CN114252436A - Method for identifying natural fucosylated glycosaminoglycan - Google Patents
Method for identifying natural fucosylated glycosaminoglycan Download PDFInfo
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- 229920002683 Glycosaminoglycan Polymers 0.000 title claims abstract description 53
- 238000000034 method Methods 0.000 title claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000000243 solution Substances 0.000 claims abstract description 41
- 239000013558 reference substance Substances 0.000 claims abstract description 29
- 239000012488 sample solution Substances 0.000 claims abstract description 25
- 238000012360 testing method Methods 0.000 claims abstract description 25
- 239000012490 blank solution Substances 0.000 claims abstract description 17
- 239000012085 test solution Substances 0.000 claims abstract description 16
- 239000012088 reference solution Substances 0.000 claims abstract description 15
- 238000005303 weighing Methods 0.000 claims abstract description 15
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000010438 heat treatment Methods 0.000 claims abstract description 13
- 238000010521 absorption reaction Methods 0.000 claims abstract description 12
- LMYVDPWHMJXPIY-UHFFFAOYSA-N 2-(9h-carbazol-1-yl)ethanol Chemical compound C12=CC=CC=C2NC2=C1C=CC=C2CCO LMYVDPWHMJXPIY-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 10
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000001816 cooling Methods 0.000 claims abstract description 9
- 229940097043 glucuronic acid Drugs 0.000 claims abstract description 7
- 239000008213 purified water Substances 0.000 claims abstract description 6
- 239000003086 colorant Substances 0.000 claims abstract description 5
- 238000002474 experimental method Methods 0.000 claims abstract description 5
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims abstract 3
- 239000000523 sample Substances 0.000 claims description 28
- 238000001514 detection method Methods 0.000 claims description 14
- 239000000126 substance Substances 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims 2
- IAJILQKETJEXLJ-KLVWXMOXSA-N (2s,3r,4r,5r)-2,3,4,5-tetrahydroxy-6-oxohexanoic acid Chemical compound O=C[C@H](O)[C@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-KLVWXMOXSA-N 0.000 abstract description 7
- 238000007865 diluting Methods 0.000 abstract description 5
- 159000000000 sodium salts Chemical class 0.000 abstract description 4
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 6
- 229920001282 polysaccharide Polymers 0.000 description 5
- 239000005017 polysaccharide Substances 0.000 description 5
- 241000251511 Holothuroidea Species 0.000 description 4
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 4
- 241000258955 Echinodermata Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000004255 ion exchange chromatography Methods 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000005904 alkaline hydrolysis reaction Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000009776 industrial production Methods 0.000 description 2
- SCVFZCLFOSHCOH-UHFFFAOYSA-M potassium acetate Chemical compound [K+].CC([O-])=O SCVFZCLFOSHCOH-UHFFFAOYSA-M 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000007670 refining Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- MSWZFWKMSRAUBD-UHFFFAOYSA-N 2-Amino-2-Deoxy-Hexose Chemical compound NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 229920002567 Chondroitin Polymers 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 125000002446 fucosyl group Chemical group C1([C@@H](O)[C@H](O)[C@H](O)[C@@H](O1)C)* 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- -1 polysaccharide compounds Chemical class 0.000 description 1
- 235000011056 potassium acetate Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
- G01N21/33—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using ultraviolet light
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- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
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Abstract
The invention belongs to the technical field of medicines, and relates to a method for identifying natural fucosylated glycosaminoglycan, in particular to a method for identifying hexuronic acid in natural fucosylated glycosaminoglycan sodium salt, which comprises the following steps: weighing natural fucosylated glycosaminoglycan, placing the natural fucosylated glycosaminoglycan in a volumetric flask, adding water to dissolve the natural fucosylated glycosaminoglycan and diluting the natural fucosylated glycosaminoglycan to a scale, and shaking the natural fucosylated glycosaminoglycan uniformly to serve as a test solution; weighing glucuronic acid reference substance, and preparing reference solution by the same method; taking purified water as a blank solution; taking a sample solution, placing the sample solution in a test tube or a colorimetric tube with a plug scale, adding concentrated sulfuric acid under an ice bath condition, heating in a water bath for 20 minutes, cooling to room temperature, adding a 1% carbazole ethanol solution, uniformly mixing by vortex, and standing at room temperature; and taking the reference solution and the blank solution for experiment by the same method, observing and recording the colors of the reference solution, the test solution and the blank solution, carrying out ultraviolet full-wavelength scanning on the reference solution and the test solution, confirming whether absorption exists between 524nm and 528nm, and recording the absorption wavelength.
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to an identification method of natural fucosylated glycosaminoglycan.
Background
NFG (Natural Fucosylated Glycosaminoglycan, hereinafter referred to as NFG) is extracted from sea cucumber animals of marine life and is prepared by further refining through ion exchange chromatography, and is a key starting material for semi-synthesis of bulk drugs. Fucosylated Glycosaminoglycans (FG) are only one Glycosaminoglycan with a specific chemical structure and pharmacological activity that has been found in echinoderms so far, which has a chondroitin sulfate-like backbone and in which a Fucosyl (Fuc) side chain-substituted Glycosaminoglycan analogue, which is complicated in chemical structure but has a specific pharmaceutical activity, is present. The LFG (Low Molecular Weight Fucosylated Glycosaminoglycan, hereinafter referred to as LFG) is obtained by depolymerizing natural Fucosylated Glycosaminoglycan, and the identification of NFG can effectively control the quality of LFG and ensure the clinical application of the LFG.
The natural FG obtained by conventional extraction from the echinoderm body wall usually contains dextran, fucosan (Fucan) and/or hexosamine-containing polysaccharide compounds having a molecular weight distribution close to that of the natural FG. These polysaccharide components that are incorporated in natural FG are difficult to remove cleanly by gel chromatography, ultrafiltration, and even ion exchange chromatography. The natural FG extract extracted from echinoderm usually contains other polysaccharides in an amount of about 10 to 20% by mass. These polysaccharide components can be degraded during depolymerization of natural FG and then removed by separation means, so that FG needs to be detected and monitored efficiently and sensitively during extraction of the depolymerized product of natural FG.
Disclosure of Invention
The invention aims to provide an identification method of natural fucosylated glycosaminoglycan, which can realize the rapid detection of the natural fucosylated glycosaminoglycan in the industrial production process and adopts the following technical scheme:
a method for identifying natural fucosylated glycosaminoglycan is used for identifying hexuronic acid in the sodium salt of the natural fucosylated glycosaminoglycan, and comprises the following steps:
1) weighing natural fucosylated glycosaminoglycan, placing the natural fucosylated glycosaminoglycan in a volumetric flask, adding water to dissolve the natural fucosylated glycosaminoglycan and diluting the natural fucosylated glycosaminoglycan to a scale, and shaking the natural fucosylated glycosaminoglycan uniformly to serve as a test solution; weighing a glucuronic acid reference substance, placing the reference substance in a volumetric flask, adding water to dissolve the reference substance, diluting the reference substance to a scale, and shaking up the reference substance to obtain a reference substance solution; taking purified water as a blank solution;
2) taking a sample solution, placing the sample solution in a test tube or a colorimetric tube with a plug scale, adding concentrated sulfuric acid under an ice bath condition, heating in a water bath for 15-25 minutes, cooling to room temperature, adding a 1% carbazole ethanol solution, uniformly mixing in a vortex mode, and standing at room temperature; taking the reference solution and the blank solution, carrying out the experiment by the same method, observing the colors of the reference solution, the test solution and the blank solution, recording the color of the solution,
3) and (3) carrying out ultraviolet full-wavelength scanning on the reference solution and the test solution, confirming whether absorption exists between 524nm and 528nm or not, and recording the absorption wavelength.
The concentration range of the natural fucosylated glycosaminoglycan detectable in the present invention is: the minimum detection concentration of 0.3-1.5 mg/ml hexuronic acid is as follows: 0.1mg/ml
Preferably, the method for identifying the natural fucosylated glycosaminoglycan comprises the following steps of: the method for identifying the hexuronic acid in the sodium salt of the natural fucosylated glycosaminoglycan comprises the following steps: weighing 10mg of natural fucosylated glycosaminoglycan, putting the natural fucosylated glycosaminoglycan into a 10ml volumetric flask, adding water to dissolve and dilute the natural fucosylated glycosaminoglycan to a scale, and shaking the solution uniformly to be used as a test solution; weighing 15mg of glucuronic acid reference substance, putting the weighed reference substance into a 50ml volumetric flask, adding water to dissolve and dilute the reference substance to a scale, and shaking up the reference substance to obtain a reference substance solution; taking purified water as a blank solution; taking 1ml of sample solution, placing the sample solution into a 25ml test tube or a colorimetric tube with a plug scale, adding 7ml of concentrated sulfuric acid under an ice bath condition, heating in a water bath at 85 ℃ for 20 minutes, cooling to room temperature, adding 0.25ml of 1% carbazole ethanol solution, uniformly mixing in a vortex mode, and standing at room temperature for 2 hours; and taking 1ml of the reference solution and the blank solution, carrying out an experiment by the same method, observing the colors of the reference solution, the test solution and the blank solution, recording the color of the solutions, carrying out ultraviolet full-wavelength scanning on the reference solution and the test solution, confirming whether the absorption exists between 524nm and 528nm, and recording the absorption wavelength.
Has the advantages that: the method is simple to operate, has strong specificity, can effectively identify the natural fucosylated glycosaminoglycan, and can realize the rapid detection of products in industrial production.
Drawings
FIG. 1 the color development results of the experiment in example 1 are blank, reference, test 20161209, test 20161229 and test 20161202 from left to right.
FIG. 2 example 1 control ultraviolet spectra
FIG. 3 ultraviolet-visible spectrum of sample 20161209 of example 1.
FIG. 4 ultraviolet-visible spectrum of sample 20161202 of example 1.
FIG. 5 ultraviolet-visible spectrum of sample 20161229 of example 1.
FIG. 6 shows the results of color development of test solutions of different concentrations.
FIG. 7 color development results for different concentrations of hexuronic acid control.
FIG. 885 ℃ water bath condition shows the color development results of the test articles with different heating times.
FIG. 9 shows the change of the test article developing solution left at room temperature for various periods of time.
Detailed Description
1. Devices, reagents, controls and samples
1.1 Primary device information
Table 1 main device information table
1.2 Primary reagent information
TABLE 2 Main reagent information Table
1.3 reference information
TABLE 3 reference information sheet
1.4 sample information
Table 4 NFG sample trial production information summary sheet
2. NFG preparation method
NFG is prepared from sea cucumber animal by alkaline hydrolysis, enzymolysis, extraction, and refining by ion exchange chromatography. The method specifically comprises the following steps:
1. extraction: taking dried sea cucumber, mechanically slicing, crushing, adding pure water for soaking overnight, adding compound protease under the condition of 50 ℃ water bath, stirring and uniformly mixing, and stirring for enzymolysis for 4 hours. Subsequently, sodium hydroxide solution was added to a final concentration of about 0.5M, and alkaline hydrolysis was carried out at 60 ℃ for 1 hour. Then, the pH value is adjusted to be neutral by hydrochloric acid solution, centrifugation is carried out, ethanol is added into supernate to enable the final concentration to reach 70% (v/v), standing is carried out overnight, and centrifugation is carried out to obtain sediment.
2. And (3) purification: re-dissolving the precipitate in the step 1 with 10 times (w/w) of pure water, centrifuging to remove insoluble substances, adding potassium acetate into the supernatant to a final concentration of about 1.0M, adding ethanol to make the final concentration of ethanol in the solution reach about 60% (v/v), standing at room temperature for 4h, and centrifuging to obtain the precipitate. Washing the obtained precipitate with about 2 times of 80% ethanol (v/v) twice, and drying under reduced pressure to obtain sea cucumber polysaccharide component containing fucosylated glycosaminoglycan.
Dissolving the component in pure water, loading the component into a DEAE-cellulose chromatographic column, carrying out gradient elution by using 0.5-2M NaCl aqueous solution, collecting eluent, monitoring the flow part containing the acidic mucopolysaccharide, combining the flow parts containing the acidic mucopolysaccharide, dialyzing by using a 30kD dialysis membrane, and freeze-drying to obtain the natural fucosylated glycosaminoglycan test sample.
3. Identification method
1) Solution preparation
Weighing 10mg of natural fucosylated glycosaminoglycan samples 20161202, 20161209 and 20161229 in batches, respectively placing in 10ml volumetric flasks, adding water for dissolving, diluting to scale, and shaking up to obtain a sample solution; weighing 15mg of glucuronic acid reference substance, putting the weighed reference substance into a 50ml volumetric flask, adding water to dissolve and dilute the reference substance to a scale, and shaking up the reference substance to obtain a reference substance solution; taking purified water as a blank solution;
2) sample processing
Taking 1ml of a sample solution, placing the sample solution in a 25ml test tube or a colorimetric tube with a plug scale, adding 7ml of concentrated sulfuric acid under an ice bath condition, heating in a water bath at 85 ℃ for 20 minutes, cooling to room temperature, adding 0.25ml of 1% carbazole ethanol solution, uniformly mixing in a vortex mode, and standing at room temperature for 2 hours; and treating the reference solution and 1ml of blank solution according to the method, observing the colors of the reference solution, the test solution and the blank solution, and recording the color of the solutions.
3) Sample detection
And (3) carrying out ultraviolet full-wavelength scanning on the reference solution and the test solution, confirming whether absorption exists between 524nm and 528nm or not, and recording the absorption wavelength.
2. The result of the detection
The three batches of test solution and glucuronic acid reference solution are treated by the same method, and show obvious purple red, the test solution has absorption in the wavelength range of 524-528 nm, the blank does not develop color, the statistics of related results are shown in table 5, the developing spectrum is shown in figure 1, and the ultraviolet visible spectrum is shown in figures 2-5.
TABLE 5 NFG sample hexuronic acid identification results Table
Example 2 detection of sodium salt of Natural fucosylated glycosaminoglycan
1. Identification method
1) Solution preparation
Weighing appropriate amount of natural fucosylated glycosaminoglycan sample, adding water to dissolve 0.3mg, 1.0mg and 1.5mg per 1ml, and shaking to obtain sample solution.
2) Sample detection
Taking 1ml of sample solution, placing the sample solution into a 25ml test tube or a colorimetric tube with a plug scale, adding 7ml of concentrated sulfuric acid under an ice bath condition, heating in a water bath at 85 ℃ for 20 minutes, cooling to room temperature, adding 0.25ml of 1% carbazole ethanol solution, uniformly mixing by vortex, standing at room temperature for 2 hours, and recording the color of the solution.
3) Results
The color of the test sample deepens along with the increase of the concentration through detection, but the color difference of the test sample with the concentration of 1.0mg/ml and the test sample with the concentration of 1.5mg/ml is not obvious, which indicates that the reaction is complete after the concentration reaches a certain degree, and the color is not deepened any more. Therefore, 1.0mg/ml is selected as the test concentration of the test sample. The results are shown in FIG. 6.
Example 3 minimum assay concentration of Hexauronic acid
1. Identification method
1) Solution preparation
Weighing appropriate amount of hexuronic acid reference, adding water to obtain solution containing 0.1mg, 0.2mg, and 0.5mg per 1ml, and shaking to obtain sample solution.
2) Sample detection
Respectively placing the sample solution into 25ml test tubes or colorimetric tubes with plug scales, adding 7ml concentrated sulfuric acid under ice bath condition, heating in 85 ℃ water bath for 20 minutes, cooling to room temperature, adding 0.25ml of 1% carbazole ethanol solution, uniformly mixing by vortex, standing for 2 hours at room temperature, and recording the color of the solution.
3) Results
The concentration of the detected reference substance is 0.1mg/ml, and the detection can be carried out. The results are shown in FIG. 7.
Example 485 ℃ Water bath heating time study
1) Solution preparation
Weighing 10mg of natural fucosylated glycosaminoglycan sample, placing the sample in a 10ml volumetric flask, adding water to dissolve and dilute the sample to a scale, shaking up, and preparing three parts in parallel to be used as a sample solution.
2) Sample detection
Taking 1ml of each sample solution, respectively placing the sample solution into a 25ml test tube or a colorimetric tube with a plug scale, adding 7ml of concentrated sulfuric acid under an ice bath condition, respectively heating in a water bath at 85 ℃ for 15, 20 and 25 minutes, cooling to room temperature, adding 0.25ml of 1% carbazole ethanol solution, uniformly mixing by vortex, standing at room temperature for 2 hours, and recording the color of the solution.
3) Results
The color of the test sample is not obviously changed along with the increase of the heating time through detection, which shows that the result is not influenced by heating in water bath at 85 ℃ for 15-25 minutes. The results are shown in FIG. 8.
Example 5 Room temperature standing time examination
1) Solution preparation
Weighing 10mg of a natural fucosylated glycosaminoglycan sample, placing the natural fucosylated glycosaminoglycan sample in a 10ml volumetric flask, adding water to dissolve the natural fucosylated glycosaminoglycan sample, diluting the natural fucosylated glycosaminoglycan sample to a scale, shaking the solution uniformly, and preparing 2 parts of the solution in parallel to serve as a sample solution.
2) Sample detection
Taking 1ml of each sample solution, respectively placing the sample solution into a 25ml test tube or a colorimetric tube with a plug scale, adding 7ml of concentrated sulfuric acid under an ice bath condition, respectively heating in a water bath at 85 ℃ for 20 minutes, cooling to room temperature, then adding 0.25ml of 1% carbazole ethanol solution, uniformly mixing in a vortex mode, respectively standing at room temperature for 1 hour and 2 hours, and recording the color of the solution.
3) Results
The color of the test article deepens with the increase of the standing time. The results are shown in FIG. 9.
Claims (6)
1. A method for identifying natural fucosylated glycosaminoglycan comprises the following steps:
1) weighing natural fucosylated glycosaminoglycan, placing the natural fucosylated glycosaminoglycan in a volumetric flask, adding water to dissolve and dilute the natural fucosylated glycosaminoglycan, and shaking up the natural fucosylated glycosaminoglycan to obtain a sample solution; weighing a glucuronic acid reference substance, putting the reference substance into a volumetric flask, adding water to dissolve and dilute the reference substance, and shaking up the reference substance to obtain a reference substance solution; taking purified water as a blank solution;
2) placing a sample solution into a test tube or a colorimetric tube with a plug scale, adding concentrated sulfuric acid under an ice bath condition, heating in a water bath for 15-25 minutes, cooling to room temperature, adding a 1% carbazole ethanol solution, uniformly mixing in a vortex mode, and standing at room temperature; taking a reference substance solution and a blank solution for carrying out an experiment by the same method, observing the colors of the reference substance solution, the test substance solution and the blank solution, and recording the color of the solutions;
3) and (3) carrying out ultraviolet full-wavelength scanning on the reference solution and the test solution, confirming whether absorption exists between 524nm and 528nm or not, and recording the absorption wavelength.
2. The method for identifying natural fucosylated glycosaminoglycan according to claim 1, wherein in the step 1), 10mg of the natural fucosylated glycosaminoglycan is weighed, placed in a 10ml volumetric flask, dissolved by adding water and diluted to a scale, and shaken up to be used as a test solution; weighing 15mg of glucuronic acid reference substance, placing the weighed reference substance in a 50ml volumetric flask, adding water to dissolve and dilute the reference substance to a scale, and shaking up to obtain the reference substance solution. Purified water was taken as a blank solution.
3. The method for identifying natural fucosylated glycosaminoglycan according to claim 1, wherein in the step 2), 1ml of a sample solution is taken and placed in a 25ml test tube or a colorimetric tube with a plug scale, 7ml of concentrated sulfuric acid is added under an ice bath condition, the sample solution is heated in a water bath at 85 ℃ for 20 minutes, after being cooled to room temperature, 0.25ml of 1% carbazole ethanol solution is added, the mixture is uniformly mixed in a vortex mode, and the mixture is kept standing at room temperature for 2 hours; the control solution and 1ml of blank solution were used in the same manner.
4. The method of claim 1, wherein the control solution and the sample solution have absorption peaks at 524nm to 528 nm.
5. The method for identifying natural fucosylated glycosaminoglycan according to claim 1, wherein the detection concentration range of the test sample is: 0.3-1.5 mg/ml.
6. The method of claim 1, wherein the sample has a minimum color concentration of 0.1 mg/ml.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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