Purification Of Ganglioside GD1a From Porcine Brain

Ganglioside is an acidic glycosphingolipid,which is composed of sphingol,fatty acid and oligosaccharide chain.It is known that various molecular species exist.The gangliosides can be divided into different types,according to the types of the oiosaccharide and the numbers of sialic acid and its location on the oligosaccharide chain.Ganglioside GDla is a kind of gangliosides which oligosaccharide chain contains two sialic acids.Its molecular formula is(NeuNAc2-3Ga1?1-3GalNAc?1-4(NeuNAc2-3)Gal?1-4Glc?1-1Cer).GD1a has lots of biological activities and widely usage.The artificial synthesis of the activity GD1a has great difficulty.According to the actual condition of our country,it has important practical significance to separate and purify high purity GD1a from pig brain tissues.In order to explore the extraction approach to obtain high-purity GD1a from pig brain tissues,this paper explores the following contents:1.Exploration the extraction method of the gangliosides:It used the method of Folch partition chromatography to extract gangliosides,which is modified by Svennerholm and Fredman.This article modified the progress to obtain purer GD1a.Finally the optimum progress is extracting the gangliosides from the dewatering tissue at room temperature.The result of experiment is analyzed by thin layer chromatography(TLC),high performance liquid chromatography(HPLC)and sialic acid content determination.From the result we get that using the acetone powder which is made of fresh tissue is better than fresh tissue to extract gangliosides and the room temperature is better than 4?.The extraction rate is about 1.71%.Sialic acid content is 3.90%,and the yield of gangliosidesialic acid is up to 0.0667%under the extraction condition of ambient temperation and aetone power.2.Exploration the purification method of gangliosides:the cude lipids obtained by the method of Folch partition chromatography contains neutral lipid.The gangliosides are negativelipids,which can use ion exchange chromatography to seprate from neutral lipid.We use DEAE-Cellulose and DEAE-Sephadex A-25 ion exchange chromatography to purify gangliosides.By TLC analysis it can be seen that it is hard to separate neutral lipids with DEAE-Cellulose.The DEAE-Cellulose can't adsorb gangliosides.DEAE-Sephadex A-25 has good ability to adsorb the ganglioside and have a great separation between the neutral lipids and negative lipids.The acidic lipids also contain phospholipids,sulfur fat acid etc.So the alkali treatment can remove those acid complex lipids.Further purify is taken by using silica gel column chromatography,and the total gangliosides is obtained.The results analysis using TLC,HPLC show that the total gangliosides contain lots types of gangliosides including GM1,GD1a and GT1b.Sialic acid content is up to19.7%.The total yield is 1.20%.3.Exploration the separation method of GD1a:due to the difference of sialic acid quantity,the ion exchange chromatography can be used to isolate various types of ganglioside.DEAE-Sephadex A-25 is used to seprarte the different types of the ganglioside by ammonium acetate methanol solution to gradient elute.In 0.15 M fraction 340 mg GD1a is obtained and its purity is up to 35.0%.