CN1799552A - Single sialic acid tetrahexose ganglioside preparation method - Google Patents

Abstract

The invention relates to a method for preparing mono sialic acid hexose ganglioside sodium agent, comprising removing integument and blood vessel from the fresh or frozen pig brain or cattle brain, pushing for dewatering with cold acetone for several times, filtering with organic membrane, pouring out the acetone, and treating into acetone powder, packing, and conserving; extracting clarified solution from acetone powder, filtering with ultra-filter membrane and getting ultra-filtrate, extracting with tobacco extract, sodium filtering with sodium filter membrane to remove small molecule, abandoning the filtrate, getting clean condensed liquid of the mono sialic acid hexose ganglioside sodium agent; sterilizing and getting the refine product. The invention changes the polarity of the extracting organic solvent, which makes the ganglioside be released almost fully into the solution. The invention employs the ionic sodium filter membrane to separate and converse the ganglioside depending on the its number difference in sialic acid and carboxyl groups, and employs modern membrane separation technique to replace filling material and column suction technique, which saves large quantity of solvent and time, and increases productivity.

Description

The preparation method of Monostalotetrahexosylgangliside preparation of sodium

Technical field

The present invention relates to the preparation method of medicine, separation and Extraction is the preparation method of the glycolipid class material of main component with Monostalotetrahexosylgangliside sodium (GM1) from animal tissue specifically.

Background technology

Ganglioside (Gangliosides, GLS) be the acid glycolipid that a class contains sialic acid (N-n acetylneuraminic acid n), it is the important component of mammalian cell membrane, it is by the baroque membrane glycolipid that contains sialic acid residues of gang's isomery, and its molecule all is made up of a hydrophobic ceramide part and a hydrophilic sialyloligosaccharide group.The existing water solublity of ganglioside molecule has fat-solublely again, is a kind of amphipathic molecule; Press the position of sialic acid residues and the length of oligosaccharide core space, ganglioside is divided into different kinds, wherein GM1 is a Monostalotetrahexosylgangliside.It has multiple important biological function, has become the heat subject of current life science, and Monostalotetrahexosylgangliside sodium has been used for the nerve injury treatment of diseases clinically.For single ganglioside, GM1, GD1b and GT1 concentrate on the presynaptic caudacoria.Ganglioside is positioned at neuronal cell film double-decker skin, and ceramide one end embeds in the film, and oligonucleotide chain one end stretches out cell membrane and charges into external environment outward.This investigation on asymmetric distribution of ganglioside and their chemical property difference especially easily react to each other it, thereby serve as the key player in the cell membrane activities with various extracellulars information.Content is a little less than average level in pericaryon for ganglioside, and content is higher than average level in synaptosome.

The major physiological effect of Ganglioside GM1 in nervous system is: 1. the growth, differentiation and development and the neuranagenesis that promote neurocyte; 2. participate in the synapse transmission, keep the normal function of brain, participate in various learning and memory activities; 3. at cell and cell, play mediation in the interaction process between cell and microorganism and cell and substrate; 4. range protein function in the adjusting cell membrane, as ion channel, EGF-R ELISA etc.Experimental results show that exogenous ganglioside especially Monostalotetrahexosylgangliside sodium can embed neuron membrane, some function of imitation endogenous ganglioside, regulate membrane-mediated cell function, and the potential mechanism that replaces stops the development that damages after stimulating central nervous system injury, protects int nervous tissue; The neuronic growth and the activity thereof of In vitro culture be can also influence simultaneously, its survival and growth promoted.Formally sell the nineties by the GM1 injection (trade name Sygen) that Italian Fidia company extracts from cow brain tissue in China.Because GM1 can pass through blood brain barrier, in brain and myeloid tissue, disperse fast after the injection, therefore the GM1 therapeutic effect is very remarkable clinically.Be mainly used in treatment people's vascular or traumatic central nervous system injury, as apoplexy, acute spinal cord injury and cerebral trauma.Replenish ganglioside, ruined cranial nerve cell is repaired in the accumulation of the lipofuscin of degrading on the one hand on the other hand, delays neurocyte aging, enhancing brain power thereby reach, and recovers brain function.

Ganglioside and GM1 extract and existing document of separation and purification and patent report.Its main extracting method is: Medulla sus domestica or Medulla Bovis seu Bubali are mixed through strand, make acetone powder with the acetone dehydration.Reuse chloroform, methanol, water mixed organic solvents extract [Svennerholm.et al Biochemical et BiophsicaActa, 617-109 (1980)].The extract ganglioside changes water [J.Folch, The Journal ofBiological Chemistry, 226,497-509 (1957)] over to.With DEAE-Sephadex-A25 purification [R.W.Ledeen, et al.Journal of Neurochemistry, 21,829 (1973)].Silica gel column chromatography, the GM1 yield reaches 14%.The useful a cyclodextrin of the separation of ganglioside technology (EP00469352A1) is also used large aperture resin absorption technology (CN85102590).The useful sialidase facture of ganglioside seborrheic alopecia sialic acid (Richard Kuhn, etal., Chemische berichte, 1963.96.886).Useful heat treated method (US4868292) and microbial treatment method (Fukano Y, et al., Appl EnvironMicrobiol, 1997.63.1861).GM1 purification process Momoi TaKashi has set up anion-exchange chromatography technology (Biochim Biophys Acta, 1976,411,488).The affinity chromatograph technology (Krishna Kant, etal J.Chromato, 1989.494:289) etc.

Laboratory adopts DEAE-Sephadex and latrobeads bulky grain silica-gel sphere to carry out the column chromatography for separation extraction more both at home and abroad.The shortcoming of said method is that the organic solvent consumption is big, and is time-consuming, and two kinds of fillers all need import, so the cost height.

Summary of the invention

The objective of the invention is to found with modern biomembrane is the method for the mass preparation ganglioside of core.

Concrete preparation method is:

1) removes surperficial peplos and blood vessel etc. with fresh (or frozen) pig or Medulla Bovis seu Bubali, the cold acetone that adds 1～10 times of volume at ambient temperature through 3～6 times the press-dehydrating of pressing, is used the multilamellar filtered through gauze, with the aperture is the clarification of 0.1～1.0um micropore organic membrane filter, incline acetone after, the cerebral tissue after the dehydration is ground, put 60-80 ℃ of oven dry (can not use naked light), be crushed to fine powder with dry grinding, get acetone powder, airtight packing is preserved standby;

2) get acetone powder 5.0Kg, add and use chloroform: isopropyl alcohol: water=10～50: 20～80: 0.1～10 extracting solution 5～50Kg that is mixed with stirred 1～3 hour, filter clear liquor, adding 0.1～10 purified water again divides and carries, the supernatant is that 1000～100,000 ultrafilter membrane carries out ultrafiltration with the molecular weight that dams, the ultrafiltrate that obtains, carry through the extract collection, extract is a chloroform: methanol: 0.1～4.0mol/L acetate (sodium salt, potassium salt etc.)=1～4: 1.5～8: 0.1～1.5 is mixed with; Carefully put upside down the back static layering twice; Getting the upper strata liquid reuse molecular weight that dams is that 100～1000 NF membrane carries out getting final product when nanofiltration concentrates 1/5～1/10 volume, removes micromolecule, abandons permeate, collects limpid Monostalotetrahexosylgangliside sodium concentrated solution; Get highly finished product through degerming.Through this step separation and purification GM1 through its purity of efficient liquid phase chromatographic analysis greater than 95%.

3) Zhi Bei concentrated solution after rare joining, the fill finished product, make sterile preparation, both for reaching the Monostalotetrahexosylgangliside sodium injection of national drug quality standard.

Or 2) concentrated solution of step gained, after the microporous filter membrane aseptic filtration in 0.2um aperture, packing, cryogenic vacuum lyophilization, become milky or linen Monostalotetrahexosylgangliside sodium dry powder, be the aseptic freeze-dried injectable powder of Monostalotetrahexosylgangliside sodium.

The present invention has changed the polarity of extracting organic solvent, and ganglioside almost all is discharged in the solution.Ganglioside all enters the upper strata water after measured, and other dispensed materials such as pigment and most of phospholipid, thioester are to lower floor's organic facies.The present invention selects for use the ion-type NF membrane with its separation and conversion according to the difference of ganglioside at sialic acid and acylamino-group number.Through this step separation and purification GM1 through its purity of efficient liquid phase chromatographic analysis greater than 95%.The present invention adopts modern membrane separation technique to replace above-mentioned two kinds of fillers, replaces column chromatography technology to succeed, and saves a large amount of solvents and time, has improved productive rate.

The specific embodiment

Embodiment 1

1) removes surperficial peplos and blood vessel etc. with fresh (or frozen) Medulla sus domestica, the cold acetone that adds 2 times of volumes at ambient temperature through 3 times the press-dehydrating of pressing, is used the multilamellar filtered through gauze, with the aperture is the clarification of 0.2um micropore organic membrane filter, incline acetone after, the cerebral tissue after the dehydration is ground, put 60～80 ℃ of oven dry (can not use naked light), be crushed to fine powder with dry grinding, get acetone powder, airtight packing is preserved standby;

2) get acetone powder 5.0kg, add chloroform: isopropyl alcohol: the extracting solution 6kg that the volume ratio of water=10: 20: 0.1 is mixed with stirred 1～3 hour, filter clear liquor, the purified water that adds 0.2kg is again divided and is carried, the supernatant is that 3000 ultrafilter membrane carries out ultrafiltration with the molecular weight that dams, obtain ultrafiltrate, carry through extract collection, extract is a chloroform: methanol: the volume ratio of 0.1～4.0mol/L sodium acetate=1: 1.5: 0.1 is formulated; Getting the upper strata liquid reuse molecular weight that dams is that 300 NF membrane carries out getting final product when nanofiltration is concentrated into 1/5 volume, abandons permeate, the Monostalotetrahexosylgangliside sodium concentrated solution of collecting limpidly;

3) concentrated solution of above-mentioned preparation after rare joining, the fill finished product, make sterile preparation, both for reaching the Monostalotetrahexosylgangliside sodium injection of national drug quality standard.

Embodiment 2

1) removes surperficial peplos and blood vessel etc. with fresh (or frozen) Medulla Bovis seu Bubali, the cold acetone that adds 10 times of volumes at ambient temperature through 6 times the press-dehydrating of pressing, is used the multilamellar filtered through gauze, with the aperture is the clarification of 1.0um micropore organic membrane filter, incline acetone after, the cerebral tissue after the dehydration is ground, put 60～80 ℃ of oven dry (can not use naked light), be crushed to fine powder with dry grinding, get acetone powder, airtight packing is preserved standby;

2) get acetone powder 5.0Kg, add chloroform: isopropyl alcohol: the extracting solution 50kg that the volume ratio of water=50: 80: 10 is mixed with stirred 1～3 hour, filter clear liquor, adding the 10kg purified water again divides and carries, the supernatant is that 100,000 ultrafilter membrane carries out ultrafiltration with the molecular weight that dams, obtain ultrafiltrate, carry through extract collection, extract is a chloroform: methanol: the volume ratio of 0.1～4.0mol/L sodium acetate=4: 8: 1.5 is formulated; Getting the upper strata liquid reuse molecular weight that dams is that 1000 NF membrane carries out getting final product when nanofiltration is concentrated into 1/10 volume, abandons permeate, the Monostalotetrahexosylgangliside sodium concentrated solution of collecting limpidly;

3) after the microporous filter membrane aseptic filtration in 0.2um aperture, packing, cryogenic vacuum lyophilization, become milky or linen Monostalotetrahexosylgangliside sodium dry powder, get highly finished product through degerming, be the aseptic freeze-dried injectable powder of Monostalotetrahexosylgangliside sodium.

Claims (1)

1. the preparation method of a Monostalotetrahexosylgangliside preparation of sodium is characterized in that:

1) removes surperficial peplos and blood vessel etc. with fresh or frozen Medulla sus domestica or Medulla Bovis seu Bubali, the cold acetone that adds 1～10 times of volume at ambient temperature, through 3～6 times the press-dehydrating of pressing, use the multilamellar filtered through gauze, be the clarification of 0.1～1.0um micropore organic membrane filter with the aperture, incline acetone after, cerebral tissue after the dehydration is ground, put 60-80 ℃ of oven dry, be crushed to fine powder with dry grinding, get the airtight packing of acetone powder, preserve standby;

2) get acetone powder 5.0Kg, add chloroform: isopropyl alcohol: water=10～50: 20～80: extracting solution 5～50Kg that 0.1～10 volume ratio is mixed with stirred 1～3 hour, get clear liquor, the purified water that adds 0.1～10Kg is again divided and is carried, the supernatant is that 0.1 ten thousand～100,000 ultrafilter membrane carries out ultrafiltration with the molecular weight that dams, the ultrafiltrate that obtains, carry through the extract collection, extract is a chloroform: methanol: 0.1～4.0mol/L acetate=1～4: 1.5～8: 0.1～1.5 volume ratio is formulated; Carefully put upside down the back static layering twice; Getting the upper strata liquid reuse molecular weight that dams is that 100～1000 NF membrane carries out getting final product when nanofiltration concentrates 1/5～1/10 volume, removes micromolecule, abandons permeate, the Monostalotetrahexosylgangliside sodium concentrated solution of collecting limpidly; Get highly finished product through degerming.Through this step separation and purification GM1 through its purity of efficient liquid phase chromatographic analysis greater than 95%.

3) Pei Zhi concentrated solution after rare joining, the fill finished product, make sterile preparation, both for reaching the Monostalotetrahexosylgangliside sodium injection of national drug quality standard.

Or 2) concentrated solution of step gained, after the microporous filter membrane aseptic filtration in 0.2um aperture, packing, cryogenic vacuum lyophilization, become milky or linen Monostalotetrahexosylgangliside sodium dry powder, be the aseptic freeze-dried injectable powder of Monostalotetrahexosylgangliside sodium.