Phosphatidic Acid Extraction And Purification And Physiological Activity

ONE The Aim of the Study:Phosphatidic acid (PA) is a small phospholipid commonly found in cell membranes. It also serves as a central intermediate for the synthesis of membrane and storage lipids. Recently, PA has also been implicated in various cellular processes such as signal transduction, membrane trafficking, hormone secretion by endocrine cells, and cytoskeletal rearrangement. As such, PA is regarded as essential for the survival, proliferation, and reproduction of cells or organisms. Because of its important biological functions, it is of great interest to use PA to improve human health. Indeed, when consumed as a supplementation of soy lecithin PA and phosphatidylserine complex, for instance, PA has been shown to improve pituitary adrenal reactivity and psychological response to mental and emotional stress. It has also been used as a drug or supplement in some cosmetics.To support commercial application of PA, large quantifies of PA of high purity need to be produced from readily available sources. Soybean phospholipids contain relatively high level of PA. Pure PA was obtained using TLC, HPLC, phosphatidase D, chemically synthesize and extraction with a supercritical solvent. These analytic methods, however, can only produce small quantities of PA for research purposes or the amount of PA we get is too small and can only use for science research. As such, an efficient process is still needed to prepare large quantifies of PA of high purity. Here we report the development of a new process that permits the effective extraction of PA to high purity (>99%) from powdered phospholipids derived soybeans. This process can be readily adapted to industrial PA production at commercial scales.TWO Methods and Results of StudyWe use solvent to get high purity phosphatidic acid. In this process, by ethanol extraction, methanol extraction, lipid-lipid extraction, metal ion precipitation and acetone washing, we get hight purity PA sample.The PA extracted was analyzed by TLC. The mobile phase consisted of chloroform– ethanol– triethylamine - water (30:35:34: 8). The TLC was allowed to run for 2 hours. A bromothymol blue solution was used for color development and visualization. The Rf of both the extracted PA and the PA reference was determined to be 0.51.The extracted PA was further confirmed by HPLC analysis. The analytical column (4.00mm×300mm, hypersil SiO2 5μm) has a silica normal-phase. The detection wavelength was set at 210 nm. Chromatographic separation was carried out using a mobile phase of hexane-isopropanol-H₂O (5:3:0.4) at a constant flow rate of 1.5 ml/min. Exhibiting a single major peak of the same retention time as the PA reference, the PA in the PA extract was further confirmed and appeared to be of high purity. Estimated from the peak areas, the purity of the PA extract was approximately 99.1%.The fatty acid components of the extracted PA were identified by gas chromatography-mass spectrometry (GC-MS) analysis. Two fatty acids were identified in the PA extract: stearic acid (C18:0) and oleic acid (C18:1 cis-9), which account for 43.6% and 56.4%, respectively.In the process we also investigated physiological activity of PA. That is the role of exogenous PA ON [Ca²⁺] in cultured original rat hippocampal nerve terminals by flu-3 technique.the result is that PA caused an evident increase on [Ca²⁺] in the hippocampal nerve terminals.THREE Innovation and Significance of Research FruitThe innovation:solvent is used in the process to get high purity PA. Exogenous PA can inctrase [Ca²⁺] in the rat hippocampal nerve terminals.The significance:A process for isolating phosphatidic acid of C18 fatty acids from powered soybean phospholipids was described. This process can be adapted to industrual production of PA.