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Antifungal metabolites from Chaetomium globosum, an endophytic fungus in Ginkgo biloba
Biochemical Systematics and Ecology 39 (2011) 876–879
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Antifungal metabolites from Chaetomium globosum, an endophytic fungus in Ginkgo biloba Hu-Qiang Li a, Xiao-Jun Li a, You-Lin Wang a, Qiang Zhang a, An-Ling Zhang a, Jin-Ming Gao a, *, Xing-Chang Zhang b, ** a
Research Centre for Natural Medicinal Chemistry, College of Science, Northwest A&F University, Yangling, Shaanxi 712100, PR China State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Science (CAS), Yangling, Shaanxi 712100, PR China b
a r t i c l e i n f o
.
Article history: Received 11 April 2011 Accepted 25 June 2011 Available online 28 July 2011 Keywords: Ginkgo biloba Chaetomium globosum Endophytic fungus Antifungal activity Epipolythiodioxopiperazine Gliotoxin
1. Subject and source Endophytes are microorganisms that have been recognized as important sources of a variety of structurally novel active secondary metabolites with anticancer, antimicrobial and other biological activities (Strobel, 2003; Krohn et al., 2007). Chaetomium is a large genus of the fungal family Chaetomiaceae (Ascomycota) with over an hundred marine- and terrestrial-derived species (Udagawa et al., 1997). To date, more than 200 compounds have been reported from this genus. As a part of study into the chemical diversity and biological activity of compounds associated with plant-endophytic fungal interactions (Qin et al., 2009a,b; Yang et al., 2011), a fungal strain Chaetomium globosum (NM0066) was separated from the healthy leaves of the medicinal plant Ginkgo biloba, growing in Linyi, Shandong province, China. It was characterized, based on morphological studies, and has been deposited at Research Centre for Natural Medicinal Chemistry, College of Science, Northwest A&F University. We undertook chemical and biological investigations of this fungus because of the significant antifungal activity of the crude EtOAc extract against some phytopathogenic fungi.
2. Previous work In the past decades, various strains of C. globosum have been reported as endophytes of some plants. Cultivation of endophytic C. globosum produced a series of compounds such as chaetoglobosins G (Qin et al., 2009a), chaetoglobosins Q, R * Corresponding author. Tel.: þ86 29 87092335. ** Corresponding author. E-mail addresses: [email protected] (J.-M. Gao), [email protected] (X.-C. Zhang). 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.06.019
H.-Q. Li et al. / Biochemical Systematics and Ecology 39 (2011) 876–879
877
and T (Jiao et al., 2004), chaetoglobosins U, V and W (Ding et al., 2006; Zhang et al., 2010), cytoglobosins A–G (Cui et al., 2010); azaphilones, chaetoviridins A and C (Qin et al., 2009a); pyrones, chaetoglocins A and B (Ge et al., 2011); orsellides, globosumones A–C (Bashyal et al., 2005); steroids (Qin et al., 2009b). Some of the compounds have been reported to possess significant biological activities, such as cytotoxic, enzyme inhibitory and antibiotic (Gunatilaka, 2006; Scherlach et al., 2010). However, little is known about the anti-phytopathogenic activity of secondary metabolites from C. globosum isolated from leaves of G. biloba. 3. Present study 3.1. Fermentation The fungus C. globosum was grown on PDA plates at 28 C for 6 days, and the fresh mycelium was inoculated into 500 mL flasks containing 200 mL PDA medium composed of glucose 20 g, MgSO4$7H2O 1.5 g, K2HPO4 3 g, yeast extract 1 g and 1 L water, followed by shaking in an incubator at 130 r/min at 28 C for 6 days.
3.2. Extraction and isolation The culture broth (110 L) was filtered to give the mycelium and culture filtrate. The culture filtrate obtained was subsequently extracted with EtOAc at room temperature, then evaporation of solvent under reduced pressure gave a residue (22 g). The mycelium was dried naturally, and ultrasonically extracted four times by EtOAc, then evaporation of solvent under reduced pressure to provide another crude extract (28 g). The EtOAc extract (28 g) of the mycelium was fractionated on a silica gel column using petroleum ether (PE)-EtOAc (100:0, 98:2, 95:5, 9:1, 8:2, 7:3) to give six fractions (Fr.1–Fr.6). Fr.1 (93 mg) was subjected to repeated column chromatography (CC) (Sephadex LH-20, CHCl3:MeOH 1:1, then MeOH; silica gel, n-hexane) to yield 1 (15 mg). Fr. 2 (1.2 g) was separated by CC on silica gel with PE:EtOAc 8:2, followed by Sephadex LH-20 (MeOH), and then crystallization from EtOAc to afford 2 (19 mg). Fr. 4 (940 mg) was fractionated by a silica gel column eluted with PE-acetone (8:2, 85:15, 7:3) to give two fractions (Fr. 4–1 and Fr. 4–2). Fr.4–1 was purified by repeated CC (silica gel, CHCl3:acetone 85:15; silica gel, PE-acetone 7:3; Sephadex LH-20, MeOH) to obtain 3 (20 mg); Fr. 4–2 was purified by repeated CC (Sephadex LH-20, CHCl3:MeOH 1:1, then MeOH; silica gel, PE-acetone 7:3) to provide 4 (10 mg) and 7 (8 mg). Fr.6 (460 mg) was separated by CC (silica gel, PEacetone 7:3; Sephadex LH-20, CHCl3:MeOH 1:1; silica gel, CHCl3:acetone 95:5; Sephadex LH-20, MeOH) to produce 5 (10 mg). The EtOAc extract (22 g) of the filtrate was fractionated on a silica gel column eluted with CHCl3-MeOH (100:0, 98:2, 95:5, 9:1, 8:2, 7:3, 6:4) to give seven fractions (Fr.7–Fr.13). Fr.7 (525 mg) was subjected to repeated CC (silica gel, PE:acetone 7:3; Sephadex LH-20, MeOH, then CHCl3:MeOH 1:1) to yield 6 (8 mg). Fr.8 (800 mg) was separated by repeated CC (silica gel, CHCl3:MeOH 100:0–90:10; Sephadex LH-20, CHCl3:MeOH 1:1) and then crystallization from EtOAc to give 8 (10 mg). Fr. 10 (1.2 g) was purified by repeated CC (silica gel, petroleum ether:EtOAc 7:3; Sephadex LH-20, MeOH; silica gel, CHCl3:MeOH 97:3) to afford 9 (10 mg) and 10 (15 mg). Fr. 12 (1.6 g) was fractionated on CC (silica gel, CHCl3:MeOH:H2O 90:10:1; Sephadex LH-20, MeOH, then CHCl3:MeOH 1:1) to give 11 (17 mg). Fr. 13 (960 mg) was separated by CC (silica gel, CHCl3:MeOH:H2O 90:10:1; Sephadex LH-20, MeOH, then CHCl3:MeOH 1:1) to afford 12 (13 mg). The structures of compounds 1–12 were identified, respectively, as squalene (1) (Masuda et al., 1989), ergosterol peroxide (2) (Zhang et al., 2009b), methylthiogliotoxin (3) (Lee et al., 2001), chaetoglobosin G (4) (Sekita et al., 1982a,b), (22E,24R)ergosta-7,22-diene-3b,5a,6b-triol (5) (Gao et al., 2001), fumitremorgin C (6) (Cui et al., 1996), chaetoglobosin C (7) (Sekita et al., 1983), gliotoxin (8) (Lee et al., 2001), 2,3,4-trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran (9) (Chen et al., 2002), pseurotin A (10) (Aoki et al., 2004), 4-aminophenylacetic acid (11) (Ahmed et al., 2006) and 3,4-dihydroxyphenylacetic acid (12) (Katherine, 1972) by a combination of spectroscopic methods (MS, 1D- and 2D- NMR) and comparison with literature data (Fig. 1). The in vitro antifungal activity of the isolated compounds 1–12 was evaluated by mycelial growth inhibitory rate method (Zhang et al., 2009a) using Fusarium oxysporum f. sp. vasinfectum, Fusarium graminearum, Fusarium sulphureum, Cercospora sorghi, Botrytis cinerea and Alternaria alternata as the pathogenic fungi. Hymexazol was used as a positive drug. 4. Ecological significance In the current study, the cultures of an endophyte C. globosum found in G. biloba produced twelve metabolites 1–12. To our knowledge, this is the first time that eight compounds (1, 3, 6 and 8–12) have been reported from the genus Chaetomium. Among them, fumitremorgin C (6) is an indole mycotoxin, which was also isolated from an endophytic Aspergillus fumigatus CY018 (Liu et al., 2004). Gliotoxin (8), the first and best-known epipolythiodioxopiperazine toxin, was isolated from culture broths of Gliocladium fimbriatum (Weindling and Emerson, 1936) and later also from other microorganisms such as Trichoderma, Aspergillus and Penicillium (Weindling, 1941; Gardiner et al., 2005; Lewis et al., 2005). It shows strong antibacterial and antiviral activity (Johnson et al., 1943; Rightsel et al., 1964), is immunosuppressive (Yamada et al., 2000) and causes apoptotic and necrotic cell death (Waring and Beaver, 1996).
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H.-Q. Li et al. / Biochemical Systematics and Ecology 39 (2011) 876–879
HO
O
HO
O
N H O HOH2C
2
1
SMe O N SMe
3
OH H N O HO O
N H
H HO
O
OH OH
4
5
O H
O
H
N H3CO
N H
N H
O
N O HO
H
O
N H
6
H HO O
O O OH 9
O
OH O 10
H N
O
OH
8
NH2
HO
S S N
O
7
OH
N
OH
HO
O
OH OCH3
O
O OH
OH
11
12
Fig. 1. Compounds (1–12) isolated from Chaetomium globosum.
Microorganisms, particular fungi, when associated with plants have been recognized as a rich source of compounds with phytotoxic and plant growth regulating-properties (Hussain et al., 2007). In order to exploit its ecological niche, endophytes need to compete with other endophytes and pathogens; secondary metabolites that have antibiotic properties can play an important role in this process (Gunatilaka, 2006). Accordingly, most secondary metabolites from endophytes indicated antibiotic activities against microorganisms (You et al., 2009). An in vitro growth inhibition bioassay, that measured the rate of mycelia growth of a range of phytopathogenic fungi, was used to evaluate the antifungal activity of the 12 isolated metabolites. Compounds 1–7 and 9–12 were found to be inactive against the fungi at 100 mg/ml, whereas gliotoxin (8) showed good antifungal activity against F. sulphureum, A. alternata and C. sorghi with EC50 values of 68.5, 36.8 and 59.8 mg/mL, respectively. The potency is comparable with the positive control hymexazol, with EC50 values of 100.3, 32.8 and 64.3 mg/mL, respectively. In contrast, gliotoxin (8) presented a moderate inhibitory activity toward F. oxysporum f. sp. vasinfectum, B. cinerea and F. graminearum with EC50 values of 93.7, 106.6 and 100 mg/mL, respectively. This efficacy is weaker than the positive control with EC50 values of 47.2, 83.3 and 70.4 mg/mL, respectively. Interestingly, methylthiogliotoxin (3) did not show any activity, which may be attributable to the absence of a disulphide bridge in the molecule, as compared with gliotoxin (8). This suggests that the disulphide bridge is essential for antifungal activity. The pronounced antifungal activity against the phytopathogenic fungi of gliotoxin (8) suggests that the endophytic fungus C. globosum present in leaves of the plant G. biloba could protect the host by producing metabolites, which may be toxic or even lethal to phytopathogens.
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Acknowledgments This work was supported by the National Natural Science Foundation of China (No. 30770237, 30971882) and by the Program for New Century Excellent Talents in University (NCET-05-0852), and the Programs for changjiang scholars and innovative Research Team in University (IRT0749). References Aoki, S.Y., Oi, T., Shimizu, K., Shiraki, R., Takao, K.I., Tadano, K.I., 2004. Bull. Chem. Soc. Jpn. 77, 1703. Ahmed, H.B., Fawzy, M.A., Ahmed, M.E.A., Fathy, A.E., Mohammed, A.A.E.N., Gamal, E.S., 2006. Acta Pharm. 56, 273. Bashyal, B.P., Wijeratne, E.M., Faeth, S.H., Gunatilaka, A.A., 2005. J. Nat. Prod. 68, 724. Cui, C.M., Li, X.M., Li, C.S., Proksch, P., Wang, B.G., 2010. J. Nat. Prod. 73, 729. Chen, C.H., Shaw, C.Y., Chen, C.C., Tsai, Y.C., 2002. J. Nat. Prod. 65, 740. Cui, C.B., Kakeya, H., Osada, H., 1996. J. Antibiot. 49, 534. Ding, G., Song, Y.C., Chen, J.R., Xu, C., Ge, H.M., Wang, X.T., Tan, R.X., 2006. J. Nat. Prod. 69, 302. Gardiner, D.M., Waring, P., Howle, B.J., 2005. Microbiology 151, 1021. Gao, J., Hu, L., Liu, J., 2001. Steroids 66, 771. Ge, H.M., Zhang, Q., Xu, S.H., Guo, Z.K., Song, Y.C., Huang, W.Y., Tan, R.X., 2011. Planta Med. 77, 277. Gunatilaka, A.A.L., 2006. J. Nat. Prod. 69, 509. Hussain, H., Krohn, K., Ullah, Z., Draeger, S., Schulz, B., 2007. Biochem. Syst. Ecol. 35, 898. Jiao, W., Feng, Y., Blunt, J.W., Cole, A.L., Munro, M.H., 2004. J. Nat. Prod. 67, 1722. Johnson, J.R., Bruce, F.W., Dutcher, J.D., 1943. J. Am. Chem. Soc. 65, 2005. Katherine, N.S., 1972. J. Magn. Reson. 6, 55. Krohn, K., Kock, I., Elsasser, B., Florke, U., Schulz, B., Draeger, S., Pescitelli, G., Antus, S., Kurtan, T., 2007. Eur. J. Org. Chem., 1123. Lee, H.J., Lee, J.H., Hwang, B.Y., Kim, H.S., Lee, J.J., 2001. Arch. Pharm. Res. 24, 397. Lewis, R.E., Wiederhold, N.P., Lionakis, M.S., Prince, R.A., Kontoyiannis, D.P., 2005. J. Clin. Microbiol. 43, 6120. Liu, J.Y., Song, Y.C., Zhang, Z., Wang, L., Guo, Z.J., Zou, W.X., Tan, R.X., 2004. J. Biotechnol. 114, 279. Masuda, K., Shiojima, K., Ageta, H., 1989. Chem. Pharm. Bull. 37, 1140. Qin, J.C., Zhang, Y.M., Gao, J.M., Bai, M.S., Yang, S.X., Laatsch, H., Zhang, A.L., 2009a. Bioorg. Med. Chem. Lett. 19, 1572. Qin, J.C., Gao, J.-M., Zhang, Y.-M., Yang, S.-X., Bai, M.-S., Ma, Y.-T., Laatsch, H., 2009b. Steroids 74, 786. Rightsel, W.A., Schneider, H.G., Sloan, B.L., Graf, P.R., Miller, F.A., Bartz, Q.R., Ehrlich, J., Dixon, G.J., 1964. Nature 204, 1333. Strobel, G.A., 2003. Microbes Infect. 5, 535. Sekita, S., Yoshihira, K., Natori, S., Udagawa, S., Sakabe, F., Kurata, H., Umeda, M., 1982a. Chem. Pharm. Bull. 30, 1609. Sekita, S., Yoshihira, K., Natori, S., Kuwano, H., 1982b. Chem. Pharm. Bull. 30, 1618. Sekita, S., Yoshihira, K., Natori, S., 1983. Chem. Pharm. Bull. 31, 490. Scherlach, K., Boettger, D., Remme, N., Hertweck, C., 2010. Nat. Prod. Rep. 27, 869. Udagawa, S., Toyasaki, N., Yaguchi, T., 1997. Mycoscience 38, 399. Waring, P.J., Beaver, J., 1996. Gen. Pharmacol 27, 1311. Weindling, R., 1941. Phytopathology 31, 991. Weindling, R., Emerson, O.H., 1936. Phytopathology 26, 1068. Yamada, A., Kataoka, T., Nagai, K., 2000. Immunol. Lett. 71, 27. Yang, S.-X., Gao, J.-M., Zhang, Q., Laatsch, H., . Bioorg. Med. Chem. Lett. 21, 1887. You, F., Han, T., Wu, J.-Z., Huang, B.-K., Qin, L.-P., 2009. Biochem. Syst. Ecol. 37, 162. Zhang, J.-W., Gao, J.-M., Xu, T., Zhang, X.-C., Ma, Y.-T., Jarussophon, S., Yasuo, K., 2009a. Chem. Biodivers 6, 838. Zhang, A.-L., Li, Y.-L., Ma, Y.-T., Gao, J.-M., Jia, A.-Q., 2009b. Biochem. Syst. Ecol. 37, 756. Zhang, J., Ge, H.M., Jiao, R.H., Li, J., Peng, H., Wang, Y.R., Wu, J.H., Song, Y.C., Tan, R.X., 2010. Planta. Med. 76, 1910.
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Antifungal metabolites from Chaetomium globosum, an endophytic fungus in Ginkgo biloba Hu-Qiang Li a, Xiao-Jun Li a, You-Lin Wang a, Qiang Zhang a, An-Ling Zhang a, Jin-Ming Gao a, *, Xing-Chang Zhang b, ** a
Research Centre for Natural Medicinal Chemistry, College of Science, Northwest A&F University, Yangling, Shaanxi 712100, PR China State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, Institute of Soil and Water Conservation, Chinese Academy of Science (CAS), Yangling, Shaanxi 712100, PR China b
a r t i c l e i n f o
.
Article history: Received 11 April 2011 Accepted 25 June 2011 Available online 28 July 2011 Keywords: Ginkgo biloba Chaetomium globosum Endophytic fungus Antifungal activity Epipolythiodioxopiperazine Gliotoxin
1. Subject and source Endophytes are microorganisms that have been recognized as important sources of a variety of structurally novel active secondary metabolites with anticancer, antimicrobial and other biological activities (Strobel, 2003; Krohn et al., 2007). Chaetomium is a large genus of the fungal family Chaetomiaceae (Ascomycota) with over an hundred marine- and terrestrial-derived species (Udagawa et al., 1997). To date, more than 200 compounds have been reported from this genus. As a part of study into the chemical diversity and biological activity of compounds associated with plant-endophytic fungal interactions (Qin et al., 2009a,b; Yang et al., 2011), a fungal strain Chaetomium globosum (NM0066) was separated from the healthy leaves of the medicinal plant Ginkgo biloba, growing in Linyi, Shandong province, China. It was characterized, based on morphological studies, and has been deposited at Research Centre for Natural Medicinal Chemistry, College of Science, Northwest A&F University. We undertook chemical and biological investigations of this fungus because of the significant antifungal activity of the crude EtOAc extract against some phytopathogenic fungi.
2. Previous work In the past decades, various strains of C. globosum have been reported as endophytes of some plants. Cultivation of endophytic C. globosum produced a series of compounds such as chaetoglobosins G (Qin et al., 2009a), chaetoglobosins Q, R * Corresponding author. Tel.: þ86 29 87092335. ** Corresponding author. E-mail addresses: [email protected] (J.-M. Gao), [email protected] (X.-C. Zhang). 0305-1978/$ – see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2011.06.019
H.-Q. Li et al. / Biochemical Systematics and Ecology 39 (2011) 876–879
877
and T (Jiao et al., 2004), chaetoglobosins U, V and W (Ding et al., 2006; Zhang et al., 2010), cytoglobosins A–G (Cui et al., 2010); azaphilones, chaetoviridins A and C (Qin et al., 2009a); pyrones, chaetoglocins A and B (Ge et al., 2011); orsellides, globosumones A–C (Bashyal et al., 2005); steroids (Qin et al., 2009b). Some of the compounds have been reported to possess significant biological activities, such as cytotoxic, enzyme inhibitory and antibiotic (Gunatilaka, 2006; Scherlach et al., 2010). However, little is known about the anti-phytopathogenic activity of secondary metabolites from C. globosum isolated from leaves of G. biloba. 3. Present study 3.1. Fermentation The fungus C. globosum was grown on PDA plates at 28 C for 6 days, and the fresh mycelium was inoculated into 500 mL flasks containing 200 mL PDA medium composed of glucose 20 g, MgSO4$7H2O 1.5 g, K2HPO4 3 g, yeast extract 1 g and 1 L water, followed by shaking in an incubator at 130 r/min at 28 C for 6 days.
3.2. Extraction and isolation The culture broth (110 L) was filtered to give the mycelium and culture filtrate. The culture filtrate obtained was subsequently extracted with EtOAc at room temperature, then evaporation of solvent under reduced pressure gave a residue (22 g). The mycelium was dried naturally, and ultrasonically extracted four times by EtOAc, then evaporation of solvent under reduced pressure to provide another crude extract (28 g). The EtOAc extract (28 g) of the mycelium was fractionated on a silica gel column using petroleum ether (PE)-EtOAc (100:0, 98:2, 95:5, 9:1, 8:2, 7:3) to give six fractions (Fr.1–Fr.6). Fr.1 (93 mg) was subjected to repeated column chromatography (CC) (Sephadex LH-20, CHCl3:MeOH 1:1, then MeOH; silica gel, n-hexane) to yield 1 (15 mg). Fr. 2 (1.2 g) was separated by CC on silica gel with PE:EtOAc 8:2, followed by Sephadex LH-20 (MeOH), and then crystallization from EtOAc to afford 2 (19 mg). Fr. 4 (940 mg) was fractionated by a silica gel column eluted with PE-acetone (8:2, 85:15, 7:3) to give two fractions (Fr. 4–1 and Fr. 4–2). Fr.4–1 was purified by repeated CC (silica gel, CHCl3:acetone 85:15; silica gel, PE-acetone 7:3; Sephadex LH-20, MeOH) to obtain 3 (20 mg); Fr. 4–2 was purified by repeated CC (Sephadex LH-20, CHCl3:MeOH 1:1, then MeOH; silica gel, PE-acetone 7:3) to provide 4 (10 mg) and 7 (8 mg). Fr.6 (460 mg) was separated by CC (silica gel, PEacetone 7:3; Sephadex LH-20, CHCl3:MeOH 1:1; silica gel, CHCl3:acetone 95:5; Sephadex LH-20, MeOH) to produce 5 (10 mg). The EtOAc extract (22 g) of the filtrate was fractionated on a silica gel column eluted with CHCl3-MeOH (100:0, 98:2, 95:5, 9:1, 8:2, 7:3, 6:4) to give seven fractions (Fr.7–Fr.13). Fr.7 (525 mg) was subjected to repeated CC (silica gel, PE:acetone 7:3; Sephadex LH-20, MeOH, then CHCl3:MeOH 1:1) to yield 6 (8 mg). Fr.8 (800 mg) was separated by repeated CC (silica gel, CHCl3:MeOH 100:0–90:10; Sephadex LH-20, CHCl3:MeOH 1:1) and then crystallization from EtOAc to give 8 (10 mg). Fr. 10 (1.2 g) was purified by repeated CC (silica gel, petroleum ether:EtOAc 7:3; Sephadex LH-20, MeOH; silica gel, CHCl3:MeOH 97:3) to afford 9 (10 mg) and 10 (15 mg). Fr. 12 (1.6 g) was fractionated on CC (silica gel, CHCl3:MeOH:H2O 90:10:1; Sephadex LH-20, MeOH, then CHCl3:MeOH 1:1) to give 11 (17 mg). Fr. 13 (960 mg) was separated by CC (silica gel, CHCl3:MeOH:H2O 90:10:1; Sephadex LH-20, MeOH, then CHCl3:MeOH 1:1) to afford 12 (13 mg). The structures of compounds 1–12 were identified, respectively, as squalene (1) (Masuda et al., 1989), ergosterol peroxide (2) (Zhang et al., 2009b), methylthiogliotoxin (3) (Lee et al., 2001), chaetoglobosin G (4) (Sekita et al., 1982a,b), (22E,24R)ergosta-7,22-diene-3b,5a,6b-triol (5) (Gao et al., 2001), fumitremorgin C (6) (Cui et al., 1996), chaetoglobosin C (7) (Sekita et al., 1983), gliotoxin (8) (Lee et al., 2001), 2,3,4-trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran (9) (Chen et al., 2002), pseurotin A (10) (Aoki et al., 2004), 4-aminophenylacetic acid (11) (Ahmed et al., 2006) and 3,4-dihydroxyphenylacetic acid (12) (Katherine, 1972) by a combination of spectroscopic methods (MS, 1D- and 2D- NMR) and comparison with literature data (Fig. 1). The in vitro antifungal activity of the isolated compounds 1–12 was evaluated by mycelial growth inhibitory rate method (Zhang et al., 2009a) using Fusarium oxysporum f. sp. vasinfectum, Fusarium graminearum, Fusarium sulphureum, Cercospora sorghi, Botrytis cinerea and Alternaria alternata as the pathogenic fungi. Hymexazol was used as a positive drug. 4. Ecological significance In the current study, the cultures of an endophyte C. globosum found in G. biloba produced twelve metabolites 1–12. To our knowledge, this is the first time that eight compounds (1, 3, 6 and 8–12) have been reported from the genus Chaetomium. Among them, fumitremorgin C (6) is an indole mycotoxin, which was also isolated from an endophytic Aspergillus fumigatus CY018 (Liu et al., 2004). Gliotoxin (8), the first and best-known epipolythiodioxopiperazine toxin, was isolated from culture broths of Gliocladium fimbriatum (Weindling and Emerson, 1936) and later also from other microorganisms such as Trichoderma, Aspergillus and Penicillium (Weindling, 1941; Gardiner et al., 2005; Lewis et al., 2005). It shows strong antibacterial and antiviral activity (Johnson et al., 1943; Rightsel et al., 1964), is immunosuppressive (Yamada et al., 2000) and causes apoptotic and necrotic cell death (Waring and Beaver, 1996).
878
H.-Q. Li et al. / Biochemical Systematics and Ecology 39 (2011) 876–879
HO
O
HO
O
N H O HOH2C
2
1
SMe O N SMe
3
OH H N O HO O
N H
H HO
O
OH OH
4
5
O H
O
H
N H3CO
N H
N H
O
N O HO
H
O
N H
6
H HO O
O O OH 9
O
OH O 10
H N
O
OH
8
NH2
HO
S S N
O
7
OH
N
OH
HO
O
OH OCH3
O
O OH
OH
11
12
Fig. 1. Compounds (1–12) isolated from Chaetomium globosum.
Microorganisms, particular fungi, when associated with plants have been recognized as a rich source of compounds with phytotoxic and plant growth regulating-properties (Hussain et al., 2007). In order to exploit its ecological niche, endophytes need to compete with other endophytes and pathogens; secondary metabolites that have antibiotic properties can play an important role in this process (Gunatilaka, 2006). Accordingly, most secondary metabolites from endophytes indicated antibiotic activities against microorganisms (You et al., 2009). An in vitro growth inhibition bioassay, that measured the rate of mycelia growth of a range of phytopathogenic fungi, was used to evaluate the antifungal activity of the 12 isolated metabolites. Compounds 1–7 and 9–12 were found to be inactive against the fungi at 100 mg/ml, whereas gliotoxin (8) showed good antifungal activity against F. sulphureum, A. alternata and C. sorghi with EC50 values of 68.5, 36.8 and 59.8 mg/mL, respectively. The potency is comparable with the positive control hymexazol, with EC50 values of 100.3, 32.8 and 64.3 mg/mL, respectively. In contrast, gliotoxin (8) presented a moderate inhibitory activity toward F. oxysporum f. sp. vasinfectum, B. cinerea and F. graminearum with EC50 values of 93.7, 106.6 and 100 mg/mL, respectively. This efficacy is weaker than the positive control with EC50 values of 47.2, 83.3 and 70.4 mg/mL, respectively. Interestingly, methylthiogliotoxin (3) did not show any activity, which may be attributable to the absence of a disulphide bridge in the molecule, as compared with gliotoxin (8). This suggests that the disulphide bridge is essential for antifungal activity. The pronounced antifungal activity against the phytopathogenic fungi of gliotoxin (8) suggests that the endophytic fungus C. globosum present in leaves of the plant G. biloba could protect the host by producing metabolites, which may be toxic or even lethal to phytopathogens.
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