Preparation And Purification Of DHA And 2-DHA-MAG And Their Regulation Of Lipid Metabolism In HepG2 Cells

Docosahexaenoic acid(DHA)belongs to n-3 polyunsaturated fatty acids,which plays important roles in the maintenance of human nutrition and health and the prevention and treatment of diseases.Most of the concentrated n-3 PUFA products in the market today are in ethyl ester(EE)form,but free fatty acid form and acylglycerol form are better absorbed.In addition,the position of DHA located on triacylglycerols affects its physiological functions.Based on the metabolism of triacylglycerols,triacylglycerols with different distributions of DHA generate different forms of DHA,namely free fatty acid form and 2 MAG form(2-DHAMAG).Therefore,high purity DHA and 2-DHA-MAG were prepared.The acyl migration of 2-MAG was also studied.Meanwhile,DHA and 2-DHA-MAG represented two metabolites of triacylglycerols with different DHA distributions,and the effects of them on lipid metabolism in HepG2 cells were investigated.The main contents are as follows:Firstly,low temperature solvent crystallization and urea complexation were carried out to concentrate DHA from the hydrolysis product of tuna oil.Acetonitrile was chosen as crystallization solvent and it reduced crystallization time effectively.Under the optimum conditions as FFA/acetonitrile of 1:10(w/v),crystallization temperature of-60°C and time of 1 h,the content of DHA increased from 26.85% to 56.31%,with the recovery of about 60%.Enrichment of DHA from tuna oil fatty acid by urea complexation was also optimized.Under the optimum conditions as fatty acid/urea/ethanol of 1:3:12(w/w/v),complexation temperature of-8°C and complexation time of 10 h,the content of DHA enriched from 26.85% to 77.21%,with the recovery of about 50%.Under the same conditions,the DHA content of the hydrolysis product of algal oil(Schizochytrium sp.)increased from 48.20% to 79.82% and 83.12%,respectively.Next,the final product with 95.23% DHA in a recovery of 49.33% was achieved by preparative HPLC equipped with a 5 cm diameter pillar.Secondly,2-MAG rich in DHA was prepared by enzymatic ethanolysis of tuna oil and algal oil(Schizochytrium sp.).The effects of lipase type,oil to ethanol molar ratio,reaction time,reaction temperature and lipase load on the content of 2-MAG in the crude product were investigated.Under the optimal conditions(molar ratio of TAG to ethanol of 1:60 at 30 °C for 2 h with 8% Lipozyme 435 as catalyst),the content of 2-MAG in the crude reaction products of fish oil and algal oil ranged from 22.10% to 27.40% and 26.50% to 30.95%,respectively.After ethanolysis reaction,solvent extraction with hexane and 85% ethanol was performed to extract 2-MAG,increasing its content to more than 94% with a recovery of about 70%.The DHA contents in the fish oil and algal oil 2-MAG were 53.22% and 74.76%,respectively.Subsequently,low temperature crystallization and molecular distillation were applied to concentrate DHA in 2-MAG form.Under the 2-MAG/acetonitrile of 1:10(w/v),crystallization temperature of-40°C for 10 h,the DHA content of tuna oil MAG increased to 67.63%,and the 2-MAG content remained more than 90%.By molecular distillation,the DHA content of tuna oil MAG increased to 59.95%,but the 2-MAG content decreased to 69.81% due to acyl migration.Thirdly,acyl migrations of 2-MAG at different temperatures(25°C,40°C,50°C)and in different solvents(hexane,ethanol,tert-butanol,acetone,acetonitrile,dichloromethane)were studied,which were in accordance with the first-order reversible reaction model.In solventfree system,with the increase of temperature by 25°C,the degree of acyl migrations increased 5.6 times.Different solvents had different influences on the acyl migrations,and the order of acyl migration rate constants was hexane>dichloromethane>ethanol≈acetone≈acetonitrile>tertbutanol.A significant positive correlation was observed between acyl migration rate constants and log P value of solvents(R=0.932 for 40°C and R=0.939 for 50°C,p<0.01).Then the distribution of fatty acids in the 2-MAG fraction and 1-MAG fraction were investigated.At 40°C,in solvent systems(ethanol,acetone,acetonitrile,tert-butanol)with little acyl migration,the contents of DHA were higher in the 2-MAG fraction than that in 1-MAG fraction,because of the low acyl migration rate of DHA.At 50°C,the difference of DHA contents distributed in 2-MAG and 1-MAG fraction decreased in the same solvent systems.This might result from increasing acyl migration rate of DHA with higher temperature.Finally,the effects of DHA and 2-DHA-MAG on the lipid metabolism of HepG2 cells were studied.DHA and 2-DHA-MAG at concentrations below 50 μmol/L were not toxic to cells.They were both well incorporated to HepG2 cells,and the DHA contents in HepG2 cells were without significant differences.Compared to DHA,2-DHA-MAG can decrease the contents of total triglyceride and cholesterol more effectively,and the contents of total triglyceride and cholesterol decreased by 25.21% and 32.64% in the oleic acid induced HepG2 cells,respectively.DHA in 2-MAG form significantly increased the expression of genes involved fatty acid oxidation(PPARα and CPT-1)and meanwhile inhibited the expression of gene involved in cholesterol synthesis(HMGCR).In summary,the methods for purification of DHA were established and the content of DHA after purification was 95.23%.Then synthesis and purification of 2-DHA-MAG were carried out,achieving the product with more than 94% 2-MAG and about 74% DHA.Temperature and solvents had great influences on the acyl migration of 2-MAG.According to cell experiment,2-DHA-MAG performed better in decreasing the triglyceride and cholesterol contents than DHA,which indicated DHA at the sn-2 position enhanced the regulation of lipid metabolism.It can provide theoretical support for developing novel DHA product.