Study On The Preparation And Properties Of Ornithine Phytosteryl Ester Hydrochloride

Phytosterols,as a kind of important natural compounds,have significant functions of lowering cholesterol,lowering blood lipid,anti-cancer and anti-inflammatory.The water-solubility and oil-solubility of phytosterols are much low due to their unique chemical structure,which limits their use in food systems.In this study,ornithine phytosteryl ester hydrochloride was prepared by a mild and high-efficiency chemical modification method,which improved the hydrophilicity of phytosterols.The water solubility,emulsifying properties,bioavailability and cholesterol lowering activity of the target products was also studied.In this work,ornithine phytosteryl ester hydrochloride was synthesized for the first time,through N,N'-bis[tert-butoloxycarbonyl(BOC)]-ornithine phytosteryl ester as an intermediate.This method also involved the esterification of phytosterols with N,N'-bis(BOC)-ornithine and the deprotection of BOC group.The optimal process for the synthesis of N,N'-bis(BOC)-ornithine phytosterol ester was determined by single factor experiments and response surface method.With N,N'-dicyclohexylcarbodiimide(DCC),1-Hydroxybenzoyltriazole(HOBt)and4-dimethylaminopyridine(DMAP)as the catalyses,N,N'-bis(BOC)-ornithine to phytosterols in 1.5:1.0 molar ratio was added into reaction system followed by the addition of N,N'-bis(BOC)-ornithine/DCC/HOBt/DMAP in 1.00:1.20:1.20:0.08 molar ratio.The feeding method adopted a two-step method.Firstly,DCC and HOBt was dissolved in dichloromethane followed by the addition of N,N'-bis(BOC)-ornithine for 2 h activated reaction at 0~oC.Then DMAP and phytosterols were added at 25°C for 6 h fully reacting.Under this conditions,the yield of N,N'-bis(BOC)-ornithine phytosteryl ester could reach at 90%.And more than 99%of BOC group deprotection degree was achieved using HCl/ethyl acetate deprotection method in with less by-products was presented.Based on this processing method,the final product ornithine phytosteryl ester hydrochloride was obtained.The reaction products were firstly analyzed and separated.The HPLC mobile phase for bis(BOC)-ornithine phytosteryl ester and ornithine phytosteryl ester hydrochloride analysis was methanol/formic acid(1000/1,v/v).And the silica gel column chromatographic eluent for N,N'-bis(BOC)-ornithine phytosteryl ester was formic acid/petroleum ether(60~oC-90~oC)/ethyl acetate(0.02/12/8,v/v/v).The purity of the final product was 99%after separation and purification.The intermediate and final products were analyzed by infrared spectroscopy(FT-IR),mass spectrometry(MS)and nuclear magnetic resonance spectroscopy(NMR).The results characterized the structures of N,N'-bis(BOC)-ornithine phytosteryl ester and ornithine phytosteryl ester hydrochloride.The melting and crystallization characteristics,thermal stability,antioxidant activity,water solubility and emulsifying properties of ornithine phytosteryl ester hydrochloride were determined.The results showed that the melting temperature of ornithine phytosteryl ester hydrochloride was higher than that phytosterols,but the crystallization temperature was lower than phytosterols.The thermal stability of ornithine phytosteryl ester hydrochloride was higher than phytosterols,which means thermal processing could not affect its chemical structure.The antioxidant activity of ornithine phytosteryl ester hydrochloride did not change much.The solubility of ornithine phytosteryl ester hydrochloride in water was 0.74 mg/mL,higher than phytosterols.The emulsifying property of the ornithine phytosteryl ester hydrochloride was greatly improved,which showed a twice emulsifying activity increase of phytosterols,and more than twice increase for the emulsifying stability of phytosterols.The bioavailability and cholesterol-reducing activity of ornithine phytosteryl ester hydrochloride were studied.The bioavailability of ornithine phytosteryl ester hydrochloride was 4.5%,higher than phytosterols by 42.5%,and 65.0%of ornithine phytosteryl ester hydrochloride could be hydrolyzed in digestion test.The results indicated that ornithine phytosteryl ester hydrochloride could be decomposed in the body and the bioavailability was significantly improved.The cholesterol-reducing activity was investigated in vitro model,showing that ornithine phytosteryl ester hydrochloride could reduce the dissolution capacity of cholesterol in the bile salt micelles solution.It is proposed that ornithine phytosteryl ester hydrochloride showed a higher cholesterol-reducing activity than phytosterols.The results indicated that ornithine phytosteryl ester hydrochloride could increase the cholesterol-reducing activity of phytosterols.