| Pinus koraiensis seed oil is rich in polyunsaturated fatty acids needed by human body,including linolenic acid,linoleic acid,pinolenic acid,palmitic acid,eicosapentaenoic acid,etc.Among them,pinolenic acid is the unique octadecadecantaenoic acid in the pinus koraiensis seed oil,which accounts for 14%-19%of the total fatty acids and has many functions such as lowering blood lipids,immunity and anti-inflammatory,enhancing insulin sensitivity and weight loss.Due to the structure of pinolenic acid contains three unsaturated double bonds,its stability is poor and it’s difficult to be separated and purified.However,the function of unsaturated fatty acid ethyl ester is almost the same as that of unsaturated fatty acid,and the former has stronger stability.In this study,the derivatization of pinolenic acid made it has good stability,and the efficient separation of pinolenic acid in pinus koraiensis seed oil was also realized.Pinolenic acid ethyl ester can be used as a precursor of pinolenic acid to exert its application in the fields of medicine and food.In this study,pinus koraiensis seed oil was used as the raw material.The process of ethyl esterification of pinus koraiensis seed oil fatty acids was first explored.Secondly,the oxidation stability of pinus koraiensis seed oil and pinus koraiensis seed oil fatty acid ethyl esters was compared.Then,the urea inclusion method was used to enrich and separate the pinolenic acid ethyl ester from the pinus koraiensis seed oil fatty acid ethyl esters.Finally,the lipid-lowering effect of the pinolenic acid ethyl ester was preliminarily evaluated in mice.The main research findings are as follows:(1)The physical and chemical properties of pinus koraiensis oil:Appearance is light yellow liquid:acid value is 1.08 mg(KOH)/g;peroxide value is 6.95 mmol/kg;saponification value is 159.18 mg/g;iodine value is 156.09 g/100g;average relative molecular weight is 1064.71.(2)The pinus koraiensis oil fatty acid ethyl esters were prepared by the alcohol transesterification method.Through single factor experiment combined with orthogonal experiment,the best process parameters for the ethyl esterification of pinus koraiensis seed oil were the alcohol-oil molar ratio 8:1,catalyst dosage 0.6%(calculated by the quality of pinus koraiensis oil),transesterification temperature 75℃,transesterification reaction time 2 h.Under these conditions,the ethyl esterification conversion of pinus koraiensis seed oil can reach 93.88%.(3)Through single factor experiments,four factors including the mass ratio of urea and the pinus koraiensis seed oil fatty acid ethyl esters,the ratio of ethanol to urea,the inclusion temperature,and the inclusion time in the urea inclusion method have been investigated the effects for the content of pinolenic acid.Then Box-Behnken response surface analysis was combined to determine the best experimental parameters for the urea inclusion method:the mass ratio of urea to the pinus koraiensis seed oil ethyl ester was 3:1,the ratio of ethanol to urea was 6:1(mL:g),the inclusion temperature-13℃ and inclusion time 24 h.Three sets of confirmatory tests were carried out under these conditions,and the average GC content of pinolenic acid ethyl ester was 94.75%and the recovery rate was 66.25%.The samples after urea inclusion enrichment and separation were qualitative analyzed by fourier infrared spectroscopy and hydrogen nuclear magnetic resonance spectroscopy.(4)High-purity pinolenic ethyl ester has a significant lipid-lowering activity on high-fat-induced model mice,and can significantly reduce plasma total cholesterol(TC),total triglycerides(TG),low-density lipoprotein(LDL-C)levels and elevate high-density lipoprotein(HDL-C)while inhibiting the expression of lipid synthesis-related proteins in liver tissues and promoting the expression of lipid oxidized proteins.In summary,this study realized the derivatization and separation of pinolenic acid in pinus koraiensis seed oil.High-purity pinolenic acid ethyl ester had been obtained.This study provided a feasible method for the efficient enrichment of pinolenic acid in pinus koraiensis seed oil.Preliminary evaluation of the lipid-lowering activity of the derivatized product of pinolenic acid has provided theoretical basis and technical support for the rational development and in-depth utilization of the resources of pinus koraiensis and related products of pinolenic acid. |
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