Brazilian Journal of Microbiology (2012): 224-229 ISSN 1517-8382 ANTI-MYCOBACTERIUM TUBERCULOSIS ACTIVITY OF FUNGUS PHOMOPSIS STIPATA Karina Andrade de Prince1,3, Renata Sordi2, Fernando Rogério Pavan1, Adolfo Carlos Barreto Santos1, Angela R. Araujo2, Sergio R.A. Leite2, Clarice Q. F. Leite1* 1 Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, Araraquara, SP, Brasil; 2 Instituto de Química, Universidade Estadual Paulista, Araraquara, SP, Brasil; 3 Faculdades Integradas Pitágoras, Montes Claros, MG, Brasil. Submitted: July 22, 2010; Approved: June 06, 2011. ABSTRACT Our purpose was to determine the anti-Mycobacterium tuberculosis activity of the metabolites produced by the endophitic fungus Phomopsis stipata (Lib.) B. Sutton, (Diaporthaceae), cultivated in different media. The antimycobacterial activity was assessed through the Resazurin Microtiter Assay (REMA) and the cytotoxicity test performed on macrophage cell line. The extracts derived from fungi grown on Corn Medium and Potato Dextrose Broth presented the smallest values of Minimum Inhibitory Concentration (MIC) and low cytotoxicity, which implies a high selectivity index. This is the first report on the chemical composition and antitubercular activity of metabolites of P. stipata, as well as the influence of culture medium on these properties. Key words: Phomopsis stipata, antimycobacterial activity, Mycobacterium tuberculosis. INTRODUCTION developing countries, where the situation is more severe. The emergence of strains resistant to most drugs currently used as Tuberculosis (TB) is a respiratory disease caused by Mycobacterium tuberculosis, transmitted from person to antituberculosis makes urgent the search for new synthetic or natural agents against this disease (35). person, which affects approximately 32% of world population. One approach to this is to search in the nature, especially In Brazil, 80.000 cases are registered annually with incidence for the rich flora of the tropics and its endophytic fungi. rate of 37,1/100,000 inhabitants, according to data from the Natural products and/or their semi-synthetic derivatives can Ministry of Health (9). Among the infected individuals in the lead to novel antimycobacterial drugs and might have world, about eight million a year develop active TB and nearly important role in the chemotherapy of tuberculosis in the future two million die (33). Advancements in health care facilities (26). Thus, some reports have demonstrated the importance of offered a goal to eradicate tuberculosis (TB) by the end of the performing screening of natural products with activity against 20th century, but it reemerged because of the global resistance M. tuberculosis (17, 13, 20, 32). against antitubercular drugs (22). The advent multidrug The vegetal species Styrax camporum Pohl (Styracaceae) resistance (MDR), extensive drug resistance (XDR) infection, occurs predominantly in Brazilian central cerrado (8), which is concerns health authorities around the world, especially in one of the major biogeographical regions in the world, with *Corresponding Author. Mailing address: Rodovia Araraquara - Jaú KM 01, CEP 14801-902, Araraquara – SP, Brazil.; Tel.: +55 (16) 33016953 +55(16)33518285.; E-mail: leitecqf@fcfar.unesp.br 224 Prince, K.A. et al. Anti-M. tuberculosis activity more than 7000 native species of vascular plants (21). This min and aqueous ethanol (70 %) for 1 min. A second washing plant is widely used in folk medicine to treat gastric and with water and soap was performed and finally the leaves were intestinal diseases. Different authors evaluated the anti- immersed in sterile water for 10 min. The sterilized leaves ulcerative potentiality and toxicity of crude extracts and were cut into 2 cm2 pieces and deposited on Petri dishes fractions of this plant and their effectiveness to treat peptic containing Potato Dextrose Agar (PDA) and gentamicin sulfate ulcer (2, 3) as well its antioxidant activity (15). (0.5 ug/mL), 4 pieces for dish. The material was incubated at The endophytic fungi from genus Phomopsis Sacc. & 25°C for 10 days and the endophyte P. stipata was isolated by Roum., (Diaporthaceae) are known as rich sources of replication and preserved in sterile water (19). The fungus was secondary bioactive metabolites of different chemical natures identified by Dr. Ludwig H. Pfenning using rRNA internal (29, 30). A Phomopsis sp isolated from Thailand forest transcribed spacer (ITS) region and deposited in the Micology presented metabolites with anti-M. tuberculosis activity (5). Collection of the Federal University of Lavras, Lavras, MG, This genus of endophyte had never before been isolated from Brazil. the Brazilian cerrado plants. The present study describes the isolation of P. stipata Cultive of the Endophytic Fungus (Lib.) B. Sutton, (Diaporthaceae) from leaves of S. camporum The fungus P. stipata was cultivated in different and the determination of the anti-M. tuberculosis activity, commercial media from Difco (Difcotm laboratories, Detroit, cytotoxicity and selectivity index (SI) of crude extracts this MI, USA) (Potato Dextrose Broth - PD, Yeast Medium - YM, endophytic fungus, cultured on different media. We also Nutrient Broth - NB and Czapek Medium – CZ) and in a identified the main classes of compounds present in extracts by homemade corn medium (ECorn). The commercial media were (HPLC) prepared as recommended by the manufacturer (Difcotm) and fractionation and analysis by Nuclear magnetic resonance the corn medium by 3 times autoclavation of 90g of corn in 80 (NMR). mL of distilled water. The culture media were maintained, High-performance liquid chromatography under agitation, in an incubator for 28 days at 25°C. All MATERIAL AND METHODS cultivation was done in duplicate. After this, the cultures containing the secondary metabolites secreted by the fungus Plant Material were separated from mycelia by vacuum filtration and The leaves from species plant S. camparum were collected submitted to extraction with ethyl acetate (Synth® laboratories, Ecological Mogi-Guaçu, Diadema, SP, Brazil). The ethyl acetate solutions were Campininha Farm (22°17' S, 47°07' W), São Paulo State, evaporated under reduced pressure, resulting the following Brazil, and identified by Dr. Inês Cordeiro (Botanic Institute of masses of the dry crude extracts, in mg: 57.2 for EPD, 20.1 for São Paulo Agriculture Secretary). The specimen was classified EYM, 19.2 for ENB, 17.9 for ECZ, and 67.0 for ECorn. at Experimental Station of and deposited in the herbarium of the same institution under the number Young 07-SP. Chemical analysis Each crude extract was submitted to RP-HPLC-DAD Isolation of the Endophytic Fungus (Reverse Phase - High Performance Liquid Chromatography - For isolation of the endophytic fungus, adult and healthy Diode Array Detector) with analytical column Phenomenex leaves were selected and submitted to surface sterilization. C18 in exploratory gradient, using MeOH : H2O (95:5 w/w) to They were first washed with water and soap, and then (0:100 w/w) as elution system, flow of 1.0 mL/min (total time immersed in a 1% aqueous sodium hypochlorite solution for 5 of 40 min) and detection at λ = 253 nm. The extracts were also 225 Prince, K.A. et al. Anti-M. tuberculosis activity analyzed by NMR spectroscopy The NMR spectra in acceptable range of isoniazid MIC is from 0.015 to 0.03 g/mL deuterated chloroform (CDCl3) were obtained using a Varian (24,27). 1 INOVA 500 spectrometer, operating at 500MHz for H and at 150MHz for 13C. Cytotoxicity assay The in vitro cytotoxicity (IC50) assay was determined on Anti - M. tuberculosis activity assay macrophage cell line J774. The cells were routinely maintained The anti-M. tuberculosis activity of the crude extracts in Complete Medium (RPMI-1640 supplemented with 10% (ECorn, EPD, ECZ, EYM and ENB) were determined in heat inactivated fetal bovine serum (FBS); 100 U/mL penicillin triplicate using the Resazurin Microtiter Assay (REMA) as and 100 g/mL streptomycin), at 37oC in a humidified 5% CO2 analytical method (24, 34). Stock solutions of the tested atmosphere. After reaching confluence, the cells were detached compounds were prepared in dimethyl sulfoxide (DMSO) and and counted. For the cytotoxicity assay, 1 x 105 cells/mL were diluted seeded in 200 in broth medium Middlebrook 7H9 (Difco), L of complete medium in 96-well plates tm supplemented with oleic acid, albumin, dextrose, and catalase (NUNC , Thermo Fisher Scientific, USA). The plates were (OADC enrichment - BBL/Becton-Dikinson, Sparks, MD, incubated at 37oC under a 5% CO2 atmosphere for 24 h, to USA), to obtain final drugs concentration ranges of 15.6 to allow cell adhesion prior to drug testing. The crude extracts 2000 g/mL. The isoniazid was dissolved in distilled water, as (ECorn, EPD and ECZ) were dissolved in DMSO and recommended by the manufacturer (Difco laboratories, Detroit, submitted at two fold dilution from 2000 to 15.6 g/mL. Cells MI, USA), and used as a standard drug. MTB H37Rv ATCC were exposed to the compounds for a 24 h period. Resazurin 27294 was grown for 7 to 10 days in Middlebrook 7H9 broth solution was added to cell cultures and incubated for 6 h. The supplemented with OADC, plus 0.05% Tween 80 to avoid fluorescence measurements (530 nm excitation filter and 590 clumps. Cultures were centrifuged for 15 min at 3,150 x g, nm emission filter) were performed in a SPECTRAfluor Plus washed twice, and resuspended in phosphate-buffered saline (Tecan) microfluorimeter. The IC50 value was defined as the and aliquots were frozen at -80°C. After 2 days, an aliquot was highest drug concentration at which 50% of the cells are viable thawed to determine the viability and the CFU after freezing. relative to the control (1, 28, 27). MTB H37Rv (ATCC 27294) was thawed and added to the test compounds, yielding a final testing volume of 200 µL with 4 2x10 CFU/mL. Microplates with serial dilutions of each compound were incubated for 7 days at 37°C, after resazurin ® (Sigma-Aldrich Selectivity Index The selectivity index (SI) was obtained by the ratio between IC50 and MIC values (23). St. Louis, MO, USA) was added to test viability. Wells that turned from blue to pink, with the development of fluorescence, indicated growth of bacterial General 1 H NMR (500 MHz) experiments were recorded on a cells, while maintenance of the blue colour indicated bacterial VARIAN DRX-500 spectrometer, using the non-deuterated inhibition (24,27). The fluorescence was read (530 nm signal as reference. TLC was performed using Merck silica gel excitation a 60 (230 mesh) and precoated silica gel 60 PF254. Spots on TLC Switzerland) plates were visualized under UV light and by spraying with microfluorimeter. The Minimum Inhibitory Concentration anisaldehyde-H2SO4 reagent followed by heating at 120 ºC. (MIC) was defined as the lowest concentration resulting in Analytical HPLC-DAD was carried out on a Varian (Palo Alto, 90% inhibition of growth of MTB (6). As a standard test, the CA, USA) ProStar system consisting of a ternary pump model MIC of isoniazid was determined on each microplate. The 240, a diode array detector model 330 and an auto-sampler filter SPECTRAfluor and Plus 590 nm ® (Tecan emission Männedorf, filter) in 226 Prince, K.A. et al. Anti-M. tuberculosis activity model 410, controlled by a Star chromatography workstation a concentration of 200 µg/mL and according to Cantrell et al. (version 5.3), using a Phenomenex C18 column (250 mm x 4.6 (4) the isolated compounds that exhibit a MIC of 64 g/mL or mm, 5µm). lower are defined as active. Thus, the crude extracts obtained here, mainly that coming from EPD and ECorn, are as good as RESULTS AND DISCUSSION a promising isolated compound. Although the MIC values The results of the mass yield in mg of crude extracts and obtained here are larger than that of isoniazid (0.03 g/mL), the biological activity of extracts are presented in Table 1. The these inhibitory concentrations are comparable to the MIC of ECorn showed the best mass yield of 67.0 mg and also pyrazinamide (another first-line antitubercular drug) with MIC presented the best activity against M. tuberculosis with MIC of of 50-100 g/mL (6). The promising activity of the secondary 31.25 µg/mL, followed by EPD (57.2 mg and MIC of 62.5 metabolites from endophytic fungi was verified by Silva et al. µg/mL), ECZ (17.9 mg and MIC of 125 µg/mL). For others (29, 30), against different microorganisms. From Phomopsis sp extracts it was observed smaller mass yield and MIC of 250 isolated of Thailand plants, Isaka et al. (14) founded two new µg/mL. The results indicate larger extract yield from Corn bioactive secondary metabolites (Phomoxanthones A and B) medium that probably also provided larger production of with significant in vitro antimycobacterial activity (MIC of 0.5 bioactive metabolites in this medium when the P. stipata was and 6.25 µg/mL) and Chomcheon et al. (5) identified 3- cultivated at 25ºC. Based on this, we emphasize that the Nitropropionic acid with MIC of 3.3 µM. In our case, the composition of the medium influences the metabolites activity of the extracts can be attributed to the presence of production. koninginins, identified as their major compounds. The 1 H NMR data antibacterial and mycotoxical activities of koninginins were suggested the presence of koninginins, a class of hexaketides, already described (10, 11, 12, 25, 16). However, no mention of which are natural products belonging of the group of antimycobacterial activity associated to such compounds was poliketides. Other compounds of this group, the koninginins A- reported in the last 13 years (7, 8). Analysis of the RP-HPLC-DAD and G, which are octaketides, were previously isolated from fungi of Trichoderma genus (10, 11, 12, 16, 25). The crude extracts of ECorn, EPD and ECZ also presented low cytotoxicity and the SI analysis showed that these crude For plants extracts, Tosun et al. (31) considered inactive the extracts that could not prevent M. tuberculosis growth up to extracts are 4 to 8 times more active against M. tuberculosis than against the macrophage cells (Table 1). Table 1. Results of Minimum Inhibitory Concentration (MIC), in vitro Citotoxicity (IC50), Selectivity Index (SI) and mass yield of crude extracts produced by Phomopsis stipata Mass Yield MIC IC50 SI (mg) (µ µg/mL) (µ µg/mL) (IC50/MIC) ECorn 67.0 31.25 > 250.0 8 EPD 57.2 62.5 > 500.0 8 ECZ 17.9 125.0 > 500.0 4 EYM 20.1 250.0 ND ND ENB 19.2 250.0 ND ND 0.03 ND ND Crude Extracts Isoniazid (Standard drug) ND=Not Determined 227 Prince, K.A. et al. Anti-M. tuberculosis activity 20, 535-557. CONCLUSION 8. The endophytic fungus P. stipata produced a greater Copp, B.R. & Pearce, A.N. (2007). Natural product growth inhibitors of Mycobacterium tuberculosis. Nat. Prod. Rep. 24, 278-297. 9. Cruz, H.L.A.; Montenegro, R.A.; Lima, J.F.A.; Poroca, D.R.; Lima, quantity of bioactive metabolites when cultured in PD and J.F.C.; Montenegro, L.M.L.; Crovella, S.; Schindler, H.C. (2010). Corn media, and these metabolites have showed promising Evaluation of a nested-pcr for Mycobacterium tuberculosis detection in blood and urine. Braz. J. Microbiol. 42, 321-329. anti-MTB activity. Thus, this work reinforces the affirmation 10. about the quantity and production of the metabolite being media-dependent. RP-HPLC-DAD and 1H NMR data suggested the presence of koninginins in these secondary koningii. Agric. Biol. Chem. 53, 2604-2611. 11. Cutler, H.G.; Himmelsbach, D.S.; Yagen, B.; Arrendale, R.F.; Jacyno, J.M.; Cole, P.D.; Cox, R.H. (1991). Koninginin B: a biologically active metabolites which can explain the better biological activity. congener of Koninginin A from Trichoderma koningii. J. 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