Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4725—Proteoglycans, e.g. aggreccan
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/30—Extraction; Separation; Purification by precipitation
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
  • C09H—PREPARATION OF GLUE OR GELATINE
  • C09H3/00—Isolation of glue or gelatine from raw materials, e.g. by extracting, by heating
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins

Definitions

• the present invention relates to a method for producing proteoglycan.
• 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.
• glycosaminoglycans such as chondroitin sulfate, dermatan sulfate, heparan sulfate, heparin, and keratan sulfate
• Proteoglycans are widely found 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 tissue water retention and elasticity.
• proteoglycans have been recognized, leading to their use as ingredients in functional foods and cosmetics, and further research into their use in pharmaceuticals, creating a demand for highly pure proteoglycans.
• proteoglycans have a complex structure as glycoproteins and exist in complexes with hyaluronic acid, type II collagen, and other components. Therefore, 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.
• Patent Document 1 a method of extracting proteoglycans from salmon nasal cartilage using a guanidine hydrochloride solution (Patent Document 1), a method of extracting proteoglycans using an acetic acid solution (Patent Document 2), a method of extracting proteoglycans using an alkaline solution such as sodium hydroxide (Patent Document 3), a method of extracting proteoglycans using an aqueous solution containing a surfactant such as saponin or sucrose fatty acid ester (Patent Document 4), and a method of extracting proteoglycans using an aqueous solution containing acetic acid or the like and a protease (Patent Document 5) have been reported. Also reported is a method in which crushed cartilage tissue is subjected to hot water extraction to extract proteoglycans in a complex state with hyaluronic acid and
• Patent Document 2 Furthermore, 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, and then centrifuged to obtain a semi-solid product in which the proteoglycans are concentrated. However, this also results in a low purity of proteoglycan and contains other impurities. For this reason, the method described in Patent Document 2 further increases the purity by purifying the product using a cellulose membrane with a specified molecular weight exclusion limit. Furthermore, extraction using acid has the problem of easily decomposing proteins and damaging the glycans attached to the proteoglycans.
• crude proteoglycan extract obtained by extraction with an acidic solution containing protease 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. Furthermore, the use of protease also poses the problem of decomposing the core protein of proteoglycan.
• the present invention aims to provide a simple, low-cost method for producing highly pure proteoglycan from crushed cartilage tissue, extracted material from the crushed cartilage tissue, or an extract from the crushed or extracted material.
• the present invention aims to provide a method for producing highly pure proteoglycan from a complex of proteoglycan extracted from cartilage tissue and type II collagen or the like.
• the present invention provides the following methods for producing proteoglycans.
• the present invention is an innovative method for obtaining highly purified proteoglycans by adjusting the concentration of alcohol contained in a cartilage tissue-derived sample in the presence of salt, without relying on processes requiring additional equipment such as anion exchange resins, ultrafiltration membranes, or hollow fiber membranes.
• cations derived from salts coexisting with proteoglycans render the chondroitin sulfate that constitutes the proteoglycans electrically stable or form cross-linked structures, thereby reducing their solubility.
• 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.
• FIG. 1 is a chromatogram showing the results of HPLC analysis of "Proteoglycan HG-100" (manufactured by Nippon Yakuhin 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.
• FIG. 2 shows chromatograms showing the results of HPLC analysis of the aqueous solution of the precipitate obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuhin Co., Ltd.) to a 2M aqueous sodium chloride solution, or by adding ethanol (EtOH) at different concentrations (final concentrations of 40 to 70% by volume).
• EtOH ethanol
• the peak indicated by the arrow is the peak of proteoglycan (PG).
• FIG. 2(A) is a chromatogram of an untreated sample
• FIG. 2(B) is a chromatogram of a sample obtained by adding 70% by volume of ethanol (EtOH)
• FIG. 2(C) is a chromatogram of a sample obtained by adding 60% by volume of ethanol (EtOH)
• FIG. 2(D) is a chromatogram of a sample obtained by adding 40% by volume of ethanol (EtOH).
• 3 shows chromatograms illustrating the results of HPLC analysis of aqueous solutions of precipitates obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuhin Co., Ltd.) to 2M, 2.5M, 3M, or 4M aqueous sodium chloride solutions, to which ethanol (EtOH) was added in an amount to a final concentration of 35% by volume.
• Figure 3(A) shows a chromatogram of an untreated sample
• Figure 3(B) shows a chromatogram of a sample obtained by adding Proteoglycan HG-100 to a 2M aqueous sodium chloride solution
• Figure 3(C) shows a chromatogram of a sample obtained by adding Proteoglycan HG-100 to a 2.5M aqueous sodium chloride solution
• Figure 3(D) shows a chromatogram of a sample obtained by adding Proteoglycan HG-100 to a 3M aqueous sodium chloride solution
• Figure 3(E) shows a chromatogram of a sample obtained by adding Proteoglycan HG-100 to a 4M aqueous sodium chloride solution.
• FIG. 4 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 Yakuhin Co., Ltd.) to a saturated aqueous sodium chloride solution to obtain an aqueous solution, to which ethanol was added in an amount to give a final concentration of 40% by volume.
• FIG. 5 shows chromatograms 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 at a final concentration of 40% by volume to obtain a precipitate. The resulting aqueous precipitate was then analyzed by HPLC.
• Figure 5(A) shows a chromatogram obtained when 0.5 M calcium chloride was added
• Figure 5(B) shows a chromatogram obtained when 1.0 M calcium chloride was added
• Figure 5(C) shows a chromatogram obtained when 2.0 M calcium chloride was added.
• FIG. 6 shows chromatograms obtained by adding "Proteoglycan HG-100" (manufactured by Nippon Yakuhin Co., Ltd.) to a 2 M aqueous potassium chloride solution or a 2 M aqueous sodium acetate solution to extract proteoglycan, to which ethanol (EtOH) was added to a final concentration of 40% by volume to obtain a precipitate, followed by HPLC analysis of the resulting aqueous precipitate.
• Fig. 5(A) shows a chromatogram obtained when 2.0 M sodium acetate was added
• Fig. 5(B) shows a chromatogram obtained when 2.0 M potassium chloride was added.
• FIG. 7 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 0.5 M aqueous calcium chloride solution to extract proteoglycan, to which is added isopropanol to a final concentration of 30% by volume to obtain a precipitate.
• 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.
• Figure 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 added to a final concentration of 40% by volume), with chondroitinase enzyme.
• the method for producing proteoglycan of the present invention comprises the steps of: (1) A step of immersing or adding crushed cartilage tissue, a squeezed product of the crushed cartilage tissue, or an extract of the crushed cartilage tissue or the squeezed product to an aqueous solution containing salt, and then adding alcohol at a specific concentration range to the resulting composition to precipitate proteoglycans, or a step of immersing or adding crushed cartilage tissue, a squeezed product of the crushed cartilage tissue, or an extract of the crushed cartilage tissue or the squeezed product to an aqueous solution containing salt and alcohol at a specific concentration range to precipitate proteoglycans; (2) recovering the resulting proteoglycan precipitate.
• cartilage tissue refers to not only cartilage but also tissues including the surrounding areas of cartilage, such as bone, muscle fibers, and skin.
• Examples of cartilage tissue include cartilage tissue from fish, mollusks, birds, and mammals, preferably from fish, birds, and mammals, and particularly preferably from fish.
• examples include the fins and cartilage of cartilaginous fish such as blue sharks, mako sharks, and rays that are commonly sold on the market; chicken cartilage; mammalian cartilage such as cows, pigs, and whales; and cartilage derived from mollusks such as squid and octopus.
• the nasal cartilage tissue from the salmon head known as the ice head, is preferred.
• the head is often discarded as an unused resource, so using the ice head as a raw material is beneficial from the perspective of effectively utilizing unused resources.
• crushed materials are usually obtained in a frozen state, and it is preferable to crush them as finely as possible to increase their surface area.
• a commonly used mill or blender can be used as the crushing method.
• crushing is preferably carried out while the raw materials remain frozen, taking into consideration the biological activity of the proteoglycans and type II collagen to be extracted; specifically, crushing is preferably carried out at temperatures below 0°C.
• degreasing may be performed as needed.
• degreasing 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.
• solvents used in degreasing include ethanol, hexane, and acetone.
• 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 other treatment to remove insoluble matter.
• 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 there are no particular limitations on the type of extraction process.
• crushed cartilage tissue or a product extracted from the crushed cartilage tissue can be soaked in or added to an aqueous solution of guanidine hydrochloride (Patent Document 1), an aqueous solution of acetic acid (Patent Document 2), an aqueous alkali solution 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 protease in an acidic solution such as acetic acid (Patent Document 5), to obtain an extract containing proteoglycan.
• Patent Document 1 guanidine hydrochloride
• Patent Document 2 an aqueous solution of acetic acid
• Patent Document 3 an aqueous alkali solution such as sodium hydro
• extraction When extraction is performed with an acidic aqueous solution, it can be performed at a pH of, for example, 4 to 6, preferably 5 to 5.5.
• extraction When extraction is performed with an alkaline aqueous solution, it can be performed at a pH of, for example, 8 to 11, preferably 9 to 10.
• it is preferable to perform the extraction at a pH of 6 to 8, and more preferably at a pH of 6.5 to 7.5, to avoid the effects on proteoglycans caused by acidic or alkaline conditions.
• 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.
• 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 glycans.
• proteoglycan can be extracted without using components such as guanidine hydrochloride, acid, alkali, or surfactants, there is no risk of these components remaining in the purified proteoglycan, making it possible to produce purified proteoglycan that is safer for the human body.
• Water extraction conditions vary depending on the state of the cartilage tissue being extracted (whether it is crushed or extracted, and if crushed, the degree of crushing). However, generally, conditions for extracting the above complex are set by shortening the extraction time when the temperature is increased and lengthening the extraction time when the temperature is decreased. Typically, the temperature is set within the range of 20-95°C and the extraction time is set within the range of 1-24 hours. From the standpoint of extraction efficiency, the temperature is preferably set within the range of 50-95°C and the extraction time is set within the range of 1-10 hours, and more preferably within the range of 70-95°C and the extraction time is set within the range of 3-8 hours. Furthermore, the pH is typically set within the range of 6-8, and preferably within the range of 6.5-7.5. Hot water extraction conditions are described in detail in Non-Patent Document 1, the contents of which are incorporated herein by reference.
• the above-mentioned squeezed products and extracts can be used in liquid form, but for storage they are usually freeze-dried and made into powder form, which is then dissolved in water before use.
• an amount that results in a solids content of 1 to 100 mg/mL is sufficient. From the standpoint of extraction efficiency, an amount that results in a solids content of 1 to 40 mg/mL is preferred, and an amount that results in a solids content of 1 to 10 mg/mL is particularly preferred.
• step (1) the disrupted, squeezed, or extracted cartilage tissue is immersed in or added to an aqueous solution containing salt.
• salt-derived cations are present in the disrupted, squeezed, or extracted cartilage tissue
• the chondroitin sulfate constituting the proteoglycans becomes chondroitin sulfate, becoming electrically stable or forming cross-linked structures, reducing solubility.
• the addition of a relatively low, specific concentration of alcohol reduces the polarity of the solution to a predetermined level, allowing the macromolecular proteoglycans to preferentially precipitate, thereby enabling selective precipitation of the proteoglycans.
• proteoglycans and type II collagen when raw materials in a state in which a complex containing proteoglycans and type II collagen has formed, such as disrupted cartilage tissue, squeezed cartilage tissue, or an aqueous extract of the disrupted or squeezed cartilage tissue, 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.
• a specific concentration of alcohol (described below) allows for the production of highly pure proteoglycan precipitates.
• the salt may contain a metal ion capable of forming a salt with glycosaminoglycans such as chondroitin sulfate in the solution.
• a metal ion capable of forming a salt with glycosaminoglycans such as chondroitin sulfate in the solution.
• examples include lithium salts, sodium salts, potassium salts, magnesium salts, calcium salts, and mixtures thereof. More specific examples include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium sulfate, potassium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, sodium acetate, sodium bicarbonate, sodium carbonate, disodium hydrogen phosphate, and sodium dihydrogen phosphate. These salts may be used singly or in combination.
• salts that generate polyvalent ions such as calcium chloride, facilitate the formation of crosslinked structures and promote proteoglycan precipitation. Therefore, proteoglycan precipitation can be achieved even with a low alcohol concentration, as described below.
• salts that generate monovalent ions are preferred, with sodium chloride being particularly preferred, from the standpoints of ease of redissolution after chondroitin sulfate formation, recovery rate, and ease of use.
• the salt concentration in the aqueous solution containing a salt 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 the saturated concentration, preferably selected from the range of 1.0 M to the saturated concentration, more preferably selected from the range of 2.0 M to the saturated concentration, even more preferably selected from the range of 2.5 M to the saturated concentration, still more preferably selected from the range of 3.0 M to the saturated concentration, and particularly preferably selected from the range of 3.5 M to the saturated concentration.
• the concentration is preferably selected from 2.0 M to saturated concentration, more preferably selected from 2.5 M to saturated concentration, even more preferably selected from 3.0 M to saturated concentration, even more particularly preferably selected from 3.5 M to saturated concentration, and particularly preferably selected from 4.0 M to saturated concentration.
• 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.
• the pH of the salt-containing aqueous solution is preferably near neutral to minimize damage to glycans. Specifically, a pH of 5 to 10 is preferred, with a pH of 6 to 8 being 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.
• 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 in acidic or alkaline conditions.
• 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). It may also be heated to, for example, 40 to 100°C to promote dissolution and/or extraction of proteoglycans.
• step (1) alcohol is added to a crude proteoglycan extract containing salt to precipitate the proteoglycan.
• This extract contains salt, and when a relatively low concentration of alcohol within a specific range is added to the proteoglycan extract in the presence of such salt, the polarity of the solution decreases to a certain level, selectively precipitating the macromolecules of proteoglycan, making it possible to separate the proteoglycan from coexisting components such as collagen.
• the crude proteoglycan extract containing salt may be subjected to a concentration process such as drying under reduced pressure, and alcohol may be added to the resulting concentrated extract.
• the alcohol to be added is not particularly limited, but water-soluble alcohols are preferred, such as ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-methyl-2-butanol, ethylene glycol, and glycerol.
• water-soluble alcohols such as ethanol, methanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-2-propanol, 1-pentanol, 2-methyl-2-butanol, ethylene glycol, and glycerol.
• alcohols with four or fewer carbon atoms are preferred, with ethanol and 2-propanol being particularly preferred.
• the salt concentration 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 proteoglycan in an amount that will 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 that will give a final concentration of 40 to 60% by volume.
• the above-mentioned salt concentration is 2.0 M to saturation, it is preferable to add alcohol to a composition in which the salt coexists with proteoglycan in an amount that will give a final concentration of 35 to 60% by volume, or to contain alcohol in an aqueous solution in an amount that will give a final concentration of 35 to 60% by volume.
• the above-mentioned salt concentration is 2.5 M to saturation concentration
• the salt concentration is 3.0 M to the saturated concentration
• 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.
• the step of immersing or adding crushed cartilage tissue, a product extracted from the crushed cartilage tissue, or an extract of the crushed or extracted cartilage tissue 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 immersing or adding crushed cartilage tissue, a product extracted from the crushed cartilage tissue, or an extract of the crushed or extracted cartilage tissue to an aqueous solution containing the above-mentioned salt and a specific amount of alcohol.
• 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, after leaving it as is for a certain period of time or after centrifuging, the supernatant can be removed to obtain the precipitate.
• the liquid containing the precipitate obtained in step (1) can be passed through an ultrafiltration filter to separate the precipitate.
• the precipitate can be dispersed in pure water and the dispersion subjected to dialysis to completely remove salts contained in the precipitate.
• proteoglycan HG-100 manufactured by Nippon Yakuhin Co., Ltd.
• This raw material is a powdered extract extracted from salmon nasal cartilage tissue, 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, but is obtained by extracting crushed salmon nasal cartilage tissue with hot water.
• the above starting material was added to a 2M sodium chloride aqueous solution at pH 7 at room temperature at a concentration of 10 mg/mL, and the mixture was stirred at room temperature for 1 hour to extract proteoglycan from the starting material.
• 0.5 mL of the resulting aqueous solution was placed in a microtube, and ethanol was added to a final concentration of 40% by volume, followed by vortex mixing. 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 the specimen.
• 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. The precipitates were 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.
• 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.
• Example 7 The starting material was added to a saturated sodium chloride solution at a concentration of 2 mg/mL, and the resulting solution was added with ethanol in an amount 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 lyophilized. The resulting sample was dissolved in 0.5 mL of ultrapure water to prepare the specimen.
• Example 8 to 10 The starting material was added to a 0.5M, 1M, or 2M aqueous calcium chloride solution at a concentration of 2 mg/mL. To the resulting solution, ethanol was added to a final concentration of 40% by volume to form a precipitate, which 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 the specimen.
• Example 11 to 12 The starting material was added to a 2 mg/mL solution of 2 M potassium chloride or 2 M sodium acetate, 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 lyophilized. The resulting sample was dissolved in 0.5 mL of ultrapure water to prepare the specimen.
• 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 the specimen.
• Injector Sample Injector (Model 7725) (manufactured by Reodyne) Sample injection volume: 20 ⁇ L Mobile phase: 50 mM phosphate buffer (pH 7.0) containing 0.2 M sodium chloride ⁇ Flow rate: 0.5mL/min Separation column: Shodex OH Pak SB-806M HQ (manufactured by Shoko Science Co., Ltd.) Column temperature: 40°C Pump: L-6000 Pump (Hitachi) UV detector: UV detector L-2400 (Hitachi High-Tech Corporation) ⁇ Measurement wavelength: 204nm Integrator: Chromato Integrator D-2500 (Hitachi High-Tech Corporation)
• Example 7 was filtered through a 0.45 ⁇ m membrane filter and then subjected to HPLC analysis under the following conditions.
• Injector Primeide 1210 Autosampler (Hitachi High-Tech Corporation) Sample injection volume: 20 ⁇ L 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: 0.5mL/min Separation column: Shodex OH Pak SB-806M HQ (manufactured by Shoko Science Co., Ltd.) Column temperature: 40°C UV detector: Primeide 1410 UV-detector (Hitachi High-Tech Corporation) ⁇ Measurement wavelength: 204nm
• proteoglycans can be selectively precipitated even when the starting material is added to an aqueous solution of calcium chloride, potassium chloride, or sodium acetate instead of an aqueous solution of sodium chloride and then subjected to a predetermined alcohol precipitation treatment. Furthermore, as shown in Figure 7, it was confirmed that proteoglycans were selectively precipitated when isopropanol was added to a final concentration of 30% by volume to an aqueous solution obtained by adding the starting material to an aqueous solution containing salt.
• Example 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.
• 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 ⁇ s, and a measurement temperature of 60°C.
• Example 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 ⁇ L of the solution was placed in a microtube, to which 20 ⁇ L of 0.2 M Tris-acetate buffer (pH 8.0) and 10 ⁇ L of an aqueous solution containing 0.1 units of chondroitinase were added.
• Example 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.
• the disaccharide structures of each solution after chondroitinase treatment were analyzed by HPLC analysis.
• HPLC analysis a "Docosil SP100" (manufactured by Senshu Scientific Co., Ltd.) was used as the 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.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Genetics & Genomics (AREA)
• Molecular Biology (AREA)
• Zoology (AREA)
• Medicinal Chemistry (AREA)
• Engineering & Computer Science (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Wood Science & Technology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Toxicology (AREA)
• Gastroenterology & Hepatology (AREA)
• Analytical Chemistry (AREA)
• Biotechnology (AREA)
• General Chemical & Material Sciences (AREA)
• Microbiology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• Materials Engineering (AREA)
• Polymers & Plastics (AREA)
• Peptides Or Proteins (AREA)

Priority Applications (3)

Applications Claiming Priority (2)

Publications (1)

Family

ID=96589836

Family Applications (1)

Country Status (4)

Citations (5)

Family Cites Families (5)

• 2024
  • 2024-08-22 CN CN202480014311.5A patent/CN120769920A/zh active Pending
  • 2024-08-22 WO PCT/JP2024/029854 patent/WO2025163949A1/ja active Pending
  • 2024-08-22 JP JP2025505624A patent/JP7763018B1/ja active Active
  • 2024-08-22 KR KR1020257015578A patent/KR20250120981A/ko active Pending

Patent Citations (5)

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events