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珪藻Cylindrotheca closterium藻体内に存在する赤潮ラ フィド藻Heterosigma akashiwoに対する殺藻物質の分離同 誌名 誌名 日本プランクトン学会報 ISSN ISSN 03878961 著者 著者 内田, 直行 小島, 崇宏 中園, 金吾 片桐, 律子 荒, 功一 広海, 十朗 巻/号 巻/号 57巻1号 掲載ページ 掲載ページ p. 21-29 発行年月 発行年月 2010年2月 農林水産省 農林水産技術会議事務局筑波産学連携支援センター Tsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research Council Secretariat

珪藻Cylindrotheca closterium藻体内に存在する赤潮ラ フィド …of Rt86 by LC/MS and LC/MS/MS were the same as those of a commercial cis-5,8,11,14-eicosatetraenoic acid

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  • 珪藻Cylindrotheca closterium藻体内に存在する赤潮ラフィド藻Heterosigma akashiwoに対する殺藻物質の分離同

    誌名誌名 日本プランクトン学会報

    ISSNISSN 03878961

    著者著者

    内田, 直行小島, 崇宏中園, 金吾片桐, 律子荒, 功一広海, 十朗

    巻/号巻/号 57巻1号

    掲載ページ掲載ページ p. 21-29

    発行年月発行年月 2010年2月

    農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat

  • �� Cylindrotheca closterium������������Heterosigma akashiwo�����������

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    1) ������ !"�# $252�8510 %&'()*+,-. 18662) /012345678 $250�0042 %&'(��9+:; 4643) ?@��ABCDEFG $112�0004 HIJKILMN 1�4�25

    Identification of algicidal substances in the cells of the bacillariophyceanCylindrotheca closterium acting on the red-tide raphidophycean flagellateHeterosigma akashiwo

    N6DNJ@> U8=>961)�, T6@6=>GD KDO>B61), 2), K>CIJ@D K6I6G>3),KD>8=> AG61), 6C9 JJGD H>GDB>1)

    1) Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University, Kameino 1866,

    Fujisawa, Kanagawa 252�8510, Japan2) Plant Bio PLC, Ogikubo 464, Odawara, Kanagawa 250�0042, Japan3) Chemicals Evaluation and Research Institute, Japan, Kouraku 1�4�25, Bunkyouku, Tokyo 112�0004, Japan�Corresponding author: E-mail: [email protected]

    Abstract We proposed previously that the bacillariophyceaen Cylindrotheca closterium secretedcis-6,9,12,15-octadecatetraenoic acid (ODTA), hexadecatrienoic acid (HDTA) and stearic acid-likefatty acid to the red-tide raphidophycean flagellate Heterosigma akashiwo as allelopathic sub-stances. In this paper, algicidal substances accumulated in the cells of C. closterium were identifiedto elucidate the relationship between these allelopathic substances and the algicidal substancesaccumulated in the cells.

    The cells were collected by filtration with a glass fiber filter from unialgal cultures of C.closterium under the initial stages of the stationary phase of growth. Algicidal substances wereextracted according to the method of Bligh-Dyer from cells ground with a mortar and pestle whilefrozen with liquid nitrogen. The fat-soluble extract was separated by silica gel preparativethin-layer chromatography into an algicidal fraction possessing slightly smaller Rf than that ofsaturated fatty acids such as oleic acid, suggesting that the fraction may be an algicidalunsaturated fatty acid fraction (AUFF). The AUFF, partially purified by HPLC on a gel filtrationcolumn, was separated into three aligicidal substances with retention time of 51 min (Rt51), 62min (Rt62) and 86 min (Rt86) by HPLC on a reverse phase preparative column. These threealigicidal substances (Rt51, 62 and 86) were isolated by HPLC on a reverse phase analyticalcolumn.

    LC/MS analysis of Rt51 by electrospray-ionization (negative) gave a precursor ion (M-1)� ofm/z 275. The precursor ion of m/z 275 generated product-ions with m/z 231 (corresponding tothe molecular formula of M-COOH) and 177 (M-C4H6COOH) by LC/MS/MS analysis, with theprofile being the same as that of a commercial ODTA. The acute toxicities (24h LC50) of Rt51 andODTA to H. akashiwo were 66 ng mL�1 and 66 ng mL�1, respectively.

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    �The Plankton Society of Japan 2010

    2009O 6P 2�QRS 2009O 9P 15�QT

    Bull. Plankton Soc. Japan 57(1): 21�29, 2010

  • Rt62 gave a precursor ion of m/z 301 (M-1)� by LC/MS analysis and the precursor iongenerated product-ions with m/z 257 (corresponding to the molecular formula of M-COOH) and203 (M-C4H6COOH) by LC/MS/MS analysis, of which the spectra were the same as those of acommercial cis-5,8,11,14,17-eicosapentaenoic acid (EPA). The 24h LC50 of Rt62 and EPA to H.akashiwo were 66 ng mL�1 and 73 ng mL�1, respectively.

    The spectra of Rt86 by LC/MS and LC/MS/MS were very similar to those of Rt62. Both theprecursor ion (m/z 303) and the product-ions (m/z 259 and 205), however, were larger by a factorof two in m/z than those of Rt62, suggesting that Rt86 was a fatty acid of C20 : 4. The mass spectraof Rt86 by LC/MS and LC/MS/MS were the same as those of a commercial cis-5,8,11,14-eicosatetraenoic acid (arachidonic acid, AA). The 24h LC50 of Rt86 and AA to H. akashiwo were66 ng mL�1 and 73 ng mL�1, respectively. From their mass spectra and the acute toxicities to H.akashiwo, Rt51, Rt62 and Rt86 were identified as ODTA, EPA and AA, respectively.

    The present work has shown that EPA and AA are major algicidal substances in the cells of C.closterium, and that HDTA and stearic acid-like fatty acid, major allelopathic substances in theunialgal cultures of C. closterium, are not detected in the cells as algicidal substances. On the otherhand, neither EPA nor AA were presented in the unialgal cultures as allelopathic substances inour previous paper. These results lead to the conclusion that allelopathic substances in theunialgal cultures seemed to be alternatively secreted from the cells of C. closterium.

    Key words: algicidal substance, cell, Cylindrotheca closterium, Heterosigma akashiwo, LC/MS, LC/MS/MS,ODTA, EPA, arachidonic acid, 24h LC50

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    Fig. 8. LC/MS of Rt51 and commercial cis-6,9,12,15-Octadecatetraenoic acid (ODTA) by electrospray-io-nization (negative).

    Fig. 9. LC/MS/MS of precursor ions (m/z 275) of Rt51 (A) and ODTA (B).

    Fig. 10. LC/MS of Rt62 and commercial cis-5,8,11,14,17-eicosapentaenoic acid (EPA) by electrospray-io-nization (negative).

    Fig. 11. LC/MS/MS of precursor ions (m/z 301) ofRt62 (A) and EPA (B).

    Fig. 12. LC/MS of Rt86 and commercial cis-5,8,11,14-eicosatetraenoic acid (arachidonic acid, AA) byelectrospray-ionization (negative).

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    Biondo, P. D., C. J. Field, D. V. Brindley, & M. B. Sawyer2008. The potential for treatment with dietary long-chainpolyunsaturated n-3 fatty acids during chemotherapy, J.Nutr. Biochem. 19: 787�796.

    Caro A. A. & A. I. Cederbaum 2001. Synergistic toxicity ofiron and arachidonic acid in HepG2 cells overexpressingCYP2E1. Mol. Pharmacol. 60: 742�752.������������ ���������

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    Graeme, A. D., K. V. John, M. B. Stephanie, L. Jeannie-Marie& S. W. Je#rey 1994. Essential polyunsaturated fattyacids from 14 species of diatom (bacillariophyceae). Phyto-chemistry 35: 155�161.�� !���"��#��� 1998 ��� !$"�#$%%&��&'( Heterocapsa circularisquama'�()*(+, ��) 64: 123�124.��"��-*+.�,�-. 1995 �� /01( Het-

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    Kakisawa, H., F. Asari, T. Kusumi, T. Toma, T. Sakurai, T.Oohusa, Y. Hara & M. Chihara 1988. An allelopathic fattyacid from the brown alga Cladosiphon okamuranus, Phyto-chemistry 27: 731�735.Z5��TU��[> 1990 \.�; pp. 38�55,�4VWXY]�^7_`8�a�b��� EZ[\ C4]^�_c`[a; AGH�bcd 2008��%�LMN) de�'ef)����%gRJ� /0 45: 14�16.f���g9�h���Ih�*i���ijN

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    Masia, A., S. V. Avery, M. A. Zoroddu, & G. M. Gadd 1998.Enrichment with a polyunsaturated fatty acid enhancesthe survival of Saccharomyces cerevisiae in the presence oftributyltin. FEMS Microbiol. Lett. 167: 321�326.lr s�Vtom�nu�o��pt�M�+qrs�4567�tu v 2008 v&'( Akashiwo sanguinea'�()wxy2(�'I)/0�zw� ��) 74: 598�606.lV�{�#�|}���~K 2000 /x1( Hetero-

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