Microbiological Research 159 (2004) 317—322

KEYWORDFumiquinaAntibacteractivity;Penicilliumcorylophil

0944-5013/$ - sdoi:10.1016/j.

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Antibacterial activity from Penicilliumcorylophilum Dierckx

Marley Garcia Silvaa,b, Niege Arac-ari Jacometti Cardoso Furtadob,Monica Tallarico Pupob, Maria Jose Vieira Fonsecab, Suraia Saidb,Ademar Alves da Silva Filhob, Jairo Kenupp Bastosb,�

aFaculdade de Filosofia, Ciencias e Letras de Formiga, Fundac-ao Educacional Comunitaria Formiguense, Avenida Dr.Arnaldo de Senna, 328 CEP 35570 000 Formiga MG, BrazilbFaculdade de Ciencias Farmaceuticas de Ribeirao Preto, Departamento de Ciencias Farmaceuticas, Universidade deSao Paulo, Avenida do Cafe, s/n, CEP 14040-903, Ribeirao Preto SP, Brazil

Accepted 17 June 2004

Szoline F;ial

um

ee front matter & 200micres.2004.06.003

ing author. Tel.: +55-1ess: jkbastos@fcfrp.u

SummaryA strain of Penicillium corylophilum isolated from Brazilian soil sample was submittedto different culture conditions to investigate the production of secondary metaboliteswith antimicrobial activity. The largest number of conidia was obtained after 5 days ofincubation in oat medium and the highest level of antimicrobial activity was producedwhen the fungus culture was developed in the Czapek medium. The activity againstStaphylococcus aureus was found only in the chloroform extract from Czapek culturebroth, which also showed activity against Micrococcus luteus. Fumiquinozoline F wasisolated from the active chloroform extract by using chromatographic methods. Theminimal 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 toproduce a variety of important substances for thepharmaceutical and food industries. Hence, pri-mary and secondary metabolites, such as peptides,enzymes, organic acids and antibiotics produced by

4 Elsevier GmbH. All rights rese

6-602-4162; fax: +55-16-633-194sp.br (J. Kenupp Bastos).

filamentous fungi are used for these purposes(Bennett, 1998; Demain, 2000).

The discovery and development of antibioticswas one of the most significant advances inmedicine in the 20th century. Nevertheless, manyantimicrobial agents that were used to treat avariety of human infectious diseases are now

rved.

1.

M. Garcia Silva et al.318

ineffective. Therefore, to ensure that effectivedrugs will be available in the future, it is necessaryto improve the antimicrobial use patterns and todevise strategies to identify new antibioticsthrough previously unexplored targets (Smith andJarvis, 1999).

Natural products play an important role in thediscovery of leads for the development of drugs forthe treatment of human diseases and microbialenvironment is an important source of novel activeagents (Newman et al., 2003). Many of thisproducts currently used are produced by microbialfermentation, or are derived from chemical mod-ification of a microbial product (Donadio et al.,2002). In this regard, the fermentation process is animportant tool for production of secondary meta-bolites that can not be isolated from plants andanimals, or synthesized by chemists because of theease of increasing production by environmental andgenetic manipulation (Demain, 2000).

Several secondary metabolites have been identi-fied from Penicillium corylophilum, as well as theirbiological activities. In this work, we determinedthe best procedure for the production and extrac-tion of antimicrobial metabolites from P. corylo-philum and an alkaloid derived from tryptophanwith antimicrobial activity was isolated by usingchromatographic methods.

Material and methods

Microorganisms: P. corylophilum was isolated froma soil sample collected in Sao Carlos, state of SaoPaulo, Brazil, and identified by ‘‘Fundac-ao Tropicalde Pesquisas e Tecnologia Andre Toselo’’. Thefungus is stored as a conidial suspension on silicagel (6–12mesh, grade 40, desiccant activated) at4 1C. The strains of Staphylococcus aureus ATCC25923, Micrococcus luteus ATCC 9341, Pseudomo-nas aeruginosa ATCC 27853 and Escherichia coliATCC 25922 were acquired from the ATCC.

Conidia production: Penicillium corylophilumwas submitted to four different media: oat agar(Crotti et al., 1999), malt extract (Tzean et al.,1992), potato dextrose agar—PDA (Kim et al., 1990)and Vogel medium (Vogel, 1956). The cultures wereincubated at 30 1C for 5 days. Conidia produced ineach culture were harvested with 2% of Tween 80and counted in a Neubauer hemocytometer.

Production of secondary metabolites: The pro-duction was carried out by inoculating 4� 106 con-idia/mL, obtained in oat medium, into the pre-fermentative medium (Jackson et al., 1993) at30 1C with shaking (120 rev/min) for 24 h. The

obtained mycelium mass was then inoculated intothree different fermentative media: Czapek (Atlas,1995), Jackson (Jackson et al., 1993) and Vogel(Vogel, 1956). Cultures were reincubated at 30 1Cand 120 rev/min for an additional 144 h. Foralkaloid production the fungus was grown in fiveErlenmeyer flasks containing 240mL of pre-fermen-tative medium in each and was transferred toErlenmeyer flasks containing 480mL of Czapekmedium.

Partition of the culture broth with organicsolvents and isolation of the fumiquinazoline Ffrom the chloroform extract: The culture brothswere separated by filtration, followed by threetimes partition with chloroform and buthanol insequence. All the resulting organic solvents wereevaporated under vacuum and the remaining waterfractions of the culture broths were lyophilized.The crude chloroform extract from Czapek culturebroth (3698mg), the active one, was then sub-mitted to column chromatography over 400 g ofsilica gel 60 H (70–230mesh, Merck) and elutedwith hexane-ethyl acetate at increasing propor-tions. The 7th fraction eluted with ethyl acetate(1077mg) was submitted to flash chromatography(Still et al., 1978) over 11 g of silica gel 60(230–400mesh), which was eluted with an isocraticmobile phase of hexane-acetate 3:2. Afterwards,the fourth obtained fraction (576mg) 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 IntelCeleron computer for analytical system control anddata collection and processing. Analytical chroma-tography was carried out using isocratic gradient(methanol/water/acetonitrile 11:1:8) in 25min. ACLC-ODS (M)—4.6� 250mm2 Shimadzu column wasused at a flow rate of 1.0mL/min. The spectraldata from the detector were collected within25min over the 200–400 nm range of the absorptionspectrum and the chromatograms were analyzedand plotted at 281 nm. The alkaloid was collectedbetween 16.1 and 16.5min.

General experimental procedures: A Bruker DRX-400 spectrometer, operating at 400.13MHz for 1Hand 100.62MHz for 13C was used. All spectra wererun in CDCl3 with TMS as internal standard. For aHREIMS an Autospec-Micromass-EBE was used (Uni-versidade Estadual de Campinas, UNICAMP).

Antimicrobial activity: The crude extracts wereinvestigated for antimicrobial activity by using theagar diffusion method with Petri dish templatesystem inoculated with the assayed microorgan-isms, followed by incubation at 37 1C for 24 h. Forthis procedure, aliquots of the extracts, free of

Antibacterial activity from Penicillium corylophilum Dierckx 319

solvents, were solubilized (5.0mg/mL) in 50%dimethyl sulfoxide (DMSO, v/v) aqueous solutionand applied into the template holes made on themedium surface. Negative controls were run forDMSO 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. luteusand Penicillin G (1.215 U, 50 mL, Sigma) for S.aureus were used as the positive controls. Theinoculum was prepared by culturing each organismin Antibiotic N11 agar medium (Merck) for 24 h at37 1C. The microorganisms were transferred to 0.9%NaCl until they reached the turbidity equivalent to0.5 McFarland standard. Each microorganism sus-pension (0.5%) was added in Antibiotic N11 agarmedium, and distributed over the plates. The MICvalues of the isolated alkaloid were evaluated intriplicate by microdilution broth method (Andrews,2001). The alkaloid was solubilized in DMSO(dimethyl sulfoxide) at 1mg/mL, and was dilutedin Tryptone soya broth in the range of147.5�36.8 mg/mL. The inoculum was adjusted toeach organism yielding a cell concentration of 103

colony forming units (CFU/mL). It was includedboth inoculated wells, that controls the adequacyof the broth to support the growth of the organisms

Oat agar Vogel Malt extract PDA0.0

2.5

5.0

7.5

Culture media

Con

idia

x 1

08 /mL

Figure 1. Determination of the best conditions forconidial production by P. corylophilum.

Table 1. Antimicrobial activity of different extracts from c

Microorganisms Extracts or standards

Micrococcus luteus Chloroformic

Penicillin G

Staphylococcus aureus Chloroformic

Penicillin G

and uninoculated wells, that remains free ofantimicrobial agent to check the sterility of themedium. Penicillin G (Sigma) was used as positivecontrol. The microplates (96-well) were incubatedat 37 1C for 24 h. After that, 40 mL of 2,3,5-triphenyltetrazolium chloride (0.7%) in aqueoussolution were added to indicate the viability ofmicroorganisms (Leverone et al., 1996). The MICwas determined as the lowest concentration ofdrug able to totally inhibit microorganism growth.

Results

Determination of the best conditions forconidial production

The largest number of conidia was obtained after 5days of incubation in oat agar, Vogel, malt extractand PDA media, in this order (Fig. 1).

Selection of fermentative medium forantimicrobial activity production

Different fermentative media were investigated todetermine in which broth the antimicrobial activitywas produced. The highest level of antimicrobialactivity was produced by P. corylophilum when thefungus culture was developed in the Czapekmedium, as observed for the results obtained bythe agar diffusion method (Table 1). The activitywas considered for inhibition zones wider than12mm since the template punches holes ofapproximately 10–11mm into the agar surface.The activity against S. aureus was found only in thechloroform 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 showedactivity against Gram-negative bacteria at theconcentration evaluated in this work.

ultures of Penicillium corylophilum

Fermentative medium Inhibition zones (mm)

Czapek 1470.577Jackson 1470.577Vogel 1370.577

2670.577

Czapek 1370.577Jackson 0970.577Vogel 0870.577

2070.577

Table 2. 13C (100MHz) and 1H NMR (400MHz) spectraldata for fumiquinozoline F (CDCl3)

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 s14 123.4 6.64 d (2.5)15 168.9 —16 — 5.83 br s17 49.1 2.99 q (6.6)17a 151.6 —18 — —18a 147.1 —19 19.3 1.29 d (6.6)

NH

NH

N

O

N

O 1

2

3

4

4a

5

68

8a8b

9

10

11

1212a 13 14 15 16 17

17a

18

18a

19

7

Figure 2. Structure of the fumiquinozoline F, isolatedfrom Czapek chloroform extract.

M. Garcia Silva et al.320

Identification and antimicrobial activity ofthe alkaloid from Czapek chloroform extract

The chemical structure of the isolated compound(Fig. 2) was identified from 1H and 13C NuclearMagnetic Ressonance (NMR), Attached Proton Test(APT), 1H–1H Correlation Spectroscopy (COSY),Heteronuclear Multiple Bond Coherence (HMQC),Heteronuclear Multiple Quantum Coherence(HMBC), and Mass Spectra (MS) spectral data incomparison with previous published data (Takaha-shi et al., 1995). The MS High Resolution showedthe [M+] peak m/z 358.14298. The 13C NMRspectrum of the compound showed 21 carbonsignals. The multiplicities of the carbons deter-mined by APT led to the attribution of: 8 C, 11 CH,1 CH2 and 1 CH3. Among the quaternary carbonstwo were attributed to amide carbonyls (d 168.9,C-12 and 160.7, C-5). The 1H NMR spectrum showedtwo broad singlets at d 8.00 (1H, H-13) and d 5.83(1H, H-16) indicating that these hydrogens could beattached to nitrogens. Hence, these data suggestedan alkaloid type structure for the compound(Fig. 2).

The 1H NMR spectrum also showed nine hydrogensignals between d 6.64 and 8.30. In the 1H–1H COSYexperiment the signals at d 7.35 (dd, J ¼ 8:1 and0.8 Hz, H-9), d 6.87 (ddd, J ¼ 8:1; 7.1 and 0.8 Hz, H-10), d 7.06 (ddd, J ¼ 8:1; 7.1 and 0.8 Hz, H-11) d7.23 (dd, J ¼ 8:1 and 0.8 Hz, H-12) were coupled toeach other. The hydrogen at d 6.64 (d, J ¼ 2:5Hz;H-14) was coupled to the hydrogen at d 8.00 (H-13).The chemical shifts of the carbons attached tothese hydrogens were attributed according to theHMQC data (Table 2). These data allowed us topropose an indolic moiety for the compound, whichwas corroborated by the H-C correlations observedin the HMBC experiment. The HMBC experimentshowed the correlation of the indolic carbon atd109:6 (C-8a) with the hydrogens at d 3.66 (dd,

J ¼ 14:9 and 3.3 Hz, H-80) and d 3.59 (dd, J ¼ 14:9and 5.3 Hz, H-800). In the HMQC experiment thesehydrogens were attached to the carbon at d 27.0.The 1H–1H COSY experiment showed that thesehydrogens (H-8) are coupled to each other and toanother hydrogen at d 5.63 (dd, J ¼ 5:3 and 3.3 Hz,H-7). The HMBC experiment showed the correlationof both H-8 with C-7, C-8a and C-15. These data ledus to propose an alkaloid derived from tryptophanfor the isolated compound (Bergman and Bergman,1985).

The 1H–1H COSY experiment also revealed thatthe hydrogens at d 8.30 (ddd, J ¼ 8:1; 1.5 and0.5 Hz, H-4), d 7.47 (ddd, J ¼ 8:1; 7.3 and 1.3 Hz,H-3), d 7.71 (ddd, J ¼ 8:3; 7.3 and 1.5 Hz, H-2)and d 7.53 (dd, J ¼ 8:3 and 1.5 Hz, H-1) werecoupled to each other. The aromatic carbonschemical shifts were attributed on the basis ofHMQC data. These spectral data suggested ananthranilic 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 1H NMR signals were observed at d1.29 (d, J ¼ 6:6Hz; 3H, H-19) and d 2.99 (q, J ¼

Antibacterial activity from Penicillium corylophilum Dierckx 321

6:6Hz; 1H, H-17), both coupled to each other asobserved in the 1H–1H COSY experiment. The HMBCexperiment 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 (d5.86). These data suggested an alanine moiety forthe compound. Therefore, the data allowed topropose the structure of fumiquinozoline F as analkaloid derived from the linking of the aminoacidstryptophan and alanine plus an anthranilic acidunit.

Regarding the antimicrobial activity, the MICvalues 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 wasmore active than the isolated compound for bothmicroorganisms (0.011 mg/mL and 0.023 mg/mL,respectively).

Discussion

The expression of secondary metabolites mightdepend on the culture conditions and the strains. P.corylophilum is often isolated from temperateclimates (Malmstrom et al., 2000), but in this workwe investigated a strain isolated from Brazilian soilsample, a tropical country.

According Gaden-Junior (2000), the metabolitesproduction is influenced by medium composition,nutrients availability and others aspects. Differentnitrogen and carbon sources may affect the synth-esis of enzymes involved in primary and secondarymetabolism. Microorganisms are able to use a widevariety of carbon and nitrogen sources. Never-theless, many secondary metabolic pathways arenegatively affected by these sources favorable forgrowth (Sanchez and Demain, 2002).

In the study of the secondary metabolites of theP. corylophilum, Cutler et al. (1989) isolated the3,7-dimethyl-8-hydroxy-6-metoxysocroman, fromthe mycelia using shredded wheat medium, whichwas maintained 12 days at 22 1C. By cultivation inmalt medium, strains of P. corylophilum producedthe alkaloid epoxyagroclavine I (Grabley et al.,1992).

In this work, a two step culture was used forantimicrobial active compounds production. In thefirst step, the microorganism was cultivated in pre-fermentative medium, which is rich in nutrients toincrease vegetative biomass production. The har-vested mycelium was then transferred to fermen-tative medium for secondary metabolitesproduction. The fumiquinazolina F was isolatedfrom the chloroform extract of the Czapek fermen-

tative medium, after 144 hours of incubation. Fromthe three evaluated media, the Czapek one was thebest for antimicrobial metabolites production.

Fumiquinazoline F was originally obtained from astrain of Aspergillus fumigatus, which was isolatedfrom the gastrointestinal tract of the marine fishPseudolabrus japonicus (Takahashi et al., 1995),and it was also detected in Penicillium thymicola(Larsen et al., 1998). It was the first time thatFumiquinazoline F was isolated from P. corylophi-lum. Besides, it is the first time that the anti-microbial activity of this compound is beendescribed.

The fumiquinazolines belong to a class ofcompounds possessing a wide range of biologicalactivities. For instance, fumiquinazolines A, B andC from A. fumigatus showed moderate cytotoxicityagainst the cultured P-388 lymphocytic cells (Nu-mata et al., 1992). Fumiquinazolines H and I,isolated from Acremonium sp. presented significantantifungal activity (Snider and Zeng, 2003).

Kariba et al. (2002), showed that extracts ofSchizozygia coffaeoides, containing indolines pre-sented antifungal and antibacterial activities. Theberberine, benzylisoquinoline alkaloid, isolatedfrom Hydrastis canadensis, is active against manyGram-positive and Gram-negative bacteria, as wellagainst fungi and parasites (Scazzocchio et al.,2001).

Fumiquinazoline F is derived of tryptophan, plusanthranilic acid and it showed value of MIC lowerthan berberine, canadaline and canadine for S.aureus (ATCC 25923) (Scazzocchio et al., 2001).

The identification of this class of alkaloid incultures of P. corylophilum opens new possibilitiesfor bioprospection of new compounds belonging tothis class with potential for the design of newdrugs.

Acknowledgements

The authors are grateful to CAPES for fellowshipand FAPESP for financial support (Grants # 01/14209-7 and # 99/09850-8) for financial support.

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