1. Introduction
The main beneficial health effect of fish and fish oil consumption has been attributed to their protective activity against cardiovascular diseases (CVD), which has been demonstrated by epidemiological studies [
1]. Fish oil is a good source of two important ω-3 polyunsaturated fatty acids (PUFA): eicosapentaenoic acid (EPA; 20:5(ω-3)) and docosahexaenoic acid (DHA; 22:6(ω-3)). ω-3 PUFA have been found to exert potential protective activity against thrombosis and cardiovascular diseases [
2]. Additionally, it has been suggested that other substances, apart from ω-3 PUFAs, could be responsible for the antithrombotic properties of marine fish [
3,
4]. Scientific data reported the presence of lipid micro-constituents in different fish species that have been found to exert
in vitro anti-thrombotic properties [
5,
6,
7,
8] and
in vivo anti-atherogenic activity [
9].
One of the most common global causes of sudden deaths the last decades has been CVD [
10]. PAF (1-
O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is an endogenous synthesized phospholipid compound [
11], which has been characterized as a potent inflammatory mediator with a crucial role to the mechanism of atherogenesis [
12].
Recent studies that have been conducted on polar lipid fractions of sea bass (
Dicentrarchus labrax) [
13] and gilthead sea bream (
Sparus aurata) [
14] fed with olive pomace enriched fish feed exhibited potent antithrombotic properties. Therefore, fish and fish oil are considered to be functional foodstuffs possessing protective properties against CVD.
Sardine (
Sardina pilchardus) is an important Mediterranean commercial fish species. It is a fatty fish that stores its fats as triacylglycerols in the flesh. It is also a good source of fat-soluble vitamins and high quality proteins, while sardine fillet lipids have important nutritional characteristics because of their high level of ω-3 PUFA [
15].
Several formulations of ω-3 PUFA on the market are manufactured from sardine fish oil through a complex process of purification, during which environmental pollutants and cholesterol are removed, bleaching and concentration (molecular distillation and urea complexation) of high grade sardine oil. These concentrated products contain a total concentration of 90% ω-3 ethyl esters.
Previous studies in our laboratory showed that those formulations of ω-3 PUFA include some microconstituents that can induce washed rabbit platelet aggregation or inhibit the PAF-induced platelet aggregation. Given this background work, in this paper, we have chosen to study further sardine (Sardina pilchardus) lipids and more specifically the total polar lipids (TPL) of sardine fillet.
Cod (
Gadus morhua), a coldwater marine fish, is an important source of EPA and DHA, fat-soluble vitamins and high quality protein. Cod is a lean fish that stores its reserve fats as triacylglycerols in the liver [
16,
17]. Its liver contains 50%–60% fat and accounts for 8%–12% of the total weight of the fish [
18].
Cod liver oil is a well-known “nutraceutical”, which contains a wide range of substances, including triacylglycerols, mono- and di-acylglycerols, free fatty acids, ω-3 PUFA [
19], and it is a major natural source of vitamins A and D [
20]. It is widely used as a dietary supplement. Scientific data have demonstrated that compounds with strong PAF-like and anti-PAF activity have been found in cod (
Gadus morhua) [
6]. All polar lipid fractions of cod have been found to inhibit, in a dose-dependent manner, PAF-induced aggregation or induce platelet aggregation [
6]. Bearing in mind this necessity for further studies, we have chosen to study cod liver lipids and more specifically its polar lipid fractions.
Therefore, the aim of our study was to evaluate and compare the in vitro biological activities of (a) cod liver oil produced by MERCK as a dietary supplement and (b) sardine fillet lipids extracted in our laboratory, against platelet aggregation and hence atherogenesis.
3. Experimental Section
3.1. Reagents
All reagents and solvents were of analytical grade purchased from Merck (Darmstadt, Germany). Fatty acid methyl ester standards bought individually were of GC-quality and supplied by Sigma-Aldrich (St. Louis, MO, USA), as well as bovine serum albumin (BSA) and PAF. Chromatographic material used for thin layer chromatography (TLC) was silica gel G-60 supplied by Merck and polar lipid standards used for TLC was a mix standard of hen egg yolk supplied by Sigma-Aldrich. Platelet aggregation was measured in a Chrono-Log aggregometer (model 400-VS) coupled to a Chrono-Log recorder and the gas chromatographer used was a Shimadzu CLASS-VP (GC-17A) (Kyoto, Japan) equipped with a split/splitless injector and flame ionization detector.
3.2. Sardine (Sardina pilchardus) and Cod Liver Oil Sampling
One kilogram (1 kg) of raw Greek sardines (Sardina pilchardus) were purchased from a local shop and transported to the laboratory in ice. Individual fish weighed 20 ± 2.0 g. Raw fish were washed and filleted after fish head, scales, viscera, backbone, skin and tail were removed. Then, 330 g of raw fish fillets were pooled together and that was the sardine sample.
Cod liver oil was purchased from Seven Seas Ltd, Merck.
3.3. Isolation of Fish Total Lipids of Sardine (Sardina pilchardus) and Cod Liver Oil
Total lipids (TL) of sardine fillets were extracted according to the Bligh–Dyer method [
21]. For each extraction, a sample of 110 g of fish fillet was obtained by combining several fish fillets and this sampling procedure was carried out in triplicate. One tenth of the TL samples were stored in sealed vials at −20 °C. The rest TL was further separated into total polar lipids (TPL) and total neutral lipids (TNL) using the counter-current distribution method [
22]. TNL and one tenth of TPL were stored in sealed vials at −20 °C for further analysis. The rest of TPL were further separated by preparative TLC and the obtained TLC polar lipid fractions (containing glycolipids and phospholipids) were stored in sealed vials at −20 °C for further analysis.
A quantity of 30 mL of cod liver oil was separated into TPL and TNL by counter-current distribution method [
22]. TNL and one tenth of TPL were stored in sealed vials at −20 °C for further analysis. The rest of TPL were further separated by preparative TLC and the obtained TLC polar lipid fractions were stored in sealed vials at −20 °C for further analysis.
3.4. Gas Chromatography Analysis
Fatty acid methyl esters of TPL and TNL of sardine fillets and cod liver were prepared using a solution 0.5 N KOH in CH
3OH 90% and extracted with
n-hexane. The fatty acid analysis was carried out using the internal standard method, as described extensively by Nasopoulou
et al., 2011 [
32]. The gas chromatographer used was a Shimadzu CLASS-VP (GC-17A) (Kyoto, Japan) equipped with a split/splitless injector and flame ionization detector.
Separation of fatty acid methyl esters was achieved on an Agilent J&W DB-23 fused silica capillary column (60 m × 0.251 mm i.d., 0.25 μm; Agilent). The oven temperature program was: 120 °C for 5 min, raised to 180 °C at 10 °C·min−1, then to 220 °C at 20 °C·min−1 and finally isothermal at 220 °C for 30 min. The injector and detector temperatures were maintained at 220 and 225 °C, respectively. The carrier gas was high purity helium with a linear flow rate of 1 mL·min−1 and split ratio 1:50. Fatty acid methyl esters were identified using fatty acid methyl esters standards by matching retention times of the relative peaks.
3.5. Fractionation of TPL by TLC
The TLC glass plates (20 × 20 cm) were coated with silica gel G-60 and activated by heating at 120 °C for 60 min. The thickness of the TLC plates was 1.0 mm (preparative TLC). Approximately 50 mg of TPL of sardine fillet (
Sardina pilchardus) and 25 mg of TPL of cod liver were applied to the TLC plates. A developing system consisting of chloroform:methanol:water 65:35:6 (
v/
v/
v) was utilized for the separation of TPL. The plates were stained under iodine vapors. Eight bands, either for sardine fillet or for cod liver, appeared after the separation of TPL by TLC. After the vaporization of iodine vapors, the bands were scraped and lipids were extracted from the silica gel according to the Bligh–Dyer method [
21]. The chloroform phase was evaporated to dryness under nitrogen and lipids were weighed, redissolved in 1 mL chloroform:methanol 1:1 (
v/
v) and stored at −20 °C, as described earlier [
8].
3.6. Biological Assay of the in Vitro Antithrombotic Properties
The TLC polar lipid fractions of sardine fillet and cod liver were tested for their biological activity according to the washed rabbit platelet aggregation assay [
11]. Briefly, the samples being examined and PAF were dissolved in 2.5 mg of bovine serum albumin (BSA) per mL of saline. Various amounts of the sample being examined, ranging from 1.25 to 7395 μg, were added into the aggregometer cuvette and their ability to aggregate washed rabbit platelets or to inhibit PAF-induced aggregation was determined. In order to determine the aggregatory efficiency of either PAF or the samples being examined, the maximum reversible aggregation was evaluated and the 100% aggregation was determined. The plot of the percentage of the maximum reversible aggregation (ranging from 20% to 80%)
versus different concentrations of the aggregatory agent was linear. From this curve, the concentration of the aggregatory agent, which induces 50% of the maximum reversible PAF-induced aggregation, is calculated. This value is defined as the amount of the sample that induces an equivalent to PAF EC
50, namely equivalent concentration for 50% aggregation.
In order to determine the inhibitory properties of the samples, various amounts of the sample being examined, ranging from 1.25 to 7395 μg, were added into the aggregometer cuvette and their ability to inhibit PAF-induced aggregation was determined. The platelet aggregation induced by PAF (2.95 × 10−11 M final concentration in the cuvette) was measured as PAF-induced aggregation, in washed rabbit platelets before (considered as 0% inhibition) and after the addition of various amounts of the sample being examined. Consequently, the plot of % inhibition (ranging from 20% to 80%) versus different concentrations of the sample is linear. From this curve, the concentration of the sample, which inhibited 50% PAF-induced aggregation, is calculated. This value is defined as IC50, namely inhibitory concentration for 50% inhibition.
3.7. Desensitization Experiment
Desensitization experiment was carried out according to the method of Lazanas
et al. (1988) [
33]. Briefly, platelets were desensitized by the addition of PAF or the examined aggregatory agent to the platelet suspension at a concentration that caused same size reversible aggregation. Next, stimulation was induced immediately after complete disaggregation by the addition of the same concentration of PAF or the examined aggregatory agent. It was observed that the second addition of the aggregatory agent caused less aggregation than the initial, due to the desensitization of platelets when they act through the same receptor. In cross-desensitization experiments the same experiment was repeated twice with the addition of different aggregatory agent each time [
34].
3.8. Statistical Analysis
Chemical analyses were carried out six times and all results were expressed as mean ± SD in all cases. The Wilcoxon sign test was performed to evaluate significant differences within the same group, while the Mann–Whitney U-test was performed to evaluate significant differences among different groups. Differences were considered to be statistically significant when p-value was less than 0.05. Data were analyzed using a statistical software package (SPSS for Windows, 20.0, 2012, SPSS Inc., Chicago, IL, USA).
4. Conclusions
In the research reported here, we have focused on the polar lipids of cod liver and sardine fillet, since previous studies have proved that the antithrombotic properties of foodstuffs are mainly attributed to polar lipid microconstituents. These two fish species have been chosen since sardine is the raw ingredient in the manufacture of dietary supplements of ω-3 PUFA and cod liver oil is widely used as a health supplement. The significant differences observed between the high content of TPL of sardine fillet lipids and the low TPL content of cod liver oil could be attributed to the different extraction methods that are used to deliver lipids from these two fish species. The use of solvents of high polarity in the lipid extraction of sardine tissue has increased the levels of the extracted polar lipids in comparison to the less selective way used for lipid extraction of cod and production of the nutraceutical cod liver oil. It was also shown here that neutral lipids are the dominant lipid class in cod liver oil, probably due to the wet rendering process that is used in the manufacture of cod liver oil. This process does not include the use of polar solvents and it thus leads to an extract with much lower levels of antithrombotic polar lipids [
35].
The amounts of most fatty acids in TPL of cod liver were found to be significantly lower in comparison to the amounts of fatty acids in TPL of sardine fillet. TPL of sardine fillet was also found to exert aggregatory properties, while TPL of cod liver was found to exert bimodal effect on platelets, inducing platelet aggregation at lower amounts and inhibiting the PAF-induced platelet aggregation at high ones. Conclusively, in this work, it was found that cod liver oil contains few polar lipids and these polar lipids are less in vitro antithrombotic than the corresponding ones obtained from sardine. In addition, the re-evaluation of the extraction method used in the production of cod liver oil needs to be carried out in order to increase the fraction of polar lipids obtained. This study is the first in vitro study confirming that cod liver oil contains less polar lipids and has lower activities against atherogenesis than sardine polar lipids.
Our
in vitro results should correspond to analogous favorable
in vivo results as previous studies of our group has shown,
i.e., we have found that polar lipids of sea bream have strong antithrombotic properties both
in vitro [
8] and
in vivo [
9]. Such
in vivo studies should be the best way forward to confirm that the examined lipid fractions in this paper have such antithrombotic actions that could be transferable to physiological or pathological effects. The first results, though, as presented here are encouraging and our future work will focus on
in vivo studies of the lipids of sardine and cod.
