Fatty acids from edible sea hares: anti-inflammatory capacity in LPS-stimulated RAW 264.7 cells involves iNOS modulation

Abstract

The inclusion of marine organisms in the diet is gaining importance due to their richness in health beneficial nutrients. This study evaluated the fatty acid composition and anti-inflammatory potential of lipophilic extracts of two edible sea hares, Aplysia fasciata Poiret and Aplysia punctata Cuvier. Twenty-five fatty acids were determined, nine of them not yet reported in these species. They revealed similar anti-inflammatory properties in RAW 264.7 cells stimulated with lipopolysaccharide, as ascertained by the decreased levels of ˙NO in the culture medium. A decrease of L-citrulline was also observed, indicating that the compounds may act by modulation of inducible nitric oxide synthase (iNOS). A. punctata was most effective as lipoxygenase inhibitor, probably because it contains more polyunsaturated fatty acids (PUFA) that can compete with linoleic acid for the active site, decreasing enzyme activity. The anti-inflammatory potential of A. fasciata and A. punctata is reported here for the first time.

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Introduction

The inclusion of marine organisms in the diet is gaining importance due to their richness in health beneficial nutrients. In fact, marine organisms present a huge diversity and live in complex habitats exposed to extreme conditions becoming, in this way, a unique source of bioactive compounds, which cannot be found in other organisms.¹ In recent years, great importance was demonstrated by the fatty acid composition of marine animals, especially macroinvertebrates such as, sponges, molluscs and echinoderms.² For example, research developed by our group showed that the echinoderm Marthasterias glacialis (spiny sea-star) contains a big amount of fatty acids, being an interesting source of anti-inflammatory molecules.³ Among them we can highlight the presence of long-chain polyunsaturated fatty acids (PUFA), especially those with three or more double bonds, which are a class of lipids characteristic of marine organism with important bioactivities.⁴ The ω-3 PUFA provide a myriad of health benefits, including reduction of cardiovascular diseases and anti-carcinogenic activities.^5,6 As vegetable oils do not contain long-chain PUFA and the conversion of C₁₈ PUFA to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the human body is inefficient, there has been considerable interest in finding new marine sources for these fatty acids.⁷

Sea hares are marine gastropod molluscs present in several ecosystems. The genus Aplysia is well known by neurobiologists that use these marine animals as experimental models to study memory and learned behaviour, due to their nervous system made up of a small number of nerve cells, many of them with high dimensions.⁸ These species are also known to be consumed, especially in oriental countries,^9,10 but their nutritional composition and potential health effects are nearly unknown. A non-targeted analysis of Aplysia fasciata Poiret and Aplysia punctata Cuvier was previously performed by our group.¹¹ To our knowledge, a deep study of the fatty acids composition and of the anti-inflammatory potential of Aplysia organisms was never performed.

Inflammation plays an important role in the initiation and progress of many diseases, including asthma, rheumatoid arthritis or cardiovascular diseases.^12,13 Under these pathological conditions, macrophages' stimulation leads to an increase of nitric oxide (˙NO) levels.¹⁴ ˙NO is an important inflammatory mediator synthesized from oxygen and L-arginine by inducible nitric oxide synthase (iNOS).¹⁵ This small molecule is essential for host innate immune responses to pathogens, such as bacteria, viruses, fungi and parasites. In addition, ˙NO is essential in the regulation of other physiological features, such as blood pressure, wound repair and neurotransmission. However, excessive ˙NO production can result in the development of inflammatory diseases like autoimmune disorders, infection, neoplastic diseases, liver cirrhosis, diabetes and rheumatoid arthritis.¹⁶ L-Arginine oxidation leads to the production of L-citrulline, which can be used as marker of iNOS action.^17,18

Lipoxygenase (LOX) has a primordial role in inflammation, converting arachidonic acid (AA) released from membranes into an oxidized compound, which is further metabolized to different leukotrienes.¹⁹ Leukotrienes are considered as potent inflammatory mediators, exerting their effects via binding to specific membrane and nuclear receptors.²⁰

The aim of this study was to evaluate A. fasciata and A. punctata as dietary source of fatty acids and the effects of their lipophilic extracts on the inflammatory response of LPS-stimulated macrophages. In addition, the activity against ˙NO and the inhibition of lipoxygenase in cell-free models was also assessed. Some correlations between the extracts' chemical composition and biological activities were established.

Experimental

Sample materials and chemicals

LPS from Salmonella enterica, sodium pyruvate, thiazolyl blue tetrazolium bromide (MTT), β-nicotinamide adenine dinucleotide reduced form (NADH), sodium nitroprusside (SNP), sulphanilamide, N-(1-naphthyl)ethylenediamine, dichloromethane, isooctane, methanol, and dimethyl sulfoxide (DMSO), BF₃, KOH, diacetylmonoxime, antipyrine E, H₂SO₄, linoleic acid, ethanol, soybean lipoxygenase from Glycine max (L.) Merr. (type V-S; EC 1.13.11.12) were from Sigma-Aldrich (St. Louis, MO, USA). Authentic standards of fatty acids methyl esters for GC-MS analysis were obtained from Supelco (Bellefonte, PA, USA). Dulbecco's Modified Eagle Medium (DMEM), Dulbecco's phosphate buffered saline (DPBS), heat inactivated foetal bovine serum (FBS) and Pen Strep solution (penicillin 5000 units per mL and streptomycin 5000 mg mL⁻¹) were purchased from Gibco, Invitrogen™ (Grand Island, NY, USA). Water was deionized using a Milli-Q water purification system (Millipore, Bedford, MA, USA).

The samples were randomly collected in the Portuguese coast: A. fasciata was collected in October 2009 and A. punctata in July 2010, both in the Óbidos lagoon (Foz do Arelho). After collection, specimens were placed on ice and transported to the laboratory. The organisms were then cleaned and washed with sea water. Samples were kept at −20 °C, prior to freeze-drying in a Labconco 4.5 Freezone apparatus (Kansas City, MO, USA). The dried samples were ground (particle size < 910 μm) before use.

Extraction

Extraction conditions were adapted from a previous work.²¹ Each sample (1000 ± 10.0 mg) was extracted with 20 mL of dichloromethane : methanol (1 : 1), with stirring at 200 rpm, for 20 min, at 40 °C. The extracts were then filtered through a 0.45 μm membrane (Millipore). All extractions were performed in triplicate.

Extract derivatization for GC-MS analysis

For fatty acids analysis 2.5 mL of extract were concentrated to dryness under reduced pressure (40 °C). The residue was then hydrolysed with 1 mL of KOH methanolic solution (11 g L⁻¹), at 90 °C, for 10 min.²⁰ The free fatty acids originally present and those resulting from the alkaline hydrolysis were derivatized to their methyl esters (FAME) with 1 mL of BF₃ methanolic solution (10%), at 90 °C, for 10 min. FAME were purified with 2 × 6 mL of isooctane and anhydrous sodium sulphate was added to assure the total absence of water. The resulting extract was evaporated to dryness under a stream of nitrogen and dissolved in 100 μL of isooctane. Each sea hare extract was analysed in triplicate.

GC-MS analysis

GC-MS analysis was performed following a previously established method,²² with some modifications. Derivatized extracts (2 μL) were analysed using a Varian CP-3800 gas chromatographer (USA) equipped with a Varian Saturn 4000 mass selective detector (USA) and a Saturn GC/MS workstation software version 6.8. A VF-5 ms (30 m × 0.25 mm × 0.25 μm) column (Varian) was at 40 °C for 1 min, then increased 5 °C min⁻¹ to 250 °C, 3 °C min⁻¹ to 300 °C and held for 15 min. All mass spectra were acquired in electron impact (EI) mode. Ionization was maintained off during the first 4 min, to avoid solvent overloading. The Ion Trap detector was set as follows: transfer line, manifold and trap temperatures were, respectively, 280, 50 and 180 °C. The mass ranged from 50 to 600 m/z, with a scan rate of 6 scan per s. The emission current was 50 μA and the electron multiplier was set in relative mode to auto tune procedure. The maximum ionization time was 25 000 μs, with an ionization storage level of 35 m/z. The analysis was performed in Full Scan mode.

Identification of compounds was achieved by comparison of their retention indices and mass spectra with those from pure standards, injected under the same conditions, and from NIST 05 MS Library Database. The amount of FAME present in the samples was achieved from the calibration curve of the respective standard prepared in isooctane.

Carotenoids analysis by HPLC-DAD

Carotenoids were separated on a C30 YMC column (5 μm, 250 × 4.6 mm i.d.; YMC, Japan) at room temperature, according to a previously described procedure,²³ with some modifications. The mobile phase consisted of two solvents: methanol (A) and tert-butyl methyl ether (B), starting with 95% A and using a gradient to obtain 70% at 30 min, 50% at 50 min and 95% at 60 min. The injection volume was 20 μL and the flow rate 0.9 mL min⁻¹. Elution was monitored at 450 nm. Reference substances were analysed under the same conditions.

Sterols analysis by HPLC-UV

The sterols analysis was performed as described by Lopes et al.²⁴ Briefly, the extract was hydrolysed with ethanolic KOH (10%) for 1 h, at 70 °C. The non-saponifiable fraction was extracted with n-hexane and the organic phase was dehydrated with anhydrous sodium sulphate and evaporated to dryness under reduced pressure, at 30 °C. The residue was dissolved in 1 mL of 30 : 70 (v/v) methanol : acetonitrile and then filtered through a 0.22 μm membrane (Millipore). Separation was performed using a reversed-phase Hypersil ODS (20 × 0.4 cm i.d.; Teknokroma, Barcelona, Spain) column, in isocratic mode, with methanol : acetonitrile (30 : 70) as eluent. The injection volume was 20 μL and the flow rate 0.8 mL min⁻¹. Elution was monitored at 205 nm. Reference substances were analysed under the same conditions.

RAW 264.7 macrophages culture assays

Macrophage RAW 264.7 cells were from the American Type Culture Collection (LGC Standards S.L.U., Spain) and were cultured at 37 °C, in DMEM plus 10% FBS and 2% Pen Strep solution, in a humidified atmosphere of 5% CO₂, as previously reported.²⁵ Cells were seeded in 48-well plates (90 000 cells per well) and cultured until 80–90% confluence. Cells were pre-treated with different concentrations of each sea hare extract dissolved in medium with 0.5% DMSO or vehicle, for 1 h. Afterwards, without removing the extract, 1 μg mL⁻¹ LPS (or vehicle) was added and the plates were incubated for 18 h at 37 °C, in a humidified atmosphere of 5% CO₂. Five independent assays were performed in duplicate.

MTT reduction

MTT is converted to formazan by metabolically active cells. The extent of formazan production was quantified by measuring the absorbance at 510 nm in a microplate reader (Multiskan ASCENT).²⁵ Results are expressed as percentage of the respective control (with or without LPS). Five independent experiments were performed in duplicate.

Lactate dehydrogenase (LDH) release

The LDH released into the culture medium was determined by measuring the decrease of NADH at 340 nm during the conversion of pyruvate to lactate.²⁵ Results are expressed as percentage of the respective control (with or without LPS). Five independent experiments were performed in duplicate.

˙NO determination

The nitrite resulting from the conversion of ˙NO in the culture medium was measured by the Griess reaction.²⁵ Five independent experiments were performed in duplicate. Control values were obtained in the absence of sea hare extract.

L-Citrulline determination

L-Citrulline was quantified using a previously described method,¹⁸ with some modifications. Briefly, after 18 h incubation, 250 μL of culture medium were mixed with 100 μL of a reaction mixture containing diacetylmonoxime, antipyrine E and H₂SO₄. After incubating at 96 °C for 25 min, in a block heater (Stuart SBH 200D/3, United Kingdom), the solution was cooled down to room temperature and the absorption was read at 405 nm. Five independent experiments were performed in duplicate.

˙NO scavenging in non-cellular system

The scavenging of ˙NO released from SNP was determined using the Griess reaction.²⁵ SNP was mixed with different concentrations of sea hare extract and left at room temperature for 1 h, under light. Griess reagent was added and the absorbance was read at 560 nm after 10 min incubation in the dark, at room temperature. Five independent experiments were performed in triplicate.

Inhibition of lipoxygenase

The inhibition of lipoxygenase was assessed at room temperature, following the oxidation of linoleic acid to 13-hydroperoxy linoleic acid at 234 nm, according to a described procedure,²⁶ with some modifications. 20 μL of each sea hare extract were mixed with 200 μL of phosphate buffer (pH 9) and 20 μL of soybean lipoxygenase 100 U (blank). Then, 20 μL linoleic acid (4.18 mM in ethanol) were added and the reaction was followed for 3 min. Four independent experiments were performed in triplicate.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 6 Software (San Diego, CA, USA). OneWay ANOVA and Bonferroni's test, as post-hoc test, were used to determine the statistical significance in comparison to control. Two-way ANOVA and Sidak's multiple comparison tests were used to determine the interaction between extract and LPS in cell viability and differences in L-citrulline and ˙NO release in LPS-stimulated macrophages. t-Student test was used to check for significant differences between fatty acids. Data are expressed as mean ± SEM. Values of p ≤ 0.05 were considered significant.

Results and discussion

Chemical composition

In the present work we intended to thoroughly characterize the lipophilic extracts (dichloromethane : methanol) of two edible sea hares species of Aplysia. The fatty acids profile was established by GC-MS, after sample saponification and derivatization to their respective methyl esters. The presence of fatty acids containing between 12 and 24 carbons was studied. Twenty-five fatty acids were identified and quantified (Fig. 1, Table 1), nine of them being reported for the first time in both species. The chromatographic profile of these two species was very similar, but with significant quantitative differences (p < 0.001 for all the compounds). These differences can be related with intrinsic characteristics of each species or influenced by the season and/or year of harvest, stage of development, environmental conditions, among other factors.² A. fasciata presented higher amounts of fatty acids (3.90 g kg⁻¹) than A. punctata (2.77 g kg⁻¹). Hexadecanoic acid (C16:0) was the main fatty acid identified in both species (Fig. 1, Table 1). This result could be expected, since the diet of these species is mainly composed by Ulva lactuca Linnaeus:²⁷ previous studies involving this algae species, collected in the same geographic region,²⁸ showed that it is equally characterized by the presence of high amounts of hexadecanoic acid.

Fig. 1 Fatty acids profile of A. punctata extract. Peaks' identity as in Table 1.

Table 1 Free fatty acids composition of sea hares extracts (mg kg⁻¹ dry matter)^a

Peak		Compound	A. fasciata	A. punctata
a Results are expressed as mean (standard deviation) of three determinations; the difference between the amounts of each compound in the two species is significant (p < 0.001); nq – not quantified; SFA – saturated fatty acids; MUFA – monounsaturated fatty acids; PUFA – polyunsaturated fatty acids.
1	C12:0	Dodecanoic	29.20 (0.26)	8.05 (0.23)
2	C13:0	Tridecanoic	6.33 (0.25)	0.96 (0.02)
3	C14:0	Tetradecanoic	148.21 (2.17)	79.64 (0.87)
4	C15:0	Pentadecanoic	73.24 (1.34)	25.18 (0.54)
5	C16:1	cis-9-Hexadecenoic	97.98 (1.15)	20.66 (0.19)
6	C16:0	Hexadecanoic	1572.81 (10.67)	662.44 (3.50)
7	C17:0	Heptadecanoic	113.68 (1.00)	60.03 (0.26)
8	C18:3	cis-6,9,12-Octadecatrienoic	nq	6.11 (0.01)
9	C18:2	cis-9,12-Octadecadienoic	68.11 (1.83)	81.19 (0.05)
10	C18:1	cis-9-Octadecenoic	185.71 (0.08)	232.73 (2.39)
11	C18:1	trans-9-Octadecenoic	571.21 (5.23)	65.31 (0.23)
12	C18:0	Octadecanoic	562.39 (11.47)	389.24 (2.29)
13	C20:4	cis-5,8,11,14-Eicosatetraenoic	28.22 (0.63)	206.98 (2.80)
14	C20:5	cis-5,8,11,14,17-Eicosapentaenoic	13.57 (0.05)	107.55 (3.53)
15	C20:3	cis-8,11,14-Eicosatrienoic	27.24 (0.22)	75.11 (0.90)
16	C20:2	cis-11,14-Eicosadienoic acid	6.10 (0.20)	62.84 (0.81)
17	C18:3	cis-9,12,15-Octadecatrienoic	185.74 (0.63)	126.68 (0.12)
18	C20:0	Eicosanoic	22.72 (0.25)	9.90 (0.02)
19	C21:0	Heneicosanoic	20.02 (1.33)	7.96 (0.24)
21	C22:6	cis-4,7,10,13,16,19-Docosahexaenoic	3.86 (0.06)	54.07 (0.08)
20	C22:4	cis-7,10,13,16-Docosatetraenoic	67.52 (0.60)	453.26 (4.69)
22	C22:1	cis-13-Docosaenoic	35.66 (0.68)	6.64 (0.17)
23	C22:0	Docosanoic	50.95 (0.83)	16.47 (0.21)
24	C23:0	Tricosanoic	0.48 (0.05)	3.40 (0.07)
25	C24:0	Tetracosanoic	5.41 (0.08)	5.91 (0.09)
Total			3896.36	2768.31
SFA			2605.45	1269.18
MUFA			890.56	325.34
PUFA			400.36	1173.79
ω-3			203.17	288.30
ω-6			197.19	885.49

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A. fasciata mainly contains saturated fatty acids (67%). On the other hand, considering the contents of saturated (46%) and unsaturated fatty acids (54%), A. punctata exhibits a balanced profile. Among them, cis-7,10,13,16-docosatetraenoic acid is highlighted, as it is the main fatty acid found in the nervous system of species of Aplysia genus.²⁹

Additionally, the presence of carotenoids and sterols was checked by HPLC-DAD and HPLC-UV, respectively, but such types of compounds were not detected in any of the extracts (data not shown).

Cellular viability

MTT reduction and LDH leakage assays were performed in RAW 264.7 cells exposed to the extracts, in the presence and absence of LPS, in order to define the concentration range at which the sample could be used without damaging cells. In this model the viability of cells exposed to 1 μg mL⁻¹ LPS for 18 h decreased to 76.6 ± 2.5% relatively to unexposed cells, as evaluated by the MTT assay.

As it can be seen in Fig. 2, the extracts did not revealed to be cytotoxic at the concentrations tested (15.6 to 250 μg mL⁻¹, dry matter), either in the presence or absence of LPS. Furthermore, the viability of cells stimulated with LPS increased with A. punctata extract at the three higher concentrations tested (p < 0.01 and p < 0.001), as seen in the MTT assay (Fig. 2). A. punctata extract was able to restore cellular viability to control levels in cells concomitantly exposed to LPS, while A. fasciata did not display the same capacity. This result may be partly attributed to the higher amounts of saturated fatty acids, namely hexadecanoic acid, in A. fasciata (2.4 times the amount found in A. punctata, Table 1), since it has been previously shown that hexadecanoic acid can be cytotoxic to LPS-exposed macrophages.³ Saturated fatty acids can activate Toll-like receptor-4 and induce an inflammatory response similarly to LPS.³⁰ On the other hand, ω-3 fatty acids like DHA can inhibit signalling cascades induced by saturated fatty acids and LPS.³⁰ As so, it is possible that the amount of beneficial fatty acids in A. fasciata was not sufficient to counteract the action of saturated fatty acids in LPS exposed cells.

Fig. 2 Viability of cells pre-treated for 1 h with lipophilic extracts followed by 18 h co-treatment with LPS or LPS vehicle. Results are presented as mean ± standard error of the mean of five independent experiments performed in duplicate. ** p < 0.01, *** p < 0.001 compared to the respective control (with or without LPS); ## p < 0.01; ### p < 0.001 LPS exposed cells compared with cells exposed only to the respective extract concentration.

Ambrozova and colleagues have found that ω-3 and ω-6 fatty acids were not cytotoxic to RAW 264.7 macrophages at concentrations up to 100 μM.³¹ It should be noted that the amounts of these compounds in the extracts at the concentrations tested are below the values described (at nM range); as so, no toxicity was expected. Furthermore, these concentrations are below the plasma levels that could be physiologically attained by humans consuming diets rich in DHA/EPA. Plasma concentrations of 176 and 37 μM for DHA and EPA, respectively, were measured in healthy volunteers consuming fish twice a month.³²

˙NO and L-citrulline in culture medium

The ability of the extracts to decrease ˙NO and L-citrulline levels in the culture medium of LPS-stimulated macrophages was evaluated. Bacterial LPS is the stimulant of macrophages routinely used.³¹ As an outer membrane component of Gram negative bacteria, LPS can trigger the generation of a variety of inflammatory mediators by macrophages, including ˙NO, which can exert either beneficial (e.g., bactericidal) or deleterious (e.g., DNA damage and protein oxidation) effects. The overproduction of ˙NO and other inflammatory mediators can be at the origin of inflammatory diseases.^31,33

In the assay without LPS, ˙NO and L-citrulline levels in control cells and cells exposed to sea hares extracts were very low and could not be accurately quantified (data not shown). This also was verified at low concentrations of sea hare extract in the assay with LPS (data not shown).

The increase of ˙NO and L-citrulline levels, resulting from iNOS induction by LPS, was significantly reduced by both extracts, as it can be seen in Fig. 3. The EC₅₀ corresponding to A. fasciata extract was 77 ± 7 μg mL⁻¹ for ˙NO and 72 ± 12 μg mL⁻¹ for L-citrulline, while the EC₅₀ of A. punctata extract was 71 ± 6 μg mL⁻¹ for ˙NO and 74 ± 16 μg mL⁻¹ for L-citrulline. For each extract no significant differences were seen between the decrease of L-citrulline and ˙NO levels, for the same concentrations (p > 0.05). Also, no significant differences were found between the two extracts (p > 0.05).

Fig. 3 Effect in ˙NO and L-citrulline levels of cells pre-treated for 1 h with lipophilic extracts, followed by 18 h co-treatment with 1 μg mL⁻¹ LPS. Results are presented as mean ± standard error of the mean of five independent experiments performed in duplicate.

The similar decrease in both L-citrulline and ˙NO levels may indicate that the extract primarily acts by the modulation of iNOS. This anti-inflammatory potential can be partly attributed to the presence of unsaturated fatty acids in the extracts.⁴

In general, saturated fatty acids are pro-inflammatory compounds, while unsaturated fatty acids are neutral, ω-3-PUFA exerting potent anti-inflammatory effects in RAW 264.7 macrophages and other models of inflammation.³⁴ EPA and DHA can alleviate inflammatory processes that already exist, thus highlighting the therapeutic importance of these fatty acids.⁴ ω-3-PUFA act by decreasing the production of inflammatory eicosanoids, cytokines and nitric oxide. Concerning to the last, it has been previously shown that the expression of iNOS was inhibited by EPA, DHA³³ and also α-linolenic acid.³⁵ The Aplysia extracts studied herein contained significant amounts of these three ω-3 fatty acids (203.17 and 288.30 mg kg⁻¹, dry weight, in A. fasciata and A. punctata, respectively, Table 1), which should contribute to the overall effects. The presence of higher amounts of ω-3 fatty acids in A. punctata is accompanied by the presence of higher amounts of the pro-inflammatory fatty acid cis-5,8,11,14-eicosatetraenoic (arachidonic acid) and the balance between the compounds can impart similar effects on ˙NO of A. punctata and A. fasciata extracts.⁴ Nevertheless, other compounds not determined in the extract can contribute for the observed activity, which may explain why extracts with different amounts of fatty acids displayed similar anti-inflammatory effect. Recently our group has described the anti-inflammatory potential of monogalactosyl diacylglycerols and monoacylglycerol obtained from a brown macroalgae.³⁶ The monogalactosyl diacylglycerols containing ω-3 fatty acids in their structure were more effective than the monoacylglycerol containing oleic acid, a ω-9 fatty acid.³⁶ So, ω-3 fatty acids, not only in the free form, but also in complex molecules, appear to contribute most to the anti-inflammatory activity.

˙NO scavenging in non-cellular system

In the cellular model used herein to study the anti-inflammatory potential of the extracts, the decrease of ˙NO concentration in the culture medium can be due to the interference with ˙NO production in cells under a pro-inflammatory stimulus, or to the scavenging of the ˙NO produced. So, the ability of the extracts to scavenge this reactive species was evaluated using a cell-free assay with SNP as ˙NO donor. The results showed that both samples were able to scavenge SNP-released ˙NO in a dose-dependent manner (Fig. 4).

Fig. 4 ˙NO scavenging activity of lipophilic extracts in non-cellular system. Results are presented as mean ± standard error of the mean of five independent experiments performed in triplicate.

The EC₂₅ of A. punctata (1.56 ± 0.15 mg mL⁻¹) was similar to that of A. fasciata (1.82 ± 0.28 mg mL⁻¹) (p > 0.05). This antioxidant activity of the extracts can also contribute to the observed ˙NO decreased levels in the cellular assay with LPS-stimulated macrophages (Fig. 3).

Inhibition of lipoxygenase

Lipoxygenases are enzymes involved in the synthesis of leukotrienes, which have been implicated in the pathogenesis of many human acute and chronic inflammatory diseases.³⁷ Because of the high catalytic domain similarity between plant and mammalian lipoxygenases, lipoxygenase obtained from soybean is widely accepted as a model for lipoxygenase inhibition studies.^37,38 LOXs are inducible enzymes that convert ω-3 and ω-6 fatty acids into inflammation-related signalling molecules. These molecules can have both pro- and anti-inflammatory effects, depending on the type of fatty acid from which they are metabolized.³⁹ In this cell-free assay both samples were able to inhibit lipoxygenase activity in a dose-dependent manner (Fig. 5).

Fig. 5 Inhibition of soybean lipoxygenase by lipophilic extracts in non-cellular system. Results are presented as mean ± standard error of the mean of four independent experiments performed in triplicate.

A. punctata lipophilic extract was the most active, displaying an EC₅₀ of 1.75 ± 0.09 mg mL⁻¹ compared with 3.06 ± 0.22 mg mL⁻¹ of A. fasciata (p < 0.01). As A. punctata contains higher amounts of PUFA (Table 1) it is possible that they decrease the activity of the enzyme by competing for its active site with linoleic acid, the enzyme's natural substrate.³⁸ Moreover, it is known that LOX converts AA (ω-6) into the 4-series leukotrienes and EPA (ω-3) into the 5-series leukotrienes.⁴⁰ The 4-series leukotrienes are potent signaling molecules, responsible for a number of pro-inflammatory functions, including fever, pain, bronchoconstriction, vasoconstriction, platelet aggregation and clot formation, chemotaxis, interleukin-6 (IL-6) production, and release of reactive oxygen species (ROS).^41,42 On the other hand, 5-series leukotrienes have demonstrated anti-inflammatory effects.⁴¹ So, a higher ratio of ω-6/ω-3 fatty acids is extremely correlated with much higher incidences of inflammatory disorders like heart disease, chronic inflammatory diseases, auto-immune diseases, and a variety of cancers.^43,44 Hereupon, in spite of A. punctata containing higher amounts of PUFA than A. fasciata, and can inhibit more strongly the LOX, the anti-inflammatory capacity of A. fasciata may be higher than that of A. punctata due to the presence of a low ratio ω-6/ω-3 fatty acids (0.97 for A. fasciata and 3.07 for A. punctata).

Conclusions

The extracts of two edible sea hares were characterized for their fatty acids composition. A. fasciata and A. punctata extracts revealed to contain a large amount of the essential fatty acids EPA and DHA that could have an important impact on human diet. The extracts demonstrated capacity to modulate iNOS, expressed by the reduction of ˙NO and L-citrulline levels in the culture medium of LPS-stimulated macrophages. Furthermore, both extracts inhibited lipoxygenase, pointing to their ability to reduce the production of leukotrienes. The anti-inflammatory properties may be, at least partially, attributed to fatty acids, in particular to ω-3 fatty acids.

Overall the results indicate that, in addition to their direct ingestion, A. fasciata and A. punctata may be good sources of nutraceuticals providing beneficial health effects by reducing the levels of inflammatory mediators involved in the genesis of several diseases.

Acknowledgements

This work received financial support from the European Union (FEDER funds through COMPETE) and National Funds (FCT, Fundação para a Ciência e Tecnologia) through project Pest-C/EQB/LA0006/2013. The work also received financial support from the European Union (FEDER funds) under the framework of QREN through Project NORTE-07-0124-FEDER-000069. To all financing sources the authors are greatly indebted.

Notes and references

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