目的:研究番荔枝内生真菌拟茎点霉B27的化学成分。方法:通过色谱层析柱对提取物进行分离纯化,并通过波谱解析(一维、二维的核磁共振谱和质谱)确定化合物的结构。结果:分离纯化得到5个化合物,鉴定为十元大环内酯phomolide C(1),1-甲基-8-羟基-9,10-蒽醌(2),1,8-双羟基-9,10-蒽醌(3),cytosporone C(4)和Altiloxin A(5)。结论:化合物1是新化合物;化合物1-5首次从拟茎点霉属菌株中分离得到。AIM: To study the chemical constituents of Phomopsis sp. B27, an endophytic fungal strain of Annona squamosa L. METHODS: The extract was isolated and purified by column chromatography. The structures of chemical components were deter-mined by spectroscopic analyses including 1D-, 2D-NMR and MS data. RESULTS: Five compounds were isolated and determined as a ten-membered lactone, namely phomolide C (1), 1-methoxy-8-hydroxy-9,10-anthraquinone (2), 1,8-dihydroxy-9,10-anthra-quinone (1,8-DHA) (3), cytosporone C (4) and altiloxin A (5). CONCLUSION: Compound 1 is a new ten-membered lactone; and compounds 1-5 were isolated from the genus Phomopsis for the first time.the National Science Foundation for Distinguished Young Scholars to Y. Shen. (No.30325044).~

林筱

沈月毛

鲁春华

Chinese Journal of Natural Medicines

Xiamen University Institutional Repository

   2008 年 9 月  第 6 卷  第 5 期   Chin J Nat Med  Sep. 2008  Vol. 6  No. 5  391  Chinese Journal of Natural Medicines 6 (2008) 0391−0394doi: 10.3724/SP.J.1009.2008.00391 ChineseJournal of Natural  Medicines                        One New Ten-membered Lactone from Phomopsis sp. B27, an Endophytic Fungus of Annona squamosa L. LIN Xiao, LU Chun-Hua*, SHEN Yue-Mao Key Laboratory of the Ministry of Education for Cell Biology and Tumor Cell Engineering; School of Life Sciences, Xiamen University, Xiamen 361005,. China Available online 20 Sep. 2008 【ABSTRACT】 AIM: To study the chemical constituents of Phomopsis sp. B27, an endophytic fungal strain of Annona squamosa L. METHODS: The extract was isolated and purified by column chromatography. The structures of chemical components were deter-mined by spectroscopic analyses including 1D-, 2D-NMR and MS data. RESULTS: Five compounds were isolated and determined as a ten-membered lactone, namely phomolide C (1), 1-methoxy-8-hydroxy-9,10-anthraquinone (2), 1,8-dihydroxy-9,10-anthra- quinone (1,8-DHA) (3), cytosporone C (4) and altiloxin A (5). CONCLUSION: Compound 1 is a new ten-membered lactone; and compounds 1-5 were isolated from the genus Phomopsis for the first time. 【KEY WORDS】 Phomopsis sp. B27; Chemical constituent; Spectroscopic analyses 【CLC Number】 Q936           【Document Code】  A            【Article ID】1672-3651(2008)05-0391-04   1  Introduction  Endophytic fungi can be found virtually in all terrestrial plants [1] and are viewed as an outstanding source of bioactive natural products because there are so many of them occupy-ing literally millions of unique biological niches (higher plants) growing in so many unusual environments[2].  Recently, increasing attention has been paid to plant com-mensal microorganisms as a source of new bioactive sub-stances[3-5]. During the course of our searching for new bioactive microbial metabolites, five compounds, namely phomolide C, a ten-membered lactone (1), 1-methoxy-8-hydroxy-9,10- anthraquinone (2), 1,8-dihydroxy-9,10-anthra-quinone (1,8- DHA) (3), cytosporone C (4)  and Altiloxin A (5), were isolated and identified from the endophytic fungus Phomop-sis sp. B27 isolated from the roots of Annona squamosa L. collected in Xiamen, Fujian Province. Herein, we report the isolation, structure elucidation, and antitumor and antibacte-rial activities of these compounds.                                                                      【Received on】  2008-06-04 【Foundation Item】 This project was supported by the National Science Foundation for Distinguished Young Scholars to Y. Shen. (No.30325044). 【*Corresponding author】LU Chun-Hua: Tel: 86-592-2184180, Fax: 86-592-2181722, E-mail: ahua0966@xmu.edu.cn  2  Apparatus and Material 2.1  Apparatus and Material Column chromatography: Qingdao silica gel (200-300 mesh); Merck silica gel 60 RP-18; Sephadex LH-20: Phar-macia products; TLC: Qingdao precoated plates, silica GF254 plates; NMR Spectra: Bruker DRX-600 spectrometer with TMS as internal standard; ESI-MS: Thermo-Finnigen LCQ- Advantage spectrometer. Optical rotations were obtained on a Perkin-Elmer 341 polarimeter with MeOH as solvent. The IR spectra were measured in KBr on a Nicolet FT-IR 360. 2.2  Culture Conditions and Extraction The strain B27 was cultured on PDA plates (10 L) at  28℃ for 14 days. The cultured was chopped and extracted with EtOAc-MeOH-AcOH (80 : 15 : 5, V/V/V, 10 L) at room temperature overnight for three times. The extracts were col-lected through filtration and concentrated under vacuum to remove organic solvents to produce an aqueous solution.  The aqueous solution was extracted three times with petro-leum ether (PE) and acetyl acetate (EtOAc) to afford PE ex-tract (1.2 g) and EtOAc extract (6.0 g), respectively. 2.3  Isolation The PE extract (1.2 g) was subjected to flash column chromatography over reversed-phase C-18 silica gel (30 g) eluted with a stepwise MeOH-water (30%; 50%; 70%; 100%; V/V, 400 mL for each gradient). Four fractions PE-A-D were collected. PE-C (150 mg) was subjected to column chroma-tography over normal phase silica gel (6.0 g) and eluted with LIN Xiao, et al. /Chinese Journal of Natural Medicines 2008, 6(5): 391−394  392  Chin J Nat Med  Sep.  2008  Vol. 6  No. 5     2008 年 9 月  第 6 卷  第 5 期   PE-EtOAc (20 : 1, V/V) to yield 2 (6 mg), and eluted with methanol to afford a wash-off PE-C-Me (88 mg). PE-C-Me was subjected to column chromatography over Sephadex LH-20 (40 g) eluted with methanol, and further isolated by repeated column chromatography over normal phase silica gel (2.5 g) eluted with CHCl3-MeOH (50 : 1, V/V) and nor-mal phase silica gel (0.5 g) eluted with PE-EtOAc (3 : 1, V/V) to yield 1 (7 mg). The EtOAc extract (6.0 g) was subjected to flash column chromatography over reversed-phase C-18 silica gel (180 g) eluted with a stepwise MeOH-water (30%; 50%; 70%; 100%; V/V, 2 L for each gradient) and collected as 200 mL/fraction. The collections were combined according to the TLC results to 10 fractions EA-A-J. EA-G (385 mg) was chromatographed over normal phase silica gel (6.0 g) and eluted with CHCl3-MeOH (15 : 1, V/V) to yield fraction EA-G-4. EA-G-4 (101 mg) was puri-fied by repeated column chromatography over normal phase silica gel (5.0 g) eluted with PE-EtOAc (5 : 1, V/V), and normal phase silica gel (0.8 g) eluted with chloro-form-methanol (100 : 1, V/V) and normal-phase silica gel (0.3 g) eluted with chloroform-methanol (200 : 1, V/V) to yield 5 (2 mg). EA-H (1.8 g) was subjected to column chromatography over Sephadex LH-20 (140 g) eluted with methanol to afford  10 fractions according to the TLC results. Fraction 1 (10 mg) was chromatographed over normal phase silica gel (0.5 g) and eluted with PE-EtOAc (6 : 1, V/V) to yield 4 (4 mg). EA-I (2.08 g) was subjected to column chromatography over Sephadex LH-20 (140 g) eluted with methanol to afford 12 fractions according to the TLC results. Fraction 2 (270 mg) was chromatographed over normal phase silica gel (6.0 g) and eluted with PE-EtOAc (5 : 1, V/V) to yield 3 (35 mg). 3  Results 3.1  Structure elucidation and identification  Compound 1 [α]D20 −27.5 (c 10.0, MeOH), was ob-tained as white powder. Its molecular formula was deter-mined to be C18H30O6 by positive HR-Q-TOF-ESI-MS (m/z 365.269 4 [M + Na]+, 381.245 5 [M + K]+).  The 13C-NMR (DEPT) spectra of 1 showed 18 carbon si- gnals for one methyl, nine methylenes, six methines and two quaternary carbons including one carbonyl at δ 175.6 (C-1) (Table 1). The 1H NMR spectrum showed a methyl triplet at δ 0.82 (t, J = 6.6 Hz), four oxy protons at δ 3.56, 4.31, 4.73 and 3.87, and two olefinic protons at δ 5.50 and 5.75. Addi-  Table 1  The NMR data of Compound 1 in MeOD (δ，J in Hz ) No. 1H 13C HMBC 1H-1H COSY 1  175.6s   2.36 (1H, dd/q, 10.8)  2 2.03 (1H, q,11.4)  32.4t  C-3, C-4, C-1  H-3 2.30(1H, q, 11.4)  3 1.56(1H, t, 12.0)  26.6t C-2, C-5, C-4, C-1  H-2 4 3.56(1H, d, 10.8) 75.5d  H-3, H-5 5 4.31(1H, s) 72.3d C-3, C-4, C-7, C-6 H-4, H-6 6 5.75(1H, d,15.6) 132.3d C-5, C-8, C-7 H-5, H-7 7 5.50(1H, d,15.0) 130.6d C-5, C-8, C-6 H-6 8  96.1s   9 4.73(1H, s) 70.8d C-12, C-1 H-11 2.10(1H, q,13.8)  11 1.69(1H, d,13.8)  22.9t C-12 H-11  1.45(1H, m )  12 1.34(1H, m )  24.9t C-11, C-15, C-14, C-13 C-16 H-13 13 3.87(1H, s) 69.3d C-12, C-14 H-14, H-12 1.44(1H, m )  14 1.33(1H, m )  35.8t  C-17, C-12/C-15, C-16, C-13 H-13 1.32(1H, m )   15 1.22(1H, m )  25.0t  C-17 C-18  16 1.24(2H, m ) 29.1t C-18  17 1.25(2H, m ) 22.3t   18 1.21(2H, m ) 31.6t C-17, C-16 H-19 19 0.82(3H, t, 6.6) 13.0q C-17, C-18 H-18  LIN Xiao, et al. /Chinese Journal of Natural Medicines 2008, 6(5): 391−394   2008 年 9 月  第 6 卷  第 5 期   Chin J Nat Med  Sep. 2008  Vol. 6  No. 5  393  tionally, tionally, the 1H NMR spectrum showed overlapped proton signals at δ 1.21-1.45 for seven methylenes and a broad triplet at δ 0.91 for a terminal methyl, suggesting the presence of an nonane moiety (from C-11 to C-19) which was supported by DEPT spectra and HMBC correlations. The 1H-1H-COSY spectra of 1 demonstrated the connectivities from C-2 to C-7. The HMBC correlations from H-9 to C-1 (δ 175.6) and the relatively lower-field resonance of H-9 (at δ 4.73) suggested that 1 was a 10-membered lactone. Three degrees of unsaturation attributed to carbonyl, carbon-carbon double band and the ring of lactone. The JH-6/H-7 value 15.6 Hz revealed a trans- configuration of the olefinic protons. The downshift of C-4 (δ 75.5), C-5 (δ 72.3), C-8 (δ 96.1) and C-13 (δ 69.3) revealed the presence of four more oxy carbons besides C-9. However, the acetylation of 1 revealed the presence of only three free hydroxyl groups, suggesting an epoxy ring. The epoxide moiety at C-8 and C-13 was evident from the upfield shifts of C-8 and C-13, and further sup-ported by the yield of 4,5,8-triacetyl product in acetylation. Therefore, the remaining one degree of unsaturation was assigned to the six- membered epoxy ring. Therefore, com-pound 1 was elucidated as (E)-2-hexyl-9,10,12a-trihydroxy- 2,3,4,4a,7,8,9,10-octahydropyrano[3,2-b]oxecin-6(12aH)-one. However, the relative configuration of 1 remained to be un-resolved due to insufficient NOE correlations. The structures of 2–5 were elucidated by comparison of the spectral data (1H NMR and 13C NMR) with those reported to be 1-methoxy-8- hydroxy-9,10-anthraquinone(2)[6-7], 1,8- dihydroxy-9,10-an- thraquinone(1,8-DHA) (3)[8], cytosporone C (4)[9] and Altilo- xin A (5)[10], respectively (Fig. 1). 3.2  Bioactivities of compounds 1-5 The cytotoxic activity of compounds 1-5 were measured by MTT method [11] in HepG2 and HeLa cell lines. The antimicrobial activities of 1-5 were measured by disk diffu-sion method[12], using Bacillus subtilis CMCC63501 Es-cherichia coli CMCC44103, Staphylococcus aureus ATCC9763, Candida albicans AS2.538 as indicator organ-isms. The results showed that 1-5 displayed no antitumor activities at 10 µg·mL−1 and displayed no antimicrobial activ-ity at 50 µg/disk.    Fig. 1  The selected 1H-1H COSY( ) and HMBC correlation ( ) for 1 and the structure of compounds 1 – 5  4  Discussion The genus Phomopsis is a rich source of biologically  active metabolites including antimicrotubule phomopsi- din[13], antimalarial and antitubercular phomoxanthones[14], antifungal phomoxanthone A[15] and phomodiol[16] and pho-molide A,B[17] and the plant growth regulator cytochalasin H[18]. From the extracts of the culture of the strain Phomopsis sp. B27, one new (1) and four known (2-5) compounds were isolated. These five compounds belong to four types of structures including ten-membered lactone (1), an-thraquinones (2-3), octaketide (4) and sesquiterpene (5). They were isolated from the genus Phomopsis for the first time. References [1] Saikkonen K, Faeth SH, Helander M, et al. Fungal endo-phytes: a continuum of interactions with host plants [J]. Ann Rev Ecol Syst, 1998, 29: 319-343.  [2] Strobel G, Daisy B. Bioprospecting for microbial endophytes and their natural products [J]. Microbiol Mol Biol Rev, 2003, 67(4): 491-502. [3] Tan RX, Zou WX. Endophytes, a rich source of functional metabolites [J]. Nat Prod Rep, 2001, 18(4): 448-459. [4] Bush LP, Wilkinson HH, Schardl CL. Bioprotective alka-loids of grass-fungal endophyte symbiosis [J]. Plant Physiol, 1997, 114: 1-7. [5] Lu CH, Shen YM. A new macrolide antibiotic with antitu-mor activity produced by Streptomyces sp. CS a commensal microbe of Maytenus hookeri [J]. J Antibiot, 2003, 56(4): 415-418. [6] Mahajan A, Bedi RK. Structural, optical, and electrical characterization of hot wall epitaxy grown 1-methoxy- 8-hydroxy-9, 10-anthraquinone films [J]. J Appl Phys, 2001, 89(12): 7866-7870. [7] Berger Y, Castonguay A, Brassard P.  Carbon-13 nuclear magnetic resonance studies of anthraquinones. Part II.  Hy-droxymethoxyanthraquinones, acetoxymethoxyanthraquino- nes and naturally occurring anthraquinone analogs [J]. Org Magn Resonance, 1980, 14(2): 103-108. [8] Berger Y, Castonguay A. The carbon-13 nuclear magnetic LIN Xiao, et al. /Chinese Journal of Natural Medicines 2008, 6(5): 391−394  394  Chin J Nat Med  Sep.  2008  Vol. 6  No. 5     2008 年 9 月  第 6 卷  第 5 期   resonance spectra of anthraquinone, eight polyhydroxyan-thraquinones, and eight polymethoxyanthraquinones [J]. Org Magn Resonance, 1978, 11(8): 375-377. [9] Brady SF, Wagenaar MM, Singh MP, et al. The Cyto-sporones, new octaketide antibiotics isolated from an endo-phytic fungus [J]. Org Lett, 2000, 2(25): 4043-4046. [10] Ichihara A, Sawamura S, Sakamura S. Structures of altilox-ins A and B, phytotoxins from Phoma asparagi Sacc [J]. Tetrahedron Lett, 1984, 25(30): 3209-3212. [11] Solis PN, Wright CW, Anderson MM, et al. A Microwell cytotoxicity assay using artemia salina (Brine Shrimp) [J]. Planta Med, 1993, 59(3): 250-252. [12] Kronvall G, Ringertz S. Antibiotic disk diffusion testing re-visited. Single strain regression analysis. Review article [J]. APMIS, 1991, 99(4): 295-306. [13] Kobayashi H, Meguro S, Yoshimoto T, et al. Absolute structure, biosynthesis, and anti-microtubule activity of phomopsidin, isolated from a marine-derived fungus Pho-mopsis sp. [J]. Tetrahedron, 2003, 59: 455– 459. [14] Isaka M, Jaturapat A, Rukseree K, et al.  Phomoxanthones A and B, novel xanthone dimmers from the endophytic fun-gus Phomopsis species [J]. J Nat Prod, 2001, 64(8): 1015- 1018. [15] Elsaesser B, Krohn K, Floerke U, et al. X-ray structure de-termination, absolute configuration and biological activity of phomoxanthone A [J]. Eur J Org Chem, 2005, 21: 4563- 4570. [16] Horn WS, Schwartz RE, Simmonds MSJ, et al.  Isolation and characterization of phomodiol, a new antifungal from Phomopsis [J]. Tetrahedron Lett, 1994, 35: 6037-6040. [17] Du XP, Lu CH, Li YY, et al. Three new antimicrobial me-tabolites of Phomopsis sp.[J]. J Antibiot, 2008, 61(4): 250- 253. [18] Wells JM, Cutler HG, Cole RJ. Toxicity and plant growth regulator effects of cytochalasin H isolated from Phomopsis sp. [J]. Can J Microbiol, 1976, 22(8): 1137-1143.  番荔枝内生菌拟茎点霉 B27 中分离到的一个新十元大环内酯 林  筱, 鲁春华*, 沈月毛 厦门大学生命科学学院细胞生物学与肿瘤细胞工程教育部重点实验室, 厦门 361005  【摘  要】  目的：研究番荔枝内生真菌拟茎点霉 B27 的化学成分。方法：通过色谱层析柱对提取物进行分离纯化, 并通过波谱解析(一维、二维的核磁共振谱和质谱)确定化合物的结构。结果：分离纯化得到 5 个化合物, 鉴定为十元大环内酯 phomolide C (1), 1-甲基-8-羟基-9,10-蒽醌 (2), 1,8-双羟基-9,10-蒽醌 (3), cytosporone C (4) 和 Altiloxin A (5)。结论：化合物 1 是新化合物; 化合物 1 - 5 首次从拟茎点霉属菌株中分离得到。 【关键词】  拟茎点霉 B27; 化学成分; 波谱解析                【基金项目】国家杰出青年基金(No.30325044)资助  ·征订启事·    《药物不良反应杂志》征订启事   《药物不良反应杂志》为国内外公开发行的学术性期刊, 专门报道药物不良反应及安全用药, 其内容密切结合临床, 学术性、实用性强。本刊为中国科技论文统计源期刊(中国科技核心期刊), 被多个重要数据库收录, 在全国医药界颇有影响。本刊主要栏目有：论著, 学术论坛, 调查研究, 综述, 安全合理用药, 药源性疾病, 滥用误用, 中毒救治, 病例报告, 中药不良反应, 安全信息, ADR 系列问答等。本刊读者对象主要为临床医师、药师、护师、医药院校师生、医药研究工作者, 以及药品监督管理人员。　 本刊为双月刊, 大 16 开, 80 页, 每期定价 13.80 元(全年 82.80 元)。国内邮发代号 2-420, 国外发行代号BM4886。欢迎广大读者通过当地邮局或直接向本社订阅。　 地    址：北京市长椿街 45 号 药物不良反应杂志社  邮政编码：100053 　 电    话： (010) 83198917    传    真：(010)83156049   E-mail: cadrj@sina.com 银行汇款：帐户名称：药物不良反应杂志社 帐    号：01090520500120111028565    开 户 行：北京银行营业部   

One New Ten-membered Lactone from Phomopsis sp. B27,an Endophytic Fungus of Annona squamosa L.

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One New Ten-membered Lactone from Phomopsis sp. B27,an Endophytic Fungus of Annona squamosa L.

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