Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites

Journal of Biotechnology 114 (2004) 279–287

Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites J.Y. Liua,b , Y.C. Songa , Z. Zhanga , L. Wanga , Z.J. Guob , W.X. Zoua , R.X. Tana,∗ a

State Key Laboratory of Pharmaceutical Biotechnology, Institute of Functional Biomolecules, Nanjing University, Nanjing 210093, People’s Republic of China b State Key Laboratory of Coordination Chemistry, Coordination Chemistry Institute, Nanjing University, Nanjing 210093, People’s Republic of China Received 11 December 2003; received in revised form 29 July 2004; accepted 30 July 2004

Abstract Aspergillus fumigatus CY018 was recognized as an endophytic fungus for the first time in the leaf of Cynodon dactylon. By bioassay-guided fractionation, the EtOAc extract of a solid-matrix steady culture of this fungus afforded two new metabolites, named asperfumoid (1) and asperfumin (2), together with six known bioactive compounds including monomethylsulochrin, fumigaclavine C, fumitremorgin C, physcion, helvolic acid and 5␣,8␣-epidioxy-ergosta-6,22-diene-3␤-ol as well as other four known compounds ergosta-4,22-diene-3␤-ol, ergosterol, cyclo(Ala-Leu) and cyclo(Ala-Ile). Through detailed spectroscopic analyses including HRESI-MS, homo- and hetero-nuclear correlation NMR experiments (HMQC, COSY, NOESY and HMBC), the structures of asperfumoid and asperfumin were established to be spiro-(3-hydroxyl-2,6-dimethoxyl-2,5-diene4-cyclohexone-(1,3 )-5 -methoxyl-7 -methyl-(1 H, 2 H, 4 H)-quinoline-2 ,4 -dione) and 5-hydroxyl-2-(6-hydroxyl-2-methoxyl4-methylbenzoyl)-3,6-dimethoxyl-benzoic methyl ester, respectively. All of the 12 isolates were subjected to in vitro bioactive assays against three human pathogenic fungi Candida albicans, Tricophyton rubrum and Aspergillus niger. As a result, asperfumoid, fumigaclavine C, fumitremorgin C, physcion and helvolic acid were shown to inhibit C. albicans with MICs of 75.0, 31.5, 62.5, 125.0 and 31.5 ␮g/mL, respectively. © 2004 Elsevier B.V. All rights reserved. Keywords: Aspergillus fumigatus; Cynodon dactylon; Endophyte; Antifungal; Asperfumin; Asperfumoid

1. Introduction ∗

Corresponding author. Tel.: +86 25 83592945; fax: +86 25 83302728. E-mail address: [email protected] (R.X. Tan). 0168-1656/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2004.07.008

Since a lot of microbe-produced chemicals such as antibiotics have been discovered and eventually

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utilized for the healthcare purpose of humankind, an intensifying stream of attention is being directed to the endophytes, a big reservoir of specially inhabiting microorganisms, some of which have been ascertained to possess excellent productivities (Tan and Zou, 2001; Pullen et al., 2002). In our ongoing project aiming at the characterization of structurally novel and/or substantially bioactive metabolites from specially harboring microbes (Liu et al., 2003a, 2003b, 2002; Yang et al., 2002) and herbal medicines (Zhou et al., 2003; Kong et al., 2000), we found that the EtOAc extract of the culture of the endophyte under the isolation number CY018 from the healthy leaves of Cynodon dactylon (Poaceae) was able to inhibit significantly the growth of Candida albicans. Subsequent microbial attention to the strain led to the identification of the fungus CY018 as Aspergillus fumigatus through scrutiny of its morphology and 18S rDNA sequence. A follow-up fractionation was therefore performed to afford two new (asperfumoid (1) and asperfumin (2)) along with 10 known metabolites including anti-eosinophil benzophenone monomethylsulochrin (3) (Ohashi et al., 1999), mycotoxin fumigaclavine C (4) (Cole et al., 1977), tremogenic cyclotryprostatin fumitremorgin C (5) (Cole and Cox, 1981a), cytototoxic anthraquinone physcion (6) (Kuo et al., 1997), antibacterial sterol analog helvolic acid (7) (Cole and Cox, 1981b) and antiviral ergosterol peroxide (Lindequist et al., 1989) as well as ergosterol, ergosta-4,22-diene-3␤-ol, cyclo(Ala-Leu) and cyclo(Ala-Ile). We hereby wish to report the isolation, identification and cultivation of the endophytic fungus CY018 in addition to the characterization of new and/or antifungal products it produced.

The collected plants of C. dactylon were identified by Associate Professor L. X. Zhang with a voucher specimen preserved under the number YC 01-11-34 in the herbarium of Nanjing University. The plant materials were subjected to endophyte isolation within 3 h after harvest. 2.2. Isolation of strains The endophytic fungal strain CY018 was separated from the healthy leaves of C. dactylon according to the procedure described elsewhere (Liu et al., 2001). Specifically, the leaves of C. dactylon were washed with running tap water, sterilized with 75% ethanol for 1 min and 2.5% sodium hypochlorite for 15 min, then rinsed in sterile water for three times and cut into 1 cm long segments. Both borders of sterilized segments were cut off, and the rest was incubated at 28 ± 1 ◦ C on PDA medium supplemented with 200 ␮g/mL ampicillin and 200 ␮g/mL streptomycin to inhibit the bacterial growth until the mycelium or colony originating from the newly formed surface of the segments appeared. The mycelium was purified in the same condition. Another segment of the same origin without surface sterilization was cultured as a negative control to check the presence of contaminated microbes on the segment surface. Furthermore, the endophytic nature of the isolated strains was robusted with the vitality test as described earlier (Lu et al., 2000). The purified endophytic fungi were numbered and transferred to fresh PCA slants separately and were kept at 4 ◦ C after being cultured at 28 ± 1 ◦ C for 7 days. 2.3. Identification of endophytes

2. Materials and methods 2.1. Plant ascertained as a source of the endophyte The plant material of C. dactylon was collected in early November 2001 from Yancheng Biosphere Reserve, Jiangsu Province. The aerial parts of C. dactylon are locally used as a folk remedy to stanch and treat hepatitis (Xie et al., 1996a), although it was thought to be a problem weed in at least 28 countries and a principal weed in another 29 countries (Holm et al., 1979).

The endophytic fungal strain was identified by the morphological method and reinforced by 18S rDNA sequence comparison. The morphological examination was performed by scrutinizing the fungal culture, the mechanism of spore production, and the characteristics of the spores. For inducing sporulation, each of the isolated fungal strains was separately inoculated on PDA, CMA, CA, WSA and PCA in Petri dishes. All experiments and observations were repeated at least twice. Moreover, the identification of the endophytic fungal strain CY018 was confirmed by 18S rDNA sequence comparisons (White et al., 1990; Larena et al., 1999).

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2.4. Cultivation The inoculum was prepared by introducing the periphery of 7-day-old Petri dish cultures of the title endophyte into 1000 mL flasks containing 400 mL of PDA broth. After 4 days of the incubation at 28 ± 1 ◦ C on rotary shaker at 150 rpm, 15 mL of culture liquid was transferred as the seed into 250 mL flasks each containing 15 mL of water, 15 g millet, 0.5 g yeast extract, 0.1 g tartrate sodium, 0.1 g glutamine sodium, 0.01 g copperas and 0.1 mL of pure corn oil. In the 35-day solid medium cultivation, culture temperature was set at 28 ± 1 ◦ C, and relative humidity in the incubation room at 60–70% with initial water content of the medium at approximately 67%. 2.5. Extraction and isolation The afforded fermentation product (5.3 kg, incompletely dried) was collected and extracted with 15 L of methanol for three times at room temperature. Evaporation of the solvent in vacuo gave a brown oily residue (314 g), which was suspended in water (1000 mL). The suspension was extracted successively with EtOAc (1000 mL × 3) and n-butanol (1000 mL × 3). Concentration of the EtOAc fraction in vacuo gave a brown bioactive residue (153 g), which was subjected to chromatography over silica gel column (1000 g, 200–300 mesh) eluting with a chloroform–methanol gradient (1:0, 6 L; 100:1, 7 L; 50:1 8 L; 20:1, 6 L; 10:1 6 L; 5:1 6 L; 0:1, 5 L) to give five fractions (F-1, 103.5 g; F-2, 10.1 g; F-3, 12.8 g; F-4, 10.5 g; F-5, 15.0 g). F-1 and F-5 were shown to be of no interest by TLC and antifungal test. F-2 was chromatographed further over silica gel (150 g, 200–300 mesh) using CHCl3 –MeOH mixtures of a growing polarity (200:1, 100:1, 50:1, 20:1, 10:1, each 1.2 L) to afford compounds 4 (280 mg), 5 (60 mg), ergosterol peroxide (11 mg), ergosterol (8 mg), ergosta-4,22-diene-3␤-ol (9 mg) and a mixture F-2-1 (5 g). The mixture was further separated on silica gel column (150 g, 200–300 mesh) eluting with CHCl3 –MeOH mixture (10:1, 1.5 L) to yield compounds 1 (5.0 mg), 2 (2.5 mg) and 3 (220 mg) as crystals. Repeated chromatography of F-3 on silica gel column (180 g, 200–300 mesh; CHCl3 /MeOH 100:1→10:1, each 1.2 L) followed by gel filtration over Sephadex LH-20 (CHCl3 /MeOH (1:1), 100 mL) gave compounds 6 (5.0 mg) and 7 (26 mg). F-4 was

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re-chromatographed over silica gel column (130 g, 200–300 mesh) with CHCl3 –MeOH gradient (10:1, 5:1, 5:2, each 600 mL) to give F-4-1 (1.5 g), F-4-2 (5.0 g) and F-4-3 (3.1 g). F-4-1 and F-4-3 (3.1 g) contained inseparable pigments while F-4-2 was fractionated by ODS (30 g) column with H2 O–MeOH mixtures (1:0→0:1, each 30 mL) to afford pigment mixture and a fraction which gave, upon HPLC with H2 O–MeOH (3:2), cyclo(Ala-Leu) (8 mg) and cyclo(Ala-Ile) (7 mg). 2.6. Examination of the sterile medium for the presence of 1–12 The sterile medium was extracted following exactly the procedure as that of the solid-medium culture. All isolates were confirmed to be the endophytic fungal metabolites by TLC and LC–MS examinations. 2.7. Analytical methods Melting points were determined on a Boetius micromelting point apparatus, and were uncorrected. IR spectra were recorded in KBr disks on Nexus 870 FT-spectrometer, v in cm−1 . All NMR data were collected on a Bruker DRX500 spectrometer with 1 H and 13 C nuclei observed at 500 and 125 MHz, respectively, and the chemical shifts were expressed in δ (ppm) relative to SiMe4 (the internal standard) with coupling constants J in Hz. HRESI-MS was recorded on a Mariner System 5304 instrument. Silica gel (200–300 mesh) for column chromatography was produced by Qingdao Marine Chemical Factory, Qingdao, China and ODS silica gel was from Nacalai Tesque, Kyoto, Japan. Sephadex LH-20 was from Pharmacia Biotech, Sweden. The HPLC column used in this study was Waters L-column ODS 4.6 mm × 250 mm. All other chemicals used in this study were of analytical grade. 2.8. Identification of new metabolites Asperfumoid (1): yellowish needle crystal (CHCl3 /MeOH, 2:1), mp. 284–286 ◦ C (dec.); ◦ (c 0. 45 mg/mL, CHCl3 ); IR: [α]20 D + 55.6 kBr −1 vmax (cm ) 3423.1, 3358.2, 1715.5, 1681.4, 1625.9, 1591.0, 1494.8, 1238.7, 1216.3, 976.4 and 814.3 cm−1 ; ESI-MS: m/z 360 [M + H]+ , 377 [M + NH4 ]+ and 382 [H + Na]+ ; HRESI-MS: m/z 360.1073 [M + H]+ ,

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Table 1 (500 MHz) and 13 C NMR (125 MHz) data of 1 (DMSO-d6 )

1H

Position

δC (DEPT)

1(3 )

78.0 (C) 168.4 (C) 148.7 (C) 181.8 (C) 102.7 (CH) 172.5 (C)

2 3 4 5 6 1 2 4 5 6 7 8 9 4a 8a 2-OMe 6-OMe 5 -OMe a

δH a (J in Hz)

H-5 2-OMe H-5 H-5 5.82 (s) 6-OMe 7.88 (s)

174.5 (C) 195.3 (C) 158.4 (C) 105.1 (CH) 152.5 (C) 106.0 (CH) 23.6 (CH3 ) 109.7 (C) 148.7 (C) 51.7 (CH3 ) 57.5 (CH3 ) 56.5 (CH3 )

HMBC

6.35 (s) 6.49 (s) 2.44 (s)

H-8 H-6 H-6 , 5 -OMe H-9 H-8 , H-9 H-9 H-8 H-6

3.44 (s) 3.69 (s) 3.96 (s)

Assigned by HMQC.

calcd. for C18 H18 NO7 , 360.1083; 1 H and 13 C NMR spectral data (see Table 1). Asperfumin (2): yellowish needle crystal (CHCl3 /MeOH, 2:1), mp. 252–255◦ (dec.); −1 vkBr max (cm ) 3421.8, 2918.0, 2849.2, 1719.6, 1633.6, 1592.1, 1465.0, 1349.5, 1219.6 and 835.1 cm−1 ; ESI-MS: m/z 377 [M + H]+ , 399 [H + Na]+ ; HRESIMS: m/z 375.1085 [M − H]− , calcd. for C19 H19 O8 : 375.1080; 1 H and 13 C NMR spectral data (see Table 2). 2.9. Antifungal activities tests Antifungal activities of compounds 1–12 were assayed in vitro using the three human pathogenic fungi including C. albicans, T. rubrum and A. niger by the method outlined elsewhere (Barchiesi et al., 2000).

3. Results A total of 37 strains fungi were isolated from the healthy leaves of C. dactylon, suggesting that C. dactylon was a potential source of endophytic fungi. The isolated fungus CY018 was identified according the following morphological characters. Colonies of

CY018 on CA grew slow, attaining 42–45 mm in diameter in 14 days at 24 ◦ C, with a regular and even margin, dark greenish from the center where formed spores, pale green conidiophore comparatively short and curved often, with smooth surface, conidial head column, long or short. Vesicles were like flask and green in color. Phailides were monolayered, tight and inserted on the top of vesicle. Conidia were spherical, green with rough surface and 2.5–3 ␮m in diameter. Appressoria on CA were absent. Newly isolated mycelium grew well on PDA, and easily produced fruiting bodies. Colonies with a regular margin attained 35–45 mm in diameter after incubation on PDA at 28 ◦ C for 4 days and took on weak green in PDA plates. Conidiophores were upright, simple, terminating in a globose swelling, bearing phialides at the apex; conidia (phialospores) 1-celled, globose. These morphological characteristics allowed the identification of the endophytic fungus as A. fumigatus (Barnett and Hunter, 1998; Frisvad and Samson, 1990), which was reinforced by the sequence of its 18S rDNA that gave a 99% sequence similarity to those accessible at the BLASTN of A. fumigatus. It should be emphasized that cultivation time and water content are among the key factors for biosyntheses of the isolated metabolites in the solid-state cultivation. In the present study, cultivation time was allotted depending on the growth state of the fungus indicated by the sporulation and accumulation of the pigments. At early stage of cultivation, the inoculum was small, gray grain with white and short mycelium, which became dark green 3 days later, and turned to be dark green powder after the next 7-day culture. And finally the white fructification appeared after the subsequent 10-day cultivation, followed by maintaining in the state for 10–15 days more. And the moisture content in the starting medium ranging from 60 to 70% was suitable for the production of the mentioned metabolites. On the basis of spectral and physical data, the 10 known metabolites were identified as monomethylsulochrin (3) (Turner, 1965; Ma et al., 2004), fumigaclavine C (4) (Cole et al., 1977), fumitremorgin C (5) (Cole and Cox, 1981a), physcion (6) (Xiang et al., 2001), helvolic acid (7) (Cole and Cox, 1981b), 5␣,8␣epidioxy-ergosta-6,22-diene-3␤-ol (Ma et al., 1994), ergosterol (Cushley and Filipenko, 1976), ergosta4,22-diene-3␤-ol (Seldes et al., 1988), cyclo(Ala-Leu) and cyclo(Ala-Ile) (Xiong et al., 2002), respectively.

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Table 2 (500 MHz) and 13 C NMR (125 MHz) data of 2 and 3 (CDCl3 /MeOD-d3 )

1H

Position

2 δC

1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 1-OMe 4-OMe 7-OMe 3 -OMe 6-OHb 7 -OHb a b

166.4 124.0 128.5 152.8 102.5 151.5 139.0 197.0 110.2 161.0 103.0 148.3 110.2 163.1 22.1 51.9 56.0 61.9 55.5

3 δH a

HMBC

δC

1-MeO H-5

167.2 137.1 128.7 158.4 103.5 157.3 108.2 200.8 110.8 161.5 103.6 148.6 110.8 164.1 22.4 52.2 56.2

H-5, 4-MeO 6.65 (s)

6.14 (br s) 6.42 (br s) 2.31 (s) 3.60 (s) 3.69 (s) 3.83 (s) 3.48 (s) 12.00 (br s) 12.67 (br s)

H-5, 7-MeO H-5 H-6 3 -OMe H-6 , H-8 H-8’ H-4 , H-8 H-4 , H-6

55.8

δH a

6.66 (d,2.0) 7.02 (d,2.0)

6.13 (br s) 6.42 (br s) 2.30 (s) 3.69 (s) 3.70 (s) 3.40 (s) 13.03 (s)

Assigned by HMQC. Acquired in CDCl3 .

The molecular weight of compound 1 was deduced to be 359 by the quasimolecuar ions at m/z 360 [M + H]+ , 377 [M + NH4 ]+ and 382 [H + Na]+ in its positive mode ESI mass spectrum. Its molecular formula was established to be C18 H17 NO7 according to the precise protonated molecular weight at m/z 360.1073 [M + H]+ (calcd. for C18 H18 NO7 : 360.1083) in its high-resolution ESI-MS. This was well consistent with a total of 18 carbon resonance lines in the 13 C NMR spectrum of 1. Furthermore, the intense IR absorption bands of 1 at 3423.1, 3358.2, 1715.5, 1681.4, 1625.9, 1591.0 and 1494.8 cm−1 implied the presence of hydroxyl(s), amide(s), carbonyl(s), double bond(s) and phenyl(s). A subsequent scrutiny of its 1 H and 13 C NMR data (edited with DEPT pulse sequences and the HMQC experiment) revealed the presence of one phenyl-linked methyl (Me-7 ), three methoxyls (on C-2, C-6 and C-5 ), three carbonyls (C-4, C-2 and C-4 ), one sp3 -hybridized quaternary carbon (C-1), and a total of 10 sp2 -hybridized carbons including three methines (C-5, C-6 and C-8 ) and seven quaternary carbons (C-2, C-3, C-6, C-5 , C-7 ,

C-4a and C-8a ) (Table 1). These structural fragments altogether accounted for nine degrees of unsaturation. Accordingly, two more rings had to be proposed for 1 in addition to the ascertained phenyl nucleus. In the HMBC (Table 1) and NOSEY (Fig. 1) spectra of 1, HMBC correlations of methoxyl hydrogens to C-2, C-5 and C-6 confirmed the proposed 2,5 ,6trimethoxy groups. And the presence of methyl at C-7 , established by HMBC correlations of H-9 to C-6 and C-8 , was reinforced by the NOESY correlations of H-9 with H-6 and H-8 . Furthermore, the discernible HMBC correlation between H-6 and C-4 , along with the magnitude (195.3 ppm) of the

Fig. 1. The NOESY correlations of 1.

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chemical shift of C-4 , suggested the connection of C-4 to C-4a . The NOESY correlation between H-8 and H-1 , together with the HMBC correlation of H-8 to C-2 , confirmed the presence of imide. Moreover, the HMBC correlation of H-5 to C-1, C-3, C-4 and C-6, combined with the tricyclic nature of 1, demonstrated that C-2, C-6, C-2 and C-4 had to anchor on C-1 to form a spiral structure. In conclusion, the structure of compound 1 was established as spiro-(3-hydroxyl-2,6-dimethoxyl-2,5-diene-4-cyclohexone-(1,3 )-5 -methoxyl-7 -methyl-(1 H, 2 H, 4 H)quinoline-2 ,4 -dione). It is noteworthy that none of the reported natural products bears the carbon skeleton of compound 1. We have named metabolite 1 asperfumoid after the scientific name of its producing organism. Compound 2 was afforded as yellowish needle crystals. Its [M-H]- peak at m/z 375.1085 in its high-resolution ESI-MS, indicated that its molecular formula was C19 H20 O8 (calc. for C19 H19 O8 : 375.1080). The intense IR absorption bands at 3421.8, 1719.6, 1633.6, 1592.1 and 1465.0 cm−1 suggested the presence of hydroxyl(s), carbonyl(s) and phenyl(s). In its 1 H NMR spectrum, a broaden singlet at δ 12.67, disappeared upon addition of CD3 OD, required the presence of a hydroxyl group stably hydrogen-bonded to a carbonyl. Furthermore, three sets of signals including a phenyl-carried methyl singlet at δ 2.31, three one-hydrogen aromatic singlets at δ 6.65, 6.42 and 6.14, and four methoxy singlets at δ 3.83, 3.69, 3.60 and 3.48 were closely similar to those of 3 (Ma et al., 2004). However, the doublet (J = 2.0 Hz) of H-7 at δ 7.02 in the 1 H NMR spectrum of 3 was replaced by a three-proton singlet at δ 3.83 in that of 2, implying that 2 was a methoxylated derivative of 3. This assumption was consistent with an additional carbon resonance line at δ 61.9 in the 13 C NMR spectrum of 2, and the fact that the molecular weight of 2 was 30 amu higher than that of 3 (Ma et al., 2004). This deduction was subsequently reinforced by the 2D NMR experiments including NOESY, HMBC and HMQC, allowing the exact assignment of all 1 H and 13 C NMR signals (Table 2). Moreover, the substitution pattern of compound 2 was confirmed by its NOESY spectrum where the anticipated NOE correlations were discerned between the four proton pairs H-5/4-OMe, H-4 /3 -OMe, H-4 /H-8 and H-6 /H-8 (Fig. 2). Thus, the structure of compound 2 was determined to be 5-hydroxyl-2-(6-

Fig. 2. The NOESY correlations of 2.

hydroxyl-2-methoxyl-4-methylbenzoyl)-3,6-dimethoxyl-benzoic methyl ester, we named asperfumin. The previous improperly assigned 1 H and 13 C NMR signals of fumigaclavine C were corrected as well through a combination of 1D and 2D NMR techniques (1 H and 13 C NMR, DEPT, HMQC and HMBC) (Table 3). Furthermore, the optical rotation ([α]22 D ) was determined to be −88.8◦ (c. 0.006 g/mL in CHCl3 ), ◦ close to that ([α]22 D : −90 ) in (Cole and Cox, 1981a) confirming its optical identity. All 12 metabolites were tested in vitro for the antifungal activity against the human pathogenic fungi T. rubrum, C. albicans and A. niger, and the results are listed in Table 4. Compounds 1 and 4–7 inhibited the growth of C. albicans with MICs of 75.0, 31.5, 62.5, 125.0 and 31.5 ␮g/mL, respectively. The MICs of ketonazole used as a positive reference in the study against T. rubrum, C. albicans and A. niger were 250.0, 31.5 and 31.5 ␮g/mL, respectively.

4. Discussion A number of A. fumigatus strains have been isolated with considerable genetic variability (J´anos and Genetic, 2003) from saltern (Tepˇsiˇc et al., 1997), molded silage (Cole et al., 1977) and aspergillosissuffering patients with its pathobiology reviewed elsewhere (Latg´e, 2001). In our efforts of screening for the suitable microorganism(s) that can produce new and/or bioactive chemicals, we found that A. fumigatus residing in C. dactylon is a versatile producer of new and bioactive metabolites. To our knowledge, the present paper described the isolation of A. fumigatus as an endophytic fungus for the first time. The possibility that it could be a contaminating microbe was excluded by the vitality test (Lu et al., 2000). As reported (Pullen et al., 2002; Tan and Zou, 2001), an endophyte in one plant could be a pathogen of the other depending on the

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Table 3 (500 MHz) and 13 C NMR (125 MHz) spectral data (CDCl3 ) of 4

1H

δC (DEPT)

Position 1 2 3 4

136.6 (C) 105.9 (C) 27.9 (CH2 )

5 7

61.5 (CH) 57.5 (CH2 )

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

32.9 (CH) 71.3 (CH) 39.2 (CH) 129.0 (C) 112.6 (CH) 107.7 (CH) 122.0 (CH) 132.1 (C) 127.9 (C) 43.4 (CH3 ) 16.5 (CH3 ) 170.9 (C) 21.1 (CH3 ) 38.9 (C) 27.3 (CH3 ) 27.1 (CH3 ) 145.6 (CH) 111.7 (CH2 )

δH a (J in Hz)

HMBC

7.97 (s)

a

H-1, H-22, H-23, H-4 H-1, H-4 ␣ 2.71 (t, 11.0) ␤ 3.57 (br d, 11.0) 2.68 (dd, 11.0, 8.5) ␣ 2.67 (dd, 12.0, 9.0) ␤ 2.76 (dd, 12.0, 3.5) 2.15 (dd, 3.5, 2.0) 5.72 (br s) 3.36 (br d, 8.5)

H-4, H-10, H-17 H-17, H-18 H-18 H-5, H-18 H-4, H-12 H-10, H-13 H-14 H-12 H-12 H-1, H-13 H-1, H-4, H-12, H-14 H-5

6.77 (d, 6.5) 7.09 (dd, 7.5, 6.5) 7.12 (d, 7.5)

2.49 (s) 1.36 (d, 7.0)

H-20 1.91 (s) H-22, H-23, H-24, H-25c, H-25t H-23 H-22 H-22, H-23, H-25c, H-25t

1.57 (s) 1.57 (s) 6.14 (dd, 17.0 11.0) c 5.17 (d, 11.0) t 5.17 (d, 17.0)

Assigned by HMQC.

Table 4 The in vitro antifugal MICs (␮g/mL) of compounds 1–7

Tr Ca An a

Ka

1

2

3

4

5

6

7

250.0 31.5 31.5

>300 75.0 >150

>300 >150 >150

>300 >150 >150

>300 31.5 >150

>300 62.5 >150

>300 125.0 >150

>300 31.5 >150

K: ketonazole used as positive control; Tr: T. rubrum; Ca: C. albicans; An: A. niger.

balance between pathogenicity and endophytism of the microorganism in the different hosts. In order to ascertain whether any of the 12 isolates obtained in this study was the constituent of the millet and yeast extract in the substrate, the EtOAc extract of the sterile medium treated equally but without inoculation of the microorganism was subjected to an LC–MS comparison showing that all the isolates were indeed produced by the title endophytic fungus. Grass endophytes are known to produce alkaloids that may play an important role in the host–herbivore and host–microbe chemical interactions affecting presumably the hosts’ adaptability to stresses such as

drought, high salinity, heavy metal intoxication, and microbial infection (Clay, 1990; Tan and Zou, 2001). In this study, two indole-derived mycotoxins including fumigaclavine C (4) and fumitremorgin C (5) were characterized from the culture of an endophytic fungus although they were isolated previously from A. fumigatus strains in molded silage (Cole et al., 1977) and saltern (Tepˇsiˇc et al., 1997). However, fumigaclavine C and fumitremorgin C were newly demonstrated to be potential antifungal agents in the present study. Furthermore, the antifungal action of compounds 6 and 7 were confirmed against the human pathogen C. albicans.

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The structure of the new benzophenone 2, close to that of 3, was assigned by a successful interpretation of its spectra (ESI-MS, IR, 1 H and 13 C NMR, HMQC, HMBC and NOESY). Previously, benzophenone derivatives, sharing the carbon framework with metabolites 2 and 3, have been reported from the fungi belong to the genera Aspergillus (Turner, 1965; Kiriyama et al., 1977; Inamori et al., 1983), Penicillium (Mahmoodian and Stickings, 1964), Rhizoctonia (Ma et al., 2004) and Oospora (Curtis et al., 1966). Some of this type of compounds have been shown to be anti-Helicobacter pylori (Ma et al., 2004), and to inhibit eosinophils, which may play important roles in allergic diseases such as asthma and atopic dermatitis (Ohashi et al., 1999). During the formation of the metabolites 1–5, this endophytic fungus can biosynthesize a cytototoxic anthraquinone phytochemical physcion (6), a plant constituent previously isolated as an antifungal agent from Rheum emodi (Polygonaceae) (Agarwal et al., 2000) and as a tumor cell growth inhibitor from Polygonum hypoleucum (Polygonaceae) (Kuo et al., 1997). This biologically important plant anthraquinone was also detected in the some Leguminosae representatives such as Senna multiglandulosa, S. sophera, S. didymobotrya (Alemayehu and Abegaz, 1996) and Cassia obtusifolia (Xie et al., 1996b). Interestingly we have hereby disclosed a microbial source of the important phytochemical physcion (5). As formulated elsewhere (Chen et al., 1995), the co-characterization of anthraquinone and benzophenones from the endophytic culture confirmed that both types of metabolites are included in the same biosynthetic pathway with the former being the precursor of the latter. Thus, a smart manipulation of the pathway would lead to a scaled-up fermentation production of some important anthraquinones. This seems more workable than the desired microbial production of taxol, which is still out of sight although, its producing endophyte has been reported a decade ago (Stierle et al., 1993). In conclusion, the endophytic fungus A. fumigatus CY018 inside the healthy leaves of C. dactylon was a versatile producer of the new (asperfumoid and asperfumin) and variously bioactive (asperfumoid, fumigaclavine C, monomethylsulochrin, fumitremorgin C, helvolic acid and ergosterol peroxide) metabolites while providing a microbial source of physcion, a biologically important phytochemical. Moreover, the

production of benzophenone metabolites by some C. dactylon endophytes such as A. fumigatus CY018 and Rhizoctonia sp. Cy064 (Ma et al., 2004) offers an alternative source of benzophenone derivatives, which could be valuable candidates for the discovery of new drugs for anti-Helicobacter pylori (Ma et al., 2004), anti-inflammatory (Khanum et al., 2004), anti-allergic (Ohashi et al., 1999) and antineoplastic purposes (Pettit et al., 2000).

Acknowledgment The work was co-financed by grants from National Natural Science Foundation of China (Nos. 30171104 and 30270034) and from the Ministry of Science & Technology—National Marine 863 projects (Nos. 2003AA624010 and 2003AA620411).

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