Bull. Soc. Sea Water Sci., Jpn., 60, 195 -198 (2006)

Bulletin of the Society of

Sea Water Science, Japan

Note

Content and Composition of Brominated Compounds in Marine Sponges

Yukiho YAMAOKA *1, Sinji OHTA *2, Marvelisa L. CARIVIONA * 3 and Jose M. OCLARIT *3

AbstractMarine sponges were collected from the Seto Inland Sea and the Mindanao Sea. Brominated compounds in 10 species of marine

sponges were investigated using GC-MS. Eleven compounds were identified and determined by GC-MS in Dysidea sp.: 2, 4-

dibromophenol (1), 2-(4'-dibromophenoxy)-3-bromophenol (3), 2-(4' -dibromophenoxy)-3, 5-dibromophenol (4), 2-(4'-

dibromophenoxy)-4, 6-dibromophenol (5), 2-(2', 4'-dibromophenoxy)-3, 5- dibromophenol (6), 2-(2',4'- dibromophenoxy)-4, 6-

dibromophenol (7), 3, 5-dibromo-2-(3', 5'-dibromo, 2'-methoxyphenoxy) phenol (8), 2-(2', 4'-dibromophenoxy)-4-chloro, 3, 5-

dibromophenol (9), 2-(2', 4'-dibromophenoxy)-3, 4, 5-tribromophenol (10), and 2-(2', 4'-dibromophenoxy)- 4, 5, 6-

tribromophenol (11). These major components in the brominated compounds of Dysidea sp. and Phyllospongia sp. were identified as:

(4), (6) and (10), (6) and (7) respectively. We detected no brominated compounds in other marine sponges.

Key Words: Brominated compounds, GC-MS, Marine sponges; Dysidea sp., Phyllospongia sp.

1. Introduction

Numerous bromophenolic and chlorophenolic

compounds have been isolated as natural products;

marine life is the largest source of organohalogen

compounds'). Brominated compounds with antibacterial

activity have been isolated from various sponges 2-5). The

marine sponge Dysidea sp. (family Dysideidae, Order

Dendroceratida) occurs in two chemotypes: one

chemotype contains only polybrominated diphenyl ethers,

whereas the second chemotype contains polychlorinated

amino acid derivatives and sesquiterpenes 1.9). In

addition, 2, 4-dibromophenoxy-2,4-dibromophenol and its

analo-gues, collectively called polybrominated diphenyl

ethers, have been isolated from Dysidea herbacea, Dysidea

chlorea and Phyllospongia foliascens. They have unique

biological activities toward microorganisms 3). Recently, it

was suggested that production of polybrominated

diphenyl ethers of Dysidea sp. is caused by a

cyanobacterium (Oscillatoria spongeliae) and not by the

sponge or symbiotic heterotrophic bacteria 6 7) For a

successful search for such a sponge, it is necessary to use

a sensitive analytical technique that permits

determination of small amounts (picogram-scale) of

necessary metabolites. The most reliable and sensitive

technique is GC-MS 7). This paper describes the

identification and determination of brominated

compounds of marine sponges using GC-MS.

2. Experiments

Thorecta, Dysidea, Theonella, Cinachyra, Acanthella,

Phyllospongia, Aaptos (Magsaysay Islet in Bohol Sea,

Philippines) and Haliclona permollis, Halichondria

japonica, Halichondria okadai (Seto Inland Sea, Japan)

were collected during skin diving excursions off the

islands' shores. The marine sponge sample (ca. 5 g,

freeze-dried weight) was extracted with CHC13 : methanol=2:1(500ml×3 times) .The extract was evaporated

under reduced pressure. The　 extract(500 mg)was

chromatographed on a column(60×2.5 cm diam.) of

silica gel using eluents of increasing polarity from hexane

through ether to ethyl acetate. GC-MS analyses were

carried out with a GC-MS QP-5000 (Shimadzu Corp.)

fitted with a fused silica DB-5 capillary column (0.22 mm×30 m , 0.25μm film thickness; J&W Scientific Inc.)

with helium gas at a flow rate of 1 ml/min. The sample

was injected in the splitless mode (sampling time, 5 min).

The respective temperatures of the GC injection port and

ionization chamber were 290℃ and 280℃. Temperature

programs for the column oven were as follows: program,

60℃ for 1 min, elevated to 130℃ at 20℃/min, then to

210℃ at 10℃/min, then to 260℃ at℃/min, then to

300℃ at 10℃/min;it was finally maintained at 300℃.

3. Results and discussion

Hexane/ether (1 / 1) fractions that contained phenolic

*1 National Institute of Advanced Industrial Science and Technology, Hiroshima 737 - 0197, Japan *2

Nagahama Institute of Bio-Science and Technology, Siga 526-0829, Japan *3 Department of Biological Sciences

, MSU-Illigan Institute of Technology, Illigan City, Philippines

195

196Bull. Soc. Sea Water Sci.,Vol. 60, No.3 (2006)

compounds were combined and analyzed using GC-MS.

Fig. 1 shows a typical gas chromatogram (TIC) of the

hexane/ether fractions in the gray-green sponge Dysidea

sp. The retention times and mass spectra of known

compounds coincide with those of peak No. 1 and 3-11 on

GC-TIC (Table 1). Furthermore, the compound identities

were confirmed by comparing their spectral data with

those of published values 3,5). Table 1 and Fig. 1 show

eleven compounds identified in Dysidea by full-scan GC-

MS: 2, 4-dibromophenol (1), 2 - (4' - dibromophenoxy) - 3 -

bromophenol (3), 2 - (4' -dibromophenoxy) - 3, 5 -

dibromophenol (4), 2 - (4' - dibromophenoxy) - 4, 6 -

dibromophenol (5), 2 - (2', 4' - dibromophenoxy) - 3, 5 -

dibromophenol (6), 2 - (2', 4' - dibromophenoxy) - 4, 6 -

dibromophenol (7), 3, 5 - dibromo - 2 - (3', 5' - dibromo, 2' -

methoxyphenoxy) phenol (8), 2 - (2', 4' - dibromo-

phenoxy) - 4 - chloro, 3, 5 - dibromophenol (9), 2 - (2', 4' - dibromophenoxy) - 3, 4, 5 - tribromophenol (10), and 2 -

(2', 4' - dibromophenoxy) - 4, 5, 6 - tribromophenol (11). In

addition, fractions eluted with hexane/ether in

Phyllospongia sp. contained brominated compounds (6)

and (7) (Table 2). On the other hand, brominated

compounds were not detected in Thorecta, Theonella,

Cinachyra, Acanthella, Aaptos, Haliclona permollis,

Halichondria japonica, or Halichondria okadai. Already,

the identity of brominated compounds (6, 7, 10, and 11) in

hexane/ether fractions has been confirmed using 3H and 13C NMR, and EI- MS 8). A collection of Dysidea herbacea

Retention time (min)

Fig.1 Gas chromatogram (TIC) and structure of the brominated compounds in Dysidea sp. using GC-MS method.

Column conditions, J&W fused silica DB-5 capillary column(0.22 mm×30m, 0.25μm film thickness).

Temperature programs for the column oven were: program, 60℃ for 1 min; elevated to 130℃ at 20℃/min;

then to 210℃ at 10℃/min;then to 260℃ at 5℃/min;then to 300℃ at 10℃/min;and finally maintained at

300℃. Injection temp. 290℃. Ionization temp. 280℃. Carrier gas-He,1ml/min. The sample was injected in

splitless mode(sampling time,5min)

Tabel 1 Identification of brominated compounds in the Dysidea sp. by GC-MS

Y. YAMAOKA S. OHTA M. L. CARMONA J. M. OCLARIT: Content and Composition of Brominated Compounds in Marine Sponges 197

Tabel 2 Contents of brominated compounds in Dysidea sp. by GC-MS analysis

μg/g dry weight

from the Caroline Islands in the Pacific Ocean yielded a

series of brominated compounds (3, 4, 7, 10 and 11), the

structures of which were established through syntheses

of various representatives 2, 10). In addition, four known

brominated compounds (6, 7, 8 and 10) have been

isolated from an undescribed Dysidea sp. collected at

Setawan Atoll, Micronesia11). Identification of ten

brominated compounds at a time from one sponge is

unprecedented. From the gas chromatography pattern,

changes from the dimeric brominated compounds to

trimeric isomers, tetrameric isomers, and pentameric

isomers are presumed. Brominated compounds of (9)

contain chlorine and are presumed to be involved in the

change of (11) into a tetramer. Major components (1-11)

of brominated compounds were determined by standard

curves on the SIM for addition of each brominated

compound to CH2C12 solutions. Based on the GC-MS-SIM

data, the amounts of respective brominated compounds

were estimated roughly as follows (Table 2): (6) 10.6; (7)

1.3; (4) 2.5; (5) 0.9; (10) 6.5; (11) 1.4; and (9)0.02 mg/kg

dry weight. In addition, Phyllospongia contained (6) 0.2

and (7) 0.1 mg/kg dry weight. These major components

in brominated compounds in Dysidea and Phyllospongia

were identified as (6), (7). Sharma et al.2), Carte et al.3),

and Handayani et al.5) isolated many brominated

compounds as antibacterial substances from Dysidea

herbacea. Also the adovementioned difference in

brominated compounds might be attributable to

differences among sponge species. Previous studies have

attributed this content variation in Dysidea herbacea to

different algal or bacterial symbionts associated with

sponges 0. Kuniyoshi et al.12) isolated (6) and (7) as

antibacterial substances from the green alga Cladophora

fascicularis. Vetter et al.13,14) detected (6) in dolphin and

mammals from Australians. Melcher et al.15) recognized

brominated compounds as critical residues in foodstuff

(e.g.fish ) and environmental samples(e.g. marine

mammals and birds) Unfortunately, no toxicologic data

exist for brominated compounds natural products. This is

deplorable in view of the high concentrations and these

brominated compounds can reach in higher organisms of

the marine food web 15). Details have not been elucidated

yet: the bromination process must be examined in thefuture.

References

1) Y. Naya, "Marine Natural Products," NipponKagakukai, 25, pp.88-110 (1979) (Japanese)

2) G.M. Sharma and S. Vig, "Studies on theAntimicrobial Substances of Sponge. VI Structures ofTwo Antibacterial Substances Isolated from theMarine Sponge Dysidea Herbacea", TetrahedronLetters, pp.1715-1718 (1972)

3) B. Carte and D. J. Faulkner, "PolybrominatedDiphenyl Ethers from Dysidea herbacea, Dysideachlorea and Phyllospongia foliascens", TetrahedronLetters, 37, pp.2335-2339 (1981)

4) S. Hirsch, A. Rudi and Y. Kashman, "New Avaroneand Avarol Derivatives from the Marine SpongeDysidea cinerea", J. Nat. Pro. 54, pp.92-97 (1991)

5) D. Handayani, R.A. Edrada, P. Proksch, V. Wray, L.Witte, R.W.M. Van Soest, A. Kunzmann, andSoedarsono, "Four New Bioactive PolybrominatedDiphenyl Ethers of the Sponge Dysidea herbacea fromWest Sumatra, Indonesia", J. Nat. Prod., 60, pp.1313-1316 (1997)

6) G.B. Elyakov, T. Kuznetsova, V.V. Mikhailov, 1.1.Maltsev, V.G. Voinov and S.A. Fedoreyev,"Brominated Diphenyl Ethers from a Marine

Bacterium Associated with the Sponge Dysidea sp".,Experientia, 47, pp.632-633 (1991)

7) V.G. Voinov, Y.N. EL'kin, T.A. Kuznetsova, I.I.Mal'tsev, V.V. Mikhailov and V.A. Sasunkevich, "Useof Mass Spectrometry for the Detection andIdentification of Bromine-containing Diphenyl

198 Bull. Soc. Sea Water Sci.,Vol. 60, No.3 (2006)

Ethers", J. Chromatogr., 586, pp.361-362 (1991)8) R.M. Malaluan, M.L. Carmona, J.M. Oclarit, Y.

Yamaoka and S. Ohta, "Extraction of bioactiveMetabolite from the Marine Sponge Dysidea sp.Using Supercritical Carbon Dioxide, The 8thAPCCbE Congress Proceedings 2, pp.893-897 (1999)

9) W. Hofheinz and W.E. Oberhansli, "Dysidin, a NovelChlorine-containing Natural Product from the SpongeDysidea herbacea", Helvetica Chimica Acta, 60, pp.660-669 (1977)

10) B.R. Capon, E.L. Ghisalberti, P.R. Jefferies, B.W.Skelton and A.H. White, "Structural Studies ofHalogenated Diphenyl Ether from a Marine Sponge",

J. C. S. Perkin, 1 pp.2464-2467 (1981)11) X. Fu, F.J. Schmitz, M. Govindan, S.A. Abbas, K.

Hanson, P.A. Horton, P. Crews, M. Laney and R.C.Schatzman, "Enzyme Inhibitors: New and KnownPolybrominated Phenols and Diphenylethers fromFour Indo-Pacific Dysidea sponges", J. Nat. Prod., 58,

pp.1384-1391 (1995)12) M.Kuniyoshi, K.Yamada and T.Higa, "Abiologically

Active Diphenyl Ether from the Green AlgaCladophora fascicularis", Experientia, 41, pp.523-524(1985)

13) W. Vetter, E. Stoll, M. J. Garson, S. J. Fahey, C. Gausand J. F. Muller, "Sponge Halogenated NaturalProducts Found at Parts-Per-Million Levels in MarineMammals", Environ. Toxicol. Chem., 21, pp.2014-2019 (2002)

14) W. Vetter, E. Scholz, C. Gause, J. F. Muller and D.Haynes, "Anthropogenic and Natural OrganohalogenCompound in Blubber of Dolphins and Dugongs

(Dugong dugon) from Northeastern Australia", Arch.Environ. Contam. Toxicol., 41, pp.221-231 (2001)

15) J. Melcher, D. Olbrich, G. Marsh, C. Gause, J. F.Muller and W. Vetter, "Identification and

Quantification of the Halogenated Natural ProductBC-3", Organohalogen Compounds, 66, pp.431-436

(2004)

(平 成18年3月13日 受 付,平 成18年3月17日 採 用 決 定

Received Mar. 13, 2006, Accepted Mar. 17, 2006 )

GC-MSに よる海綿中の臭素化合物 の同定

山 岡 到 保*1,太 田 伸 二*2,マ ル ビ リサ ・カ ル モ ナ*3,ホ セ ・オ ク ラ リ ッ ト*3

要 昏

瀬 戸 内海 及 び フ ィ リピ ンで10種 類 の海 綿 を採 取 し,ポ リ臭 素化 合物 を研 究 した.Dysidea sp.か ら,2,4-dibromophenol,

2-(4'-dibromophenoxy)-3-bromophenol, 2-(4'-dibromophenoxy)-3,5-dibromophenol, 2-(4'-dibromophenoxy)-4,6-

dibromophenol, 2-(2',4'-dibromophenoxy)-3,5-dibromophenol, 2-(2',4'-dibromophenoxy)-4,6-dibromophenol, 3,5-

dibromo-2-(3',5'-dibromo, 2'-methoxyphenoxy) phenol, 2-(2',4'-dibromophenoxy)-4-chloro-3,5-dibromophenol, 2-(2',4'-

dibromophenoxy)-3,4,5-tribromophenol‚Æ2-(2',4'-dibromophenoxy)-4,5,6-tribromophenol 0)10 種類の臭素化合物をGC-

MSで 同 定 ・定 量 した.Dysideasp.中 の主 な臭 素 化合 物 は, 2-(2',4'-dibromophenoxy)-4,6-dibromophenol(6), 2-(2',4'-

dibromophenoxy)-3,5-dibromophenol(7), 2-(4'-bromophenoxy)-3,5-dibromophenol.‚Æ2-(2',4'-dibromophenoxy)-3,4,5-

tribromophenol‚Å, Phyllospongia sp. ‚Å‚Í, (6 ) と(7)で あ った.他 の海 綿 で は,ポ リ臭素 化合 物 は検 出 され なか っ た.

キー ワ ー ド:海 綿,臭 素 化 合物,含 量,組 成

*1独 立行政法人 産業技術総合研究所(〒737-0197広 島県呉市広末広2-2-2)

*2長 浜バイオ大学(〒526-0829滋 賀県長浜市 田村 町1266)

*3 Department of Biological Sciences, MSU-Illigan Institute of Technology, Illigan City, Philippines