Method for analyzing proteoglycan

Abstract

Provided is a method for analyzing proteoglycan, which enables simple analysis of proteoglycan in a sample.　Specifically, provided is a method for analyzing proteoglycan, comprising immersing or adding a sample containing proteoglycan into an aqueous solution containing a salt to obtain a liquid composition; and adding an alcohol to the liquid composition at a specific, relatively low concentration to obtain a purified precipitate, which is then subjected to proteoglycan analysis.

Description

Proteoglycan analysis method

[0001] The present invention relates to a method for analyzing proteoglycans, which involves a specific pretreatment.

[0002] Proteoglycans are a type of glycoprotein in the broad sense, in which sulfated polysaccharides called glycosaminoglycans, such as chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate, are covalently bound to a core protein that forms a core structure. Proteoglycans are widely present in the skin and cartilage of fish, mollusks, birds, and mammals, and as the main component of the extracellular matrix, they form complexes with fibrous matrix proteins such as hyaluronic acid and type II collagen, playing an important role in maintaining the water retention and elasticity of tissues.

[0003] In recent years, there have been reports that proteoglycans are effective in improving knee joint health and skin beauty, and their use in foods such as supplements and cosmetics is progressing, and their use in pharmaceuticals is also being considered. Accordingly, there is a need for a simple method for analyzing proteoglycans in products containing proteoglycans, their raw materials, or intermediate products.

[0004] However, products containing proteoglycans are manufactured through processes such as extraction from cartilage tissue, etc., and contain various additives depending on the type of product, such as food or cosmetics. Therefore, in addition to proteoglycans, they also contain other contaminating proteins derived from collagen or tissues, or contaminants added to foods, such as supplements. The presence of these contaminants makes it difficult to measure proteoglycans.

[0005] In this regard, a method for quantifying proteoglycans using a molecular weight cutoff membrane and size exclusion chromatography has been proposed (Patent Document 1). However, this method requires repeated concentration and purification using the molecular weight cutoff membrane, so a simpler method is desired.

[0006] A method for quantifying proteoglycans using antibodies has also been disclosed (Patent Document 2), but this method is expensive and there are problems with its quantitation when contaminating proteins are present in the sample.

[0007] Furthermore, a method for analyzing proteoglycans has been proposed, which includes a step of passing a solution containing proteoglycans through a strongly basic anion exchange resin, followed by a step of passing salt ion solutions containing chloride ions at different concentrations through the resin (Patent Document 3). However, a simpler method is still desired for industrial use.

[0008] Patent Publication No. 2018-151207, Patent Publication No. 2016-160226, Patent Publication No. 2020-134435

[0009] Journal of the Japanese Society of Food Chemistry, Vol. 29(2), 104-113 (2022)

[0010] In contrast to the above-mentioned prior art, an object of the present invention is to provide a method for analyzing proteoglycans that can easily analyze proteoglycans in a sample and that can utilize a variety of detection systems.

[0011] The present inventors discovered that when a sample containing proteoglycan was immersed in or added to an aqueous solution containing salt, and alcohol was added to the resulting liquid composition at a relatively low specific concentration, proteoglycan was selectively precipitated, and that by measuring proteoglycan using this precipitate, proteoglycan could be detected while avoiding the influence of contaminants, leading to the present invention.

[0012] Based on these findings, the present invention provides the following methods. [1] A method for analyzing proteoglycans in a sample, comprising: (1) immersing or adding the sample in an aqueous solution containing salt, and adding alcohol to the resulting liquid composition in an amount to a final concentration of 10 to 60% by volume to precipitate proteoglycans, or immersing or adding the sample in an aqueous solution containing salt and 10 to 60% by volume of alcohol to precipitate proteoglycans, and (2) recovering the resulting precipitate and analyzing the proteoglycans. [2] The method described in [1], wherein the alcohol is added to the liquid composition in an amount to a final concentration of 20 to 60% by volume. [3] The method described in [1], wherein the alcohol is added to the liquid composition in an amount to a final concentration of 30 to 60% by volume. [4] The method described in any of [1] to [3], wherein the aqueous solution containing salt contains salt at a concentration of 0.5 M to saturation. [5] The method according to any one of [1] to [3], wherein the aqueous solution containing the salt contains the salt at a concentration of 2.0 M to saturation. [6] The method according to any one of [1] to [5], wherein the salt comprises one or a combination of two or more selected from lithium salts, sodium salts, potassium salts, calcium salts, and magnesium salts. [7] The method according to any one of [1] to [6], wherein the alcohol comprises one or a combination of two or more selected from ethanol and isopropanol. [8] The method according to any one of [1] to [7], wherein the proteoglycan is measured by HPLC analysis. [9] The method according to any one of [1] to [8], wherein step (1) is repeated multiple times (e.g., two or three times).

[0013] The above-mentioned step (1) allows selective precipitation of proteoglycans in a sample without relying on additional equipment such as an anion exchange resin, an ultrafiltration membrane, or a hollow fiber membrane, and this purified precipitate can be used to easily detect proteoglycans while avoiding the influence of contaminants. Without being bound by theory, the phenomenon occurring in step (1) can be briefly explained as follows: cations derived from salts coexisting with proteoglycans render glycosaminoglycans such as chondroitin sulfate, which constitute proteoglycans, electrically stable or form cross-linked structures, thereby reducing their solubility. Adding a relatively low, specific concentration of alcohol to a solution containing proteoglycans in this state reduces the polarity of the solution to a predetermined level, resulting in the preferential precipitation of the macromolecular proteoglycans.

[0014]

[0033] Figure 1 shows a chromatogram obtained by subjecting a cartilage proteoglycan (PG) standard solution to gel filtration HPLC analysis before treatment with chondroitinase. Figure 2 shows a chromatogram obtained by subjecting a cartilage proteoglycan (PG) standard solution to gel filtration HPLC analysis after treatment with chondroitinase. Figure 3 shows a chromatogram obtained by subjecting a cartilage proteoglycan (PG) sample solution to gel filtration HPLC analysis before treatment with chondroitinase. Figure 4 shows a chromatogram obtained by subjecting a cartilage proteoglycan (PG) sample solution to gel filtration HPLC analysis after treatment with chondroitinase. Figure 5 shows a chromatogram obtained by subjecting an untreated solution prepared by dissolving a proteoglycan (PG) powder sample in a mobile phase (50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride) to gel filtration HPLC analysis.

[0039] Figure 1 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in 2.0 M aqueous sodium chloride solution, adding ethanol to a final concentration of 40% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis. Figure 2 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in 4.5 M aqueous sodium chloride solution, adding ethanol to a final concentration of 40% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis. Figure 3 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in saturated aqueous sodium chloride solution, adding ethanol to a final concentration of 40% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis.

[0039] Figure 1 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in a 0.5 M aqueous calcium chloride solution, adding ethanol to a final concentration of 40% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis. Figure 2 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in a 1.0 M aqueous calcium chloride solution, adding ethanol to a final concentration of 40% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis.

[0033] Figure 1 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in 2.0 M aqueous calcium chloride solution, adding ethanol to a final concentration of 40% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis. Figure 2 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in 2.0 M aqueous potassium chloride solution, adding ethanol to a final concentration of 40% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis. Figure 3 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in 2.0 M aqueous sodium acetate solution, adding ethanol to a final concentration of 40% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis.

[0039] Figure 1 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in 0.5 M aqueous calcium chloride solution, adding isopropanol to a final concentration of 30% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis.

[0040] Figure 1 shows a chromatogram obtained by subjecting a proteoglycan sample solution prepared by dissolving a proteoglycan (PG) powder sample in 4.5 M aqueous sodium chloride solution, adding ethanol to a final concentration of 60% by volume, and dissolving the resulting precipitate in the mobile phase, followed by gel filtration HPLC analysis.

[0041] Figure 1 shows a calibration curve prepared from data obtained by subjecting a dilution series of a cartilage proteoglycan (PG) standard solution to gel filtration HPLC analysis.

[0042] Figure 1 shows a gel filtration HPLC chromatogram of a cartilage proteoglycan solution in a salmon nasal cartilage sample, followed by gel filtration HPLC chromatograms of untreated or purified cartilage proteoglycan solutions obtained from commercial product A. Figure 18(A) shows a gel filtration HPLC chromatogram of an untreated cartilage proteoglycan solution, and Figure 18(B) shows a gel filtration HPLC chromatogram of a purified cartilage proteoglycan solution. Figure 19(A) shows a gel filtration HPLC chromatogram of an untreated cartilage proteoglycan solution, and Figure 19(B) shows a gel filtration HPLC chromatogram of a purified cartilage proteoglycan solution.Figure 20 shows gel filtration HPLC chromatograms of untreated or purified cartilage proteoglycan solutions obtained from commercial product C. Figure 20(A) shows a gel filtration HPLC chromatogram of untreated cartilage proteoglycan solution, and Figure 20(B) shows a gel filtration HPLC chromatogram of purified cartilage proteoglycan solution. Figure 20 shows gel filtration HPLC chromatograms of untreated or purified cartilage proteoglycan solutions obtained from commercial product D. Figure 21(A) shows a gel filtration HPLC chromatogram of untreated cartilage proteoglycan solution, and Figure 21(B) shows a gel filtration HPLC chromatogram of purified cartilage proteoglycan solution. Figure 20 shows gel filtration HPLC chromatograms of untreated or purified cartilage proteoglycan solutions obtained from commercial product E. FIG. 22(A) shows a gel filtration HPLC chromatogram of an untreated cartilage proteoglycan solution, and FIG. 22(B) shows a gel filtration HPLC chromatogram of a purified cartilage proteoglycan solution.

[0015] The present invention will be described in detail below with reference to the preferred embodiments, but the present invention should not be understood as being limited to the following embodiments.

[0016] In one embodiment, the present invention relates to a method for measuring proteoglycans by subjecting a sample to a predetermined pretreatment to selectively precipitate proteoglycans and using the purified precipitate obtained.

[0017] As used herein, the term "sample" is not particularly limited except that it contains proteoglycans. It may be derived from tissues of any organism, such as fish, mollusks, birds, or mammals, such as bone, muscle fibers, or skin. Proteoglycans derived from cartilage or its surrounding areas are particularly preferred. The term "sample" may also include a disrupted tissue of such proteoglycan-containing tissue, a squeezed product of the disrupted tissue, or an extract of the disrupted or squeezed product. The term "sample" also encompasses products such as foods, cosmetics, and pharmaceuticals made using the disrupted tissue, squeezed product, or extract. Therefore, the "sample" may take various forms, such as solid, powder, granules, paste, or liquid.

[0018] The sample pretreatment includes the following steps: (1) immersing or adding the sample to an aqueous solution containing a salt; and (2) adding alcohol to the resulting liquid composition in an amount that results in a specific final concentration to precipitate proteoglycans. The salt used in step (1) may be any salt containing a metal ion that can form a salt with glycosaminoglycans, such as chondroitin sulfate, that constitute the proteoglycans in the solution. Examples include lithium salts, sodium salts, potassium salts, magnesium salts, calcium salts, and mixtures thereof. More specific 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, and sodium dihydrogen phosphate. These salts can be used alone or in combination. Since a neutral pH is desirable for the aqueous solution, sodium chloride and calcium chloride are particularly preferred.

[0019] The salt concentration in the aqueous solution may be selected depending on the final alcohol concentration and the type of salt, as described below, and is typically selected from the range of 0.5 M to saturation. Furthermore, for salts that generate monovalent ions, such as sodium chloride, the salt concentration is preferably selected from the range of 2.0 M to saturation, more preferably 3.0 M to saturation, even more preferably 4.0 M to saturation, and particularly preferably 4.5 M to saturation. On the other hand, for salts that generate polyvalent ions (particularly divalent ions), such as calcium chloride, proteoglycan precipitation can be obtained at lower concentrations, and therefore the salt concentration is preferably selected from the range of 0.5 M to 3.0 M, more preferably 0.5 M to 2.0 M. However, calcium chloride increases viscosity at high concentrations, and therefore the salt concentration is preferably selected from the range of 0.5 M to 2.0 M.

[0020] The pH of the aqueous solution containing the salt is preferably near neutral in order to minimize damage to sugar chains. Specifically, a pH of 5 to 10 is preferred, and a pH of 6 to 8 is more preferred. However, when the sample is an acidic aqueous solution, the aqueous solution containing the salt may be adjusted to, for example, a pH of 4 to 6, preferably a pH of 5 to 5.5. Furthermore, when the sample is an alkaline aqueous solution, the aqueous solution containing the salt may be adjusted to, for example, a pH of 8 to 11, preferably a pH of 9 to 10.

[0021] The temperature of the salt-containing aqueous solution is not particularly limited and can be, for example, room temperature (e.g., 10 to 40°C). Alternatively, to promote dissolution and / or extraction of proteoglycans, the solution may be heated, for example, to 40 to 100°C.

[0022] In one embodiment, the crude proteoglycan extract containing salt may be concentrated by drying under reduced pressure or the like, and the resulting concentrated extract may be subjected to step (2).

[0023] The alcohol used in step (2) is preferably a water-soluble alcohol, and examples thereof include 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. The amount of alcohol added depends on the type and concentration of the salt used; typically, it is an amount that results in 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.

[0024] The step of immersing or adding a sample to an aqueous solution containing a salt and the step of adding an amount of alcohol to the resulting liquid composition to achieve a specific final concentration to precipitate proteoglycans may be carried out separately, or they may be carried out in a single step by immersing or adding a sample to an aqueous solution containing the above-mentioned salt and a specific amount of alcohol.

[0025] As demonstrated in the examples below, adding alcohol to the above-mentioned predetermined concentration selectively precipitates proteoglycans, and the resulting purified precipitate can be used to measure proteoglycans while avoiding the influence of contaminants. Furthermore, because this pretreatment removes contaminants from the measurement sample, proteoglycan analysis using the resulting purified precipitate does not require a step to avoid contaminants, and various existing proteoglycan analysis methods can be utilized. For example, qualitative or quantitative analysis of proteoglycans can be performed using gel filtration HPLC (see, e.g., Patent Document 1 and Non-Patent Document 1), HPLC using an anion exchange resin (see Patent Document 3), electrophoresis (see, e.g., Non-Patent Document 1), or detection using anti-proteoglycan antibodies (polyclonal or monoclonal antibodies) (see, e.g., Patent Document 2).

[0026] For example, in the gel filtration HPLC method, a column corresponding to the molecular weight of cartilage proteoglycan is selected, and a detector capable of detecting cartilage proteoglycan is sufficient. For example, in the case of a UV detector, detection can be performed at a UV wavelength of 200 to 280 nm, with 200 to 220 nm being particularly preferred. A differential refractive index detector can also be used to detect cartilage proteoglycan.

[0027] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

[0028] 1. Preparation of Cartilage Proteoglycan-Containing Powder 500 g of nasal cartilage extracted from salmon heads frozen at -25°C was prepared and finely pulverized into chips using an electric mixer. The starting material was added to 1,250 g of water and extracted at 95°C for 3.5 hours while stirring. The resulting extract was filtered through a stainless steel mesh (150 μm) to remove insoluble matter. The extract was then concentrated using a rotary evaporator, and the concentrate was powdered using a vacuum freeze dryer to obtain a cartilage proteoglycan-containing powder.

[0029] 2. Confirmation of Cartilage Proteoglycan Peak in Gel Filtration HPLC Analysis (Preparation of Cartilage Proteoglycan Sample Solution) 350 mg of cartilage proteoglycan-containing powder was collected and added to a 4.5 M aqueous sodium chloride solution at a concentration of 10 mg / mL to prepare a liquid composition. The liquid composition was centrifuged (10,000 rpm, 15 minutes), and 30 mL of the supernatant was collected in a microtube. 20 mL of ethanol was added (final concentration: 40% by volume), vortexed, centrifuged (10,000 rpm, 15 minutes), and the supernatant was discarded. The precipitate was again dissolved in 30 mL of 4.5 M aqueous sodium chloride solution, and 20 mL of ethanol was added (final concentration: 40% by volume). The mixture was centrifuged and stirred, and the supernatant was discarded. The purified precipitate was dissolved in water and desalted in a centrifuge tube equipped with an ultrafiltration membrane (Amicon Ultra-15, MWCO 100,000). The resulting solution was freeze-dried to obtain a powder sample. 10 mg of the powder sample was dissolved in water to a constant volume of 5 mL, and filtered through a 0.45 μm filter to obtain a cartilage proteoglycan sample solution.

[0030] (Preparation of cartilage proteoglycan standard solution) "Proteoglycan, derived from salmon nasal cartilage" (Wako) was used as a standard substance for cartilage proteoglycan. Approximately 10 mg of this standard substance was dissolved in water to a final volume of 5 mL, and filtered through a 0.45 μm filter to prepare a cartilage proteoglycan standard solution.

[0031] (Confirmation of cartilage proteoglycan peaks by chondroitinase treatment) Cartilage proteoglycan sample solution and cartilage proteoglycan standard solution were each placed in a 40 μL microtube. 40 μL of 0.2 M Tris-acetate buffer (pH 8.0) and 20 μL of chondroitinase ABC solution adjusted to 0.1 U were added to each microtube and stirred. After heating at 37°C for 16 hours, the samples were inactivated in a boiling water bath for 3 minutes to obtain an enzyme-treated group. On the other hand, an enzyme-untreated group was prepared by adding 20 μL of 0.2 M Tris-acetate buffer (pH 8.0) instead of the chondroitinase ABC solution (total volume: 60 μL). Using 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride as the mobile phase, the enzyme-treated and enzyme-untreated groups were subjected to gel filtration HPLC analysis under the following conditions. Injector: Primeaide 1210 Autosampler (Hitachi High-Tech Corporation) Sample injection volume: 20 μL Pump: Primeaide 1110 Pump (Hitachi High-Tech Corporation) 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 (Shoko Science Co., Ltd.) Column temperature: 40°C UV detector: Primeaide 1410 UV-detector (Hitachi High-Tech Corporation) Measurement wavelength: 204 nm

[0032] (Test Results) Figures 1 and 2 show chromatograms obtained by subjecting a cartilage proteoglycan standard solution to gel filtration HPLC analysis before and after treatment with chondroitinase. Figures 3 and 4 show chromatograms obtained by subjecting a cartilage proteoglycan sample solution to gel filtration HPLC analysis before and after treatment with chondroitinase. As shown in Figures 1 and 2, the peak observed in the enzyme-untreated group of the cartilage proteoglycan standard solution disappeared in the enzyme-treated group, confirming that this peak is derived from cartilage proteoglycan. Similarly, the peak observed in the enzyme-untreated group of the cartilage proteoglycan sample solution disappeared in the enzyme-treated group, confirming that this peak represents cartilage proteoglycan. The following tests were performed assuming that the peak detected around retention times of 12 to 16 minutes from the chromatograms shown in Figures 1 to 4 represents proteoglycan. In the chromatograms obtained in each test, the proteoglycan peaks are indicated by arrows.

[0033] 3. Preparation of Purified Cartilage Proteoglycan and HPLC Analysis Using It (Investigation of the Type and Concentration of Salt and Alcohol) [Example 1] 10 mg of the above-described cartilage proteoglycan-containing powder was collected and added to a 2.0 M aqueous sodium chloride solution at a concentration of 2.0 mg / mL to prepare a liquid composition. The liquid composition was centrifuged (15,000 rpm, 5 minutes), and 500 μL of the supernatant was collected in a microtube. 333 μL of ethanol (final concentration: 40% by volume) was added to the mixture, vortexed, and centrifuged (15,000 rpm, 30 minutes), and the supernatant was discarded. The precipitate was again dissolved by adding 500 μL of 2.0 M aqueous sodium chloride solution, and 333 μL of ethanol (final concentration: 40% by volume) was added. The mixture was stirred and centrifuged, and the supernatant was discarded. Then, 500 μL of 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride was added to the precipitate, and the mixture was filtered through a 0.45 μm filter to obtain a purified cartilage proteoglycan solution. 10 mg of the cartilage proteoglycan-containing 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.

[0034] The purified cartilage proteoglycan solution and the untreated solution were subjected to gel filtration HPLC analysis under the following conditions: Injector: Primeide 1210 Autosampler (Hitachi High-Tech Corporation) Sample injection volume: 20 μL Pump: Primeide 1110 Pump (Hitachi High-Tech Corporation) 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 (Shoko Science Co., Ltd.) Column temperature: 40°C UV detector: Primeide 1410 UV-detector (Hitachi High-Tech Corporation) Wavelength: 204 nm

[0035] [Examples 2 to 10] A cartilage proteoglycan-containing powder was dissolved in an aqueous solution of the salt shown below, the resulting liquid composition was centrifuged, 500 μL of the supernatant was collected in a microtube, and the alcohol shown below was added to this, but other than this, purified cartilage proteoglycan solutions were obtained in the same manner as in Example 1. The obtained purified cartilage proteoglycan solutions and the untreated solution were subjected to gel filtration HPLC analysis in the same manner as in Example 1.

[0036] The results are shown in Figures 5 to 15. As shown in Figure 5, in the chromatogram obtained by subjecting the untreated solution to gel filtration HPLC, peaks of cartilage proteoglycan and impurities were confirmed. On the other hand, as shown in Figures 6 to 13, when the cartilage proteoglycan-containing powder was dissolved in solutions of various salt concentrations and then precipitated with ethanol, the cartilage proteoglycan peak was isolated or the degree of separation was improved in all cases, demonstrating that cartilage proteoglycan could be analyzed without being affected by impurities. Furthermore, as shown in Figures 14 to 15, when precipitation was performed with different concentrations of ethanol or different types of alcohol at different concentrations, the cartilage proteoglycan peak was isolated or the impurity peaks were almost completely removed in all cases, demonstrating that cartilage proteoglycan could be analyzed without being affected by impurities.

[0037] 4. Quantification of Cartilage Proteoglycans in Salmon Nasal Cartilage [Example 11] 50 mg of the above-mentioned cartilage proteoglycan-containing powder was collected and dissolved in 4.5 M sodium chloride solution, and the volume was adjusted to 25 mL. The sample solution was centrifuged (15,000 rpm, 5 minutes), and 500 μL of the supernatant was collected in a microtube. 333 μL of ethanol (final concentration: 40% by volume) was added, vortexed, and centrifuged (15,000 rpm, 30 minutes). The supernatant was discarded. Another 500 μL of 4.5 M sodium chloride solution was added to dissolve the sample, and 333 μL of ethanol (final concentration: 40% by volume) was added. The mixture was stirred and centrifuged, and the supernatant was discarded. The precipitate was then diluted with 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride, adjusted to 5 mL, and filtered through a 0.45 μm filter to obtain a purified cartilage proteoglycan solution. "Proteoglycan, derived from salmon nasal cartilage" (Wako) was used as a standard substance for cartilage proteoglycan. 10 mg of this standard substance was dissolved in 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride to a final volume of 5 mL, and diluted with the phosphate buffer to 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL. The solution was then filtered through a 0.45 μm filter to prepare a dilution series of cartilage proteoglycan standard solutions.

[0038] A dilution series of the purified cartilage proteoglycan solution and the cartilage proteoglycan standard solution was subjected to gel filtration HPLC analysis under the following conditions. Injector: Primeide 1210 Autosampler (Hitachi High-Tech Corporation) Sample injection volume: 50 μL Pump: Primeide 1110 Pump (Hitachi High-Tech Corporation) 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 (Shoko Science Co., Ltd.) Column temperature: 40°C UV detector: Primeide 1410 UV-detector (Hitachi High-Tech Corporation) Wavelength: 204 nm A calibration curve was created from the peak area of ​​the chromatogram obtained for each dilution of the standard solution and the concentration of the standard substance in each dilution. The created calibration curve is shown in Figure 16. Furthermore, the area of ​​the peak in the chromatogram obtained for the purified cartilage proteoglycan solution was applied to the calibration curve, and the amount of cartilage proteoglycan was determined to be 48.4 mg / 100 mg. Figure 17 shows the chromatogram of the purified cartilage proteoglycan solution.

[0039] 5. Quantification of Cartilage Proteoglycan in Food Compositions [Examples 12-15] (Cartilage Proteoglycan Purification) Commercially available products A (tablets), B (tablets), C (tablets), and D (granules) containing cartilage proteoglycan were crushed in a mortar, and 300 mg (Commercially available product A), 1160 mg (Commercially available product B), 380 mg (Commercially available product C), and 3000 mg (Commercially available product D) of the crushed product were dissolved in 4.5 M sodium chloride solution and adjusted to a constant volume of 25 mL. The sample solution was centrifuged (15,000 rpm, 5 minutes), and 500 μL of the supernatant was transferred to a microtube. 333 μL of ethanol (final concentration: 40% by volume) was added to the mixture, which was then vortexed and centrifuged (15,000 rpm, 30 minutes). The supernatant was then discarded. The precipitate was dissolved in 500 μL of 4.5 M aqueous sodium chloride solution, 333 μL of ethanol (final concentration: 40% by volume) was added, and the mixture was stirred and centrifuged. The supernatant was discarded. The precipitate was then diluted to 5 mL with 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride and filtered through a 0.45 μm filter to obtain a purified cartilage proteoglycan solution. The same amount of commercially available ground product was dissolved in 4.5 M sodium chloride solution, adjusted to 25 mL, and centrifuged (15,000 rpm, 5 minutes). The resulting supernatant (500 μL) was then diluted to 5 mL and filtered through a 0.45 μm filter to obtain a raw cartilage proteoglycan solution.

[0040] (Quantification of proteoglycan) The purified cartilage proteoglycan solution obtained from each product was subjected to gel filtration HPLC analysis in the same manner as in Example 11. Figures 18 to 21 show chromatograms of the purified cartilage proteoglycan solution obtained from commercial products A to D and the untreated cartilage proteoglycan solution. As shown in Figures 18(B) to 21(B), no peaks other than cartilage proteoglycan were observed in the chromatograms of the purified cartilage proteoglycan solutions obtained from any of the commercial products. The peak areas of the obtained chromatograms were applied to the calibration curve prepared in Example 11, and the amounts of cartilage proteoglycan were 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).

[0041] Example 15 (Purification of Cartilage Proteoglycan) 1560 mg of the contents of commercially available product E (soft capsule) containing cartilage proteoglycan was collected in a glass test tube. 10 mL of acetone was added, mixed, and then centrifuged and the supernatant discarded. This procedure was repeated three times to remove hydrophobic components. After drying under reduced pressure, the residue was dissolved in 4.5 M sodium chloride solution and adjusted to a constant volume of 25 mL. The sample solution was centrifuged (15,000 rpm, 5 minutes), and 500 μL of the supernatant was collected in a microtube. 333 μL of ethanol (final concentration: 40% by volume) was added, stirred with a vortex, centrifuged (15,000 rpm, 30 minutes), and the supernatant was discarded. 500 μL of 4.5 M sodium chloride aqueous solution was added again to dissolve the contents, and 333 μL of ethanol (final concentration: 40% by volume) was added. The mixture was stirred and centrifuged, and the supernatant was discarded. The precipitate was then diluted to 5 mL with 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride and filtered through a 0.45 μm filter. The residue from the same amount of commercially available product was dissolved in 4.5 M sodium chloride solution and diluted to 25 mL. The sample solution was centrifuged (15,000 rpm, 5 minutes) to obtain a supernatant (500 μL), which was then diluted to 5 mL and filtered through a 0.45 μm filter to obtain an untreated cartilage proteoglycan solution.

[0042] (Gel filtration HPLC) The purified cartilage proteoglycan solution obtained from product E was subjected to gel filtration HPLC analysis in the same manner as in Example 11. Figure 22 shows chromatograms of the purified cartilage proteoglycan solution obtained from commercial product E and the untreated cartilage proteoglycan solution. As shown in Figure 22 (B), no peaks other than cartilage proteoglycan were observed in the chromatogram. The peak volumes of the obtained chromatograms were applied to the calibration curve prepared in Example 11, and the amount of cartilage proteoglycan was determined to be 6.2 mg / 1 tablet (commercial product E).

[0043] The present invention provides a simple and inexpensive method for analyzing cartilage proteoglycans, which is expected to be useful for quality control of proteoglycans in the food, cosmetics, pharmaceutical, and other industries.

Claims

1. A method for analyzing proteoglycans in a sample, comprising: (1) immersing or adding the sample to an aqueous solution containing salt, and adding alcohol to the resulting liquid composition in an amount to a final concentration of 10 to 60% by volume to precipitate the proteoglycans; or immersing or adding the sample to an aqueous solution containing salt and 10 to 60% by volume of alcohol to precipitate the proteoglycans; and (2) recovering the resulting precipitate and analyzing the proteoglycans.

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

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

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

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

6. The method of any one of claims 1 to 5, wherein the salt comprises one or a combination of two or more selected from 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 a combination of two or more selected from ethanol and isopropanol.

8. The method according to any one of claims 1 to 7, wherein the measurement of the proteoglycans is carried out by HPLC analysis.

9. The method according to any one of claims 1 to 8, wherein step (1) is repeated multiple times.

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