Method for analyzing proteoglycan

Abstract

Provided is a method for analyzing proteoglycan, whereby proteoglycan in a sample can be easily analyzed. A sample containing proteoglycan is immersed or added to an aqueous solution containing a salt, and a purified precipitate obtained by adding an alcohol at a low specific concentration to the obtained liquid composition is subjected to proteoglycan analysis.

Description

Technical Field

[0001] This invention relates to a method for analyzing proteoglycans with specific pretreatment. Background Technology

[0002] Proteoglycans are a broad category of glycoproteins formed by the covalent bonding of sulfated polysaccharides such as chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratin sulfate with core proteins that form the core structure. Proteoglycans are widely found in the skin and cartilage of fish, mollusks, birds, and mammals. As a major component of the extracellular matrix, they form complexes with fibrous matrix proteins such as hyaluronic acid and type II collagen, playing a crucial role in maintaining tissue water retention and elasticity.

[0003] In recent years, reports have shown that proteoglycans are effective in improving knee joints and skin health, leading to their application in nutritional supplements and cosmetics, and further research into their pharmaceutical applications. Therefore, it is hoped that a simple method can be developed to analyze proteoglycans in products, their raw materials, or intermediates.

[0004] However, because products containing proteoglycans are manufactured through processes such as extraction from cartilage tissue, and because food or cosmetic products contain various additives depending on the type of product, in addition to proteoglycans, they also contain other impurities such as proteins derived from collagen or tissues or nutritional supplements mixed in with food. The presence of these impurities makes it difficult to detect proteoglycans.

[0005] Regarding this, a quantitative method for proteoglycans using molecular weight fractionation membranes and size exclusion chromatography has been proposed previously (Patent Document 1). However, since this method requires repeated concentration and purification using molecular weight fractionation membranes, a simpler method is needed.

[0006] In addition, a method for quantifying proteoglycans using antibodies has been disclosed (Patent Document 2), but it suffers from high costs and quantification issues when proteins are present in the sample.

[0007] In addition, a method for analyzing proteoglycans has been proposed (Patent Document 3), which includes the steps of passing a solution containing proteoglycans into a strongly basic anion exchange resin and then sequentially passing chloride ion solutions containing different concentrations of chloride ions. However, for industrial applications, a simpler method is still desired.

[0008] Existing technical documents Patent documents Patent Document 1: Japanese Patent Application Publication No. 2018-151207 Patent Document 2: Japanese Patent Publication No. 2016-160226 Patent Document 3 Japanese Patent Publication No. 2020-134435 Non-patent literature Non-patent literature 1 Journal of the Japanese Society of Food Chemistry, Vol.29(2), 104-113 (2022) Summary of the Invention The problem that the invention aims to solve The present invention addresses the aforementioned prior art by providing a method for analyzing proteoglycans in samples that is simple to use and can be analyzed using various detection systems.

[0009] Methods for solving problems The inventors impregnated or added a sample containing proteoglycan to an aqueous solution containing salt. When alcohol was added to the resulting liquid composition at a low specific concentration, it was surprisingly found that the proteoglycan was selectively precipitated. By using this precipitate to detect proteoglycan, the influence of impurities could be avoided, thus completing the present invention.

[0010] Based on this insight, the present invention provides the following method.

[0011] [1] Methods for analyzing proteoglycans in samples include: (1) The step of immersing or adding the sample to an aqueous solution containing salt, adding alcohol to the resulting liquid composition in an amount that is 10 to 60% by volume to precipitate proteoglycan; or, the step of immersing or adding the sample to an aqueous solution containing salt and 10 to 60% by volume alcohol to precipitate proteoglycan. (2) The process of analyzing the proteoglycans from the recovered precipitate.

[0012] [2] The alcohol is added to the liquid composition in an amount that results in a final concentration of 20 to 60% by volume, according to the method described in [1].

[0013] [3] The alcohol is added to the liquid composition in an amount that results in a final concentration of 30 to 60% by volume, according to the method described in [1].

[0014] [4] According to any one of [1] to [3], the aqueous solution containing the salt contains a salt concentration of 0.5 M to saturation concentration.

[0015] [5] According to any one of [1] to [3], the aqueous solution containing the salt contains a salt concentration of 2.0 M to saturation.

[0016] [6] According to any one of [1] to [5], the salt comprises one or more of lithium salts, sodium salts, potassium salts, calcium salts and magnesium salts.

[0017] [7] According to any one of [1] to [6], the alcohol comprises one or more combinations selected from ethanol and isopropanol.

[0018] [8] The detection of the proteoglycan is carried out by HPLC analysis according to any one of [1] to [7].

[0019] [9] The process (1) is repeated multiple times (e.g., 2 or 3 times) according to any one of [1] to [8].

[0020] Through the above process (1), proteoglycans in the sample can be selectively precipitated without relying on additional equipment such as anion exchange resin, ultrafiltration membrane or hollow fiber membrane. By using the purified precipitate, the influence of impurities can be easily avoided and proteoglycans can be detected.

[0021] Not limited to theory, simply explaining the phenomenon produced in process (1), it can be considered that through the cations of salts coexisting with proteoglycans, glycosaminoglycans such as chondroitin sulfate that constitute proteoglycans become electrically stable or form cross-linked structures, thereby reducing solubility. When a lower concentration of alcohol is added to a solution in which proteoglycans exist in this state, the polarity of the solution will decrease to a specified level, and proteoglycans, as macromolecules, will preferentially precipitate. Attached Figure Description

[0022] Figure 1 This represents a chromatogram obtained by gel filtration HPLC analysis of the chondroitin proteoglycan (PG) standard solution before treatment with chondroitinase.

[0023] Figure 2 This represents the chromatogram obtained by gel filtration HPLC analysis of a chondroitin proteoglycan (PG) standard solution treated with chondroitinase.

[0024] Figure 3 This represents the chromatogram obtained by gel filtration HPLC analysis of the chondroitin (PG) sample solution before treatment with chondroitinase.

[0025] Figure 4 This represents the chromatogram obtained by gel filtration HPLC analysis of a chondroitin (PG) sample solution after treatment with chondroitinase.

[0026] Figure 5 This represents the chromatogram obtained by gel filtration HPLC analysis of an untreated solution prepared by dissolving a proteoglycan (PG) powder sample in a mobile phase (50 mM phosphate buffer containing 0.2 M sodium chloride (pH 7.0)).

[0027] Figure 6 The method involves dissolving a proteoglycan (PG) powder sample in a 2.0M sodium chloride aqueous solution, adding ethanol to achieve a final concentration of 40% by volume to generate a precipitate, dissolving the precipitate in the mobile phase to prepare a proteoglycan sample solution, and then performing gel filtration HPLC analysis on the proteoglycan sample solution to obtain the chromatogram.

[0028] Figure 7 The method involves dissolving a proteoglycan (PG) powder sample in a 4.5M sodium chloride aqueous solution, adding ethanol to achieve a final concentration of 40% by volume to generate a precipitate, dissolving the precipitate in the mobile phase to prepare a proteoglycan sample solution, and then performing gel filtration HPLC analysis on the proteoglycan sample solution to obtain the chromatogram.

[0029] Figure 8 This indicates that a proteoglycan (PG) powder sample was dissolved in a saturated sodium chloride aqueous solution, and ethanol was added to achieve a final concentration of 40% by volume to generate a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram of the proteoglycan sample solution was obtained by gel filtration HPLC analysis.

[0030] Figure 9 The method involves dissolving a proteoglycan (PG) powder sample in a 0.5M calcium chloride aqueous solution, adding ethanol to achieve a final concentration of 40% by volume to generate a precipitate, dissolving the precipitate in the mobile phase to prepare a proteoglycan sample solution, and then performing gel filtration HPLC analysis on the proteoglycan sample solution to obtain the chromatogram.

[0031] Figure 10 The method involves dissolving a proteoglycan (PG) powder sample in a 1.0M calcium chloride aqueous solution, adding ethanol to achieve a final concentration of 40% by volume to generate a precipitate, dissolving the precipitate in the mobile phase to prepare a proteoglycan sample solution, and then performing gel filtration HPLC analysis on the proteoglycan sample solution to obtain the chromatogram.

[0032] Figure 11 The method involves dissolving a proteoglycan (PG) powder sample in a 2.0M calcium chloride aqueous solution, adding ethanol to achieve a final concentration of 40% by volume to generate a precipitate, dissolving the precipitate in the mobile phase to prepare a proteoglycan sample solution, and then performing gel filtration HPLC analysis on the proteoglycan sample solution to obtain the chromatogram.

[0033] Figure 12The method involves dissolving a proteoglycan (PG) powder sample in a 2.0M potassium chloride aqueous solution, adding ethanol to achieve a final concentration of 40% by volume to form a precipitate, dissolving the precipitate in the mobile phase to prepare a proteoglycan sample solution, and then performing gel filtration HPLC analysis on the proteoglycan sample solution to obtain the chromatogram.

[0034] Figure 13 The method involves dissolving a proteoglycan (PG) powder sample in a 2.0M sodium acetate aqueous solution, adding ethanol to achieve a final concentration of 40% by volume to generate a precipitate, dissolving the precipitate in the mobile phase to prepare a proteoglycan sample solution, and then performing gel filtration HPLC analysis on the proteoglycan sample solution to obtain the chromatogram.

[0035] Figure 14 This indicates that a proteoglycan (PG) powder sample was dissolved in a 0.5M calcium chloride aqueous solution, and isopropanol was added to achieve a final concentration of 30% by volume to form a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram of the proteoglycan sample solution was obtained by gel filtration HPLC analysis.

[0036] Figure 15 The method involves dissolving a proteoglycan (PG) powder sample in a 4.5M sodium chloride aqueous solution, adding ethanol to achieve a final concentration of 60% by volume to generate a precipitate, dissolving the precipitate in the mobile phase to prepare a proteoglycan sample solution, and then performing gel filtration HPLC analysis on the proteoglycan sample solution to obtain the chromatogram.

[0037] Figure 16 This represents a standard curve prepared based on data obtained from gel filtration HPLC analysis of a series of diluted chondroitin (PG) standard solutions.

[0038] Figure 17 The gel filtration HPLC chromatogram shows the chondroitin proteoglycan solution in a salmon nasal cartilage sample.

[0039] Figure 18 This represents a gel filtration HPLC chromatogram of an untreated or purified chondroitin proteoglycan solution obtained from commercially available product A. Figure 18 (A) shows the gel filtration HPLC chromatogram of the untreated chondroitin proteoglycan solution. Figure 18 (B) shows the gel filtration HPLC chromatogram of the purified chondroitin proteoglycan solution.

[0040] Figure 19 This represents a gel filtration HPLC chromatogram of an untreated or purified chondroitin proteoglycan solution obtained from commercially available product B. Figure 19 (A) shows the gel filtration HPLC chromatogram of the untreated chondroitin proteoglycan solution. Figure 19(B) shows the gel filtration HPLC chromatogram of the purified chondroitin proteoglycan solution.

[0041] Figure 20 This represents a gel filtration HPLC chromatogram of an untreated or purified chondroitin proteoglycan solution obtained from commercially available product C. Figure 20 (A) shows the gel filtration HPLC chromatogram of the untreated chondroitin proteoglycan solution. Figure 20 (B) shows the gel filtration HPLC chromatogram of the purified chondroitin proteoglycan solution.

[0042] Figure 21 This represents a gel filtration HPLC chromatogram of an untreated or purified chondroitin proteoglycan solution obtained from commercially available product D. Figure 21 (A) shows the gel filtration HPLC chromatogram of the untreated chondroitin proteoglycan solution. Figure 21 (B) shows the gel filtration HPLC chromatogram of the purified chondroitin proteoglycan solution.

[0043] Figure 22 This represents a gel filtration HPLC chromatogram of an untreated or purified chondroitin proteoglycan solution obtained from commercially available product E. Figure 22 (A) shows the gel filtration HPLC chromatogram of the untreated chondroitin proteoglycan solution. Figure 22 (B) shows the gel filtration HPLC chromatogram of the purified chondroitin proteoglycan solution. Detailed Implementation

[0044] The embodiments of the present invention will be described in detail below. However, the present invention should not be construed as being limited to the following embodiments.

[0045] In one embodiment of the present invention, there is a method for performing a prescribed pretreatment on a sample to selectively precipitate proteoglycans, and for detecting proteoglycans using the resulting purified precipitate.

[0046] In this application, the term "sample" is not particularly limited except for containing proteoglycans, and can be derived from tissues of all organisms, such as fish, mollusks, birds, or mammals, including bone, muscle fibers, and skin. Particularly, proteoglycans derived from cartilage or its peripheral components are preferred. Furthermore, "sample" can include fragments of such proteoglycan-containing tissue, extracts of such fragments, or extracts of such fragments or extracts. Additionally, "sample" includes products such as food, cosmetics, and pharmaceuticals made using these fragments, extracts, or extracts. Therefore, "sample" can be in various forms, including solid, powder, granules, paste, and liquid.

[0047] Sample pretreatment includes: (1) The process of immersing or adding the sample to an aqueous solution containing salt; (2) The step of adding alcohol to the obtained liquid composition in an amount that becomes a specific final concentration to precipitate proteoglycans.

[0048] The salt used in step (1) can be any salt whose solution contains metal ions that form salts with glycosaminoglycans such as chondroitin sulfate that constitute proteoglycans. Examples include lithium salts, sodium salts, potassium salts, magnesium salts, calcium salts, and mixtures thereof. More specifically, examples include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, sodium acetate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, sodium dihydrogen phosphate, etc. One or more of these can be used, and sodium chloride or calcium chloride is particularly preferred from the perspective of wanting the aqueous solution to have a neutral pH.

[0049] The salt concentration in the aqueous solution can be selected based on the final concentration of the alcohol (described later) and the type of salt, typically within the range of 0.5 M to saturation concentration. Furthermore, if the salt is sodium chloride or similar salt that generates monovalent ions, a concentration of 2.0 M to saturation concentration is preferred, more preferably 3.0 M to saturation concentration, further preferably 4.0 M to saturation concentration, and particularly preferably 4.5 M to saturation concentration. On the other hand, if the salt is calcium chloride or similar salt that generates polyvalent ions (especially divalent ions), since proteoglycan precipitation can be obtained at a lower concentration, a concentration of 0.5 M to 3.0 M is preferred, more preferably 0.5 M to 2.0 M. However, since the viscosity of calcium chloride increases at high concentrations, a concentration of 0.5 M to 2.0 M is more preferable.

[0050] From the viewpoint of minimizing sugar chain damage, the pH of the aqueous solution containing salt is preferably near neutral. Specifically, pH 5 to 10 is preferred, and pH 6 to 8 is more preferred. However, if the sample is an acidic aqueous solution, the aqueous solution containing salt can be set to, for example, pH 4 to 6, preferably pH 5 to 5.5. Furthermore, if the sample is an alkaline aqueous solution, the aqueous solution containing salt can be set to, for example, pH 8 to 11, preferably pH 9 to 10.

[0051] There are no particular restrictions on the temperature of the aqueous solution containing salt; for example, it can be at room temperature (e.g., 10–40°C). Additionally, heating, for example, to promote the dissolution and / or extraction of proteoglycans, can be used.

[0052] In one approach, the crude proteoglycan extract containing salt can be concentrated by vacuum drying or the like, and the concentrated extract can be processed in step (2).

[0053] As the alcohol used in step (2), a water-soluble alcohol is preferred, such as ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-methyl-2-butanol, ethylene glycol, and glycerol. Ethanol or isopropanol, which are commonly used, are particularly preferred.

[0054] Depending on the type or concentration of the salt used, the amount of alcohol added is usually sufficient to achieve a final concentration of 10 to 60% by volume, preferably 20 to 60% by volume, more preferably 30 to 60% by volume, even more preferably 35 to 50% by volume, and particularly preferably 40 to 45% by volume.

[0055] The steps of immersing or adding the sample to an aqueous solution containing salt and adding alcohol to the resulting liquid composition to achieve a specific final concentration to precipitate proteoglycans can be performed separately or in one step, by immersing or adding the sample to an aqueous solution containing the aforementioned salt and a specific amount of alcohol.

[0056] As demonstrated in the examples described below, by adding an alcohol at the specified concentration, proteoglycans can be selectively precipitated. By using the purified precipitate obtained, the influence of impurities can be avoided when detecting proteoglycans.

[0057] Furthermore, since this pretreatment yields a test sample free of impurities, the analysis of proteoglycans using the purified precipitate does not require a step to avoid impurities, allowing the use of various existing proteoglycan analysis methods. For example, qualitative or quantitative analysis of proteoglycans can be performed using gel filtration HPLC (e.g., see Patent Document 1 and Non-Patent Document 1), HPLC using anion exchange resin (see Patent Document 3), electrophoresis (e.g., see Non-Patent Document 1), or detection based on anti-proteoglycan antibodies (polyclonal antibodies, monoclonal antibodies) (e.g., see Patent Document 2).

[0058] For example, in gel filtration HPLC, a chromatographic column corresponding to the molecular weight of chondroitin is selected, and the detector only needs to be able to detect chondroitin. For example, in the case of a UV detector, detection can be performed at a UV wavelength of 200–280 nm, and 200–220 nm is particularly preferred. Furthermore, a differential refractive index detector can also be used for the detection of chondroitin.

[0059] [Example] The present invention will be further described in detail below through embodiments, but the present invention is not limited to these embodiments.

[0060] 1. Preparation of powder containing chondroitin proteoglycan Prepare 500g of nasal cartilage extracted from salmon heads, frozen at -25℃. Grind the cartilage into fine fragments using an electric mixer. Add the starting material to 1250g of water and extract at 95℃ for 3.5 hours while stirring. Filter the resulting extract through a 150μm stainless steel mesh to remove insoluble matter. Then, concentrate the extract using a rotary evaporator and freeze-dry it to obtain a powder containing chondroitin proteoglycans.

[0061] 2. Confirm the peak of chondroitin proteoglycan in gel filtration HPLC analysis. (Preparation of chondroitin proteoglycan sample solution) Take 350 mg of chondroitin powder and add it to a 4.5 M sodium chloride aqueous solution at a concentration of 10 mg / mL to prepare a liquid composition. Centrifuge the liquid composition (10,000 rpm, 15 minutes) and collect 30 mL of the supernatant in a microtube. Add 20 mL of ethanol (final concentration 40% by volume), stir with a vortex mixer, and centrifuge (10,000 rpm, 15 minutes), discarding the supernatant. Dissolve the precipitate again with 30 mL of 4.5 M sodium chloride aqueous solution, add 20 mL of ethanol (final concentration 40% by volume), centrifuge, stir, and discard the supernatant.

[0062] The purified precipitate was dissolved in water and then added to an ultrafiltration tube (Amicon Ultra-15, MWCO 100000) equipped with an ultrafiltration membrane for desalination. The resulting solution was freeze-dried to obtain a powder sample. 10 mg of the powder sample was dissolved in water and brought to a final volume of 5 mL. The solution was then filtered through a 0.45 μm filter to obtain the chondroitin proteoglycan sample solution.

[0063] (Preparation of chondroitin proteoglycan standard solution) "Protein glucan derived from salmon nasal cartilage" (Wako) was used as the standard substance for cartilage protein glucan. Approximately 10 mg of this standard substance was dissolved in water, and the volume was adjusted to 5 mL. The solution was then filtered through a 0.45 μm filter to obtain the cartilage protein glucan standard solution.

[0064] (Confirmation of the chondroitin proteoglycan peak treated with chondroitinase) 40 μL each of the chondroitin proteoglycan sample solution and the chondroitin proteoglycan standard solution were placed in microtubes. 40 μL of 0.2 M Tris-acetic acid buffer (pH 8.0) and 20 μL of chondroitin ABC solution adjusted to 0.1 U were added to each microtube and the mixture was stirred. After heating at 37°C for 16 hours, the mixture was placed in a boiling water bath for 3 minutes to inactivate the enzyme, resulting in the enzyme-treated group. Conversely, a solution containing 20 μL of 0.2 M Tris-acetic acid buffer (pH 8.0) instead of the chondroitin ABC solution (therefore, the total volume was 60 μL) was used as the untreated group.

[0065] Using 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride as the mobile phase, gel filtration HPLC analysis was performed on the enzyme-treated and untreated groups under the following conditions.

[0066] Sample injector: Primaide 1210 Autosampler (manufactured by Hitachi High Technology Co., Ltd.) Sample injection volume: 20 μL Pump: Primaide 1110 Pump (manufactured by Hitachi High Technology Co., Ltd.) Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride. Flow rate: 0.5 mL / min Separation column: Shodex OH Pak SB-806M HQ (manufactured by Shodex Science & Technology Co., Ltd.) Column temperature: 40℃ UV detector: Primaide 1410 UV-detector (manufactured by Hitachi High Technology Co., Ltd.) Measurement wavelength: 204nm (Experimental Results) Figure 1 and Figure 2 This represents the chromatograms obtained by gel filtration HPLC analysis of the chondroitin proteoglycan standard solution before and after treatment with chondroitinase. Furthermore, Figure 3 and Figure 4 The chromatograms are obtained by gel filtration HPLC analysis of the chondroitin proteoglycan sample solution before and after treatment with chondroitinase.

[0067] like Figure 1 and Figure 2As shown, the peak visible in the untreated group of the chondroitin standard solution disappeared in the enzyme-treated group, confirming that the peak originated from chondroitin. Similarly, in the chondroitin sample solution, the peak visible in the untreated group disappeared in the enzyme-treated group, confirming that the peak represented chondroitin. According to... Figures 1 to 4 The chromatograms shown assume that peaks detected around 12–16 minutes of retention time represent proteoglycans. The following experiments were conducted. In the chromatograms obtained from each experiment, arrows indicate proteoglycan peaks.

[0068] 3. Preparation of purified chondroitin proteoglycan and its HPLC analysis (study on the types and concentrations of salts and alcohols). [Example 1] Take 10 mg of the above-mentioned chondroitin powder and add it to a 2.0 M sodium chloride aqueous solution at a concentration of 2.0 mg / mL to prepare a liquid composition. Centrifuge the liquid composition (15000 rpm, 5 min) and collect 500 μL of the supernatant in a microtube. Add 333 μL of ethanol (final concentration 40% by volume), stir with a vortex mixer, and centrifuge (15000 rpm, 30 min), discarding the supernatant. Add 500 μL of 2.0 M sodium chloride aqueous solution to the precipitate again to dissolve it, add 333 μL of ethanol (final concentration 40% by volume), stir, centrifuge, and discard the supernatant. Then, add 500 μL of 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride to the precipitate, filter through a 0.45 μm filter to obtain a purified chondroitin solution. In addition, 10 mg of chondroitin powder was added to 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride at a concentration of 2.0 mg / mL, stirred, and filtered through a 0.45 μm filter to obtain an untreated solution.

[0069] The purified chondroitin proteoglycan solution and the untreated solution were subjected to gel filtration HPLC analysis under the following conditions.

[0070] Sample injector: Primaide 1210 Autosampler (manufactured by Hitachi High Technology Co., Ltd.) Sample injection volume: 20 μL Pump: Primaide 1110 Pump (manufactured by Hitachi High Technology Co., Ltd.) Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride. Flow rate: 0.5 mL / min Separation column: Shodex OH Pak SB-806M HQ (manufactured by Shodex Science & Technology Co., Ltd.) Column temperature: 40℃ UV detector: Primaide 1410 UV-detector (manufactured by Hitachi High Technology Co., Ltd.) Measurement wavelength: 204nm [Examples 2-10] The powder containing chondroitin was dissolved in an aqueous solution of the salts shown below. The resulting liquid composition was centrifuged, and 500 μL of the supernatant was placed in a microtube. An alcohol as shown below was added to the microtube, and the purified chondroitin solution was obtained in the same manner as in Example 1. Furthermore, the purified chondroitin solution and the untreated solution were analyzed by gel filtration HPLC in the same manner as in Example 1.

[0071] [Table 1]

[0072] The results are as follows Figures 5-15 As shown. Figure 5 As shown, in the HPLC chromatogram of the untreated solution after gel filtration, peaks of inclusions were also confirmed in addition to chondroitin proteoglycans. On the other hand, as... Figures 6 to 13 As shown, when chondroitin powder containing chondroitin was dissolved in solutions of various salt concentrations and subjected to ethanol precipitation, the chondroitin peak was separated or its resolution was improved under all conditions, enabling analysis of chondroitin unaffected by impurities.

[0073] In addition, such as Figures 14-15 As shown, precipitation treatment with different concentrations of ethanol or different types of alcohols at different concentrations resulted in the separation of chondroitin peaks or near-removal of impurity peaks in all cases, enabling analysis of chondroitin unaffected by impurities.

[0074] 4. Quantitative analysis of chondroitin proteoglycans in salmon nasal cartilage [Example 11] Take 50 mg of the above-mentioned chondroitin powder and dissolve it in 4.5 M sodium chloride solution, bringing the volume to 25 mL. Centrifuge the sample solution (15000 rpm, 5 min), and take 500 μL of the supernatant into a microtube. Add 333 μL of ethanol (final concentration 40% by volume), stir with a vortex mixer, and centrifuge (15000 rpm, 30 min), discarding the supernatant. Dissolve the precipitate again in 500 μL of 4.5 M sodium chloride aqueous solution, add 333 μL of ethanol (final concentration 40% by volume), stir, centrifuge, and discard the supernatant. Then, add 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride to the precipitate and bring the volume to 5 mL. Filter through a 0.45 μm filter to obtain a purified chondroitin solution.

[0075] "Protein glucan, derived from salmon nasal cartilage" (Wako) was used as the standard substance for cartilage protein glucan. 10 mg of this standard substance was dissolved in 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride and brought to a final volume of 5 mL. This solution was then diluted with the same phosphate buffer to concentrations of 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL. The solutions were filtered through a 0.45 μm filter to prepare a series of dilutions of the cartilage protein glucan standard solution.

[0076] The purified chondroitin proteoglycan solution and the diluted series of chondroitin proteoglycan standard solutions were analyzed by gel filtration HPLC under the following conditions.

[0077] Sample injector: Primaide 1210 Autosampler (manufactured by Hitachi High Technology Co., Ltd.) Sample injection volume: 50 μL Pump: Primaide 1110 Pump (manufactured by Hitachi High Technology Co., Ltd.) Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride. Flow rate: 0.5 mL / min Separation column: Shodex OH Pak SB-806M HQ (manufactured by Shodex Science & Technology Co., Ltd.) Column temperature: 40℃ UV detector: Primaide 1410 UV-detector (manufactured by Hitachi High Technology Co., Ltd.) Measurement wavelength: 204nm A standard curve was constructed based on the peak areas of the chromatograms obtained for each dilution of the standard solution and the concentration of the standard substance in each dilution. The constructed standard curve is shown below. Figure 16 As shown.

[0078] In addition, the peak area of ​​the chromatogram obtained from the purified chondroitin solution was used as the standard curve to determine the amount of chondroitin as 48.4 mg / 100 mg. Figure 17 This is a chromatogram showing the purified chondroitin proteoglycan solution.

[0079] 5. Quantitative analysis of chondroitin proteoglycans in food compositions [Examples 12-15] (Purification process of chondroitin) Commercially available products A (tablets), B (tablets), C (tablets), and D (granules) containing chondroitin proteoglycans were pulverized using a mortar and pestle. 300 mg (product A), 1160 mg (product B), 380 mg (product C), and 3000 mg (product D) of the pulverized material were dissolved in 4.5 M sodium chloride solution and brought to a final volume of 25 mL. The solution was centrifuged (15000 rpm, 5 min), and 500 μL of the supernatant was transferred to a microtube. 333 μL of ethanol (final concentration 40% by volume) was added, and the mixture was stirred using a vortex mixer and centrifuged (15000 rpm, 30 min). The supernatant was discarded. Another 500 μL of 4.5 M sodium chloride solution was added to dissolve the pulverized material, followed by 333 μL of ethanol (final concentration 40% by volume). The mixture was stirred, centrifuged, and the supernatant was discarded. Then, 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride was added to the precipitate and the volume was adjusted to 5 mL. The solution was then filtered through a 0.45 μm filter to obtain a purified chondroitin proteoglycan solution. Separately, an equal volume of commercially available pulverized material was dissolved in 4.5 M sodium chloride solution and the volume was adjusted to 25 mL. 500 μL of the supernatant obtained by centrifuging (15000 rpm, 5 min) was then adjusted to 5 mL and filtered through a 0.45 μm filter to obtain an untreated chondroitin proteoglycan solution.

[0080] (Quantification of proteoglycans) The purified chondroitin proteoglycan solutions obtained from each product were subjected to gel filtration HPLC analysis in the same manner as in Example 11. Figures 18 to 21 This shows the chromatograms of purified chondroitin solutions and untreated chondroitin solutions obtained from commercially available products A through D. (Example) Figure 18 (B) to Figure 21As shown in (B), no peaks other than chondroitin were found in the chromatograms of purified chondroitin solutions obtained from any commercially available product. The peak areas of the obtained chromatograms were applied to the standard curve prepared in Example 11, and the chondroitin content was determined to be 23.7 mg / tablet (commercial product A), 10.3 mg / tablet (commercial product B), 20.8 mg / tablet (commercial product C), and 10.1 mg / packet (commercial product D).

[0081] [Example 15] (Purification process of chondroitin) Take 1560 mg of the contents of commercially available product E (soft capsules) containing chondroitin proteoglycan and place it in a glass test tube. Add 10 mL of acetone and mix. Repeat the centrifugation process three times, discarding the supernatant, to remove hydrophobic components. After drying under reduced pressure, dissolve the residue in 4.5 M sodium chloride solution and bring the volume to 25 mL. Centrifuge the sample solution (15000 rpm, 5 min). Take 500 μL of the supernatant and place it in a microtube. Add 333 μL of ethanol (final concentration 40% by volume), stir with a vortex mixer, and centrifuge (15000 rpm, 30 min). Discard the supernatant. Add another 500 μL of 4.5 M sodium chloride aqueous solution to dissolve the residue, add 333 μL of ethanol (final concentration 40% by volume), stir, centrifuge, and discard the supernatant. Then, 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride was added to the precipitate and the volume was adjusted to 5 mL. The solution was then filtered through a 0.45 μm filter. Additionally, the residue obtained from an equal volume of the contents of a commercially available product was dissolved in 4.5 M sodium chloride solution and the volume was adjusted to 25 mL. 500 μL of the supernatant obtained by centrifuging (15000 rpm, 5 min) of the sample solution was then adjusted to 5 mL and filtered through a 0.45 μm filter to obtain an untreated chondroitin proteoglycan solution.

[0082] (Gel filtration HPLC) The purified chondroitin proteoglycan solution obtained from product E was subjected to gel filtration HPLC analysis in the same manner as in Example 11. Figure 22 The chromatograms show the purified chondroitin proteoglycan solution and the untreated chondroitin proteoglycan solution obtained from commercially available product E. Figure 22 As shown in (B), no peaks other than chondroitin were found in the chromatogram. The peak volumes of the obtained chromatogram were applied to the standard curve prepared in Example 11 to determine the amount of chondroitin as 6.2 mg / 1 capsule (commercially available product E).

[0083] Industrial availability This invention provides a simple and inexpensive method for analyzing cartilage proteoglycans. This method holds promise for quality management of proteoglycans in the food, cosmetics, and pharmaceutical industries.

Claims

1. A method for analyzing proteoglycans in a sample, including: (1) The step of immersing or adding the sample to an aqueous solution containing salt, adding alcohol to the resulting liquid composition in an amount that is 10 to 60% by volume to precipitate proteoglycan; or, the step of immersing or adding the sample to an aqueous solution containing salt and 10 to 60% by volume alcohol to precipitate proteoglycan. (2) The process of analyzing the proteoglycans from the recovered precipitate.

2. The method according to claim 1, wherein the alcohol is added to the liquid composition in an amount that results in a final concentration of 20 to 60% by volume.

3. The method according to claim 1, wherein the alcohol is added to the liquid composition in an amount that results in a final concentration of 30 to 60% by volume.

4. The method according to any one of claims 1 to 3, wherein the aqueous solution containing the salt contains a salt concentration of 0.5 M to saturation concentration.

5. The method according to any one of claims 1 to 3, wherein the aqueous solution containing the salt contains a salt concentration of 2.0 M to saturation level.

6. The method according to any one of claims 1 to 5, wherein the salt comprises one or more of lithium salts, sodium salts, potassium salts, calcium salts, and magnesium salts.

7. The method according to any one of claims 1 to 6, wherein the alcohol comprises one or more combinations selected from ethanol and isopropanol.

8. The method according to any one of claims 1 to 7, wherein the proteoglycan is detected by HPLC analysis.

9. The method according to any one of claims 1 to 8, wherein step (1) is performed repeatedly.

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