ASIALOGANGLIOSIDE-GM2

Preparation method of Gls 1. Using dog red blood cells as raw material, proceed according to the method of Ledcen et al. and Kanfer et al. The total lipids were extracted sequentially with a mixture of chloroform/methanol (2:1, 1:1, 1:2). Treated by DEAE-Sephadex A-25 ion exchange column to remove neutral lipids. After removing the phospholipids with alkaline water, dialysis, and Iatrobeads adsorption column chromatography, the total Gls is obtained. Method 2. Preparation of crude Gls using pig brain as raw material. Take 500 g of fresh pig brain, add 10 times the volume of the mixed solution of equal volume of chloroform and methanol, and then homogenize in the ultrasonic cell pulverizer of the electric homogenizer, at 4℃ Stir and extract overnight. Centrifuge to get the supernatant. The precipitate is homogenized again with the above-mentioned mixed solution. Repeatedly extract 3 times. Combine the supernatants and concentrate to 1/4 of the original volume by evaporation under reduced pressure at 40℃. Overnight, filter, add a mixed solution of 5% NaCl: methanol=1:1 (volume ratio) according to 1/5 volume of the filtrate for Folch distribution, transfer Gls into the water phase, collect the upper layer, divide the lower layer several times, combine the upper layer The liquid was evaporated and concentrated under reduced pressure at 40°C to remove methanol. The concentrated solution was dialyzed at 4°C until there was no Cl-, and freeze-dried to obtain crude Gls. Fresh pig brain [mixed solution]→[4℃, extract 3 times] centrifugation→supernatant [concentration]→concentrate[refrigeration]→[-20℃]filtration→filtrate[mixed solution]→[Floch distribution] supernatant [Concentration, dialysis]→[Freeze drying] Crude Gls Centrifugal Liquid Chromatography Packing Take 170g of activated chromatography silica gel, add 700ml of mixed solution at the ratio of chloroform:methanol=7:1 (volume ratio), and mix well. Slowly inject into the hole of a centrifugal liquid chromatography disc with a diameter of 30cm and a height of 1cm at a speed of 400r/min. After the disc is filled with silica gel, add the mixture at the ratio of chloroform: methanol: water = 7:3:0.5 (volume ratio) The solution is eluted to remove impurities until the eluate is colorless. Sample loading, elution and collection. Dissolve the crude Gls in 50-80ml 0.1mol/L NaOH methanol solution. After incubating at 37°C for 2h (dephospholipid), neutralize to neutrality with 0.5mol/L HCl methanol solution and dialyze. Dry cold to get Gls dry powder. The Gls dry powder was dissolved in a mixed solution of chloroform: methanol = 7:1 (volume ratio), centrifuged to remove insoluble matter, and the supernatant was taken for injection. After the sample is loaded, elute with 3 times the volume of the above solution, then use 5 times the volume of the chloroform: methanol: water = 6: 4: 1 (volume ratio) mixed solution to elute and collect the eluate , Concentrated, freeze-dried, weighed to obtain Gls. Crude Gls [treatment] → Gls dry powder [mixed solution] → [centrifugation] supernatant [injection, elution, concentration] → [freeze drying] Gls Sephadex LH-20 chromatography take Sephadex LH-20, add 5 times the volume 0.3mol/L KOH methanol: water=95:5 (volume ratio) mixed solution, soak overnight at 4℃ to remove fine particles, the next day, change to methanol: water=95:5 (volume ratio) mixed solution to remove KOH, pour the processed Sephadex LH-20 into the chromatography column (inner diameter 3cm, column length 100cm) to the bed height of about 85cm, add methanol: water = 95: 5 (volume ratio) mixed solution balance, the liquid will be centrifuged Gls obtained by chromatograph (Clc) was dissolved in 10ml of a mixed solution of 0.3 mol/L KOH in chloroform:methanol=95:5 (volume ratio), incubated at 37°C for 2h, centrifuged, and the supernatant was chromatographed. Elute and collect with a mixed solution of methanol: water=95:5 (volume ratio). After the collected solution becomes alkaline, stop the collection, combine the tubes, concentrate, and freeze-dry to obtain the refined Gls, which is combined with sialic acid ( The content of LBSA) is 30.1% and contains 5 kinds of Gls, namely 19.5% for GM1, 13.8% for GD3, 27.8% for GD1a, 14.2% for GD1b and 19.3% for GT1b. Gls[Mixed Solution]→[Centrifugation]→Supernatant [Sampling, Elution, Concentration]→Gls Fine Crude Gls The extraction method is basically similar to Yu's method, except that it is refrigerated overnight at -20℃. The purpose of this step is Removal of neutral lipids is beneficial to save the solvent consumption, time and increase the content of Gls in subsequent steps. The content of lipid-binding sialic acid is determined to be 12%, but it can only reach 8%-9% without refrigeration, and the yield is 0.35%. Similar to Katsumi’s 0.34%, higher than Xia’s 0.25%. Centrifugal liquid chromatography (Clc) for Gls purification is a new method. The basic principle of the instrument is to improve the separation effect according to the different quality of each component in the mixed sample, the different distribution effects in the stationary phase and the mobile phase, and the different centrifugal force. Method 3. Using bovine brain as the raw material, the chloroform-methanol extract of bovine brain was extracted with water to separate gangliosides from fat-soluble components, then phospholipids were removed by alkaline water, and the adsorption resin column was applied to separate water-soluble impurities. Then make product identification. Results: The purity of the prepared ganglioside was 26% with sialic acid content; composition: GM116%, GD36%, GD1a45%, GD1b10%, GT1a14%, GT1b17%. 2. The preparation of GM3 (using dog red blood cells as raw material) was carried out according to the method of Ledcen et al. and Klenk et al. Extract with a mixture of chloroform/methanol, 5 times of Folch partitioning, and retain the aqueous phase. The monosialic acid fraction was separated by DEAE-Sephadex A-25 ion exchange column chromatography, and then acetylation, Florisil adsorption column chromatography, deacetylation, dialysis, and lyophilization were performed to obtain pure GM3. Relevant data show that: GM1 is extracted and purified from pig brain; GM2 is extracted from rabbit liver; GM1 and GD3 are extracted from pig liver; GD1a and GT1b are contained in dog liver.

GM1 is mainly used to treat central neuropathy caused by a variety of causes, including common cerebral spinal cord injury, cerebrovascular accident, Parkinson~"s disease, and various causes of hypoxic-ischemic encephalopathy (neonatal hypoxic-ischemic encephalopathy, Drowning, CO poisoning, etc.).