Research On The Separation And Purification Of Active Component Of Egg Yolk Lecithin And Its Effects On Memory Improvement

As an important component of biological membrane,lecithin possesses a lot of nutritional and healthy functions,such as lipometabolism regulation,cardiovascular diseases prevention and brain function enhancement.Our country has a large number of poultry resources,thus it’s necessary to study egg-yolk lecithin in-depth to improve added value of poultry products and promote poultry industy development.This study researched the solvent extraction optimized by single factor tests and response surface methodology,purification using silica gel-based column chromatography,memory-enhancing effect by animal model,neuroprotective effect by cell model of egg-yolk lecithin and its active components,phosphatidylcholine（PC）and phosphatidylethanolamine（PE）.The results were as follows:（1）Solvent extraction of egg yolk lecithin was optimized by single factor tests and response surface methodology.The content of PC and PE in egg yolk lecithin was detected by high performance liquid chromatography combined with evaporation light scattering detector（HPLC-ELSD）.The corresponding optimal output of PC was obtained at ethanol concentration of 91.1%and extraction temperature of 39.5 ^oC to achieve the highest content（226.78±0.18μg/mL）of PC.The optimum extraction conditions were found to be a ratio of ethanol to n-hexane of 4.6:1,an extraction temperature of 40.7 ^oC,and an ethanol concentration of 98%,to achieve the highest PE content（58.94μg/mL）.（2）Memory-enhancing effect of egg-yolk lecithin against scopolamine-induced dementia in mice was performed by behavioral experiments,enzymatic index and histopathologic indices.The results of water maze test and step-through test showed that the latency time and error time of lecithin group were markedly（P<0.05）reduced compared with model group.The results of memory-related enzymes in brain tissue showed that egg-yolk lecithin could significantly（P<0.05）downregulate the acetylcholinesterase（AChE）activity and upregulate the choline acetyltransferase（ChAT）activity.Hematoxylin and eosin（HE）stain of cerebral cortex and hippocampus of lecithin group showed that the atrophy of cells were restored to some extent compared with model group.In addition,egg-yolk lecithin had no significant effect on blood sugar and blood fat analyzed by blood index,body composition test and HE staining of liver and colon in mice.Animal experiments showed that egg-yolk lecithin could enhancing memory by decreasing AChE activity and increasing ChAT activity without affecting blood sugar and lipid in the mice.（3）Based on the result that egg-yolk lecithin has memory enhancement function,the purification and molecular structure profiling of active components in egg-yolk lecithin was study in the chapte,which could provide a basis for the follow-up study of their biological activity.Purification and molecular structure profiling of PC and PE in egg yolk lecithin was implemented by column chromatography（silica gel）and matrix-assisted laser desorption/ionization-time of flight mass spectrometry（MALDI-TOF MS）combined with gas chromatography-mass spectrometry（GC-MS）.PE was successfully purified using isocratic elution with a mixed solvent eluent（chloroform-methanol-acetic acid=18:5:1）.HPLC-ELSD results indicated that the PE purity obtained by column chromatography reached up to 98%.Purification of PC was performed using gradient elution.The chloroform-methanol-acetic acid mixture at a ratio of 18:5:1（v/v/v）was used as the first elution,then polarity of the mobile phase was increased by changing the proportion of the chloroform-methanol-acetic acid to 10:5:1（v/v/v）.PC was successfully purified and the purity reached up to 98%detected by HPLC-ELSD.The molecular species and structure of PC and PE were characterized by MALDI-TOF MS combined with GC-MS.PC was identified as 16:0/16:0-PC,16:0/16:1^Δ9-PC,16:0/18:0-PC,16:0/18:1^Δ9-PC,16:0/18:2^Δ9,12-PC,18:0/18:1^Δ9-PC,18:0/18:2^Δ9,12-PC,18:1/18:2^Δ9,12-PC and 18:0/20:4^Δ5,8,11,14-PC,and PE was identified as 16:0/18:1^Δ9-PE,16:0/18:2^Δ9,12-PE,16:0/20:4^Δ5,8,11,14-PE,18:0/18:1^Δ9-PE,18:0/18:2^Δ9,12-PE,and 18:0/20:4^Δ5,8,11,14-PE.（4）Neuroprotective effect of egg-yolk lecithin and its active components inhibited scopolamine-induced cytotoxicity in PC12 cells.The ability of purified PC and PE to scavenge DPPH free radicals was evaluated in vitro using electron paramagnetic resonance（EPR）.The intensity of the EPR signals of DPPH free radicals markedly decreased after addition to PC and PE.The results suggested the PC and PE exhibited a high radical-scavenging activity in dose-dependent manner.In the study,intracellular AChE activity,monoamine oxidase（MAO）activity and malondialdehyde（MDA）level were used to study the neuroprotective effect of egg-yolk PC and PE against scopolamine-induced neurotoxicity of PC12 cells.The resulted that incubation with 4 mg/mL of scopolamine for 4h reduced survival rate to 50.30±2.65%of the control value,which induced a moderate damage.Pretreatment of PC12 cells with egg-yolk lecithin,PC standard,purified PC,PE standard and purified PE（0.2 mg/mL）before addition of scopolamine caused a marked（P<0.05）decrease in the AChE activity,MAO activity and MDA level compared with only scopolamine-treated cells.The result indicated that means egg-yolk lecithin,purified PC and purified PE might play neuroprotective effects by inhibiting neurotoxicity and oxidative stress induced by scopolamine in PC12 cells.These results as mentioned above suggested that the egg-yolk lecithin and its active components played a neuroprotective effect by inhibiting and oxidative stress induced by scopolamine via decreasing the AChE activity,MAO activity and MDA level to enhance memory,which indicate egg-yolk lecithin and its active components are potential nutrients to alleviate memory impairment.