Biologically Active Polyketides Produced by Penicillium janthinellum Isolated as an Endophytic Fungus from Fruits of Melia azedarach

Penicillium janthinellum, isolado como um fungo endofítico dos frutos de Melia azedarach, foi cultivado por 20 dias em milho branco triturado e autoclavado, onde os policetídeos conhecidos citrinina, emodina, 1,6,8-triidróxi-3-hidroximetilantraquinona, e uma nova antraquinona modificada, denominada janthinona, foram produzidos e isolados por procedimentos cromatográficos clássicos e identificados por extensivos estudos espectroscópicos, principalmente RMN 1D e 2D e EM. Essas substâncias foram ensaiadas contra diversas bactérias. Citrinina foi ensaiada pela primeira vez contra Leishmania e inibiu 100% o crescimento de cepas depois de 48h a uma concentração de 40 μg mL.


Introduction
Endophytic microorganisms are isolated from internal plant tissue and can be present even at the cell level. 1,2The biochemistry of these associations is a fascinating and novel field of research.The colonization of the host plant by endophytes may be mediated by interesting secondary metabolites. 3In general these compounds show insecticidal and antimicrobial activities, which may give the host some protection against further invasion. 1,3e have been studying the chemistry of microorganisms associated with Meliaceae plants.A large collection of fungi have been isolated from the fruits, leaves, stems and roots of Melia azedarach. 46][7] Herein we report our chemical investigations of another Penicillium, identified as P. janthinellum, cultivated on sterilized white corn.This fungus produced polyketides, basically hydroxyanthraquinones, ergosterol and poliols.Citrinin was tested against bacteria and Leishmania mexicana, showing promising inhibition properties.
The mass spectrum of compound 3, obtained by electrospray (ESI) in the negative ion mode, contains an abundant [M-H] -ion at m/z 283.This information, along with the 1 H and 13  The IR (ν max 1653 and 1742 cm -1 ) and 13 C NMR (δ 180.0 and 169.6) spectroscopic data indicated that compound 3 has a conjugated ketone and a conjugated lactone in its molecular structure.The position of the lactone function was based on the HMBC correlation detected between H-5 (whose signal was unequivocally ascribed by 1 H-1 H COSY) and C-10.Other important HMBC are shown below in Figure 2. The NMR signal of the hydrogen H-7 (δ 7.30) showed a nuclear Overhauser effect (NOE) with the methoxyl at δ 4.03 confirming structure 3 (Figure 1) for this modified anthraquinone.It was named janthinone, and appears to be a new natural product.Similar lactones (with C-11 oxidized) were produced by a Brazilian strain of Aspergillus versicolor. 10ompound 4 (citrinin) is a well known mycotoxin produced mainly by Penicillium citrinum and several Aspergillus species. 11This polyketide was produced in good yields by P. janthinellum obtained from the host plant M. azedarach.The identification of 4 was achieved mainly by 1 H and 13 C NMR, which showed that it occurs in the p-quinone form. 12he antibacterial activity of citrinin ( 4) and the anthraquinones 1-3 was examined in the presence of Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, and the results are shown in Table 1.Emodin and citrinin were almost completely inactive against E. coli, but shown promising activity against P. aeruginosa and B. subtilis.Compound 3, the new lactone janthinone, was considered inactive in all tests (>500 µg mL -1 ).The growth inhibition property of citrinin (4) against Leishmania mexicana was also studied (Table 2).Inhibition rates at very low concentration of citrinin (5 µg mL -1 ) were observed.The highest growth inhibition was observed after 48 h of inoculation of the microorganism in the medium containing 40 µg mL -1 of citrinin.

General procedures
UV spectra were obtained in CH 2 Cl 2 solution on a "+" : Growth like in the negative control; "-" : bacteriostatic effect (growth in subculture); "=" : bactericidal effect (not grow in subculture).HEWLETT PACKARD 8452-A spectrophotometer, and IR spectra were measured with a BOMEN MB-102 spectrophotometer in KBr pellets.Low-resolution APCIMS and ESIMS data were acquired in positive ion mode, using a MICROMASS QUATTRO-LC instrument equipped with an ESI/APCI "Z-spray" ion source. 1 H and 13 C NMR experiments were recorded on a BRUKER DRX-400 spectrometer with Acetone-d 6 or CDCl 3 as the solvent and TMS as the internal standard.

Plant material
Fruits of Melia azedarach were collected on the campus of the Federal University of São Carlos, São Carlos, Brazil.Samples (dried leaves and fruits) of the plant are deposited in the Herbarium of Department of Botanic at the University to be included in their catalogue.Microorganism P. janthinellum was obtained from the collection of the Laboratório de Bioquímica Micromolecular of the Chemistry Department at Universidade Federal de São Carlos.This collection contains isolates recently obtained 4 from Melia azedarach.P. janthinellum is identified by the number LaBioMi-018.

White corn culture of P. janthinellum and isolation of the polyketides
45 Erlenmeyer flasks (500 mL), containing 90g of white corn ("Yoki") and 75 mL of distilled water per flask, were autoclaved twice at 121 o C for 40 min.A small disc of the PDA medium from the Petri dish containing mycelium of P. janthinellum was transferred under sterile conditions to 42 Erlenmeyer flasks containing sterilized corn.Three flasks were kept for control purposes.After 20 days of growth at 25 °C, methanol (200 mL) was added to each flask and allowed to stand for 5 h, and then it was filtered by gravity.The methanol was evaporated under reduced pressure, producing a yellowish residue (14.2 g).Part of this residue (10.0 g), was subjected to a low-pressure silica gel CC eluted with n-hexane, ethyl acetate and methanol gradient.The medium polarity fractions eluted with ethyl acetate were repeatedly chromatographed on silica gel CC [n-hexane:ethyl acetate:methanol (60:35:05), isocratic ) and preparative TLC [chloroform:methanol (96:04)] and the polyketides, emodin (80.1 mg) (1), 1,6,8trihydroxy-3-hydroxymethylanthraquinone (10.2 mg) (2), and janthinone (80.1 mg) (3), were finally purified.Citrinin (180.2 mg) (4) crystallized from a methanol solution of a fraction eluted with ethyl acetate:methanol (95:05) from the first silica gel CC.

Antibacterial bioassay
The susceptibilities of microorganisms to the test polyketides were determined by microbroth dilution assay as recommended 13 by the Subcommittee on Antifungal Susceptibility Testing of the US National Committee for Clinical Laboratory Standards (NCCLS).They were performed on 96 well plates with 100 µL of Mueller Hinton Broth (MHB), 100 µL of test compound and 5 µL of test bacteria at 1.0 x 10 7 UFC mL -1 , followed by incubation at 37 o C (24h).The test substances obtained from the fungal culture were dissolved in dimethylsulfoxide at initial concentration 500 µg mL -1 .The test microorganisms were Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis (obtained from Universidade de Maringá -PR, Brazil).Bioactivity was recorded as absence of red coloration in the wells after addition of 10 µL of 2,3,5-triphenyltetrazolium chloride.The tested microorganisms were subcultured on MHB plates.The activities of the test compounds were classified as bacteriostatic or bactericidal effects according the behavior of the microorganisms in these subcultures.Penicillin, vancomycin and tetracycline (25 µg mL -1 each) were used as positive controls; the cultivation medium (MHB only) was used as negative control.

Leishmanicidal bioassay
Auxenic culture of Leishmania mexicana (MNYC/BZ/ 62/M379, Universidade de Maringá -PR, Brazil) in exponential phase of growth was incubated at 33 o C in modified UM-54 medium along with 5, 10, 20, and 40 mg mL -1 of citrinin (4), dissolved in DMSO (0.1 % final concentration), in a 24 wells ELISA plate.The inhibitions of growth, reported as percentage of death 14 (Table 2), were measured in a Neubauer chamber, by counting dead microorganisms after 24, 48 and 72 h compared with the controls (only medium without the test compound).

Figure 1 .
Figure 1.Polyketides isolated from the fungi biomass produced by P. janthinellum over white-corn.

Figure 2 .
Figure 2. Important long range correlations detected for janthinone (3) by means of HMBC NMR experiment.

Table 1 .
Growth behavior of bacteria in the presence of different concentrations of polyketides produced by P. janthinellum

Table 2 .
Growth inhibition rate of Leishmania mexicana in the presence of different concentrations of citrinin(4)