versión impresa ISSN 1516-8913
Braz. arch. biol. technol. v.48 n.spe Curitiba jun. 2005
ENGINEERING AND TECHNOLOGY
Daniel GranatoI; Domingos Sávio NunesI, *; Patrícia Póvoa de MattosII; Ester de Moura RiosIII; Adeline GlinskiIII; Luciana Collares RodriguesIII; Gerson Zanusso JúniorIII
IUniversidade Estadual de Ponta Grossa; Departamento de Química
IIEmbrapa Florestas; C. P. 319; 83411-000; Colombo - PR - Brasil
IIIDepartamento de Biologia; Campus de Uvaranas; 84030-900; email@example.com; Ponta Grossa - PR - Brasil
This study aimed chemical characterization and microbiological evaluation of extracts obtained from the waste of woods marketed in Paraná State: Peroba-Rosa (Aspidosperma sp.), Roxinho (Peltogyne sp.), Jatobá(Hymenaea sp.), Curupixá (Micropholis sp.), Itaúba (Mezilaurus sp.), Cedrilho (Erisma sp.) and Imbúia (Licaria sp.), whose botanical identifications were based on anatomical studies. The extracts were prepared with different solvents, analyzed by TLC and UV/VIS techniques, and tested against: Proteus mirabilis ATCC15290, Staphylococcus aureus ATCC25923, Escherichia coli ATCC25922, Enterobacter aerogenes ATCC13048, Micrococcus luteus ATCC9341, Klebsiella pneumoniae ATCC13883, Pseudomonas aeroginosa ATCC27853, Staphylococcus aureus, Streptococcus mutans and Bacillus cereus isolated from the clinic. The ethanol extract from Peroba-rosa containing alkaloids showed activity against P. mirabilis. Itaúba, Jatobá and Imbúia methanol extracts containing phenolics, and the Roxinho ethyl acetate extract containing terpenoids and phenolics were active against K. pneumoniae, M. luteus, E. coli, S. aureus and P. mirabilis. P. aeroginosa, S. mutans and E. aerogenes were resistant to the extracts.
Key words: Tropical woods, microbiological evaluation, chemical profile of woods
Este estudo visa a caracterização química e a avaliação da atividade antimicrobiana de extratos obtidos a partir de rejeitos resultantes do beneficiamento de madeiras nobres comercializadas no Paraná: Peroba-Rosa (Aspidosperma sp.), Roxinho (Peltogyne sp.), Jatobá (Hymenaea sp.), Curupixá (Micropholis sp.), Itaúba (Mezilaurus sp.), Cedrilho (Erisma sp.) e Imbúia-do-Norte (Licaria sp.), cujas identificações botânicas basearam-se em estudos anatômicos. Os extratos foram preparados com diversos solventes, analisados por CCD e espectrometria UV/VIS, testando-se contra: Proteus mirabilis ATCC15290, Escherichia coli ATCC25922, Enterobacter aerogenes ATCC13048, Staphylococcus aureus ATCC25923, Micrococcus luteus ATCC9341, Klebsiella pneumoniae ATCC13883, Pseudomonas aeroginosa ATCC27853, e Staphylococcus aureus, Streptococcus mutans e Bacillus cereus isolados da clínica. O extrato etanólico de Peroba-Rosa, contendo alcalóides, apresentou atividade contra P. mirabilis. Os extratos metanólicos do Jatobá, Itaúba e Imbúia, contendo fenóis, e o extrato de Roxinho obtido com acetato de etila contendo fenóis e terpenóides, foram ativos contra K. pneumoniae, M. luteus, E. coli, S. aureus e P. mirabilis. Nenhum dos extratos foi ativo contra P. aeroginosa, S. mutans e E. aerogenes.
The native wood exploration in Brazil is frequently predatory, and the explored areas have been transformed into pasture or simply abandoned. Currently, Paraná State is a major wood exporter in the country with USS 500,000.00 in the year of 2002 (AIMEX, 2004). This business is mainly based on Eucalyptus and Pinus species, but part of such export and the demand of the regional industry is met by the exploration of natural forests, especially of the Amazon Forest (Agência Estado, 2003).
The mechanical processing of these woods generates approximately 14% of lumber from the total volume of the logs. The residues are partially used to generate energy or they are displaced at inadequate areas, generating serious environmental problems (Mayan et al., 2003). The search for useful applications for these rejects of this industry is still a hard task to carry through. The secondary metabolites found in the lumbers can add value to such crude materials or it can be transformed into novel by-products of these residues, but it is necessary to study the specific chemical composition of each lumber.
Many woods of high commercial value come from plant species with few or none chemical/biological study about their secondary metabolites. In a recent bibliographical search (ISI WEB OF KNOWLEDGE, 2003), only a few old chemical studies about Peroba-rosa (Aspidosperma polyneuron, alkaloids), Jatobá (Hymenaea courbaril, flavonoids and terpenoids), Imbúia-do-Norte (Licaria sp., neolignoids), Roxinho (Peltogyne sp., peltogynoids) and Itaúba (Mezilaurus itauba, neolignoids and essential oil) were found. The majority of these studies mentions the chemical composition of other parts of the tree, not of the wood itself. The woods of Cedrilho (Erisma sp.) and Curupixá (Micropholis sp.) were the most explored Amazonian species in the last years (AIMEX, 2004), but not even one scientific study on the secondary metabolites of these two botanical genera could be found.
A troublesome factor for the accomplishment of studies on rejects of the wood industry is that the logs arrive there only with a commercial identification or a popular name. The popular denomination of the Brazilian native trees is changeable from region to region. Sometimes different common names represent the same species, and same name can represent different species (Camargos et al., 2001). In these cases, the botanical identification of the genus can be made by anatomical analysis of the wood and by verifying characteristics such as color, flavor or smell (USDA, 2004). The intensification of scientific research on the wood of native species would lead to a more realistic valuation of their environmental role by those who explore them commercially. The chemical study of the secondary metabolites of woods and their microbiological activities may lead to a comprehensive understanding of their defenses against plagues, providing the scientific basis to the development of new protective products for white woods, for example.
MATERIALS AND METHODS
Wood samples of seven species were supplied by a wood industry of the city of Ponta Grossa - PR, which are commercially identified as Peroba-rosa, Cedrilho, Imbúia-do-Norte, Itaúba, Roxinho, Jatobá and Curupixá. The botanical identification of the samples was made by macroscopical analysis of their color and anatomical characteristics, following the identification key of Mainieri (1983). For the microscopical analysis, permanent blades with safra-blau coloration were prepared by histologic cuts in the three plans (transversal, longitudinal tangential and longitudinal radial). The identifications were confirmed by comparison with samples of the Embrapa Florestas collection and with descriptions contained in specialized literature (Mainieri and Chimelo, 1989; Metcalfe and Chalk, 1965).
Preparation of the crude extracts
Lumber of each wood sample (100 g) of were dried at 40 ºC during 3 days. After that the samples were extracted exhaustively in Soxhlet apparatus with dichloromethane and later with methanol, and then the solvents were evaporated under vacuum. Roxinho was also extracted with ethyl acetate and methanol. Peroba-rosa was extracted only with ethanol. Itaúba's lumber was extracted with a mixture of 80% chloroform and 20% methanol, and then with pure methanol.
TLC and UV/VIS Analyses
In the thin layer chromatography (TLC) analyses of the extracts, 5 µL of sample solution made up from 10 mg of crude extract for 1 mL of solvent were applied on silica gel HF-254 plates eluted with different systems of solvents. After the development, the chromatograms were observed under UV light of 254 and 360 nm, and then sprayed with specific reagents for terpenoids (anisaldehyde/H2SO4), phenolics (FeCl3) and alkaloids (Dragendorff). Qualitative UV/VIS spectra of the extracts were measured in methanol. Each measure of spectrum was repeated after adding two drops of a NaOH solution (5%), to confirm the presence of phenolic compounds by the occurrence of batochromic shifts. The spectrum in acid medium was also obtained with the extract of Peroba-Rosa for a comparison with literature.
Microbiological activity of the extracts
The method used for the microbiological evaluation was the diffusion in solid-medium by plate-cavity (Cleeland and Squires, 1991). The bacterial suspensions were standardized by the 0.5 MacFarland tube and inoculated by "pour-plate" on Tryptic Soy Agar medium (Oplustil et al., 2000). In cavities of 6 mm located in the middle of the culture medium, the dissolved extracts were placed using a concentration of 1 mg/cavity. After incubation at 37 ºC for 24 h, the diameter of the halo of inhibition around the cavity was measured. For the microbiological tests, the extracts were dissolved in DMSO. The tested bacteria used in this study were: S. aureus ATCC 25923, P. mirabilis ATCC 15290, E. coli ATCC 25922, E. aerogenes ATCC 13048, B. cereus and S. mutans isolated from the clinic. The extracts from Roxinho wood obtained with ethyl acetate, Jatobá's and Itaúba's methanolic extract, Peroba's ethanolic extract and Itaúba's extract obtained with CHCl3/MeOH 20% were tested against M. luteus ATCC 9314, K. pneumoniae ATCC 13883, P. aeroginosa ATCC 27853 and S. aureus isolated from the clinic. The experiments have been carried out three times using equivalent volumes of DMSO as negative control.
RESULTS AND DISCUSSION
The wood samples were identified as botanical genera: Peroba-rosa (Aspidosperma sp., Apocynaceae), Cedrilho (Erisma sp., Vochysiaceae), Imbúia-do-Norte (Licaria sp., Lauraceae), Itaúba (Mezilaurus sp., Lauraceae), Roxinho (Peltogyne sp., Caesalpinaceae), Jatobá (Hymenaea sp., Caesalpinaceae) and Curupixá (Micropholis sp., Sapotaceae). The yield of the extracts that have been tested against the bacteria are shown in Table 1.
In Table 2 the results of the TLC analyses and detection of compound classes are summarized. In some cases, these results supported the identification of the botanical genus carried through by the anatomical studies of the woods.
Strong alkaloid presence in the crude ethanol extract of Peroba-Rosa was observed, that was a natural class of compounds occurring in all species of the Aspidosperma genus. UV spectra of this extract showed great similarity with the literature values for polyneuridine, the main indole alkaloid isolated from Aspidosperma polyneuron (Antonaccio et al., 1962). These results strongly suggested that wood sample of Peroba-Rosa came from A. polyneuron. This tree is found in the Atlantic Forest in Bahia, Espírito Santo, São Paulo and Paraná States; it also occurs in Mato Grosso do Sul, Minas Gerais, Goiás, Mato Grosso and Rondônia States, in the semideciduous latifoliated forest (Lorenzi, 1992).
The observed phenolic compounds in the Itaúba extracts probably corresponded to lignoids already isolated from the Mezilaurus genus (Yaez et al., 1986); bisbenzylisoquinoline alkaloids were found in this botanical genus (Hisses et al., 1983).
Lignoids are also characteristic of species from the Licaria genus, here represented by the Imbúia-do-Norte (Braz Filho et al., 1981). The volatile monoterpenoid anethole, isolated from Licaria species, is known for its antimicrobial properties (Himejima and Kubo, 1992). Some species of the Peltogyne genus, the one to that Roxinho belongs, have their wood endowed with violet coloration due to phenolic compounds known as peltogynoids, which are flavonoids (Almeida et al., 1974). The trees of the Hymenaea genus (Jatobá) are characterized by its resins with high ratio of diterpene acids, which are strong antimicrobials (Abdel-Kader et al., 2002); flavonoids of varied structures are also common in Hymenaea species (Pettit et al., 2003).
The results of the microbiological tests indicated that ethanol extract of Peroba-rosa had strong inhibitory effect against Proteus mirabilis. Methanol extracts of Itaúba, Jatobá and Imbúia-do-Norte, as well as the extracts of Roxinho obtained with ethyl acetate and Itaúba with CHCl3/MeOH 20%, all inhibited the growth of the Gram positive and Gram negative bacteria to some extent. The Curupixá's, Roxinho's and Cedrilho's methanol extracts showed weak or no antibacterial activity.
The main relative results of the microbiological tests with the extracts are summarized in Table 3; the values, given in mm, are the diameters of inhibition halos provoked by the presence of each extract in the culture medium. E. aerogenes, P. aeroginosa and S. mutans were resistant to all the extracts tested.
We are grateful to the Programa PIBIC UEPG/CNPq for an IC grant (Daniel Granato) and to CNPq for a Research grant (Domingos Sávio Nunes, 308591/2003-8). We also thank to Dr. Paulo C. Botosso at Embrapa Florestas, Colombo - PR, for his participation in the botanical identification.
Abdel-Kader, M.; Berger, J.; Slebodnick, C.; Hoch, J.; Malone, S.; Wisse, J.; Werkhoven, M.; Mamber, S. and Kingston, D. (2002), Isolation and absolute configuration of ent-halimane diterpenoids from Hymenaea courbaril from the Suriname rain forest. J. Nat. Prod., 65, 11-15. [ Links ]
Agência Estado (2004), Greenpeace denuncia fraude em exportação de mogno. Available at: http://www.estadao.com.br/ciencia/noticias/2002/jun/14/315.htm. Access on: 2 Mar. 2004. [ Links ]
Almeida, M. E.; Gottlieb, O. R.; Regodeso, J. and Teixeira, M. A. (1974), Chemistry of Brazilian Leguminosae .18. New peltogynoids from three Peltogyne species. Phytochem., 13, 1225-1228. [ Links ]
Antonaccio, L.; Pereira, N.; Gilbert, B.; Vorbrueggen, H.; Budzikiewicz, H.; Wilson, J.; Duhram, L. and Djerassi, C. (1962), Polyneuridine, a new alkaloid from Aspidosperma polyneuron and some observations on mass spectra of indole alkaloids. J. Amer. Chem. Soc., 84, 2161-2170. [ Links ]
Braz Filho, R.; Carvalho, M. G.; Gottlieb, O. R.; Maia, J. G. S. and Silva, M. L. (1981), Neolignans from Licaria rigida. Phytochem., 20, 2049-2050. [ Links ]
Camargos, J. A. A.; Coradin, V. T. R.; Czarneski, C. M.; Oliveira, D. and Meguerditchian, I. (2001), Catálogo de árvores do Brasil. 2. ed. rev. Brasília : IBAMA. [ Links ]
Cleeland, R. and Squires, E. (1991), Evaluation of new antimicrobials in vivo and in experimental animal infections. In: Lorian, V. (Ed.). Antibiotics in Laboratory Medicine. 3. ed. Baltimore : William and Wilkins. Chapter 21. [ Links ]
Himejima, M. and Kubo, I. (1992) Antimicrobial agents from Licaria puchuri major Mez and their synergistic effect with polygodial. J. Nat. Prod., 55, 620-625. [ Links ]
Lorenzi, H. (1992), Árvores Brasileiras - Manual de identificação e cultivo de plantas arbóreas nativas do Brasil. Nova Odessa : Editora Plantarum. [ Links ]
Maia, C. M. B. F.; Budziak, C. R.; Paixão, R. E. and Mangrich, A. S. (2003), Compostagem de resíduos florestais: um guia para produção de húmus através da reciclagem e aproveitamento de resíduos florestais. Colombo : EMBRAPA. (Série Documentos; 87). [ Links ]
Mainieri, C. (1983), Manual de identificação das principais madeiras comerciais brasileiras. São Paulo : IPT. [ Links ]
Mainieri, C. and Chimelo, J. P. (1981), Fichas de características das madeiras brasileiras. São Paulo : IPT. [ Links ]
Metcalfe, C. R. and Chalk, L. (1965), Anatomy of the dicotyledons: leaves, stem and wood in relation to taxonomy with notes on economic uses. Oxford : Clarendon Press. [ Links ]
Oplustil, C. P.; Zoccoli, C. M.; Tobouti, N. R. and Sinto, S. I. (2000), Procedimentos básicos em microbiologia clínica. São Paulo : SARVIER. [ Links ]
Pettit, G.; Meng, Y.; Stevenson, C.; Doubek, D.; Knight, J.; Cichacz, Z; Pettit, R.; Chapuis, J. and Schmidt, J. (2003), Isolation and structure of palstatin from the Amazon tree Hymenaea palustris. J. Nat. Prod., 66, 259-262. [ Links ]
Silva, R.; Nagem, T. J.; Mesquita, A. L. and Gottlieb, O. R. (1983), g-Lactones from Mezilaurus synandra. Phytochem., 22, 772-773. [ Links ]
Yaez, R. X.; Diaz, A. M. P. and Diaz, P. P. (1986), Neolignans from Mezilaurus itauba. Phytochem., 25, 1953-1956. [ Links ]
Received: September 29, 2004;
Revised: February 25, 2005;
Accepted: March 25, 2005.
* Author for correspondence