Method for producing proteoglycan

Abstract

Provided is a method for simply and inexpensively producing a high-purity proteoglycan from a crushed product of cartilage tissue, a squeezed product of the crushed product, or an extract of the crushed product or the squeezed product.　In the present invention, a crushed product of cartilage tissue, a squeezed product of the crushed product, or an extract of the crushed product or the squeezed product is immersed in or added to an aqueous solution containing a salt, and an alcohol is added to the obtained liquid composition in such an amount as to have a final concentration of 10-60 vol%, whereby a proteoglycan is precipitated. Alternatively, a crushed product of cartilage tissue, a squeezed product of the crushed product, or an extract of the crushed product or the squeezed product is immersed in or added to an aqueous solution containing a salt and an alcohol in such an amount as to have a final concentration of 10-60 vol%, whereby a proteoglycan is precipitated.

Description

Method for producing proteoglycan

[0001] The present invention relates to a method for producing proteoglycans.

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

[0003] In recent years, the diverse physiological functions of proteoglycans have been recognized, and their use as ingredients in functional foods and cosmetics has progressed. Furthermore, research is also underway into their use in pharmaceuticals, creating a demand for highly pure proteoglycans.

[0004] However, as mentioned above, proteoglycans have a complex structure as glycoproteins and exist in complexes with hyaluronic acid, type II collagen, etc., so it is often difficult to extract proteoglycans while maintaining their original structure. Therefore, in pharmaceuticals, functional foods, and cosmetics, only chondroitin sulfate has been extracted from the core protein of proteoglycans and used.

[0005] On the other hand, attempts have also been made to recover proteoglycans without decomposing core proteins.For example, the following methods have been reported: extraction from salmon nasal cartilage using a guanidine hydrochloride solution (Patent Document 1), extraction using an acetic acid solution (Patent Document 2), extraction using an alkaline solution such as sodium hydroxide (Patent Document 3), extraction using an aqueous solution containing a surfactant such as saponin or sucrose fatty acid ester (Patent Document 4), and extraction using an aqueous solution containing an acidic solution containing acetic acid or the like and a protease (Patent Document 5).In addition, a method has been reported in which crushed cartilage tissue is subjected to hot water extraction to extract proteoglycans in the state of forming a complex with hyaluronic acid and type II collagen (Non-Patent Document 1).

[0006] However, in the method described in Patent Document 1, the crude proteoglycan extract obtained by extraction with guanidine hydrochloride is diluted with two volumes of water, and then three volumes of ethanol (approximately 75%) and 1.3% (w / v) potassium acetate are added, followed by centrifugation to obtain a precipitated fraction. However, this precipitated fraction has low proteoglycan purity and contains other impurities. For this reason, the method described in Patent Document 1 further refines the extract with an anion exchange resin to increase its purity.

[0007] In the method described in Patent Document 2, a three-fold volume of salt-saturated ethanol (approximately 75%) is added to the crude proteoglycan extract obtained by extraction with acetic acid, followed by centrifugation to obtain a semi-solid product containing concentrated proteoglycans. However, this also results in a low purity of proteoglycans and contains other impurities. For this reason, the method described in Patent Document 2 further refines the proteoglycans using a cellulose membrane with a predetermined molecular weight exclusion limit to increase the purity. Furthermore, extraction using acid has the drawback of easily decomposing proteins and damaging the sugar chains attached to proteoglycans.

[0008] In addition, in the method described in Patent Document 3, a crude proteoglycan extract obtained by extraction with an alkaline solution such as caustic soda is centrifuged to recover a liquid phase containing proteoglycans, but this liquid phase also contains many impurities. For this reason, even in the method described in Patent Document 3, this liquid phase is further subjected to ultrafiltration to increase purity. Furthermore, extraction using alkali has problems such as protein degradation and susceptibility to damage to the sugar chains bound to proteoglycans.

[0009] In the method described in Patent Document 4, a crude proteoglycan extract obtained by extraction with a saponin or sucrose fatty acid ester aqueous solution is centrifuged to obtain a liquid phase containing proteoglycan, but this liquid phase also contains many impurities. For this reason, the method described in Patent Document 4 also further increases the purity by subjecting this liquid phase to an ultrafiltration membrane with a specific molecular weight exclusion.

[0010] In the method described in Patent Document 5, a crude proteoglycan extract obtained by extraction with a solution containing protease in an acidic solution is purified by removing lipids using an oil-absorbing mat and then removing substances with a molecular weight of 50,000 or less using a hollow fiber membrane with a molecular weight cutoff of 50,000. The use of protease also poses the problem of decomposing the core protein of proteoglycan.

[0011] As described above, conventional methods for producing proteoglycans have either made it difficult to obtain highly purified proteoglycans or required additional steps such as the use of an anion exchange resin, an ultrafiltration membrane, or a hollow fiber membrane to increase the purity. Therefore, there has been a demand for a method for producing highly purified proteoglycans using simpler steps.

[0012] Furthermore, with regard to the method for obtaining proteoglycan in a state in which it has formed a complex with hyaluronic acid and type II collagen, there has been a demand for a means for extracting highly pure proteoglycan from the obtained complex.

[0013] JP 2001-172296 A JP 2002-69097 A JP 4219974 A JP 2014-9164 A JP 2020-127397 A

[0014] Applied Glycoscience Vol. 7 No. 1 23-28 (2017)

[0015] An object of the present invention is to provide a simple and low-cost method for producing high-purity proteoglycan from a cartilage tissue homogenate, a product extracted from the homogenate, or an extract of the homogenate or product. In one embodiment, an object of the present invention is to provide a method for producing high-purity proteoglycan from a complex of proteoglycan extracted from cartilage tissue and type II collagen or the like.

[0016] While investigating various methods for extracting proteoglycans in order to solve the above-mentioned problems, the inventors discovered that when crushed cartilage tissue, a squeezed product of the crushed tissue, or an extract of the crushed or squeezed product was immersed in or added to an aqueous solution containing salt, and then a relatively low specific concentration of alcohol was added to the resulting liquid composition, they were surprised to find that proteoglycans specifically precipitated, resulting in a highly pure proteoglycan precipitate, which led to the present invention.

[0017] That is, the present invention provides the following methods for producing proteoglycan: [1] A method for producing proteoglycan, comprising the steps of: immersing or adding a disrupted cartilage tissue, a squeezed product of the disrupted cartilage tissue, or an extract of the disrupted or squeezed product in an aqueous solution containing salt, and adding alcohol to the resulting liquid composition in an amount to give a final concentration of 10 to 60% by volume, thereby precipitating proteoglycan; or immersing or adding a disrupted cartilage tissue, a squeezed product of the disrupted cartilage tissue, or an extract of the disrupted or squeezed product in an aqueous solution containing salt and alcohol in an amount to give a final concentration of 10 to 60% by volume, thereby precipitating proteoglycan; and recovering the resulting precipitate. [2] The method according to [1], wherein the salt concentration of the aqueous solution containing salt is selected from the range of 0.5 M to saturation, depending on the type of salt and the amount or content of the alcohol added. [3] The method according to [1] or [2], wherein the salt concentration of the aqueous solution containing the salt is 1.5 M to saturation, and the alcohol is added to the composition in an amount to give a final concentration of 40 to 60% by volume, or the alcohol is contained in the aqueous solution in an amount to give a final concentration of 40 to 60% by volume. [4] The method according to [1] or [2], wherein the salt concentration of the aqueous solution containing the salt is 2.5 M to saturation, and the alcohol is added to the composition in an amount to give a final concentration of 30 to 60% by volume, or the alcohol is contained in the aqueous solution in an amount to give a final concentration of 30 to 60% by volume. [5] The method according to [1] or [2], wherein the salt concentration of the aqueous solution containing the salt is 3.0 M to saturation, and the alcohol is added to the composition in an amount to give a final concentration of 20 to 60% by volume, or the alcohol is contained in the aqueous solution in an amount to give a final concentration of 20 to 60% by volume. [6] The method according to any one of [1] to [5], wherein the pH of the aqueous solution containing the salt is 5 to 10. [7] The method according to any one of [1] to [6], wherein the extract is a water extract of the crushed or 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 [1] to [7], wherein the cartilage tissue is salmon nasal cartilage tissue.[9] The method according to any one of [1] to [8], 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 any one of [1] to [9], wherein the alcohol comprises one or a combination of two or more selected from the group consisting of ethanol and 2-propanol.

[0018] The present invention is an innovative method for obtaining highly purified proteoglycans by adjusting the alcohol concentration in a cartilage tissue-derived sample containing salts, without relying on processes requiring additional equipment such as anion exchange resins, ultrafiltration membranes, or hollow fiber membranes. In the present invention, cations derived from salts coexisting with proteoglycans render the chondroitin sulfates constituting the proteoglycans electrically stable or form crosslinked structures, thereby reducing their solubility. Furthermore, in the complexes obtained by water extraction or other methods, the cations suppress the interaction between the proteoglycans and fibrous matrix proteins such as type II collagen. Adding a relatively low specific concentration of alcohol (described below) to a solution containing proteoglycans in this state reduces the polarity of the solution to a predetermined level, allowing the macromolecular proteoglycans to preferentially precipitate, thereby enabling the purification of the proteoglycans.

[0019] Figure 1 shows chromatograms of HPLC analysis of "Proteoglycan HG-100" (manufactured by Nippon Pharmaceutical Co., Ltd.) using ultrapure water or 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride as the mobile phase. It has been shown that proteoglycan and type II collagen form a complex in ultrapure water, but the complex is separated by the addition of salt. Figure 2 shows chromatograms of HPLC analysis of aqueous solutions of precipitates obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Pharmaceutical Co., Ltd.) to a 2 M aqueous sodium chloride solution, and then adding ethanol (EtOH) without or with different concentrations of ethanol (EtOH) (final concentrations ranging from 40 to 70% by volume). In each chromatogram, the peak indicated by the arrow is the proteoglycan (PG) peak. 2(A) is a chromatogram of an untreated sample, 2(B) is a chromatogram of a sample obtained by adding 70% by volume of ethanol (EtOH), 2(C) is a chromatogram of a sample obtained by adding 60% by volume of ethanol (EtOH), and 2(D) is a chromatogram of a sample obtained by adding 40% by volume of ethanol (EtOH). Figure 3 is a chromatogram showing the results of HPLC analysis of an aqueous solution of the precipitate obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuhin Co., Ltd.) to a 2M, 2.5M, 3M, or 4M aqueous sodium chloride solution, to which ethanol (EtOH) was added in an amount to give a final concentration of 35% by volume. 3(A) is a chromatogram of an untreated sample, 3(B) is a chromatogram of a sample obtained by adding 2M aqueous sodium chloride solution, 3(C) is a chromatogram of a sample obtained by adding 2.5M aqueous sodium chloride solution, 3(D) is a chromatogram of a sample obtained by adding 3M aqueous sodium chloride solution, and 3(E) is a chromatogram of a sample obtained by adding 4M aqueous sodium chloride solution. The values ​​in parentheses are the ratios of the area of ​​the PG peak in each graph when EtOH is added to the area of ​​the PG peak in the graph when EtOH is not added, which is set to 100.FIG. 4 is a chromatogram showing the results of HPLC analysis of an aqueous solution of the precipitate obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuhin Co., Ltd.) to a saturated sodium chloride aqueous solution, to which ethanol was added in an amount to give a final concentration of 40% by volume. FIG. 5 is a chromatogram showing the results of HPLC analysis of an aqueous solution of the precipitate obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuhin Co., Ltd.) to aqueous calcium chloride solutions of different concentrations to extract proteoglycan, to which ethanol (EtOH) was added to give a final concentration of 40% by volume. FIG. 5(A) is a chromatogram showing the results of ... FIG. 6 shows a chromatogram of the result of HPLC analysis of an aqueous solution of the precipitate obtained by adding ethanol (EtOH) to a final concentration of 40% by volume to a solution obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Pharmaceutical Co., Ltd.) to a 2M aqueous potassium chloride solution or a 2M aqueous sodium acetate solution to extract proteoglycan, and then adding ethanol (EtOH) to a final concentration of 40% by volume. FIG. 5(A) shows a chromatogram of the result of adding 2.0M sodium acetate, and FIG. 5(B) shows a chromatogram of the result of adding 2.0M potassium chloride. FIG. 7 shows a chromatogram of the result of HPLC analysis of an aqueous solution of the precipitate obtained by adding isopropanol to a final concentration of 30% by volume to a solution obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Pharmaceutical Co., Ltd.) to a 0.5M aqueous calcium chloride solution to extract proteoglycan. FIG. 8 shows the NMR spectrum of purified PG obtained by the method of Example 1 (EtOH was added to a final concentration of 40% by volume). FIG. 9 is a chromatogram showing the results of HPLC analysis of purified PG obtained by the method of Example 1 (EtOH was added to a final concentration of 40% by volume) before and after treatment with chondroitinase enzyme.FIG. 10 is a chromatogram showing the results of HPLC analysis of the unsaturated disaccharides produced by treating a commercially available proteoglycan reagent, "proteoglycan, derived from salmon nasal cartilage" (manufactured by Wako, Wako PG), and purified PG obtained by the method of Example 1 (EtOH was added to a final concentration of 40% by volume), with chondroitinase.

[0020] The present invention will be described in detail below with reference to the preferred embodiments, but the present invention should not be understood as being limited to the following embodiments.

[0021] As described above, the method for producing proteoglycan of the present invention comprises the following steps: (1) immersing or adding crushed cartilage tissue, a squeezed product of the crushed cartilage tissue, or an extract of the crushed or squeezed product in an aqueous solution containing salt, and adding alcohol at a specific concentration range to the resulting composition to precipitate proteoglycan; or immersing or adding crushed cartilage tissue, a squeezed product of the crushed cartilage tissue, or an extract of the crushed or squeezed product in an aqueous solution containing salt and alcohol at a specific concentration range to precipitate proteoglycan; and (2) recovering the resulting proteoglycan precipitate.

[0022] As used herein, the term "cartilage tissue" refers to tissue including not only cartilage but also the surrounding areas of the cartilage, such as bone, muscle fibers, skin, etc. Examples of cartilage tissue include cartilage tissue of fish, mollusks, birds, or mammals, preferably fish, birds, and mammals, and particularly fish.

[0023] More specifically, examples include the fins and cartilage of cartilaginous fish such as blue sharks, mako sharks, and rays, which are commonly available on the market; chicken cartilage; mammalian cartilage such as cows, pigs, and whales; and cartilage derived from mollusks such as squid and octopus. In particular, the nasal cartilage tissue from the salmon head, known as the "ice head," is preferred. When salmon is processed into various products, its head is often discarded as an unused resource. Therefore, using the ice head as a raw material is beneficial from the perspective of effectively utilizing unused resources.

[0024] These raw materials are usually obtained in a frozen state, and are preferably crushed as finely as possible to increase the surface area. A commonly used mill or blender may be used as the crushing method. Furthermore, crushing is preferably performed while maintaining the frozen state of the raw materials, taking into consideration the biological activity of the proteoglycans and type II collagen to be extracted, and specifically, crushing is preferably performed at 0°C or below.

[0025] To improve the extraction efficiency, delipidation may be performed as needed. Examples of delipidation include exposing the cartilage tissue to water for at least one hour (in this case, it is preferable to subsequently crush the cartilage tissue and subject it to extraction as needed), and immersing the crushed tissue in an organic solvent. Examples of solvents used in delipidation include ethanol, hexane, and acetone.

[0026] In this specification, the term "squeezed product" refers to a liquid composition derived from cartilage tissue, which is produced when the tissue is crushed or obtained by squeezing crushed cartilage tissue, or a clarified liquid composition obtained by subjecting the liquid composition to centrifugation, filtration, or the like to remove insoluble matter.

[0027] In this specification, the term "extract" refers to a composition obtained by subjecting crushed cartilage tissue or a product extracted from the crushed cartilage tissue to any extraction process, and the type of extraction process is not particularly limited. For example, a proteoglycan-containing extract can be obtained by immersing or adding crushed cartilage tissue or a product extracted from the crushed cartilage tissue in an aqueous solution of guanidine hydrochloride (Patent Document 1), an aqueous solution of acetic acid (Patent Document 2), an aqueous solution of alkali such as 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 a protease in an acidic solution such as acetic acid (Patent Document 5). These extraction methods and extracts are described in detail in Patent Documents 1 to 5, the contents of which are incorporated herein by reference.

[0028] When extraction is performed with an acidic aqueous solution, extraction can be performed, for example, at a pH of 4 to 6, preferably at a pH of 5 to 5.5. When extraction is performed with an alkaline aqueous solution, extraction can be performed, for example, at a pH of 8 to 11, preferably at a pH of 9 to 10. When extraction is performed without using other acidic or alkaline aqueous solutions, extraction is preferably performed at a pH of 6 to 8, more preferably at a pH of 6.5 to 7.5, in order to avoid the effects on proteoglycans under acidic or alkaline conditions.

[0029] Furthermore, an extract containing a complex comprising proteoglycan and type II collagen can be obtained by soaking or adding water to crushed cartilage tissue or a product extracted from the crushed tissue. When extraction is performed with water, the extraction can be performed without using acid, alkali, or protease, providing a method for producing proteoglycan with little risk of protein degradation or damage to sugar chains. Furthermore, because proteoglycan can be extracted without using components such as guanidine hydrochloride, acid, alkali, or surfactant, there is no risk of these components remaining in the purified proteoglycan, and purified proteoglycan that is safer for the human body can be produced.

[0030] The conditions for water extraction vary depending on the state of the cartilage tissue to be extracted (whether it is crushed or extracted, and if crushed, the degree of crushing). However, in general, conditions for extracting the complex are set by shortening the extraction time when the temperature is increased and lengthening the extraction time when the temperature is decreased. The conditions are typically set at a temperature of 20 to 95°C and a time of 1 to 24 hours. From the viewpoint of extraction efficiency, the temperature is preferably set at 50 to 95°C and a time of 1 to 10 hours, and more preferably at 70 to 95°C and a time of 3 to 8 hours. The pH is typically set at 6 to 8, preferably 6.5 to 7.5. Hot water extraction conditions are described in detail in Non-Patent Document 1, the contents of which are incorporated herein by reference.

[0031] The above-mentioned squeezed products and extracts can be used in liquid form as they are, but are usually freeze-dried and made into powder form for storage, and then dissolved in water before use.

[0032] There are no particular restrictions on the amount of the crushed material, squeezed material, or extract to be immersed in or added to the aqueous salt solution described below, and it is usually sufficient to use an amount that results in a solid content of 1 to 100 mg / mL. From the standpoint of extraction efficiency, it is preferable to use an amount that results in a solid content of 1 to 40 mg / mL, and it is particularly preferable to use an amount that results in a solid content of 1 to 10 mg / mL.

[0033] In step (1), the disrupted, squeezed, or extracted cartilage tissue is immersed in or added to an aqueous solution containing salt. In the presence of salt-derived cations, the chondroitin sulfate constituting the proteoglycans in the disrupted, squeezed, or extract converts to chondroitin sulfate, becoming electrically stable or forming crosslinked structures, reducing the solubility. Subsequently, the addition of a relatively low specific concentration of alcohol (described below) reduces the polarity of the solution to a predetermined level, allowing the macromolecular proteoglycans to preferentially precipitate, thereby enabling selective precipitation of the proteoglycans. Furthermore, when raw materials in a state in which a complex containing proteoglycans and type II collagen has formed, such as disrupted cartilage tissue, squeezed products from the disrupted cartilage tissue, or aqueous extracts of the disrupted or squeezed products, are added to or immersed in an aqueous solution containing salt, the proteoglycans can be dissociated from fibrous matrix proteins such as type II collagen, and the addition of a specific concentration of alcohol (described below) allows the production of highly pure proteoglycan precipitates.

[0034] The salt may be any salt containing a metal ion capable of forming a salt with glycosaminoglycans such as chondroitin sulfate in the solution, such as lithium salts, sodium salts, potassium salts, magnesium salts, calcium salts, and mixtures thereof. 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. may be used alone or in combination. Among these, the use of a salt that generates polyvalent ions (especially divalent ions), such as calcium chloride, facilitates the formation of crosslinked structures and promotes proteoglycan precipitation. Therefore, proteoglycan precipitation can be achieved even with a low alcohol concentration, as described below. On the other hand, salts that generate monovalent ions are preferred, particularly sodium chloride, because they are easily redissolved after forming chondroitin sulfate, and from the standpoints of recovery rate and operability, and are particularly preferred.

[0035] The salt concentration in the salt-containing aqueous solution may be selected depending on the final concentration of the alcohol and the type (valence) of the salt, which will be described later, and is usually selected from the range of 0.5 M to saturation concentration, preferably selected from 1.0 M to saturation concentration, more preferably selected from 2.0 M to saturation concentration, even more preferably selected from 2.5 M to saturation concentration, still more preferably selected from 3.0 M to saturation concentration, and particularly preferably selected from 3.5 M to saturation concentration. In the case of an aqueous solution of a salt that generates monovalent ions, such as sodium chloride, the salt concentration is preferably selected from the range of 2.0 M to saturation concentration, more preferably selected from 2.5 M to saturation concentration, even more preferably selected from 3.0 M to saturation concentration, still more particularly preferably selected from 3.5 M to saturation concentration, and particularly preferably selected from 4.0 M to saturation concentration. On the other hand, in the case of an aqueous solution of a salt that generates polyvalent ions, particularly divalent ions, such as calcium chloride, the concentration is preferably selected from 0.5 M to 3.5 M, more preferably selected from 0.5 M to 3.0 M, even more preferably selected from 0.5 M to 2.5 M, and particularly preferably selected from 0.5 M to 2.0 M.

[0036] The pH of the salt-containing aqueous solution is preferably near neutral in order to minimize damage to sugar chains. Specifically, a pH of 5 to 10 is preferred, and a pH of 6 to 8 is more preferred. Furthermore, depending on the pH conditions of the various extraction methods described above, when extraction is performed with an acidic aqueous solution, the salt-containing aqueous solution can be adjusted to, for example, a pH of 4 to 6, preferably a pH of 5 to 5.5. When extraction is performed with an alkaline aqueous solution, the salt-containing aqueous solution can be adjusted to, for example, a pH of 8 to 11, preferably a pH of 9 to 10. When extraction is performed without using another acidic or alkaline aqueous solution, the salt-containing aqueous solution is also preferably adjusted to a pH of 6 to 8, more preferably a pH of 6.5 to 7.5, in order to avoid the effects on proteoglycans under acidic or alkaline conditions.

[0037] The temperature of the salt-containing aqueous solution is not particularly limited and can be, for example, room temperature (e.g., 10 to 40°C). Alternatively, the solution may be heated to, for example, 40 to 100°C to promote dissolution and / or extraction of proteoglycans.

[0038] In step (1), proteoglycans are precipitated by adding alcohol to a crude proteoglycan extract containing salt. This extract contains salt, and when alcohol is added to the proteoglycan extract in a relatively low concentration range in the presence of such salt, the polarity of the solution is reduced to a predetermined level, selectively precipitating the macromolecules, proteoglycans, and enabling the separation of the proteoglycans from coexisting components such as collagen.

[0039] In one embodiment, a crude proteoglycan extract containing salt may be subjected to a step of concentrating the extract by drying under reduced pressure or the like, and alcohol may be added to the resulting concentrated extract.

[0040] The alcohol to be added is not particularly limited, but 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. Among these, alcohols having 4 or less carbon atoms are preferred, with ethanol and 2-propanol being particularly preferred.

[0041] As a result of extensive investigations into the selective precipitation of proteoglycans, the amount of alcohol added is determined to be, depending on the concentration and type (valence) of the salt, preferably an amount to give a final concentration of 10 to 60% by volume, more preferably an amount to give a final concentration of 20 to 55% by volume, even more preferably an amount to give a final concentration of 30 to 50% by volume, and particularly preferably an amount to give a final concentration of 35 to 45% by volume. More specifically, when the salt concentration is 1.5 M to saturation, it is preferable to add alcohol to a composition in which the salt coexists with proteoglycans in an amount to give a final concentration of 40 to 60% by volume, or to include alcohol in an aqueous solution (aqueous solution containing salt and alcohol) in an amount to give a final concentration of 40 to 60% by volume. Furthermore, when the salt concentration is 2.0 M to saturation, it is preferable to add alcohol to the composition in which the salt coexists with proteoglycan in an amount to give a final concentration of 35 to 60% by volume, or to include alcohol in the aqueous solution in an amount to give a final concentration of 35 to 60% by volume. When the salt concentration is 2.5 M to saturation, it is preferable to add alcohol to the composition in which the salt coexists with proteoglycan in an amount to give a final concentration of 30 to 60% by volume, or to include alcohol in the aqueous solution in an amount to give a final concentration of 30 to 60% by volume. When the salt concentration is 3.0 M to saturation, it is preferable to add alcohol to the composition in which the salt coexists with proteoglycan in an amount to give a final concentration of 20 to 60% by volume, or to include alcohol in the aqueous solution in an amount to give a final concentration of 20 to 60% by volume. Furthermore, in the case of a composition in which a salt that generates polyvalent ions (particularly divalent ions), such as a calcium salt, coexists with proteoglycan, it is preferable to add alcohol to the composition in which the salt coexists with proteoglycan in an amount that will give a final concentration of 20 to 60% by volume, preferably 30 to 60% by volume, while adjusting the salt concentration to 0.5 M to the saturated concentration, or to add alcohol to an aqueous solution (aqueous solution containing a salt and alcohol) in an amount that will give a final concentration of 20 to 60% by volume, preferably 30 to 60% by volume.The term "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 calculated as an alcohol equivalent value.

[0042] The step of soaking or adding crushed cartilage tissue, a squeezed product of the crushed cartilage tissue, or an extract of the crushed or squeezed product to an aqueous solution containing a salt and the step of adding a specific amount of alcohol may be carried out separately, or they may be carried out in a single step by soaking or adding crushed cartilage tissue, a squeezed product of the crushed cartilage tissue, or an extract of the crushed or squeezed product to an aqueous solution containing the above-mentioned salt and a specific amount of alcohol.

[0043] In step (2), the resulting precipitate is recovered. The resulting precipitate can be separated from the supernatant and recovered by methods commonly known in the art. For example, the precipitate can be obtained by leaving the mixture for a certain period of time or by centrifuging it, and then removing the supernatant. Alternatively, the liquid containing the precipitate obtained in step (1) can be passed through an ultrafiltration filter to separate the precipitate. These steps can be repeated by adding the separated precipitate to pure water, thereby obtaining proteoglycan of higher purity.

[0044] If necessary, the precipitate may be dispersed in pure water, and the resulting dispersion may be subjected to dialysis to completely remove salts contained in the precipitate.

[0045] The present invention will be described in detail below with reference to examples, but the present invention is not limited to the following examples.

[0046] 1. Production of Purified Proteoglycan [Example 1] "Proteoglycan HG-100" (manufactured by Nippon Yakuhin Co., Ltd.) was used as the starting material. This material is a powdered extract extracted from salmon nasal cartilage tissue, and is extracted in a state in which a complex containing proteoglycan and type II collagen is formed. Such an extract cannot be obtained by extraction in a solution containing added acid or alkali, and is obtained by extracting crushed salmon nasal cartilage tissue with hot water.

[0047] The starting material was added to a 2 M aqueous sodium chloride solution at pH 7 at a concentration of 10 mg / mL at room temperature, and the mixture was stirred at room temperature for 1 hour to extract proteoglycan from the starting material.

[0048] 0.5 mL of the resulting solution was placed in a microtube, and ethanol was added to a final concentration of 40% by volume. The mixture was then vortexed. The supernatant was removed by centrifugation (15,000 rpm, 15 minutes), and the precipitate was freeze-dried. The resulting sample was dissolved in 0.5 mL of ultrapure water to prepare a specimen.

[0049] To confirm the state of proteoglycan when a complex containing proteoglycan and type II collagen is allowed to coexist with salt, "Proteoglycan HG-100" (manufactured by Nippon Yakuhin Co., Ltd.) was subjected to HPLC analysis using ultrapure water or 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride as the mobile phase. For HPLC analysis, "Shodex OH Pak 806M HQ" (manufactured by Showa Denko K.K.) was used as the separation column, and detection was performed using a UV detector at a wavelength of 204 nm. As shown in Figure 1, it was confirmed that when a complex containing proteoglycan and type II collagen is allowed to coexist with salt, the proteoglycan dissociates from the complex.

[0050] [Example 2 and Comparative Example 1] The starting material was added to a 2 M aqueous sodium chloride solution at a concentration of 10 mg / mL, and ethanol was added to the resulting aqueous solution to give a final concentration of 60% by volume and 70% by volume, respectively, to form precipitates. The precipitates were obtained and freeze-dried in the same manner as in Example 1. The obtained samples were dissolved in 0.5 mL of ultrapure water to prepare specimens.

[0051] Examples 3 to 6 The starting material was added to a 2 M, 2.5 M, 3 M, or 4 M aqueous sodium chloride solution at a concentration of 10 mg / mL, and ethanol was added to the resulting solution to give a final concentration of 35% by volume to form a precipitate. The precipitate was obtained and 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 specimen.

[0052] Example 7: The starting material was added to a saturated sodium chloride solution at a concentration of 2 mg / mL, and ethanol was added to the resulting solution to a final concentration of 40% by volume to form a precipitate. The precipitate was then centrifuged (15,000 rpm, 30 minutes) to remove the supernatant. 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 to prepare a specimen.

[0053] [Examples 8 to 10] The starting material was added to a 0.5 M, 1 M, or 2 M aqueous calcium chloride solution at a concentration of 2 mg / mL, and ethanol was added to the resulting solution to produce a precipitate at a final concentration of 40% by volume. The precipitate was then centrifuged (15,000 rpm, 30 minutes) and the supernatant was removed. This alcohol precipitation procedure was repeated to obtain a precipitate, which was then lyophilized. The resulting sample was dissolved in 0.5 mL of ultrapure water to prepare a specimen.

[0054] [Examples 11-12] The starting material was added to a 2 mg / mL concentration in a 2 M potassium chloride or 2 M sodium acetate aqueous solution, and ethanol was added to the resulting aqueous solution to a final concentration of 40% by volume to form a precipitate. The precipitate was then centrifuged (15,000 rpm, 30 minutes) and the supernatant was removed. This alcohol precipitation procedure was repeated to obtain a precipitate, which was then lyophilized. The resulting sample was dissolved in 0.5 mL of ultrapure water to prepare a specimen.

[0055] [Example 13] The starting material was added to a 0.5 M calcium chloride aqueous solution at a concentration of 2 mg / mL, and isopropanol was added to the resulting solution to give a final concentration of 30% by volume to form a precipitate. The precipitate was then centrifuged (15,000 rpm, 30 minutes) to remove the supernatant. This alcohol precipitation procedure was repeated to obtain a precipitate, which was then lyophilized. The resulting sample was dissolved in 0.5 mL of ultrapure water to prepare a specimen.

[0056] 2. Evaluation of the Effects of Ethanol Concentration and Sodium Chloride Concentration on Proteoglycan Purity Each of the samples in Examples 1, 2, Comparative Example 1, and 3 to 6 was filtered through a 0.45 μm membrane filter and then subjected to HPLC analysis under the following conditions.

[0057] Injector: Sample Injector (Model 7725) (manufactured by Reodyne) Sample injection volume: 20 μL 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 Pump: L-6000 Pump (manufactured by Hitachi, Ltd.) UV detector: UV detector L-2400 (manufactured by Hitachi High-Tech Corporation) Measurement wavelength: 204 nm Integrator: Chromato Integrator D-2500 (manufactured by Hitachi High-Tech Corporation)

[0058] The sample of Example 7 was filtered through a 0.45 μm membrane filter and then subjected to HPLC analysis under the following conditions: Injector: Primeide 1210 Autosampler (Hitachi High-Tech Corporation) Sample injection volume: 20 μL Pump: Primeide 1110 Pump (Hitachi High-Tech Corporation) Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride Flow rate: 0.5 mL / min Separation column: Shodex OH Pak SB-806M HQ (Shoko Science Co., Ltd.) Column temperature: 40° C. UV detector: Primeide 1410 UV-detector (Hitachi High-Tech Corporation) Measurement wavelength: 204 nm

[0059] The results are shown in Figures 2, 3, and 4. As shown in Figure 2, the area ratio of the peak corresponding to proteoglycan changed depending on the ethanol concentration, and it was confirmed that proteoglycan was selectively precipitated at ethanol concentrations of 40 to 60% by volume. Furthermore, as shown in Figures 3 and 4, the area of ​​the peak corresponding to proteoglycan increased depending on the sodium chloride concentration. When the results shown in Figure 2 were also evaluated, it was confirmed that the use of a high-concentration sodium chloride solution increased the recovery rate of proteoglycan even in fractions with lower ethanol concentrations.

[0060] 3. Evaluation of methods using different salts and alcohols Each sample in Examples 8 to 13 was filtered through a 0.45 μm membrane filter and then subjected to HPLC analysis under the following conditions. Injector: Primeaide 1210 Autosampler (Hitachi High-Tech Corporation) Sample injection volume: 20 μL Pump: Primeaide 1110 Pump (Hitachi High-Tech Corporation) Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride Flow rate: 0.5 mL / min Separation column: Shodex OH Pak SB-806M HQ (Shoko Science Co., Ltd.) Column temperature: 40°C UV detector: Primeaide 1410 UV-detector (Hitachi High-Tech Corporation) Measurement wavelength: 204 nm

[0061] The results are shown in Figures 5 to 7. As shown in Figures 5 and 6, it was confirmed that proteoglycan could be selectively precipitated by adding the starting material to an aqueous solution of calcium chloride, potassium chloride, or sodium acetate instead of an aqueous sodium chloride solution and performing the specified alcohol precipitation treatment. Furthermore, as shown in Figure 7, it was confirmed that proteoglycan could be selectively precipitated by adding isopropanol to a final concentration of 30% by volume to an aqueous solution obtained by adding the starting material to an aqueous solution containing salt. Furthermore, as shown in Figures 5 and 7, the precipitates obtained by adding the starting material to 0.5 M, 1 M, or 2 M aqueous calcium chloride solutions and performing the specified alcohol precipitation treatment yielded peaks of approximately the same shape and size in HPLC analysis, confirming that PG could be selectively precipitated within this range.

[0062] 4. Evaluation of Purity of Purified Proteoglycan by NMR Analysis The specimen obtained in Example 1 was desalted by ultrafiltration (Amicon Ultra 30k, manufactured by Merck), and approximately 5 mg of the freeze-dried sample was weighed out, dissolved in heavy water, and subjected to NMR analysis.

[0063] The NMR analysis was performed using a JEOL ECX 600 manufactured by JEOL Ltd., with a resonance frequency of 600 MHz, an observation width of 10,000 Hz, an accumulation count of 300, a pulse width of 12 μs, and a measurement temperature of 60°C.

[0064] In the NMR spectrum shown in Figure 8, signal peaks attributable to N-acetylgalactosamine and glucuronic acid, which constitute the chondroitin sulfate of proteoglycan, as well as signals attributable to amino acids that constitute the core protein of proteoglycan, were observed. On the other hand, no signal peaks attributable to other compounds, such as nucleic acids or lipids, were observed. This confirmed that highly pure proteoglycan was recovered.

[0065] 5. Evaluation of Purity of Purified Proteoglycan by HPLC Analysis Before and After Chondroitinase Treatment The sample obtained in Example 1 was desalted by ultrafiltration (Amicon Ultra 30k, Merck). The lyophilized sample was dissolved in ultrapure water at a concentration of 2 mg / mL. 20 μL of the solution was placed in a microtube, to which 20 μL of 0.2 M Tris-acetate buffer (pH 8.0) and 10 μL of an aqueous solution containing 0.1 units of chondroitinase were added. This mixture was incubated at 37°C for 16 hours to decompose the chondroitin sulfate into its constituent unsaturated disaccharides, and then heated at 100°C for 3 minutes to inactivate the enzyme. After cooling, the change in the proteoglycan peak was confirmed by HPLC under the conditions described in "2. Evaluation of the Effect of Ethanol Concentration and Sodium Chloride Concentration on Proteoglycan Purity."

[0066] The results are shown in Figure 9. As is clear from the chromatograms before and after enzyme treatment in Figure 9, all peaks corresponding to proteoglycan on the chromatogram disappeared after chondroitinase treatment, confirming that all chondroitin sulfate chains in the proteoglycan were decomposed. From these results, it can be seen that the sample obtained in Example 1 contains highly purified proteoglycan.

[0067] 6. Evaluation of Purity of Purified Proteoglycan by Analysis of Proteoglycan Disaccharide Structures The specimen obtained in Example 1 was desalted by ultrafiltration (Amicon Ultra 30k, Merck), and the lyophilized sample was dissolved in ultrapure water at a concentration of 2 mg / mL. 20 μL of this solution and a commercially available proteoglycan reagent, "Proteoglycan, Salmon Nasal Cartilage Derived" (Wako), dissolved in ultrapure water at a concentration of 10 mg / mL were each placed in a microtube, to which 20 μL of 0.2 M Tris-acetate buffer (pH 8.0) and 10 μL of an aqueous solution containing 0.1 units of chondroitinase were added. This mixture was incubated at 37°C for 16 hours to decompose the chondroitin sulfate into its constituent unsaturated disaccharides, and then heated at 100°C for 3 minutes to inactivate the enzyme.

[0068] The disaccharide structures of each solution after chondroitinase treatment were analyzed by HPLC analysis. For HPLC analysis, a "Docosil SP100" (manufactured by Senshu Scientific Co., Ltd.) was used as a separation column, and measurements were performed by concentration gradient elution 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. A UV detector was used for detection, and measurements were taken at a wavelength of 232 nm.

[0069] The results are shown in Figure 10. From the results shown in Figure 10, it can be seen that the solution prepared from the specimen obtained in Example 1 contained the disaccharide structures of salmon-derived proteoglycan, ΔDi-0S, ΔDi-4S, ΔDi-6S, ΔDi-2S, and 6S (ΔDi-S), which are found in commercially available proteoglycan reagents. Ｄ ) was confirmed to be an equivalent refined product.

[0070] 7. Summary of Evaluation Results The above test results demonstrate that highly purified proteoglycans can be obtained by the method of the present invention.

Claims

1. A method for producing proteoglycan, comprising the steps of: immersing or adding crushed cartilage tissue, a squeezed product of the crushed cartilage tissue, or an extract of the crushed or squeezed product 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 proteoglycan; or immersing or adding crushed cartilage tissue, a squeezed product of the crushed cartilage tissue, or an extract of the crushed or squeezed product in an aqueous solution containing salt and alcohol in an amount to give a final concentration of 10 to 60% by volume to precipitate proteoglycan; and recovering the resulting 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 saturated 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 1.5 M to saturation, and the alcohol is added to the composition in an amount that will give a final concentration of 40 to 60% by volume, or the alcohol is contained in the aqueous solution in an amount that will give a final concentration of 40 to 60% by volume.

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

5. The method according to claim 1 or 2, wherein the salt concentration of the aqueous solution containing the salt is 3.0 M to saturation, and the alcohol is added to the composition in an amount that will give a final concentration of 20 to 60% by volume, or the alcohol is contained in the aqueous solution in an amount that will give a final concentration of 20 to 60% by volume.

6. 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 any one of claims 1 to 6, wherein the extract is a water extract of the crushed or squeezed material, 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 of any one of claims 1 to 8, wherein the salt comprises one or a combination of two or more selected from lithium salts, sodium salts, potassium salts, magnesium salts, and calcium salts.

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

Images