The Preparation Process Of Ultrasonic Saponification And High-speed Countercurrent Chromatography Studies Of Lutein

Lutein is one of the natural pigments, easy to be oxidized in the light, airconditions. The antioxidant of lutein make it has a variety of pharmacologicalactivities, lutein esters is the mostly form existing in the nature. When lutein ester isconverted to lutein by saponification reaction, its absorption and utilization issignificantly improved in body. In this paper, marigold particles were used as the rawmaterial, we systematically studied the extraction, purification, characterization oflutein and its antioxidant activity by using of ultrasonic technology, high performanceliquid chromatography, mass spectrometry, high-speed countercurrentchromatography and cell model. The study includes the following aspects:1. Comparing with bath saponification reaction, the ultrasonic saponification hadshort reaction time, high efficiency and a complete saponification reactioncompared with bath saponification reaction; Four factors and three levels were setfor the orthogonal experiment, the optimum conditions of the saponification ofultrasound were determined. The ultrasound saponification time was20min, KOHconcentration was1.5mol/L, solid-liquid ratio was1:10, saponificationtemperature was50℃,the yield of lutein above1.420%.2. Identified the substances of ultrasonic extract composition before and aftersaponification by mass spectrometry. According to the validation guidelines oftraditional Chinese medicine quality standard analysis method of the2010editionof Chinese pharmacopoeia,we make a systematic study on methodology on theHPLC analysis method and the stability of lutein.3. Pure lutein was prepared by high-speed counter current chromatography(HSCCC).In accordance with its polarity, lutein was weak polar molecule, a opportunetwo-phase solvent system for the HSCCC was selected. The experimental resultsshowed that the solvent system of n-hexane: ethanol: water is good, each peak hadgood resolution within150min,the purity of lutein reached more than99.0%. 4. The assess of antioxidant activity of lutein was carried out by using of Africangreen monkey kidney cells(VERO cells)and the effects of lutein concentrations onthe cell damaged by using H2O2were investigatived. Experimental results showthat, cell survival was significantly improved when the lutein exist. It is provedthat lutein can protect VERO cells, reducing oxidative damage induced by H2O2.The paper optimized the process of lutein prepared by using of ultrasoundsaponification, as well as purification methods of countercurrent chromatography, thepreliminary study of quality standards,the study of activity of the anti-oxidative andstability. Our studies have provided reference for the further conduct research anddevelopment of lutein.