中国南海黑乳海参两种共附生真菌中活性次生代谢产物的研究
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摘要
海洋生物迥异的生态环境导致其次生代谢产物无论结构还是生物功能与陆地生物均有极大不同，因而成为新药发现不可替代的宝贵源泉。近年来，海洋无脊椎动物共附生微生物由于能够产生丰富多样的具有多种生物活性的结构新颖的次生代谢产物更是成为了海洋活性物质研究的热点。因而海洋无脊椎动物共附生微生物也为天然药物的研究提供了丰富的潜在资源。
在寻找海洋活性次生代谢产物的过程中，本课题对中国南海黑乳海参（Holothurianobilis Selenka）两种共附生真菌（Dendrodochium.sp.和Phialemonium.sp.）中活性次生代谢产物进行了研究。应用正、反相硅胶柱层析、Sephadex LH-20凝胶柱层析和HPLC等多种现代色谱分离技术，对黑乳海参上述两种活性菌株提取物进行了系统的分离纯化，得到了32个化合物，运用NMR、HRMS、CD、UV、IR等多种现代光谱技术鉴定了其中的27个化合物的结构，并运用CD/TDDFT法及改进的mosher法等对部分化合物的绝对构型进行了确定。这27个化合物可分为三类：1）多羟基环己醇类化合物（DEN-1到DEN-4）；2）十二元大环内酯环类化合物（DEN-5到DEN-19）；3）苯衍生物（DEN-20到DEN-27）。其中前两类化合物共计19个，均为新化合物。
对分离得到的部分化合物（DEN-1到DEN-4）进行了体外抗菌活性测试和肿瘤细胞生长抑制活性测试。体外抗菌活性测试实验结果表明除了DEN-4未见抗真菌活性，其他三个化合物均一定的抗真菌活性，但活性较弱，有关结果有待进一步深入研究。肿瘤细胞生长抑制活性实验表明化合物对人骨肉瘤细胞（MG-63）和人肺癌细胞（A549）有一定的抑制活性，而对人结肠癌细胞（Lovo）没有表现出抑制活性，有关结果有待进一步深入研究。
本课题采用活性追踪的方法，运用多种现代色谱分离技术，对中国南海黑乳海参两种共附生真菌中活性次生代谢产物进行了研究，得到了一系列新次生代谢产物；运用波谱学与化学相结合的方法对其结构进行了确定。体外活性试验中，部分化合物显示一定的抗菌及肿瘤细胞生长抑制活性。特别有意思的是，十二元内酯环类化学成分在自然界中极为罕见，迄今为止仅报道12个，全部来自海洋来源真菌中。本研究为海洋天然产物研究积累了新的资料，对我国丰富的海洋资源开发具有重要的意义。
Differing hugely in entironment makes the marine organism’s secondarymetabolite biosynthesis and response system huge difference of the terrestrial biological,,marine organisms show significant difference from terricolous organisms in secondarymetabolites concerning their chemistry and bioactivity, and are therefore regarded asirreplaceable resources in new drug discovery.In recent years, growing interest in marinenatural products has led to the discovery of an increasing number of bioactive secondarymetabolites from marine fungi and make it become a hot research area. And marine fungihave been a rich source of pharmaceutically important compounds.
In the course of searching for biologically active substances from marine sources,we chemically investigated the metabolites of fungi Dendrodochium.sp.(internal strain no.10087) and Phialemonium.sp.(internal strain no.10082) associated with the sea cucumberHolothuria nobilis Selenka, collected from the South China Sea.32compounds wereisolated by using a variety of modern chromatographic techniques, including normal phasesilica gel, reverse phase silica gel and Sephadex LH-20, and HPLC. Structures of27compounds were elucidented on the basis of modern spectroscopic method, includingNMR、HRMS、CD、UV、IR. Absolurte configuration of some compounds were determinedby TDDEFT/CD calculation and modified Mosher’s methods. These compounds can besorted into three types, namely polyhydroxy cyclohexanols,12-membered microlactone,and benzene derivatives.
Compounds isolated from fungi associated with the sea cucumber Holothuria nobilisSelenka were obtained in vitro anti-fungal activity, results show that the screening ofanti-fungal compounds have a certain activity. Also did antitumor activity, showed havecertain activity to MG-63cell and A549cell, but no activity to Lovo cell. Our studiesfocused on chemical constituents of the fungi Dendrodochium.sp. and Phialemonium.sp.and their bioactivities have established a foundation for further research, and providedimportant leading compounds for the development of new antifungal drugs.
在寻找海洋活性次生代谢产物的过程中，本课题对中国南海黑乳海参（Holothurianobilis Selenka）两种共附生真菌（Dendrodochium.sp.和Phialemonium.sp.）中活性次生代谢产物进行了研究。应用正、反相硅胶柱层析、Sephadex LH-20凝胶柱层析和HPLC等多种现代色谱分离技术，对黑乳海参上述两种活性菌株提取物进行了系统的分离纯化，得到了32个化合物，运用NMR、HRMS、CD、UV、IR等多种现代光谱技术鉴定了其中的27个化合物的结构，并运用CD/TDDFT法及改进的mosher法等对部分化合物的绝对构型进行了确定。这27个化合物可分为三类：1）多羟基环己醇类化合物（DEN-1到DEN-4）；2）十二元大环内酯环类化合物（DEN-5到DEN-19）；3）苯衍生物（DEN-20到DEN-27）。其中前两类化合物共计19个，均为新化合物。
对分离得到的部分化合物（DEN-1到DEN-4）进行了体外抗菌活性测试和肿瘤细胞生长抑制活性测试。体外抗菌活性测试实验结果表明除了DEN-4未见抗真菌活性，其他三个化合物均一定的抗真菌活性，但活性较弱，有关结果有待进一步深入研究。肿瘤细胞生长抑制活性实验表明化合物对人骨肉瘤细胞（MG-63）和人肺癌细胞（A549）有一定的抑制活性，而对人结肠癌细胞（Lovo）没有表现出抑制活性，有关结果有待进一步深入研究。
本课题采用活性追踪的方法，运用多种现代色谱分离技术，对中国南海黑乳海参两种共附生真菌中活性次生代谢产物进行了研究，得到了一系列新次生代谢产物；运用波谱学与化学相结合的方法对其结构进行了确定。体外活性试验中，部分化合物显示一定的抗菌及肿瘤细胞生长抑制活性。特别有意思的是，十二元内酯环类化学成分在自然界中极为罕见，迄今为止仅报道12个，全部来自海洋来源真菌中。本研究为海洋天然产物研究积累了新的资料，对我国丰富的海洋资源开发具有重要的意义。
Differing hugely in entironment makes the marine organism’s secondarymetabolite biosynthesis and response system huge difference of the terrestrial biological,,marine organisms show significant difference from terricolous organisms in secondarymetabolites concerning their chemistry and bioactivity, and are therefore regarded asirreplaceable resources in new drug discovery.In recent years, growing interest in marinenatural products has led to the discovery of an increasing number of bioactive secondarymetabolites from marine fungi and make it become a hot research area. And marine fungihave been a rich source of pharmaceutically important compounds.
In the course of searching for biologically active substances from marine sources,we chemically investigated the metabolites of fungi Dendrodochium.sp.(internal strain no.10087) and Phialemonium.sp.(internal strain no.10082) associated with the sea cucumberHolothuria nobilis Selenka, collected from the South China Sea.32compounds wereisolated by using a variety of modern chromatographic techniques, including normal phasesilica gel, reverse phase silica gel and Sephadex LH-20, and HPLC. Structures of27compounds were elucidented on the basis of modern spectroscopic method, includingNMR、HRMS、CD、UV、IR. Absolurte configuration of some compounds were determinedby TDDEFT/CD calculation and modified Mosher’s methods. These compounds can besorted into three types, namely polyhydroxy cyclohexanols,12-membered microlactone,and benzene derivatives.
Compounds isolated from fungi associated with the sea cucumber Holothuria nobilisSelenka were obtained in vitro anti-fungal activity, results show that the screening ofanti-fungal compounds have a certain activity. Also did antitumor activity, showed havecertain activity to MG-63cell and A549cell, but no activity to Lovo cell. Our studiesfocused on chemical constituents of the fungi Dendrodochium.sp. and Phialemonium.sp.and their bioactivities have established a foundation for further research, and providedimportant leading compounds for the development of new antifungal drugs.
引文
[1] John W. Blunt，Brent R. Copp，Wan-Ping Hu，et al. Northcotec and Michèle R. Prinsep[J]. Marine natural products. Nat. Prod. Rep..2008,25(35):35-37.
[2] Cheng Yuanrong, Zheng wei.Research progress in bioactive microbial Products[J].Chinese Journal of Antibiotics,2002,27(10):632-640.
[3] Shamil Sh. Afiyatullov, Tatyana A. Kuznetsova,Vladimir V. Isakov, Mikhail V. Pivkin,Nina G. Prokof’eva, and George B. Elyakov..New Diterpenic Altrosides of the FungusAcremonium striatisporum Isolated from a Sea Cucumber [J]. J. Nat. Prod.2000,63,848-850
[4] Shamil Sh. Afiyatullov, Tatyana A. Kuznetsova,Vladimir V. Isakov, Mikhail V. Pivkin,Nina G. Prokofeva, and George B. Elyakov. New Diterpene Glycosides of the FungusAcremonium striatisporum Isolated from a Sea Cucumber [J]. J. Nat. Prod.2002,65,641-644.
[5] Shamil Sh. Afiyatullov, Tatyana A. Kuznetsova,Vladimir V. Isakov, Mikhail V. Pivkin,Nina G. Prokof’eva, and George B. Elyakov. New Glycosides of the Fungus Acremoniumstriatisporum Isolated from a Sea Cucumber [J]. J. Nat. Prod.2004,67,1047-1051
[6] Shamil Sh. Afiyatullov, Tatyana A. Kuznetsova, Vladimir V. Isakov, Mikhail V. Pivkin,Nina G. Prokof’eva, and George B. Elyakov. Additions and Corrections [J]. J. Nat. Prod.2005,68,1308.
[7]夏雪奎，刘新，张永刚，等。海参共附生微生物HS-1Epicoccum spp.次级代谢产物的研究[J].中药材.33(10):1577-1579.
[8]刘昌衡,夏雪奎,齐君,等海参共生微生物HS23A lternaria sp1次级代谢产物的研究[J].中药材.2010,33(12):1875-1877
[9] Sheo B. Singh, Kithsiri Herath, Ziqiang Guan, et al. Integramides A and B, Two NovelNon-Ribosomal Linear Peptides Containing Nine Cr-Methyl Amino Acids Produced byFungal Fermentations That Are Inhibitors of HIV-1Integrase [J]. Organic letters.2002.4(9):1431-1434.
[10] M. Gennaro, P. Gonthier and G. Nicolotti. Fungal Endophytic Communities inHealthy and Declining Quercus robur L. and Q. cerris L. Trees in Northern Italy [J]. J.Phytopathology.2003,151:529–534
[11] Leonardo Petruzzi, Antonio Bevilacqua, Claudio Ciccarone,etal. Use ofmicrofungi inthe treatment of oak chips: possible effects on wine [J]. J Sci Food Agric,2010.90:2617–2626.
[12] Raquel Jadulco, Peter Proksch, Victor Wray,et al. New Macrolides and FuranCarboxylic Acid Derivative from the Sponge-Derived Fungus Cladosporium herbarum [J].J. Nat. Prod.2001,64:527-530.
[13] Shiri Gesner, Naama Cohen, Micha Ilan, at al. Pandangolide1a, a Metabolite of theSponge-Associated Fungus Cladosporium sp., and the Absolute Stereochemistry ofPandangolide1and iso-Cladospolide B.[J]. J. Nat. Prod.2005,68,1350-1353
[14] Cameron J. Smith, Darren Abbanat, Valerie S. Bernan, et al. Novel PolyketideMetabolites from a Species of Marine Fungi [J]. J. Nat. Prod.2000,63,142-145.
[15] Ping Jiao, Dale C. Swenson, James B, et al. Chloriolide, a12-Membered Macrolidefrom Chloridium Wirescens var. chlamydosporum (NRR L37636)[J]. J. Nat. Prod.2006,69,636-639.
[16] Yuguo Du, Qi Chen, and Robert J. Linhardt. The First Total Synthesis of SporiolideA [J]. J. Org. Chem.2006,71,8446-8451.
[17] Chun Y i Chou, Duen Ren Hou. Synthesis of (+)-Cladospolide C[J]. J. Org. Chem.2006,71,9887-9890.
[18] Yalan Xing and George A. O’Doherty. De Novo Asymmetric Synthesis ofCladospolide B-D: Structural Reassignment of Cladospolide D via the Synthesis of itsEnantiomer [J]. Organic letters.2009,11(5):1107-1110.
[19] Birkinshaw and Raistrick, Biochem. J.,1936,30,801
[20] Bowden, Lythgoe, and Marsden1959,1662-1669
[21] Georges Hareau, et al. Asymetric synthesis of Palitantin from the (5R)–tert-butyldimethylsiloxy-2-cyclohexenone [J]. Tetrahedron.1999,40:7493-7496.
[22]Tridib Mahapatra, Samik Nanda. Asymmetric synthesis of palitantin by an enzymaticand organocatalytic approach [J]. Tetrahedron: Asymmetry.2009,20:610-615.
[23] Yasuo Kimura, Takashi Mizuno and Atsumi Shimada. Penienone and penihydrone,New plant growth regulators produced by the Fungus Penicillium. Sp. No.13[J].Tetrahedran Letters.1997,38(3):469-472.
[24] Hyang Burm Lee and Hyuncheol Oh. Two New Fungal Metabolites from anEpiphytic Fungus Paraphaeosphaeria Species [J]. Bull. Korean Chem. Soc.2006,27(5):779-782.
[25] Goverdhan Mehta and Subhrangsu Roy. Enantioselective Total Synthesis of(+)-Eupenoxide and (+)-Phomoxide: Revision of Structures and Assignmentof Absolute Configuration [J]. Organtic letters.2004,6(14):2389-2392.
[2] Cheng Yuanrong, Zheng wei.Research progress in bioactive microbial Products[J].Chinese Journal of Antibiotics,2002,27(10):632-640.
[3] Shamil Sh. Afiyatullov, Tatyana A. Kuznetsova,Vladimir V. Isakov, Mikhail V. Pivkin,Nina G. Prokof’eva, and George B. Elyakov..New Diterpenic Altrosides of the FungusAcremonium striatisporum Isolated from a Sea Cucumber [J]. J. Nat. Prod.2000,63,848-850
[4] Shamil Sh. Afiyatullov, Tatyana A. Kuznetsova,Vladimir V. Isakov, Mikhail V. Pivkin,Nina G. Prokofeva, and George B. Elyakov. New Diterpene Glycosides of the FungusAcremonium striatisporum Isolated from a Sea Cucumber [J]. J. Nat. Prod.2002,65,641-644.
[5] Shamil Sh. Afiyatullov, Tatyana A. Kuznetsova,Vladimir V. Isakov, Mikhail V. Pivkin,Nina G. Prokof’eva, and George B. Elyakov. New Glycosides of the Fungus Acremoniumstriatisporum Isolated from a Sea Cucumber [J]. J. Nat. Prod.2004,67,1047-1051
[6] Shamil Sh. Afiyatullov, Tatyana A. Kuznetsova, Vladimir V. Isakov, Mikhail V. Pivkin,Nina G. Prokof’eva, and George B. Elyakov. Additions and Corrections [J]. J. Nat. Prod.2005,68,1308.
[7]夏雪奎，刘新，张永刚，等。海参共附生微生物HS-1Epicoccum spp.次级代谢产物的研究[J].中药材.33(10):1577-1579.
[8]刘昌衡,夏雪奎,齐君,等海参共生微生物HS23A lternaria sp1次级代谢产物的研究[J].中药材.2010,33(12):1875-1877
[9] Sheo B. Singh, Kithsiri Herath, Ziqiang Guan, et al. Integramides A and B, Two NovelNon-Ribosomal Linear Peptides Containing Nine Cr-Methyl Amino Acids Produced byFungal Fermentations That Are Inhibitors of HIV-1Integrase [J]. Organic letters.2002.4(9):1431-1434.
[10] M. Gennaro, P. Gonthier and G. Nicolotti. Fungal Endophytic Communities inHealthy and Declining Quercus robur L. and Q. cerris L. Trees in Northern Italy [J]. J.Phytopathology.2003,151:529–534
[11] Leonardo Petruzzi, Antonio Bevilacqua, Claudio Ciccarone,etal. Use ofmicrofungi inthe treatment of oak chips: possible effects on wine [J]. J Sci Food Agric,2010.90:2617–2626.
[12] Raquel Jadulco, Peter Proksch, Victor Wray,et al. New Macrolides and FuranCarboxylic Acid Derivative from the Sponge-Derived Fungus Cladosporium herbarum [J].J. Nat. Prod.2001,64:527-530.
[13] Shiri Gesner, Naama Cohen, Micha Ilan, at al. Pandangolide1a, a Metabolite of theSponge-Associated Fungus Cladosporium sp., and the Absolute Stereochemistry ofPandangolide1and iso-Cladospolide B.[J]. J. Nat. Prod.2005,68,1350-1353
[14] Cameron J. Smith, Darren Abbanat, Valerie S. Bernan, et al. Novel PolyketideMetabolites from a Species of Marine Fungi [J]. J. Nat. Prod.2000,63,142-145.
[15] Ping Jiao, Dale C. Swenson, James B, et al. Chloriolide, a12-Membered Macrolidefrom Chloridium Wirescens var. chlamydosporum (NRR L37636)[J]. J. Nat. Prod.2006,69,636-639.
[16] Yuguo Du, Qi Chen, and Robert J. Linhardt. The First Total Synthesis of SporiolideA [J]. J. Org. Chem.2006,71,8446-8451.
[17] Chun Y i Chou, Duen Ren Hou. Synthesis of (+)-Cladospolide C[J]. J. Org. Chem.2006,71,9887-9890.
[18] Yalan Xing and George A. O’Doherty. De Novo Asymmetric Synthesis ofCladospolide B-D: Structural Reassignment of Cladospolide D via the Synthesis of itsEnantiomer [J]. Organic letters.2009,11(5):1107-1110.
[19] Birkinshaw and Raistrick, Biochem. J.,1936,30,801
[20] Bowden, Lythgoe, and Marsden1959,1662-1669
[21] Georges Hareau, et al. Asymetric synthesis of Palitantin from the (5R)–tert-butyldimethylsiloxy-2-cyclohexenone [J]. Tetrahedron.1999,40:7493-7496.
[22]Tridib Mahapatra, Samik Nanda. Asymmetric synthesis of palitantin by an enzymaticand organocatalytic approach [J]. Tetrahedron: Asymmetry.2009,20:610-615.
[23] Yasuo Kimura, Takashi Mizuno and Atsumi Shimada. Penienone and penihydrone,New plant growth regulators produced by the Fungus Penicillium. Sp. No.13[J].Tetrahedran Letters.1997,38(3):469-472.
[24] Hyang Burm Lee and Hyuncheol Oh. Two New Fungal Metabolites from anEpiphytic Fungus Paraphaeosphaeria Species [J]. Bull. Korean Chem. Soc.2006,27(5):779-782.
[25] Goverdhan Mehta and Subhrangsu Roy. Enantioselective Total Synthesis of(+)-Eupenoxide and (+)-Phomoxide: Revision of Structures and Assignmentof Absolute Configuration [J]. Organtic letters.2004,6(14):2389-2392.