CN112812142A - Method for refining monosialotetrahexosyl ganglioside sodium - Google Patents
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Abstract
The invention belongs to the technical field of medical biology, and particularly relates to a method for refining high-purity monosialotetrahexosyl ganglioside sodium. The method comprises the following steps: (1) the ganglioside pig brain extract is firstly purified by positive amorphous silica gel, and then separated by octyl bonded silica gel reverse filler to obtain a crude product of ganglioside; (2) the crude ganglioside is ion exchanged with cationic resin and refined with 10k hollow fiber ultrafiltering membrane to obtain high purity monosialotetrahexosyl ganglioside sodium. The monosialotetrahexosyl ganglioside sodium prepared by the method has higher purity and higher product quality, and is suitable for popularization and application.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a method for refining monosialotetrahexosyl ganglioside sodium.
Background
Monosialotetrahexosylganglioside (GM1), a sialic acid-containing glycosphingolipid species, is the only known ganglioside that can penetrate the blood brain barrier. It is widely present in mammalian cell membranes, most abundant in nervous system, embedded in the cell bilayer lipid membrane structure, and is one of the important constituents of nerve cells, accounting for about 10% of total lipid. GM1 plays an essential role in the development, growth, differentiation and regeneration of the human nervous system. Experiments and clinical reports for many years show that GM1 can promote the recovery of damaged nerves, has the function of promoting nerve remodeling, and is one of the main substances involved in the repair of neuropathy.
The medicinal GM1 prepared in the prior art can be extracted from mammalian brain, and has positive industrial value. The content of GM1 in brain tissue of different mammal sources is different, but there are data showing that GM1 extracted from pig brain tissue is completely consistent with human GM1 in structure and composition, especially the type of sialic acid. Therefore, GM1 was extracted from animal brain tissue as a starting material and was free from immunogenicity and abnormal toxic reactions. According to the literature report, the GM1 content in the bovine brain is higher than that of other livestock, but in view of the serious harm of bovine-derived viruses, such as mad cow disease, to the human health, the relatively safe porcine brain is generally selected as the raw material.
The preparation method of the monosialotetrahexosyl ganglioside sodium generally comprises two steps of extraction and purification; for example, chinese patent application CN103524572A discloses a method for preparing high-purity monosialotetrahexosyl ganglioside sodium, which is prepared by taking fresh pig brain as a raw material and performing an extraction step and a purification step, wherein the extraction step comprises an acetone powder preparation step, an extraction step of tissue total lipid, a folch distribution step, a virus removal step, and an acid hydrolysis virus inactivation step; the purification step comprises a desalting step, a silica gel chromatography step, a reverse phase chromatography step, a concentration step, an acetone recrystallization step and a freeze-drying step. Chinese patent application CN102731584A discloses a method for preparing high-purity monosialotetrahexosylganglioside (GM1), which specifically comprises the steps of preparation of pig brain acetone powder, preparation of total ganglioside concentrated solution and acid hydrolysis.
Although the preparation method in the prior art achieves certain technical effects, more or less quality problems still exist. Because the clinically used monosialotetrahexosylganglioside sodium has great influence on human bodies and higher requirement on use purity, the method has important significance on the improvement of the refining technology of the monosialotetrahexosylganglioside sodium.
Disclosure of Invention
In order to overcome the technical problems, the invention provides a method for refining monosialotetrahexosyl ganglioside sodium. The prepared monosialotetrahexosyl ganglioside sodium has higher purity and higher product quality, and is suitable for popularization and application.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
a method for refining monosialotetrahexosylganglioside sodium comprises the following steps:
(1) after the ganglioside pig brain extract is firstly purified by positive amorphous silica gel, the ganglioside pig brain extract is separated by octyl bonded silica gel reverse filler to obtain a crude product of ganglioside;
(2) the crude ganglioside is ion exchanged with cationic resin and refined with 10k hollow fiber ultrafiltering membrane to obtain high purity monosialotetrahexosyl ganglioside sodium.
Preferably, the method for refining the monosialotetrahexosylganglioside sodium comprises the following steps:
(1) extracting, extracting and hydrolyzing the pig brain to obtain a pig brain extract for later use;
(2) homogenizing amorphous silica gel with methanol, loading onto column, balancing with mixed solution of chloroform, methanol and water as mobile phase, dissolving medulla sus domestica extract with water under heating, eluting with mobile phase, collecting eluate, concentrating under reduced pressure to viscous, adding acetone, standing, and precipitating to obtain ganglioside crude product 1;
(3) after dissolving the crude ganglioside 1, eluting with a mixed solution of trifluoroacetic acid methanol solution and water by using a high-pressure dynamic axial compression column reversed-phase filler, collecting eluent, and detecting the eluent according to HPLC (high performance liquid chromatography); mixing the eluates, concentrating under reduced pressure to viscous, adding acetone, standing for precipitation to obtain crude ganglioside 2;
(4) dissolving the crude product 2 with purified water, adding active carbon, stirring, filtering, concentrating, adding acetone, and precipitating to obtain a crude product 3 of the nervoside;
(5) loading the column with cationic resin, washing the cationic resin column with hydrochloric acid aqueous solution, dissolving crude ganglioside 3, loading, eluting with hydrochloric acid aqueous solution, collecting eluate, ultrafiltering, stopping ultrafiltering when conductivity of ultrafiltrate is detected to be less than or equal to 10us/cm to obtain ultrafiltrate, concentrating ultrafiltrate under reduced pressure to viscous liquid, adding acetone, standing for precipitation, filtering, and drying to obtain high-purity monosialotetrahexosylganglioside sodium.
Preferably, in the step (2), the mesh number of the amorphous silica gel is 200-300 meshes;
preferably, in the step (2), the volume ratio of the trichloromethane to the methanol to the water is 65-68: 30-33: 3-6;
preferably, in the step (2), the volume ratio of the trichloromethane to the methanol to the water is 68: 33: 6;
preferably, in the step (2), the heating temperature is 30-60 ℃;
preferably, in the step (2), the heating temperature is 50 ℃;
preferably, in the step (2), the ratio of the pig brain extract to water is water: pig brain extract ═ 1L: 80-120g (v: w);
preferably, in the step (2), the ratio of the pig brain extract to water is water: pig brain extract ═ 1L:100g (v: w);
preferably, in the step (2), the elution of the mobile phase is carried out at a flow rate of 20-60 mL/min;
preferably, in the step (2), the elution of the mobile phase is carried out at a flow rate of 40 mL/min;
preferably, in the step (2), the volume of the added acetone is 5 to 10 times of the volume of the concentrated solution.
Preferably, in the step (2), the volume of the added acetone is 10 times of the volume of the concentrated solution.
Preferably, in the step (3), the reverse phase filler is octyl bonded silica gel reverse phase filler with the particle size of 10 μm and the pore diameter of
The volume ratio of the 0.5% trifluoroacetic acid methanol solution to the water is (68-70): (30-32) a mobile phase, the column specification is 50 x 1000mm, and the ultraviolet detection wavelength is 205 nm;
preferably, in step (3), the elution is performed at a flow rate of 60-100 mL/min;
preferably, in step (3), the elution is performed at a flow rate of 80 mL/min;
preferably, in the step (3), the chromatographic conditions for detecting by high performance liquid chromatography are that octadecylsilane chemically bonded silica is used as a filler, acetonitrile (32:68), which is 0.01mol/L potassium dihydrogen phosphate solution, is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate was 1.2ml per minute; the detection wavelength is 205 nm;
preferably, in the step (3), the volume of the added acetone is 5 to 10 times of the volume of the concentrated solution.
Preferably, in the step (3), the volume of the acetone is 10 times of the volume of the concentrated solution;
preferably, in the step (4), the activated carbon is needle activated carbon, and the adding amount is 3-5% of the mass of the crude product 2.
Preferably, in the step (4), the activated carbon is needle activated carbon, and the adding amount is 5% of the mass of the crude product 2.
Preferably, in the step (5), the hydrochloric acid aqueous solution is hydrochloric acid: purified water ═ 1L: 5-10L;
preferably, in the step (5), the hydrochloric acid aqueous solution is hydrochloric acid: purified water ═ 1L: 5L;
preferably, in the step (5), the washing of the cationic resin column with the aqueous hydrochloric acid solution is performed for 1 to 3 hours at a flow rate of 30 to 70 ml/min;
preferably, in the step (5), the washing of the cationic resin column with the aqueous hydrochloric acid solution is performed at a flow rate of 50ml/min for 1 h;
preferably, in step (5), the ultrafiltration is performed using a 10k hollow fiber ultrafiltration membrane;
preferably, in the step (5), the volume of the added acetone is 5-10 times of the volume of the viscous liquid.
Preferably, in the step (5), the volume of the added acetone is 10 times of the volume of the viscous liquid.
Compared with the prior art, the invention has the technical advantages that:
(1) the method comprises the steps of firstly purifying a pig brain extract by using 200-mesh 300-mesh amorphous silica gel to obtain a ganglioside mixed component (containing about 90% of mixed GM1 component), adopting a TLC control method, then adopting a high-pressure dynamic axial compression column, an octyl bonded silica gel reverse phase filler, a trifluoroacetic acid methanol solution and a mobile phase purification technology of water to respectively obtain monosialotetrahexosylganglioside GM1A and GM1B components, and then mixing. Can effectively separate and remove sensitization impurities included between GMA and GM1B, has high separation degree, and can obtain high-purity (99%) monosialotetrahexosylganglioside.
(2) The refining method provided by the invention has the advantages of short production period, quick production process, low cost and easy large-scale industrial production operation.
Drawings
FIG. 1: HPLC profile of the product obtained in example 1;
FIG. 2: HPLC profile of the product obtained in example 2;
FIG. 3: HPLC profile of the product obtained in example 3;
FIG. 4: HPLC profile of the product obtained in example 4;
FIG. 5: HPLC profile of the product obtained in example 5;
the invention will now be further described with reference to the accompanying drawings and examples.
Detailed Description
The present invention will be described below with reference to specific examples to make the technical aspects of the present invention easier to understand and grasp, but the present invention is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.
Example 1
(1) Extracting, extracting and hydrolyzing pig brain to obtain pig brain extract for later use;
(2) homogenizing 200 mesh amorphous silica gel 2kg, 6L methanol, loading into column (column specification of 100 × 800mm), balancing with mobile phase containing chloroform, methanol and water at volume ratio of 68: 33: 6, dissolving medulla Sus Domestica extract with water at 50 deg.C, wherein the ratio of medulla Sus Domestica extract to water is about: eluting with the mobile phase at a flow rate of 40mL/min, collecting the eluent, collecting one barrel per 2L, collecting the eluent by stages according to TLC detection results, concentrating the eluent under reduced pressure to be viscous, adding acetone with the volume of 10 times of the volume of the concentrated solution, standing and precipitating to obtain crude ganglioside 1;
(3) dissolving the crude ganglioside 1, and using high-pressure dynamic axial compression column and reverse phase filler (the grain diameter of octyl bonded silica gel reverse phase filler is 10 μm, and the pore diameter is
A mobile phase of 0.5% trifluoroacetic acid in methanol and water in a volume ratio of 70: 30, a column specification of 50X 1000mm, and an ultraviolet detection wavelength of 205nm), eluting at a flow rate of 80mL/min, collecting the eluate, collecting one vial per 1000mL, collecting the eluates of GM1A and GM1B in stages (GM 1A was collected as the first peak position and GM1B was collected as the second peak position shown in FIG. 1, and the following examples were all treated in the same manner), and subjecting the eluate to HPLC high performance liquid chromatography (chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler, acetonitrile (32:68) which is 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate was 1.2ml per minute; detecting at 205nm detection wavelength), mixing the eluates of GM1A and GM1B, concentrating under reduced pressure to viscous, adding acetone 10 times the volume of the concentrated solution, standing for precipitation, and standing for precipitation to obtain crude ganglioside 2;
(4) dissolving the crude product 2 with purified water, adding 5% (m/m) of active carbon for injection, stirring for 30min, filtering, concentrating, adding acetone, and precipitating to obtain crude product 3 of the nervoside ester;
(5) packing the column (column specification is 50X 300mm) with 001X 7 strong acid type cation resin, washing the cation exchange column with hydrochloric acid water solution (hydrochloric acid: purified water is 1L:5L) at the flow rate of 50ml/min for 1 hour, dissolving and loading the crude ganglioside 3, eluting with hydrochloric acid water solution, collecting eluent, ultrafiltering with 10k hollow fiber ultrafiltration membrane until the conductivity of the ultrafiltrate is detected to be less than or equal to 10us/cm, obtaining ultrafiltrate, concentrating the ultrafiltrate under reduced pressure to be viscous, adding 10 times of acetone, standing for precipitation, filtering the precipitation solution, and drying to obtain the high-purity monosialotetrahexosylganglioside sodium of the invention. The HPLC purity of the monosialotetrahexosylganglioside sodium was determined to be 99.8%. The HPLC spectrum of the obtained product is shown in FIG. 1.
Example 2
(1) Extracting medulla Sus Domestica, extracting, hydrolyzing to obtain medulla Sus Domestica extract
(2) Homogenizing 300 mesh amorphous silica gel 2kg, 6L methanol, loading into column (column specification of 100 × 800mm), balancing with mobile phase containing chloroform, methanol and water at volume ratio of 68: 33: 6, dissolving medulla Sus Domestica extract with water at 50 deg.C, wherein the ratio of medulla Sus Domestica extract to water is about: eluting with the mobile phase at a flow rate of 40mL/min, collecting the eluent, collecting one barrel per 2L, collecting the eluent by stages according to TLC detection results, concentrating the eluent under reduced pressure to be viscous, adding acetone with the volume of 10 times of the volume of the concentrated solution, standing and precipitating to obtain crude ganglioside 1;
(3) after the ganglioside crude product 1 is dissolved, a high-pressure dynamic axial compression column and a reverse phase filler (the grain diameter of the octyl bonded silica gel reverse phase filler is 10 mu m, the pore diameter is
A mobile phase in which the volume ratio of 0.5% trifluoroacetic acid in methanol to water is 75: 25, the column specification is 50 × 1000mm, the ultraviolet detection wavelength is 205nm), elution is performed at a flow rate of 80mL/min, an eluate is collected, one bottle per 1000mL, eluents of GM1A and GM1B are collected in stages, and the eluate is subjected to high performance liquid chromatography according to HPLC (chromatographic conditions: by usingOctadecylsilane chemically bonded silica is used as a filler, acetonitrile (32:68) which is 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate was 1.2ml per minute; detection wavelength of 205nm), mixing the eluates of GM1A and GM1B, concentrating under reduced pressure to viscous, adding acetone with volume of 10 times of the concentrated solution, standing for precipitation, and standing for precipitation to obtain crude ganglioside 2;
(4) dissolving the crude product 2 with purified water, adding 5% (m/m) of active carbon for injection, stirring for 30min, filtering, concentrating, adding acetone, and precipitating to obtain crude product 3 of the nervone glycoside;
(3) packing the column (column specification is 50X 300mm) with 001X 7 strong acid type cation resin, washing the cation exchange column with hydrochloric acid water solution (hydrochloric acid: purified water is 1L:5L) at the flow rate of 50ml/min for 1 hour, dissolving and loading the crude ganglioside 3, eluting with hydrochloric acid water solution, collecting eluent, ultrafiltering with 10k hollow fiber ultrafiltration membrane until the conductivity of the ultrafiltrate is detected to be less than or equal to 10us/cm, obtaining ultrafiltrate, concentrating the ultrafiltrate under reduced pressure to be viscous, adding 10 times of acetone, standing for precipitation, filtering the precipitation solution, and drying to obtain the high-purity monosialotetrahexosylganglioside sodium of the invention. The HPLC purity of the monosialotetrahexosylganglioside sodium was determined to be 99.0%. The HPLC spectrum of the obtained product is shown in FIG. 2.
Example 3
(1) Extracting, extracting and hydrolyzing the pig brain to obtain a pig brain extract for later use;
(2) homogenizing 200 mesh amorphous silica gel 2kg, 6L methanol, loading into column (column specification of 100 × 800mm), balancing with mobile phase containing chloroform, methanol and water at volume ratio of 68: 33: 6, dissolving medulla Sus Domestica extract with water at 50 deg.C, wherein the ratio of medulla Sus Domestica extract to water is about: eluting with the mobile phase at a flow rate of 40mL/min, collecting the eluent, collecting one barrel per 2L, collecting the eluent by stages according to TLC detection results, concentrating the eluent under reduced pressure to be viscous, adding acetone with the volume of 10 times of the volume of the concentrated solution, standing and precipitating to obtain crude ganglioside 1;
(3) After the ganglioside crude product 1 is dissolved, a high-pressure dynamic axial compression column and a reverse phase filler (the grain diameter of the octyl bonded silica gel reverse phase filler is 10 mu m, the pore diameter is
A mobile phase in which the volume ratio of 0.5% trifluoroacetic acid in methanol to water is 68: 32, the column specification is 50X 1000mm, the ultraviolet detection wavelength is 205nm), elution is performed at a flow rate of 80mL/min, an eluate is collected, one bottle per 1000mL, eluents of GM1A and GM1B are collected in stages, and the eluate is subjected to HPLC high performance liquid chromatography (chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler, acetonitrile (32:68) which is 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate was 1.2ml per minute; detection wavelength of 205nm), mixing the eluates of GM1A and GM1B, concentrating under reduced pressure to viscous, adding acetone with volume of 10 times of the concentrated solution, standing for precipitation, and standing for precipitation to obtain crude ganglioside 2;
(4) dissolving the crude product 2 with purified water, adding 5% (m/m) of active carbon for injection, stirring for 30min, filtering, concentrating, adding acetone, and precipitating to obtain crude product 3 of the nervoside ester;
(5) packing 001 × 7 strong acid type cation resin into a column (the column specification is 50 × 300mm), washing the cation exchange column with hydrochloric acid aqueous solution (hydrochloric acid: purified water is 1L:5L) at the flow rate of 50ml/min for 1 hour, dissolving and loading the crude ganglioside 3, eluting with hydrochloric acid aqueous solution, collecting eluent, ultrafiltering with a 10k hollow fiber ultrafiltration membrane until the conductivity of the ultrafiltrate is detected to be less than or equal to 10us/cm, stopping the ultrafiltration to obtain ultrafiltrate, concentrating the ultrafiltrate under reduced pressure to be viscous, adding 10 times of acetone, standing for precipitation, filtering and drying the precipitate to obtain the high-purity monosialotetrahexosylganglioside sodium of the invention. The HPLC purity of the monosialotetrahexosylganglioside sodium was determined to be 99.2%. The HPLC spectrum of the obtained product is shown in FIG. 3.
Example 4
(1) Extracting medulla Sus Domestica, extracting, hydrolyzing to obtain medulla Sus Domestica extract
(2) 2kg of 300-mesh amorphous silica gel is taken, homogenized by 6L of methanol, and then loaded on a column (the column specification is 100 x 800mm), and the column is prepared by using chloroform, methanol and water in a volume ratio of 65: 30: 3, balancing the mobile phase, taking the pig brain extract, heating the pig brain extract with water to 30 ℃ for dissolving, wherein the adding ratio of the pig brain extract to the water is about: eluting with the mobile phase at a flow rate of 20mL/min, collecting the eluent, collecting one barrel per 2L, collecting the eluent by stages according to TLC detection results, concentrating the eluent under reduced pressure to be viscous, adding acetone with the volume 5 times of that of the concentrated solution, standing and precipitating to obtain a crude product 1 of ganglioside;
(3) after the ganglioside crude product 1 is dissolved, a high-pressure dynamic axial compression column and a reverse phase filler (the grain diameter of the octyl bonded silica gel reverse phase filler is 10 mu m, the pore diameter is
A mobile phase in which a 0.5% trifluoroacetic acid methanol solution and water are in a volume ratio of 70: 30, a column specification is 50 × 1000mm, an ultraviolet detection wavelength is 205nm), elution is performed at a flow rate of 60mL/min, an eluate is collected, one bottle per 1000mL, eluents of GM1A and GM1B are collected in stages, and the eluate is subjected to high performance liquid chromatography according to HPLC (chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler, acetonitrile (32:68) which is 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate was 1.2ml per minute; detection wavelength of 205nm), mixing the eluates of GM1A and GM1B, concentrating under reduced pressure to viscous, adding acetone with volume 5 times of the concentrated solution, standing for precipitation, and standing for precipitation to obtain crude ganglioside 2;
(4) dissolving the crude product 2 with purified water, adding 3% (m/m) of active carbon for injection, stirring for 30min, filtering, concentrating, adding acetone, and precipitating to obtain crude product 3 of the nervone glycoside;
(3) packing 001 × 7 strong acid type cation resin into a column (the column specification is 50 × 300mm), washing the cation exchange column with hydrochloric acid aqueous solution (hydrochloric acid: purified water is 1L:10L) at the flow rate of 30ml/min for 3 hours, dissolving and sampling the crude ganglioside 3, eluting with hydrochloric acid aqueous solution, collecting eluent, ultrafiltering with a 10k hollow fiber ultrafiltration membrane until the conductivity of the ultrafiltrate is detected to be less than or equal to 10us/cm, stopping the ultrafiltration to obtain ultrafiltrate, concentrating the ultrafiltrate under reduced pressure to be viscous, adding 5 times of acetone, standing for precipitation, filtering and drying the precipitate to obtain the high-purity monosialotetrahexosylganglioside sodium of the invention. The HPLC purity of the monosialotetrahexosylganglioside sodium was determined to be 99.6%. The HPLC spectrum of the obtained product is shown in FIG. 4.
Example 5
(1) Extracting medulla Sus Domestica, extracting, hydrolyzing to obtain medulla Sus Domestica extract
(2) 2kg of 300-mesh amorphous silica gel is taken, homogenized by 6L of methanol, and then loaded on a column (the column specification is 100 x 800mm), and the column is prepared by using chloroform, methanol and water in a volume ratio of 68: 30: 3, balancing the mobile phase, taking the pig brain extract, heating the pig brain extract with water to 60 ℃ for dissolving, wherein the adding ratio of the pig brain extract to the water is about: pig brain extract ═ 1L: 120g (v: w), eluting with the mobile phase at the flow rate of 60mL/min, collecting the eluent, collecting one barrel per 2L, collecting the eluent in stages according to the TLC detection result, concentrating the eluent under reduced pressure until the eluent is viscous, adding acetone with the volume of 5 times of that of the concentrated solution, standing and precipitating to obtain a crude product 1 of ganglioside;
(3) after the ganglioside crude product 1 is dissolved, a high-pressure dynamic axial compression column and a reverse phase filler (the grain diameter of the octyl bonded silica gel reverse phase filler is 10 mu m, the pore diameter is
A mobile phase in which a 0.5% trifluoroacetic acid methanol solution and water are in a volume ratio of 70: 30, a column specification is 50 × 1000mm, an ultraviolet detection wavelength is 205nm), elution is performed at a flow rate of 100mL/min, an eluate is collected, one bottle per 1000mL, eluents of GM1A and GM1B are collected in stages, and the eluate is subjected to high performance liquid chromatography according to HPLC (chromatographic conditions: octadecylsilane chemically bonded silica is used as a filler, acetonitrile (32:68) which is 0.01mol/L potassium dihydrogen phosphate solution is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate was 1.2ml per minute; detection wavelength of 205nm), mixing GM1A and GM1B eluates, concentrating under reduced pressure to viscous, adding acetone 5 times of the concentrated solution, standing for precipitationStanding and precipitating to obtain a crude product 2 of ganglioside;
(4) dissolving the crude product 2 with purified water, adding 5% (m/m) of active carbon for injection, stirring for 30min, filtering, concentrating, adding acetone, and precipitating to obtain crude product 3 of the nervone glycoside;
(3) packing the column (column specification is 50X 300mm) with 001X 7 strong acid type cation resin, washing the cation exchange column with hydrochloric acid aqueous solution (hydrochloric acid: purified water is 1L:10L) at the flow rate of 70ml/min for 3 hours, dissolving and loading the crude ganglioside 3, eluting with hydrochloric acid aqueous solution, collecting eluent, ultrafiltering with 10k hollow fiber ultrafiltration membrane until the conductivity of the ultrafiltrate is detected to be less than or equal to 10us/cm, obtaining ultrafiltrate, concentrating the ultrafiltrate under reduced pressure to be viscous, adding 10 times of acetone, standing for precipitation, filtering the precipitation solution, and drying to obtain the high-purity monosialotetrahexosylganglioside sodium of the invention. The HPLC purity of the monosialotetrahexosylganglioside sodium was determined to be 99.5%. The HPLC spectrum of the obtained product is shown in FIG. 5.
The above detailed description is specific to one possible embodiment of the present invention, and the embodiment is not intended to limit the scope of the present invention, and all equivalent implementations or modifications without departing from the scope of the present invention should be included in the technical scope of the present invention.
Claims (10)
1. A method for refining monosialotetrahexosylganglioside sodium comprises the following steps:
(1) the ganglioside pig brain extract is firstly purified by positive amorphous silica gel, and then separated by octyl bonded silica gel reverse filler to obtain a crude product of ganglioside;
(2) the crude ganglioside is ion exchanged with cationic resin and refined with 10k hollow fiber ultrafiltering membrane to obtain high purity monosialotetrahexosyl ganglioside sodium.
2. The method of refining monosialotetrahexosylganglioside according to claim 1, comprising the steps of:
(1) extracting, extracting and hydrolyzing the pig brain to obtain a pig brain extract for later use;
(2) homogenizing amorphous silica gel with methanol, loading onto column, balancing with mixed solution of chloroform, methanol and water as mobile phase, dissolving medulla sus domestica extract with water under heating, eluting with mobile phase, collecting eluate, concentrating under reduced pressure to viscous, adding acetone, standing, and precipitating to obtain ganglioside crude product 1;
(3) after dissolving the crude ganglioside 1, eluting with a mixed solution of trifluoroacetic acid methanol solution and water by using a high-pressure dynamic axial compression column reversed-phase filler, collecting eluent, and detecting the eluent according to HPLC (high performance liquid chromatography); mixing the eluates, concentrating under reduced pressure to viscous, adding acetone, standing for precipitation to obtain crude ganglioside 2;
(4) dissolving the crude product 2 with purified water, adding active carbon, stirring, filtering, concentrating, adding acetone, and precipitating to obtain a crude product 3 of the nervoside;
(5) loading the column with cationic resin, washing the cationic resin column with hydrochloric acid aqueous solution, dissolving crude ganglioside 3, loading, eluting with hydrochloric acid aqueous solution, collecting eluate, ultrafiltering, stopping ultrafiltering when conductivity of ultrafiltrate is detected to be less than or equal to 10us/cm to obtain ultrafiltrate, concentrating ultrafiltrate under reduced pressure to viscous liquid, adding acetone, standing for precipitation, filtering, and drying to obtain high-purity monosialotetrahexosylganglioside sodium.
3. The method for purifying monosialotetrahexosylganglioside sodium according to claim 2, wherein in step (2), the mesh size of the amorphous silica gel is 200-300 mesh; the volume ratio of the trichloromethane to the methanol to the water is 65-68: 30-33: 3-6; the heating temperature is 30-60 ℃; the adding proportion of the pig brain extract to water is as follows: pig brain extract ═ 1L: 80-120g (v: w); eluting the mobile phase at a flow rate of 20-60 mL/min; the volume of the added acetone is 5-10 times of the volume of the concentrated solution.
4. The method for purifying monosialotetrahexosylganglioside sodium according to claim 2, wherein in step (2), the volume ratio of chloroform to methanol to water is 68: 33: 6;
the heating temperature is 50 ℃; the adding proportion of the pig brain extract to water is as follows: pig brain extract ═ 1L:100g (v: w); eluting the mobile phase at a flow rate of 40 mL/min; the volume of acetone added was 10 times the volume of the concentrate.
5. The method for refining monosialotetrahexosylganglioside sodium according to claim 2, wherein in step (3), the reverse phase filler is octyl bonded silica gel reverse phase filler having a particle size of 10 μm and a pore size of
The volume ratio of the 0.5% trifluoroacetic acid methanol solution to the water is (68-70): (30-32) a mobile phase, the column specification is 50 x 1000mm, and the ultraviolet detection wavelength is 205 nm; the elution is carried out at a flow rate of 60-100 mL/min; the volume of the added acetone is 5-10 times of the volume of the concentrated solution.
6. The method for refining monosialotetrahexosylganglioside sodium according to claim 2, wherein in step (3), the elution is performed at a flow rate of 80 mL/min; in the step (3), the chromatographic conditions for detecting by the high performance liquid chromatography are that octadecylsilane chemically bonded silica is used as a filler, acetonitrile (32:68), which is 0.01mol/L potassium dihydrogen phosphate solution, is used as a mobile phase A, and acetonitrile is used as a mobile phase B; the flow rate was 1.2ml per minute; the detection wavelength is 205 nm; in the step (3), the volume of the acetone is 10 times of the volume of the concentrated solution.
7. The method for refining monosialotetrahexosylganglioside sodium according to claim 2, wherein in step (4), the activated carbon is activated carbon for injection and is added in an amount of 3 to 5% by mass based on 2% by mass of the crude product.
8. The method for refining monosialotetrahexosylganglioside sodium according to claim 2, wherein in step (4), the activated carbon is activated carbon for injection and is added in an amount of 5% by mass based on 2% by mass of the crude product.
9. The method for purifying monosialotetrahexosylganglioside sodium according to claim 2, wherein in step (5), the aqueous hydrochloric acid solution is hydrochloric acid: purified water ═ 1L: 5-10L;
the cation resin column is flushed by hydrochloric acid aqueous solution at the flow rate of 30-70ml/min for 1-3 h; the volume of the added acetone is 5-10 times of the volume of the viscous liquid.
10. The method for purifying monosialotetrahexosylganglioside sodium according to claim 2, wherein in step (5), the aqueous hydrochloric acid solution is hydrochloric acid: purified water ═ 1L: 5L;
the washing of the cationic resin column with the hydrochloric acid aqueous solution is carried out for 1h at a flow rate of 50 ml/min;
the ultrafiltration is carried out by using a 10k hollow fiber ultrafiltration membrane; the volume of the added acetone is 10 times of the volume of the viscous liquid.
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