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Method for separating and purifying pig ganglioside

A technology for the separation and purification of gangliosides, which is applied in the field of high-efficiency separation and purification of porcine gangliosides, can solve the problems of low yields, and achieve the effects of high yields, high recovery rates, and stable yields

Inactive Publication Date: 2019-05-03
SUZHOU NANOMICRO TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The yield is too low, so it is necessary to do further research on the isolation, purification and preparation of porcine gangliosides

Method used

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  • Method for separating and purifying pig ganglioside
  • Method for separating and purifying pig ganglioside
  • Method for separating and purifying pig ganglioside

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Take the crude extract of porcine gangliosides with a purity of 53.25%, dissolve the crude product with a mobile phase of chloroform:methanol:water=70:28:4.5, and the concentration of porcine gangliosides in the solution is 10 mg / mL. After the solution was clarified, it was filtered with a 0.45 μm membrane filter, and the filtrate was collected for later use. A chromatographic column of 4.6 × 250 mm was used, and UniSil normal phase silica gel microspheres (produced by Suzhou Nawei Technology Co., Ltd.) were used as the column filler, and the column volume was 4.2 ml. Pre-column pretreatment was performed on the chromatographic column, first with 10% aqueous phase for impurity removal and regeneration, then with organic phase for replacement equilibrium, and finally with 4.5% aqueous phase for equilibrium. Then carry out isocratic elution according to the mobile phase of chloroform:methanol:water=70:28:4.5, the flow rate is controlled at 1.5ml / min, the solution of the p...

Embodiment 2

[0031] Take the crude extract of porcine ganglioside with a purity of 54.24%, use 80% ethanol-salt solution as phase A, and use 100% ethanol as phase B. The crude product was dissolved with 100% ethanol, and the concentration of porcine ganglioside in the solution was 5 mg / mL. After standing for two days, the supernatant was filtered through a 0.45 μm membrane filter, and the filtrate was collected for later use. A chromatographic column of 4.6 × 250mm was used, and UniQ-30S was used as the packing material of the chromatographic column, and the packing volume was 4.2ml. Equilibrate with phase B. Set the 100% B-0% B of 0min-60min for gradient elution, control the flow rate at 1.0ml / min, collect the solution of the target peak in sections, summarize the component solutions that meet the requirements, and analyze them by high performance liquid chromatography. The purity of porcine ganglioside in the eluate is 99.1%, and the yield is 30.2%.

Embodiment 3

[0033] Take the crude extract of porcine ganglioside with a purity of 53.21%, use 1.0‰ phosphoric acid-water solution as phase A, and use 100% acetonitrile as phase B. The crude product was dissolved with 50% acetonitrile, and the concentration of porcine ganglioside in the solution was 2 mg / mL. Filter with a 0.45 μm membrane filter and collect the filtrate for later use. A chromatographic column of 4.6×250mm was used, and UniPS 10-300 was used as the column filler, and the packed volume was 4.2ml. Equilibrate with 2% Phase B. Wash with 2% B for 0min-15min, set 2%B-100%B gradient elution for 15min-65min, control the flow rate at 1.0ml / min, collect the solution of the target peak in sections, and summarize the components that meet the requirements. After HPLC analysis, the impurities of porcine ganglioside in the eluent could not be removed, and the experimental results were poor.

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Abstract

The invention provides a method for efficiently separating and purifying porcine ganglioside, which comprises the steps of: dissolving and filtering a crude extract of the pig ganglioside; applying afiltered solution to a chromatography column filled with a homogenous normal-phase silica gel for chromatography; using ultrapure water and an organic solvent as a flow phase for elution of a target product; collecting the solution of the target peak in stages, and collecting the component liquid meeting the requirements. The method is applied to the deep purifying of the pig ganglioside, and onlyneeds one-step chromatography to satisfy the requirement that the pig ganglioside purity is greater than 99% and the yield is greater than 93.0%. The purification yield is high and stable, and the separation method of the invention is simple and convenient, can be used for large-scale production, and the production cost is greatly reduced.

Description

technical field [0001] The invention relates to the field of medicine purification, in particular to a method for efficiently separating and purifying porcine gangliosides. Background technique [0002] Porcine ganglioside (ganglioside) exists more in the cell membrane, and its relationship with membrane function has attracted attention. The exotoxins of bacteria tetanus and cholera can specifically bind to specific porcine ganglioside molecules. Soluble in water and organic solvents. In water, it exists in the state of micelles aggregated by millions of molecules. Porcine ganglioside has a significant role in promoting nerve regeneration. Nerve growth factor (NGF) and porcine ganglioside (GM) are essential substances for the regeneration and development of brain nerves. GM mediates NGF to enhance its activity dozens of times and form new Rich neural network, repair and promote the development of cranial nerves again. [0003] The structural formula is as follows: [00...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H15/10C07H1/08
Inventor 江必旺王希陈强强吴永跃
Owner SUZHOU NANOMICRO TECH CO LTD
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