Studies On Isolation And Purification Of Plasmalogens In Sea Foods

Plasmalogens are widely found in blood, muscle and vitals of mammals and birds, especially have highest content in brain tissue. Because of the special vinyl-ether bond structure on sn-1 position, plasmalogens have many physiological and biochemical activities. With the development of molecular biology in the pathogenesis of various human diseases and studies on phospholipid omics, the physiological activities of plasmalogens are proved. In addition to the mammals, plasmalogens are found to be exist in sea cucumbers and shellfish and lower marine creatures such as sea urchins and starfish. This paper firstly described the composition and content of plasmalogens in marine shellfish, and finally chose mussels as the research object due to the high content of plasmalogens and low-value of it. Further we probed into the changes of plasmalogens in different processes of it to find the suitable pretreatment conditions in industrial extraction of the plasmalogens. After that, the paper continued to explore with enzymatic method of extraction and purification of plasmalogens in mussels and column chromatography method of extraction and purification of plasmalogens in Cucumaria frondosa respectively. The established method is used to extract plasmalogens in mussels, Cucumaria frondosa and bovine brain. Finally, five kinds of plasmalogens were studied on the antioxidant function in vitro.Plasmalogens exist widely in marine shellfishes, six kinds of shellfish in the study were all found to have high content of pPE and aPC, the species with the highest percentage of aPC is rapana bezona Linnaeus,followed by mussels, and the lowest is sinonovacula constricta. The percentage of total pPE is generally slightly below aPC. the species with highest content of aPC are scapharca subcrenata and mussels, followed by rapana bezona linnaeus and sinonovacula constricta, and the lowest are chlamys farreri and Ruditapes philippinarum. Overall, aPC and pPE content in mussels is relatively high in six kinds of shellfish, respectively 38.79% and 24.29% of the total aPC and pPE. The primary molecular species in pPE are p18:0/20:5, p18:0/22:2 and p20:1/22:6, the primary molecular species in aPC are a16:0/22:6 and a16:0/22:5. At the same time, shellfish is also a good source of PUFA, especially rich in DHA and EPA phospholipids. Principal component analysis (PCA) can do analysis of a variety of differences in biological samples on the basis of phospholipid composition, and intuitively reflected in two-dimensional figure, due to its funcion on analysis of the variable contribution of different molecular species, it is considered as a fast and efficient method on variance analysis. After analysis, mussels are selected as the most suitable material by rich in plasmalogens and ether phosphatide and low-value, for extraction and purification and further explore the biological activity of the plasmalogens.The content of pFE is not only higher than that of pPC in mussels, but also shows better stability than the latter in processing. After boiling, microwave, hot air and cold air drying method, plasmalogens content is only slightly reduced. Boiled and hot air drying processing caused relatively obvious loss, which is about 15%-20%, the destruction of plasmalogens caused by microwave drying and cold air drying to is not serious, and PUFA content is stable, so cold air drying was chosen for raw materials handling. In the use of the phospholipase Ai hydrolysis method to enrich pPE and aPC, through the condition of single factor experiment, we ultimately determine to choose the 16 ml/g (n-hexane/buffer=1/1, v/v) reaction system volume, with enzyme quantity is 8 times quality, reaction was under 50? for 4 hours for the enrichment of the pPE optimum technological conditions. To extract phospholipids, column chromatography method was carried out by 2 times the volume of cold acetone to precipitate phospholipids, further using 5 times column volume chloroform and 3 times column volume chloroform/methanol (9/1, v/v) and eventually three times of methanol can be collected for purification of phospholipids with high purity (97.11%), continue to use chloroform/methanol/water (60/35/8, v/v/v) and chloroform/methanol (2/3, v/v) as gradient elution, phospholipids purity can reach to 99.42% at last. The experimental research on column chromatography separation methods of pPE in PE directly, found that n-hexane/isopropanol/water system of mobile phase on the glycol based bonded silica filler for PE and acetal PE has a certain trend of separation, and 90% purity of plasmalogens can be collected.Experiment finally studied on the effect of 5 test substances on protection of injured PC12 cells induced by H2O2 which were extracted by method described above. Plasmalogens have ability to protect PC 12 cell oxidative damage induced by H2O2, plasmalogens can inhibit the accumulation of intracellular ROS, improve T-AOC and SOD activity in cells, thereby enhancing cell against effect of cell killing induced by H2O2. and the effect of plasmalogens from source of marine is better than that of land source, and pPE is better than the aPC. Plasmalogens are absorbed on the form of overall or on the form of lyso-plasmalogen after hydrolysis of the sn-2 position fatty acid. When using corn oil as the carrier, and take samples of 10 mg, its total biological accessibility was 21.03%, among them 45.88% were in the form of the pPE, and 54.12% were in form of lyso-plasmalogen.