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Biochemical Systematics and Ecology 36 (2008) 223e226 www.elsevier.com/locate/biochemsyseco

Sesquiterpenes from the introduced red seaweed Laurencia caduciramulosa (Rhodomelaceae, Ceramiales) Vale´ria Cassano a, Joel Campos De-Paula a, Mutue Toyota Fujii b, Bernardo Antonio Perez Da Gama c, Vale´ria Laneuville Teixeira c,* a

Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcaˆntara Gomes, Departamento de Biologia Vegetal, Rua S~ao Francisco Xavier, 524, Maracan~a, 20550-013 Rio de Janeiro, Brazil b Instituto de Botaˆnica, Sec¸~ao de Ficologia, Av. Miguel Este´fano, 3687, 04301-012 S~ao Paulo, SP, Brazil c Universidade Federal Fluminense, Instituto de Biologia, Departamento de Biologia Marinha, Caixa Postal 100.644, 24001-970 Nitero´i, RJ, Brazil Received 15 June 2007; accepted 22 July 2007

Keywords: Laurencia caduciramulosa; Rhodophyta; Sesquiterpenes; Chemotaxonomy; Antifouling

1. Subject and source In continuation to our phycochemical studies of Brazilian red algae of the genus Laurencia J.V. Lamouroux (Davyt et al., 2001, 2006; Carvalho et al., 2003, 2006; Da Gama et al., 2003) we focused the present work on the analysis of the chemical constituents from Laurencia caduciramulosa Masuda et Kawaguchi in Masuda et al., 1997. The specimens were collected at Ilha Grande Bay, Rio de Janeiro, Brazil: Angra dos Reis, Ilha Grande Island, Lagoa Azul’s Island (23 050 0600 S, 44 140 2800 W), in 29 July 2005 (HRJ 10457, SP 365680) and Piraquara de Fora Cove, Velho’s Beach (23 010 1200 S, 44 260 1200 W), in 19 April 2006 (HRJ 10652, SP 371294). Voucher specimens are deposited in the herbaria of the Universidade do Estado do Rio de Janeiro (HRJ) and Instituto de Botaˆnica, S~ao Paulo, Brazil (SP). The type material of L. caduciramulosa provided by the Herbarium of the Hokkaido University was analyzed for morphological comparisons (holotype: SAP 062086, type locality: Hon Tre Island, Tien Hai Islands, Hatien, Kien Giang Province, Vietnam). 2. Previous study L. caduciramulosa was originally described for Vietnam (Masuda et al., 1997). Its geographical distribution was expanded to Malaysia (Masuda et al., 2001), Mediterranean Sea [Furnari et al., 2001 (Italy); Klein and Verlaque, 2005 (France)], and Indonesia (Liao et al., 2004). Klein and Verlaque (2005) suggested the hypothesis of its recent introduction into the Mediterranean Sea. L. caduciramulosa was reported for the first time in the Atlantic Ocean at 10 different sites in Ilha Grande Bay, State of Rio de Janeiro, Brazil by Cassano et al. (2006), when the hypothesis of its * Corresponding author. Fax: þ55 21 2629 2292. E-mail address: [email protected] (V.L. Teixeira). 0305-1978/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2007.07.005

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recent introduction into Brazilian coastal environments was suggested to have occurred through transoceanic shipping transport. Sesquiterpenes are usually the most abundant compounds in Laurencia species, followed by acetogenins, diterpenes, and triterpenes (Pereira and Teixeira, 1999). In regard to L. caduciramulosa from the type locality, the species was chemically characterized by the following halogenated secondary metabolites: aplysiadiol (diterpene), deoxyprepacifenol (sesquiterpene), laurenenyne A and B (C15 acetogenins) (Masuda et al., 1997). 3. Present study Air-dried specimens (32 g for Angra dos Reis, Ilha Grande Island, Lagoa Azul’s Island and 50 g for Piraquara de Fora Cove, Velho’s Beach) of L. caduciramulosa were extracted with CH2Cl2 at room temperature, separately, to yield two crude extracts (1 and 2, respectively). Evaporation of the crude extract 1 yielded 191 mg of a brownish residue. The extract 1 was examined by TLC and eluted with 20% EtOAc in CH2Cl2. Analytical TLC was performed on Merck Kieselgel GF254 layers, and the compounds were detected by spraying with a 2% solution of Ce(SO4)2 in 2 N H2SO4 followed by heating at 100  C. The extract 1 was subjected to silica gel-column chromatography (70e230 mesh e Merck), eluted with CH2Cl2, EtOAc, and MeOH, resulting in 50 fractions (100 ml each). The second fraction eluted with CH2Cl2 presented a 1:1 mixture of two sesquiterpenes. The 1HNMR spectrum revealed two para-disposed aromatic protons at d 6.51 (s, 1H) and 7.10 (s, 2H), two ortho-disposed aromatic protons at d 6.71 (d, J ¼ 7.8 Hz, 1H) and 7.01 (d, J ¼ 7.8 Hz, 1H), aromatic methyl groups at d 2.27 (s, 3H) and 2.29 (s, 3H), and aliphatic methyl groups at d 1.36 (s, 3H), 1.37 (s, 3H), 1.22 (s, 3H), 1.21 (s, 3H), 0.72 (d, J ¼ 7.2 Hz, 3H) and 0.71 (d, J ¼ 6.9 Hz, 3H). The compounds were identified as laurane cyclic ethers (1 and 2) by comparison of physical and spectroscopic data with reported values (Gochfeld and Hamann, 2001; Nemoto et al., 1995). These ethers possess a six-membered oxide ring rather than the five-membered ring of the aplysin series. Filiformin (1) and debromofiliformin (2) can be considered as transformation products of allolaurinterol (3) and debromoallolaurinterol (4), spontaneously cyclized under acidic conditions (Erickson, 1983). The seventh fraction eluted with CH2Cl2 yielded crude pacifenol (5). Evaporation of the second crude extract yielded 660 mg of brownish residues then subjected to silica gel-column chromatography (70e230 mesh e Merck) (elution with n-hexane, CH2Cl2, EtOAc, and MeOH), resulting 93 fractions. The fraction eluted with hexane/CH2Cl2 (2:8) yielded pure pacifenol (5). Pacifenol was identified by comparison of physical and spectroscopic data with reported values (Argandon˜a et al., 1993). Compounds 1e4 were observed as minority components of the crude extract. H

H

H

OH

O

O

Br

H

H

Br

H H

H

1

2

3

H Br

OH

O H

Cl

H H

4

Br OH

5

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The crude dichloromethane extract was assayed for its antifouling properties using the mussel Perna perna Linnaeus, 1978, as described by Epifanio et al. (2006). The natural concentration of the extract obtained from L. caduciramulosa was tested in the laboratory using the ‘mussel test’, a screening method for antifouling substances whose results have been shown to have good reproducibility in more ecologically relevant field experiments (Da Gama et al., 2003 and references therein). Antifouling activity was measured by the property of juvenile mussels producing byssal threads in order to attach to preferred substrata, consisting of water-resistant filter paper circles (9 cm diameter) soaked in solvent alone (control filter) or in a natural concentration of extract (determined as the extract equivalent to the dry weight of organism ¼ DW of filter paper). After 12 h, all records of attachment (three juvenile mussels per petri dish, 10 replicates per treatment) were checked, mussels were placed in mesh bags tagged according to treatment, and suspended in a sea aquarium for 24 h to check for mortality due to exposure to the test substances. After the trials, treated filter papers were taken from dishes, allowed to air dry and then reextracted and applied to a TLC plate for comparison with the original extracts. 4. Chemotaxonomic significance The species is characteristically small (up to 5 cm high), with a well-developed stolon-like basal system and abundant deciduous branchlets at the upper portion of the erect axes that function as propagules. The five compounds detected from Brazilian specimens of L. caduciramulosa are reported for the first time in Laurencia species from the Brazilian coast. Only pacifenol has been previously isolated from the Brazilian sea hare Aplysia dactilomela Rang, 1828 (Kaiser et al., 2001). The compounds described by Masuda et al. (1997) for L. caduciramulosa from Vietnam were not found in Brazilian material. L. caduciramulosa from Brazil produces only sesquiterpenes differing from the mixture of sesquiterpenes and diterpenes reported for this species from Vietnam. The analysis of the type material revealed that there are no morphological differences between L. caduciramulosa from Brazil and Vietnam. This fact has drawn our attention to a possible mixture of material in L. caduciramulosa from Vietnam, since the simultaneous production of sesquiterpenes and diterpenes is an uncommon feature in the Laurencia complex. 5. Ecological significance The results revealed that the crude extract from L. caduciramulosa exhibited significant antifouling activity relative to controls with solvent alone (77% inhibition, Fig. 1). This is particularly interesting given that this is an introduced 16 Mean

14

n = 10 p = 0.005

±SE ±SD

Mussel attachment

12 10 8 6 4 2 0 -2 -4

Control

L. caduciramulosa

Treatments Fig. 1. Mussel attachment (mean number of mussel byssal threads attached  standard error e SE, standard deviation e SD) to control and L. caduciramulosa treatments. Significant difference indicated is from the Wilcoxon signed ranks test (n ¼ number of replicates per treatment).

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species, which could successfully inhibit further colonization of the habitat once established by the production and possibly release of antifouling compounds, whose nature remains to be elucidated. Acknowledgments We wish to thank Michio Masuda for the loan of the type specimen. M.T.F., V.L.T. and B.A.P.G. thank CNPq for Research Productivity fellowships. This study was partially supported by CNPq (Proc. 478941/2006-4) and by the Coordenac¸~ao de Aperfeic¸oamento de Pessoal de Nı´vel Superior (Capes) to V.C. References Argandon˜a, V.H., San-Martı´n, A., Rovirosa, J., 1993. Phytochemistry 32, 1159. Carvalho, L.R., Fujii, M.T., Roque, N.F., Lago, J.H.G., 2003. Tetrahedron Lett. 44, 2637. Carvalho, L.R., Fujii, M.T., Roque, N.F., Lago, J.H.G., 2006. Phytochemistry 67, 1331. Cassano, V., de Sze´chy, M.T.M., Fujii, M.T., 2006. Cryptogam. Algol. 27, 265. Da Gama, B.A.P., Pereira, R.C., Soares, A.R., Teixeira, V.L., Yoneshigue-Valentin, Y., 2003. Biofouling 19, 161. Davyt, D., Fernandez, R., Suescun, L., Mombru´, A.W., Saldan˜a, J., Domı´nguez, L., Fujii, M.T., Manta, E., 2006. J. Nat. Prod. 69, 1113. Davyt, D., Fernandez, R., Suescun, L., Mombru´, A.W., Saldan˜a, J., Dominguez, L., Coll, J., Fujii, M.T., Manta, M., 2001. J. Nat. Prod. 64, 1552. Epifanio, R.A., Da Gama, B.A.P., Pereira, R.C., 2006. Biochem. Syst. Ecol. 34, 446. Erickson, K.L., 1983. In: Scheuer (Ed.), Marine Natural Products e Chemical and Biological Perspectives. Academic Press, New York, p. 131. Furnari, G., Cormarci, M., Serio, D., 2001. Cryptogam. Algol. 22, 331. Gochfeld, D.J., Hamann, M.T., 2001. J. Nat. Prod. 64, 1477. Kaiser, C.R., Pitombo, L.F., Pinto, A.C., 2001. Magn. Reson. Chem. 39, 147. Klein, J., Verlaque, M., 2005. Cryptogam. Algol. 26, 209. Liao, M., Uy, F.A., Heyrosa, N.A., 2004. Raffles Bull. Zool. 11, 19. Masuda, M., Kawaguchi, S., Takahashi, Y., Matsuo, Y., Suzuki, M., 1997. Cryptogam. Algol. 18, 1. Masuda, M., Abe, T., Kawaguchi, S., Phang, S.M., 2001. Bot. Mar. 44, 467. Nemoto, H., Miyata, J., Hakamata, H., Nagamochi, N., Fukumoto, K., 1995. Tetrahedron 51, 5511. Pereira, R.C., Teixeira, V.L., 1999. Quı´m. Nova 22, 369.