Method for producing proteoglycan

Abstract

Provided is a method for simply and inexpensively producing high-purity proteoglycan from a crushed cartilage tissue product, an extract of the crushed product, or an extract of the crushed product or the extract. The present invention relates to a method for producing a chondroglycan, which comprises the steps of: immersing or adding a crushed cartilage tissue, a squeezed product of the crushed product, or an extract of the crushed product or the squeezed product into an aqueous solution containing a salt, and adding an alcohol to the obtained liquid composition in an amount of a final concentration of 10-60 vol% to precipitate a proteoglycan; alternatively, a crushed product of cartilage tissue, an extract of the crushed product, or an extract of the crushed product or the extract is immersed or added into an aqueous solution containing a salt and an alcohol in an amount of a final concentration of 10-60 vol%, and proteoglycan is precipitated.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for producing a proteoglycan. BACKGROUND

[0002] A proteoglycan is a kind of a general glycoprotein in which a sulfated polysaccharide called glycosaminoglycan such as chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, keratan sulfate, etc. is covalently bound to a core protein that forms a core structure. Proteoglycans are widely present in the skin, cartilage, etc. of fish, mollusk, birds, and mammals, and as a main component of an extracellular matrix, form a complex with matrix proteins such as fibrous hyaluronic acid and type II collagen, and play an important role in maintaining the water retention and elasticity of tissues.

[0003] In recent years, various physiological functions of proteoglycans have been discovered, and the application of proteoglycans as a raw material for functional foods and cosmetics has been developed, and further, the application of proteoglycans in pharmaceuticals has been studied, and proteoglycans with high purity are required.

[0004] However, as described above, proteoglycans have a complex structure as a glycoprotein, and exist in a complex with hyaluronic acid and type II collagen, etc., and thus it is difficult to extract proteoglycans while maintaining the original structure, and in the case of pharmaceuticals, functional foods, and cosmetics, only chondroitin sulfate is extracted from the core protein of proteoglycans and applied.

[0005] On the other hand, attempts have also been made to recover proteoglycans without decomposing the core protein. For example, various methods have been reported, such as a method of extracting from the nasal cartilage of salmon using a guanidine hydrochloride solution (Patent Literature 1), a method of extracting using an acetic acid solution (Patent Literature 2), a method of extracting using an alkali solution such as sodium hydroxide (Patent Literature 3), a method of extracting using an aqueous solution to which a surfactant such as saponin or sucrose fatty acid ester is added (Patent Literature 4), and a method of extracting using an aqueous solution to which a protease is added in an acidic solution containing acetic acid or the like (Patent Literature 5).

[0006] In addition, a method of obtaining proteoglycans in a state of forming a complex with hyaluronic acid and type II collagen by performing hot water extraction on a crushed product of cartilage tissue has also been reported (Non-Patent Literature 1).

[0007] However, in the method described in Patent Literature 1, after diluting the crude proteoglycan extract obtained by extraction with a guanidine hydrochloride solution with 2 times the amount of water, 3 times the amount of ethanol (about 75%) and 1.3% (w / v) of potassium acetate are added, and a precipitate fraction is obtained by centrifugal separation, but the proteoglycan purity in the precipitate fraction is low, and other impurities are also contained. Therefore, in the method described in Patent Literature 1, purification with an anion exchange resin is also performed to improve the purity.

[0008] In addition, in the method that patent documentation 2 is recorded in writing, also in the crude proteoglycan extract that obtains with acetic acid extraction, add the salt saturated ethanol (about 75%) of 3 times of amounts, carry out centrifugation, obtain the semisolid thing after proteoglycan concentration, this also is that the purity of proteoglycan is low, and comprises other impurities.Therefore, in the method that patent documentation 2 is recorded in writing, also purify and improve purity with the cellulose membrane of prescribed molecular weight cut-off.In addition, in using the extraction of acid, also there is the problem that protein decomposition and the sugar chain that is combined with proteoglycan are easily damaged.

[0009] In addition, in the method that patent documentation 3 is recorded in writing, the crude proteoglycan extract that obtains with alkaline solutions such as sodium hydroxide is extracted and centrifuged, and the liquid phase that comprises proteoglycan is reclaimed, but this liquid phase also comprises a lot of impurities.Therefore, in the method that patent documentation 3 is recorded in writing, also further this liquid phase is carried out ultrafiltration to improve purity.In addition, in using the extraction of alkali, also there is the problem that protein decomposition and the sugar chain that is combined with proteoglycan are easily damaged.

[0010] In addition, in the method for patent documentation 4 records, the crude proteoglycan extract obtained by extracting saponin or sucrose fatty acid ester aqueous solution is centrifuged and obtained comprising the liquid phase of proteoglycan, but this liquid phase also comprises a lot of impurities.Therefore, in the method for patent documentation 4 records, also further this liquid phase is improved purity with the ultrafiltration membrane of specific molecular cut-off.

[0011] In addition, the method described in Patent Document 5 involves extracting a crude proteoglycan extract with a solution containing protease in an acidic solution, removing lipids using an oil absorbent pad, and then using a hollow fiber membrane with a molecular weight cutoff of 50,000 to remove substances with a molecular weight of 50,000 or less to improve purity. Furthermore, the use of protease also presents the problem of decomposing the core protein of the proteoglycan.

[0012] Therefore, existing methods for producing proteoglycans are difficult to obtain high-purity proteoglycans, or in order to improve the purity, additional steps such as anion exchange resins, ultrafiltration membranes or hollow fiber membranes are required. Therefore, there is a demand for a method for producing high-purity proteoglycans using a simpler process.

[0013] Furthermore, there is a need for a method for obtaining proteoglycan in a complex state with hyaluronic acid and type II collagen, and a method for obtaining highly pure proteoglycan from the obtained complex.

[0014] Prior art literature

[0015] Patent Literature

[0016] Patent Document 1: Japanese Patent Application Laid-Open No. 2001-172296

[0017] Patent Document 2: Japanese Patent Application Laid-Open No. 2002-69097

[0018] Patent Document 3: Japanese Patent No. 4219974

[0019] Patent Document 4: Japanese Patent Application Laid-Open No. 2014-9164

[0020] Patent Document 5: Japanese Patent Application Laid-Open No. 2020-127397

[0021] Non-patent literature

[0022] Non-patent document 1 Applied Carbohydrate Science, Vol. 7, No. 1, 23-28 (2017) Summary of the Invention

[0023] Problems to be solved by the invention

[0024] The present invention aims to provide a method for producing high-purity proteoglycans simply and inexpensively using a crushed cartilage tissue product, a squeezed product of the crushed product, or an extract of the crushed product or squeezed product. Furthermore, in one embodiment, the present invention aims to provide a method for producing high-purity proteoglycans using a complex of proteoglycans extracted from cartilage tissue and type II collagen, etc.

[0025] Means for solving problems

[0026] In order to solve the above-mentioned problems, the present inventors studied various methods for extracting proteoglycans and found that when a crushed cartilage tissue, an extract of the crushed material, or an extract of the crushed material or the squeezed material is immersed in or added to an aqueous solution containing salt, and alcohol is added to the resulting liquid composition at a relatively low specific concentration, surprisingly, proteoglycans are specifically precipitated, and a precipitate of proteoglycan with high purity can be obtained, thereby completing the present invention.

[0027] That is, the present invention provides the following method for producing proteoglycan.

[0028] [1] A method for producing proteoglycan, comprising: immersing or adding a crushed cartilage tissue, a squeezed product of the crushed material, or an extract of the crushed material or squeezed material in 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 a crushed cartilage tissue, a squeezed product of the crushed material, or an extract of the crushed material or squeezed material in an aqueous solution containing a salt and an alcohol in an amount to give a final concentration of 10 to 60% by volume to precipitate proteoglycan;

[0029] The step of recovering the generated precipitate.

[0030] [2] The method according to [1], the salt concentration of the aqueous solution containing the salt is selected from the range of 0.5 M to saturated concentration, depending on the kind of the salt, and the amount or content of the alcohol to be added.

[0031] [3] The method according to [1] or [2], the salt concentration of the aqueous solution containing the salt is 1.5 M to saturated concentration; the alcohol is added to the composition in an amount to become a final concentration of 40 to 60% by volume; or the aqueous solution contains the alcohol in an amount to become a final concentration of 40 to 60% by volume.

[0032] [4] The method according to [1] or [2], the salt concentration of the aqueous solution containing the salt is 2.5 M to saturated concentration; the alcohol is added to the composition in an amount to become a final concentration of 30 to 60% by volume; or the aqueous solution contains the alcohol in an amount to become a final concentration of 30 to 60% by volume.

[0033] [5] The method according to [1] or [2], the salt concentration of the aqueous solution containing the salt is 3.0 M to saturated concentration; the alcohol is added to the composition in an amount to become a final concentration of 20 to 60% by volume; or the aqueous solution contains the alcohol in an amount to become a final concentration of 20 to 60% by volume.

[0034] [6] The method according to any one of [1] to [5], the pH of the aqueous solution containing the salt is 5 to 10.

[0035] [7] The method according to any one of [1] to [6], the extract is a water extract of the broken or pressed product, and the pH of the aqueous solution containing the salt is 6.5 to 7.5.

[0036] [8] The method according to any one of [1] to [7], the cartilage tissue is salmon nasal cartilage tissue.

[0037] [9] The method according to any one of [1] to [8], the salt comprises one or a combination of two or more selected from the group consisting of lithium salt, sodium salt, potassium salt, magnesium salt, and calcium salt.

[0038]

[10] The method according to any one of [1] to [9], the alcohol comprises one or a combination of two or more selected from the group consisting of ethanol and 2-propanol.

[0039] The present application is a breakthrough method that does not rely on a process requiring additional devices such as anion exchange resin, ultrafiltration membrane, or hollow fiber membrane, and that obtains high-purity proteoglycans by adjusting the concentration of alcohol contained in a sample derived from cartilage tissue in the presence of a salt.

[0040] In the present application, by the cation derived from the salt coexisting with the proteoglycan, the chondroitin sulfate constituting the proteoglycan becomes in an electrically stable state, or the solubility is reduced due to the formation of a crosslinked structure. Further, in the above complex obtained by water extraction or the like, by the above cation, the interaction of the proteoglycan with the matrix protein such as type II collagen and the like is inhibited. If an alcohol of a specific concentration which is relatively low as described later is added to a solution in which the proteoglycan exists in this state, the polarity of the solution is reduced to a prescribed level, and in this state, the proteoglycan as a macromolecule is preferentially precipitated, and thus the purification of the proteoglycan becomes possible. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is a chromatogram showing the results of HPLC analysis of "Proteoglycan HG-100" (manufactured by Nippon Shinyaku Co., Ltd.) using ultrapure water or 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride as a mobile phase. It is shown that although the proteoglycan forms a complex with type II collagen in ultrapure water, the complex is dissociated by a salt.

[0042] Figure 2 is a chromatogram showing the results of HPLC analysis of a precipitate obtained by adding an aqueous solution to which no ethanol (EtOH) was added or ethanol (EtOH) of different concentrations having a final concentration of 40 to 70% by volume to an aqueous solution in which "Proteoglycan HG-100" (manufactured by Nippon Shinyaku Co., Ltd.) was added to 2 M sodium chloride aqueous solution. In each chromatogram, the peak indicated by an arrow is a peak of the proteoglycan (PG). Figure 2 (A) is a chromatogram of an untreated sample; Figure 2 (B) is a chromatogram of a sample obtained by adding 70% by volume of ethanol (EtOH); Figure 2 (C) is a chromatogram of a sample obtained by adding 60% by volume of ethanol (EtOH); Figure 2 (D) is a chromatogram of a sample obtained by adding 40% by volume of ethanol (EtOH).

[0043] Figure 3 is a chromatogram showing the results of HPLC analysis of a precipitate obtained by adding ethanol (EtOH) having a final concentration of 35% by volume in an amount to an aqueous solution in which "Proteoglycan HG-100" (manufactured by Nippon Shinyaku Co., Ltd.) was added to 2 M, 2.5 M, 3 M, or 4 M sodium chloride aqueous solution. In each chromatogram, the peak indicated by an arrow is a peak of the proteoglycan (PG). Figure 3 (A) is a chromatogram of an untreated sample;

[0044] Figure 3 (B) is a chromatogram of a sample obtained by adding to 2 M sodium chloride aqueous solution; Figure 3(C) is a chromatogram of a sample obtained by adding to a 2.5 M sodium chloride aqueous solution; Figure 3 (D) is a chromatogram of a sample obtained by adding a 3 M sodium chloride aqueous solution; Figure 3 (E) is a chromatogram of a sample added to a 4 M sodium chloride aqueous solution. The area in parentheses indicates the ratio of the PG peak area in each graph with EtOH added to the area of ​​the PG peak in the graph without EtOH added as 100.

[0045] Figure 4 This is a chromatogram showing the results of HPLC analysis of the aqueous solution of the precipitate obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuin Co., Ltd.) to a saturated sodium chloride aqueous solution, adding ethanol in an amount to a final concentration of 40 volume %, and performing HPLC analysis on the aqueous solution of the obtained precipitate.

[0046] Figure 5 This is a chromatogram showing the results of HPLC analysis of the aqueous solution of the precipitate obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuin Co., Ltd.) to a solution in which proteoglycan was extracted by adding ethanol (EtOH) to a final concentration of 40% by volume. Figure 5 (A) is the chromatogram when 0.5 M calcium chloride was added; Figure 5 (B) is the chromatogram when 1.0 M calcium chloride was added; Figure 5 (C) is a chromatogram obtained when 2.0 M calcium chloride was added.

[0047] Figure 6 This is a chromatogram showing the results of HPLC analysis of the aqueous solution of the precipitate obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuin Co., Ltd.) to a solution in which proteoglycan is extracted, adding ethanol (EtOH) to a final concentration of 40% by volume. Figure 5 (A) is the chromatogram when 2.0 M sodium acetate was added; Figure 5 (B) is a chromatogram obtained when 2.0 M potassium chloride was added.

[0048] Figure 7 This is a chromatogram showing the results of HPLC analysis of an aqueous solution of a precipitate obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuin Co., Ltd.) to a solution in which proteoglycan was extracted by adding isopropyl alcohol to a final concentration of 30% by volume.

[0049] Figure 8 The NMR spectrum of purified PG obtained by the method of Example 1 (EtOH was added at a final concentration of 40% by volume) is shown.

[0050] Figure 9 This is a chromatogram showing the results of HPLC analysis of the purified PG obtained by the method of Example 1 (EtOH was added at a final concentration of 40% by volume) before and after treatment with chondroitinase.

[0051] Figure 10 This is a chromatogram showing the results of HPLC analysis of the unsaturated disaccharides generated by treating the commercially available proteoglycan reagent "Proteoglycan from salmon nasal cartilage" (Wako PG) with chondroitinase and the purified PG obtained by the method of Example 1 (EtOH was added at a final concentration of 40% by volume). DETAILED DESCRIPTION

[0052] The embodiments of the present invention will be described in detail. However, the present invention should not be construed as being limited to the following embodiments.

[0053] The method for producing proteoglycan of the present invention, as described above, comprises:

[0054] (1) A step of immersing or adding a crushed cartilage tissue, a squeezed product of the crushed material, or an extract of the crushed material or squeezed material into an aqueous solution containing salt, and adding alcohol to the resulting composition at a concentration within a specific range to precipitate proteoglycans; or a step of immersing or adding a crushed cartilage tissue, a squeezed product of the crushed material, or an extract of the crushed material or squeezed material into an aqueous solution containing salt and alcohol at a concentration within a specific range to precipitate proteoglycans;

[0055] (2) A step of recovering the generated proteoglycan precipitate.

[0056] In the present specification, "cartilage tissue" refers not only to cartilage but also to tissues surrounding cartilage such as bone, muscle fiber, skin, and the like.

[0057] Examples of the cartilage tissue include cartilage tissue of fish, mollusks, birds, and mammals. Fish, birds, and mammals are preferred, and fish are particularly preferred.

[0058] More specifically, examples include the fins and cartilage of commercially available cartilaginous fish such as mako sharks, blue sharks, and stingrays; chicken cartilage; cartilage from mammals such as cattle, pigs, and whales; and cartilage from mollusks such as squid and octopus. Nasal cartilage tissue from the head of salmon, also known as icehead, is particularly preferred. Salmon heads are often discarded as unused resources when processed into various products. Therefore, using icehead as a raw material is beneficial from the perspective of effectively utilizing unused resources.

[0059] These raw materials are typically obtained in a frozen state and are preferably crushed as finely as possible to increase their surface area. A commonly used mill or blender can be used for crushing. Furthermore, considering the biological activity of the proteoglycans and type II collagen to be extracted, crushing is preferably performed while maintaining the raw materials in a frozen state, preferably below 0°C.

[0060] To improve extraction efficiency, a degreasing treatment may be performed as needed. Examples of degreasing treatments include exposing the cartilage tissue to water for at least one hour (in this case, the cartilage tissue is preferably then crushed and extracted as needed), and immersing the crushed material in an organic solvent. Examples of solvents used in degreasing treatments include ethanol, hexane, and acetone.

[0061] In this application, "extracted material" includes a liquid composition derived from cartilage tissue produced when the cartilage tissue is crushed, or obtained by squeezing the crushed cartilage tissue; or a clear liquid composition obtained by removing insoluble matter from the liquid composition by centrifugation or filtration.

[0062] In this specification, "extract" refers to a composition obtained by performing any extraction treatment on the crushed material of cartilage tissue or the squeezed material of the crushed material, and there is no particular restriction on the type of extraction treatment. For example, the crushed material of cartilage tissue or the squeezed material of the crushed material can be immersed in or added to an alkaline aqueous solution such as guanidine hydrochloride aqueous solution (patent document 1), an acetic acid aqueous solution (patent document 2), sodium hydroxide (patent document 3), an aqueous solution containing a surfactant such as saponin or sucrose fatty acid ester (patent document 4), or an aqueous solution containing protease in an acidic solution containing acetic acid (patent document 5) to obtain an extract containing proteoglycan. These extraction methods and extracts are described in detail in patent documents 1 to 5, and their contents are incorporated into this specification by reference.

[0063] When extraction is performed with an acidic aqueous solution, for example, extraction can be performed at pH 4 to 6, preferably pH 5 to 5.5. Furthermore, when extraction is performed with an alkaline aqueous solution, for example, extraction can be performed at pH 8 to 11, preferably pH 9 to 10. When extraction is performed without using an acidic or alkaline aqueous solution, extraction is preferably performed at pH 6 to 8, more preferably pH 6.5 to 7.5, in order to avoid the effects of acidic or alkaline conditions on proteoglycans.

[0064] Furthermore, an extract containing a complex of proteoglycans and type II collagen can be obtained by immersing or adding crushed cartilage tissue or a squeezed extract thereof in water. Extraction with water eliminates the need for acids, alkalis, or proteases, providing a method for producing proteoglycans with minimal risk of protein degradation or sugar chain damage. Furthermore, since proteoglycans can be extracted without the use of ingredients such as guanidine hydrochloride, acids, alkalis, and surfactants, there is no risk of these ingredients remaining in the purified proteoglycans, enabling the production of purified proteoglycans that are safer for the human body.

[0065] The conditions for water extraction vary depending on the state of the cartilage tissue being extracted (whether it is a crushed material or a squeezed material, and the degree of crushing in the case of a crushed material). Generally, when the temperature is increased, the extraction time is shortened, and when the temperature is lowered, the extraction time is extended, thereby setting the conditions for extracting the above-mentioned complex. Generally, the extraction time is set in the range of 1 hour to 24 hours within the temperature range of 20 to 95°C; from the perspective of extraction efficiency, it is preferably set in the range of 1 hour to 10 hours within the temperature range of 50 to 95°C; more preferably, it is set in the range of 3 hours to 8 hours within the temperature range of 70 to 95°C. In addition, the pH is generally set to pH 6 to 8, and preferably to pH 6.5 to 7.5. The hot water extraction conditions are described in detail in Non-Patent Document 1, the contents of which are incorporated into this specification by reference.

[0066] The above-mentioned squeezed products and extracts can also be used directly in liquid form, but usually they are freeze-dried for preservation and made into powder form, which is then dissolved in water before use.

[0067] The amount of the crushed product, squeezed product, or extract to be immersed in or added to the aqueous salt solution described below is not particularly limited, but is generally an amount of 1 to 100 mg / mL in terms of solid content. From the viewpoint of extraction efficiency, an amount of 1 to 40 mg / mL in terms of solid content is preferred, and an amount of 1 to 10 mg / mL in terms of solid content is particularly preferred.

[0068] In step (1), the crushed material, squeezed material or extract is immersed in or added to an aqueous solution containing salt. If the proteoglycan in the crushed material, squeezed material or extract coexists with cations derived from salt, the chondroitin sulfate constituting the proteoglycan will become a chondroitin sulfate salt, becoming electrically stable, or the solubility will be reduced by forming a cross-linked structure. Thereafter, if a relatively low specific concentration of alcohol described later is added, the polarity of the solution will be reduced to a predetermined level, and the proteoglycan as a large molecule will be preferentially precipitated, thereby enabling selective precipitation of the proteoglycan. In addition, when a raw material in the form of a complex containing proteoglycan and type II collagen, such as a crushed material of cartilage tissue, a squeezed material of the crushed material, or a water extract of the crushed material or squeezed material, is added or immersed in an aqueous solution containing salt, the proteoglycan can be dissociated from fibrous matrix proteins such as type II collagen, and by adding a specific concentration of alcohol described later, a highly pure proteoglycan precipitate can be obtained.

[0069] As salt, as long as it is a salt containing a metal ion that can form a salt with glycosaminoglycans such as chondroitin sulfate in its solution, for example, lithium salts, sodium salts, potassium salts, magnesium salts, calcium salts and their mixtures can be listed. More specifically, 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. can be used. They can use one or more than two kinds. Among them, when using calcium chloride and other salts that generate multivalent ions (particularly divalent ions), it is easy to form a cross-linked structure, which can promote the precipitation of proteoglycans. Therefore, even if the concentration of the alcohol described later is reduced, the precipitation of proteoglycans can also be obtained. On the other hand, it is easy to dissolve again after forming the chondroitin sulfate salt. From the viewpoint of recovery rate and operability, it is preferred to generate a salt of a monovalent ion, particularly preferably sodium chloride.

[0070] The salt concentration in the aqueous solution containing the salt can be selected according to the final concentration of the alcohol described later and the type (valence) of the salt, and is usually selected from the range of 0.5 M to saturation concentration, preferably from 1.0 M to saturation concentration, more preferably from 2.0 M to saturation concentration, further preferably from 2.5 M to saturation concentration, further preferably from 3.0 M to saturation concentration, and particularly preferably from 3.5 M to saturation concentration.

[0071] Furthermore, in the case of aqueous solutions of salts that generate monovalent ions, such as sodium chloride, the concentration is preferably selected from 2.0 M to saturation, more preferably from 2.5 M to saturation, even more preferably from 3.0 M to saturation, even more preferably from 3.5 M to saturation, and particularly preferably from 4.0 M to saturation. On the other hand, in the case of aqueous solutions of salts that generate polyvalent, particularly divalent, ions, such as calcium chloride, the concentration is preferably selected from 0.5 M to 3.5 M, more preferably from 0.5 M to 3.0 M, even more preferably from 0.5 M to 2.5 M, and particularly preferably from 0.5 M to 2.0 M.

[0072] From the viewpoint of less damage to sugar chains, the pH of the aqueous solution containing salt is preferably near neutral. Specifically, it is preferably pH 5 to 10, and more preferably pH 6 to 8. In addition, according to the pH conditions of the various extraction methods described above, when extracting with an acidic aqueous solution, the aqueous solution containing salt can be set to, for example, pH 4 to 6, and preferably pH 5 to 5.5. In addition, when extracting with an alkaline aqueous solution, the aqueous solution containing salt can be set to, for example, pH 8 to 11, and preferably pH 9 to 10. When other extractions are performed without using an acidic aqueous solution or an alkaline aqueous solution, in order to avoid the influence of proteoglycans in an acidic or alkaline state, the aqueous solution containing salt can also be preferably set to pH 6 to 8, and more preferably to pH 6.5 to 7.5.

[0073] The temperature of the salt-containing aqueous solution is not particularly limited and may be, for example, room temperature (eg, 10 to 40° C.) or heated, for example, to 40 to 100° C., to promote the dissolution and / or extraction of proteoglycans.

[0074] In step (1), alcohol is added to a crude proteoglycan extract in the presence of a salt to precipitate the proteoglycan. The extract contains salt, and in the presence of such salt, when alcohol is added to the proteoglycan extract at a relatively low concentration within a specific range, the polarity of the solution decreases to a predetermined level, selectively precipitating the macromolecular proteoglycan, thereby making it possible to separate the proteoglycan from coexisting components such as collagen.

[0075] In one embodiment, the crude proteoglycan extract in the presence of a salt may be concentrated by drying under reduced pressure or the like, and alcohol may be added to the concentrated extract obtained.

[0076] The alcohol to be added is not particularly limited, but water-soluble alcohols are preferred, 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. Among them, alcohols having 4 or fewer carbon atoms are preferred, with ethanol and 2-propanol being particularly preferred.

[0077] As a result of in-depth studies based on the viewpoint of selectively precipitating proteoglycans, the amount of alcohol added is generally preferably an amount to make the final concentration of 10 to 60 volume %, more preferably an amount to make the final concentration of 20 to 55 volume %, further preferably an amount to make the final concentration of 30 to 50 volume %, and particularly preferably an amount to make the final concentration of 35 to 45 volume %.

[0078] More specifically, when the above-mentioned salt concentration is from 1.5 M to saturation concentration, it is preferred that alcohol is added to the composition in which salt and proteoglycan coexist in an amount to achieve a final concentration of 40 to 60% by volume, or that the aqueous solution (an aqueous solution containing salt and alcohol) contains alcohol in an amount to achieve a final concentration of 40 to 60% by volume.

[0079] When the salt concentration is from 2.0 M to saturation, it is preferred to add alcohol to the composition containing salt and proteoglycan in an amount to give a final concentration of 35 to 60% by volume, or to allow the aqueous solution to contain alcohol in an amount to give a final concentration of 35 to 60% by volume.

[0080] When the salt concentration is from 2.5 M to saturation, it is preferred to add alcohol to the composition containing salt and proteoglycan in an amount to give a final concentration of 30 to 60% by volume, or to allow the aqueous solution to contain alcohol in an amount to give a final concentration of 30 to 60% by volume.

[0081] When the salt concentration is from 3.0 M to saturation, it is preferred to add alcohol in an amount to give a final concentration of 20 to 60% by volume, or to allow the aqueous solution to contain alcohol in an amount to give a final concentration of 20 to 60% by volume.

[0082] In addition, in the case of a composition in which a salt that generates multivalent ions (especially divalent ions) such as a calcium salt coexists with proteoglycan, while adjusting the salt concentration to 0.5 M to a saturation concentration, an alcohol is added to the composition in which the salt and proteoglycan coexist in an amount to give a final concentration of 20 to 60% by volume, preferably an amount to give a final concentration of 30 to 60% by volume, or an aqueous solution (an aqueous solution containing salt and alcohol) is made to contain alcohol in an amount to give a final concentration of 20 to 60% by volume, preferably an amount to give a final concentration of 30 to 60% by volume.

[0083] The "final concentration" refers to the alcohol concentration (volume %) in the composition in which proteoglycan is precipitated in the presence of salt and alcohol. When less than 100% alcohol is used, the final concentration is a value calculated as an alcohol conversion value.

[0084] The step of immersing or adding the crushed cartilage tissue, a squeezed product of the crushed product, or an extract of the crushed product or squeezed product in an aqueous solution containing a salt and the step of adding a specific amount of alcohol may be carried out separately, or may be carried out in a single step of immersing or adding the crushed cartilage tissue, a squeezed product of the crushed product, or an extract of the crushed product or squeezed product in an aqueous solution containing the above-mentioned salt and a specific amount of alcohol.

[0085] In step (2), the obtained precipitate is recovered. The obtained precipitate can be separated and recovered from the supernatant by methods commonly known in the art. For example, the precipitate can be obtained by directly leaving it for a certain period of time, or by centrifuging it, and then removing the supernatant. In addition, the precipitate can be separated by passing the liquid containing the precipitate obtained in step (1) through an ultrafiltration membrane filter. These steps can be repeated by adding the separated precipitate to pure water, thereby obtaining proteoglycans with higher purity.

[0086] Furthermore, if necessary, the precipitate may be dispersed in pure water and the dispersion may be dialyzed to completely remove salts contained in the precipitate.

[0087] [Example]

[0088] Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

[0089] 1. Preparation of Purified Proteoglycans

[0090] [Example 1]

[0091] As a starting material, "Proteoglycan HG-100" (manufactured by Nippon Yakuin Co., Ltd.) was used.

[0092] This raw material is a powdered extract from salmon nasal cartilage tissue, extracted in a complex containing proteoglycans and type II collagen. This extract cannot be obtained through extraction with acidic or alkaline solutions, but is obtained by extracting the crushed salmon nasal cartilage tissue with hot water.

[0093] The above starting material was added to a 2 M sodium chloride aqueous solution at pH 7 at a concentration of 10 mg / mL at room temperature, and stirred at room temperature for 1 hour to extract proteoglycan from the starting material.

[0094] 0.5 mL of the resulting aqueous solution was added to a microtube, and ethanol was added to a final concentration of 40% by volume. The mixture was mixed using a vortex shaker. The supernatant was removed by centrifugation (15,000 rpm for 15 minutes), and the precipitate was freeze-dried. The resulting sample was dissolved in 0.5 mL of ultrapure water as a test sample.

[0095] To confirm the state of proteoglycan in a complex containing proteoglycan and type II collagen in the presence of salt, HPLC analysis was performed on "Proteoglycan HG-100" (manufactured by Nippon Yakuin Co., Ltd.) using ultrapure water or 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride as the mobile phase.

[0096] In the HPLC analysis, "Shodex OH Pak 806M HQ" (manufactured by Showa Denko KK) was used as a separation column, and a UV detector was used for detection, with measurement performed at a wavelength of 204 nm.

[0097] like Figure 1 As shown, it was confirmed that when a complex containing proteoglycan and type II collagen is allowed to coexist with salt, proteoglycan is dissociated from the complex.

[0098] [Example 2 and Comparative Example 1]

[0099] To an aqueous solution obtained by adding the starting material at a concentration of 10 mg / mL to a 2M sodium chloride aqueous solution, ethanol was added to final concentrations of 60% by volume and 70% by volume, respectively, to produce a precipitate. Precipitates were obtained in the same manner as in Example 1, and freeze-dried. Separately, the obtained sample was dissolved in 0.5 mL of ultrapure water to serve as a test sample.

[0100] [Examples 3 to 6]

[0101] A precipitate was obtained by adding the starting material to an aqueous solution obtained at a concentration of 10 mg / mL in a 2M, 2.5M, 3M, or 4M sodium chloride aqueous solution, and then adding ethanol to a final concentration of 35% by volume to produce a precipitate. The precipitate was then freeze-dried in the same manner as in Example 1. The obtained sample was dissolved in 0.5 mL of ultrapure water to prepare a test sample.

[0102] [Example 7]

[0103] To the aqueous solution obtained by adding the starting material at a concentration of 2 mg / mL to a saturated sodium chloride aqueous solution, ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then centrifuged (15,000 rpm for 30 minutes) and the supernatant removed. This alcohol precipitation procedure was repeated to obtain a precipitate, which was then freeze-dried. The resulting sample was dissolved in 0.5 mL of ultrapure water as a test sample.

[0104] [Examples 8 to 10]

[0105] In 0.5M, 1M or 2M calcium chloride aqueous solution, the concentration of the starting raw material was added to the aqueous solution obtained at 2mg / mL, and the ethanol of the amount of 40 volume % was added to produce a precipitate. The supernatant was removed by centrifugation (15000rpm, 30 minutes). The alcohol precipitation operation was performed again to obtain a precipitate, which was then freeze-dried. The sample obtained was dissolved in 0.5mL of ultrapure water as a test sample.

[0106] [Examples 11-12]

[0107] To the aqueous solution obtained by adding the starting material at a concentration of 2 mg / mL to a 2M potassium chloride or 2M sodium acetate aqueous solution, ethanol was added to a final concentration of 40% by volume to produce a precipitate. The precipitate was then centrifuged (15,000 rpm for 30 minutes) and the supernatant removed. This alcohol precipitation procedure was repeated to obtain a precipitate, which was then freeze-dried. The resulting sample was dissolved in 0.5 mL of ultrapure water as a test sample.

[0108] [Example 13]

[0109] In the aqueous solution obtained by adding starting raw material with the concentration of 2mg / mL in 0.5M calcium chloride aqueous solution, adding the isopropyl alcohol of the amount of 30 volume % to produce precipitate, centrifugation (15000rpm, 30 minutes) removed supernatant. Carry out this alcohol precipitation operation again, obtain precipitate, and carry out freeze drying. The sample dissolution obtained is used as the detection sample in ultrapure water 0.5mL.

[0110] 2. Evaluation of the Effects of Ethanol and Sodium Chloride Concentrations on Proteoglycan Purity

[0111] Each test sample of Example 1, Example 2, Comparative Example 1, and Examples 3 to 6 was filtered through a 0.45 μm membrane filter and then subjected to HPLC analysis under the following conditions.

[0112] Sample Injector: Sample Injector (Model 7725) (manufactured by Reodyne)

[0113] Sample injection volume: 20μL

[0114] Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride

[0115] Flow rate: 0.5mL / min

[0116] Separation column: Shodex OH Pak SB-806M HQ (manufactured by Shoko Scientific Co., Ltd.)

[0117] Column temperature: 40°C

[0118] Pump: L-6000 Pump (made by Hitachi, Ltd.)

[0119] UV detector: UV detector L-2400 (manufactured by Hitachi High-Technologies Corporation)

[0120] Measurement wavelength: 204nm

[0121] Integrator: Chromato Integrator D-2500 (manufactured by Hitachi High-Technologies Corporation)

[0122] Furthermore, the test sample of Example 7 was filtered through a 0.45 μm membrane filter and then subjected to HPLC analysis under the following conditions.

[0123] Sampler: Primaide 1210 Autosampler (manufactured by Hitachi High-Technologies Corporation)

[0124] Sample injection volume: 20μL

[0125] Pump: Primaide 1110 Pump (manufactured by Hitachi High-Technologies Corporation)

[0126] Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride

[0127] Flow rate: 0.5mL / min

[0128] Separation column: Shodex OH Pak SB-806M HQ (manufactured by Shoko Scientific Co., Ltd.)

[0129] · Column temperature: 40°C

[0130] UV detector: Primaide 1410 UV-detector (manufactured by Hitachi High-Technologies Corporation)

[0131] Measurement wavelength: 204nm

[0132] The results are as follows Figure 2 、 Figure 3 as well as Figure 4 As shown. Figure 2 As shown, it was confirmed that the area ratio of the peak corresponding to proteoglycan changed depending on the ethanol concentration, and proteoglycan was selectively precipitated at an ethanol concentration of 40 to 60 vol%.

[0133] In addition, if Figure 3 as well as Figure 4 As shown in the figure, it was confirmed that the area of ​​the peak corresponding to proteoglycan increases depending on the concentration of sodium chloride. Figure 2As shown in the results, by using a high concentration of sodium chloride solution, the recovery rate of proteoglycans was higher even in the ethanol fraction with a lower concentration.

[0134] 3. Evaluation of methods using different salts and alcohols

[0135] Each test sample of Examples 8 to 13 was filtered through a 0.45 μm membrane filter and then subjected to HPLC analysis under the following conditions.

[0136] Sampler: Primaide 1210 Autosampler (manufactured by Hitachi High-Technologies Corporation)

[0137] Sample injection volume: 20μL

[0138] Pump: Primaide 1110 Pump (manufactured by Hitachi High-Technologies Corporation)

[0139] Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride

[0140] Flow rate: 0.5mL / min

[0141] Separation column: Shodex OH Pak SB-806M HQ (manufactured by Shoko Scientific Co., Ltd.)

[0142] Column temperature: 40°C

[0143] UV detector: Primaide 1410 UV-detector (manufactured by Hitachi High-Technologies Corporation)

[0144] Measurement wavelength: 204nm

[0145] The results are as follows Figures 5 to 7 shown.

[0146] like Figure 5 as well as Figure 6 As shown, it was confirmed that proteoglycan can be selectively precipitated and obtained by adding the starting material to an aqueous solution of calcium chloride, potassium chloride or sodium acetate instead of an aqueous solution of sodium chloride and then performing a predetermined alcohol precipitation treatment.

[0147] In addition, if Figure 7 As shown, it was confirmed that proteoglycan was selectively precipitated even when isopropanol was added to a final concentration of 30% by volume in an aqueous solution obtained by adding a starting material to an aqueous solution containing a salt.

[0148] In addition, if Figure 5 as well as Figure 7As shown, it was confirmed that in the precipitates obtained by adding the starting material to an aqueous solution obtained by 0.5 M, 1 M, or 2 M calcium chloride aqueous solution and then subjecting the obtained solution to a predetermined alcohol precipitation treatment, peaks of substantially the same shape and size were obtained by HPLC analysis, and PG was selectively precipitated within this range.

[0149] 4. Evaluating the Purity of Purified Proteoglycans by NMR Analysis

[0150] About 5 mg of the test sample obtained in Example 1 was weighed, desalted by ultrafiltration (Amicon Ultra 30k, manufactured by Merck), and freeze-dried, and dissolved in deuterated water for NMR analysis.

[0151] NMR analysis used JEOL ECX 600 manufactured by JEOL Ltd. with a resonance frequency of 600 MHz, an observation width of 10,000 Hz, an accumulation number of 300 times, a pulse width of 12 μs, and a measurement temperature of 60°C.

[0152] exist Figure 8 In the NMR spectrogram shown, N-acetylgalactosamine and the signal peak of glucuronic acid that derive from the chondroitin sulfate constituting proteoglycan and the amino acid whose signal that belongs to the core protein constituting proteoglycan are observed.On the other hand, do not observe the signal peak that derives from other compounds, for example nucleic acid or lipid.Thus, can confirm that highly purified proteoglycan has been recovered.

[0153] 5. Purity of purified proteoglycans evaluated by HPLC analysis before and after chondroitinase treatment

[0154] The detection sample that obtains among the embodiment 1 is carried out desalination and the sample after lyophilization by ultrafiltration (Amicon Ultra 30k, Merck system) is dissolved in ultrapure water with the concentration of 2mg / mL, its 20 μ L are added in micropipe, to the aqueous solution of the chondroitinase that contains 0.1 unit of the Tris-acetic acid buffer (pH8.0) of 0.2M that wherein adds 20 μ l and 10 μ L.This mixed solution was cultivated 16 hours down at 37 ℃, after being decomposed into the unsaturated disaccharide that constitutes chondroitin sulfate, in order to make enzyme inactivation, heated 3 minutes down at 100 ℃.After the cooling, under the condition of putting down in writing in " 2. the influence of evaluation ethanol concentration and sodium chloride concentration on the purity of proteoglycan ", confirm that the peak of proteoglycan changes by HPLC.

[0155] The results are as follows Figure 9 As shown. Figure 9The chromatograms before and after the enzyme treatment show that the peaks corresponding to proteoglycans on the chromatograms disappear completely after the chondroitinase treatment, and the chondroitin sulfate chains in the proteoglycans are completely decomposed. Based on this result, it can be understood that the test sample obtained in Example 1 contains highly purified proteoglycans.

[0156] 6. Evaluating the Purity of Purified Proteoglycans by Disaccharide Structure Analysis of Proteoglycans

[0157] The test sample obtained in Example 1 was desalted and freeze-dried by ultrafiltration (Amicon Ultra 30k, Merck system) and dissolved in ultrapure water at a concentration of 2 mg / mL. 20 μL of this solution and a solution obtained by dissolving "proteoglycan, derived from salmon nasal cartilage" (Wako system), a commercially available proteoglycan reagent, in ultrapure water at a concentration of 10 mg / mL were collected and added to a microtube. 20 μL of 0.2M Tris-acetate buffer (pH 8.0) and 10 μL of an aqueous solution containing 0.1 units of chondroitinase were added thereto. The mixed solution was cultured at 37°C for 16 hours and then decomposed into unsaturated disaccharides constituting chondroitin sulfate, and then heated at 100°C for 3 minutes to inactivate the enzyme.

[0158] Disaccharide structure analysis was performed by HPLC using each solution after chondroitinase treatment. The HPLC analysis used a DOCOSIL SP100 (manufactured by Izumi Shu Scientific Co., Ltd.) separation column, and concentration gradient elution was performed using (A) a 12% methanol solution containing 1.2 mM tetrabutylammonium and (B) a 12% methanol solution containing 1.2 mM tetrabutylammonium and 0.2 M sodium chloride. Detection was performed using a UV detector at a wavelength of 232 nm.

[0159] The results are as follows Figure 10 As shown. Figure 10 The results shown in the figure indicate that the disaccharide structures of salmon-derived proteoglycans, namely ΔDi-0S, ΔDi-4S, ΔDi-6S, ΔDi-2S, and 6S (ΔDi-S) that are found in commercially available proteoglycan reagents, were confirmed in the solution prepared from the test sample obtained in Example 1. D ), confirming that they were identical purified products.

[0160] 7. Summary of evaluation results

[0161] The above test results confirm that the method of the present invention can obtain highly purified proteoglycan.

Claims

1. A method for producing proteoglycan, comprising: A step of immersing or adding a crushed cartilage tissue, a squeezed product of the crushed material, or an extract of the crushed material or squeezed material in an aqueous solution containing salt, and adding alcohol to the resulting liquid composition at a final concentration of 10 to 60% by volume to precipitate proteoglycans; or a step of immersing or adding a crushed cartilage tissue, a squeezed product of the crushed material, or an extract of the crushed material or squeezed material in an aqueous solution containing salt and alcohol in an amount to a final concentration of 10 to 60% by volume to precipitate proteoglycans; The step of recovering the generated precipitate.

2. The method according to claim 1, wherein the salt concentration of the aqueous solution containing the salt is selected from the range of 0.5 M to saturation concentration depending on the type of the salt and the amount or content of the alcohol added.

3. The method according to claim 1 or 2, wherein the salt concentration of the aqueous solution containing the salt is from 1.5 M to saturation concentration; the alcohol is added to the composition in an amount to give a final concentration of 40 to 60 volume %; or the alcohol is contained in the aqueous solution in an amount to give a final concentration of 40 to 60 volume %.

4. The method according to claim 1 or 2, wherein the salt concentration of the aqueous solution containing the salt is from 2.5 M to saturation concentration; the alcohol is added to the composition in an amount to give a final concentration of 30 to 60 volume %; or the alcohol is contained in the aqueous solution in an amount to give a final concentration of 30 to 60 volume %.

5. The method according to claim 1 or 2, wherein the salt concentration of the aqueous solution containing the salt is from 3.0 M to saturation concentration; the alcohol is added to the composition in an amount to give a final concentration of 20 to 60 volume %; or the alcohol is contained in the aqueous solution in an amount to give a final concentration of 20 to 60 volume %. The method according to any one of claims 1 to 5, wherein the pH of the aqueous solution containing the salt is 5 to 10. 7 . The method according to claim 1 , wherein the extract is a water extract of the crushed product or the squeezed product, and the pH of the aqueous solution containing the salt is 6.5 to 7.

5.

8. The method according to any one of claims 1 to 7, wherein the cartilage tissue is salmon nasal cartilage tissue. 9 . The method according to claim 1 , wherein the salt comprises one or a combination of two or more selected from the group consisting of lithium salts, sodium salts, potassium salts, magnesium salts, and calcium salts. 10 . The method according to claim 1 , wherein the alcohol comprises one or a combination of two or more selected from ethanol and 2-propanol.

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