Synthesis of Soybean Sterol Esters and Their Bioavailabilities

Author:Yang Fu Ming

Supervisor:ma zuo

Database:Doctor

Degree Year:2018

Download:175

Pages:150

Size:1971K

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Soybean sterol as a kind of phytosterol is extracted from deodorization distillate of soybean oil.Soybean sterol is composed of β-sitosterol,stigmasterol,campesterol and other kinds of phytosterol.Soybean sterol has several kinds of biological functions,like lowering cholesterol,anti-inflammatory,anti-cancer,reducing blood fat.Soybean sterol is insoluble in water,and its solubility is very low in edible oil.Those properties limit its application in food and health food products.Soybean sterol ester is synthesized by soybean sterol and organic acids.The biological properties of soybean sterol esters are similar as soybean sterol in human body.The solubility of soybean sterol esters is better than that of soybean sterol in vegetable oil.In this paper,soybean sterol acetate,soybean sterol oleate and soybean sterol linoleate were synthesized by soybean sterol and fatty acids without chemical catalyst.For the synthesized soybean sterol esters,the detection method,physiochemical properties,structural characteristics,in vitro and in vivo bioavailabilities were studied.A HPLC-UV method was established to simultaneous detect 3 soybean sterols and 9 soybean sterol esters.Symmetry-C18 chromatographic column was used to separate soybean sterols and their esters.The mobile phase was acetonitrile and acetone(1︰3,V/V)with flow rate at 1.0 m L/min.The UV wavelength was set at 210 nm,and the column temperature was 30℃.Under this chromatographic condition,3 soybean sterols and 9 soybean sterol esters can be effectively detected in 25 min.Based on the methodological results,limit of detection,limit of quantity,accuracy and precision were 0.20-1.37 μg/m L,0.67-4.57 μg/m L,96.3%-104.7%,and 0.71%-3.55%,respectively.Standard recovery and substrate recovery were 96.07%-111.98% and 94.43%-105.11%,respectively.In the range of 0.01-1.0mg/m L,the linear relation is perfect.Soybean sterol acetate was synthesized by soybean sterol and acetic anhydride without chemical catalyst.The synthesis condition was that the mole ratio of soybean sterol and acetic anhydride was 1︰1,and the reaction was performed at 135°C for 1.5 h.The esterification rate of soybean sterol and yield of acetate were 98.72% and 98.39%,and the final purity was 98.46%.The Acyl chloride method was used to synthesis soybean sterol oleate and soybean sterol linoleate.The synthesis condition of soybean sterol oleate was that mole ratio of soybean sterol and oleoyl chloride was 1:1.1,and the reaction was performed at 90°C for 1 h.The esterification rate of soybean sterol and yield of oleate were 96.84% and 92.05%,and the final purity was 97.51%.The synthesis condition of soybean sterol linoleate was that mole ratio of soybean sterol and linoleoyl chloride was 1:1.2,and the reaction was performed at 80°C for 1.5h.The esterification rate of soybean sterol and yield of linoleate were 94.04% and 87.65%,and the final purity was 95.57%.The structures of soybean sterol esters were characterized by FT-IR,GC-MS and HPLC-MS.The results indicated that almost no free-OH was found in the structure of soybean sterol esters,but new C=O and C-O-C groups were formed in the structure of soybean sterol esters.Characteristic ions and molecular mass of β-sitosterol esters,stigmasterol esters,campesterol esters were in accordance with the theoretical molecular mass,which proved that soybean sterol acetate,soybean sterol oleate and soybean sterol linoleate were synthesized.Thermodynamic properties of soybean sterol esters were analyzed using DSC.Melting and crystallization temperatures of soybean sterol esters were lower than that of soybean sterol.The melting and crystallization temperatures of soybean sterol acetate were 136.9°C and 113.4°C,respectively.The melting and crystallization temperatures of soybean sterol oleate were 22.8°C and 12.9°C,respectively.The melting and crystallization temperatures of soybean sterol linoleate were 12.4°C and-4.2°C,respectively.In soybean oil,solubility of soybean sterol acetate,soybean sterol oleate and soybean sterol linoleate were respectively 3.68%,32.90%,and 33.81% at ambient temperature,but the solubility of soybean sterol was 1.43%.Soybean sterol esters had better p H stabilities(p H 2.0-12.0)and thermal stabilities(210°C).Soybean sterol oleate and linoleate had poor oxide stability compared with soybean sterol acetate.In vitro simulated gastrointestinal tract,the bioavailabilities of soybean sterol,soybean sterol acetate,soybean sterol oleate,soybean sterol linoleate were 1.59%,2.93%,14.72%,16.64% respectively.In Caco-2 cells,the absorptions of soybean sterol,soybean sterol acetate,soybean sterol oleate,soybean sterol linoleate were 4.84 μg/g,25.84 μg/g,75.93 μg/g,78.89 μg/g,respectively.The transported mechanisms of soybean sterol and soybean sterol esters into Caco-2 cells were passive diffusion and P-glycoprotein efflux.In rat vivo test,soybean sterol and soybean sterol esters reached the maximum absorption at 3 h.About 70% of soybean sterol acetate was hydrolyzed into soybean sterol and acetic acid,more than 99% of soybean sterol oleate and linoleate were hydrolyzed into soybean sterol and oleic acid/linoleic acid in the rat body.Soybean sterols were mainly distributed in blood,heart,liver,spleen,kidney and lung after absorption,and its largest distribution was in the liver.95% of soybean sterol/soybean sterol acetate,and 80%-85% of soybean sterol oleate/linoleate was not absorbed in the body and were excreted through feces.The bioavailabilities of soybean sterol and soybean sterol acetate were lower than 5%,and the bioavailabilities of soybean sterol oleate and linoleate were in the range of 15%-20%.