corylophilum Dierckx Marley Garcia Silva a,b , Niege Arac-ari Jacometti Cardoso Furtado b , Monica Tallarico Pupo b , Maria Jose Vieira Fonseca b , Suraia Said b , Ademar Alves da Silva Filho b , Jairo Kenupp Bastos b, a Faculdade de Filosoa, Ciencias e Letras de Formiga, Fundac-ao Educacional Comunitaria Formiguense, Avenida Dr. Arnaldo de Senna, 328 CEP 35570 000 Formiga MG, Brazil b Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Departamento de Ciencias Farmaceuticas, Universidade de Sao Paulo, Avenida do Cafe, s/n, CEP 14040-903, Ribeirao Preto SP, Brazil Accepted 17 June 2004 Summary A strain of Penicillium corylophilum isolated from Brazilian soil sample was submitted to different culture conditions to investigate the production of secondary metabolites with antimicrobial activity. The largest number of conidia was obtained after 5 days of incubation in oat medium and the highest level of antimicrobial activity was produced when the fungus culture was developed in the Czapek medium. The activity against Staphylococcus aureus was found only in the chloroform extract from Czapek culture broth, which also showed activity against Micrococcus luteus. Fumiquinozoline F was isolated from the active chloroform extract by using chromatographic methods. The minimal inhibitory concentration (MIC) values for M. luteus and S. aureus were 99 mg/ mL and 137 mg/mL, respectively. & 2004 Elsevier GmbH. All rights reserved. Introduction Microorganisms have been traditionally used to produce a variety of important substances for the pharmaceutical and food industries. Hence, pri- mary and secondary metabolites, such as peptides, enzymes, organic acids and antibiotics produced by lamentous fungi are used for these purposes (Bennett, 1998; Demain, 2000). The discovery and development of antibiotics was one of the most signicant advances in medicine in the 20th century. Nevertheless, many antimicrobial agents that were used to treat a variety of human infectious diseases are now www.elsevier.de/micres KEYWORDS Fumiquinazoline F; Antibacterial activity; Penicillium corylophilum 0944-5013/$ - see front matter & 2004 Elsevier GmbH. All rights reserved. doi:10.1016/j.micres.2004.06.003
E-mail address: jkbastos@fcfrp.usp.br (J. Kenupp Bastos). ineffective. Therefore, to ensure that effective drugs will be available in the future, it is necessary to improve the antimicrobial use patterns and to devise strategies to identify new antibiotics through previously unexplored targets (Smith and Jarvis, 1999). Natural products play an important role in the discovery of leads for the development of drugs for the treatment of human diseases and microbial environment is an important source of novel active agents (Newman et al., 2003). Many of this products currently used are produced by microbial fermentation, or are derived from chemical mod- ication of a microbial product (Donadio et al., 2002). In this regard, the fermentation process is an important tool for production of secondary meta- bolites that can not be isolated from plants and animals, or synthesized by chemists because of the ease of increasing production by environmental and genetic manipulation (Demain, 2000). Several secondary metabolites have been identi- ed from Penicillium corylophilum, as well as their biological activities. In this work, we determined the best procedure for the production and extrac- tion of antimicrobial metabolites from P. corylo- philum and an alkaloid derived from tryptophan with antimicrobial activity was isolated by using chromatographic methods. Material and methods Microorganisms: P. corylophilum was isolated from a soil sample collected in Sa o Carlos, state of Sao Paulo, Brazil, and identied by Fundac-ao Tropical de Pesquisas e Tecnologia Andre Toselo. The fungus is stored as a conidial suspension on silica gel (612 mesh, grade 40, desiccant activated) at 4 1C. The strains of Staphylococcus aureus ATCC 25923, Micrococcus luteus ATCC 9341, Pseudomo- nas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922 were acquired from the ATCC. Conidia production: Penicillium corylophilum was submitted to four different media: oat agar (Crotti et al., 1999), malt extract (Tzean et al., 1992), potato dextrose agarPDA (Kim et al., 1990) and Vogel medium (Vogel, 1956). The cultures were incubated at 30 1C for 5 days. Conidia produced in each culture were harvested with 2% of Tween 80 and counted in a Neubauer hemocytometer. Production of secondary metabolites: The pro- duction was carried out by inoculating 4 10 6 con- idia/mL, obtained in oat medium, into the pre- fermentative medium (Jackson et al., 1993) at 30 1C with shaking (120 rev/min) for 24 h. The obtained mycelium mass was then inoculated into three different fermentative media: Czapek (Atlas, 1995), Jackson (Jackson et al., 1993) and Vogel (Vogel, 1956). Cultures were reincubated at 30 1C and 120 rev/min for an additional 144 h. For alkaloid production the fungus was grown in ve Erlenmeyer asks containing 240 mL of pre-fermen- tative medium in each and was transferred to Erlenmeyer asks containing 480 mL of Czapek medium. Partition of the culture broth with organic solvents and isolation of the fumiquinazoline F from the chloroform extract: The culture broths were separated by ltration, followed by three times partition with chloroform and buthanol in sequence. All the resulting organic solvents were evaporated under vacuum and the remaining water fractions of the culture broths were lyophilized. The crude chloroform extract from Czapek culture broth (3698 mg), the active one, was then sub- mitted to column chromatography over 400 g of silica gel 60 H (70230 mesh, Merck) and eluted with hexane-ethyl acetate at increasing propor- tions. The 7th fraction eluted with ethyl acetate (1077 mg) was submitted to ash chromatography (Still et al., 1978) over 11 g of silica gel 60 (230400 mesh), which was eluted with an isocratic mobile phase of hexane-acetate 3:2. Afterwards, the fourth obtained fraction (576 mg) was sub- mitted to HPLC analysis. Instrumentations con- sisted of a Shimadzu (SCL-10Avp, Japan) multisolvent delivery system, Shimadzu SPD- M10Avp Photodiode Array Detector, and an Intel Celeron computer for analytical system control and data collection and processing. Analytical chroma- tography was carried out using isocratic gradient (methanol/water/acetonitrile 11:1:8) in 25 min. A CLC-ODS (M)4.6 250 mm 2 Shimadzu column was used at a ow rate of 1.0 mL/min. The spectral data from the detector were collected within 25 min over the 200400 nm range of the absorption spectrum and the chromatograms were analyzed and plotted at 281 nm. The alkaloid was collected between 16.1 and 16.5 min. General experimental procedures: A Bruker DRX- 400 spectrometer, operating at 400.13 MHz for 1 H and 100.62 MHz for 13 C was used. All spectra were run in CDCl 3 with TMS as internal standard. For a HREIMS an Autospec-Micromass-EBE was used (Uni- versidade Estadual de Campinas, UNICAMP). Antimicrobial activity: The crude extracts were investigated for antimicrobial activity by using the agar diffusion method with Petri dish template system inoculated with the assayed microorgan- isms, followed by incubation at 37 1C for 24 h. For this procedure, aliquots of the extracts, free of M. Garcia Silva et al. 318 solvents, were solubilized (5.0 mg/mL) in 50% dimethyl sulfoxide (DMSO, v/v) aqueous solution and applied into the template holes made on the medium surface. Negative controls were run for DMSO aqueous solution, chloroform and buthanol. Streptomycin (77.7 U, 50 mL, Sigma) for Gram- negative bacteria (P. aeruginosa and E. coli), Penicillin G (0.6075 U, 50 mL, Sigma) for M. luteus and Penicillin G (1.215 U, 50 mL, Sigma) for S. aureus were used as the positive controls. The inoculum was prepared by culturing each organism in Antibiotic N11 agar medium (Merck) for 24 h at 37 1C. The microorganisms were transferred to 0.9% NaCl until they reached the turbidity equivalent to 0.5 McFarland standard. Each microorganism sus- pension (0.5%) was added in Antibiotic N11 agar medium, and distributed over the plates. The MIC values of the isolated alkaloid were evaluated in triplicate by microdilution broth method (Andrews, 2001). The alkaloid was solubilized in DMSO (dimethyl sulfoxide) at 1 mg/mL, and was diluted in Tryptone soya broth in the range of 147.536.8 mg/mL. The inoculum was adjusted to each organism yielding a cell concentration of 10 3 colony forming units (CFU/mL). It was included both inoculated wells, that controls the adequacy of the broth to support the growth of the organisms and uninoculated wells, that remains free of antimicrobial agent to check the sterility of the medium. Penicillin G (Sigma) was used as positive control. The microplates (96-well) were incubated at 37 1C for 24 h. After that, 40 mL of 2,3,5- triphenyltetrazolium chloride (0.7%) in aqueous solution were added to indicate the viability of microorganisms (Leverone et al., 1996). The MIC was determined as the lowest concentration of drug able to totally inhibit microorganism growth. Results Determination of the best conditions for conidial production The largest number of conidia was obtained after 5 days of incubation in oat agar, Vogel, malt extract and PDA media, in this order (Fig. 1). Selection of fermentative medium for antimicrobial activity production Different fermentative media were investigated to determine in which broth the antimicrobial activity was produced. The highest level of antimicrobial activity was produced by P. corylophilum when the fungus culture was developed in the Czapek medium, as observed for the results obtained by the agar diffusion method (Table 1). The activity was considered for inhibition zones wider than 12 mm since the template punches holes of approximately 1011 mm into the agar surface. The activity against S. aureus was found only in the chloroform extract from Czapek culture broth. Independently of the culture medium, the anti- microbial activity was detected only in the chloro- form extracts. Moreover, no extracts showed activity against Gram-negative bacteria at the concentration evaluated in this work. Oat agar Vogel Malt extract PDA 0.0 2.5 5.0 7.5 Culture media C o n i d i a
x
1 0 8 / m L Figure 1. Determination of the best conditions for conidial production by P. corylophilum. Table 1. Antimicrobial activity of different extracts from cultures of Penicillium corylophilum Microorganisms Extracts or standards Fermentative medium Inhibition zones (mm) Micrococcus luteus Chloroformic Czapek 1470.577 Jackson 1470.577 Vogel 1370.577 Penicillin G 2670.577 Staphylococcus aureus Chloroformic Czapek 1370.577 Jackson 0970.577 Vogel 0870.577 Penicillin G 2070.577 Antibacterial activity from Penicillium corylophilum Dierckx 319 Identication and antimicrobial activity of the alkaloid from Czapek chloroform extract The chemical structure of the isolated compound (Fig. 2) was identied from 1 H and 13 C Nuclear Magnetic Ressonance (NMR), Attached Proton Test (APT), 1 H 1 H Correlation Spectroscopy (COSY), Heteronuclear Multiple Bond Coherence (HMQC), Heteronuclear Multiple Quantum Coherence (HMBC), and Mass Spectra (MS) spectral data in comparison with previous published data (Takaha- shi et al., 1995). The MS High Resolution showed the [M + ] peak m/z 358.14298. The 13 C NMR spectrum of the compound showed 21 carbon signals. The multiplicities of the carbons deter- mined by APT led to the attribution of: 8 C, 11 CH, 1 CH 2 and 1 CH 3 . Among the quaternary carbons two were attributed to amide carbonyls (d 168.9, C-12 and 160.7, C-5). The 1 H NMR spectrum showed two broad singlets at d 8.00 (1H, H-13) and d 5.83 (1H, H-16) indicating that these hydrogens could be attached to nitrogens. Hence, these data suggested an alkaloid type structure for the compound (Fig. 2). The 1 H NMR spectrum also showed nine hydrogen signals between d 6.64 and 8.30. In the 1 H 1 H COSY experiment the signals at d 7.35 (dd, J 8:1 and 0.8 Hz, H-9), d 6.87 (ddd, J 8:1; 7.1 and 0.8 Hz, H- 10), d 7.06 (ddd, J 8:1; 7.1 and 0.8 Hz, H-11) d 7.23 (dd, J 8:1 and 0.8 Hz, H-12) were coupled to each other. The hydrogen at d 6.64 (d, J 2:5 Hz; H-14) was coupled to the hydrogen at d 8.00 (H-13). The chemical shifts of the carbons attached to these hydrogens were attributed according to the HMQC data (Table 2). These data allowed us to propose an indolic moiety for the compound, which was corroborated by the H-C correlations observed in the HMBC experiment. The HMBC experiment showed the correlation of the indolic carbon at d109:6 (C-8a) with the hydrogens at d 3.66 (dd, J 14:9 and 3.3 Hz, H-8 0 ) and d 3.59 (dd, J 14:9 and 5.3 Hz, H-8 00 ). In the HMQC experiment these hydrogens were attached to the carbon at d 27.0. The 1 H 1 H COSY experiment showed that these hydrogens (H-8) are coupled to each other and to another hydrogen at d 5.63 (dd, J 5:3 and 3.3 Hz, H-7). The HMBC experiment showed the correlation of both H-8 with C-7, C-8a and C-15. These data led us to propose an alkaloid derived from tryptophan for the isolated compound (Bergman and Bergman, 1985). The 1 H 1 H COSY experiment also revealed that the hydrogens at d 8.30 (ddd, J 8:1; 1.5 and 0.5 Hz, H-4), d 7.47 (ddd, J 8:1; 7.3 and 1.3 Hz, H-3), d 7.71 (ddd, J 8:3; 7.3 and 1.5 Hz, H-2) and d 7.53 (dd, J 8:3 and 1.5 Hz, H-1) were coupled to each other. The aromatic carbons chemical shifts were attributed on the basis of HMQC data. These spectral data suggested an anthranilic acid derivative moiety. It was corrobo- rated by the correlation of the carbon at d 160.7 (C-5) with H-4 (d 8.30). The remaining 1 H NMR signals were observed at d 1.29 (d, J 6:6 Hz; 3 H, H-19) and d 2.99 (q, J Table 2. 13 C (100 MHz) and 1 H NMR (400 MHz) spectral data for fumiquinozoline F (CDCl 3 ) Position C H 1 127.3 7.53 dd (8.3; 0.5) 2 134.6 7.71 ddd (8.3; 7.3; 1.5) 3 127.1 7.47 ddd (8.1; 7.3; 1.3) 4 126.9 8.30 ddd (8.1; 1.5; 0.5) 4a 120.3 5 160.7 6 7 55.6 5.63 dd (5.3; 3.3) 8 27.0 3.66 dd (14.9; 3.3) 3.59 dd (14.9; 5.3) 8a 109.6 8b 127.2 9 118.6 7.35 dd (8.1; 0.8) 10 121.6 6.87 ddd (8.1; 7.1; 0.8) 11 122.6 7.06 ddd (8.1; 7.1; 0.8) 12 111.1 7.23 dd (8.1; 0.8) 12a 136.0 13 8.00 br s 14 123.4 6.64 d (2.5) 15 168.9 16 5.83 br s 17 49.1 2.99 q (6.6) 17a 151.6 18 18a 147.1 19 19.3 1.29 d (6.6) N H N H N O N O 1 2 3 4 4a 5 6 8 8a 8b 9 10 11 12 12a 13 14 15 16 17 17a 18 18a 19 7 Figure 2. Structure of the fumiquinozoline F, isolated from Czapek chloroform extract. M. Garcia Silva et al. 320 6:6 Hz; 1 H, H-17), both coupled to each other as observed in the 1 H 1 H COSY experiment. The HMBC experiment showed the following correlations: H- 19 (d 1.29) correlated with a carbon at d 151.6 (C- 17a) and C-19 (d 19.3) correlated with H-16 (d 5.86). These data suggested an alanine moiety for the compound. Therefore, the data allowed to propose the structure of fumiquinozoline F as an alkaloid derived from the linking of the aminoacids tryptophan and alanine plus an anthranilic acid unit. Regarding the antimicrobial activity, the MIC values of this compound against M. luteus and S. aureus were of 99 mg/mL and 137 mg/mL, respec- tively. Nevertheless, the penicillin G standard was more active than the isolated compound for both microorganisms (0.011 mg/mL and 0.023 mg/mL, respectively). Discussion The expression of secondary metabolites might depend on the culture conditions and the strains. P. corylophilum is often isolated from temperate climates (Malmstrom et al., 2000), but in this work we investigated a strain isolated from Brazilian soil sample, a tropical country. According Gaden-Junior (2000), the metabolites production is inuenced by medium composition, nutrients availability and others aspects. Different nitrogen and carbon sources may affect the synth- esis of enzymes involved in primary and secondary metabolism. Microorganisms are able to use a wide variety of carbon and nitrogen sources. Never- theless, many secondary metabolic pathways are negatively affected by these sources favorable for growth (Sanchez and Demain, 2002). In the study of the secondary metabolites of the P. corylophilum, Cutler et al. (1989) isolated the 3,7-dimethyl-8-hydroxy-6-metoxysocroman, from the mycelia using shredded wheat medium, which was maintained 12 days at 22 1C. By cultivation in malt medium, strains of P. corylophilum produced the alkaloid epoxyagroclavine I (Grabley et al., 1992). In this work, a two step culture was used for antimicrobial active compounds production. In the rst step, the microorganism was cultivated in pre- fermentative medium, which is rich in nutrients to increase vegetative biomass production. The har- vested mycelium was then transferred to fermen- tative medium for secondary metabolites production. The fumiquinazolina F was isolated from the chloroform extract of the Czapek fermen- tative medium, after 144 hours of incubation. From the three evaluated media, the Czapek one was the best for antimicrobial metabolites production. Fumiquinazoline F was originally obtained from a strain of Aspergillus fumigatus, which was isolated from the gastrointestinal tract of the marine sh Pseudolabrus japonicus (Takahashi et al., 1995), and it was also detected in Penicillium thymicola (Larsen et al., 1998). It was the rst time that Fumiquinazoline F was isolated from P. corylophi- lum. Besides, it is the rst time that the anti- microbial activity of this compound is been described. The fumiquinazolines belong to a class of compounds possessing a wide range of biological activities. For instance, fumiquinazolines A, B and C from A. fumigatus showed moderate cytotoxicity against the cultured P-388 lymphocytic cells (Nu- mata et al., 1992). Fumiquinazolines H and I, isolated from Acremonium sp. presented signicant antifungal activity (Snider and Zeng, 2003). Kariba et al. (2002), showed that extracts of Schizozygia coffaeoides, containing indolines pre- sented antifungal and antibacterial activities. The berberine, benzylisoquinoline alkaloid, isolated from Hydrastis canadensis, is active against many Gram-positive and Gram-negative bacteria, as well against fungi and parasites (Scazzocchio et al., 2001). Fumiquinazoline F is derived of tryptophan, plus anthranilic acid and it showed value of MIC lower than berberine, canadaline and canadine for S. aureus (ATCC 25923) (Scazzocchio et al., 2001). 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Garcia Silva et al. 322