NPC Natural Product Communications Chemical Variation in the Diterpenes from the Brazilian Brown Alga Dictyota mertensii (Dictyotaceae, Phaeophyta) 2007 Vol. 2 No. 1 13 - 15 Odinéia do Socorro Pamplona Freitasa, Aline Santos de Oliveirab, Joel Campos De-Paulac, Renato Crespo Pereirab, Diana Negrão Cavalcantib and Valéria Laneuville Teixeirab,* a Pós-Graduação em Química Orgânica, Instituto de Química, Universidade Federal Fluminese, Niterói, RJ, 24020-150, Brazil b Pós-Graduação em Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, CP 100.644, Niterói, RJ, 24001-970, Brazil c Departamento de Biologia Vegetal, Instituto de Biologia Roberto Alcântara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, 20550-013, Brazil gbmvalt@vm.uff.br Received: July 13th, 2006; Accepted: July 29th, 2006 Crude extracts of specimens from five populations of the Brazilian brown seaweed Dictyota mertensii (Martius) Kützing were analysed by HRGC-MS. A total of seven diterpenoids were identified: pachydictyol A (1), isopachydictyol A (2), dictyol B and its natural acetate (3-4), dictyoxide (5), dictyol C (6), dictyol H (7), and fucosterol (8), the last being the most abundant metabolite in four of the extracts. The results confirm that small-scale local variability in the chemicals found in D. mertensii probably is more significant than the overall latitudinal differences commonly observed in some marine organisms. Keywords: Dictyota mertensii, Dictyotaceae, diterpenes, chemotaxonomy, quantification, HRGC-MS. In order to continue our chemotaxonomic and geographical studies of the genus Dictyota Lamouroux (Dictyotales, Phaeophyta) [1-6], we examined the chemical variation in the diterpenes from D. mertensii (Martius) Kützing in five localities along the Brazilian coast, using an HRGC-MS technique. In previous studies of this alga [7-8], the prenylated guaianes pachydictyol A, dictyol H and dictyol C were isolated. The wide distribution of this seaweed along the Brazilian coast makes possible the study of geographic variations in the production of diterpenes, an important characteristic in the establishment of the use of diterpenes as Dictyota chemomarkers and in chemical marine ecology studies. On the other hand, this species has taxonomic limits very well established by morphological aspects easily visible in the field. D. mertensii is distributed in the Atlantic Ocean from the coast of Florida to that of Rio de Janeiro, including the Bahamas, Greater Antilles, Lesser Antilles, Southern Caribbean, Canary Islands and Cape Verde Islands [9-11]. This species is also cited for the African coast (Gabon) [10], for South-west Asia (Philippines) and the Pacific Islands (Fiji) [12,13]. De Clerck [14] noted that the name usually applied to this algal species, D. dentata Lamouroux, is illegitimate. Crude extracts of D. mertensii from five different localities were analyzed by HRGC-MS. This analysis revealed the presence of peaks in the mass spectra compatible with the fragmentation patterns of fatty acids (myristic, palmitic and oleic acids), natural fatty acid esters (myristic acid ethyl ester, palmitic acid methyl and ethyl esters, and steraric acid ethyl ester), seven diterpenes (1-7) and fucosterol, generally the major sterol of Dictyotaceae. The mass spectra of the diterpenes are summarized in Table 1. 14 Natural Product Communications Vol. 2 (1) 2007 Freitas et al. Table 1: Principal peaks observed in the mass spectra (m/z and relative intensity in parenthesis) of the chemical constituents from D. mertensii. * Rt * 7.4 [M]+ 288 9.4 9.6 288 288 11.8 306 13.1 304 14.1 362 14.2 346 Principal peaks 288 (12); 270 (4); 255 (5); 227 (3); 188 (22); 177 (22); 175 (13); 159 (40); 121 (70); 107 (100); 91 (64 ); 69 (90); 55 (60); 41 (100) 288(22); 270 (23); 255 (5); 203 (28); 175 (16); 159 (78); 105 (72); 91 (58); 69 (76); 41 (100) 288(12); 270 (6); 255 (7); 227 (4); 188 (20); 173 (20); 159 (96); 145 (42); 119 (70); 107 (59); 91 (61); 69 (71); 55 (61); 41 (100) 306(5); 288 (37); 270 (12); 255 (28); 245 (6); 203 (27); 185 (25); 177 (39); 159 (69); 145 (22); 121 (37); 119 (72); 81 (53); 69 (78); 41 (100) 304 (1); 286 (50); 268 (10); 253 (4); 225 (9); 215 (9); 197 (22); 186 (22); 173 (28); 157 (100); 155 (9); 142 (27); 129 (29); 105 (30); 91 (35); 79 (27); 69 (45); 55 (29); 41 (45) 302 (14); 284 (14); 274 (3); 269 (4); 261 (5); 243 (8); 241 (4); 225 (5); 213 (9); 203 (8); 199 (14); 185 (46); 157 (20); 143 (52); 133 (24); 125 (62); 121 (33); 109 (33); 107 (42); 93 (27); 91 (28); 81 (47); 71 (31); 55 (20); 43 (100) 346 (0,1); 328 (5); 286 (25); 268 (28); 253 (6); 225 (20); 201 (11); 197 (28); 186 (31); 173 (18); 157 (100); 142 (25); 129 (27); 117 (21); 105 (31); 91 (32); 81 (25); 69 (54); 55 (27); 43 (45) Compound Dictyoxide (5) Pachydictyol A (1) Isopachydictyol A (2) Dictyol C (6) Dictyol B (3) Dictyol H (7) Dictyol B acetate (4) Retention time in minutes Table 2: Percentage composition of the diterpenes and fucosterol in specimens of D. mertenssi from the 5 different localities. Identified compounds Dictyoxide Pachydictyol A Isopachydictyol A Dictyol B Dictyol C Dictyol H Dictyol B acetate Fucosterol Undetermined compounds, fatty acids and natural fatty acids esters Praia Rasa (RJ) trace trace trace none 0.9 4.9 none 8.1 86.1 Regions in the Brazilian littoral Praia Preta (RJ) Itapuã (BA) Riacho Doce (AL) 1.3 2.1 1.4 0.8 1.5 1.7 trace 0.5 1.7 19.8 22.1 5.3 4.2 4.2 1.5 none none none 2.7 2.1 2.1 22.7 12.7 7.1 48.5 54.8 79.2 The populations of D. mertensii analyzed presented various diterpenes as major metabolites, all prenylated guaianes, which would result from cyclization of geranyl-geraniol between positions 1 and 10 [1-4]. Pachydictyol A, isopachydictyol A, and dictyol C were the most frequent diterpenes, occurring in the collections from all the localities (Table 2). However, fucosterol was the most abundant product in four of the extracts analyzed, which corroborates previous studies [15]. Dictyol-H, the diterpene proposed as the taxonomic marker of D. mertensii, was only observed in specimens of the populations of Atol das Rocas (3.5% of the crude extract) and Praia Rasa (4.9%). In the remaining populations, dictyol B was the major diterpene (19.8, 22.1 and 5.3% of the crude extracts of the Praia Preta, Itapuã, and Riacho Doce collections, respectively). These results gave evidence that phenotypic differences between specimens from 5 different populations of D. mertensii along the Brazilian coast may be chemically based. Because of the variability in the presence and abundance of the diterpenes found in D. mertensii, we supposed that this variation could be influenced by local environmental factors. Atol das Rocas (RN) trace 2.3 trace none 4.4 3.5 none 18.3 71.5 Experimental Algal material: Specimens of D. mertensii were collected during July 1999/February 2005 from Praia Rasa, Búzios, in the north (lat. 22º44’49”S, long. 41º52’54”W) and Praia Preta, Ilha Grande, in the south of Rio de Janeiro State (lat. 23º00’24”S, long. 44º19’05”W), at Praia de Itapuã, Salvador, Bahia State (lat. 12º58’16”S, long. 38º30’39”W), at Praia do Riacho Doce, Maceió, Alagoas State (lat. 09º39’57”S, long. 35º44’07”W, and at Atol das Rocas reef, Rio Grande do Norte State (lat. 03º51' S, long. 33º40' W), the only atoll in the South Atlantic. The algae were collected at depths ranging from 2 to 5 m. Voucher specimens are deposited in the Herbarium of the Universidade do Estado do Rio de Janeiro (HRJ). Extraction: The air-dried algal material was extracted with either dichloromethane or acetone (only specimens from Atol das Rocas), at room temperature (25oC). The solvent was evaporated under reduced pressure yielding brownish residues. Aliquots of the crude extracts (20 mg for each population) were filtered on silica gel (elution with 30 mL of dichloromethane/ethyl acetate 9:1). Diterpenes from Dictyota mertensii HRGC-MS analysis: An aliquot of each extract was diluted in an appropriate volume of ethyl acetate and analysed by HRGC-MS on a HP 6890 series GC system, coupled to a HP 5973 mass selective detector in the electron impact mode (70 eV), equipped with an HP-1 MS capillary column (30 m x 0.25 mm; film thickness 0.25 µm). Injector and detector temperatures were set at 270oC and 290oC, respectively. The temperature program was kept at 160oC, then programmed to 260oC at a rate of 4oC/min and finally raised at a rate of 15oC/min to 290oC for 15 min. Hydrogen was the carrier gas at a flow rate of 1 mL/min. Diluted samples were injected manually in the split mode (1/10). Results were Natural Product Communications Vol. 2 (1) 2007 15 obtained from Frd area percent data. The chemical components were identified based on comparisons of their mass spectra data with those of standards and/or literature data, by co-injection of these samples in the HRGC, and from Wiley 275 library data of the HRGC-MS system. Acknowledgments - We are grateful to CNPq for financial support and for Productivity Fellowships to RCP and VLT. DNC, OSPF and ASO thank CAPES for providing ProDoc, PhD and Master degree fellowships, respectively. The authors also thank Dr Claudia Rezende and Angelo Pinto for the use of HRGC and HRGC-MS equipment. References [1] Vallim MA, De-Paula JC, Pereira RC, Teixeira VL (2005) The diterpenes from Dictyotacean marine brown algae in the Tropical Atlantic American region. Biochemical Systematics and Ecology, 33, 1-16. [2] De-Paula JC, Pedrini AG, Pinheiro MD, Pereira RC, Teixeira VL. 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