C. krusei., C. parapsilosis., and C. tropicalis.. In the analysis of anti-inflammatory activity, a 500 mg/kg body weight dose of the extracts of C. podantha., N. falcifolia., P. elegans., and H. gardneriana., administered orally (by gavage), reduced the volume of the inflammatory exudates in rats induced by intrapleural injection. H. gardneriana. also inhibited migration of leukocytes to the lesion site. Crude extract of M. arenosa. was ineffective on the intensity of the inflammatory response. Regarding antimicrobial activity, the best results were obtained with N. falcifolia. and M. arenosa. against the Gram-positive bacteria tested. N. falcifolia. was active against B. subtilis. with a minimum inhibitory concentration (MIC) of 39 µg/ml and a minimum bactericidal concentration (MBC) of 75 µg/ml and M. arenosa. with MIC of 625 µg/ml against B. subtilis. and MIC and MBC of 625 µg/ml against S. aureus.. The results obtained demonstrate the importance of pharmacological studies with neotropical plants, and further research into the specific components responsible for the observed bioactivities is under way.
Introduction
In terms of biodiversity, Brazil is one of the richest countries on the planet, with more than 20% of the world biodiversity located in its forests. It accounts for more than 55,000 classified vegetable species; however, relatively few of these plants have been submitted to biological or pharmacological tests (Embrapa, Citation1996; Suffredini et al., Citation2004; Wilson, Citation1988).
In the general floristic research carried out by Souza et al. (Citation2004) on the phytodiversity of the Upper Paraná River floodplain (Porto Rico, Paraná State, Brazil), specifically that carried out on the vascular plants of that area, 117 families and 652 species were registered. Many of these plants are used in folk medicine, but few of them have been studied scientifically.
Plants produce bioactive molecules that allow them to interact with other organisms in their environment. Many of these substances are important in their defense against herbivores and contribute to their resistance to diseases. Plants, therefore, can be promising sources of antimicrobial agents.
Several vegetable species have been used popularly for the treatment of different types of infections and inflammatory processes. Besides research on antimicrobial substances, there has also been a significant search, by the pharmaceutical industry and research institutes from around the world, for anti-inflammatory agents that, besides being effective, present low toxicity and reduced adverse effects. Research with plants used in traditional medicine, or plants related to them, can lead to an increase in the diversity of therapeutic agents.
Therefore, considering the importance of studies on tropical species, assays were performed, for the verification of the anti-inflammatory and antimicrobial activities, with the ethanol extracts of five naturally occurring species of the area: Cayaponia podantha. Cogn. (Cucurbitaceae), popularly known as “taiuiá,” Helicteres gardneriana. St. Hil. & Naud. (Sterculiaceae), “sacarrolha,” Melochia arenosa. Benth. (Sterculiaceae), “malva,” Nectandra falcifolia. (Nees) Castiglioni (Lauraceae), “canelinha,” and Paullinia elegans. Cambess. (Sapindaceae), “cipó-timbó.”
Materials and Methods
Plant material
Plant material of the five species was collected in March 2000 from different areas of the Upper Paraná River floodplain: C. podantha. (leaves) and H. gardneriana. (aerial parts) from the Ivinhema River, municipal district of Jatei (MS, Brazil), M. arenosa. (aerial parts) from Garças' Lake, municipal district of Bataiporã (MS, Brazil), N. falcifolia. (leaves) from the Baía River, municipal district of Taquaruçu (MS, Brazil), and P. elegans. (leaves) from Figueira's Pond, municipal district of Porto Rico (PR, Brazil). Voucher specimens of each species have been deposited in the collection of the HNUP Herbarium (Nupelia, Universidade Estadual de Maringá, PR, Brazil; register numbers 1281; 2844; 1834; 1421, and 463, respectively).
Plant extraction
Plant material from each species was dried by airflow at 40°C and powdered in a knife mill: C. podantha. 200 g; H. gardneriana. 450 g; M. arenosa. 600 g; N. falcifolia. 580 g; and P. elegans. 395 g. Crude extracts were prepared by percolation with ethanol at room temperature and were dried under reduced pressure, obtaining 15.8, 32.7, 71.0, 64.0, and 55.8 g of ethanol extracts, respectively.
Anti-inflammatory activity
Animals
Male Wistar rats weighting 180–200 g were used.The animals were fasted for a period of 14 h before the single administration, orally (by gavage), of the extract solutions (500 mg/kg of corporal weight). Simultaneously, groups of control animals, treated with dimethylsulfoxide (DMSO; used in the dissolution of the extracts) and with indomethacin [the reference nonsteroidal anti-inflammatory drug, which was administered orally (by gavage) in the dose of 5 mg/kg] were evaluated.
Carrageenan-induced pleurisy
Pleurisy was induced by intrapleural injection of carrageenan (200 µg), on the right side of the mediastinum between the third and fourth ribs, according to the method of Vinegar et al. (Citation1973). The carrageenan was used in suspension form at 0.2% in sterile apyrogenic solution of NaCl at 0.9%. Four hours after induction, the rats were sacrificed and the skin and pectoral muscle retracted, leaving the rib cage exposed. A longitudinal incision was made between the third and fifth ribs on each side of the mediastinum. The chest was opened and exudate was collected by aspiration and transferred to a conical centrifuge tube. The total volume of exudate was determined and a 50-µl aliquot used to determine the total leukocyte count in a Neubauer chamber. For differential cell counts, red blood cells were lysed by the addition of Turk's solution (Costacurta, Citation1969), and the remaining fluid was centrifuged at 1500 rpm for 10 min and cells resuspended in 0.1 ml of rat plasma. Exudate smears were prepared, air-dried, and fixed with Rosenfeld stain (Rosenfeld, Citation1947).
Statistical analysis
The statistical analysis was carried out using the program GraphPad Prism, applying the Student's t.-test. Statistical significance was set at p < 0.05.
Antimicrobial activity
Microorganisms and growth conditions
The following bacteria were used: Staphylococcus aureus. ATCC 25923, Bacillus subtilis. ATCC 6623, Escherichia coli. ATCC 25922, and Pseudomonas aeruginosa. ATCC 15442. Cultures of these microorganisms were grown in nutrient broth (Difco Laboratories, Detroit, MI, USA) at 37°C and maintained on nutrient agar slants at 4°C. The following yeasts, obtained from clinical material, were used: Candida albicans., C. krusei., C. parapsilosis., and C. tropicalis..
Reference antibiotics and antifungals
The following reference antibiotics and antifungals were used: chloramphenicol, vancomycin, tetracycline, penicillin, and nystatin (Sigma Chemical Co., St. Louis, MO, USA).
Susceptibility testing
The antimicrobial properties of the ethanol extracts were evaluated by the broth microdilution procedure (NCCLS, Citation2000Citation2002). The extracts were diluted in DMSO and Mueller-Hinton broth (1:9). Stock solutions of the extracts and of the reference antibiotics were serially diluted in Mueller-Hinton broth (Merck S.A., São Paulo, SP, Brazil). A volume of 100 µl of each dilution was added to the wells of a 96-well plate containing 100 µl of the same medium. Each inoculum was prepared in phosphate saline 0.01 M, pH 7.2, at a density adjusted to a 0.5 McFarland turbid standard [108 colony-forming units (CFU)/ml] and diluted 1:100 for the procedure. Next, 5 µl of the standardized microorganism suspensions were added in each well and the plates incubated to 37°C for 24–48 h. Growth inhibition was evidenced by the absence of microbial growth in the medium, enabling determination of the minimal inhibitory concentrations (MICs), that is, the lowest concentration of the sample capable of inhibiting in vitro. microbial growth. Sample controls (Mueller-Hinton broth + microbial sample) and solvent controls (Mueller-Hinton broth + solvent + microbial sample) were carried out in parallel. Minimal bactericidal concentrations (MBCs) were determined by subculturing 10 µl of the content of the well with the lowest concentration at which growth was not observed in Mueller-Hinton agar. After incubation at 37°C for 24–48 h, inhibition was evaluated. For the evaluation of antifungal activity, Sabouraud broth was used as culture medium containing 104 to 106 CFU/ml of inoculum. After incubation to 37°C for 48 h, inhibition was evaluated.
Results
Anti-inflammatory activity
In the evaluation of anti-inflammatory activity of the five species, the ethanol extract of M. arenosa., administered orally (by gavage), in a daily dose of 500 mg/kg of body weight, did not provoke an effect on the intensity of the inflammatory response induced by the intrapleural injection of carrageenan in rats. The results are presented in . On the other hand, treatment of the animals with the ethanol extracts of H. gardneriana., C. podantha., P. elegans., and N. falcifolia., in the dose of 500 mg/kg, by oral route, reduced the volume of inflammatory exudates significantly, compared with the control animals. The reductions were of 36.5%, 34.0%, 30.0%, and 25.0%, respectively. Indomethacin (5 mg/kg), used as the reference drug, caused a reduction of 60% in the volume of inflammatory exudate.
Figure 1 Effect of the ethanol extracts of Cayaponia podantha. (Cp), Helicteres gardneriana. (Hg), Melochia arenosa. (Ma), Nectandra falcifolia. (Nf), and Paullinia elegans. (Pe), administered by oral route (gavage) in the dose of 500 mg/kg of body weight, and of indomethacin (Indo), in the dose of 5 mg/kg, on the volume of exudate obtained in rats after carrageenan injection into the pleural cavity. *p < 0.05 compared with control groups (C) and **p < 0.05 compared with the indomethacin group (Student's t.-test).
As observed, the ethanol extract of H. gardneriana., under the conditions used, not only reduced the volume of inflammatory exudates but also inhibited the migration leukocytes to the location of the lesion, as evidenced by the reduction of cells present in the exudate ().
Table 1.. Effect of the ethanol extracts of Cayaponia podantha. (Cp), Helicteres gardneriana. (Hg), Melochia arenosa. (Ma), Nectandra falcifolia. (Nf), and Paullinia elegans. (Pe), administered by oral route (gavage), in the dose of 500 mg/kg of body weight, and of indomethacin, in the dose of 5 mg/kg, on the total and differential leukocytes counts.
Antimicrobial activity
In relation to antimicrobial activity, the best results were obtained for N. falcifolia. and M. arenosa. against the Gram-positive bacteria tested (). The ethanol extract of N. falcifolia. was active against B. subtilis., with MIC of 39 µg/ml and MBC of 78 µg/ml. The extract of M. arenosa. presented a MIC of 625 µg/ml against B. subtilis. and MIC and MBC of 625 µg/ml against S. aureus..
Table 2.. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the ethanol extracts of Cayaponia podantha. (Cp), Helicteres gardneriana. (Hg), Melochia arenosa. (Ma), Nectandra falcifolia. (Nf), and Paullinia elegans. (Pe).
The extracts showed no antifungal activity against the yeasts tested, under the conditions used.
Discussion
Medicinal plants and phytopreparations play an important role in the treatment and prevention of various human diseases, mainly in the developing countries, where modern health services are limited (Lovkova et al., Citation2001; Katewa et al., Citation2004). Research with plants used in folk medicine, or related to them, has increased all over the world, especially in developing countries, as an alternative form of health care.
In this paper, five naturally occurring Brazilian species, all belonging to genera with several species used in traditional medicine, were studied.
Among the species of the genus Cayaponia. used in folk medicine, C. espelina. Cogn. and C. tayuya. (Vell.) Cogn. (antisyphilitic, anti–snake venom, tonic, diuretic, anti-asthmatic, purgative, and depurative effects and to combat diarrhea, bronchitis, pain, and epilepsy), C. pilosa. Cogn. (antisyphilitic, emmenagogue, and purgative effects), and C. cabocla. M. (as a purgative, depurative in cutaneous diseases, and emmenagogue) are noteworthy. Pharmacological studies have demonstrated the anti-inflammatory activity of C. tayuya. (Correa, Citation1984; Rodrigues & Carvalho, Citation2001; Lorenzi, Citation2002; Recio et al., Citation2004) and recently, in the assays performed by Truiti et al. (Citation2005), the trypanocidal activity of C. podantha. has been verified.
Examples of popularly used species of the genus Helicteres. are H. angustifolia. (analgesic, anti-inflammatory, and antibacterial effects), H. sacarolha. Juss. (depurative and in syphilitic inflammations), H. isora. L. (as an expectorant, demulcent, astringent, antigalactagogue, anthelmintic, for the relief of the flu, in treatment of chronic nephritis, dysentery, gastrospasm, empyema, stomach affections, and diabetes), and H. ovata. Lam. (depurative, emollient, and antisyphilitic). Pharmacological studies have demonstrated the hypoglycemic and hypolipidemic activities of H. isora. L. (Correa, Citation1984; Chang et al., Citation2001; Kamiya et al., Citation2001; Chakrabarti et al., Citation2002; Venkatesh et al., Citation2003Citation2004; Katewa et al., Citation2004).
Some species of the genus Melochia. that have been used in traditional medicine are M. corchorifolia. L. (dysentery, abdominal swellings, and water-snake bites), M. umbellata. (Houtt.) Stapf (deobstruent), and M. pyramidata. L. (bronchitis and cough) (Correa, Citation1984; Bhakuni et al., Citation1987; Lorenzi, Citation1991). In the research carried out by Truiti et al. (Citation2005), M. arenosa. presented trypanocidal and molluscicidal activities.
Species of the genus Nectandra. have been used in folk medicine for the relief of pain, arthritis, rheumatism, and diarrhea and also as antifungals, and pharmacological studies have demonstrated the antimalarial activity of N. cuspidata. Nees, vascular and antimalarial activities of N. salicifolia. Nees, antitumoral activity of N. rigida. Nees, analgesic and trypanocidal activities of N. megapotamica. (Spreng) Chodat et Hassler (Le Quesne et al., Citation1980; Correa, Citation1984; Böhlke et al., Citation1996; Slish et al., Citation1999; Muñoz et al., Citation2000; Silva Filho et al., Citation2004aCitationb), and antileishmanial activity of N. falcifolia., verified by Truiti et al. (Citation2005).
Paullinia cupana. H.B.K. and P. yoco. Schultes & Killip are traditionally used to prepare stimulating beverages and P. pinnata. L. for the treatment of human malaria. Many other qualities are attributed to the famous Brazilian guaraná, P. cupana., such as, aphrodisiac, tonic, antidiarrheic, and diuretic. Tests in animals have shown an antifatigue effect and improvement of mental performance from this plant, and an antioxidant effect in vitro. has also been verified (Mattei et al., Citation1998; Abourashed et al., Citation1999; Carlini, Citation2003; Weckerle et al., Citation2003).
In this study, compared with the controls, oral administration of ethanol extracts of H. gardneriana., C. podantha., P. elegans., and N. falcifolia. inhibited development of the inflammatory response induced by pleural injection of carrageenan.
The ethanol extract of H. gardneriana. also inhibited leukocyte migration to the location of the lesion. Interestingly, this reduction was due to the decrease in the number of polymorphonuclear cells. On the other hand, no significant reduction in the number of leukocytes was observed for the other extracts or for indomethacin (reference drug).
Steroidal and nonsteroidal drugs are commonly used in the treatment of inflammatory diseases. Despite their widespread use, nonsteroidal drugs are frequently associated with adverse effects; the most common being gastrointestinal bleeding (Gepdiremen et al., Citation2004). Plants with anti-inflammatory activity could increase therapeutic agent diversity and effectiveness while giving reduced adverse effects.
The data of this study demonstrate that the extracts of H. gardneriana., C. podantha., P. elegans., and N. falcifolia. presented important anti-inflammatory activity, which can be researched further in the future.
In the analysis of antimicrobial activity, the best results were obtained with the extract of the leaves of N. falcifolia., which demonstrated activity against B. subtilis. in concentrations below 100 µg/ml, presenting MIC of 39 µg/ml and MBC of 75 µg/ml. The concentration of 100 µg/ml is currently considered the appropriate concentration for an antimicrobial extract (Suffredini et al., Citation2004). The extract of the aerial parts of M. arenosa. showed moderate activity against S. aureus., presenting MIC and MBC of 625 µg/ml, and against B. subtilis., presenting MIC of 625 µg/ml. These results justify further research into these plants.
The incidence of severe nosocomial infections caused by Gram-positive bacteria has increased in the past decades. This increase has been accompanied by the increased resistance of these microorganisms to various antimicrobials. The recent emergence of bacterial infections and resistant strains has stimulated the investigation of plants as a source of new anti-infective agents (Truiti et al., Citation2003).
A large number of new antibiotics introduced on the market have been obtained from natural or semisynthetic resources (Suffredini et al., Citation2004). It is also important to note that besides the direct use of vegetable extracts, or substances isolated from them, there is also the possibility of their association with antibiotics in such a way as to obtain a synergic effect, leading to new options for the treatment of infectious processes.
The relevance of studies on tropical vegetable species is based on the countless results that have so far been obtained. In addition to the increase in therapeutic options, the isolation of biologically active substances is also worthy of note. The five species studied here have previously been evaluated regarding antiprotozoal and molluscicidal activities (Truiti et al., Citation2005), and this study complements the research on the biological properties of these plants. Based on the results obtained, further research into the specific components responsible for the observed bioactivities is already under way.
Acknowledgments
The authors are grateful to Dr. Carmen L. Cristóbal and Dr. Maria Silvia Feruci, Instituto de Botanica del Nordeste, Corrientes, Argentina, and to Dr. João Batista Baitelo, Instituto Florestal, São Paulo, SP, Brazil, for identifying the plants.
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