Fatty Acid Composition, Phospholipid Molecules, and Bioactivities of Lipids of the Mud Crab Scylla paramamosain

Graduate University of Sciences and Technology, Vietnam Academy of Science and Technology (VAST), 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Vietnam Food Administration, Ministry of Health, 138-Giang Vo, Ba Dinh, Hanoi, Vietnam Institute of Natural Products Chemistry, VAST, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam Center for Research and Technology Transfer, VAST, 18-Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam


Introduction
Crab species of the genus Scylla are known as mud crabs and include the four species of S. serrata (Forskål), S. tranquebarica (Fabricius), S. olivacea (Herbst), and S. paramamosain (Estampador) [1]. ese crabs are widely distributed in warm waters such as the tropical waters of the Indian and Pacific oceans [2]. e cultured mud crab is highly nutritious and is an important source of income for farmers in coastal communities because of the rising demand for crab meat in both domestic and export markets. S. paramamosain has been studied in ailand and China and is the most common species of mud crab in Vietnam [3][4][5]. However, there have been no studies of this crab during its moulting period, which is an important aspect of the crab life cycle that creates a special kind of crab, i.e., soft-shell crab.
A soft-shell crab is the physiological form of any crab after moulting when the crab replaces its old skeleton with a new, larger, and decalcified soft skeleton. Soft-shell crab is very nutritious with high mineral, protein, and healthy fatty acid contents [6]. Lipids are an important nutrient source for the growth of organisms, especially crustaceans, because they are precursors of hormones that promote the moulting process [7,8]. Phospholipids, which are lipid components, are believed to affect the growth and metabolism of crabs [9,10]. e phospholipid content of crustaceans typically ranges from 1% to 3% [11]. e absence of phospholipids in the diet is detrimental to crustaceans and results in moult death syndrome, which is indicated by death during or suddenly after moulting [12]. In the crab structure, phospholipids maintain the structural integrity of biological membranes and are precursors of important steroids [13].
Phospholipids are also essential for human dietary because their fatty acid residues are delivered to incorporate with cell membrane after digestion and absorption in the intestinal lumen [14]. Phospholipids consist of phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidylcholine, in which, phosphatidylcholine is most abundant with more than 50% of phospholipid content in the intestine [15]. Phospholipid was shown multi-bioactivities in human, such as anti-inflammat anti-inflammatory, anticancer, and antitumor activities. Recent studies indicated that the fatty acid composition of phospholipids has decided their bioactivity [16,17]. In report of Gaudry, 2014, prostate cancer patients took marine phospholipid containing high omega-3 fatty acids and evaluated the effect on the disease treatment [16]. is demonstrated that marine phospholipids were effective in increasing the n-3/n-6FA ratio significantly in cells and reducing the biosynthesis of proinflammatory eicosanoids in tumor cells, thus reducing metastatic progression of prostate cancer and inhibiting inflammation [14,16,17].
Herein, we detail the total lipids, fatty acids, and phospholipids present in soft-shell mud crabs found in Vietnam. Many molecular types were characterised. is study also evaluated the anti-inflammatory and cytotoxic activities of the polar and neutral lipid fractions; the polar lipid fraction contained the phospholipids.

Lipid Extraction and Fractionation.
Samples of S. paramamosain were collected at an experimental farm by Dr. Bui i Bich Ngoc, the Research Institute for Aquaculture No. 3. e moulting crab weight ranged from 100 to 110 g. Crabs were peeled within 2 h of collection, frozen, and transferred to the Institute of Natural Products Chemistry. Lipids were extracted from the crab samples according to the methods of Bligh and Dyer in combination with ultrasound [18,19]. A sample (10 g) was ground and added to 30 mL of methanol-chloroform (MeOH-CHCl 3 ; 2 : 1 v/v). e mixture was then exposed to three successive 30-minute ultrasound treatments. Filtration provided the first solution, and the residue was soaked in 10 mL CHCl 3 and the ultrasound treatment was repeated. e second solution after filtering was combined with the first solution, and 8 mL of water (H 2 O) was added. e resulting solution was vigorously shaken and transferred to a separating funnel. e lower organic phase containing the lipids was treated with Na 2 SO 4 to absorb H 2 O. en, this anhydrous phase was filtered and evaporated to dryness to provide the total lipids. e total lipids were dissolved in CHCl 3 and stored at -5°C. An aliquot of this total lipid solution (200 mg) was dissolved in CHCl 3 and fractionated on silica columns (J. T. Baker, Phillipsburg, NJ, USA). e silica gel was saturated with CHCl 3 , and the lipid aliquot was applied to the column. Chloroform (32 mL) was used to elute the neutral lipids, and four column volumes (56 mL) of MeOH-H 2 O (97 : 3 v/v) were used to elute the polar lipids, which contained the phospholipids. e obtained fractions were dried in Ar gas and stored at −35°C until analysed.

Fatty Acid Analysis. Fatty acid methyl esters (FAMEs)
were obtained by treating the lipids with 2% H 2 SO 4 in CH 3 OH and incubating at 80°C for 2 h. en, n-hexane (2 mL) and H 2 O (0.1 mL) were added, and the solution was vigorously shaken. e upper organic phase was purified by thin-layer chromatography using hexane : diethyl ether (95 : 5 v/v). e FAME content was determined by gas chromatography at 210°C using a Shimadzu GC-2010 (Kyoto, Japan) instrument equipped with a flame ionisation detector on an Equity 5 capillary GC column (Merck; length × internal diameter � 30 m × 0.25 mm; film thickness � 0.25 μm). Helium was used as the carrier gas at 20 mL/min. e initial oven temperature of 160°C was ramped at 2°C/min to 240°C and held at this temperature for 20 min. e injector and detector temperatures were maintained at 250°C [20]. FAMEs were analysed by gas chromatographymass spectrometry (GC-MS) to identify the fatty acid structures. e spectra were compared with the NIST library and fatty acid mass spectra archive [21].

Analysis of the Molecular Species of Phospholipids.
e chemical structures and relative amounts of molecular species of phospholipids were analysed by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS). Phospholipids were separated from the polar lipid fraction using HPLC using a solvent system with a constant triethylamine [(C 2 H 5 ) 3 N] content, i.e., (C 2 H 5 ) 3 N/formic acid (0.08 : 1, v/v) [22].
is composition was maintained for 1 min before returning to 5% of mixture B over 10 min when it was maintained at 5% for another 4 min (total run time was 40 min). e flow rate was 0.2 mL/min. Polar lipids were analysed by HRMS with processing software (LC Solutions ver. 3.60.361, Shimadzu). Quantification of the individual molecular species within each polar lipid class was carried out by calculating the peak areas of the individual extracted ion chromatograms [23]. e nitric oxide (NO) assay was performed as described previously with slight modification [24]. Following preincubation of RAW 264.7 cells (2 × 10 5 cells/mL) with LPS (1 μg/mL) for 24 h, the quantity of nitrite in the culture medium was measured as an indicator of NO production. Amounts of nitrite, a stable metabolite of NO, were measured using Griess reagent (1% sulphanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid). In brief, 100 μL of cell culture medium was mixed with 100 μL of Griess reagent. Subsequently, the mixture was incubated at room temperature for 10 min and the absorbance at 540 nm was measured in a microplate reader. Fresh culture medium was used as a blank in every experiment. e quantity of nitrite was determined from a sodium nitrite standard curve.
Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method. First, 180 μL of RAW 264.7 cells at suitable concentration were dropped into 96-well plates in the presence of lipid extracts at concentrations of 100 μg/mL and then incubated at 37°C with 5% CO 2 . After 72 h, MTT (5 µg/mL) was added to the medium for 4 h. Finally, the supernatant was removed and the formazan crystals were dissolved in DMSO. Absorbance was measured at 540 nm. e percentage of dead cells was determined relative to the control samples.

Cytotoxicity Assay with Five Cancer Cell Lines.
Cytotoxicity of the lipid sample was evaluated against five human cancer cell lines using the sulphorhodamine B (SRB) cytotoxicity assay [25]. Five cancer cell lines were SK-LU-1 human lung cancer cell line, HL-60 human leukemia cell line, HT-29 human colon cancer cell line, HepG2 human liver cancer cell line, and MCF7 human breast cancer cell line whichprovided by Prof. Dr. D.V. Delfino, Perugia University, Italy. Each cancer cell line was trypsinised and incubated with lipid samples at concentrations of 0.8, 4, 20, and 100 μg/mL in 96-well plates at 37°C for 48 h. Following this, cells were fixed with trichloroacetic acid for 1 h at room temperature (28 ± 2°C). SRB solution (100 μL of 0.4% w/v in 1% acetic acid) was added to each well. After 30 min, the unbound SRB was removed by washing with 1% acetic acid. e samples were then air-dried at room temperature (28 ± 2°C). e protein bound stain was solubilised with 10 mM Tris base (pH 10.2), and the absorbance was measured at 515 nm using a microplate reader (SpectraMax Plus 384; Molecular Devices, Sunnyvale, CA, USA).

Total Lipid Content and Fatty Acid Components.
e total lipid (TL) content of S. paramamosain was 1.62 ± 0.08%.

Molecular Species of Phospholipids.
Phospholipids form the basis of cellular structures. Because of their amphiphilic properties, they have a natural tendency to form bilayers in aqueous systems [28]. e phospholipid structure was determined using the HRMS fragmentation data of phospholipid standards as described previously [20]. In S. paramamosain, the content of polar lipids containing glycolipids and phospholipids was 40.02% of the total lipids. Glycolipids are the main component of photosynthetic organelles in plants, while phospholipids are present in the cell membranes of both animals and plants [29,30]. In this study, we focused on determining phospholipid structures using molecular analysis. ere were six types of  (Figure 1). For ethanolamine glycerophospholipid or PE, the ESI-HMRS signals of negative ions and corresponding positive ions were analysed simultaneously [19,20]. e negative fragment ion signal was always more intense than that of the positive ion ( Figure 1S) spectrum displayed four ion fragments, i.e., two fragments at m/z 303.2290 and m/z 259.2420, respectively, corresponding to ion fragments of the C20 : 4 acid and of the C20 : 4 acid that had lost a CO 2 molecule. us, the final LPE had the structure with C20 : 4 acid. e MS − and MS 2− data thus indicated that all three LPE forms lost the acyl radical and then continued to lose a CO 2 molecule.
Negative ion [M-H] − signals corresponding to inositol glycerophospholipid or PI were observed in the ESI-MS1 spectra. Many signals of different fragments were observed in the MS 2− spectrum, of which some corresponded to the initial molecular ions that had lost small fragments such as a fatty acid molecule, a ketone radical, two fatty acids, or an inositol branch (C 6 H 10 O 5 ). In addition to signals of the PI ions, we always observed signals corresponding to two fatty acid ions derived from the PI molecule. e most intense signal in the MSspectrum of the PI class appeared at m/z 883.5236 and corresponded to the empirical formula C 47 H 81 O 13 P. e number of oxygen atoms in the formula indicated that the PI molecule had the diacyl form. e MS 2− spectrum displayed signals at m/z 281.2461 and 581.3024, which corresponded to the ion of the C18 : 0 acid and to the PI molecule that had lost the anion of the C20 : 5 acid, respectively. e signal at m/z 297.0349 corresponded to the [C 9 H 14 O 9 P] fragment, which was formed when the PI molecule lost two fatty acids. is specific ion fragment signal was characteristic of the PI class. Based on the obtained ion fragments (Figure 4S), the PI molecular structure was identified as PI 18 : 0/20 : 5.

4
Journal of Chemistry Nine other PI molecules were similarly identified. Of these, PI 18 : 0/20 : 4 was the most abundant at 26.24% (Table 4). e second-, third-, and fourth-ranked positions belonged to PI 19 : 0/20 : 4 and PI 18 : 1/20 : 5 with contents of 18.40% and 11.89%, respectively. e remaining molecules had contents that were less than 10%. Notably, all PI molecules except PI 18 : 0/21 : 4 always contained a longchain acyl 20 C tail with four or five double bonds. e MS 2− spectra of serine glycerophospholipid and PS compounds always showed the loss of the neutral fragment corresponding to the serine group C 3 H 5 NO 2 (calculated m/z 87.0320) from the [M-H] − ions. is is a characteristic signal of PS molecules in ESI-MS spectra. In the ESI-MS spectra of the PS class of S. paramamosain, the most intense signals appeared at m/z 808.4996, corresponding to the empirical formula C 44 H 76 NO 10 Figure 5S shows the MS 2− spectrum in detail. ese results established that the PS molecule had the PS 18 : 0/20 : 5 structure.
We also identified 12 different types of PS molecules in the polar lipid fraction. e remaining form had low content (<10%) excepting the signal at m/z 810.5141 with content of 28.08% (Table 5).
For choline glycerophospholipid or PC, the MS + spectra showed characteristic signals of positive ions [M + H] + while the MS − spectra displayed signals  Figure 1: Structural formula of phospholipid classes.   (Table 6). Notably, the most intense signal at m/z 760.5906 in the MS + spectrum (10.59%) corresponded to the empirical formula C 42 H 82 NO 8 P (different by 0.00582; four double bonds). is was the first form of diacyl PC to be identified in this sample with both C16 : 0 and C18 :1 fatty acids. e MS 2− spectrum displayed signals corresponding to fragments of the C18 :1 acid at m/z 281.2479 and the C16 : 0 acid at m/z 255.2276. e signal at m/z 480.3151 corresponded to an ion fragment of PC that had lost a ketone group and a methyl group, i.e., M-[R-CH�C�O]-CH 3 . Based on the observed fragments, the PC molecule was identified as the diacyl PC 16 : 0/18 :1. Seven of the 21 identified molecular forms were similarly characterised using the MS 2spectrum. Table 6 lists the PC molecules. e LPC class has a similar structure as that of the PC group, but a hydrogen atom is present instead of an alkyl/ alkenyl group. e LPC molecules were identified from the signals corresponding to the [M-CH 3 ] − and [M-RCOO] − ions in the MS − spectrum. Of these, the LPC 20 : 5 molecule was characterised by the signal at m/z 526.2520; it had the highest content of 43.36%. is signal corresponded to the empirical formula C 28 H 48 NO 7 P (different by 0.00536; five double bonds). e MS 2− spectrum displayed peaks at m/z 301.2146 corresponding to the anion of the C20 : 5 acid and m/z 283.2101 and 257.2242 corresponding to the C20 : 5 acid that had lost a H 2 O and CO 2 molecule, respectively ( Figure 7S). e LPC molecule with a second-ranked content of 38.00% had the structure 22 : 6. It displayed a signal at m/z 552.2672, which corresponded to the calculated formula C 30 H 50 NO 7 P (different by 0.00485; six double bonds). e last LPC molecule with the lowest content of 18.64% contained the 20 : 4 acid, which corresponded to the formula C 28 H 52 NO 7 P. Table 7 provides the data that were used to identify the LPC molecules.

Anti-Inflammatory Activity.
Lipids play important roles in many organisms. We wanted to study the impact of lipid components on human health. We first studied the biological activity of the neutralised and polar lipids; the latter contained phospholipids. Proinflammatory agents, such as LPS, can significantly increase the number of macrophages that produce NO [31,32]. High levels of NO cause a variety of pathophysiological processes including inflammation [33] and carcinogenesis [34]. In this study, we evaluated the NO inhibitory activity of lipid fractions using an LPS-stimulated RAW 264.7 cell assay.   Marine organisms are a potential source of secondary metabolites that can be developed into cancer therapies [36]. In previous work, haemolymph collected from the walking legs of the Dromia dehaani crab was evaluated for cytotoxicity toward rhabdomyosarcoma, HepG2, A549, MCF-7, and HT-29 cell lines [37]. e IC 50 values ranged from 75 to 100 μg/mL; the highest activity was exhibited toward the MCF-7 cell line. In other work, mangrove crab soup   inhibited growth of Jurkat leukemic T-lymphocyte cells in dose-and time-dependent manners [38]. Compared to these studies, the cytotoxicity of S. paramamosain polar lipids had an IC 50 similar to that of crab haemolymph. We suggest that crab polar lipids merit further investigation as anticancer food supplements.

Conclusions
We report profiles of the composition and molecular forms of phospholipids obtained from the mud crab S. paramamosain. Six types of phospholipids containing 54 different molecular forms were identified. ese molecules were mainly made of the C16 : 0; C18 : 0; C20 : 4; C20 : 5; and C22 : 6 fatty acids. Notably, different contents were measured for the 21 common fatty acids found in the lipid fraction. e variety of fatty acid composition created a high nutritional value of phospholipid which was widely used as a food additive in a range of products.
We also report, for the first time, anti-inflammatory and cytotoxic effects of crab lipid and phospholipid. e antiinflammatory activities (IC 50 ) of the total and polar lipids were 71.5 and 68.6 μg/mL, respectively. e polar lipid sample containing phospholipids also presented high cytotoxic activity (IC 50 ) toward five cancer cell lines, ranging from 85.4 to 95.8 μg/mL. us, we suggest that crab polar lipids merit further investigation about function and bioactivities of each phospholipid class and furthermore evaluate potential application of phospholipids in the clinical therapy.

Data Availability
e data used to support the findings of this study are included within the article and the supplementary information file.

Conflicts of Interest
e authors declare that they have no conflicts of interest.