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World Journal of Microbiology & Biotechnology 2005 21: 27–32 DOI: 10.1007/s11274-004-1552-6

 Springer 2005

Secondary metabolites from endophytic fungi isolated from the Chilean gymnosperm Prumnopitys andina (Lleuque) G. Schmeda-Hirschmann1,*, E. Hormazabal1, L. Astudillo1, J. Rodriguez2 and C. Theoduloz2 1 Laboratorio de Quı´mica de Productos Naturales, Instituto de Quı´mica de Recursos Naturales, Universidad de Talca, Casilla 747, Talca, Chile 2 Departamento de Ciencias Ba´sicas Biome´dicas, Facultad de Ciencias de la Salud, Universidad de Talca, Casilla 747, Talca, Chile *Author for correspondence: Tel.: +56-71-200-288, Fax: +56-71-200-448, E-mail: [email protected] Received 30 December 2003; accepted 19 May 2004

Keywords: Endophytes, gliovictin, mellein, Penicillium janczewskii, peniprequinolone, Prumnopitys andina

Summary Two endophytic fungi isolated from the phloem of the Chilean gymnosperm Prumnopitys andina were cultured in liquid potato-dextrose medium. The secondary metabolites were isolated and identified by spectroscopic methods. The fungus E-3 which could not be identified yielded 4-(2-hydroxyethyl)phenol, p-hydroxybenzaldehyde and the isochromanone mellein. The second fungus, identified as Penicillium janczewskii K.M. Zalessky yielded peniprequinolone and gliovictin, reported for the first time for this species. This is the first report on secondary metabolites produced by endophytic fungi from Chilean gymnosperms.

Introduction Endophytic fungi are eucaryotic organisms that live inside plant tissues and behave as plant hosts (Petrini 1996). The association is symbiotic and both organisms profit from the relationship. Endophytes are presumably ubiquitous in plants, with populations dependent on host species and location, and they have been recognized as a valuable source of novel bioactive metabolites (Bills & Polishook 1991). Some endophytes isolated from gymnosperms have been shown to produce secondary metabolites with very strong fungicidal and bactericidal activity (Tan & Zou 2001). In Chile, the native gymnosperms comprise 4 families including 8 genera and 9 species, most of them restricted to the southern part of the country and the eastern Andean slopes in Argentina. The genus Prumnopitys (Podocarpaceae) is monotypic and indigenous with a distribution area ranging from the province of Linares to the province of Cautin in central Chile. The tree grows at an altitude of 500–1000 m over the sea level. According to the conservation status, the species has to be considered rare (Marticorena & Rodriguez 1995). The search for active microbial compounds from endophytes in Chilean gymnosperms is a challenge, since the native species belonging to this plant group have a restricted distribution, unique evolutionary traits and their conservation status is rare. The aim of the present work was to isolate and to take into culture endophytic fungi from the Podocarpacea

Prumnopitys andina (Poepp. ex Endl.) de Laub (Lleuque) as well as to identify their active secondary metabolites. Materials and methods Isolation and culture of the endophytic fungi The fungi were isolated from wood pieces collected on the western Andean slopes near Las Trancas, Chillan. The samples were surface-disinfected with 5% sodium hypochlorite and 70% ethanol and placed in PDA medium (potato-dextrose agar: 20 g dehydrated mashed potatoes, 20 g glucose, 15 g agar in 1 l water, pH 5.5) supplemented with antibiotics (penicillin G, 30 mg/l, streptomycin 30 mg/l) and cultivated at 25 C according to the methodology described by Bills (1996). Microorganisms were cultured in potato dextrose (PD) medium, prepared with 20 g dehydrated mashed potatoes, 20 g glucose in 1 l water, pH 5.5. Microorganisms were identified by Prof Eduardo Piontelli, Universidad Cato´lica de Valparaı´ so and they are kept at the microbial strain collection from the Universidad de Talca. Fungal culture and isolation of secondary metabolites Nuclear Magnetic Resonance (NMR) spectra were obtained with a Bruker spectrometer operating at 400 MHz for protons and 100 MHz for 13C. Mass spectra were measured in a Varian unit at 70 eV.

28 Infrared (IR) spectra on KBr discs were run on a Bruker FT-IR spectrophotometer. The fungus E-3 is mitosporic, hyaline, probably phyalidic but could not be identified or related with similar microorganisms. The fungus was cultured in liquid potato-dextrose broth in Erlenmeyer flasks (7.2 l) at 25 C under constant shaking (150 rev/min). Once the glucose in the medium was consumed (17 days), mycelium and culture broth were separated by filtration and extracted separately with EtOAc (3 · 2 l). Some 668 mg of EtOAc-solubles were obtained from the culture filtrate and 1.66 g from the mycelium. The extract obtained from the culture medium was dissolved in MeOH. A soluble portion and 100 mg of a precipitate were obtained. The precipitate was identified as ergosterol by spectroscopic methods and comparison with a standard sample. The extract obtained from the culture medium (550 mg) yielded after gel permeation in Sephadex LH20 (MeOH) and preparative TLC on silica gel (PE: EtOAc 1:1) the phenolics 4-(2-hydroxyethyl)phenol 1 (3 mg, Rf 0.42), p-hydroxybenzaldehyde 2 (3 mg, Rf 0.77) and the isocromanone mellein 3 (3.5 mg, Rf 0.90). The structure of the isolated compounds was determined by spectroscopic methods, mainly NMR and mass spectroscopy. From the less polar fractions, the following fatty acids were identified by GC/MS of the corresponding methyl esters: tetradecanoic acid (C14), hexadecanoic acid (C16), octadecenoic acid (C18:1), octadecanoic acid (C18) and octadecadienoic acid (C 18:2). The structure of the isolated compounds is presented in Figure 1.

G. Schmeda-Hirschmann et al. Compound 3 Mellein Molecular formula: C10H10O3 . 1H-NMR (400 MHz; CDCl3, d-values, j in Hz): 4.76 dq (7.2; 6.3) (1 H) (H-3); 2.95 d (7.2) (2 H) (H-4); 6.72 dd (7.2; 1.0) (1 H) (H-5); 7.44 dd (8.2; 7.7) (1 H) (H-6); 6.92 dd (8.2; 0.9) (1 H) (H-7); 1.55 d (6.3) (3 H) (H-11). Penicillium janczewskii K.M. Zalessky was isolated from the phloem of Prumnopitys andina. The fungus was cultured in potato-dextrose medium in Erlenmeyer flasks to a total volumen of 7.2 l at 25 C under agitation (150 rev/min). The glucose was consumed after 23 days and the medium was extracted with EtOAc yielding 1.955 g of EtOAc-solubles. The crude extract was chromatographed on silica gel (column length 70 cm, internal diameter 3.5 cm) with a petroleum ether (PE) – PE:Ethyl acetate (EtOAc) gradient. Some 23 fractions were collected and pooled as follows: PE (500 ml, fractions 1–4); PE:EtOAc 9:1 (750 ml; fractions 5–8); PE:EtOAc 85:15 (750 ml; fractions 9–11); PE:EtOAc 8:2 (1 l; fractions 12–16); PE:EtOAc 75:25 (400 ml; fractions 17–18); EtOAc (1 l; fractions 19–23). Fractions 13 and 14 were pooled (100 mg) and the main compounds isolated by preparative TLC (SiO2, PE:EtOAc 7:3, two developments) yielding 30 mg of peniprequinolone 4, identified by its specroscopic data which fully agree with those reported by Kusano et al. (2000). Fractions 16–17 yielded after preparative TLC (SiO2, PE:EtOAc 1:1), 35 mg of gliovictin 5. The Rf values of compounds 4 and 5 (SiO2, PE:EtOAc 1:1) were 0.50 and 0.35, respectively. The 1H-NMR data of gliovictin are in agreement with those reported by Dorn

Figure 1. Structure of the compound isolated from endophytic fungi of Prumnopitys andina.

29

Metabolites from endophytic fungi

Table 1. 1H- , 13C-NMR and HMBC data of Peniprequinolone (compound 4) (400 and 100 MHz, respectively, CDCl3, dH and dC in ppm, j in Hz). H 2 3 4 5 6 7 8 9 10 11 12, 16 13, 15 14 17 18 19 20 21 3-OMe 14-OMe 1-NH 4-OH 6-OH

C

3.69 s

7.00 d (8) 6.39 d (8)

7.18 d (9) 6.86 d (9) 3.31 dd (15.9; 7.3); 3.22 dd (15.9; 7.1) 5.30 tq (7.2, 0.9) 1.74 1.70 3.55 3.77 8.96 4.55 9.07 4.68

d (0.9) br s s s s br s s OH

& Arigoni (1974) while the 13C-NMR data were reported by Shin & Fenical (1987). The NMR spectral data of compounds 4–6 are presented in Tables 1 and 2 and the assignation was based in correlation experiments. The complete spectral data of compound 6 is presented for the first time. The structure of the compounds 4–6 is presented in the Figure 1. Compound 4 Peniprequinolone. Molecular formula C22H25NO5. [a]D ¼ )1.0 (c ¼ 1.6, CHCl3). The 1H- and 13C-NMR data are presented in Table 1. NOESY experiments are in full agreement with those reported by Kusano et al. (2000). Compound 5 3-(Hydroxymethyl)-1,4-dimethyl-3,6-bis(methylthio)-6benzyl-2,5-piperazinedione (Gliovictin). Molecular formula: C16H22 N2O3S2. [a]D ¼ )21.8 (c ¼ 0.7, CHCl3). Treatment of 5 with Ac2O in pyridine and usual workup afforded the corresponding acetate 6. The 1H- and 13 C-NMR data are presented in Table 2. Compound 6 Gliovictin acetate. Molecular formula: C18H24 N2O4S2. [a]D ¼ )20.2 (c ¼ 0.65, CHCl3). Antibacterial and antifungal activity Extracts were assessed for antimicrobial activity by the agar diffusion method at a concentration of 100 lg/disk towards the following microorganisms: Gram-positive bacteria: Bacillus brevis, Bacillus subtilis, Micrococcus

166.50 84.76 79.13 110.81 155.94 126.05 130.39 106.75 133.05 129.88 128.25 114.63 160.60 28.09 122.68 133.61 26.14 18.13 59.25 55.63

HMBC s d s s s s d d s s d d s t d s q q q q

9.08, 3.69 s 9.07, 3.55 (strong) 7.18, 3.69 9.07, 8.96, 6.39, 3.69 s 8.96, 7.00 d, 3.31; 3.22 8.96, 6.39, 3.31, 3.22. 28.09 t 126.05, 110.81, 79.13; 9.06 4.68, 6.86 160.6, 129.88, 79.08 160.6, 128 d 7.18, 6.86, 3.77 155.9, 133.05, 126.05, 122.68

133.05, 122 133.05, 122 84.7 150 s 155.6, 126.06, 110.8 167.2, 133.61, 110.8, 84.7 79, 129.88

luteus, Staphylococcus aureus, Streptococcus pyogenes. Gram-negative bacteria: Citrobacter sp., Enterococcus faecalis, Erwinia carotovora, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Providencia sp., Pseudomonas sp., Pseudomonas syringae, Salmonella sp., Shigella sp. Fungi: Alternaria alternata, Botrytis cinerea, Candida albicans, Mucor miehei, Paecilomyces variotii, Penicillium notatum. The activity of the extracts was estimated by the growth inhibition (in mm) as follows: 15 mm: strong activity. As a reference, the commercial antifungal Benlate (benomyl, methyl-1-(butylcarbamoyl)-2-benzimidazol carbamate, 50% active ingredient, 50 lg benomyl/disk) and chloramphenicol (30 lg/disk) were used. Cytotoxicity assay The cytotoxic effect of the compounds, expressed as cell viability, was assessed on two cell lines: the permanent fibroblast cell line derived from human lung (MRC-5) (ATCC Nr CCL-171) and the human gastric adenocarcinoma cells (AGS) (ATCC Nr CRL-1739). MRC-5 fibroblasts were grown as monolayers in minimum essential Eagle medium (MEM), with Earle’s salts, 2 mM L -glutamine and 1.5 g/l sodium bicarbonate, supplemented with 10% heat-inactivated foetal calf serum (FCS), 100 IU/ml penicillin and 100 lg/ml streptomycin in a humidified incubator with 5% CO2 in air at 37 C. AGS cells were grown in Ham F-12K (Kaighn’s modification) medium in the conditions described for MRC-5 fibroblasts. Cells were seeded at a density of 2.5 · 103 cells per well in 96-well plates. Confluent

1.72, 4.37, 4.46

7.29

3.31, 4.37, 4.46 2.29, 3.09, 4.37

cultures were treated with medium containing the compounds at concentrations ranging from 3.8 up to 1000 lM. The substance was firstly dissolved in DMSO and then in the medium. The final concentration of DMSO in the test medium and controls was 1%. Cells were exposed for 24 h to the test medium with or without the compound (control). Each drug concentration was tested in quadruplicate, and repeated three times in separate experiments. At the end of incubation, the neutral red uptake (NRU) assay was carried out as described by Rodrı´ guez & Haun (1999). To calculate the IC50 values the results were transformed to percentage of controls and the IC50 values were graphically obtained from the dose–response curves.

dd (6.9, 1.5) m m s s s

d (11.7) 4.46 d (11.7) d (14.5) 3.92 d (14.5)

30.96 163.66 68.25 29.70 164.89 72.30 68.20 42.25 134.66 131.76 129.82 127.67 14.30 14.31 20.70 169.96 71.98 74.14 – –

3.17: 74.14, 130 3.75, 3.17 3.75, 3.17

3.31 – – 3.09 – – 4.37 3.22 – 7.19 7.29 7.25 2.29 2.38 1.72 – 74.14 3.89, 3.15, 3.34, 3.06 3.06, 2.17 71.98 3.89, 3.15, 3.34, 3.06 3.75, 3.34, 3.17, 2.34

dd (7.6; 2.4) m m s s 7.15 7.30 7.29 2.17 2.34 – –

– 3.89 d (11.9); 3.15 d (11.9) 3.75 d (13.8); 3.17 d (13.8)

31.40 q 165.97 s 71.98 s 29.76 q 165.97 s 74.14 s 64.74 t 42.77 t 134.55 s 130.49 d 129.10 d 128.28 d 13.81 q 14.69 q – – 3.34 s – – 3.06 s

HMBC C

s

H H

s

Compound 6 Compound 5

1 N-Me 2 3 4 N-Me 5 6 1¢ 1¢¢ 2¢¢ 3¢¢, 7¢¢ 4¢¢, 6¢¢ 5¢¢ 3 S-Me 6 S-Me OAc

C

Table 2. 1H- ,

13

C-NMR and HMBC data of compounds 5 and 6 (400 and 100 MHz, respectively, CDCl3, dH and dC in ppm, j in Hz).

C

q s s q s s t t s d d d q q q s

HMBC

2.29, 2.38

G. Schmeda-Hirschmann et al.

3.22, 3.92, 3.09 2.38, 3.31, 3.22, 3.92

30

Results and discussion The gymnosperm tree Prumnopitys andina (Poepp. ex Endl). de Laub. presents association with endophytic fungi, some of them able to be taken into culture from wood samples. Two endophytic fungi were cultured in liquid medium and several secondary metabolites were isolated and identified by spectroscopic methods. From the isolate E-3, simple phenolics (compounds 1–2), fatty acids and mellein 3 were obtained. The extract from the culture medium of this fungus presented a weak activity towards the following Grampositive bacteria: Bacillus subtilis, Micrococcus luteus and Staphylococcus aureus, the Gram-negative Citrobacter sp., Klebsiella pneumoniae, Proteus mirabilis, Providencia sp., Pseudomonas sp., Salmonella sp., Shigella sp. and the filamentous fungi Paecilomyces variotii and Penicillium notatum. Furthermore, the crude culture medium extract was active towards the following dermatophytic fungi: Epidermophyton floccosum, Trichophyton rubrum, Trichophyton mentagrophytes, Microsporum canis and Microsporum gypseum, with MIC’s of 100, 100, 125, 125 and 250 lg/ml, respectively. Mellein was previously isolated from endophytic fungi comprising Plectophomella sp., Pezicula livida and Cryptosporiopsis malicorticis (Krohn et al. 1997). The occurrence of the compound in the phytopathogenic fungi Septoria nodarum and Botryosphaeria obtusa has been reported (Parisi et al. 1993). Mellein presents antibacterial activity (Krohn et al. 1997), phytotoxic effects (Parisi et al. 1993) and is active against the protease of hepatitis C virus C (Dai et al. 2001). 5-Carboxymellein was recently reported from the marine fungus Halorosellinia oceanica by Chinworrungsee et al. (2001) and exhibited moderate cytotoxicity against KB and BC-1 cell lines as well as an antimalarial effect. The culture extract of Penicillium janczewskii displayed a chemical pattern characterized by the synthesis of nitrogenous compounds, including the quinolone peniprequinolone 4 and the dioxopiperazine derivative gliovictin 5. Peniprequinolone was previously isolated from Penicillium cf. simplicissimum by Kusano et al. (2000). At

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Metabolites from endophytic fungi 1000 mg/l, the compound was active towards the nematode Pratylenchus penetrans with 82.4% nematicidal effect and accelerated the root growth of rice seedlings in concentrations from 100 to 300 mg/l. Related compounds comprises Penigequinolone A and its 19-epimer, Penigequinolone B (Kimura et al. 1996). Penigequinolone A has been reported as a pollen growth inhibitor. Gliovictin was first reported as a metabolite from Helminthosporium victoriae by Dorn & Arigoni (1974). A closely related compound, the epitetrathiodioxopiperazine derivative hyalodendrin was described by Struntz et al. (1975) from a Hyalodendron sp. In the same article, Struntz et al. (1975) refers to the configuration of hyalodendrin at positions 3 and 6 in Hyalodendron and Penicillium turbatum. While in Hyalodendron the configuration at both positions was shown to be S, the products synthesized by Penicillium turbatum had the configuration R. The synthesis of (±)-gliovictin was achieved by Williams & Rastetter (1980). The 13C-NMR data of gliovictin were first reported by Shin & Fenical (1987) from the ()) compound isolated from culture broths of the marine deuteromycete Asteromyces cruciatus. Related compounds were isolated from Gliocladium virens (Kirby et al. 1988) and Phoma lingam (Pedras et al. 1990). Diketopiperazine alkaloids were reported from Penicillium fellutanum by Kozlovsky et al. (2000). Penicillium janczewskii has been described as a griseofulvin producer (Dewick 1997) and synthesizes a fumagillin derivative active towards the yeast MetAP2 (methionine aminopeptidase type 2) (Kwon et al. 2000). Kozlovskii et al. (1997) reported the isolation of griseofulvin, dechlorogriseofulvin, patulin, aurantioclavine ergoalkaloids and diketopiperazines of the roquefortine group from different isolates of the microorganism. The production of enzymes by the fungus has been described by several authors, including extracellular inulinases (Pessoni et al. 1999) and catalase (Mikhailova et al. 2001). The biosynthesis of indolediterpenoid mycotoxins by P. janczewskii has been studied by Mantle & Penn (1989) and Penn & Mantle (1994). The effect of Penicillium janczewskii and its metabolites on systemic acquired resistance to stem rot caused by Rhizoctonia solani has been reported by Madi & Katan (1998). Previous chemical studies on Prumnopitys andina reported the isolation of several main compounds belonging to the abietane and pimarane diterpenes (Becerra et al. 2002). From the closely related species Podocarpus saligna phenolic diterpenoids (Cambie et al. 1984), lignans and norditerpene dilactones (Matlin et al. 1984a, b) were reported. Those compounds are typical ‘plant’ compounds and do not show any relation to the metabolites produced by the endophytes of Prumnopitys andina. The cytotoxicity of compounds 3–6 was assessed in AGS cells and fibroblasts (Table 3). Under our experimental conditions, the cytotoxicity (IC50) of peniprequ-

Table 3. Cytotoxicity of compounds 3–6 on AGS cells and fibroblasts (data are presented as IC50 (lM)). Compound

AGS IC50 (lM)

Fibroblasts IC50 (lM)

Mellein 3 Peniprequinolone 4 Gliovictin 5 Gliovictin acetate 6

>1000 89 475 >1000

>1000 116 681 >1000

inolone towards fibroblasts was 116 lM while the compound presented an IC50 of 89 lM against AGS cells. The cytotoxicity of gliovictin 5 ranged between 475 and 681 lM against AGS cells and fibroblasts, respectively. Acetylation of gliovictin (compound 6) reduced the cytotoxic effect indicating that the free hydroxy group is required for the toxicity. The cytotoxicity of mellein 3 proved to be low towards both cell lines. At 100 lg/disk, peniprequinolone and gliovictin were active towards Bacillus subtilis with an inhibition area of 16– 17 mm with less effect against B. brevis (9–11 mm). The acetate 6 was inactive against both microorganisms. This is the first report on the occurrence of peniprequinolone and gliovictin in Penicillium janczewskii. The compounds produced by our microorganism clearly differ from those obtained by other authors (Dewick 1997; Kozlovskii et al. 1997; Kwon et al. 2000). Mellein has previously been isolated from other endophytic and phytopathogenic fungi. The biological activities and chemical diversity of the compounds synthetized by the two endophytes from Prumnopitys andina point to the potential of native endophytes as a source of active natural products. Further research work on other endophytes from Chilean gymnosperms is in progress.

Acknowledgements E. Hormaza´bal thanks the Universidad de Talca for a Doctoral grant. We thank the Centro de Investigacio´n en Biotecnologı´ a Silvoagrı´ cola de la Universidad de Talca and the Programa ‘Investigacio´n y Desarrollo de Productos Bioactivos’ for financial support.

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Metabolites from endophytic fungi

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