Methods for Analysing Proteoglycans

Abstract

The present invention provides a method for analyzing proteoglycan, which can easily analyze proteoglycan in a sample. A sample containing proteoglycan is immersed in or added to an aqueous solution containing a salt, and a relatively low specific concentration of alcohol is added to the resulting liquid composition to obtain a purified precipitate, which is then subjected to proteoglycan analysis.

Description

[Technical field]

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

[0002] Proteoglycans are a type of glycoprotein in the broad sense of the term, 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 joints and beautifying the skin, 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 to establish a method for easily analyzing proteoglycans in products that contain proteoglycans, their raw materials, or intermediate products.

[0004] However, products containing proteoglycan 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 proteoglycan, they contain collagen or other contaminating proteins derived from tissues, or contaminants that are added to foods, such as supplements, and there is a problem that the presence of these contaminants makes it difficult to measure proteoglycan.

[0005] In this regard, a method for quantifying proteoglycan 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, and therefore a simpler method is desired.

[0006] A method for quantifying proteoglycan using antibodies has also been disclosed (Patent Document 2), but this method is expensive and problems have been pointed out regarding the feasibility of quantification when contaminating proteins are present in the sample.

[0007] In addition, a method for analyzing proteoglycans has been proposed, which includes a step of passing a solution containing proteoglycan through a strongly basic anion exchange resin, and then passing salt ion solutions containing chloride ions at different concentrations through the resin in sequence (Patent Document 3). However, for industrial-level use, a simpler method is still desired. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent Publication 2018-151207 [Patent Document 2] Special table 2016-160226 [Patent Document 3] Special table 2020-134435 [Non-patent literature]

[0009] [Non-Patent Document 1] Journal of the Japanese Society of Food Chemistry, Vol.29(2), 104-113(2022) Summary of the Invention [Problem to be solved by the invention]

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

[0011] The inventors of the present invention discovered that when a sample containing proteoglycan was immersed in or added to an aqueous solution containing salt, and then 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 contaminating substances, thereby completing the present invention.

[0012] Based on this finding, the present invention provides the following method. [1] A method for analyzing proteoglycans in a sample, comprising: (1) immersing or adding the sample to an aqueous solution containing a salt, and adding alcohol to the resulting liquid composition in an amount to give a final concentration of 10 to 60% by volume to precipitate proteoglycan, or immersing or adding the sample to an aqueous solution containing a salt and 10 to 60% by volume of alcohol to precipitate proteoglycan; (2) recovering the resulting precipitate and analyzing the proteoglycans; A method comprising: [2] The method according to [1], wherein the alcohol is added to the liquid composition in an amount to give a final concentration of 20 to 60% by volume. [3] The method according to [1], wherein the alcohol is added to the liquid composition in an amount to give a final concentration of 30 to 60% by volume. [4] The method according to any one of [1] to [3], wherein the aqueous solution containing the salt contains the salt at a concentration of from 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 from 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 the group consisting of 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 step (1) allows proteoglycan in a sample to be selectively precipitated without relying on steps requiring 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 proteoglycan while avoiding the influence of contaminating substances. Without wishing to be bound by theory, the phenomenon that occurs in step (1) can be briefly explained as follows: glycosaminoglycans such as chondroitin sulfate that constitute proteoglycans become electrically stable or form cross-linked structures due to cations derived from salts coexisting with proteoglycans, thereby reducing their solubility; when a relatively low specific concentration of alcohol is added to a solution containing proteoglycans in such a state, the polarity of the solution is reduced to a certain level, and the proteoglycans, which are macromolecules, are preferentially precipitated. [Brief description of the drawings]

[0014] [Figure 1] The chromatogram obtained by subjecting a standard solution of cartilage proteoglycan (PG) to gel filtration HPLC analysis before treatment with chondroitinase is shown. [Diagram 2] The chromatogram shown is obtained by subjecting a standard solution of cartilage proteoglycan (PG) to gel filtration HPLC analysis after treating it with chondroitinase. [Diagram 3] The figure shows a chromatogram obtained by subjecting a cartilage proteoglycan (PG) sample solution to gel filtration HPLC analysis before treatment with chondroitinase. [Figure 4] The chromatogram obtained by subjecting a cartilage proteoglycan (PG) sample solution to gel filtration HPLC analysis after treatment with chondroitinase is shown. [Diagram 5]The figure 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. [Figure 6] A proteoglycan (PG) powder sample was dissolved in a 2.0 M aqueous sodium chloride solution, and ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 7] A proteoglycan (PG) powder sample was dissolved in 4.5 M sodium chloride aqueous solution, and ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 8] A proteoglycan (PG) powder sample was dissolved in a saturated aqueous solution of sodium chloride, and ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 9] A proteoglycan (PG) powder sample was dissolved in 0.5 M calcium chloride aqueous solution, and ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 10] A proteoglycan (PG) powder sample was dissolved in 1.0 M calcium chloride aqueous solution, and ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 11] A proteoglycan (PG) powder sample was dissolved in a 2.0 M calcium chloride aqueous solution, and ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 12]A proteoglycan (PG) powder sample was dissolved in a 2.0 M potassium chloride aqueous solution, and ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 13] A proteoglycan (PG) powder sample was dissolved in 2.0 M aqueous sodium acetate solution, and ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 14] A proteoglycan (PG) powder sample was dissolved in 0.5 M calcium chloride aqueous solution, and isopropanol was added to a final concentration of 30% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 15] A proteoglycan (PG) powder sample was dissolved in 4.5 M sodium chloride aqueous solution, and ethanol was added to a final concentration of 60% by volume to produce a precipitate. The precipitate was then dissolved in the mobile phase to prepare a proteoglycan sample solution. The chromatogram obtained was then subjected to gel filtration HPLC analysis. [Figure 16] The figure 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. [Figure 17] Gel filtration HPLC chromatogram of cartilage proteoglycan solution from salmon nasal cartilage sample. [Figure 18] 18 shows gel filtration HPLC chromatograms of untreated or purified cartilage proteoglycan solutions obtained from commercial product A. Fig. 18(A) shows a gel filtration HPLC chromatogram of an untreated cartilage proteoglycan solution, and Fig. 18(B) shows a gel filtration HPLC chromatogram of a purified cartilage proteoglycan solution. [Figure 19]19 shows gel filtration HPLC chromatograms of untreated or purified cartilage proteoglycan solutions obtained from commercial product B. Fig. 19(A) shows a gel filtration HPLC chromatogram of an untreated cartilage proteoglycan solution, and Fig. 19(B) shows a gel filtration HPLC chromatogram of a purified cartilage proteoglycan solution. [Figure 20] 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 an untreated cartilage proteoglycan solution, and Figure 20(B) shows a gel filtration HPLC chromatogram of a purified cartilage proteoglycan solution. [Figure 21] 21 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 an untreated cartilage proteoglycan solution, and Figure 21(B) shows a gel filtration HPLC chromatogram of a purified cartilage proteoglycan solution. [Figure 22] 22 shows gel filtration HPLC chromatograms of untreated or purified cartilage proteoglycan solutions obtained from commercial product E. Figure 22(A) shows a gel filtration HPLC chromatogram of an untreated cartilage proteoglycan solution, and Figure 22(B) shows a gel filtration HPLC chromatogram of a purified cartilage proteoglycan solution. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in detail below with reference to the preferred embodiments. However, 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 proteoglycan by subjecting a sample to a predetermined pretreatment to selectively precipitate proteoglycan and using the resulting purified precipitate.

[0017] In the present specification, the term "sample" is not particularly limited except that it contains proteoglycan, and may be derived from tissues of any organism, such as fish, mollusks, birds, or mammals, such as bones, muscle fibers, or skin, and proteoglycans derived from cartilage or the surrounding areas of cartilage are particularly preferred. The term "sample" may also include a disrupted tissue containing such proteoglycan, a squeezed product of the disrupted tissue, or an extract of the disrupted or squeezed product. The term "sample" also includes products such as foods, cosmetics, and pharmaceuticals that are manufactured using the disrupted tissue, squeezed product, or extract. Thus, the "sample" may take various forms, such as a solid, powder, granule, paste, or liquid.

[0018] The sample pretreatment is as follows: (1) immersing or adding a sample to an aqueous solution containing a salt; (2) adding alcohol to the resulting liquid composition in an amount to give a specific final concentration to precipitate the proteoglycan; Includes. The salt used in step (1) may be any salt containing a metal ion that can form a salt with glycosaminoglycan such as chondroitin sulfate, which constitutes proteoglycan in the solution, and examples thereof 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. These salts may be used alone or in combination, and it is particularly preferred to use sodium chloride or calcium chloride since it is desirable for the aqueous solution to have a neutral pH.

[0019] The salt concentration in the aqueous solution may be selected according to the final concentration of the alcohol and the type of salt, which will be described later, and is usually selected from the range of 0.5M to saturation concentration. In addition, in the case of a salt that generates monovalent ions such as sodium chloride, the salt concentration is preferably selected from 2.0M to saturation concentration, more preferably 3.0M to saturation concentration, even more preferably 4.0M to saturation concentration, and particularly preferably 4.5M to saturation concentration. On the other hand, in the case of a salt that generates polyvalent ions (particularly divalent ions) such as calcium chloride, the salt concentration is preferably selected from 0.5M to 3.0M, more preferably 0.5M to 2.0M, since proteoglycan precipitation can be obtained at a lower concentration. However, calcium chloride increases viscosity at high concentrations, so the salt concentration is more preferably selected from 0.5M to 2.0M.

[0020] The pH of the aqueous solution containing the salt is preferably near neutral in terms of minimizing damage to sugar chains. Specifically, the pH is preferably 5 to 10, more preferably 6 to 8. However, when the sample is an acidic aqueous solution, the aqueous solution containing the salt may be adjusted to, for example, pH 4 to 6, preferably pH 5 to 5.5. When the sample is an alkaline aqueous solution, the aqueous solution containing the salt may be adjusted to, for example, pH 8 to 11, preferably pH 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.). In order to promote dissolution and / or extraction of proteoglycan, the solution may be heated to, for example, 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, such as ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-methyl-2-butanol, ethylene glycol, glycerol, etc. In particular, ethanol or isopropanol, which are commonly used, are preferred. The amount of alcohol added may vary depending on the type and concentration of the salt used, usually to give 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 proteoglycan may be carried out separately, or the steps 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 will be demonstrated in the examples described below, proteoglycans can be selectively precipitated by adding alcohol to the above-mentioned specified concentration, and the resulting purified precipitate can be used to measure proteoglycans while avoiding the influence of contaminating substances. In addition, since the measurement sample is obtained by removing impurities by this pretreatment, a step of avoiding impurities is not necessary in the analysis of proteoglycans using the purified precipitate obtained, and various existing methods for analyzing proteoglycans can be used. For example, qualitative or quantitative analysis of proteoglycans can be performed using gel filtration HPLC (see, for example, Patent Document 1 and Non-Patent Document 1), HPLC using an anion exchange resin (see Patent Document 3), electrophoresis (see, for example, Non-Patent Document 1), detection with anti-proteoglycan antibodies (polyclonal antibodies, monoclonal antibodies) (see, for example, Patent Document 2), etc.

[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 may be used. For example, in the case of a UV detector, detection can be performed at a UV wavelength of 200 to 280 nm, and in particular, 200 to 220 nm is preferably selected. A differential refractive index detector can also be used to detect cartilage proteoglycan. EXAMPLES

[0027] The present invention will be described 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 500g of nasal cartilage was prepared from the head of salmon frozen at -25℃, and was finely crushed into chips using an electric mixer to serve as the raw material. The starting material was added to 1250g of water, and extracted at 95℃ for 3.5 hours while stirring. The resulting extract was filtered through a stainless steel mesh (150μm) to remove insoluble matter. The extract was then concentrated using a rotary evaporator, and the concentrated liquid was powdered using a vacuum freeze dryer to obtain a cartilage proteoglycan-containing powder.

[0029] 2. Confirmation of cartilage proteoglycan peaks in gel filtration HPLC analysis (Preparation of cartilage proteoglycan sample solution) A liquid composition was prepared by collecting 350 mg of cartilage proteoglycan-containing powder and adding it to a 4.5 M sodium chloride aqueous solution at a concentration of 10 mg / mL. 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 thereto (final concentration 40% by volume), stirred with a vortex, centrifuged (10,000 rpm, 15 minutes), and the supernatant was discarded. Again, 30 mL of 4.5 M sodium chloride aqueous solution was added to the precipitate to dissolve it, and 20 mL of ethanol was added (final concentration 40% by volume), centrifuged, 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), and 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) As a standard substance for cartilage proteoglycan, "Proteoglycan, derived from salmon nasal cartilage" (Wako) was used. Approximately 10 mg of this standard substance was dissolved in water to a constant volume of 5 mL, and filtered through a 0.45 μm filter to prepare a cartilage proteoglycan standard solution.

[0031] (Confirmation of cartilage proteoglycan peak by chondroitinase treatment) Cartilage proteoglycan sample solution and cartilage proteoglycan standard solution were collected in 40 μL microtubes. 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, the enzyme-untreated group was prepared by adding 20 μL of 0.2 M Tris-acetate buffer (pH 8.0) (total amount of 60 μL) instead of the chondroitinase ABC solution. Using 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride as the mobile phase, the enzyme-treated group and the enzyme-untreated group were subjected to gel filtration HPLC analysis under the following conditions. Injector: Primaide 1210 Autosampler (Hitachi High-Tech Corporation) Sample injection volume: 20μL Pump: Primaide 1110 Pump (Hitachi High-Tech Corporation) Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride ·Flow rate: 0.5mL / min Separation column: Shodex OH Pak SB-806M HQ (Shoko Science Co., Ltd.) Column temperature: 40℃ UV detector: Primaide 1410 UV-detector (Hitachi High-Tech Corporation) ·Measurement wavelength: 204nm

[0032] (Test Results) Figures 1 and 2 show chromatograms obtained by subjecting a standard solution of cartilage proteoglycan to gel filtration HPLC analysis before and after treatment with chondroitinase, while Figures 3 and 4 show chromatograms obtained by subjecting a sample solution of cartilage proteoglycan 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 in the cartilage proteoglycan standard solution disappeared in the enzyme-treated group, and it was confirmed that this peak originated from cartilage proteoglycan. Similarly, the peak observed in the enzyme-untreated group in the cartilage proteoglycan sample solution disappeared in the enzyme-treated group, and it was confirmed that this peak represents cartilage proteoglycan. From the chromatograms shown in Figures 1 to 4, the peak detected at a retention time of about 12 to 16 minutes represents proteoglycan, and the following tests were carried out. In the chromatograms obtained in each test, the proteoglycan peaks are indicated by arrows.

[0033] 3. Preparation of purified cartilage proteoglycan and its HPLC analysis (examination of the type and concentration of salt and alcohol) [Example 1] 10 mg of the above-mentioned cartilage proteoglycan-containing powder was collected and added to an aqueous solution of 2.0 M sodium chloride 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 thereto, stirred by vortexing, centrifuged (15,000 rpm, 30 minutes), and the supernatant was discarded. Again, 500 μL of an aqueous solution of 2.0 M sodium chloride was added to the precipitate to dissolve it, 333 μL of ethanol (final concentration 40% by volume) was added, stirred, centrifuged, and the supernatant was discarded. Thereafter, 500 μL of a 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride was added to the precipitate, and filtered through a 0.45 μm filter to obtain a purified cartilage proteoglycan solution. Also, 10 mg of the cartilage proteoglycan-containing powder was added to a 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: Primaide 1210 Autosampler (manufactured by Hitachi High-Tech Corporation) · Sample injection volume: 20 μL · Pump: Primaide 1110 Pump (manufactured by 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 (manufactured by Shoko Science Co., Ltd.) · Column temperature: 40 °C · UV detector: Primaide 1410 UV-detector (manufactured by 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 following alcohol was added to this, but other than that, a purified cartilage proteoglycan solution was obtained in the same manner as in Example 1. The resulting purified cartilage proteoglycan solution and the untreated solution were subjected to gel filtration HPLC analysis in the same manner as in Example 1. [Table 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 impurities were confirmed together with cartilage proteoglycan. On the other hand, as shown in Figures 6 to 13, when the powder containing cartilage proteoglycan was dissolved in solutions of various salt concentrations and subjected to ethanol precipitation, the peak of cartilage proteoglycan was isolated or the degree of separation was improved in all cases, indicating that cartilage proteoglycan can be analyzed without being affected by impurities. Furthermore, as shown in Figures 14 and 15, when precipitation treatment was performed using ethanol of different concentrations or different types of alcohol of different concentrations, in each case, the cartilage proteoglycan peak was isolated or the contaminant peaks were almost completely removed, demonstrating that cartilage proteoglycan could be analyzed without being affected by contaminants.

[0037] 4. Quantitative analysis of cartilage proteoglycan in salmon nasal cartilage [Example 11] 50 mg of the above-mentioned cartilage proteoglycan-containing powder was collected, 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, to which 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, and 333 μL of ethanol (final concentration 40% by volume) was added, stirred, centrifuged, and the supernatant was discarded. Thereafter, 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride was added to the precipitate to adjust the volume to 5 mL, and filtered through a 0.45 μm filter to obtain a purified cartilage proteoglycan solution. As a standard substance for cartilage proteoglycan, "Proteoglycan, derived from salmon nasal cartilage" (Wako) was used. 10 mg of this standard substance was dissolved in 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride to a constant volume of 5 mL, and diluted with the phosphate buffer to 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL, and filtered through a 0.45 μm filter to prepare a dilution series of cartilage proteoglycan standard solution.

[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: Primaide 1210 Autosampler (Hitachi High-Tech Corporation) Sample injection volume: 50μL Pump: Primaide 1110 Pump (Hitachi High-Tech Corporation) Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride ·Flow rate: 0.5mL / min Separation column: Shodex OH Pak SB-806M HQ (Shoko Science Co., Ltd.) Column temperature: 40℃ UV detector: Primaide 1410 UV-detector (Hitachi High-Tech Corporation) ·Wavelength: 204nm A calibration curve was created from the peak areas of the chromatograms obtained for each dilution of the standard solution and the concentrations of the standard substances 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 Proteoglycans in Food Compositions [Examples 12 to 15] (Cartilage proteoglycan purification process) Cartilage proteoglycan-containing commercial products A (tablets), B (tablets), C (tablets), and D (granules) were crushed in a mortar, and 300 mg (commercial product A), 1160 mg (commercial product B), 380 mg (commercial product C), and 3000 mg (commercial product D) of the crushed products were collected, 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, to which 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, and 333 μL of ethanol (final concentration 40% by volume) was added, stirred, centrifuged, and the supernatant was discarded. The precipitate was then mixed with 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride to give a final volume of 5 mL, and filtered through a 0.45 μm filter to obtain a purified cartilage proteoglycan solution. The same amount of commercially available pulverized material was dissolved in 4.5 M sodium chloride solution and the final volume was adjusted to 25 mL. The sample solution was centrifuged (15,000 rpm, 5 min), and 500 μL of the resulting supernatant was adjusted to 5 mL and filtered through a 0.45 μm filter to obtain a non-treated cartilage proteoglycan solution.

[0040] (Quantitative determination of proteoglycans) 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 the 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 areas of the peaks in 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 / 1 tablet (commercial product A), 10.3 mg / 1 tablet (commercial product B), 20.8 mg / 1 tablet (commercial product C), and 10.1 mg / 1 packet (commercial product D).

[0041] [Example 15] (Cartilage proteoglycan purification process) 1560 mg of the contents of a commercially available product E (soft capsule) containing cartilage proteoglycan was collected in a glass test tube. After adding 10 mL of acetone and mixing, the capsule was centrifuged and the supernatant was discarded. This operation 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 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, to which 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 solution, and 333 μL of ethanol (final concentration 40% by volume) was added, 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 to make a final volume of 5 mL, and the solution was filtered through a 0.45 μm filter. Also, the residue obtained from the same amount of the commercially available product was dissolved in 4.5 M sodium chloride solution and made to a final volume of 25 mL. The sample solution was centrifuged (15,000 rpm, 5 minutes) to obtain a supernatant of 500 μL, which was made to a final volume of 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. FIG. 22 shows the chromatograms of the purified cartilage proteoglycan solution obtained from commercial product E and the untreated cartilage proteoglycan solution. As shown in FIG. 22(B), no peaks other than cartilage proteoglycan were observed in the chromatogram. The volume of the peak in the obtained chromatogram was 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). [Industrial Applicability]

[0043] The present invention provides a method for analyzing cartilage proteoglycan simply and inexpensively. Such a method is expected to be used for quality control of proteoglycan in the food, cosmetics, pharmaceutical, and other industries.

Claims

1. A method for analyzing proteoglycans in a sample, (1) A step of immersing or adding the sample to an aqueous solution containing salt, and then adding alcohol to the resulting liquid composition in an amount that results in a final concentration of 10 to 60% by volume to precipitate the proteoglycan, or a step of immersing or adding the sample to an aqueous solution containing salt and 10 to 60% by volume of alcohol to precipitate the proteoglycan, (2) A step of collecting the obtained precipitate and analyzing the proteoglycans. Methods that include...

2. The method according to claim 1, comprising the steps of immersing or adding the sample to an aqueous solution containing a salt, adding alcohol to the resulting liquid composition in an amount that results in a final concentration of 20 to 60% by volume to precipitate the proteoglycan, or immersing or adding the sample to an aqueous solution containing a salt and 20 to 60% by volume of alcohol to precipitate the proteoglycan.

3. The method according to claim 1, comprising the steps of immersing or adding the sample to an aqueous solution containing a salt, adding alcohol to the resulting liquid composition in an amount that results in a final concentration of 30 to 60% by volume to precipitate the proteoglycan, or immersing or adding the sample to an aqueous solution containing a salt and 30 to 60% by volume of alcohol to precipitate the proteoglycan.

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

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

6. The method according to any one of claims 1 to 3, wherein the salt comprises one or more combinations selected from lithium salts, sodium salts, potassium salts, calcium salts, and magnesium salts.

7. The method according to any one of claims 1 to 3, 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 3, wherein the measurement of the proteoglycan is performed by HPLC analysis.

9. The method according to any one of claims 1 to 3, wherein step (1) is repeated multiple times.

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