Exploring Montagu’s crab: Primary and secondary metabolites and enzyme inhibition

2.4.1 Derivatization process

Trimethylsilylation process was performed as reported by Pereira et al. (2012). Briefly, 100 μL of ethanol extract was transferred to a glass vial and 100 μL of each internal standard (norvaline, methyl linolelaidate and desmosterol, at a final concentration of 12.00, 100.00 and 80.00 μg/mL, respectively) was added. The solvent was evaporated under a nitrogen stream and 100 μL of the derivatization reagent, MSTFA, was added to the residue. The vial was capped, vortexed and heated for 30 min in a dry block heater, maintained at 60 °C.

2.4.2 GC–MS analysis

GC–MS analysis was performed with a Varian CP-3800 gas chromatograph coupled to a Varian Saturn 4000 mass selective ion trap detector (USA) and a Saturn GC/MS workstation software version 6.8, with a VF-5 ms (30 m × 0.25 mm × 0.25 μm) column (VARIAN), in accordance with a method previously described by Pereira et al. (2012). A CombiPAL automatic autosampler (Varian, Palo Alto, CA) was used for all experiments. All mass spectra were acquired in the electron impact (EI) mode. Identification of compounds was achieved by comparison of their retention time and mass spectra with those from pure trimethylsilyl (TMS) standard derivatives analysed under the same conditions, and from NIST05 MS Library Database. For quantification purposes, each sample was injected in triplicate and, excepting for palmitoelaidic acid (compound 12) which was quantified as palmitic acid (compound 13), and for the pair linoleic acid plus linolenic acid (compound 15 + 16) which were quantified together as compound 16, the amount of each metabolite was determined from the calibration curves of the respective standards. All compounds were quantified in Full Scan mode, with the exception of linoleic (m/z 262, 337 and 352), linolenic (m/z 191, 335 and 350) and oleic (m/z 264, 339 and 354) acids that were quantified by the area obtained from the re-processed chromatogram, using the characteristic m/z fragments.

2.4.3 GC–MS method validation

2.5.1 HPLC–DAD analysis

The determination of carotenoids was based on a procedure described by Mariutti et al. (2012), with some modifications. The separation was performed on a C30 YMC column (5 μm, 250 × 4.6 mm i.d.; YMC, Japan) at room temperature. The data were processed on Unipoint System software (Gilson Medical Electronics, Villiers le Bel, France). Elution was monitored at 450 nm. All carotenoids were identified by comparing their chromatographic behaviour (retention time) and UV/Vis spectra with reference standards analysed under the same sample conditions. Peak purity of each peak was thoroughly checked by software contrast facilities (Unipoint System Software). The quantification of each carotenoid was determined from the calibration curves of the respective standards. The extract was injected in triplicate.

2.5.2 HPLC–DAD method validation

2.6.1 Cholinesterases

The acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) inhibitory activities were determined spectrophotometrically in a Multiskan Ascent plate reader (Thermo Electron Corporation), using 96-well plates. This determination was based on Ellman’s method, as previously described (Vinholes et al., 2011). Briefly, in each well the mixture consisted of acetylthiocholine iodide (15 mM)/S-butyrylthiocholine chloride (15 mM) in water, DTNB in buffer C (50 mM Tris–HCl, pH 8, containing 0.1 M NaCl and 0.02 M MgCl·6H₂O), buffer B (50 mM Tris–HCl, pH 8, containing 0.1% bovine serum albumin (BSA)), different concentrations of sample (50.00, 25.00, 12.50, 6.25 and 3.25 mg/mL) dissolved in a solution of 10% methanol in buffer A (50 mM Tris–HCl, pH 8) and AChE (0.44 U/mL)/BuChE (0.1 U/mL). The absorbance was measured at 405 nm and the rates of the reactions were calculated by Ascent Software version 2.6 (Thermo Labsystems Oy). Three independent assays were performed in triplicate. Results were compared with those of galantamine (positive control).

2.6.2 MAO-A

The effect on MAO-A activity was measured spectrophotometrically in a Multiskan Ascent plate reader (Thermo Electron Corporation), using 96-well plates, as previously described (Grosso et al., 2013). Briefly, in each well the mixture consisted of chromogenic solution (0.8 mM vanillic acid, 417 mM 4-aminoantipyrine and 8 U/mL horseradish peroxidase in 0.2 M potassium phosphate buffer pH 7.6), 3 mM tyramine and different concentrations of L. incisus ethanolic extract (50.00, 25.00, 12.50, 6.25 and 3.25 mg/mL) dissolved in 0.2 M potassium phosphate buffer (pH 7.6), and MAO-A (8 U/mL). The kinetic curves were obtained at 490 nm and three independent assays were performed in triplicate. Results were compared with those of clorgyline (positive control).

2.6.3 α-Glucosidase

The spectrophotometric microassay used to evaluate the α-glucosidase inhibition was performed in a Multiskan Ascent plate reader (Thermo Electron Corporation), based on the reaction with PNP-G, as previously reported (Vinholes et al., 2011). Briefly, each well contained 2 mM PNP-G in 100 mM potassium phosphate buffer (pH 7.0) and different concentrations of L. incisus ethanolic extract (50.00, 25.00, 12.50, 6.25 and 3.25 mg/mL) in buffer with 10% of DMSO. The reaction was initiated by the addition of the enzyme solution (56.6 mU/mL). The plates were incubated at 37 °C for 10 min. The absorbance of 4-nitrophenol released from PNP-G at 400 nm was measured spectrophotometrically in a Multiskan Ascent plate reader (Thermo Electron Corporation). Three independent assays were performed in triplicate. Results were compared with those of acarbose (positive control).

3.1.1 Amino acids

Amino acids are essential to human life, being important in brain metabolism imbalances, as neurotransmitters, hormones, metabolic intermediates and alimentary supplements. Furthermore, taking into account that some of these compounds cannot be synthesized by humans (essential ones), a diet enriched with these metabolites is essential to human health (Wu, 2009).

The characterization of the free amino acid profile of L. incisus ethanol extract revealed the presence of nine compounds, comprising essential (valine, leucine, isoleucine, threonine and phenylalanine) and non-essential ones (alanine, glycine, proline and serine) (Table 1 and Fig. 1), which, as far as we know, are reported for the first time in this crab. The calibration plots showed a good correlation, as indicated by correlation coefficient values higher than 0.98 for all amino acids (Table 1). Calculated LOD and LOQ are shown in Table 1. Alanine was clearly the major compound, followed by glycine, corresponding to 33% and 28% of the total of amino acids, respectively (Table 1). Alanine was already reported to be the main compound in other marine organisms, namely in the gastropod molluscs Aplysia fasciata Poiret and Aplysia punctata Cuvier, and in the echinoderm Holothuria forskali Chiaje, while in the echinoderms Paracentrotus lividus Lamarck and Marthasterias glacialis Linnaeus, glycine was clearly the most abundant one (Pereira et al., 2012, 2013). Serine, threonine and phenylalanine were detected in vestigial amounts in L. incisus ethanol extract (Table 1).

Close

Figure 1 GC–MS profile of L. incisus ethanol extract. Peaks’ identity as in Table 1. Internal standards: N, norvaline; M, methyl linolelaidate; D, desmosterol.

Export to PPT

3.1.2 Fatty acids

Fatty acids act like hormones or their precursors, help the digestion process, and are a source of metabolic energy (Burtis and Ashwood, 1996). Thirteen fatty acids were identified, being distributed by saturated fatty acids (SFA) (myristic, pentadecanoic, palmitic, margaric and stearic acids), monounsaturated fatty acids (MUFA) (palmitoelaidic, oleic and cis-11-eicosaenoic acids) and polyunsaturated fatty acids (PUFA) (linoleic, linolenic, arachidonic, eicosapentaenoic and docosahexaenoic acids) (Table 2 and Fig. 1). To our knowledge, this is the first report on the fatty acid composition of L. incisus. A good correlation (correlation coefficient values higher than 0.98 for all fatty acids) was found (Table 2). The LOD and LOQ for the analysed compounds are indicated in Table 2.

L. incisus ethanol extract is essentially constituted by MUFA (ca. 66% of total fatty acids), followed by PUFA (ca. 20% of total fatty acids) (Table 2), unlike what happens with other crabs, such as Carcinus maenas L., Carcinus mediterraneus Czerniavsky, Paralithodes camtschaticus Tilesius, Paralithodes platypus Brandt, Chionoecetes opilio Fabricius, Chionoecetes japonicus Rathbun and Chionoecetes angulatus Rathbun, which are mainly composed by PUFA, followed by MUFA (Naczk et al., 2004; Cherif et al., 2008; Latyshev et al., 2009). The predominance of unsaturated fatty acids is also observed in other marine organisms, namely M. glacialis, A. punctata, P. lividus and H. forskali (Pereira et al., 2012, 2013), but not in the mollusc A. fasciata (Pereira et al., 2013; Pereira et al., 2015).

Oleic acid was clearly the most abundant MUFA, which is in agreement with data found for C. maenas and C. mediterraneus and for crabs from Paralithodes and Chionoecetes genus. Nevertheless, the relative content of oleic acid in L. incisus (ca. 61% of total fatty acids) is about three times higher than that found in other crabs (Naczk et al., 2004; Cherif et al., 2008; Latyshev et al., 2009). Oleic acid is included in the omega-9 family and is not essential for humans, once they possess all the enzymes required for its synthesis. Several studies revealed the capacity of this compound to reduce the incidence of cardiovascular diseases, as well as its anti-diabetic and anti-inflammatory properties (Studer et al., 2005).

Regarding PUFA, linoleic and linolenic acids were the main compounds (ca. 55% of PUFA total contents) (Table 1), unlike what happened in C. mediterraneus and C. maenas, in which arachidonic and eicosapentaenoic acids were the main PUFA, respectively (Naczk et al., 2004; Cherif et al., 2008). Latyshev and collaborators (2009) demonstrated that eicosapentaenoic acid was the major PUFA in crabs from Paralithodes and Chionoecetes genus. Linoleic acid must be obtained from the diet as it cannot be synthesized by the human organism. This compound originates the omega-6 fatty acids series, namely γ-linolenic and arachidonic acids (Voet and Voet, 2004). Therefore, this marine organism can constitute an alternative source of PUFA.

SFA were the compounds present in lower amounts (ca. 14% of total fatty acids), palmitic acid being the major one (ca. 62% of SFA total contents) (Table 2), which is in agreement with the results found for C. maenas, C. mediterraneus and crabs from Paralithodes and Chionoecetes genus (Naczk et al., 2004; Cherif et al., 2008; Latyshev et al., 2009). This SFA is recognized by its antibacterial properties and by its capacity to cause apoptosis in neuroblastoma cells (Zheng et al., 2005; Pereira et al., 2014).

3.1.3 Sterols

As it happens for the other compounds mentioned above, sterols are also important to human health mainly due to their capacity to inhibit intestinal cholesterol absorption (Moghadasian, 2000). Furthermore, they are recognized for their anticarcinogenic, anti-inflammatory and antidiabetic activities (Fernandes and Cabral, 2007).

Cholesterol was the only sterol identified in L. incisus ethanol extract (Table 3), which is in accordance with data obtained for green crab C. maenas (Skonberg and Perkins, 2002). A linear relationship was obtained with a coefficient value higher than 0.99 (Table 3). The presence of this metabolite in marine organisms is not a surprise, since it constitutes a chemical defence against predation and unfavourable environmental conditions (Bernsdorff and Winter, 2003; Pereira et al., 2013).

3.1.4 Carotenoids

Carotenoids are natural pigments commonly found in marine organisms (Pereira et al., 2014). Five compounds, namely fucoxanthin, astaxanthin, lutein, zeaxanthin and β-carotene (Table 3 and Fig. 2), were found in the ethanol extract of L. incisus. As far as we know, all these metabolites are described for the first time in this species. The calibration plots showed a good correlation, as indicated by correlation coefficient values higher than 0.99 for all identified carotenoids (Table 3). The LOD and LOQ for the analysed metabolites are presented in Table 3.

Close

Figure 2 HPLC–DAD profile of L. incisus ethanol extract. Detection at 450 nm. Peaks’ identity as in Table 1.

Export to PPT

This sample is characterized by the presence of high amounts of xanthophylls, namely astaxanthin, which corresponds to 57% of the total of carotenoids (Table 3). C. maenas has also been described to be essentially constituted by xanthophylls, namely by astaxanthin diester, followed by astaxanthin monoester (Naczk et al., 2004). Astaxanthin, a carotenoid present in high quantities in marine organisms (Yuan et al., 2013), is known for its capacity to scavenge free radicals and reactive oxygen species. In fact, its antioxidant activity is about 10 times higher than that revealed by zeaxanthin, lutein and β-carotene (Yuan et al., 2013), which can be explained by the presence of hydroxyl and carbonyl groups in the ionone ring (Liu and Osawa, 2007). Additionally, this metabolite is known for its anti-inflammatory, antidiabetic and anticancer properties (Miki, 1991; Naguib, 2000).

Lutein and β-carotene are minor metabolites, corresponding to 3% and 5% of the total of carotenoids, respectively. These compounds are known as antioxidant agents. Furthermore, some studies had shown that due to its antioxidant activity, β-carotene is able to increase memory (Dhingra and Bansal, 2014).

3.2.1 Cholinesterases

Alzheimeŕs disease (AD) is characterized by the degeneration of cholinergic neurons in specific areas of the brain, associated with higher intellectual functions, memory and consciousness (Houghton et al., 2006). Taking into account that its treatment is based on the cholinergic hypothesis, which correlates the cognitive decline observed with the loss of the neurotransmitter acetylcholine in the synaptic cleft, the search of cholinesterases (acetyl- or butyrylcholinesterase) inhibitors is a challenge (Çokuğraş, 2003; Greig et al., 2002). Under the assay conditions, the ethanol extract of L. incisus was able to inhibit both cholinesterases in a dose-dependent manner, being more active against BuChE (IC₅₀ value of 4.177 mg/mL) (Fig. 3A). It was also observed that this extract was less effective than galantamine (positive control tested under the same conditions), which displayed IC₅₀ values of 3 μg/mL against AChE and 9 μg/mL against BuChE (data not shown). Nevertheless, these results are promising when compared with other marine organisms tested under the same conditions, for which no inhibitory capacity was noticed, namely several Chlorophyta, Rhodophyta and Phaeophyta macroalgae species (Andrade et al., 2013).

Close

Figure 3 In vitro enzymes inhibition by L. incisus ethanol extract. (A) Cholinesterases; (B) MAO-A; (C) α-glucosidase. Values show mean ± SEM of 3 independent experiments performed in triplicate.

Export to PPT

3.2.2 MAO-A

Depression is other important public health problem and one of the leading causes of disease burden worldwide. Currently, there are many drugs for the treatment of this disorder, among which we can highlight MAO-A inhibitors. MAO-A is a catabolic enzyme responsible for the breakdown of monoamine neurotransmitter in the central nervous system (Shih et al., 2009; Youdim et al., 2006). L. incisus ethanol extract inhibited MAO-A in a concentration-dependent way (IC₅₀ of 4.311 mg/mL) (Fig. 3B). Comparing this result with the one obtained for clorgyline (positive control tested under same conditions), which showed an IC₅₀ of 12.10 ng/mL (data not shown), it was verified that the ethanol extract of L. incisus was much less active.

3.2.3 α-Glucosidase

α-Glucosidase is the enzyme responsible for the breakdown of α-glycosidic bonds of complex carbohydrates, resulting in less complex molecules that can be absorbed. Therefore, its inhibitors could retard the use of dietary carbohydrates, suppressing postprandial hyperglycaemia, being normally used to control the levels of blood glucose in patients with diabetes mellitus (Li et al., 2010). L. incisus ethanol extract revealed some capacity to inhibit this enzyme in a concentration-dependent way (Fig. 3C). It was observed that this extract was less effective than acarbose (positive control tested under the same conditions), which showed an IC₅₀ of 0.30 mg/mL. Nonetheless, this matrix was more active than several macroalgae, for which lower or no capacity was found (Andrade et al., 2013).