| Sargassum horneri contains polysaccharides,dietary fiber,plant sterols,polyunsaturated fatty acids and other active components.At present,studies on S.horneri at home and abroad mainly focus on macromolecular active substances such as polysaccharides and dietary fiber,while there are relatively few studies on low-polarity small molecule compounds such as polyunsaturated fatty acids.Studies have shown that polyunsaturated fatty acids are nutrients needed by the human body and have physiological functions such as lowering blood lipid,lowering blood glucose and delaying aging.However,the content of polyunsaturated fatty acids in S.horneri is low,and its structure and physicochemical properties are similar.Therefore,in this paper,modern separation and analysis methods such as ultrasound-assisted extraction,liquid-mass analysis and high-speed counter-current chromatography(HSCCC)were used to explore and establish efficient extraction,analysis and separation methods of polyunsaturated fatty acids from S.horneri.The main research contents and results are as follows:1.The ultrasonic assisted extraction method of polyunsaturated fatty acids from S.horneri was established.The effects of different extraction solvents,solid-liquid ratio,ultrasonic time and ultrasonic power on the extraction of polyunsaturated fatty acids were investigated.On the basis of single factor experiment and taking the peak area of arachidonic acid as the reference index,response surface analysis(RSM)was used to optimize the optimum conditions of ultrasonic assisted extraction.The optimal extraction process of S.horneri polyunsaturated fatty acids was optimized as follows: liquid-solid ratio of 21 mL/g,ultrasonic time of 21 min and ultrasonic power of 500 W.Under these conditions,the peak area of polyunsaturated fatty acids is only 1.2% different from the predicted value,which indicates that the response surface model and its results are accurate and reliable.2.A rapid qualitative and quantitative analysis method of unsaturated fatty acids in S.horneri by liquid-mass analysis was established.High-performance liquid chromatography-high resolution time-of-flight mass spectrometry(HPLC-ESI-TOF-MS)wasused to detect the fatty acid extract of S.horneri,and 8 fatty acid compounds were identified.?-Linolenic acid and arachidonic acid in the extracts of S.horneri unsaturated fatty acids were quantitatively analyzed by high-performance liquid chromatography-tandem quaternary mass spectrometry.?-Linolenic acid has a good linear relationship in the range of 0.5~25?g/mL.Arachidonic acid has a good linear relationship in the range of 0.1~25 ?g/mL.The recoveries of the two compounds were above 90% and the RSD value was less than 6%.The content of ?-linolenic acid and arachidonic acid were 0.82% and 2.45% respectively.The results showed that HPLC-tandem mass spectrometry had high sensitivity,high recovery and good precision,and could accurately analyze ?-linolenic acid and arachidonic acid in S.horneri unsaturated fatty acid extract.3.?-Linolenic acid and arachidonic acid monomers in S.horneri were isolated and purified by high-speed counter-current chromatography and semi-preparative liquid chromatography.By measuring the distribution coefficient,n-hexane-methanol-water(0.1%TFA)(10:6.5:3.5,v/v/v)was determined as a two-phase solvent system for countercurrent chromatography.The effects of rotational speed and mobile phase velocity on the retention rate of stationary phase were investigated systematically.?-Linolenic acid and arachidonic acid were separated by high-speed counter-current chromatography.The purity of ?-linolenic acid and arachidonic acid analyzed by HPLC were 72.7% and 80.8%,respectively.After purified by semi-preparative liquid chromatography,the purity of ?-linolenic acid and arachidonic acid were 91.5% and 96.6%,respectively.The results show that high-speed counter-current chromatography combined with semi-preparative liquid chromatography is an efficient and economical preparative chromatography method,which is very suitable for the separation and purification of S.horneri unsaturated fatty acids. |
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