| Phytosterol is a kind of important bioactive compound, which is widely used in food, medical and cosmetic industries. The physiological functions of phytosterol and sterol esters include:they can lower the concentration of cholesterol in the blood, prevent prostate disease, be used as anticancer substance, with anti-inflammatory and antipyretic effect, with skin care and beauty effect, promote animal growth and body health. Phytosterol have poor oil solubility, we need to change the form of phytosterol without changing the physiological function in order to facilitate the application in food industry. Phytosterol esters meet with this requirement, they greatly improve the oil solubility of phytosterol so that we can add them into the food with oil.The mail research contents of this paper are as follows:1)Comparison of normal and reverse phase column in phytosterol detection by high performance liquid chromatography(HPLC)Both normal phase and reverse phase columns in HPLC-UV were applied to detect phytosterols, respectively. The normal phase chromatographic conditions were as follows:using hypersil SiO2(4.6mm×250mm,5μm) as column and V(hexane): V(isopropanol)=99:1as mobile phase, column temperature of35℃, flow rate of1.0mL/min, and detection wavelength of205nm.Reverse phase chromatographic conditions were as follows:using Merck RP-18(4.6mm X250mm,5μm) as column and pure methanol as mobile phase, column temperature of35癈, flow rate of1.0mL/min, and detection wavelength of205nm.As a result, high sensitivity was observed for phytosterol detection using UV detector in both system. It was revealed that the normal phase system could not separate individual phytosterol compounds, while the reverse phase system exhibited good separation capacity, In terms of retention time, the normal phase system needed much less time as compared with reverse one. In addition, phytosterols were much more solube in the eluent of the normal phase system.It is concluded that the normal phase column is more suitable than reverse one for quantification of total phytosterols despite of lower separation ability. 2)Esterification catalysts screening and esterification systhesis studya)An efficient approach based on the synthesis of phytosterol esters with a metal oxide-surfactant-combined catalyst was developed. We screened out the best catalyst through studying the catalytic effect of surfactant, metal oxides and their combinations. The reaction conditions were further optimized by catalyst dose, substrate molar ratio, reaction temperature and reaction time. A high yield of phytosterol esters (94.00%) was obtained under optimum conditions:the catalyst is1%MgO-2%span-combined-catalyst,2:1molar ratio of palmitic acid to phytosterols,150℃and5h. IR and NMR were adopted to confirm the chemical structrure of esters. We apply the optimized reaction system to different carbon chain length fatty acid to see get their esterification rate.b)A green and efficient approach based on the synthesis of phytosterol esters with a metal oxide-sodium bicarbonate-combined catalyst was developed. Because sodium bicarbonate is food friendly, it brings advantages over existing catalysts. The reaction conditions were optimized by the dose of sodium bicarbonate and reaction time. Then we screened out the best catalyst through studying the catalytic effect of other carbonate, and the combination of metal oxide and sodium bicarbonate. A high yield of phytosterol esters (90.10%) was obtained under optimum conditions:the catalyst is2%ZnO-2%sodium bicarbonate-combined-catalyst,2:1molar ratio of palmitic acid to phytosterols,150℃and4h. We apply the optimized reaction system to four saturated fatty acid with different carbon chain lengths (decanoic acid, lauric acid, myristic acid and stearic acid) and two unsaturated (oleic acid and linoleic acid)to get their esterification rate.3)Esterification kineticsWe study on the esterification kinetics of phytosterol with palmitic acid (the molar ratio is1:1). The reaction conditions are:1%MgO-2%span-combined as catalyst,150℃as reaction temperature and5h as reaction time. Under different temperatures, sterol and sterol esters were sampled from the system at a period time, and their changes with time were determined by HPLC to obtain the reaction order and the parameters of the kinetics. Kinetic constants at different temperatures were obtained. The results indicated that the activitation engery of the reaction was about111.42kJ/mol,and the rate equation of esterification could be expressed as r=1.2997x1010exp[111.42/(RT)]cAcB. It showed that the reaction met the second order reaction, and any side reaction was not found.4) A highly sensitive quantification of phytosterols through an inexpensive derivatizationPhytosterol have weak UV absorption ability in long wavelength,254nm for instance. In order to change this situation, a highly sensitive method for quantification of phytosterols based on HPLC has been developed by derivatization with the benzoyl chromophore. Introduction of the chromophore, benzoyl group, to phytosterols via simple and inexpensive derivatization greatly improved the UV response at254nm. Quantification of phytosterols was effectively performed by HPLC analysis with methyl benzoate as the internal standard after derivatization. This new method demonstrated outstanding yield of recovery (>95%) and excellent sensitivity (ng level) and was applicable for sterols from either plant or animal sources. This method is generally useful in phytosterol studies. |
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