Profiling of Heterobranchia Sea Slugs from Portuguese

molecules Article Profiling of Heterobranchia Sea Slugs from Portuguese Coastal Waters as Producers of Anti-Cancer and Anti-Inflammatory Agents Nelson G. M. Gomes 1,* ID ,Fátima Fernandes 1, Áurea Madureira-Carvalho 1,2 ID , Patrícia Valentão 1 ID , Alexandre Lobo-da-Cunha 3,4, Gonçalo Calado 5,6 and Paula B. Andrade 1,* ID 1 REQUIMTE/LAQV, Laboratory of Pharmacognosy, Departament of Chemistry, Faculty of Pharmacy, University of Porto, R. Jorge Viterbo Ferreira, no. 228, 4050-313 Porto, Portugal; mfgfernandes@gmail.com (F.F.); aurea.carvalho@iucs.cespu.pt (Á.M.-C.); valentao@ff.up.pt (P.V.) 2 IINFACTS—Institute of Research and Advanced Training in Health Sciences and Technologies, Department of Sciences, University Institute of Health Sciences (IUCS), CESPU, CRL, Central de Gandra Street, 1317, 4585-116 Gandra PRD, Portugal 3 Departamento de Microscopia, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto, 4050-313 Porto, Portugal; alcunha@icbas.up.pt 4 Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR), 4450-208 Matosinhos, Portugal 5 Departamento de Ciências da Vida, Universidade Lusófona de Humanidades e Tecnologia, 1749-024 Lisboa, Portugal; goncalo.calado@ulusofona.pt 6 MARE NOVA. Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Departamento de Ciências e Engenharia do Ambiente, Campus da Caparica, 2829-516 Caparica, Portugal * Correspondence: ngomes@ff.up.pt (N.G.M.G.); pandrade@ff.up.pt (P.B.A.); Tel.: +35-122-042-8500 (N.G.M.G.); +35-122-042-8654 (P.B.A.) Received: 15 March 2018; Accepted: 25 April 2018; Published: 27 April 2018 Abstract: Bioprospection of marine invertebrates has been predominantly biased by the biological richness of tropical regions, thus neglecting macro-organisms from temperate ecosystems. Species that were not the object of studies on their biochemical composition include the Heterobranchia gastropods Armina maculata, Armina tigrina and Aglaja tricolorata, inhabitants of the Portuguese Atlantic coastal waters. Here, we present for the first time the fatty acid profile of neutral lipids and homarine content of these three species. Qualitative and quantitative differences in the fatty acid content among species points to the existence of a fatty acid profile of neutral lipids, particularly of each genus. The results from cytotoxicity assays, using the acetonic extracts of the gastropods on human gastric adenocarcinoma (AGS) and human lung adenocarcinoma (A549) cell lines, revealed a pronounced cytotoxic effect of the A. tigrina extract on both cell lines (IC50 values of 68.75 and 69.77 µg mL−1 for AGS and A549, respectively). It is worth noting the significant reduction of NO levels in LPS-challenged RAW 264.7 macrophages exposed to A. tricolorata extract, at concentrations as low as 125 µg mL−1. Keywords: Armina spp.; Aglaja tricolorata; sea slugs; fatty acids; PUFA; EPA; DHA; homarine 1. Introduction The increasing number of reports on the structural variety and pharmacological properties of naturally occurring compounds obtained from marine invertebrates [1,2] has been driving the focus on several taxonomic groups, namely Heterobranchia mollusks [3,4]. Molecules 2018, 23, 1027; doi:10.3390/molecules23051027 www.mdpi.com/journal/molecules Molecules 2018, 23, 1027 2 of 15 Driven by the richer biological diversity compared to temperate regions, marine organisms from tropical ecosystems have been preferentially targeted as sources of therapeutically useful chemical agents [5]. Despite the geographical trends, bioprospection of temperate environments has been rewarding, leading to the discovery of a significant number of compounds from marine invertebrates [6]. Several species of nudibranchs, such as Armina spp., are widely distributed in the Mediterranean and Atlantic coasts, namely Armina maculata Rafinesque, 1814 and Armina tigrina Rafinesque, 1814 [7]. The cephalaspidean Aglaja tricolorata Renier, 1807 shares a similar geographical distribution, being also recorded along the Atlantic coast [8]. Despite their ubiquitous distribution, inhabiting sub-tropical and temperate shores [9], the above-mentioned species are considered scarce and/or seldom found, the chemical investigation of both genera being virtually absent [10]. There is a single study on the isolation of briarane and cembrane diterpenoids from A. maculata, and their subsequent identification in its prey Veretillum cynomorium [11]. As building blocks of several biologically relevant molecules, fatty acids act as biochemical mediators, energy sources and structural components [12], being obtained through heterotrophic and autotrophic pathways. They are frequently used as chemotaxonomic and chemo-ecological markers in some taxonomic groups [13]. While the fatty acid profile of a marine invertebrate is predominantly determined by its dietary sources, environmental and biotic factors can influence on the profile, providing valuable information on its ecological traits and supporting their chemotaxonomic discrimination [13,14]. Several experimental and epidemiological studies have demonstrated that specific classes of fatty acids, namely polyunsaturated fatty acids (PUFA), exhibit anti-cancer effects and immunoactive functions [15,16], playing also a pivotal role on the inflammatory process as precursors of eicosanoids [17]. Given the regression of the shell, and lack of an acquired immune system, some mollusks have evolved alternative mechanisms of defense, being active producers of bioactive metabolites for circulation in the hemolymph, as well as toxic and deterrent secretions, frequently containing molecules exhibiting anti-inflammatory and cytotoxic properties [18]. In addition to fatty acids, the biosynthetic machinery of marine macro-organisms enables the production of secondary metabolites falling into a wide range of structural classes. With the current study, we aimed to investigate the chemical profiles of A. maculata, A. tigrina and A. tricolorata, providing further insights on their metabolome. Considering that Heterobranchia sea slugs rely mainly on chemical defenses through the production of intrinsically active molecules, it was also our aim to assess their cytotoxicity towards human gastric (AGS) and human lung (A549) adenocarcinoma cell lines, as well as the anti-inflammatory potential, evaluated through the interference with the production of nitric oxide (NO) in lipopolysaccharide (LPS)-challenged RAW 264.7 macrophages. 2. Results 2.1. Chemical Characterization 2.1.1. Fatty Acid Profiles As shown in Table1, specimens under study are characterized by a complex fatty acid profile, their quantification revealing total fatty acid concentrations ranging from 136.21 to 165.81 µg mg−1 dry extract. In A. maculata and A. tigrina extracts, 23 and 26 fatty acids were characterized, respectively, while, in A. tricolorata extract, 18 fatty acids were identified, indicating clear differences between the two genera. Concerning the main constituents, the fatty acids contents of A. maculata and A. tigrina were rather similar, C16:0, C18:0, C20:4n-6, and C20:5n-3 being identified as the main components (>10%, each), accounting for approximately 63% of the total fatty acid content. Nonetheless, the saturated fatty acids (SFA) C21:0 and C22:0, and the PUFA C18:2n-6t were detected only in A. tigrina extract. While C16:0, C18:0 and C20:5n-3 were also identified as main components, the acetone extract of A. tricolorata exhibited high contents of C20:2n-6 (ca. 12%) and C22:6n-3 (ca. 13%). Moreover, distinct proportions of the different classes of fatty acids was also evidenced for the two genera (Figure1). Molecules 2018, 23, 1027 3 of 15 A. maculata and A. tigrina presented a similar qualitative profile concerning the SFA, monounsaturated fatty acids (MUFA) and PUFA content, while A. tricolorata was significantly richer in PUFA (ca. 57%). The cephalaspidean A. tricolorata exhibited a significantly higher proportion of n-3 PUFA (n-6/n-3 < 1), in contrast with the nudibranchs A. maculata and A. tigrina with a n-6/n-3 ratio higher than 1.75 (Table1). Table 1. Fatty acid content of A. maculata, A. tigrina and A. tricolorata extracts (µg mg−1 dry extract) 1. Compound A. maculata A. tigrina A. tricolorata µg mg−1 Relative µg mg−1 Relative µg mg−1 Relative dry extract % dry extract % dry extract % SFA C12:0 nq nq nq nq - - C13:0 nq nq nq nq - - C14:0 1.20 (0.05) 0.75 1.64 (0.04) 1.20 1.18 (0.16) 0.71 C15:0 1.14 (0.04) 0.87 1.14 (0.06) 0.84 1.69 (0.07) 1.02 C16:0 30.43 (2.82) 19.08 25.73 (1.10) 18.89 21.28 (1.67) 12.84 C17:0 14.60 (0.38) 9.16 13.38 (0.50) 9.82 9.71 (0.61) 5.86 C18:0 18.42 (0.49) 11.55 15.20 (0.69) 11.16 15.99 (1.05) 9.64 C20:0 1.36 (0.02) 0.85 3.70 (0.11) 2.72 nq nq C21:0 - - nq nq - - C22:0 - - 2.79 (0.14) 2.05 - - MUFA C14:1n-5 nq nq nq nq nq nq C15:1n-5 1.39 (0.14) 0.87 1.48 (0.17) 1.09 - - C16:1n-7 1.72 (0.07) 1.08 2.78 (0.33) 2.04 3.46 (0.17) 2.09 C17:1n-7 1.13 (0.17) 0.71 1.79 (0.11) 1.32 - - C18:1n-9c 3.91 (0.20) 2.45 3.91 (0.42) 2.87 9.81 (0.64) 5.92 C18:1n-9t 3.56 (0.28) 2.23 1.71 (0.22) 1.25 7.43 (0.46) 4.48 C22:1n-9 1.07 (0.14) 0.67 nq nq - - PUFA C18:2n-6c 4.27 (0.27) 2.68 2.36 (0.15) 1.74 3.16 (0.14) 1.91 C18:2n-6t - - nq nq - - C20:2n-6 11.05 (1.23) 6.93 6.68 (0.89) 4.91 19.38 (0.81) 11.69 C18:3n-6c nq nq nq nq 2.18 (0.09) 1.31 C18:3n-3c 4.37 (0.44) 2.74 1.99 (0.24) 1.46 4.20 (0.25) 2.53 C20:3n-6 5.95 (0.72) 3.73 4.30 (0.78) 3.16 9.63 (0.33) 5.81 C20:4n-6 30.34 (2.18) 19.03 25.54 (0.93) 18.75 11.38 (0.72) 6.87 C20:5n-3 20.00 (1.22) 12.54 18.42 (0.87) 13.52 24.02 (1.59) 14.49 C22:6n-3

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