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Secondary Metabolites of Fusarium sp, an Endophytic

By Beatrice Black,2014-09-08 19:25
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Secondary Metabolites of Fusarium sp, an Endophytic

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    Secondary Metabolites of Fusarium sp., an Endophytic

     Fungus in Ficus carica112Zhang Hongchi, Ma Yangmin, Liu Rui

    5 (1. Key laboratory of Auxiliary Chemistry and technology for Chemical Industry, Ministry of

    Education. Shaanxi university of Science and technology, Xi'an 710021;

    2. College of Life Science, Shanxi Datong University, ShanXi DaTong 037009) Abstract: Objective: To study on secondary metabolites from an endophytic fungus of Ficus carica. Method: The compounds were isolated by column chromatography and identified on the basis of

    10 physic-chemical constants and spectral analysis. Result: Eight compounds were obtained and elucidated as rgosterol (1), cerevisterol (2), ergosterol peroxide (3), allantoin (4), fumitremorgin B (5), verruculogen (6), fumitremorgin C (7), and cyclotryprostatins B (8). Conclusion: Among them, compound 4, 6 and 8 were isolated from endophytic fungi for the first time.

    Keywords: Natural metabolites; Endophytic fungi; Ficus carica; Fusarium sp

    15

    0 Introduction

    Endophytic fungi that inhabit normal tissues of the host plants without causing apparent pathogenic symptoms have been proven to be a rich source of new biologically active natural products because as a group they inhabit a relatively untapped ecological environment, and their

    20 secondary metabolism is activated by their metabolic interactions with their hosts [1]. Natural products from endophytic fungi have been observed to inhibit or kill a wide variety of harmful microorganisms including, but not limited to, phytopathogens, as well as bacteria, fungi, viruses, and protozoans that affect humans and animals [2].

    In the course of our search for biologically active metabolites from endophytic fungi of

    25 Chinese medicinal plants, a subculture of an isolate of Fusarium sp., obtained from roots of Ficus

    carica, was cultivated in on potato dextrose agar (PDA). An ethyl acetate extract of the culture showed significant antimicrobial activity. This prompted us to carry out secondary metabolites studies on this fungus, which resulted in the isolation of twelve compounds. Herein we describe the isolation and structural elucidation of its secondary metabolites.

     30 1 Experimental Section

1.1 Fungal Material and Identification

    The fungal strain FR20 was isolated from the roots of Ficus carica, collected in the Qinling

    Mountains, Shaanxi Province, China, on September 10, 2010. By classical microscopic analysis, the fungus was identified as a member of the genus Fusarium. The fungal strain has been

    35 preserved at the Research Centre for Natural Product, College of Chemistry & Chemical Engineering, Shaanxi University of Science & Technology, Shaanxi Province, China.

1.2 General Experimental Procedures

    113The H- and C-NMR spectra were recorded on Bruker ADVANCE 400 MHz spectrometer,

    with TMS as an internal standard. Melting points were obtained on a Fisher-Johns apparatus and

     40 are uncorrected. EI-MS were recorded on a JEOL SX102A mass spectrometer. Column

     Foundations: 高等学校博士学科点专项科研基金 the Specialized Research Fund for the Doctoral Program of

    Higher Education (No.20116125110001)

     Brief author introduction:张弘弛,(1980-),在读博士,主要从事微生物代谢产物方面的工作。

     Correspondance author: 马养民,?1963-?,教授,博导,主要从事天然产物化学和有机合成的研究。E-mail:

    mym63@sina.com

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    chromatography was performed with silica gel (200-300 mesh, Qingdao Marine Chemical Inc.

    Qingdao, China) and Sephadex LH-20 (Amersham Biosciences Inc. Shanghai, China).

    1.3 Fermention, Extraction and Isolation

    Starter cultures were maintained on PDA medium at 28 ?C for 7 days. Plugs of agar 45 supporting mycelial growth were cut and transferred aseptically to 1000 mL Erlenmeyer flasks

    containing 400 mL of liquid Czapek medium at 28 ?C on a rotary shaker for 15 days. The fungal

    culture (40 L) was filtered through cheesecloth. The filtrate was concentrated to 3.5 L below 60?C

    and then extracted five times with ethyl acetate (4.5 L). The dried mycelium (55 ?C, 51 g) was

    extracted three times with methanol (4 L). All extracts were concentrated at reduced pressure to 50 afford 13.9 g of a dark brown crude extract.

    The crude extract was subjected to silica gel column chromatography eluting successively

    with ethyl acetate/methanol gradient (1:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1: 1, 0:1) and yielded eight

    fractions (A-H). Fraction A was separated by column chromatography on silica gel with a gradient

    of ethyl acetate in petroleum ether to give five subfractions (A1-5). Fraction A1 was separated by 55 silica gel column chromatography with elution with petroleum ether/ethyl acetate (2:1) to give

    pure compound 1 and compound 2. Fraction A3 was separated by silica gel column

    chromatography with elution with petroleum ether/ethyl acetate (1:2) to give pure compound 3,

    together with crude compound 4. The crude compound 4 was then recrystallized from petroleum

    ether/ethyl acetate to give the pure compound 4. Fraction B was separated by column 60 chromatography on silica gel with a gradient of ethyl acetate in petroleum ether to give five

    subfractions (B1-5). Fraction B2 was repeatedly subjected to silica gel column chromatography

    with a gradient of ethyl acetate in petroleum ether and Sephadex LH-20 with ethyl

    acetate/methanol (1:1) to yield compound 5 and compound 6. Similarly compound 7 and

    compound 8 was obtained from the fraction B3 subjected to repeated chromatographic 65 purifications using petroleum ether/ethyl acetate.

    2 Results and Discussion

    The ethyl acetate extract was subjected to chromatography on silica gel eluting successively

    with different mobile phases which resulted in the isolation and characterization of eight

    compounds. The compounds were subjected to characterization using nuclear magnetic resonance 70 spectroscopy. The secondary metabolites were identified as ergosterol (1), cerevisterol (2),

    ergosterol peroxide (3), allantoin (4), fumitremorgin B (5), verruculogen (6), fumitremorgin C (7),

    cyclotryprostatins B (8) (Figure 1), by comparison of their spectral data with the reported data in

    the literature.

    +1Compound 1: CHO, Colorless needle, mp 155-157 ?C, El-MS m/z 398 [M]. H-NMR 2846

    75 (400 MHz, CDCl): δ : 3.66 (1H, m, H-3), 5.59 (1H, dd, J = 8.2 Hz, 4.0 Hz, H-6), 5.41 (1H, dd, J 3

    = 4.0 Hz, 2.1 Hz, H-7), 0.65 (3H, s, H-18), 0.94 (3H, s, H-19), 1.05 (3H, d, J = 4.0 Hz, H-21),

    5.18 (1H, dd, J = 16.0 Hz, 8.2 Hz, H-22), 5.22 (1H, dd, J = 16.0 Hz, 8.2 Hz, H-23), 0.85 (3H, d, J

    13= 8.2 Hz, H-26), 0.83 (3H, d, J = 8.0 Hz, H-27), 0.93 (3H, d, J = 8.0 Hz, H-28). C-NMR (100

    MHz, CDCl): δ: 38.4 (C-1), 32.0 (C-2), 70.5 (C-3), 40.8 (C-4), 139.8 (C-5), 119.6 (C-6), 116.3 3

    80 (C-7), 141.4 (C-8), 46.2 (C-9), 37.0 (C-10), 21.1 (C-11), 39.1 (C-12), 42.8 (C-13), 54.6 (C-14),

    23.0 (C-15), 28.3 (C-16), 55.7 (C-17), 12.1 (C-18), 17.6 (C-19), 40.5 (C-20), 21.1 (C-21), 132.0

    (C-22), 135.6 (C-23), 42.8 (C-24), 33.1 (C-25), 19.7 (C-26), 20.0 (C-27), 16.3 (C-28). According

    to their spectroscopic data, the compound was determined to be ergosterol [3].

    +1Compound 2: CHO, Colorless needle, mp 240-242 ?C, El-MS m/z 430 [M]. H-NMR 28463

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85 ): δ: 4.08 (1H, m, H-3), 3.62 (1H, d, J = 4.8 Hz, H-6), 5.35 (1H, m, H-7), 0.60 (400 MHz, CDCl3 (3H, s, H-18), 1.09 (3H, s, H-19), 1.03 (3H, d, J = 6.5 Hz, H-21), 5.17 (1H, dd, J = 15.0 Hz, 8.0 Hz, H-22), 5.23 (1H, dd, J = 15.0 Hz, 7.5 Hz, H-23), 0.84 (3H, d, J = 7.0 Hz, H-26), 0.82 (3H, d, J 13 = 7.0 Hz, H-27), 0.91 (3H, d, J = 7.0 Hz, H-28). C-NMR (100 MHz, CDCl): δ: 32.8 (C-1), 30.6 3 (C-2), 67.5 (C-3), 39.7 (C-4), 78.1 (C-5), 76.4 (C-6), 115.1 (C-7), 143.9 (C-8), 43.7 (C-9), 37.4

    (C-10), 22.3 (C-11), 39.6 (C-12), 43.9 (C-13), 55.2 (C-14), 23.2 (C-15), 28.8 (C-16), 56.3 (C-17), 90 , 19.8 (C-21), 132.3 (C-22), 135.9 (C-23), 43.2 (C-24), 33.4 12.5 (C-18), 18.4 (C-19), 40.8 (C-20)

     (C-25), 20.1 (C-26), 21.3 (C-27), 17.8 (C-28). The compound was identified as cerevisterol by comparison spectroscopic data with literature data [4].

     +1Compound 3: CHO, Colorless needle, mp 175-177 ?C, EI-MS m/z428[M]. H-NMR 28443

    (400 MHz, CDCl): δ: 4.06-3.93 (1H, m, H-3), 6.28 (1H, d, J = 8.5 Hz, H-6), 6.53 (1H, d, J = 8.5 95 3 H-18), 0.92 (3H, d, J = 6.8 Hz, H-19), 1.02 (3H, d, J = 6.6 Hz, H-21), 5.15 Hz, H-7), 0.90 (3H, s, (1H, dd, J = 15.2, 7.4 Hz, H-22), 5.24 (1H, dd, J = 15.2, 7.4 Hz, H-23), 0.83 (3H, s, H-26), 0.82 13 (3H, s, H-27), 0.84 (3H, s, H-28). C-NMR (100 MHz, CDCl): δ: 34.65 (C-1), 30.08 (C-2), 3 66.45 (C-3), 36.88 (C-4), 82.16 (C-5), 135.40 (C-6), 130.73 (C-7), 79.42 (C-8), 51.02 (C-9), 36.93

    (C-10), 20.61 (C-11), 39.76 (C-12), 44.54 (C-13), 51.65 (C-14), 23.38 (C-15), 28.66 (C-16), 56.14 100

     (C-17), 12.86 (C-18), 18.17 (C-19), 39.30 (C-20), 20.87 (C-21), 135.19 (C-22), 132.27 (C-23), 42.75 (C-24), 33.04 (C-25), 19.95 (C-26), 19.64 (C-27), 17.55 (C-28). Compound 3 was identified

     as ergosterol peroxide by comparison spectroscopic data with literature data [5]. 1Compound 4: CHNO, white powder, mp 230-232 ?C. H-NMR (400 MHz, DMSO-d): δ: 46436

    8.07 (1H, s, 1-NH), 10.55 (1H, s, 3-NH), 6.89 (1H, d, J = 8.2 Hz, 4-NH), 5.80 (2H, s, 6-NH2), 105 13 5.25 (1H, d, J = 8.2 Hz, H-4). C-NMR (100 MHz, DMSO-d): δ: 157.21 (C-2), 62.86 (C-4), 6 174.06 (C-5), 157.80 (C-6). Compound 4 was identified as allantoin by comparison spectroscopic data with literature data [6]. 1Compound 5: CHNO, white needle. mp 205-207 ?C. H-NMR (400 MHz, CDCl): δ: 2733353

    7.85 (1H, d, J = 8.6 Hz, H-4), 6.79 (1H, d, J = 8.6 Hz, H-5), 6.69 (1H, s, H-7), 5.77 (1H, s, H-8), 110

     4.45 (1H, t, J = 8.1 Hz, H-12), 2.47 (1H, m, H-13a), 2.08 (1H, m, H-13b), 2.10 (1H, m, H-14a), 1.93 (1H, m, H-14b), 3.63 (2H, d, J = 8.2 Hz, H-15), 5.98 (1H, d, J = 10.0 Hz, H-18), 4.71 (1H, d, J = 10.1 Hz, H-19), 1.99 (3H, s, H-21), 1.63 (3H, s, H-22), 4.53 (2H, s, H-23), 5.03 (1H, s, H-24), 1.85 (3H, s, H-26), 1.70 (3H, s, H-27), 3.84 (3H, s, 6-OMe), 4.74 (1H, s, 8-OH), 4.17 (1H, s, 139-OH). C-NMR (100 MHz, CDCl): δ: 131.13 (C-2), 104.42 (C-3), 120.52 (C-3a), 121.34 (C-4), 115 3 109.29 (C-5), 156.17 (C-6), 93.82 (C-7), 137.87 (C-7a), 68.95 (C-8), 82.98 (C-9), 170.51 (C-11), 58.76 (C-12), 28.93 (C-13), 22.61 (C-14), 45.26 (C-15), 166.25 (C-17), 49.03 (C-18), 122.97 (C-19), 135.22 (C-20), 18.37 (C-21), 25.71 (C-22), 41.77 (C-23), 120.29 (C-24), 134.61 (C-25), 18.20 (C-26), 25.55 (C-27), 55.72 (6-OMe). Based on its spectroscopic data, the compound was

    considered to be fumitremorgin B [7]. 120 1 Compound 6: CHNO, white needle. H-NMR (400 MHz, CDCl): δ: 7.95 (1H, d, J = 8.6 2733373 Hz, H-4), 6.88 (1H, d, J = 8.6 Hz, H-5), 6.58 (1H, s, H-7), 5.66 (1H, s, H-8), 4.45 (1H, t, J = 8.1 Hz, H-12), 2.45 (1H, m, H-13a), 2.10 (1H, m, H-13b), 2.08 (1H, m, H-14a), 1.94 (1H, m, H-14b), 3.63 (2H, d, J = 8.2 Hz, H-15), 6.11 (1H, d, J = 10.0 Hz, H-18), 2.00 (1H, m, H-19a), 1.63 (1H, d,

    J = 10.0 Hz, H-19b), 1.99 (3H, s, H-21), 1.02 (3H, s, H-22), 6.73 (1H, s, H-23), 5.03 (1H, s, H-24), 125

     1.77 (3H, s, H-26), 1.70 (3H, s, H-27), 3.84 (3H, s, 6-OMe), 4.74 (1H, s, 8-OH), 4.04 (1H, s, 139-OH). C-NMR (100 MHz, CDCl): δ: 131.13 (C-2), 105.50 (C-3), 120.87 (C-3a), 121.64 (C-4), 3 109.29 (C-5), 156.36 (C-6), 93.82 (C-7), 136.19 (C-7a), 68.96 (C-8), 82.58 (C-9), 166.10 (C-11), 58.76 (C-12), 28.94 (C-13), 22.61 (C-14), 51.11 (C-15), 170.71 (C-17), 49.03 (C-18), 45.29

    (C-19), 82.09 (C-20), 25.71 (C-21), 27.00 (C-22), 85.82 (C-23), 118.40 (C-24), 143.11 (C-25), 130

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     18.90 (C-26) , 24.09 (C-27), 55.72 (6-OMe). According to their spectroscopic data, the compound was determined to be verruculogen [8]. 1 HNO, white needle. mp 259-260 ?C. H-NMR (400 MHz, CDCl): δ:Compound 7: C 2225333 7.89 (1H, s, H-1), 7.43 (1H, d, J = 8.6 Hz, H-4), 6.82 (1H, dd, J = 8.6, 2.2 Hz, 4.0 Hz, H-5), 6.86

    135 (1H, d, J = 2.1 Hz, H-7), 3.52 (1H, dd, J = 15.9, 4.9 Hz, H-8a), 3.11(1H, dd, J = 15.8, 11.6 Hz,

     H-8b), 4.18 (1H, dd, J = 11.6, 4.8 Hz, H-9), 4.11(1H, t, J = 8.1 Hz, H-12), 2.41 (1H, m, H-13a), 2.24 (1H, m, H-13b), 2.07 (1H, m, H-14a), 1.95 (1H, m, H-14b), 3.65 (1H, d, J = 4.8 Hz, H-15a), 3.63 (1H, d, J = 5.4 Hz, H-15b), 5.99 (1H, d, J = 9.5 Hz, H-18), 4.91 (1H, d, J = 9.5 Hz, H-19), 131.99 (3H, s, H-21), 1.65 (3H, s, H-22), 3.83 (3H, s, 6-OMe). C-NMR (100 MHz, CDCl): δ: 3

    132.19 (C-2), 106.09 (C-3), 120.68 (C-3a), 118.84 (C-4), 109.40 (C-5), 156.41 (C-6), 95.26 (C-7), 140

     136.99 (C-7a), 21.90 (C-8), 56.76 (C-9), 169.53 (C-11), 59.21 (C-12), 28.56 (C-13), 23.04 (C-14), 45.41 (C-15), 165.77 (C-17), 50.99 (C-18), 124.09 (C-19), 134.03 (C-20), 18.07 (C-21), 25.72 (C-22), 55.75 (6-OMe). The compound was identified as fumitremorgin C by comparison spectroscopic data with literature data [9]. 1145 Compound 8: CH-NMR (400 MHz, CDCl): δ: 8.10 (1H, s, H-1),HNO, white needle. 2327353 7.43 (1H, d, J = 8.6 Hz, H-4), 6.81 (1H, d, J = 8.6 Hz, H-5), 6.85 (1H, s, H-7), 4.7 (1H, s, H-8), 4.42 (1H, dd, J = 15.9, 5.0 Hz, H-12), 2.51 (1H, m, H-13a), 2.05(1H, m, H-13b), 2.19(1H, m, H-14a), 1.96 (1H, m, H-14b), 3.75 (1H, m, H-15a), 3.73 (1H, m, H-15b), 6.66 (1H, d, J = 9.5 Hz, H-18), 5.62 (1H, d, J = 9.5 Hz, H-19), 2.24 (3H, s, H-21), 1.79 (3H, s, H-22), 3.82(3H, s, 6-OMe), 13150 3.34(3H, s, 8-OMe). C-NMR (100 MHz, CDCl): δ: 133.75 (C-2), 105.22 (C-3), 122.60 (C-3a), 3 118.68 (C-4), 109.99 (C-5), 156.49 (C-6), 95.23 (C-7), 136.69 (C-7a), 76.83 (C-8), 84.77 (C-9), 167.03 (C-11), 59.93 (C-12), 29.69 (C-13), 22.05 (C-14), 45.82 (C-15), 165.73 (C-17), 49.11 (C-18), 123.54 (C-19), 137.92 (C-20), 18.10 (C-21), 26.03 (C-22), 55.77 (6-OMe), 56.57 (8-OMe). Based on its spectroscopic data, the compound was considered to be cyclotryprostatins B [10].

     155

     O H N N HCO N3H H H O 7 Fig. 1 Compounds 1-8 isolated from Fusarium solani of Ficus carica 160

    3 Conclusions

    Among these compounds, compound 4, 6 and 8 were isolated from endophytic fungi for the

    first time. Moreover, compounds 5-8 are fungal toxins, these compounds have important

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     biological activity and can be applied to medicine and agriculture. The present study of secondary

    metabolites revealed that Fusarium sp., an endophytic fungi from Ficus carica is the sources for 165 the production of fungal toxins. Acknowledgements This work was cofinanced by the Specialized Research Fund for the Doctoral Program of Higher Education (20116125110001).

     170

     References [1] B. Schulz, C. Boyle, S. Draeger, A.K. Rommert, K. Krohn, Endophytic fungi: a source of novel biologically active secondary metabolites[J], Mycol. Res, 2002, 48, 996-1004. [2] G.. Strobel, B. Daisy, U. Castillo, J. Harper, Natural Products from Endophytic Microorganisms[J], J. Nat. 175 Prod., 2004, 67, 257-268.

     [3] F. De Simone, F. Senatore, D. Sica and F. Zollo, Sterols from some basidiomycetes[J], Phytochemistry, 1979, 18, 1572-1573. [4] S. Gupta, C. Montllor, Y.S. Hwang, Isolation of Novel Beauvericin Analogues from the Fungus Beauveria bassiana[J], J. Nat. Prod., 1995, 58, 733-738.

    180 [5] L. A. A. Gunatilaka, Y. Gopichand, F. J. Schmitz, C. Djerassi, Minor and trace sterols in marine invertebrates.

     26. Isolation and structure elucidation of nine new 5α,8α-epidioxy sterols from four marine organisms[J]. J. Org. Chem., 1981, 46, 3860-3866. [6] F. Yin, L. Hu, R. Pan, Novel Dammarane-Type Glycosides from Gynostemma pentaphyllum[J], Chem. Pharm. Bull, 2004, 52, 1440-1444.

    185 [7] M. Yamazaki, K. Sasago and K. Miyaki, The structure of fumitremorgin B ( FTB ) , a tremorgenic toxin from

     Aspergillus fumigatus Fresp[J]. J. Chem. Soc.,Chem. Commun, 1974, 10, 408-409. [8] J. Fayos, D. Lokensgard, J. Clardy, R.J. Cole and J.W. Kirksey, Structure of verruculogen, a tremor producing peroxide from Penicillium verruculosum[J]. J. Am. Chem. Soc., 1974, 96, 6785-6787. [9] C. Cui, H. Kakeya, H. Osada, Novel mammalian cell cycle inhibitor, tryprostatins A, B and other

    Diketopiperazines produced by Aspergillus fumigatus II. physicochemical properties and structures[J]. J. Antibiot, 190

    1996, 49, 534-540. [10] C.B. Cui, H. gakeya and H. Osada, Novel mammalian cell cycle inhibitor, cyclotryprostatins A-D, produced by Aspergillus fionigatus, which inhibit mammalian cell cycle at G2/M phase[J]. Teterahedron, 1997, 53, 59-72.

     一株无花果内生真菌 Fusarium sp.的代谢产物 195 112 张弘弛,马养民,刘瑞 1. 教育部轻化工助剂化学与技术重点实验室,陕西科技大学化学与化工学院, 西安 710021 2. 山西大同大学农学与生命科学学院,山西 大同 037009 摘要!目的!研 究无花果内生真菌 Fusarium sp.的代谢产物。方法!采用反复硅胶柱色谱法、 Sephadex LH-20 200 凝胶色谱法等进行分离纯化,并通过理化常数测定和光谱分析鉴定其化学结 构。 结果!从无 花果内生真菌 Fusarium sp 的发酵液中分离得到 8 个代谢产物,经波谱解析, 分别为 gosterol (1), cerevisterol (2), ergosterol peroxide (3), allantoin (4), fumitremorgin B (5), verruculogen (6), fumitremorgin C (7), and cyclotryprostatins B (8). 结论!化合物 46 8 首次从无花果内生真菌中分离得到。205 关键词!天然化合物,内生真菌,无花果,镰刀霉

     中图分类号!O629.3

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