| Phytosterol is a kind of natural active substances in plants, which has similar chemical structure to cholesterol. Phytosterol has biological activity which can lower cholesterol in the human body blood, and be used as an anti-cancer and anti-inflammatory to improve health.Therefore, phytosterol is widely applied to medical pharmaceutical, food and cosmetics.Due to poor ability of solubility in water and fat, it’s limited in the scope of its application. Fatty acids of phytosterol ester can successfully meet with this requirement of good solubility. Thus, we want to study a new efficient way to synthesize phytosterol ester. Considering that fatty acids and phytosterol are both mixtures, in order to make it simple and clear to study the esterification, we selected oleic acid and stigmasterol as the reactants model to get the phytosterol ester.The mail research contents of this paper are as follows:1. The analysis methods of stigmasterol and stigmasterol esterWe set up stigmasterol and stigmasterol oleate gas chromatography methods. We used the DB-5ht chromatographic column (30 m*0.25 mm*0.25 μm).The GC conditions were:FID detector:350℃;the carrier:N2;the temperature of the column: 180℃-350℃. GC-MS and GC external calibration methods were used in this work to get the quantitative and qualitative analyses of stigmasterol and stigmasterol oleate. The results of GC were:stigmasterol for 22.262 min, and stigmasterol oleate for 19.543 min.2. The research of catalyst screening and esterificationBy the selection of the catalyst and the optimization of reaction conditions, we explored the best synthesis route of stigmasterol oleate with the esterification yield as the index. Stigmasterol oleate synthesized from stigmasterol and oleic acid using the composite catalysts of potassium bisulfate and metal oxide was investigated. By comparing with other sulfates, potassium bisulfate was proved to be more suitable for the esterification of oleic acid and stigmasterol in an efficient and safe way. The catalyst comparative experiments of MgO, Al2O3, ZnO, CaO with potassium bisulfate suggested that the composite catalyst of KHSO4-ZnO was the best catalyst in terms of the esterification yields. When the esterification reaction was carried out at 155℃ for 7h with oleic acid and stigmasterol in a molar ratio of 1.8:1 and 3.0% KHSO4 and 1.0% ZnO catalyst (based on the mass of stigmasterol) under the protection of nitrogen, the highest esterification yield was 93.26±2.88%, and the conversion rate was 97.05±3.64% with the selectivity of 96.09±3.17%.3. The separation and purification of stigmasterol oleateThe stigmasterol oleate with the purity of 99% was made by twice column chromatography separation with the mobile phase of cyclohexaneand ethyl acetate (12:1, v:v). The chemical structure of stigmasterol oleate was characterized by GC-MS, GC, EA, FTIR and NMR.We compared the solubility of stigmasterol and stigmasterol oleate in various vegetable oil and various organic solvents, with the result of the selectivity of n-hexane on stigmasterol oleate reaching 216.47 and the selectivity of ethanol on stigmasterol reaching 10.29. Thus, we chose n-hexane and anhydrous ethanol as the composite solvent for extraction to get the target product. With the recovery rate and purity rate of stigmasterol ester as the index, the selected separation process was using the n-hexaneand ethanol (50℃) as the composite solvent to extract the crude product and using potassium bicarbonate solution (50℃) to remove the oleic acid and the catalyst. The optimal separation conditions finally were:the volume ratio of n-hexaneand ethanol for 1:1.5 (v:v) and the quality-volume ratio of n-hexaneand crude products for 8 (v:m, mL/g).According to the optimized process conditions for purification, the best results was with the recovery rate of 92.23±3.35%, and the purity rate of 94.87±4.26%. |
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