WO2020004216A1 - Procédé d'analyse de mucopolysaccharide acide ou d'un sel de celui-ci contenu dans un échantillon de test, procédé de détermination quantitative de sulfate de chondroïtine ou d'un sel de celui-ci, et procédé de test de contrôle de qualité de produit - Google Patents

Procédé d'analyse de mucopolysaccharide acide ou d'un sel de celui-ci contenu dans un échantillon de test, procédé de détermination quantitative de sulfate de chondroïtine ou d'un sel de celui-ci, et procédé de test de contrôle de qualité de produit Download PDF

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WO2020004216A1
WO2020004216A1 PCT/JP2019/024482 JP2019024482W WO2020004216A1 WO 2020004216 A1 WO2020004216 A1 WO 2020004216A1 JP 2019024482 W JP2019024482 W JP 2019024482W WO 2020004216 A1 WO2020004216 A1 WO 2020004216A1
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salt
test
acidic mucopolysaccharide
test sample
membrane
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PCT/JP2019/024482
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English (en)
Japanese (ja)
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笠島　直樹
亜由太 船木
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サントリーホールディングス株式会社
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Priority to CN201980043220.3A priority Critical patent/CN112352156A/zh
Priority to JP2020527457A priority patent/JP7384793B2/ja
Publication of WO2020004216A1 publication Critical patent/WO2020004216A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/27—Association of two or more measuring systems or cells, each measuring a different parameter, where the measurement results may be either used independently, the systems or cells being physically associated, or combined to produce a value for a further parameter
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis

Definitions

  • the present invention relates to a method for analyzing an acidic mucopolysaccharide or a salt thereof contained in a test sample.
  • the present invention also relates to a method for quantifying chondroitin sulfate or a salt thereof contained in a test sample.
  • the present invention also relates to a quality control test method for a product containing the acidic mucopolysaccharide or a salt thereof.
  • the acidic mucopolysaccharide is a mucopolysaccharide having an acidic group such as a carboxylic acid in the structure.
  • Acidic mucopolysaccharides are classified into chondroitin sulfate, hyaluronic acid, dermatan sulfate and the like according to the type and combination of uronic acid (or galactose) and amino sugar constituting the disaccharide unit. Since acidic mucopolysaccharides exhibit physiological activity in vivo, their qualitative and quantitative determinations are useful in predicting or determining the effects expected of health foods and pharmaceuticals, and in quality control of products.
  • Chondroitin sulfate is an acidic mucopolysaccharide having a repeating structure of a disaccharide unit in which D-glucuronic acid is bound to N-acetyl-D-galactosamine, and in which some of the hydroxyl groups of N-acetyl-D-galactosamine are sulfated. It is.
  • an HPLC method is known in which chondroitin sulfate is hydrolyzed with chondroitinase ABC, and the amount of unsaturated disaccharide produced is measured by HPLC.
  • chondroitin sulfate include barium sulfate gravimetric method for detecting compounds having a sulfate group, carbazole sulfuric acid method for detecting glucuronic acid and desulfide, and Alcian blue colorimetric method for detecting acidic mucopolysaccharide. There is an analysis method.
  • Non-Patent Document 1 a sample containing an acidic mucopolysaccharide such as chondroitin sulfate was electrophoresed on a nitrocellulose membrane Immobilon (product name, Millipore) and subjected to Alcian blue staining to separate and identify the acidic mucopolysaccharide. It is described.
  • the membrane used in Non-Patent Document 1 is a mixed membrane of nitrocellulose and cellulose acetate.
  • the HPLC method is known as a method for quantifying chondroitin sulfate, but when analyzing a product or the like by the HPLC method, excipients or other raw materials (eg, a polyphenol that inhibits an enzyme) mixed with the product are used. There was a problem that it was easily affected by.
  • the HPLC method requires an expensive and large-sized HPLC device and the like, and a hydrolyzing enzyme used for decomposing chondroitin sulfate and a standard product for identifying chondroitin sulfate are also expensive. It may not be sold. For this reason, there is a demand for a method capable of easily and simply analyzing an acidic mucopolysaccharide or a salt thereof at a lower cost without using such a large-sized apparatus.
  • a quantification method other than the HPLC method is also known as described above, the barium sulfate gravimetric method has a problem in specificity because a sulfate group other than the acidic mucopolysaccharide is also quantified.
  • Non-Patent Document 1 a plurality of types of acidic mucopolysaccharides are separated by electrophoresis, and the band density on the membrane is measured by a densitometer. It is thought that it is difficult to carry out with high accuracy.
  • An object of the present invention is to provide an analysis method capable of confirming and quantifying the type of an acidic mucopolysaccharide or a salt thereof contained in a sample containing an acidic mucopolysaccharide or a salt thereof in a simple step.
  • the present inventors have intensively studied to solve the above problems, and among them, a colorimetric method (colorimetric method) such as the Alcian Blue colorimetric method, which is one of methods for quantifying acidic mucopolysaccharide.
  • a colorimetric method colorimetric method
  • Alcian Blue colorimetric method which is one of methods for quantifying acidic mucopolysaccharide.
  • the total amount and quality of the acidic mucopolysaccharide or its salt contained in the test sample can be evaluated by a simple process.
  • the analysis method combining the qualitative test and the quantitative test as described above can also be used for quantification of the acidic mucopolysaccharide or its salt when the test sample contains only one kind of acidic mucopolysaccharide or its salt.
  • a method for analyzing an acidic mucopolysaccharide or a salt thereof contained in a test sample by combining a qualitative test and a quantitative test by a colorimetric method has not been reported so far.
  • the present invention relates to the following analysis method and the like.
  • a test sample containing an acidic mucopolysaccharide or a salt thereof is subjected to a qualitative test and a quantitative test, and the qualitative test is performed by subjecting the test sample to electrophoresis using a membrane.
  • the membrane after electrophoresis, a staining step of staining with Alcian blue, and, from the band pattern obtained by the staining, including a detection step of confirming the type of acidic mucopolysaccharide or salt thereof in the test sample,
  • the quantitative test is included in the test sample, including a colorimetric determination step of quantifying the acidic mucopolysaccharide or a salt thereof in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or a salt thereof. For analyzing acidic mucopolysaccharides or salts thereof.
  • test sample containing chondroitin sulfate or a salt thereof to a qualitative test and a quantitative test
  • the qualitative test comprises subjecting the test sample to electrophoresis using a membrane;
  • the membrane after the electrophoresis, a staining step of staining with Alcian blue, and a detection step of confirming the type of acidic mucopolysaccharide or a salt thereof in the test sample from the band pattern obtained by the staining
  • the test includes a colorimetric quantification step of quantifying the acidic mucopolysaccharide or a salt thereof in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or a salt thereof.
  • the quantitative result obtained by the quantitative test is converted to the chondroitin sulfate. Determination method of to the content of its salts, chondroitin sulfate or a salt thereof contained in the test sample.
  • a quality control test method for a product containing the acidic mucopolysaccharide or a salt thereof Using the above product as a test sample, a test sample analysis step of analyzing by the analysis method according to any one of the above [1] to [8], a standard sample containing an acidic mucopolysaccharide or a salt thereof whose concentration and type are known.
  • the standard sample analysis step of analyzing by the above analysis method, and comparing the qualitative test result and the quantitative test result of the test sample obtained in the above analysis with the qualitative test result and the quantitative test result of the standard sample A quality control test method for a product containing an acidic mucopolysaccharide or a salt thereof, comprising an evaluation step of evaluating the quality of the product.
  • the analysis method which can confirm and quantify the kind of acidic mucopolysaccharide or its salt contained in the sample containing acidic mucopolysaccharide or its salt by a simple process is provided.
  • INDUSTRIAL APPLICABILITY The analysis method of the present invention is useful for analysis of acidic mucopolysaccharide or a salt thereof contained in foods, pharmaceuticals, and the like, quality control of products containing the acidic mucopolysaccharide or a salt thereof, and the like.
  • FIG. 1 is a photograph showing a band pattern of an acidic mucopolysaccharide preparation and a shark cartilage raw material (I) obtained by electrophoresis using a cellulose acetate membrane.
  • FIG. 2 is a photograph showing a band pattern of an acidic mucopolysaccharide preparation and shark cartilage raw material (II) obtained by electrophoresis using a cellulose acetate membrane.
  • FIG. 3 is a photograph showing a band pattern of an acidic mucopolysaccharide preparation and shark cartilage raw material (I) obtained by electrophoresis using a nitrocellulose membrane.
  • FIG. 1 is a photograph showing a band pattern of an acidic mucopolysaccharide preparation and a shark cartilage raw material (I) obtained by electrophoresis using a cellulose acetate membrane.
  • FIG. 2 is a photograph showing a band pattern of an acidic mucopolysaccharide preparation and shark cartilage raw material (II) obtained by electrophor
  • FIG. 4 is a photograph showing band patterns of an acidic mucopolysaccharide preparation and a shark cartilage raw material (II) obtained by electrophoresis using a nitrocellulose membrane.
  • FIG. 5 is a photograph showing band patterns of an enzyme-treated product of a shark cartilage raw material and an enzyme-treated product of an acidic mucopolysaccharide preparation obtained by electrophoresis using a cellulose acetate membrane or a nitrocellulose membrane ((a)). : Cellulose acetate membrane, (b): nitrocellulose membrane).
  • FIG. 4 is a photograph showing band patterns of an acidic mucopolysaccharide preparation and a shark cartilage raw material (II) obtained by electrophoresis using a nitrocellulose membrane.
  • FIG. 5 is a photograph showing band patterns of an enzyme-treated product of a shark cartilage raw material and an enzyme-treated product of an acidic mucopolysaccharide preparation obtained by electrophoresis using a cellulose acetate
  • FIG. 6 is a photograph showing a band pattern obtained by electrophoresing a sample of a raw material of shark cartilage and chondroitin sulfate C sodium without enzyme treatment, a sample treated with actinase, and a sample treated with actinase E and chondroitinase ABC on a cellulose acetate membrane.
  • FIG. 7 is a schematic diagram of the band pattern of the photograph shown in FIG. 1 ((a): a schematic diagram of FIG. 1 (a), (b): a schematic diagram of FIG. 1 (b)).
  • FIG. 8 is a schematic diagram of the band pattern of the photograph shown in FIG. 2 ((a): a schematic diagram of FIG. 2 (a), (b): a schematic diagram of FIG. 2 (b)).
  • FIG. 9 is a schematic diagram of the band pattern of the photograph shown in FIG. 3 ((a): a schematic diagram of FIG. 3 (a), (b): a schematic diagram of FIG. 3 (b)).
  • FIG. 10 is a schematic diagram of the band pattern of the photograph shown in FIG. 4 ((a): a schematic diagram of FIG. 4 (a), (b): a schematic diagram of FIG. 4 (b)).
  • FIG. 11 is a schematic diagram of the band pattern of the photograph shown in FIG. 5 ((a): a schematic diagram of FIG. 5 (a), (b): a schematic diagram of FIG. 5 (b)).
  • FIG. 12 is a schematic diagram of the band pattern of the photograph shown in FIG.
  • the analysis method of the present invention is a method for analyzing an acidic mucopolysaccharide or a salt thereof contained in a test sample, including subjecting the test sample containing the acidic mucopolysaccharide or a salt thereof to a qualitative test and a quantitative test.
  • the test sample in the qualitative test, is subjected to electrophoresis using a membrane, an electrophoretic membrane is stained with Alcian blue, and a band obtained by the staining is obtained.
  • the quantitative test includes a colorimetric quantification step of quantifying the acidic mucopolysaccharide or a salt thereof in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or a salt thereof.
  • a dye capable of detecting an acidic mucopolysaccharide or a salt thereof is also simply referred to as a dye.
  • the acidic mucopolysaccharide is an acidic polysaccharide in which uronic acid (iduronic acid or glucuronic acid) or galactose has a repeating structure of a disaccharide unit bonded to an amino sugar, and has an acidic group (carboxyl group, sulfate group) in the structure. It is.
  • Acid mucopolysaccharides usually have an unbranched linear skeleton. Acidic mucopolysaccharides include chondroitin sulfate, hyaluronic acid, dermatan sulfate, heparin, heparan sulfate, keratan sulfate, and the like.
  • the test sample in the analysis method of the present invention may be any as long as it contains at least one kind of acidic mucopolysaccharide or a salt thereof.
  • the salt is not particularly limited, and examples thereof include alkali metal salts such as sodium and potassium; and alkaline earth metal salts such as calcium and magnesium. Preferred are alkali metal salts, and more preferred are sodium salts.
  • the acidic mucopolysaccharide is preferably at least one selected from the group consisting of chondroitin sulfate, hyaluronic acid, dermatan sulfate, heparin and heparan sulfate.
  • the analysis method of the present invention is suitably used for analyzing a test sample containing one or more of these acidic mucopolysaccharides or salts thereof.
  • chondroitin sulfate or hyaluronic acid is more preferred as the acidic mucopolysaccharide, and chondroitin sulfate is more preferred.
  • chondroitin sulfate refers to chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, and chondroitin sulfate E, and may be one type of these or two or more types.
  • Dermatan sulfate (chondroitin sulfate B) is not included in chondroitin sulfate in the present invention.
  • chondroitin sulfate chondroitin sulfate C is preferable.
  • chondroitin sulfate C includes a case where chondroitin sulfate C is contained as a main component and at least one other chondroitin sulfate is contained.
  • Test samples containing acidic mucopolysaccharides or salts thereof include, for example, foods (including health foods), pharmaceuticals, cosmetics, and their raw materials (eg, shark cartilage extract, porcine cartilage extract, squid cartilage extract, salmon) Cartilage extract).
  • the combination of the above qualitative test and quantitative test enables confirmation and quantification of the type of acidic mucopolysaccharide or a salt thereof in the test sample in a simple step.
  • the type of acidic mucopolysaccharide or a salt thereof refers to the type of acidic mucopolysaccharide contained in the acidic mucopolysaccharide or a salt thereof.
  • the analysis method of the present invention can be used, for example, to confirm that the test sample contains one or more acid mucopolysaccharides or salts thereof, and to quantify the acid mucopolysaccharides or salts thereof in the test sample. It can be performed.
  • the fact that the acidic mucopolysaccharide or a salt thereof is one (or two or more) means that the acidic mucopolysaccharide or the salt thereof contains one (or two or more) acidic mucopolysaccharides.
  • the test sample contains only one kind of acidic mucopolysaccharide or a salt thereof
  • confirmation (identification) of the type of the acidic mucopolysaccharide or a salt thereof, and the acid mucopolysaccharide in the test sample are performed.
  • quantification of a salt thereof can be performed.
  • the analysis method of the present invention when the acidic mucopolysaccharide or salt thereof contained in the test sample is two or more, confirmation of the type of acidic mucopolysaccharide or salt thereof contained in the test sample, and their Can be used to determine the total amount.
  • the order in which the qualitative test and the quantitative test are performed is not particularly limited. After conducting a qualitative test and confirming the type of acidic mucopolysaccharide or salt thereof contained in the test sample, a quantitative test may be performed, and the acid mucopolysaccharide or salt thereof in the test sample was quantified by a quantitative test. Thereafter, a qualitative test may be performed, or a qualitative test and a quantitative test may be performed in parallel. In one embodiment, it is preferable to perform a quantitative test after performing a qualitative test.
  • the type of acidic mucopolysaccharide or salt thereof contained in the test sample can be confirmed by performing the electrophoresis step, the staining step, and the detection step in this order.
  • the test sample is subjected to electrophoresis using a membrane (membrane electrophoresis).
  • a membrane membrane electrophoresis
  • a porous membrane usable for electrophoresis can be used.
  • a film include a nitrocellulose film, a cellulose acetate film, and a mixed film of nitrocellulose and cellulose acetate.
  • the above-mentioned membrane is preferably a nitrocellulose membrane, or a mixed membrane of nitrocellulose and cellulose acetate.
  • a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate When a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate is used, the separation of chondroitin sulfate or a salt thereof from other acidic mucopolysaccharide or a salt thereof is particularly good. Therefore, electrophoresis using a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate is advantageous in detecting chondroitin sulfate or a salt thereof.
  • the test sample contains chondroitin sulfate or its salt, or when confirming the presence of chondroitin sulfate or its salt in the test sample, use a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate. Is preferred.
  • a cellulose acetate membrane it is preferable to use a cellulose acetate membrane.
  • a cellulose acetate membrane When a cellulose acetate membrane is used, the separation of hyaluronic acid or a salt thereof from other acidic mucopolysaccharide or a salt thereof is good.
  • a test sample contains hyaluronic acid or a salt thereof, or when the presence of hyaluronic acid or a salt thereof in a test sample is confirmed, it is preferable to use a cellulose acetate membrane.
  • separation of chondroitin sulfate or a salt thereof from other acidic mucopolysaccharide or a salt thereof is good.
  • a test sample is subjected to chondroitinase ABC treatment as described below, and the band pattern of the obtained enzyme-treated product and the chondroitinase ABC treatment It is also preferable to compare with the band pattern of the test sample which has not been subjected to the above.
  • the pore size of the membrane is not particularly limited.
  • a membrane having a pore size of 0.1 to 0.6 ⁇ m can be used. If the membrane has a hydrophobic coating, it is preferable to wash the membrane with a hydrophilic solvent before use.
  • the test sample can be dissolved or suspended in a solvent, if desired, and subjected to membrane electrophoresis as a test sample solution.
  • the solvent used for preparing the test sample solution is not particularly limited, but water, electrophoresis solution, or the like can be used.
  • Electrophoresis conditions for electrophoresis are not particularly limited, and a general method can be used. It is preferable to appropriately set the electrophoresis solution used for the electrophoresis and the energizing conditions in consideration of the resolution of the obtained band pattern and the like.
  • the electrophoresis may be performed by a constant current method or a constant voltage method, and is preferably performed by a constant current method.
  • the current during the migration can be, for example, 5 to 100 mA.
  • the migration time can be appropriately set according to the magnitude of the current and the like.
  • a buffer having a pH of 3 to 7 is preferably used for the electrophoresis running solution.
  • a buffer that can be used as the electrophoresis running buffer include an acetate buffer such as a barium acetate buffer and an acetic acid-pyridine buffer. Since the separation of the acidic mucopolysaccharide or a salt thereof is good, when using a nitrocellulose membrane, a mixed membrane of nitrocellulose and cellulose acetate, a barium acetate buffer is preferable, and when using a cellulose acetate membrane, An acetate-pyridine buffer is preferred.
  • the pH can be measured with a commercially available pH meter.
  • electrophoresis is preferably performed at less than 10 ° C, more preferably at 0 to 7 ° C, and still more preferably at 0 to 5 ° C. At the time of electrophoresis, it is preferable to perform electrophoresis by setting the electrophoresis tank to the above temperature.
  • the membrane after the electrophoresis is stained with Alcian blue (staining step).
  • Alcian blue binds to an acidic mucopolysaccharide or a salt thereof, and thus can detect an acidic mucopolysaccharide or a salt thereof contained in a test sample with high sensitivity. Staining with Alcian blue also has the advantage of simple operation.
  • the quantitative analysis obtained by using Alcian Blue for the detection of acidic mucopolysaccharide or a salt thereof in the qualitative test and the colorimetric method (Alcian Blue colorimetric method) using Alcian Blue in the quantitative test described below The results reflect the content of the acidic mucopolysaccharide or salt thereof confirmed in the qualitative test.
  • Staining can be performed by immersing the membrane in an Alcian blue solution for a predetermined time, for example, 5 to 60 minutes. After staining, the membrane is destained using a destaining solution.
  • a decolorizing solution a normal decolorizing solution can be used, and an acidic aqueous solution is preferable.
  • the acidic aqueous solution include an acetic acid aqueous solution and a formic acid aqueous solution.
  • the membrane after decolorization exhibits a band pattern (also referred to as an electrophoresis pattern) composed of one or more bands having different mobilities (also referred to as moving distances) depending on the acidic mucopolysaccharide or a salt thereof. . When the test sample does not contain acidic mucopolysaccharide or its salt, no band is usually detected.
  • the type of acidic mucopolysaccharide or salt thereof in the test sample is confirmed from the band pattern obtained by the staining. The band pattern can be confirmed visually.
  • the type of acidic mucopolysaccharide or salt thereof contained in the test sample is specified (identified), and one type of acid mucopolysaccharide or salt thereof contained in the test sample is identified. Or two or more types. In one embodiment, whether the test sample contains one or more acid mucopolysaccharides or salts thereof can be confirmed by the number of bands.
  • a sample of the acidic mucopolysaccharide or a salt thereof (standard substance) or a standard sample in which the type of the acidic mucopolysaccharide or the salt contained is known is used. It is preferable to obtain a band pattern.
  • the type of the acidic mucopolysaccharide or a salt thereof can be confirmed by, for example, comparing with the moving distance of a band of a sample of the acidic mucopolysaccharide or a salt thereof.
  • a qualitative test is performed on a standard sample having a known kind of acidic mucopolysaccharide or a salt thereof by the same method, and the band pattern of the standard sample is compared with the band pattern of the test sample. Thereby, the type of acidic mucopolysaccharide or a salt thereof in the test sample can be confirmed.
  • a sample of the same acidic mucopolysaccharide or salt thereof is used as the standard, or the same origin is used. It is preferable to use a standard sample containing an acidic mucopolysaccharide or a salt thereof.
  • the acidic mucopolysaccharide or salt thereof contained in the test sample is predicted to be derived from shark cartilage
  • a sample of the acidic mucopolysaccharide or salt thereof derived from shark cartilage is used, or the sample is derived from shark cartilage. It is preferable to use a standard sample containing the acidic mucopolysaccharide or a salt thereof.
  • an enzyme-treated product of the test sample is prepared as described below, and by comparing the band patterns of the test sample and the enzyme-treated product, the acidic mucopolysaccharide or You may confirm the kind of the salt.
  • the analysis method of the present invention is suitable for analyzing chondroitin sulfate or a salt thereof derived from shark cartilage.
  • the membrane electrophoresis is performed, acidic mucopolysaccharide other than chondroitin sulfate or a salt thereof and chondroitin sulfate derived from shark cartilage or a salt thereof can be satisfactorily separated. Therefore, the qualitative test is advantageous for detecting chondroitin sulfate or a salt thereof derived from shark cartilage.
  • the analysis method of the present invention is particularly preferably used for analysis of chondroitin sulfate or a salt thereof derived from shark cartilage contained in a test sample.
  • the qualitative test may include steps other than the above-described electrophoresis step, staining step, and detection step.
  • the test sample is treated with an enzyme that degrades an acid mucopolysaccharide or a salt thereof contained in the test sample (hereinafter, also simply referred to as an acid mucopolysaccharide degrading enzyme), and the enzyme-treated product (also referred to as an enzyme-treated sample). May be performed.
  • the test sample is treated with an acid mucopolysaccharide degrading enzyme, and the resulting enzyme-treated product is also subjected to an electrophoresis step and a staining step to obtain a band pattern.
  • the band patterns of the test sample and the enzyme-treated product.
  • the present invention by comparing the moving distance or band pattern of a band obtained by electrophoresis with a sample or a standard sample as described above, The type of acidic mucopolysaccharide or salt thereof can be confirmed, and the band patterns of the electrophoresis of the test sample (unenzymatically treated) and its acid mucopolysaccharide-degrading enzyme-treated product should be compared. Thereby, the type of acidic mucopolysaccharide or a salt thereof in the test sample can be specified more reliably.
  • the acidic mucopolysaccharide degrading enzyme may be selected according to the type of acidic mucopolysaccharide or salt thereof contained in the test sample.
  • chondroitin sulfate or its salt chondroitinase ABC can be used.
  • test sample when the test sample contains chondroitin sulfate or a salt thereof, or when confirming the presence of chondroitin sulfate or a salt thereof in the test sample, the test sample is treated with chondroitinase ABC (degraded). ) To prepare a treated enzyme product.
  • a test sample containing acidic mucopolysaccharide or a salt thereof is treated with chondroitinase ABC, and the resulting enzyme-treated product is subjected to an electrophoresis step and a staining step to obtain a band pattern, By comparing the band patterns of the test sample (test sample without enzyme treatment) and the enzyme-treated product, it is possible to more accurately confirm whether or not the test sample contains chondroitin sulfate or a salt thereof. .
  • the band of the acidic mucopolysaccharide or a salt thereof detected in the test sample disappears by the enzyme treatment with chondroitinase ABC, it is confirmed that the test sample contains chondroitin sulfate or a salt thereof.
  • the comparison between the band patterns of the test sample and the enzyme-treated product can be performed in combination with the comparison with the movement distance of the band of the standard or the comparison with the band pattern of the standard sample.
  • the test sample is treated with chondroitinase ABC as described above. It is preferable to prepare an enzyme-treated product and compare the band patterns of the test sample and the enzyme-treated product.
  • the analysis method of the present invention comprises an electrophoresis step for a test sample (a test sample not treated with an acid mucopolysaccharase), an enzyme-treated product of the test sample, and a standard (or a standard sample). And performing a staining process to obtain a band pattern, and comparing the band pattern of the test sample with the band pattern of the standard (or standard sample), and the band pattern of the test sample with the enzyme-treated product. It is preferable to confirm the type of acidic mucopolysaccharide or salt thereof contained in the test sample.
  • the type of acidic mucopolysaccharide or salt thereof contained in the test sample is confirmed, and then the test sample is subjected to acid mucopolysaccharide degrading enzyme. It is preferable to prepare the enzyme-treated product by performing the treatment, obtain a band pattern of the enzyme-treated product, and compare it with the band pattern of the test sample.
  • the acid mucopolysaccharide-degrading enzyme an enzyme capable of decomposing the acidic mucopolysaccharide or a salt thereof may be used according to the type of the acid mucopolysaccharide or a salt thereof confirmed by comparison with a standard.
  • the type of the acidic mucopolysaccharide or its salt may be the type confirmed by comparison with the standard. It is confirmed.
  • the analysis method of the present invention may include a protein removal treatment step.
  • a deproteinization treatment may be performed before the treatment.
  • the degradation of the acidic mucopolysaccharide or a salt thereof by the acidic mucopolysaccharide-degrading enzyme proceeds more rapidly.
  • the protein removal treatment is not particularly limited as long as the effects of the present invention are not impaired.
  • a method of treating a test sample with a protease a method of changing the pH of a test sample (for example, setting the pH to 12 or more); To remove the protein by precipitation.
  • a method of treating with a protease is preferable because the operation is simple and the enzymatic treatment with chondroitinase ABC or the like is simple thereafter.
  • the protease those which do not decompose the acidic mucopolysaccharide or its salt can be used, and actinase E is preferable.
  • the test sample is preferably treated with actinase E before the chondroitinase ABC treatment. It is preferable to degrade the protein in the test sample with actinase E and then treat it with chondroitinase ABC, since the degradation of chondroitin sulfate or a salt thereof by the enzyme occurs more quickly.
  • Enzymatic treatment with acid mucopolysaccharide degrading enzyme such as chondroitinase ABC and actinase E can be performed by a general method, and may be selected according to the type of enzyme.
  • the pH during the treatment with an enzyme such as actinase E or chondroitinase ABC is preferably 7 to 9, and the temperature is preferably 25 to 50 ° C.
  • the treatment time with actinase E can be, for example, 10 to 24 hours.
  • the treatment with chondroitinase ABC can be performed, for example, for 0.5 to 2 hours.
  • the enzyme treatment may be deactivated as necessary, the enzyme may be removed from the sample by a known method such as centrifugation, and the sample may be subjected to membrane electrophoresis.
  • the quantitative test includes a colorimetric quantification step of quantifying the acidic mucopolysaccharide or a salt thereof in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or a salt thereof.
  • a colorimetric method using a dye for example, a dye is added to a test sample, the absorbance of a solution such as a coloring solution is measured with a spectrophotometer, and an acidic mucopolysaccharide or a salt thereof is obtained from a calibration curve prepared using a standard. Can be determined.
  • the content of the acidic mucopolysaccharide or its salt in the test sample is usually determined as the content of the acidic mucopolysaccharide or its salt used in the sample.
  • the content of the acidic mucopolysaccharide or salt thereof thus obtained can be used as a quantitative result obtained by a quantitative test.
  • the preparation may be selected according to the acid mucopolysaccharide or its salt.
  • a dye (reagent) that can be used for detecting an acidic mucopolysaccharide or a salt thereof can be used.
  • it is a dye that binds to the acidic mucopolysaccharide or a salt thereof, and more preferably, a dye that binds to the acidic mucopolysaccharide or a salt thereof and is optically detectable.
  • a dye that binds to the acidic mucopolysaccharide or a salt thereof and is optically detectable From the viewpoint of analysis accuracy and the like, Alcian Blue and carbazole are preferable as the dye, and Alcian Blue is more preferable.
  • the amount of acidic mucopolysaccharide or a salt thereof is determined by the Alcian Blue colorimetric method.
  • the Alcian blue colorimetric method can be performed by a usual method.
  • Alcian blue colorimetric method when an Alcian blue solution is added to a test sample, Alcian blue and the acidic mucopolysaccharide or a salt thereof form a complex and precipitate. The precipitate is redissolved in an alkaline solution, and the absorbance of the resulting solution is measured with a spectrophotometer.
  • the measurement wavelength is preferably from 570 to 660 nm, more preferably from 590 to 640 nm.
  • the amount of acidic mucopolysaccharide or a salt thereof can be calculated based on a calibration curve prepared in advance using a standard.
  • Alcian Blue for example, it is possible to quantify the acidic mucopolysaccharide or a salt thereof in the test sample by the method described in Examples.
  • the Alcian Blue colorimetric method has the advantages of simple operation and high reproducibility as a method for quantifying acidic mucopolysaccharide.
  • carbazole is used as the above dye, the amount of acidic mucopolysaccharide or a salt thereof can be determined by the carbazole sulfate method.
  • the carbazole sulfate method can be performed by a usual method.
  • test sample When a carbazole solution and concentrated sulfuric acid are added to the test sample, if the test sample contains uronic acid, the sample turns red-purple.
  • the absorbance of the color solution is measured with a spectrophotometer, the uronic acid content is calculated from the calibration curve of the sample, and the amount of chondroitin sulfate or a salt thereof is determined.
  • the measurement wavelength is preferably from 500 to 600 nm, more preferably from 520 to 550 nm.
  • a test sample may be treated with a protease or the like as necessary.
  • the protease those which do not decompose the acidic mucopolysaccharide or its salt can be used, and the above-mentioned actinase E and the like are preferable.
  • Alcian Blue it is more preferable to use Alcian Blue as a dye in the quantitative test.
  • Alcian Blue in qualitative and quantitative tests, the analysis accuracy is higher when analyzing products containing components other than acidic mucopolysaccharide or its salts (hereinafter, also referred to as other components).
  • Use of Alcian Blue is also preferable in that the operation in the quantitative test is simpler.
  • Alcian Blue is used in qualitative and quantitative tests, only acidic mucopolysaccharide or its salts are detected or quantified in these tests. That is, the quantitative result obtained in the quantitative test reflects the content of the acidic mucopolysaccharide or its salt confirmed in the qualitative test.
  • the quantitative result obtained in the quantitative test using the test sample is Can be determined to be the content of the acidic mucopolysaccharide or salt thereof contained in. Therefore, by combining the qualitative test and the quantitative test in this way and using the same dye (Alcian Blue) for the detection of acidic mucopolysaccharide or a salt thereof in these tests, the test sample can be prepared in a simple step. The type and the type of acidic mucopolysaccharide or salt thereof can be accurately confirmed and quantified.
  • the analysis method of the present invention can be used as a method for quantifying the acidic mucopolysaccharide or its salt.
  • foods, pharmaceuticals, cosmetics, and the like containing the acidic mucopolysaccharide or its salt, and the type and amount of the acidic mucopolysaccharide or its salt contained in these raw materials are analyzed and identified in a simple step. can do.
  • the analysis method of the present invention is also useful, for example, in quality control of such foods, pharmaceuticals, cosmetics, and raw materials thereof.
  • the analysis method of the present invention can be used as an analysis method for chondroitin sulfate or a salt thereof contained in a test sample.
  • a method for analyzing a test sample containing chondroitin sulfate or a salt thereof it is preferable to confirm the presence of chondroitin sulfate or a salt thereof in the test sample from the band pattern obtained by the staining in the detection step.
  • the analysis method of the present invention includes subjecting a test sample containing chondroitin sulfate or a salt thereof to a qualitative test and a quantitative test, wherein the qualitative test is performed by subjecting the test sample to electrophoresis using a membrane.
  • the electrophoresis step includes a colorimetric quantification step of quantifying the acidic mucopolysaccharide or a salt thereof in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or a salt thereof. May be a method for analyzing chondroitin sulfate or a salt thereof.
  • the quantification obtained by the quantitative test is performed.
  • the result can be the content of chondroitin sulfate or its salt.
  • the method for analyzing chondroitin sulfate or a salt thereof can be used as a method for quantifying chondroitin sulfate or a salt thereof in a test sample.
  • the present invention also includes the following method for quantifying chondroitin sulfate or a salt thereof in a test sample.
  • a test sample containing chondroitin sulfate or a salt thereof includes subjecting to a qualitative test and a quantitative test.
  • An acid mucopolysaccharide or a salt thereof in the test sample includes a colorimetric quantification step of quantifying the acid mucopolysaccharide or a salt thereof by a colorimetric method using a dye capable of detecting the acid mucopolysaccharide or a salt thereof.
  • the quantitative result obtained by the above quantitative test is compared with the chondroitin sulfate or its salt.
  • the content of salts, chondroitin sulfate or quantification method of a salt thereof in a test sample is the same as those in the above-described analysis method. Chondroitin sulfate and its preferred embodiments are the same as in the above-mentioned analysis method.
  • ⁇ Quality control test method for samples containing acidic mucopolysaccharide or salt thereof> In the quality control test method for a product containing an acidic mucopolysaccharide or a salt thereof according to the present invention, a product containing the acidic mucopolysaccharide or a salt thereof is used as a test sample, and the test sample is analyzed by the above-described analysis method of the present invention.
  • a sample analysis step a standard sample containing an acidic mucopolysaccharide or a salt thereof of which concentration and type are known, a standard sample analysis step of analyzing the sample by the above-described analysis method, and a qualitative test result of the test sample obtained by the analysis And evaluating the quality of the test sample by comparing the result of the quantitative test with the result of the qualitative test and the result of the quantitative test of the standard sample.
  • the product containing the acidic mucopolysaccharide or a salt thereof is not particularly limited, and examples thereof include foods, pharmaceuticals, cosmetics, and raw materials containing the acidic mucopolysaccharide or a salt thereof, which are used as test samples in the above-described analysis method.
  • any of the test sample analysis step and the standard sample analysis step may be performed first.
  • the qualitative test and quantitative test, and their preferred embodiments, are the same as those in the above-described analysis method.
  • the acidic mucopolysaccharide or a salt thereof is the same as in the above-described analysis method.
  • the standard sample is a sample containing the acidic mucopolysaccharide or a salt thereof, and the concentration (content) and type of the acidic mucopolysaccharide or the salt thereof are known.
  • the standard sample may be appropriately selected according to the test sample. For example, foods, pharmaceuticals, cosmetics, and the like containing a predetermined amount of a predetermined acidic mucopolysaccharide or a salt thereof, or a raw material thereof may be used as a standard sample.
  • the acidic mucopolysaccharide contained in the test sample is compared.
  • the type of the acidic mucopolysaccharide or a salt thereof contained in the test sample is the same as that of the standard sample, and the amount of the acidic mucopolysaccharide or the salt thereof contained in the test sample is the same as or equal to the standard sample.
  • the number is larger, it can be determined that the product as the test sample satisfies the quality standard.
  • the type of acidic mucopolysaccharide or salt thereof contained in the test sample is determined by comparing qualitative test results because the type of acidic mucopolysaccharide or salt thereof contained in the standard sample is known. Can be confirmed.
  • quality control of a product containing an acidic mucopolysaccharide or a salt thereof it is important to control the type and amount of the acidic mucopolysaccharide or a salt thereof contained in the product.
  • an acidic mucopolysaccharide or a salt thereof contained in a product can be simply and objectively analyzed, and the quality of the product can be evaluated.
  • Such a quality control method is useful in quality control of foods, pharmaceuticals, cosmetics, and raw materials containing the acidic mucopolysaccharide or a salt thereof.
  • Chem) -Shark cartilage raw material (I) (Product name: Shark cartilage extract powder, manufactured by Nippon Pharmaceutical Co., Ltd.) -Shark cartilage raw material (II) (Product name: SCP, manufactured by Maruha Nichiro Co., Ltd.) ⁇ Alcian Blue 8GX (Funakoshi Co., Ltd.) ⁇ Pyridine, acetic acid, barium acetate (all grades, manufactured by Nacalai Tesque, Inc.) ⁇ Actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.) -Chondroitinase ABC (Sigma-Aldrich)
  • the shark cartilage material (I) and the shark cartilage material (II) contain a shark cartilage extract.
  • the shark cartilage material (I) and the shark cartilage material (II) contain 20% by weight of chondroitin sulfate C (reported by the manufacturer).
  • chondroitin sulfate C sodium salt
  • heparan sulfate sodium heparin and dermatan sulfate were used as standards.
  • Actinase E solution (II) Distilled water was added to actinase E to prepare a solution having an actinase E concentration of 1.0 mg / mL. 9) Chondroitinase ABC solution Chondroitinase ABC was dissolved in distilled water and adjusted to 10 U / mL.
  • the membrane was taken out from the electrophoresis running solution (1.0 M acetic acid / pyridine buffer) and lightly dried. 5) Each sample for analysis was spotted on the cellulose acetate membrane in a band shape. 6) After pouring the electrophoresis running solution (1.0 M acetic acid / pyridine buffer) on the cathode side and the anode side of the electrophoresis tank, the cellulose acetate membrane was set on the electrophoresis tank so that the sample spot came on the cathode side. 7) The lid of the electrophoresis tank was closed and allowed to stand for 10 minutes to equilibrate the inside. 8) Electrophoresis was performed at a constant current of 11 mA for 10 minutes. 9) After completion of electrophoresis, the membrane was taken out, air-dried, immersed in an Alcian blue solution for 10 minutes, and stained. 10) The membrane was decolorized with a 1% acetic acid aqueous solution.
  • FIGS. 5 shows the results of membrane electrophoresis of the enzyme-treated product of the acidic mucopolysaccharide preparation prepared by the method (5-4) and the enzyme-treated product of shark cartilage raw material.
  • FIG. 1 is a photograph showing a band pattern of an acidic mucopolysaccharide preparation and a shark cartilage raw material (I) obtained by electrophoresis using a cellulose acetate membrane.
  • FIG. 1 (a) shows the results of electrophoresis of the acidic mucopolysaccharide preparation and the shark cartilage raw material (I), respectively.
  • FIG. 1 (b) shows the results obtained by overlaying the acidic mucopolysaccharide preparation and the shark cartilage raw material (I). This is the result of spotting and electrophoresis.
  • Table 1 shows samples of lanes 1 to 9 in FIG. 1A and lanes 10 to 15 in FIG. 1B.
  • FIG. 7 shows a schematic diagram of the band pattern of the photograph shown in FIG. 1 ((a): a schematic diagram of FIG. 1 (a), (b): a schematic diagram of FIG. 1 (b)).
  • FIG. 2 is a photograph showing a band pattern of an acidic mucopolysaccharide preparation and shark cartilage raw material (II) obtained by electrophoresis using a cellulose acetate membrane.
  • FIG. 2A shows the results of electrophoresis of the acidic mucopolysaccharide preparation and the shark cartilage raw material (II).
  • FIG. 2 (b) shows the result of electrophoresis of an acidic mucopolysaccharide preparation and a shark cartilage raw material (II) which were overlapped and spotted.
  • Table 2 shows samples of lanes 1 to 9 in FIG. 2A and lanes 10 to 15 in FIG. 2B.
  • FIG. 8 shows a schematic diagram of the band pattern of the photograph shown in FIG. 2 ((a): a schematic diagram of FIG. 2 (a), (b): a schematic diagram of FIG. 2 (b)).
  • FIG. 3 is a photograph showing the band patterns of the acidic mucopolysaccharide preparation and the shark cartilage raw material (I) obtained by electrophoresis using the nitrocellulose membrane.
  • FIG. 3 (a) shows the results of electrophoresis of the acidic mucopolysaccharide preparation and the shark cartilage raw material (I), respectively, and
  • FIG. 3 (b) shows the results obtained by overlaying the acidic mucopolysaccharide preparation and the shark cartilage raw material (I). This is the result of spotting and electrophoresis.
  • Table 3 shows samples of lanes 1 to 8 in FIG. 3A and lanes 9 to 14 in FIG. 3B.
  • FIG. 9 shows a schematic diagram of the band pattern of the photograph shown in FIG. 3 ((a): a schematic diagram of FIG. 3 (a), (b): a schematic diagram of FIG. 3 (b)).
  • FIG. 4 is a photograph showing band patterns of an acidic mucopolysaccharide preparation and a shark cartilage raw material (II) obtained by electrophoresis using a nitrocellulose membrane.
  • FIG. 4A shows the results of electrophoresis of the acidic mucopolysaccharide preparation and the shark cartilage raw material (II), respectively.
  • FIG. 4 (b) shows the result of electrophoresis by spotting the acidic mucopolysaccharide preparation and the shark cartilage raw material (II) on top of each other.
  • Table 4 shows samples of lanes 1 to 8 in FIG. 4A and lanes 9 to 14 in FIG. 4B.
  • FIG. 10 shows a schematic diagram of the band pattern of the photograph shown in FIG. 4 ((a): a schematic diagram of FIG. 4 (a), (b): a schematic diagram of FIG. 4 (b)).
  • FIG. 5 shows the results of an enzyme-treated product of shark cartilage raw material and an acidic mucopolysaccharide sample obtained by electrophoresis using a cellulose acetate membrane or a nitrocellulose membrane. It is a photograph which shows the band pattern of an enzyme-treated product ((a): a cellulose acetate membrane, (b): a nitrocellulose membrane). Table 5 shows the samples of lanes 1 to 8 in FIGS. 5 (a) and 5 (b).
  • FIG. 11 shows a schematic diagram of the band pattern of the photograph shown in FIG. 5 ((a): a schematic diagram of FIG. 5 (a), (b): a schematic diagram of FIG. 5 (b)).
  • the band stained with Alcian blue after membrane electrophoresis was the same for all shark cartilage materials (manufacturer and Lot. Difference). It was one.
  • the band observed in the shark cartilage raw material was shifted from the sodium hyaluronate, heparan sulfate, and dermatan sulfate bands. Did not match (lanes 11, 13 and 15 in FIGS. 1 and 2).
  • the band of the shark cartilage raw material matched the chondroitin sulfate sodium of the preparation (lane 12 in FIGS. 1 and 2).
  • the band spread (lane 14 in FIGS. 1 and 2).
  • electrophoresis was performed using a nitrocellulose membrane, only the band of the raw material of shark cartilage and the band of sodium chondroitin sulfate as a standard did not coincide with the bands of other acidic mucopolysaccharides (FIGS. 3 and 4). Lanes 10-14). From this, it was confirmed that the acidic mucopolysaccharide contained in the shark cartilage-derived raw material was only chondroitin sulfate, and other acidic mucopolysaccharide was not contained.
  • the bands derived from the shark cartilage materials (I) and (II) overlapped with the sodium chondroitin sulfate. Further, the band where the shark cartilage raw material was overlapped with sodium heparin had an expanded band.
  • Shark cartilage raw materials (I) and (II), and a sample of sodium chondroitin sulfate and heparin sodium were subjected to enzymatic hydrolysis with chondroitinase ABC by the method described in (5-4), and the membrane electrophoresis was performed as described above. As a result, disappearance of the bands was observed only for the shark cartilage raw materials (I) and (II) and the sample of sodium chondroitin sulfate (FIG. 5).
  • shark cartilage has been reported to contain not only chondroitin sulfate but also hyaluronic acid and dermatan sulfate as acidic mucopolysaccharides (Higashi @ K., @ Et @ al, @ PloS @ One, # 10 (6), @ e0131502 (2015).).
  • heparan sulfate and sodium heparin which are typical acidic mucopolysaccharides, were added to the standard, and membrane electrophoresis of shark cartilage raw materials (I) and (II) was performed.
  • the acidic mucopolysaccharide contained had a property to be decomposed by chondroitinase ABC. Since there is only one band stained with Alcian Blue and the migration distance matches that of chondroitin sulfate, the acidic mucopolysaccharide contained in this material is only chondroitin sulfate, and other acidic mucopolysaccharides are included. Not confirmed. Furthermore, the above band has the property of being decomposed by chondroitinase ABC, which confirms that the acidic mucopolysaccharide contained in the present raw material is only chondroitin sulfate and does not contain other acidic mucopolysaccharide.
  • quantification by a colorimetric method is carried out by utilizing the characteristics of a functional group contained in a compound, so that specificity often becomes a problem.
  • the band stained with the Alcian Blue reagent in the shark cartilage raw material evaluated this time is only chondroitin sulfate
  • the quantitative results obtained by the Alcian Blue colorimetric determination for the raw material indicate the content of chondroitin sulfate or its salt. It can be said that it reflects.
  • Example 2 For the shark cartilage raw materials (I) and (II) used in Example 1, the content of chondroitin sulfate or a salt thereof was quantified by Alcian blue colorimetry. The quantification by the Alcian blue colorimetric method is carried out according to the method described in a food hygiene magazine (Yabe Yoshie et al., Analysis of sodium chondroitin sulfate added to food, 28 (1), p13-18 (1987)). Was.
  • test solution 1 Actinase E solution A phosphate buffer was added to 0.5 g of actinase E (Kaken Pharmaceutical Co., Ltd.) to make 50 mL. 2) Alcian blue reagent solution To 1 g of Alcian blue 8GX, 4.2 mL of hydrochloric acid, 1.38 g of sodium dihydrogen phosphate and water were added to prepare 100 mL. 3) 305 mL of a 0.2 mol / L disodium phosphate solution of a phosphate buffer and 195 mL of a 0.2 mol / L monosodium phosphate solution were mixed, and water was added to make 1000 mL. The pH of this solution was 7.
  • test Solution To 2 g of the pulverized sample, 40 mL of a pH 7.0 phosphate buffer was added. Further, 5 mL of an actinase E solution (10 mg / mL) was added and a shaking operation was performed. Then, after standing at 40 ° C. overnight, the volume was adjusted to 100 mL with water. The solution filtered with filter paper was used as a test solution.
  • the amount of chondroitin sulfate or a salt thereof in the test solution was calculated.
  • the content of chondroitin sulfate or a salt thereof determined here is an amount as sodium chondroitin sulfate.
  • Example 2 The qualitative test (Example 1) confirmed that the acidic mucopolysaccharide or its salt contained in the shark cartilage raw materials (I) and (II) was chondroitin sulfate or its salt alone.
  • the quantitative result obtained in Example 2) is the content of chondroitin sulfate or its salt in the shark cartilage raw materials (I) and (II).
  • Example 1 The following test was performed to confirm that chondroitin sulfate or a salt thereof was not decomposed by actinase E. Reagents and raw materials such as chondroitin sulfate C sodium salt and shark cartilage raw materials (I) and (II) are the same as those in Example 1.
  • Example 1 Except for the above, the method described in (5-4-2) of Example 1 was used to obtain a sample of the shark cartilage raw material (I) without the enzyme treatment and a sample of the shark cartilage raw material (II) without the enzyme treatment (actinase E treatment and chondrolysis). (A sample not treated with itinase ABC).
  • FIG. 6 is a photograph showing a band pattern obtained by electrophoresing a raw material of shark cartilage and a sample of chondroitin sulfate C sodium without enzyme treatment, a sample treated with actinase, and a sample treated with enzyme with a cellulose acetate membrane.
  • Table 6 shows the samples in lanes 1 to 9 in FIG. In the column of actinase treatment, "+” is shown in the column of actinase treatment, and “-” is shown in the column of actinase treatment. In the column of chondroitinase treatment, “+” is shown in the column of chondroitinase ABC treatment, and “-” is shown in the column of chondroitinase treatment.
  • the sample amount spotted on the membrane was 5.0 ⁇ g in lanes 1 to 6, and 0.8 ⁇ g in lanes 7 to 9.
  • FIG. 12 shows a schematic diagram of the band pattern of the photograph shown in FIG.
  • Lanes 1, 4 and 7 in FIG. 6 are samples without enzyme treatment. Lanes 2, 5 and 8 are actinase treated samples. In FIG. 6, in lanes 1, 2, 4, 5, and 8, a band was detected at the same position as in lane 7 (a sample of chondroitin sulfate C sodium). Lanes 3, 6, and 9 are enzyme-treated samples treated with actinase E and chondroitinase ABC. In lanes 3, 6 and 9, the band derived from chondroitin sulfate C sodium disappeared. From these results, it was confirmed that chondroitin sulfate or a salt thereof was degraded by chondroitinase ABC but not by actinase E.

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Abstract

La présente invention concerne un procédé d'analyse d'un mucopolysaccharide acide ou d'un sel de celui-ci contenu dans un échantillon de test, le procédé comportant l'étape consistant à soumettre l'échantillon de test contenant un mucopolysaccharide acide ou un sel de celui-ci à un test qualitatif et à un test quantitatif. Le test qualitatif comprend : une étape d'électrophorèse servant à soumettre l'échantillon de test à une électrophorèse en utilisant une membrane ; une étape de coloration servant à colorer la membrane avec du bleu Alcian après l'électrophorèse ; et une étape de détection servant à confirmer le type de mucopolysaccharide acide ou du sel de celui-ci dans l'échantillon de test à partir d'un motif de bande obtenu par la coloration. Le test quantitatif comprend une étape de détermination colorimétrique servant à déterminer de manière quantitative le mucopolysaccharide acide ou le sel de celui-ci dans l'échantillon de test par colorimétrie en utilisant un colorant en mesure de détecter le mucopolysaccharide acide ou le sel de celui-ci.
PCT/JP2019/024482 2018-06-29 2019-06-20 Procédé d'analyse de mucopolysaccharide acide ou d'un sel de celui-ci contenu dans un échantillon de test, procédé de détermination quantitative de sulfate de chondroïtine ou d'un sel de celui-ci, et procédé de test de contrôle de qualité de produit WO2020004216A1 (fr)

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JP2020527457A JP7384793B2 (ja) 2018-06-29 2019-06-20 被検試料に含まれる酸性ムコ多糖又はその塩の分析方法、コンドロイチン硫酸又はその塩の定量方法及び製品の品質管理試験方法

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03191855A (ja) * 1989-12-21 1991-08-21 Olympus Optical Co Ltd 電気泳動装置
JP2005016989A (ja) * 2003-06-23 2005-01-20 Taisho Pharmaceut Co Ltd コンドロイチン硫酸又はその塩の分析方法
JP2005024336A (ja) * 2003-06-30 2005-01-27 Japan Science & Technology Corp 酸性多糖類の分析方法および酸性多糖類分析用キット
JP2018083810A (ja) * 2008-11-25 2018-05-31 ティシュージーン，インク． プライミング細胞療法

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JP4696236B2 (ja) * 2005-02-16 2011-06-08 国立大学法人広島大学 コンドロイチンの製造方法
EP2907531A1 (fr) * 2008-07-30 2015-08-19 Mesynthes Limited Méthode de séparation ou de décellularisation de couches de tissu

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH03191855A (ja) * 1989-12-21 1991-08-21 Olympus Optical Co Ltd 電気泳動装置
JP2005016989A (ja) * 2003-06-23 2005-01-20 Taisho Pharmaceut Co Ltd コンドロイチン硫酸又はその塩の分析方法
JP2005024336A (ja) * 2003-06-30 2005-01-27 Japan Science & Technology Corp 酸性多糖類の分析方法および酸性多糖類分析用キット
JP2018083810A (ja) * 2008-11-25 2018-05-31 ティシュージーン，インク． プライミング細胞療法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
VOLPI, NICOLA: "Electrophoresis Separation of Glycosaminoglycans on Nitrocellulose Membranes", ANALYTICAL BIOCHEMISTRY, vol. 240, no. 0337, 1996, pages 114 - 118 *

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