Extraction And Separation Of Phytosterols From Sargassum Horneri

Sargassum horneri is an edible brown algae,which is not only rich in minerals and many kinds of human essential amino acids,but also contains polysaccharide,protein and other biological active components.Domestic and foreign related researches mainly focuse on the large molecular substances,and the study of low polarity such as phytosterols and other small molecular compounds are relatively few.Some studies have shown that phytosterols can inhibit the absorption of cholesterol in vivo,reduce the concentration of low density lipoprotein effectively,and has anti-tumor,anti-inflammatory,anti-diabetic effects.However,the traditional analysis method is ineffective and time-consuming due to their low contents and similar structures.Hence,heat refluxing technology,high performance liquid chromatography-mass spectrometry(HPLC-MS)and high speed countercurrent chromatography(HSCCC)were applied to explore and establish the efficient method in extracting,analysing andseparating phytosterol from S.horneri in this work.The major results are shown as following:1.A method composed of refluxing and saponification was developed to extract fucosterol with 95%ethanol from S.horneri.The effect of temperature,time and liquid-solid ratio on the peak of fucosterol was studied.And the optimal process conditions were obtained by the 3 factors and 3 levels central composite design experiments:20 mL/g of liquid-solid ratio,157 min of extraction time and 85? of temperature.Under the optimized conditions,the deviation between actual and predicted values was 1.39%,which confirms that the response surface model and its results are accurate and reliable.2.A rapid detection method was established for phytosterols from S.horneri by HPLC-MS.The accurate molecular weight of some phytosterols were analyzed by high performance liquid chromatography-high resolution mass spectrometry and obtained the molecular formulas.The daughter ions were obtained by HPLC-APCI-MS/MS,and referring to the related references,10 chemical compounds were deduced.And the fragmentation pathway of the phytosterols was studied.The result indicated that the method is suitable to analyse minor phytosterols from S.horneri with its high sensitivity and fast analysis ability.3.A HSCCC method with the suitable solvents system consisting of n-hexane-acetonitrile-methanol(5:5:6,v/v)was investigated to separate and purify phytosterols from S.horneri.3.1 mg of saringosterol,19.8 mg of phytol and 23.7 mg of fucosterol were obtained from 300 mg of S.horneri within 250 min with the purities were 80.09%,93.29%and 88.13%,respectively.The structures were identified by NMR spectrum.The result suggested that HSCCC can gain target compound with high purity,greater yield but less solvent consumption,and is a chromatographic separation method with higher separation efficiency,more environmentally friendly and economic,which indicates that HSCCC is suitable for separating phytosterols from S.horneri.