Liquid Chromatography For The Separation And Purification Of Monosialogangliosides(GM1) From Pig Brain

Monosialoganglioside located at cell membrane in organism and mainly distributed in the central nervous system in mammals. As a specific receptor, gangliosides interact with some other active substance, which stimulates brain development and regulate the nervous system, also ganglioside can regenerate nerve tissues. Clinical studies show that, Monosialotetrahexosylganglioside(GM1) can protect and repaire nerve cells and, can be used for the treatment of paralysis, dementia, viability loss, senile dementia, cerebrovascular accident trauma, Parkinson and other neurological diseases. At present, the separation and purification technics of GM1is complex, and it is difficult to meet the increasing market demand, At the same time, The GM1products are often impure which is vulnerable left over polymer and other gangliosides. Therefore, it has important economic value and social significance to establish a convenient and efficient extraction method to obtain high purity GM1, and can improve the present studies on the separation of ganglioside purification methods.This project use fresh pig brain as the raw materials to get the pure GM1, including of the preparation of acetone powder, ganglioside extraction, weak anion exchange chromatography and gel exclusion chromatography, Three different methods were used to identify GM1, including amino bonded column HPLC, diol column HPLC and staining method to test produced production, using GM1standard produced by Sigma company as the control, The results showed that the physical and chemical properties of samples are highly consistent with the standard of GM1, and have the properties of high purity, and toxic substances. The establishment of method for separation and purification of GM1provides the theoretical basis for the industrial production of high purity GM1.The main research contents are as follows:①Fresh pig brain was homogenated in cold acetone, and removed large amounts of water and polarity water-solubility small molecular substances, then we got the acetone insoluble matter. The acetone insoluble matter was extracted by a certain percentage of chloroform-methanol-water, using Folch partitioning method to remove neutral material. Then the methanol-water solution was rotary evaporated remove methanol. And we got the sample solution of rough extract. Sample yield is about3.36%±0.64%. The sample was desalted by Sephadex G25, and further purified by weak anion exchange chromatography. Weak anion exchange balance system is20mmol/L Tris-HCl buffer, elution system for0%-70%1mol/L NaCl+20mmol/L Tris-HCl buffer. Yield is about (4.59±0.49)×10-2%, For gel exclusion chromatography, the elution system is20mmol/L PB buffer, to obtain pure product of GM1, extraction rate was approximately5.0mg/500g wet tissue.②Using three different methods identifying the sample, including amino bonded column, diol column and staining, GM1from Sigma as the control, results showed that the the physical and chemical properties of samples are highly consistent with the standard of GM1, and reached a high purity.