Study On Extraction And Bioactivity Of N-3PUFA Phospholipids From Marine Animals

It is well known that marine animals have large amounts of n-3 polyunsaturated fatty acids (PUFA) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). It is also known that shellfish and fish roe contain large proportions of phospholipids in total lipids. In recent years, phospholipids rich in n-3 PUFA (n-3 PUFA-PL) are found to have many novel functions other than the functionalities of PUFA.Oyster Crassostrea gigas and scallop Patinopecten yessoensis, as two kinds of the most important economic bivalves in China, now are widely cultured. The products of oyster and scallop are usually very simple and have low value. Squid Sthenoteuthis oualaniensis is thought to be the most abundant large squid in the tropical and subtropical waters of the Indo-Pacific region and Alaska pollock ( Theragra chatcogramma)is often processed in factories of Qingdao to get the meat product. However during the processing, the gonads from scallop, eggs from squid and roe from Pollock are usually picked out and have no good ways for further use.In this paper meat from oyster Crassostrea gigas, gonads from scallop Patinopecten yessoensis, eggs from squid Sthenoteuthis oualaniensis and roe from Alaska pollock were detected as materials to see if they are good sources of n-3 PUFA-PL.First, total lipids were extracted from the materials of marine animals and separated into neutral lipids, glycolipids and phospholipid. Fatty acid compositions of the lipids were assessed and compared. A high-performance liquid chromatograph equipped with an evaporative light scattering detector (ELSD) (HPLC/ELSD) was also used to assay the phospholipid compositions of the materials. The analysis of the phospholipids by HPLC/ELSD was shown to be an accurate and reproducible analytical method for the determination of cardiolipin (CL), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM) and lysophosphatidylcholine (LPC). It was found that PC was the most predominant PL in pollack roe and squid egg. Both PC and PE were the main PL in scallop ovary and spermary and oyster meat. It could be seen that the egg/gonad from marine fish and shellfish were rich in PL of different types. As a result, all the materials have large amounts of EPA and DHA in phospholipids and especially the pollock roe and squid eggs contained large amounts of n-3 PUFA-PC.Secondly, squid eggs were processed into powders. The squid-egg powder was extracted first by supercritical carbon dioxide (SC-CO₂) to remove the cholesterol and then ethanol to get the phospholipids product. The technology of using both SC-CO₂ and ethanol for extraction was proved to be practical and effective and had no pollution of poisonous organic solvent. Analysis result showed that the phospholipids product (more than 85% of phospholipids) contained over 60% of PC in weight and over 30 mol% of DHA in fatty acids.Next, the PC of DHA-PL product was analyzed to get the fatty acid composition. More than 40 mol% of DHA was found in PC. Phospholipase A1 was used to act with the PC of DHA-PL product and to hydrolyze the fatty acids at Sn-1 position and get the Sn2- acetylated LPC. The fatty acids composition of the Sn2- acetylated LPC was determined and over 50 mol% of DHA was found.To investigate the effects of the DHA-PC liposomes on antitumor and immune regulation. DHA-PL product was made as unilayer liposomes. Different dosage of DHA-PL liposomes were given to transplanted tumor-bearing mice respectively. The tumor weight,spleen/ B.W,Thymus/B.W,hemolysin content,DTH and the phagocytosis ability of peritoneal macrophage were measured respectively. Results showed that each dosage of DHA-PL liposomes could inhibit the growth of tumor in S180 bearing mice and the average of tumor-inhibitory rate was 36.87%. Furthermore they could enhance spleen/B.W (P<0.05); increase hemolysin content obviouslly (P<0.05,P<0.01); heighten delayed allergic level (P<0.05,P<0.01); promote the phagocytosis ability of peritoneal macrophage (P<0.01). Then it was concluded that DHA-PL liposomes can regulate both specific and non-specific immune function, inhibit the growth of tumor cells.