JP7384793B2 - Method for analyzing acidic mucopolysaccharide or its salt contained in a test sample, method for quantifying chondroitin sulfate or its salt, and product quality control test method - Google Patents

Description

The present invention relates to a method for analyzing acidic mucopolysaccharide or its salt contained in a test sample. The present invention also relates to a method for quantifying chondroitin sulfate or its salt contained in a test sample. The present invention also relates to a quality control testing method for products containing acidic mucopolysaccharides or salts thereof.

Acidic mucopolysaccharides are mucopolysaccharides that have acidic groups such as carboxylic acids in their structure. Acidic mucopolysaccharides are classified into chondroitin sulfate, hyaluronic acid, dermatan sulfate, etc., depending on the type and combination of uronic acid (or galactose) and amino sugars that constitute the disaccharide unit. Since acidic mucopolysaccharide exhibits physiological activity in vivo, its qualitative and quantitative determination is useful in predicting or determining the expected effects of health foods and pharmaceuticals, and in product quality control.

Chondroitin sulfate is an acidic mucopolysaccharide that has a repeating structure of disaccharide units in which D-glucuronic acid is bonded to N-acetyl-D-galactosamine, and some of the hydroxyl groups of N-acetyl-D-galactosamine are sulfated. It is.
As a method for quantifying chondroitin sulfate, 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. Methods for quantifying the amount of chondroitin sulfate other than HPLC include the barium sulfate gravimetric method to detect compounds with sulfate groups, the carbazole sulfate method to detect glucuronic acid and desulfurized products, and the Alcian blue colorimetric method to detect acidic mucopolysaccharides. There are several analysis methods.

Membrane electrophoresis is being considered as a method for separating multiple types of acidic mucopolysaccharides. Non-Patent Document 1 states that a sample containing acidic mucopolysaccharides such as chondroitin sulfate was electrophoresed on a nitrocellulose membrane Immobilon (product name, Millipore), and the acidic mucopolysaccharides were separated and identified by Alcian blue staining. It is stated that. The membrane used in this non-patent document 1 is a mixed membrane of nitrocellulose and cellulose acetate.

Volpi N. , Anal. Biochem. , 240, 114-118 (1996).

When formulating acidic mucopolysaccharide as an active ingredient in health foods or medicines, it is important to identify the type and amount of acidic mucopolysaccharide contained in the product. However, acidic mucopolysaccharides are one of the components that are difficult to analyze because their constituent units have similar structures and often have similar specificities. As mentioned above, the HPLC method is known as a method for quantifying chondroitin sulfate, but when analyzing products using the HPLC method, excipients and other raw materials (such as polyphenols that inhibit enzymes) that are blended into the product must be analyzed. The problem was that it was easily influenced by In addition, the HPLC method requires expensive and large HPLC equipment, and the hydrolase used to decompose chondroitin sulfate and standard products for identifying chondroitin sulfate are also expensive, and depending on the origin, suitable standard products may not be available. It may not be on sale. Therefore, there is a need for a method that can easily analyze acidic mucopolysaccharides or their salts at a lower cost without using such large-scale equipment. As mentioned above, quantitative methods other than the HPLC method are also known, but the barium sulfate gravimetric method has a problem in terms of specificity because sulfate groups other than acidic mucopolysaccharides are also determined. Even in quantitative determination using the carbazole sulfate method and Alcian blue colorimetric method, it is not possible to confirm the type of acidic mucopolysaccharide contained in the sample. In Non-Patent Document 1, multiple types of acidic mucopolysaccharides are separated by electrophoresis and the band density on the membrane is measured using a densitometer. It is considered difficult to perform this accurately.

An object of the present invention is to provide an analysis method that can confirm and quantify the type of acidic mucopolysaccharide or its salt contained in a sample containing acidic mucopolysaccharide or its salt through simple steps.

The present inventors have conducted extensive research to solve the above problems, and have developed colorimetric methods (colorimetric assays) such as the Alcian blue colorimetric method, which is one of the methods for quantifying acidic mucopolysaccharides. We focused on Then, the test sample containing the acidic mucopolysaccharide or its salt is subjected to membrane electrophoresis and developed, and then stained with Alcian blue. From the band pattern obtained, the acidic mucopolysaccharide or its salt contained in the sample is detected. By combining a qualitative test to confirm the type and a quantitative test to quantify the acidic mucopolysaccharide or its salt in the test sample using a colorimetric method, the total amount and quality of the acidic mucopolysaccharide or its salt contained in the test sample ( We have found that it is possible to evaluate the following types using a simple process. In this way, an analysis method that combines a qualitative test and a quantitative test can also be used to quantify the acidic mucopolysaccharide or its salt when the test sample contains only one type of acidic mucopolysaccharide or its salt. Thus, a method for analyzing acidic mucopolysaccharide or its salt contained in a test sample by combining a qualitative test and a quantitative test using a colorimetric method has not been reported so far.

That is, the present invention relates to the following analysis method.
[1] 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 includes an electrophoresis step in which the test sample is subjected to electrophoresis using a membrane; A staining step of staining the membrane after electrophoresis with Alcian blue, and a detection step of confirming the type of acidic mucopolysaccharide or its salt in the test sample from the band pattern obtained by the staining, The quantitative test includes a colorimetric determination step of quantifying the acidic mucopolysaccharide or its salt in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or its salt. A method for analyzing acidic mucopolysaccharides or their salts.
[2] The analysis method according to [1] above, wherein the acidic mucopolysaccharide is one or more selected from the group consisting of chondroitin sulfate, hyaluronic acid, dermatan sulfate, heparin, and heparan sulfate.
[3] The analysis method according to [1] or [2] above, wherein the acidic mucopolysaccharide is chondroitin sulfate.
[4] The analytical method according to any one of [1] to [3] above, wherein the dye capable of detecting the acidic mucopolysaccharide or its salt is Alcian blue or carbazole.
[5] The analytical method according to any one of [1] to [4] above, wherein the dye capable of detecting the acidic mucopolysaccharide or its salt is Alcian blue.
[6] If the above qualitative test confirms that the acidic mucopolysaccharide or its salt contained in the above test sample is one type, the quantitative results obtained from the above quantitative test are used as the acidic mucopolysaccharide or its salt. The analytical method according to any one of [1] to [5] above, wherein the content of salt is determined.
[7] The analytical method according to any one of [1] to [6] above, wherein the membrane is a nitrocellulose membrane, a cellulose acetate membrane, or a mixed membrane of nitrocellulose and cellulose acetate.
[8] The analytical method according to any one of [1] to [7] above, wherein the membrane is a nitrocellulose membrane or a cellulose acetate membrane.
[9] The test sample containing chondroitin sulfate or its salt is subjected to a qualitative test and a quantitative test, and the qualitative test includes an electrophoresis process in which the test sample is subjected to electrophoresis using a membrane, The method includes a staining step of staining the membrane after electrophoresis with Alcian blue, and a detection step of confirming the type of acidic mucopolysaccharide or its salt in the test sample from the band pattern obtained by the staining. The test includes a colorimetric determination step of quantifying the acidic mucopolysaccharide or its salt in the test sample by a colorimetric method using a dye that can detect the acidic mucopolysaccharide or its salt, and the qualitative test When it is confirmed that the acidic mucopolysaccharide or its salt contained in the test sample is only chondroitin sulfate or its salt, the quantitative result obtained by the above quantitative test is taken as the content of the chondroitin sulfate or its salt. A method for quantifying chondroitin sulfate or its salts contained in a test sample.
[10] A quality control test method for a product containing acidic mucopolysaccharide or its salt, comprising:
A test sample analysis step in which the above product is used as a test sample and is analyzed by the analysis method described in any one of [1] to [8] above, a standard sample containing an acidic mucopolysaccharide or its salt of known concentration and type is prepared. , a standard sample analysis step of analyzing by the above analysis method, and comparing the qualitative test results and quantitative test results of the test sample obtained in the above analysis with the qualitative test results and quantitative test results of the standard sample, and the above product. A quality control test method for products containing acidic mucopolysaccharides or their salts, including an evaluation step to evaluate the quality of

According to the present invention, an analysis method is provided that allows confirmation and quantification of the type of acidic mucopolysaccharide or its salt contained in a sample containing acidic mucopolysaccharide or its salt through simple steps. The analytical method of the present invention is useful in the analysis of acidic mucopolysaccharides or salts thereof contained in foods, medicines, etc., and in the quality control of products containing acidic mucopolysaccharides or salts thereof.

FIG. 1 is a photograph showing band patterns of an acidic mucopolysaccharide sample and shark cartilage raw material (I) obtained by electrophoresis using a cellulose acetate membrane. FIG. 2 is a photograph showing band patterns of an acidic mucopolysaccharide sample and shark cartilage raw material (II) obtained by electrophoresis using a cellulose acetate membrane. FIG. 3 is a photograph showing the band pattern of the acidic mucopolysaccharide sample and shark cartilage raw material (I) obtained by electrophoresis using a nitrocellulose membrane. FIG. 4 is a photograph showing the band patterns of the acidic mucopolysaccharide sample and shark cartilage raw material (II) obtained by electrophoresis using a nitrocellulose membrane. FIG. 5 is a photograph showing the band patterns of an enzyme-treated shark cartilage raw material and an enzyme-treated acidic mucopolysaccharide sample obtained by electrophoresis using a cellulose acetate membrane or a nitrocellulose membrane ((a) : cellulose acetate membrane, (b): nitrocellulose membrane). FIG. 6 is a photograph showing band patterns of shark cartilage raw material and sodium chondroitin sulfate without enzyme treatment, actinase treatment sample, and actinase E and chondroitinase ABC treatment sample electrophoresed on a cellulose acetate membrane. FIG. 7 is a schematic diagram of the band pattern of the photograph shown in FIG. 1 ((a): schematic diagram of FIG. 1(a), (b): schematic diagram of FIG. 1(b)). FIG. 8 is a schematic diagram of the band pattern of the photograph shown in FIG. 2 ((a): schematic diagram of FIG. 2(a), (b): schematic diagram of FIG. 2(b)). FIG. 9 is a schematic diagram of the band pattern of the photograph shown in FIG. 3 ((a): schematic diagram of FIG. 3(a), (b): schematic diagram of FIG. 3(b)). FIG. 10 is a schematic diagram of the band pattern of the photograph shown in FIG. 4 ((a): schematic diagram of FIG. 4(a), (b): schematic diagram of FIG. 4(b)). FIG. 11 is a schematic diagram of the band pattern of the photograph shown in FIG. 5 ((a): schematic diagram of FIG. 5(a), (b): schematic diagram of FIG. 5(b)). FIG. 12 is a schematic diagram of the band pattern of the photograph shown in FIG. 6.

<Analysis method of acidic mucopolysaccharide or its salt>
The analysis method of the present invention is a method for analyzing acidic mucopolysaccharide or a salt thereof contained in a test sample, which includes subjecting the test sample containing acidic mucopolysaccharide or a salt thereof to a qualitative test and a quantitative test. In the present invention, the qualitative test includes an electrophoresis step in which the test sample is subjected to electrophoresis using a membrane, a staining step in which the membrane after electrophoresis is stained with Alcian blue, and a band obtained by the staining. It includes a detection step of confirming the type of acidic mucopolysaccharide or its salt in the test sample from the pattern. The quantitative test includes a colorimetric determination step of quantifying the acidic mucopolysaccharide or its salt in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or its salt.
In this specification, a dye capable of detecting an acidic mucopolysaccharide or a salt thereof is also simply referred to as a dye.

Acidic mucopolysaccharides have a repeating structure of disaccharide units in which uronic acid (iduronic acid or glucuronic acid) or galactose are bonded to amino sugars, and acidic groups (carboxyl groups, sulfate groups) in the structure. It is. Acidic mucopolysaccharides usually have an unbranched linear skeleton. Examples of 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 sample as long as it contains one or more acidic mucopolysaccharides or salts thereof. The salt is not particularly limited, and examples thereof include alkali metal salts such as sodium and potassium; alkaline earth metal salts such as calcium and magnesium. Alkali metal salts are preferred, and sodium salts are more preferred.

In the present invention, the acidic mucopolysaccharide is preferably one or more 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 test samples containing one or more of these acidic mucopolysaccharides or salts thereof. Among these, as the acidic mucopolysaccharide, chondroitin sulfate or hyaluronic acid is more preferred, and chondroitin sulfate is even more preferred. The analysis method of the present invention is preferably used for analyzing test samples containing chondroitin sulfate or its salts. In the present invention, chondroitin sulfate refers to chondroitin sulfate A, chondroitin sulfate C, chondroitin sulfate D, and chondroitin sulfate E, and may be one type or two or more types thereof. Dermatan sulfate (chondroitin sulfate B) is not included in chondroitin sulfate in the present invention. As chondroitin sulfate, chondroitin sulfate C is preferred. In the present invention, chondroitin sulfate C includes chondroitin sulfate C as a main component, and also includes one or more other chondroitin sulfates.
Examples of test samples containing acidic mucopolysaccharides or salts thereof include foods (including health foods), pharmaceuticals, cosmetics, and raw materials thereof (e.g., shark cartilage extract, pig cartilage extract, squid cartilage extract, salmon cartilage extract, etc.) cartilage extract, etc.).

In the analysis method of the present invention, by combining the above qualitative test and quantitative test, the type of acidic mucopolysaccharide or its salt in the test sample can be confirmed and quantified in a simple process. In this specification, the type of acidic mucopolysaccharide or its salt refers to the type of acidic mucopolysaccharide contained in the acidic mucopolysaccharide or its salt.
The analysis method of the present invention includes, for example, confirmation that a test sample contains one or more acidic mucopolysaccharides or salts thereof, and quantification of acidic mucopolysaccharides or salts thereof in the test sample. It can be performed. When the acidic mucopolysaccharide or its salt is one type (or two or more types), it means that the acidic mucopolysaccharide or its salt contains one type (or two or more types) of the acidic mucopolysaccharide. For example, if the test sample contains one type of acidic mucopolysaccharide or its salt, confirmation (identification) of the type of acidic mucopolysaccharide or its salt, and the acidic mucopolysaccharide in the test sample. Alternatively, the salt thereof can be quantified. When the test sample contains two or more types of acidic mucopolysaccharides or salts thereof, the analysis method of the present invention is capable of confirming the types of acidic mucopolysaccharides or salts thereof contained in the test sample, and confirming their It can be used to quantify the total amount.

In the present invention, the order in which qualitative tests and quantitative tests are performed is not particularly limited. After performing a qualitative test to confirm the type of acidic mucopolysaccharide or its salt contained in the test sample, a quantitative test may be performed. After that, 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 conduct a quantitative test after performing a qualitative test.

In the above qualitative test, the type of acidic mucopolysaccharide or its salt contained in the test sample can be confirmed by performing the electrophoresis step, the staining step, and the detection step in this order.

In the electrophoresis step, the test sample is subjected to electrophoresis using a membrane (membrane electrophoresis). As the membrane, a porous membrane that can be used for electrophoresis can be used. Examples of such membranes include nitrocellulose membranes, cellulose acetate membranes, and mixed membranes of nitrocellulose and cellulose acetate. In one embodiment, the membrane is preferably a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate. When these membranes are used, in the band pattern obtained by electrophoresis, the band shape of the acidic mucopolysaccharide or its salt is sharp, and separation becomes good. Furthermore, when a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate is used, chondroitin sulfate or its salts can be particularly well separated from other acidic mucopolysaccharides or their salts. Therefore, electrophoresis using a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate is advantageous in detecting chondroitin sulfate or its salt. If the test sample contains chondroitin sulfate or its salts, or if you want to confirm the presence of chondroitin sulfate or its salts in the test sample, use a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate. It is preferable.
In another embodiment, it is preferable to use a cellulose acetate membrane. When a cellulose acetate membrane is used, hyaluronic acid or a salt thereof can be well separated from other acidic mucopolysaccharides or salts thereof. For example, when the test sample contains hyaluronic acid or its salt, or when confirming the presence of hyaluronic acid or its salt in the test sample, it is preferable to use a cellulose acetate membrane. Furthermore, when a cellulose acetate membrane is used, chondroitin sulfate or its salt can be well separated from other acidic mucopolysaccharides or its salts. When using a cellulose acetate membrane to detect chondroitin sulfate or its salts, for example, the test sample is subjected to chondroitinase ABC treatment as described below, and the band pattern of the resulting enzyme-treated product and the chondroitinase ABC treatment are It is also preferable to compare the band pattern of a test sample that has not been subjected to this process.

Although the pore size of the membrane is not particularly limited, for example, a membrane with a pore size of 0.1 to 0.6 μm can be used. If the membrane has a hydrophobic coating, it is preferred 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 to prepare the test sample solution is not particularly limited, but water, electrophoresis liquid, etc. can be used. The conditions for electrophoresis are not particularly limited, and general methods can be used. It is preferable that the electrophoresis liquid and energization conditions used for electrophoresis are appropriately set in consideration of the resolution of the obtained band pattern and the like. Electrophoresis may be performed by a constant current method or a constant voltage method, preferably by a constant current method. The current during electrophoresis can be, for example, 5 to 100 mA. The electrophoresis time can be appropriately set depending on the magnitude of the current and the like.

From the viewpoint of separating acidic mucopolysaccharides or salts thereof, a buffer solution having a pH of 3 to 7 is preferably used as the electrophoresis solution. Examples of buffers that can be used as the electrophoresis solution include acetic acid buffers such as barium acetate buffer and acetic acid-pyridine buffer. When using a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate, a barium acetate buffer is preferable because it allows good separation of acidic mucopolysaccharides or their salts, and when using a cellulose acetate membrane, Acetic acid-pyridine buffer is preferred. pH can be measured with a commercially available pH meter.

In one aspect, electrophoresis is preferably performed at a temperature of less than 10°C, more preferably 0 to 7°C, and even more preferably 0 to 5°C, from the viewpoint of more stable migration patterns. During electrophoresis, it is preferable to perform electrophoresis with the electrophoresis tank at the above temperature.

The membrane after the above electrophoresis is stained with Alcian blue (staining step). Since Alcian Blue binds to acidic mucopolysaccharides or salts thereof, it is possible to detect acidic mucopolysaccharides or salts thereof contained in a test sample with high sensitivity. Staining using Alcian blue also has the advantage of being easy to operate. In addition, when Alcian blue is used to detect acidic mucopolysaccharides or its salts in qualitative tests, and quantitative tests are performed using Alcian blue colorimetric method (Alcian blue colorimetric method) in the quantitative tests described later, the quantitative results can be obtained. The results reflect the content of acidic mucopolysaccharide or its salt confirmed in the qualitative test.

Staining can be performed by immersing the membrane in an Alcian blue solution for a predetermined period of time, for example, 5 to 60 minutes.
After staining, the membrane is decolorized using a decolorizing solution. A normal decolorizing liquid can be used as the decolorizing liquid, and an acidic aqueous solution is preferable. Examples of the acidic aqueous solution include acetic acid aqueous solution and formic acid aqueous solution. The membrane after decolorization shows a band pattern (which can also be called an electrophoretic pattern) consisting of one or more bands whose mobility (which can also be called a migration distance) differs depending on the acidic mucopolysaccharide or its salt. . If the test sample does not contain acidic mucopolysaccharide or its salt, no band is usually detected.

In the detection step, the type of acidic mucopolysaccharide or its salt in the test sample is confirmed from the band pattern obtained by the staining. The band pattern can be visually confirmed. To confirm the type of acidic mucopolysaccharide or its salt, it is necessary to specify (identify) the type of acidic mucopolysaccharide or its salt contained in the test sample, and to confirm that the test sample contains only one type of acidic mucopolysaccharide or its salt. or two or more types. In one aspect, whether the test sample contains one type of acidic mucopolysaccharide or its salt or two or more types can be confirmed by the number of bands.
In qualitative tests, in addition to the test sample, a standard sample of acidic mucopolysaccharide or its salt (standard substance), or a standard sample in which the type of acidic mucopolysaccharide or its salt contained is known, is used. Preferably, a band pattern is obtained. The type of acidic mucopolysaccharide or its salt can be confirmed, for example, by comparison with the migration distance of a band of a sample of acidic mucopolysaccharide or its salt. In another embodiment, a qualitative test is performed using the same method on a standard sample in which the type of acidic mucopolysaccharide or its salt contained is known, and the band pattern of the standard sample and the band pattern of the test sample are compared. Accordingly, the type of acidic mucopolysaccharide or its salt in the test sample can also be confirmed. In one embodiment, when the origin of the acidic mucopolysaccharide or its salt in the test sample is predicted, a specimen of the acidic mucopolysaccharide or its salt having the same origin is used as the standard, or a specimen having the same origin is used as the standard. It is preferable to use a standard sample containing acidic mucopolysaccharide or a salt thereof. For example, if the acidic mucopolysaccharide or its salt contained in the test sample is expected to be derived from shark cartilage, use a specimen of the acidic mucopolysaccharide or its salt derived from shark cartilage, or It is preferable to use a standard sample containing an acidic mucopolysaccharide or a salt thereof.
In addition to the test sample, 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, acidic mucopolysaccharide or You may want to check the type of salt.

In one embodiment, the analysis method of the present invention is suitable for analysis of chondroitin sulfate or its salt derived from shark cartilage. When the membrane electrophoresis described above is performed, acidic mucopolysaccharides other than chondroitin sulfate or salts thereof and shark cartilage-derived chondroitin sulfate or salts thereof can be well separated. Therefore, qualitative tests are advantageous for detecting chondroitin sulfate or its salts derived from shark cartilage. The analysis method of the present invention is particularly preferably used for analyzing shark cartilage-derived chondroitin sulfate or its salt contained in a test sample. In electrophoresis, when a nitrocellulose membrane or a mixed membrane of nitrocellulose and cellulose acetate is used, acidic mucopolysaccharides or salts thereof other than chondroitin sulfate can be separated particularly well from shark cartilage-derived chondroitin sulfate or its salts. .

The qualitative test may include steps other than the electrophoresis step, staining step, and detection step described above. In one embodiment, a test sample is treated with an enzyme that decomposes acidic mucopolysaccharide or its salt contained in the test sample (hereinafter also simply referred to as acidic mucopolysaccharide degrading enzyme) to produce an enzyme-treated product (also referred to as enzyme-treated sample). You may also perform a step of preparing a In this case, the test sample is treated with acidic mucopolysaccharide-degrading enzyme, and the resulting enzyme-treated product is also subjected to an electrophoresis process and a staining process to obtain a band pattern. It is preferable to compare the band patterns of the test sample) and the enzyme-treated product. In the present invention, the test sample (not subjected to enzyme treatment) is determined by comparing the migration distance or band pattern of the band obtained by electrophoresis with the specimen or standard sample as described above. The type of acidic mucopolysaccharide or its salt contained can be confirmed, but it is also possible to compare the electrophoretic band patterns of the test sample (not enzyme-treated) and its acidic mucopolysaccharide-degrading enzyme-treated product. Accordingly, the type of acidic mucopolysaccharide or its salt in the test sample can be specified more reliably. The acidic mucopolysaccharide degrading enzyme may be selected depending on the type of acidic mucopolysaccharide or its salt contained in the test sample. For example, in the case of chondroitin sulfate or its salt, chondroitinase ABC can be used. If the band of a specific acidic mucopolysaccharide or its salt detected in the test sample disappears (is not detected) in the enzyme-treated product treated with an enzyme that decomposes the acidic mucopolysaccharide or its salt, then It is confirmed that the acidic mucopolysaccharide or its salt is contained.

In one embodiment, when the test sample contains chondroitin sulfate or its salt, or when confirming the presence of chondroitin sulfate or its salt in the test sample, the test sample is treated (decomposed) with chondroitinase ABC. ) to prepare an enzyme-treated product. In addition to the test sample, a test sample containing acidic mucopolysaccharide or its salt is treated with chondroitinase ABC, and the resulting enzyme-treated product is subjected to an electrophoresis process and a staining process to obtain a band pattern. By comparing the band patterns of the test sample (test sample that has not been enzyme-treated) and the enzyme-treated product, it is possible to more accurately confirm whether the test sample contains chondroitin sulfate or its salts. . For example, if the band of acidic mucopolysaccharide or its salt detected in the test sample disappears after being enzyme-treated with chondroitinase ABC, it is confirmed that the test sample contains chondroitin sulfate or its salt. . Comparison of the band patterns of the test sample and the enzyme-treated product can be performed in combination with the above-mentioned comparison with the movement distance of the band of the standard sample or the comparison with the band pattern of the standard sample. In one embodiment, when analyzing a test sample containing chondroitin sulfate or a salt thereof, or when confirming the presence of chondroitin sulfate or a salt thereof, 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.

In one aspect, the analysis method of the present invention comprises performing an electrophoresis step on a test sample (a test sample not treated with an acid mucopolysaccharide-degrading enzyme), an enzyme-treated product of the test sample, and a specimen (or a standard sample). A staining process is performed to obtain a band pattern, and the band patterns of the test sample and standard sample (or standard sample) are compared, and the band patterns of the test sample and the enzyme-treated product are compared. It is preferable to confirm the type of acidic mucopolysaccharide or its salt contained in the test sample. In this case, for example, first the type of acidic mucopolysaccharide or its salt contained in the test sample is confirmed by comparing the band patterns of the test sample and standard, and then the test sample is treated with acidic mucopolysaccharide-degrading enzyme. It is preferable to prepare an enzyme-treated product by processing, obtain a band pattern of the enzyme-treated product, and compare it with the band pattern of the test sample. As the acidic mucopolysaccharide-degrading enzyme, an enzyme capable of degrading the acidic mucopolysaccharide or its salt may be used depending on the type of the acidic mucopolysaccharide or its salt confirmed by comparison with the standard. If the band of acidic mucopolysaccharide or its salt detected in the test sample is not detected in the enzyme-treated product, it is confirmed that the type of acidic mucopolysaccharide or its salt is the type confirmed by comparison with the standard sample. It is confirmed.

The analysis method of the present invention may include a protein removal treatment step. For example, when treating a test sample with acidic mucopolysaccharide-degrading enzyme, protein removal treatment may be performed before the treatment. When the protein removal treatment is performed, the decomposition of the acidic mucopolysaccharide or its salt by the acidic mucopolysaccharide degrading enzyme proceeds more rapidly. The protein removal treatment is not particularly limited as long as it does not impair the effects of the present invention, and includes a method of treating the test sample with protease; a method of changing the pH of the test sample (for example, increasing the pH to 12 or more); an organic solvent. Examples include a method in which the protein is removed as a precipitate by adding . Among these, the method of treatment with protease is preferred because it is easy to operate and the subsequent treatment with enzymes such as chondroitinase ABC is simple. As the protease, one that does not decompose acidic mucopolysaccharide or its salt can be used, and actinase E is preferable.

When performing the chondroitinase ABC treatment, the test sample is preferably treated with actinase E before the chondroitinase ABC treatment. It is preferable to decompose the protein in the test sample with actinase E and then treat it with chondroitinase ABC because the decomposition of chondroitin sulfate or its salt by the enzyme occurs more quickly.

Enzyme treatments such as acidic mucopolysaccharide-degrading enzymes such as chondroitinase ABC and actinase E can be carried out by general methods, and may be selected depending on the type of enzyme. For example, the pH during enzyme treatment with actinase E, chondroitinase ABC, etc. 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 carried out for, for example, 0.5 to 2 hours. When enzyme treatment is performed, the enzyme may be inactivated if 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 determination step of quantifying the acidic mucopolysaccharide or its salt in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or its salt.
In the 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 acidic mucopolysaccharide or its salt is determined from a calibration curve prepared using a standard sample. The content of can be determined. In quantitative determination by colorimetry, the content of acidic mucopolysaccharide or its salt in the test sample is usually determined as the content of acidic mucopolysaccharide or its salt used in the standard sample. The content of the acidic mucopolysaccharide or its salt obtained in this way can be used as a quantitative result obtained by a quantitative test. The standard product may be selected depending on the acidic mucopolysaccharide or its salt.
As the dye used in the quantitative test, a dye (reagent) that can be used to detect acidic mucopolysaccharide or its salt can be used. A dye that binds to an acidic mucopolysaccharide or a salt thereof is preferred, and a dye that binds to an acidic mucopolysaccharide or a salt thereof and is optically detectable is more preferred. From the viewpoint of accuracy of analysis, etc., as the dye, Alcian blue and carbazole are preferred, and Alcian blue is more preferred.

When Alcian blue is used as the dye, the acidic mucopolysaccharide or its salt is quantified by the Alcian blue colorimetric method. The Alcian Blue colorimetric method can be performed in a conventional manner. In the Alcian Blue colorimetric method, when a solution of Alcian Blue is added to a test sample, Alcian Blue and acidic mucopolysaccharide or its salt form a complex and precipitate. This precipitate is redissolved in an alkaline solution, and the absorbance of the resulting solution is measured using a spectrophotometer. The measurement wavelength is preferably 570 to 660 nm, more preferably 590 to 640 nm. From this absorbance, the amount of acidic mucopolysaccharide or its salt can be calculated based on a calibration curve prepared in advance using standard samples. When Alcian Blue is used, it is possible to quantify the acidic mucopolysaccharide or its salt in the test sample, for example, by the method described in the Examples. The Alcian blue colorimetric method has the advantages of easy operation and high reproducibility as a method for quantifying acidic mucopolysaccharides.
When carbazole is used as the dye, the amount of acidic mucopolysaccharide or its salt can be determined by the carbazole sulfate method. The carbazole sulfate method can be carried out by a conventional method. When a carbazole solution and concentrated sulfuric acid are added to a test sample, if the test sample contains uronic acid, the sample turns reddish-purple. The absorbance of the colored solution is measured with a spectrophotometer, the uronic acid content is calculated from the standard curve, and the amount of chondroitin sulfate or its salt is determined. The measurement wavelength is preferably 500 to 600 nm, more preferably 520 to 550 nm. In a quantitative test, a test sample may be treated with protease or the like, if necessary. Proteases that do not decompose acidic mucopolysaccharides or salts thereof can be used, and the above-mentioned actinase E and the like are preferred.

In the present invention, it is more preferable to use Alcian blue as the dye in the quantitative test. By using Alcian Blue in qualitative and quantitative tests, the accuracy of analysis is said to be higher when analyzing products that contain ingredients other than acidic mucopolysaccharides or their salts (hereinafter also referred to as other ingredients). There are advantages. Further, it is preferable to use Alcian Blue because the operation in quantitative tests is simpler. When Alcian Blue is used in qualitative and quantitative tests, only acidic mucopolysaccharides or their salts are detected or quantified in these tests. In other words, the quantitative results obtained in the quantitative test reflect the content of acidic mucopolysaccharide or its salt confirmed in the qualitative test. For example, if it is confirmed in a qualitative test that the acidic mucopolysaccharide or its salt contained in the test sample is one type, the quantitative results obtained in the quantitative test using the test sample are It can be determined that the content of the acidic mucopolysaccharide or its salt contained in Therefore, by combining qualitative and quantitative tests and using the same dye (Alcian blue) to detect acidic mucopolysaccharides or their salts in these tests, it is possible to detect acidic mucopolysaccharides or their salts in a simple process. The type of acidic mucopolysaccharide or its salt can be confirmed and quantified with high accuracy.

In one embodiment, when it is confirmed by the qualitative test that the acidic mucopolysaccharide or its salt contained in the test sample is one type, the quantitative result obtained by the quantitative test is Its salt content can be. Therefore, the analysis method of the present invention can be used as a method for quantifying the acidic mucopolysaccharide or its salt when the test sample contains only one type of acidic mucopolysaccharide or its salt. According to the analysis method of the present invention, it is possible to analyze and identify the types and amounts of acidic mucopolysaccharides or their salts contained in foods, medicines, cosmetics, etc., and their raw materials, using a simple process. can do. The analysis method of the present invention is also useful, for example, in quality control of such foods, pharmaceuticals, cosmetics, raw materials thereof, and the like.

The analysis method of the present invention can be used as a method for analyzing chondroitin sulfate or its salt contained in a test sample. When using 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 its salt to a qualitative test and a quantitative test, and the qualitative test includes subjecting the test sample to electrophoresis using a membrane. an electrophoresis step, a staining step of staining the membrane after electrophoresis with Alcian blue, and a detection step of confirming the type of acidic mucopolysaccharide or its salt in the test sample from the band pattern obtained by the staining. The quantitative test includes a colorimetric determination step of quantifying the acidic mucopolysaccharide or its salt in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or its salt. The method may be for analyzing chondroitin sulfate or its salt contained in In such an analysis method, if it is confirmed in the qualitative test that the acidic mucopolysaccharide or its salt contained in the test sample is only chondroitin sulfate or its salt, the quantitative test obtained in the quantitative test The result can be the content of chondroitin sulfate or its salt. The above method for analyzing chondroitin sulfate or its salt can be used as a method for quantifying chondroitin sulfate or its salt in a test sample.

The present invention also includes the following method for quantifying chondroitin sulfate or its salt in a test sample.
The test sample containing chondroitin sulfate or its salt is subjected to a qualitative test and a quantitative test, and the qualitative test includes an electrophoresis step in which the test sample is subjected to electrophoresis using a membrane, The quantitative test includes a staining step of staining the membrane with Alcian blue, and a detection step of confirming the type of acidic mucopolysaccharide or its salt in the test sample from the band pattern obtained by the staining. A colorimetric determination step of quantifying the acidic mucopolysaccharide or its salt in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or its salt; When it is confirmed that the acidic mucopolysaccharide or its salt contained is only chondroitin sulfate or its salt, the quantitative result obtained by the above quantitative test is taken as the content of the chondroitin sulfate or its salt in the test sample. A method for quantifying chondroitin sulfate or its salt.
In the above method for quantifying chondroitin sulfate or its salt, the qualitative test, quantitative test, and preferred embodiments thereof are the same as the above analysis method. Chondroitin sulfate and its preferred embodiments are the same as the above analytical method.

<Quality control test method for samples containing acidic mucopolysaccharides or their salts>
In the quality control test method for products containing acidic mucopolysaccharides or salts thereof of the present invention, a product containing acidic mucopolysaccharides or salts thereof is used as a test sample, and the test sample is analyzed by the analysis method of the present invention described above. a standard sample analysis step of analyzing a standard sample containing an acidic mucopolysaccharide or its salt of known concentration and type by the above analytical method; and a qualitative test result of the test sample obtained in the above analysis. and an evaluation step of evaluating the quality of the test sample by comparing the quantitative test results with the qualitative and quantitative test results of the standard sample.
Products containing acidic mucopolysaccharides or salts thereof are not particularly limited, and include foods, pharmaceuticals, cosmetics, raw materials thereof, etc. containing acidic mucopolysaccharides or salts thereof, which are used as test samples in the above analysis method. In the present invention, either the test sample analysis step or the standard sample analysis step may be performed first. Qualitative tests, quantitative tests, their preferred embodiments, etc. are the same as in the analysis method described above. The acidic mucopolysaccharide or its salt is analyzed using the same method as described above. The standard sample is a sample containing an acidic mucopolysaccharide or its salt, and the concentration (content) and type of the acidic mucopolysaccharide or its salt are known. The standard sample may be appropriately selected depending on the test sample. For example, foods, medicines, cosmetics, etc. containing a standard amount of a predetermined acidic mucopolysaccharide or its salt, or raw materials thereof may be used as the standard sample.

By comparing the qualitative and quantitative test results of the test sample obtained in the test sample analysis process with the qualitative and quantitative test results of the standard sample, the acidic mucopolysaccharide contained in the test sample (product) can be determined. Alternatively, it can be confirmed whether the type and amount of the salt is the same as that of the standard sample. In one embodiment, for example, the type of acidic mucopolysaccharide or its salt contained in the test sample is the same as that of the standard sample, and the amount of acidic mucopolysaccharide or its salt contained in the test sample is the same as or equal to that of the standard sample. If the number is higher than that, it can be determined that the product that is the test sample satisfies the quality standards. In one embodiment, since the type of acidic mucopolysaccharide or its salt contained in the standard sample is known, the type of acidic mucopolysaccharide or its salt contained in the test sample is determined by comparing the qualitative test results. can be confirmed. In quality control of products containing acidic mucopolysaccharides or salts thereof, it is important to control the type and amount of acidic mucopolysaccharides or salts thereof contained in the products. According to the present invention, the acidic mucopolysaccharide or its salt contained in a product can be easily 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, raw materials thereof, etc. containing acidic mucopolysaccharides or salts thereof.

EXAMPLES Hereinafter, the present invention will be explained in detail based on Examples, but the present invention is not limited thereto.

<Example 1>
After subjecting the acidic mucopolysaccharide and shark cartilage raw material to electrophoresis, they were stained with Alcian blue, and the acidic mucopolysaccharide in the shark cartilage raw material was qualitatively determined from the migration distance of the band.

(1) Equipment used for analysis ・Membrane electrophoresis device (manufactured by Nihon Eido Co., Ltd., NA-4700)
・Power supply device AE-3121 Linear Power 2000 (manufactured by ATTO)
・Cellulose acetate membrane (product name: SELECA-VSP, 6 x 11 cm, manufactured by Advantech)
・Nitrocellulose membrane (product name: Nitrocellulose membranes, 0.45 μm, 6 x 11 cm or 7 x 8.5 cm, manufactured by Bio-rad)
・Mixed membrane of nitrocellulose and cellulose acetate (product name: Nitrocellulose membrane, product number: MF membrane filters (0.45μm HA), Merck & Co., Ltd.)

(2) Reagents and raw materials used for analysis: Sodium hyaluronate (manufactured by MCH Medchemexpress)
・Sodium chondroitin sulfate C (manufactured by Nacalai Tesque Co., Ltd.)
・Heparan sulfate (manufactured by MCH Medchemexpress)
・Heparin sodium (manufactured by Santa Cruz Biotechnology)
・Dermatan sulfate (manufactured by Tronto res. Chem)
・Shark cartilage raw material (I) (Product name: Shark cartilage extract powder, manufactured by Nihon Yakuhin Co., Ltd.)
・Shark cartilage raw material (II) (product name: SCP, manufactured by Maruha Nichiro Co., Ltd.)
・Alcian Blue 8GX (manufactured by Funakoshi Co., Ltd.)
・Pyridine, acetic acid, barium acetate (all special grade, manufactured by Nacalai Tesque Co., Ltd.)
・Actinase E (manufactured by Kaken Pharmaceutical Co., Ltd.)
・Chondroitinase ABC (manufactured by Sigma-Aldrich)
Shark cartilage raw material (I) and shark cartilage raw material (II) contain shark cartilage extract. Shark cartilage raw material (I) and shark cartilage raw material (II) contain 20% by weight of chondroitin sulfate C (value reported by the manufacturer).
The above sodium hyaluronate, sodium chondroitin sulfate C, heparan sulfate, heparin sodium, and dermatan sulfate were used as standards.

(3) Preparation of reagent 1) 25% methanol aqueous solution Distilled water was added to 250 mL of methanol to make 1 L.
2) 1.0M acetic acid/pyridine buffer (acetic acid-pyridine buffer) (pH 3.5)
After adding 800 mL of distilled water to 60 g of acetic acid, the pH was adjusted to 3.5 with pyridine to make 1 L.
3) 0.1M barium acetate buffer (pH 5.0)
After adding 800 mL of distilled water to 25.5 g of barium acetate, the pH was adjusted to 5.0 with acetic acid to make 1 L.
4) Alcian blue solution (staining solution)
A 0.3% acetic acid aqueous solution was added to 5.0 g of Alcian Blue 8GX to make 1 L.
5) 1% aqueous acetic acid solution Distilled water was added to acetic acid (10 mL) to make 1 L.
6) 5% aqueous acetic acid solution Distilled water was added to acetic acid (50 mL) to make 1 L.
7) Actinase E solution (I)
Distilled water was added to actinase E to prepare a solution with an actinase E concentration of 3.4 mg/mL.
8) Actinase E solution (II)
Distilled water was added to actinase E to prepare a solution with an actinase E concentration of 1.0 mg/mL.
9) Chondroitinase ABC liquid Chondroitinase ABC was dissolved in distilled water and adjusted to 10 U/mL.

(4) Preparation of samples for analysis For the five acidic mucopolysaccharide samples (sodium hyaluronate, sodium chondroitin sulfate C, heparan sulfate, heparin sodium, and dermatan sulfate), prepare 0.1 mg/mL aqueous solutions of each in distilled water. did. For shark cartilage raw materials (I) and (II), 1.0 mg/mL aqueous solutions were each prepared in distilled water. The obtained standard aqueous solution and shark cartilage raw material aqueous solution were used as samples for analysis.

(5) Analysis procedure (5-1) Evaluation using cellulose acetate membrane 1) A line (5 mm) for spotting the sample for analysis was drawn with a pencil on the cellulose acetate membrane. More specifically, spot lines were drawn parallel to the bottom edge of the membrane at 5 mm intervals and 1 cm from the bottom edge of the membrane. The spotting lines were separated by at least 1 cm from the right and left sides of the membrane.
2) The cellulose acetate membrane was immersed in a 25% methanol aqueous solution for 10 minutes or more.
3) The cellulose acetate membrane was immersed in the electrophoresis solution (1.0M acetic acid/pyridine buffer) for 30 minutes.
4) The membrane was removed from the electrophoresis solution (1.0M acetic acid/pyridine buffer) and lightly dried.
5) Samples for analysis were spotted in bands on the cellulose acetate membrane.
6) After pouring the electrophoresis solution (1.0M acetic acid/pyridine buffer) into the cathode and anode sides of the electrophoresis tank, the cellulose acetate membrane was set in the electrophoresis tank so that the sample spot was 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 for 10 minutes at a constant current of 11 mA.
9) After electrophoresis, the membrane was taken out, air-dried, and stained in Alcian blue solution for 10 minutes.
10) The membrane was decolorized with a 1% aqueous acetic acid solution.

(5-2) Evaluation using nitrocellulose membrane 1) As in the case of the cellulose acetate membrane described above, a line (5 mm) for spotting the sample for analysis was drawn with a pencil on the nitrocellulose membrane.
2) A nitrocellulose membrane (6 x 11 cm or 7 x 8.5 cm) was immersed in the electrophoresis solution (0.1M barium acetate buffer) for 10 minutes or more.
3) The membrane was taken out from the electrophoresis solution (0.1M barium acetate buffer) and lightly dried.
4) Samples for analysis were spotted in bands on the nitrocellulose membrane.
5) After pouring the electrophoresis solution (0.1 M barium acetate buffer) into the cathode and anode sides of the electrophoresis tank, the nitrocellulose membrane was set in the electrophoresis tank so that the sample spot was on the cathode side.
6) The lid of the electrophoresis tank was closed and allowed to stand for 10 minutes to equilibrate the inside.
7) Electrophoresis was performed for 40 minutes at a constant current of 17 mA.
8) After electrophoresis, the membrane was taken out, dried slightly, and stained in an Alcian blue solution for 10 minutes.
9) The membrane was decolorized with a 5% aqueous acetic acid solution.
During electrophoresis, when the temperature of the electrophoresis tank was set to less than 10°C (for example, 4°C), the electrophoresis results became more stable.

(5-3) Evaluation using a mixed membrane of nitrocellulose and cellulose acetate In the evaluation method using a nitrocellulose membrane described in (5-2) above, a mixture of nitrocellulose and cellulose acetate was used instead of the nitrocellulose membrane. The experiment was conducted in the same manner except that a membrane was used.

(5-4) Confirmation of chondroitin sulfate by enzymatic hydrolysis using chondroitinase ABC (5-4-1) Enzymatic degradation of acidic mucopolysaccharide specimen (enzyme treatment)
1) Distilled water was added to acidic mucopolysaccharide samples of sodium chondroitin sulfate C and heparin sodium to prepare 4 mg/mL aqueous solutions, respectively.
2) 75 μL of actinase E solution (I) and 150 μL of distilled water were added to 60 μL of acidic mucopolysaccharide sample aqueous solution, and enzymatic decomposition was performed at 40° C. for 17 hours to obtain an actinase E-treated solution.
3) The actinase E-treated solution was left standing in a boiling water bath for 10 minutes to inactivate the enzyme.
4) After cooling the solution obtained in 3) to room temperature, 15 μL of chondroitinase ABC solution was added and enzymatically decomposed at 37° C. for 1 hour to obtain a chondroitinase-treated solution.
5) The chondroitinase-treated solution was left to stand in a boiling water bath for 10 minutes to deactivate the enzyme, resulting in an enzyme-treated product.
6) After cooling the enzyme-treated product obtained in 5) to room temperature, centrifuge it, dilute the supernatant 3 times with distilled water, and analyze it according to the evaluation method using cellulose acetate membrane and nitrocellulose membrane described above. went.

(5-4-2) Enzymatic decomposition of shark cartilage raw material 1) Distilled water was added to 500 mg each of shark cartilage raw material (I) and (II) to make 10 mL, and the raw material solution (solution of shark cartilage raw material (I) and A solution of shark cartilage raw material (II)) was obtained.
2) 850 μL of actinase E solution (II) was added to 100 μL of the raw material solution obtained in 1), and enzymatic decomposition was performed at 40° C. for 17 hours to obtain an actinase E-treated solution.
3) The actinase E-treated solution was left standing in a boiling water bath for 10 minutes to inactivate the enzyme.
4) After cooling the solution obtained in 3) to room temperature, 50 μL of chondroitinase ABC solution was added and enzymatically decomposed at 37° C. for 1 hour to obtain a chondroitinase-treated solution.
5) The chondroitinase-treated solution was left to stand in a boiling water bath for 10 minutes to deactivate the enzyme, resulting in an enzyme-treated product.
6) After cooling the enzyme-treated product obtained in 5) to room temperature, centrifuge it, dilute the supernatant 3 times with distilled water, and analyze it according to the evaluation method using cellulose acetate membrane and nitrocellulose membrane described above. went.
For samples without enzymatic digestion (enzyme treatment), add the same amount of distilled water instead of the enzyme solution in steps 2) and 4) of (5-4-1) and (5-4-2) above, and add other The procedure was the same as for the enzyme-treated product (enzyme-decomposed sample).

(6) Results The results of membrane electrophoresis of the acidic mucopolysaccharide preparation and shark cartilage raw material are shown in FIGS. 1 to 4. The results of membrane electrophoresis of the enzyme-treated acidic mucopolysaccharide sample and the enzyme-treated shark cartilage raw material prepared by the method (5-4) above are shown in FIG.

(6-1) Evaluation results using cellulose acetate membrane Figure 1 is a photograph showing the band patterns of the acidic mucopolysaccharide sample and shark cartilage raw material (I) obtained by electrophoresis using a cellulose acetate membrane. Figure 1(a) shows the results of electrophoresis of the acidic mucopolysaccharide specimen and shark cartilage raw material (I), and Figure 1(b) shows the results of electrophoresis of the acidic mucopolysaccharide specimen and shark cartilage raw material (I). This is the result of spotting and electrophoresis. Table 1 shows the samples in lanes 1 to 9 in FIG. 1(a) and lanes 10 to 15 in FIG. 1(b). The shark cartilage raw materials (I) in lanes 6 to 9 are from different lots, and the shark cartilage raw materials (I) used in lanes 10 to 15 are from the same lot as lane 6. In FIG. 1 and FIGS. 2 to 4 described later, the amounts of the spotted samples were 0.3 μg for the acidic mucopolysaccharide sample and 2.0 μg for the shark cartilage raw material. For reference, FIG. 7 shows a schematic diagram of the band pattern in the photograph shown in FIG. 1 ((a): schematic diagram of FIG. 1(a), (b): schematic diagram of FIG. 1(b)).

FIG. 2 is a photograph showing band patterns of an acidic mucopolysaccharide sample and shark cartilage raw material (II) obtained by electrophoresis using a cellulose acetate membrane. FIG. 2(a) shows the results of electrophoresis of the acidic mucopolysaccharide sample and shark cartilage raw material (II), respectively. FIG. 2(b) shows the results of spotting and electrophoresis of the acidic mucopolysaccharide sample and shark cartilage raw material (II) in an overlapping manner. Table 2 shows the samples in lanes 1 to 9 in FIG. 2(a) and lanes 10 to 15 in FIG. 2(b). The shark cartilage raw materials (II) in lanes 6 to 9 are from different lots, and the shark cartilage raw materials (II) used in lanes 10 to 15 are from the same lot as lane 6. For reference, FIG. 8 shows a schematic diagram of the band pattern of the photograph shown in FIG. 2 ((a): schematic diagram of FIG. 2(a), (b): schematic diagram of FIG. 2(b)).

(6-2) Evaluation results using nitrocellulose membrane Figure 3 is a photograph showing the band patterns of the acidic mucopolysaccharide sample and shark cartilage raw material (I) obtained by electrophoresis using a nitrocellulose membrane. Figure 3(a) shows the results of electrophoresis of the acidic mucopolysaccharide specimen and shark cartilage raw material (I), and Figure 3(b) shows the results of electrophoresis of the acidic mucopolysaccharide specimen and shark cartilage raw material (I). This is the result of spotting and electrophoresis. Table 3 shows the samples in lanes 1 to 8 in FIG. 3(a) and lanes 9 to 14 in FIG. 3(b). The shark cartilage raw materials (I) in lanes 6 to 8 are from different lots, and the shark cartilage raw materials (I) used in lanes 9 to 14 are from the same lot as lane 6. For reference, FIG. 9 shows a schematic diagram of the band pattern of the photograph shown in FIG. 3 ((a): schematic diagram of FIG. 3(a), (b): schematic diagram of FIG. 3(b)).

FIG. 4 is a photograph showing the band patterns of the acidic mucopolysaccharide sample and shark cartilage raw material (II) obtained by electrophoresis using a nitrocellulose membrane. FIG. 4(a) shows the results of electrophoresis of the acidic mucopolysaccharide sample and shark cartilage raw material (II), respectively. FIG. 4(b) shows the results of spotting and electrophoresis of the acidic mucopolysaccharide sample and shark cartilage raw material (II) in an overlapping manner. Table 4 shows the samples in lanes 1 to 8 in FIG. 4(a) and lanes 9 to 14 in FIG. 4(b). The shark cartilage raw materials (II) in lanes 6 to 8 are from different lots, and the shark cartilage raw materials (II) used in lanes 9 to 14 are from the same lot as lane 6. For reference, FIG. 10 shows a schematic diagram of the band pattern of the photograph shown in FIG. 4 ((a): schematic diagram of FIG. 4(a), (b): schematic diagram of FIG. 4(b)).

(6-3) Mixed membrane of nitrocellulose and cellulose acetate When using a mixed membrane of nitrocellulose and cellulose acetate, the same results (band pattern )was gotten.

(6-4) Results of confirmation of chondroitin sulfate by enzymatic hydrolysis Figure 5 shows the enzyme-treated shark cartilage raw material and acidic mucopolysaccharide specimen obtained by electrophoresis using cellulose acetate membrane or nitrocellulose membrane. It is a photograph showing the band pattern of an enzyme-treated product ((a): cellulose acetate membrane, (b): nitrocellulose membrane). Table 5 shows the samples in lanes 1 to 8 in FIGS. 5(a) and 5(b). In Table 5, "with enzyme treatment" means the above-mentioned enzyme-treated product (enzyme-decomposed sample), and "without enzyme treatment" means the sample which was not treated with actinase E and chondroitinase ABC. The sample amounts spotted during electrophoresis were 2.0 μg for the shark cartilage raw material or its enzyme-treated product, and 0.8 μg for the acidic mucopolysaccharide sample or its enzyme-treated product. For reference, FIG. 11 shows a schematic diagram of the band pattern of the photograph shown in FIG. 5 ((a): schematic diagram of FIG. 5(a), (b): schematic diagram of FIG. 5(b)).

As shown in lanes 6 to 10 of Figures 1 to 2 and lanes 6 to 9 of Figures 3 to 4, for all shark cartilage raw materials (different manufacturers and lots), the bands stained with Alcian blue after membrane electrophoresis were It was one. In electrophoresis using a cellulose acetate membrane, when shark cartilage raw material and acidic mucopolysaccharide preparation were spotted overlapping, the bands observed in shark cartilage raw material migrated at a different distance from the bands of sodium hyaluronate, heparan sulfate, and dermatan sulfate. It was found that they do not match (lanes 11, 13 and 15 in Figures 1 and 2). When the shark cartilage material and the acidic mucopolysaccharide preparation were superimposed and spotted, the bands of the shark cartilage material and the sample sodium chondroitin sulfate matched (lanes 12 in Figures 1 and 2), but the spot overlapped with heparin sodium was The band broadened (lane 14 in Figures 1 and 2). When electrophoresis was performed using a nitrocellulose membrane, only the band of the shark cartilage material and the standard sodium chondroitin sulfate band matched, but they did not match with the bands of other acidic mucopolysaccharides (Figures 3 and 4). lanes 10-14). From this, it was confirmed that the acidic mucopolysaccharide contained in the raw material derived from shark cartilage was only chondroitin sulfate, and no other acidic mucopolysaccharides were contained.

As mentioned above, in cellulose acetate membrane electrophoresis, the bands derived from shark cartilage raw materials (I) and (II) overlapped with sodium chondroitin sulfate. In addition, the band expanded in spots where shark cartilage raw material was layered with heparin sodium. Shark cartilage raw materials (I) and (II) and specimens of sodium chondroitin sulfate and sodium heparin were subjected to enzymatic hydrolysis with chondroitinase ABC according to the method described in (5-4) above, and then subjected to membrane electrophoresis as described above. As a result, disappearance of the band was observed only in the specimens of shark cartilage raw materials (I) and (II) and sodium chondroitin sulfate (FIG. 5).

It has been reported that shark cartilage contains chondroitin sulfate, hyaluronic acid, and dermatan sulfate as acidic mucopolysaccharides (Higashi K., et al, PloS One, 10(6), e0131502 (2015).) . In addition to these, heparan sulfate and heparin sodium, which are representative acidic mucopolysaccharides, were added to the preparation, and membrane electrophoresis of shark cartilage raw materials (I) and (II) was performed. When evaluated using a cellulose acetate membrane, only one band was detected from this raw material, and the migration distance matched that of the chondroitin sulfate band, but the band also partially overlapped with heparin. When the support was changed to a nitrocellulose membrane and evaluated, the migration distance of the band of this raw material matched that of chondroitin sulfate. Furthermore, in order to confirm the acidic mucopolysaccharide in the shark cartilage raw material, enzymatic hydrolysis with chondroitinase ABC was performed, and it was observed that the band of the shark cartilage raw material and chondroitin sulfate standard had disappeared. It was confirmed that the acidic mucopolysaccharide contained was susceptible to decomposition by chondroitinase ABC. Since there is only one band stained by Alcian blue and its migration distance matches that of chondroitin sulfate, the acidic mucopolysaccharide contained in this raw material is only chondroitin sulfate, and other acidic mucopolysaccharides are not included. It has been confirmed that this is not the case. Furthermore, since the above band has the property of being degraded by chondroitinase ABC, it was confirmed that the acidic mucopolysaccharide contained in this raw material was only chondroitin sulfate and no other acidic mucopolysaccharides were contained. In general, quantitative determination by colorimetry is performed by utilizing the characteristics of functional groups contained in compounds, so specificity is often a problem. However, in the shark cartilage raw material evaluated this time, the only band that is stained with the Alcian blue reagent is chondroitin sulfate, so the quantitative results obtained by Alcian blue colorimetry for the raw material do not indicate the content of chondroitin sulfate or its salts. This can be said to be a reflection of this.

<Example 2>
Regarding the shark cartilage raw materials (I) and (II) used in Example 1, the content of chondroitin sulfate or its salt was determined by the Alcian blue colorimetric method. Quantification by the Alcian blue colorimetric method was carried out in accordance with the method described in the Food Hygiene Journal (Yoshie Yabe et al., Analysis of Sodium Chondroitin Sulfate Added to Foods, 28(1), p. 13-18 (1987)). Ta.

(1) Preparation of test solution 1) Actinase E solution Phosphate buffer was added to 0.5 g of actinase E (Kaken Pharmaceutical Co., Ltd.) to make 50 mL.
2) Alcian Blue Test Solution 4.2 mL of hydrochloric acid, 1.38 g of sodium dihydrogen phosphate, and water were added to 1 g of Alcian Blue 8GX to make 100 mL.
3) Phosphate buffer 305 mL of 0.2 mol/L disodium phosphate solution and 195 mL of 0.2 mol/L monosodium phosphate solution were mixed, and water was added to make 1000 mL. The pH of this liquid was 7.

(2) Preparation of standard solution and standard solution for calibration curve 100 mg of sodium chondroitin sulfate (special grade) was accurately weighed and dissolved in water to make a constant volume of 100 mL (concentration: 1000 μg/mL). 1 mL of this solution was taken and made up to exactly 10 mL with water, which was used as a standard solution (concentration: 100 μg/mL).
This standard solution was sequentially diluted with water to 1.0 μg/mL, 4.0 μg/mL, 7.0 μg/mL, 10 μg/mL, and 20 μg/mL, which was used as a standard solution for a calibration curve.

(3) Preparation of test solution 40 mL of pH 7.0 phosphate buffer was added to 2 g of the crushed sample. Furthermore, 5 mL of actinase E solution (10 mg/mL) was added and a shaking operation was performed. After that, the mixture was left at 40° C. overnight, and then the volume was adjusted to 100 mL with water. The solution filtered through filter paper was used as the test solution.

(4) Colorimetric method 0.2 mL of Alcian blue test solution was added to 1 mL of the test solution and left for 20 minutes. The deposited precipitate was centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded, the precipitate was washed with 3 mL of water, and the centrifugation was repeated again. The obtained precipitate was dissolved in 10 mL of monoethanolamine to prepare a sample for absorbance measurement. The absorbance of the sample for absorbance measurement was measured at 615 nm using a spectrophotometer. A standard solution for the calibration curve was also used which was colored in the same manner. Using the calibration curve obtained from the absorbance of the standard solution for calibration curve, the amount of chondroitin sulfate or its salt in the test solution was calculated. The content of chondroitin sulfate or its salt determined here is the amount as sodium chondroitin sulfate.

(5) Results The content of chondroitin sulfate or its salt in shark cartilage raw material (I) was 20.6g/100g, and the content of chondroitin sulfate or its salt in shark cartilage raw material (II) was 20.9g/100g. .

From the above, it was confirmed by the qualitative test (Example 1) that all the acidic mucopolysaccharides contained in shark cartilage raw materials (I) and (II) were chondroitin sulfate (chondroitin sulfate 100%). In addition, a quantitative test using a colorimetric method (Example 2) revealed that the content of chondroitin sulfate or its salt in these shark cartilage raw materials (I) and (II) was 20.6 g/100 g and 20.9 g/100 g, respectively. I was able to confirm something. In the qualitative test (Example 1), it was confirmed that the acidic mucopolysaccharide or its salt contained in shark cartilage raw materials (I) and (II) was only chondroitin sulfate or its salt. The quantitative results obtained in Example 2) are the content of chondroitin sulfate or its salt in shark cartilage raw materials (I) and (II).
Therefore, a qualitative test involves subjecting a sample to membrane electrophoresis and confirming the type of acidic mucopolysaccharide or its salt contained in the sample from the band pattern obtained by Alcian blue staining, and a colorimetric method to confirm the type of acidic mucopolysaccharide or its salt. By performing a salt quantitative test, it was possible to confirm and quantify the type of acidic mucopolysaccharide or its salt in a sample with a simple process.

<Test Example 1>
In order to confirm that chondroitin sulfate or its salt is not decomposed by actinase E, the following test was conducted. Reagents, raw materials, etc., such as sodium chondroitin sulfate C and shark cartilage raw materials (I) and (II), were the same as in Example 1.

(1) Preparation of sample for analysis (1-1) Enzyme-degraded sample of shark cartilage raw material (actinase (+), chondroitinase (+))
An enzyme-treated sample of shark cartilage raw material (I) and an enzyme-treated sample of shark cartilage raw material (II) (actinase E and Samples treated with chondroitinase ABC) were prepared.

(1-2) Actinase-treated sample of shark cartilage raw material (actinase (+), chondroitinase (-))
A sample was prepared in the same manner as in (1-1) above, except that the shark cartilage raw material was not treated with chondroitinase ABC. Specifically, in the method described in Example 1 (5-4-2), the same amount of distilled water was added in place of the chondroitinase ABC solution in step 4). Other than the above, the actinase-treated sample of shark cartilage raw material (I) and the actinase-treated sample of shark cartilage raw material (II) (actinase E treatment were performed using the method described in Example 1 (5-4-2), Samples without Icinase ABC treatment were prepared.

(1-3) Sample of shark cartilage raw material without enzyme treatment (actinase (-), chondroitinase (-))
A sample was prepared in the same manner as in (1-1) above, except that the shark cartilage raw material was not treated with actinase E and chondroitinase ABC. Specifically, in the method described in (5-4-2) of Example 1, the same amount of distilled water was used in place of actinase E solution (II) and chondroitinase ABC solution in steps 2) and 4). added. Other than the above, samples of shark cartilage raw material (I) without enzyme treatment and samples of shark cartilage raw material (II) without enzyme treatment (actinase E treatment and chondrocyte treatment) were prepared using the method described in Example 1 (5-4-2). Samples without Ichinase ABC treatment were prepared.

(1-4) Enzyme-treated sample of chondroitin sulfate C sodium (actinase (+), chondroitinase (+))
Using the method described in Example 1 (5-4-1) (enzymatic degradation of acidic mucopolysaccharide preparation), the standard chondroitin sulfate C sodium was treated with actinase E and chondroitinase ABC to produce chondroitin sulfate C. Enzyme-treated samples of sodium were prepared.

(1-5) Actinase-treated sample of sodium chondroitin sulfate C (actinase (+), chondroitinase (-))
A sample was prepared in the same manner as in (1-4) above, except that sodium chondroitin sulfate C was not treated with chondroitinase ABC. Specifically, in the method described in Example 1 (5-4-1), the same amount of distilled water was added in place of the chondroitinase ABC solution in step 4). Except for the above, an actinase-treated sample of sodium chondroitin sulfate C was prepared by the method described in Example 1 (5-4-1).

(1-6) Sample of sodium chondroitin sulfate C without enzyme treatment (actinase (-), chondroitinase (-))
A sample was prepared in the same manner as in (1-4) above, except that sodium chondroitin sulfate C was not treated with actinase E and chondroitinase ABC. Specifically, in the method described in Example 1 (5-4-1), using chondroitin sulfate C sodium (standard product), actinase E solution (I) and chondroitin sulfate were added in steps 2) and 4). The same amount of distilled water was added instead of the Icinase ABC solution. Except for the above, a sample of chondroitin sulfate C sodium without enzyme treatment was prepared by the method described in Example 1 (5-4-1).

(2) Evaluation using cellulose acetate membrane Using the samples prepared in (1-1) to (1-6) of (1) above, the cellulose acetate membrane of (5-1) of Example 1 was used. The analysis was conducted using the evaluation method.
The results of membrane electrophoresis are shown in FIG.

FIG. 6 is a photograph showing the band patterns of shark cartilage raw material and sodium chondroitin sulfate without enzyme treatment, actinase treatment sample, and enzyme treatment sample electrophoresed on cellulose acetate membrane. Table 6 shows the samples in lanes 1-9 of FIG. Samples that were treated with actinase E were written as "+" in the actinase treatment column, and samples that were not treated were written as "-". Samples that were subjected to chondroitinase ABC treatment were written as "+" in the chondroitinase treatment column, and samples that were not treated were written as "-". The amount of sample spotted on the membrane was 5.0 μg in lanes 1 to 6 and 0.8 μg in lanes 7 to 9. For reference, FIG. 12 shows a schematic diagram of the band pattern of the photograph shown in FIG. 6.

Lanes 1, 4 and 7 in FIG. 6 are samples without enzyme treatment. Lanes 2, 5 and 8 are actinase treated samples. In lanes 1, 2, 4, 5, and 8 of FIG. 6, a band was detected at the same position as lane 7 (sodium chondroitin sulfate sample). 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 sodium chondroitin sulfate C disappeared. From these results, it was confirmed that chondroitin sulfate or its salt is decomposed by chondroitinase ABC, but not by actinase E.

Claims (6)

Including subjecting a test sample containing an acidic mucopolysaccharide or its salt to a qualitative test and a quantitative test,
The qualitative test includes an electrophoresis step in which the test sample is subjected to electrophoresis using a membrane;
A staining step of staining the membrane after electrophoresis with Alcian blue, and
A detection step of confirming the type of acidic mucopolysaccharide or its salt in the test sample from the band pattern obtained by the staining,
the acidic mucopolysaccharide is chondroitin sulfate,
The quantitative test includes a colorimetric determination step of quantifying the acidic mucopolysaccharide or its salt in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or its salt,
The dye capable of detecting the acidic mucopolysaccharide or its salt is Alcian blue,
The colorimetric determination step includes adding Alcian blue to a solution of the test sample and measuring the absorbance of the resulting colored liquid.
A method for analyzing acidic mucopolysaccharide or its salt contained in the test sample. When it is confirmed by the qualitative test that the acidic mucopolysaccharide or its salt contained in the test sample is one type, the quantitative result obtained by the quantitative test is used as the content of the acidic mucopolysaccharide or its salt. The analysis method according to claim 1. The analytical method according to claim 1 or 2, wherein the membrane is a nitrocellulose membrane, a cellulose acetate membrane, or a mixed membrane of nitrocellulose and cellulose acetate. The analytical method according to any one of claims 1 to 3, wherein the membrane is a nitrocellulose membrane or a cellulose acetate membrane. including subjecting a test sample containing chondroitin sulfate or its salt to a qualitative test and a quantitative test,
The qualitative test includes an electrophoresis step in which the test sample is subjected to electrophoresis using a membrane;
A staining step of staining the membrane after electrophoresis with Alcian blue, and
A detection step of confirming the type of acidic mucopolysaccharide or its salt in the test sample from the band pattern obtained by the staining,
The quantitative test includes a colorimetric determination step of quantifying the acidic mucopolysaccharide or its salt in the test sample by a colorimetric method using a dye capable of detecting the acidic mucopolysaccharide or its salt,
The dye capable of detecting the acidic mucopolysaccharide or its salt is Alcian blue,
The colorimetric determination step includes adding Alcian blue to a solution of the test sample and measuring the absorbance of the resulting colored liquid,
If it is confirmed by the qualitative test that the acidic mucopolysaccharide or its salt contained in the test sample is only chondroitin sulfate or its salt, the quantitative results obtained by the quantitative test are used as the chondroitin sulfate or its salt. Let the salt content be
A method for quantifying chondroitin sulfate or its salt contained in the test sample. A quality control test method for products containing acidic mucopolysaccharides or salts thereof, comprising:
A test sample analysis step of analyzing the product as a test sample by the analysis method according to any one of claims 1 to 4 ;
a standard sample analysis step of analyzing a standard sample containing an acidic mucopolysaccharide or a salt thereof whose concentration and type are known by the analysis method;
an evaluation step of evaluating the quality of the product by comparing the qualitative test results and quantitative test results of the test sample obtained in the analysis with the qualitative test results and quantitative test results of the standard sample,
the acidic mucopolysaccharide is chondroitin sulfate;
Quality control test method for products containing acidic mucopolysaccharides or their salts.

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