Preparation And Anti-Tumor Effect Of Erythritol Phytosterol Ester

Phytosterols have many important beneficial properties, such as anti-cancer, antiinflammatory, cholesterol-lowering, antioxidant capacity, immunoregulation and so on. However, their poor solubility in oil, insolubility in water and high melting point, limits their application. Previous studies have synthesized phytosterol esters to improve their solubility in oil. The present study has synthesized phytosterol esters via chemical modification to improve their solubility in water. On the basis of this modification, the anti-tumor effect of phytosterols and its modified products in vitro and their anti-cancer mechanism were investigated.Firstly, hydrophilic phytosterol esters was prepared via chemical and enzymatic ways. The highest esterification ratio was obtained under the following conditions: Lipozyme435 as catalyst and tert-butanol as solvent, Lipozyme435 dosage 100 g/L, phytosterol hemisuccinate concentration 50 mmol/L, substrate molar ratio of phytosterol hemisuccinate to sugar alcohol 1:3.5, molecular sieves dosage 80 g/L, 55℃, reaction time 120 h. Under this condition, the esterification ratio of erythritol diester is 82.0%.The phytosterol esters were separated and purified by HPLC, TLC and silica gel column chromatography. The HPLC mobile phases for monoester and diester were methanol/formic acid(1000/1, v/v). And the silica gel column chromatography and TLC eluents for phytosterol hemisuccinate and phytosterol erythritol succinate were mineral ether/acetidin/methanoic acid(13/7/0.02, v/v/v) and acetidin/carbinol /methanoic acid(18/2/0.15, v/v/v), respectively. The products were confirmed to be the goal products by MS and FT-IR.The anti-proliferative effect of different concentrations of phytosterol and its esters against tumor cells was investegated by MTT assay. The morphological changes of cells were observed with inverted microscope. The cell apoptosis proportion, the cycle distribution, the changes of mitochondrial membrane potential and the intracellular Ca2+ were measured by flow cytometry, respectively. Results showed that both phytosterols and its esters inhibited the growth of SGC-7901、HEPG2 and A549 cells, and there were dose-effect and time-effect relation in different phytosterols and its esters groups. The apoptosis ratio was significantly increased with the dose and action time. And the cell morphology appeared typical characteristics of apoptosis. The modification has reserved the ability of phytosterols to inhibiting the tumor cells and to induce its apoptosis. The cell cycle was arrested in S phase, the MMP was significantly decreased and the intracellular Ca2+ was significantly increased. Phytosterol and its esters can induce the tumor cells apoptosis through the cell cycle arrest, mitochondrial pathway, activating endonuclease and endoplasmic reticulum pathway.The solubility in water, melting, crystallizing and pyrolysis behavior of modified products were explored. Results showed that the hydrophily of phytosterol diester was significantly higher than that of phytosterol. Erythritol diester had higher crystallizing and melting temperature. The pyrolysis temperature of phytosterol erythritol succinate higher than that of phytosterol, showing that has better thermal stability.