Study On Preparation Of High-Purity Phosphatidylinositol

Phosphatidylinositol（PI）consists of phosphate 1,2-diacylglycerol and inositol.It is not only the constituent material of cell membrane,but its biological function and application in medicine have increasingly become a research hotspot.High purity PI can be used to prevent and treat Alzheimer’s disease.It can also be used in combination with common clinical anticancer drugs.It has excellent characteristics of safety,effectiveness,target and synergism.In view of the problems of high preparation cost,complicated operation of the existing high-purity PI.On the basis of optimizing the method of quantitative analysis of phospholipid,High purity PI was prepared by alkaline alcohol extraction-freeze separation-ethanol extraction and calcium salt precipitation.and the species of PI molecules in the product were identified.The specific contents are as follows:Firstly,optimized analysis conditions for quantitative analysis of PI in phospholipids by HPLC.With Si-60 column（250 mm×4.6 mm,5μm）,isopropane:isopropanol:1%acetic acid（8:8:1,v/v/v）as mobile phase A,isopropanol:hexane:0.06%ammonium acetate（6:4:0.1,v/v/v）as mobile phase B,gradient elution,The recovery rate of PI samples was98.41%¹00.97%,indicating the relative standard deviation of precision is 1.25%.At the same time,the quantitation of PI was further analyzed by ³¹P-NMR and compared with HPLC results.The results showed that both methods can be used to quantify PI in phospholipids.Secondly,PI was extracted by alkaline ethanol extraction and the extraction process was optimized.Powdered phospholipids were desugared and used as raw materials.The alkaline ethanol extract was frozen to prepare crude PI,called crude PI-1,and the residue extracted from alkaline ethanol was called crude PI-2.The PI content was used as an indicator,The optimum extraction conditions of crude product PI-1 were as follows:the extraction temperature was 40oC,and the ratio of material to liquid was at 1:25（g/mL）,98%alcohol:25%ammonia water=100:3（v/v）,the extraction time was 40 min,the freezing time was 16 h,the freezing temperature was-20oC,and the extraction times were 3 times.The crude PI-1content was 82.62%,the yield was 68.61%,the crude PI-2 content was 55.85%,and the yield was 72.62%.Then,further optimization of the crude PI-1 as raw materials,using ethanol extraction and crude PI-2 as raw materials,using calcium salt precipitation purification process.Measured by PI content and yield.The optimum extraction conditions of PI-1 were 132 min,the extraction temperature 28oC,and the ratio of material to 1:32（g/m L）.The purity of the product was 94.05%and the yield was 64.27%.The optimum extraction conditions of PI-2were as follows:,the concentration of ethanol was 56%,the volume of extraction liquid(V_n-hexane:V_{ethanol solution})was 1:2,pH 8.5,the amount of Ca²⁺added was 0.4 g/L,the extraction times were 3 times,and the final PI-2 purity was 90.26%and the yield was 59.43%.Finally,the PI molecular species of the products were identified by LC-MS.The results showed that there were 8 kinds of PI molecular types in the product PI purified from crude PI-1 and PI-2.The content of palmioyl/linoleoyl phosphatidylinositol was the highest in the two products.The content of saturated fatty acids in product PI is higher than that of raw materials,which is more conducive to the stabilization and storage of phospholipids.Moreover,the by-product PE content are relatively high and have certain application value.Through the above studies,a process for preparing a high content of phosphatidylinositol was obtained,and phosphatidylinositol in phospholipids was recovered to the the maximum.The process is simple and the product content wss high,providing a new idea for the industrial production of high levels of phosphatidylinositol.