Method for producing a hyaluronic acid complex proteoglycan-containing composition

Abstract

This invention provides a method for producing hyaluronic acid-complexed proteoglycans, which allows for the extraction of hyaluronic acid (HA), which forms a non-covalent complex with proteoglycans (PG) in vivo, along with proteoglycans (PG) from raw materials. [Solution] A method for producing the hyaluronic acid complex-type proteoglycan-containing composition, comprising: an extraction solvent addition step of adding an alkaline aqueous solution with a pH of 10 or higher and a pH of 13 or lower as an extraction solvent to an extraction raw material; an extraction process step of transferring components contained in the extraction raw material to the extraction solvent from the extraction raw material to the extraction solvent; and an extraction termination step of stopping the process with the extraction solvent, wherein the extraction termination step includes (1) adjusting the pH of the extraction solvent added to the extraction raw material, and / or (2) removing the extraction raw material from the extraction solvent by solid-liquid separation.

Description

Technical Field

[0001] The present invention relates to a proteoglycan-containing composition, and more particularly to a proteoglycan-containing composition obtained from fish-derived cartilage.

Background Art

[0002] Proteoglycan (PG), which is a major component of the extracellular matrix, is a macromolecule in which sugar chains, mainly chondroitin sulfate (CS), are bound to a core protein, and further hyaluronic acid (HA) is non-covalently bound to form a huge macromolecular complex, playing an important role in cartilage tissue and synovial fluid.

[0003] Conventionally, various methods have been used to extract proteoglycan (PG) from raw material resources. For example, Patent Document 1 discloses an invention of a method for purifying proteoglycan, characterized in that acetic acid is used as an elution solvent for crude proteoglycan. According to the purification method, cartilage-type proteoglycan can be obtained simply and at low cost without using harmful reagents. Further, Patent Document 2 discloses an invention of a proteoglycan-containing substance obtained from fish cartilage, which contains proteoglycan and acidic sugar as acidic sugar components and contains an acidic sugar component having a molecular weight of 2000 kDa or more. The proteoglycan-containing substance can be produced by degreasing fish cartilage by ethanol treatment and then performing water extraction from the degreased fish cartilage. Further, Patent Document 3 discloses an invention of a method for producing a proteoglycan-containing composition, characterized by including a freezing step of freezing raw fish-derived cartilage as a raw material and a freeze-drying step of freeze-drying the frozen product obtained in the freezing step. According to the production method, a material containing proteoglycan in a form close to its natural form can be obtained while avoiding freeze-thawing, which is a factor causing denaturation and decomposition of the raw material.

Prior Art Documents

Patent Documents

[0004] [Patent Document 1] Japanese Patent Publication No. 2002-69097 [Patent Document 2] Japanese Patent Publication No. 2016-128467 [Patent Document 3] Japanese Patent Publication No. 2020-114185 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] However, according to the inventors' research, when extracting proteoglycans (PG) from raw materials, hyaluronic acid (HA) non-covalently forms a complex with proteoglycans (PG), which are the main components of the extracellular matrix, in vivo. However, depending on the extraction conditions, this hyaluronic acid (HA) dissociates or decomposes, making it difficult to obtain a proteoglycan-containing material that retains a structure close to that in vivo.

[0006] The present invention aims to overcome these inconveniences and provide a method for producing hyaluronic acid-complexed proteoglycans, which allows for the extraction of hyaluronic acid (HA), which forms a non-covalent complex in vivo, along with proteoglycans (PG) from raw materials. [Means for solving the problem]

[0007] To achieve this objective, the present invention A method for producing a hyaluronic acid-complex proteoglycan-containing composition using fish cartilage as an extractant, An extraction solvent addition step is performed by adding an alkaline aqueous solution with a pH of 10 or higher and a pH of 13 or lower as an extraction solvent to the aforementioned extraction raw material. An extraction process comprising: an extraction process in which components contained in the extraction material are transferred from the extraction material to the extraction solvent after the extraction solvent has been added to the extraction material, The system includes an extraction termination step, which involves stopping the process with the extraction solvent, The extraction termination step includes (1) a process of adjusting the pH of the extraction solvent added to the extraction raw material, and / or (2) a process of removing the extraction raw material from the extraction solvent by solid-liquid separation, thereby providing a method for producing the hyaluronic acid complex-type proteoglycan-containing composition.

[0008] The method for producing a hyaluronic acid-complexed proteoglycan-containing composition provided by the present invention involves using an alkaline aqueous solution as the extraction solvent and including an extraction termination step to stop the treatment with the extraction solvent. This allows for efficient extraction of proteoglycans (PG) using the alkaline aqueous solution, while also suppressing the dissociation and degradation of hyaluronic acid (HA), which forms a non-covalent complex in the body, and efficiently extracting hyaluronic acid (HA) along with the proteoglycans (PG).

[0009] In the method for producing the hyaluronic acid complex-type proteoglycan-containing composition provided by the present invention, it is preferable to use an extraction solvent prepared by adding a water-soluble inorganic salt that enhances the ionic strength of the extraction solvent.

[0010] In the method for producing a hyaluronic acid complex-type proteoglycan-containing composition provided by the present invention, it is preferable that the extraction process is performed in which the extraction temperature is 10°C or lower and the extraction time is 0.01 hours or more and 2 hours or less.

[0011] In the method for producing the hyaluronic acid complex proteoglycan-containing composition provided by the present invention, it is preferable that the extraction temperature is 0°C or higher and 4°C or lower.

[0012] In the method for producing a hyaluronic acid complex-type proteoglycan-containing composition provided by the present invention, it is preferable that the hyaluronic acid complex-type proteoglycan-containing composition has a mass ratio of 50:1 to 2:1 between (1) the amount of glucuronic acid derived from glycosaminoglycans measured by the carbazole sulfuric acid method and (2) the amount of hyaluronic acid measured by a measurement method using hyaluronic acid-binding proteins that specifically bind to hyaluronic acid. [Effects of the Invention]

[0013] The present invention provides a method for producing hyaluronic acid-complexed proteoglycans. This method allows for the extraction of hyaluronic acid (HA), which forms a non-covalent complex with proteoglycans (PG) from raw materials, along with the proteoglycans (PG). [Brief explanation of the drawing]

[0014] [Figure 1] This is a step diagram showing one embodiment of a method for producing a hyaluronic acid-complex proteoglycan-containing composition according to the present invention. [Figure 2] This chart shows the amounts of glucuronic acid and hyaluronic acid derived from glycosaminoglycans, measured at intervals after the addition of the extraction solvent in the extraction process performed in Test Example 1, both normalized to their content per unit of solid content (mass%) of the sample. [Figure 3] This is a magnified view of the chart from the time the extraction solvent was added to 5 hours after Figure 2 was shown. [Figure 4] This chart shows the amounts of glucuronic acid and hyaluronic acid derived from glycosaminoglycans, measured at intervals after the addition of the extraction solvent during the extraction process in Test Example 1, normalized by the ratio of hyaluronic acid to glucuronic acid. [Figure 5] This is a magnified diagram showing the period from the addition of the extraction solvent in Figure 4 up to 5 hours later. [Figure 6]In the extraction process performed by changing the sodium chloride concentration in the extraction solvent in Test Example 2, it is a chart showing the amount of glucuronic acid derived from glycosaminoglycan measured at each elapsed time after adding the extraction solvent, normalized to the content (% by mass) per solid content of the sample. [Figure 7] It is a chart showing an enlarged view from the addition of the extraction solvent in Fig. 6 to 5 hours. [Figure 8] In the extraction process performed by changing the sodium chloride concentration in the extraction solvent in Test Example 2, it is a chart showing the amount of hyaluronic acid measured at each elapsed time after adding the extraction solvent, normalized to the content (% by mass) per solid content of the sample. [Figure 9] It is a chart showing an enlarged view from the addition of the extraction solvent in Fig. 8 to 5 hours. [Figure 10] In the extraction process performed by changing the sodium chloride concentration in the extraction solvent in Test Example 2, it is a chart showing the amounts of glucuronic acid and hyaluronic acid measured at each elapsed time after adding the extraction solvent, normalized by the ratio of hyaluronic acid to glucuronic acid. [Figure 11] It is a chart showing an enlarged view from the addition of the extraction solvent in Fig. 10 to 5 hours.

Mode for Carrying Out the Invention

[0015] Hereinafter, the present invention will be described in more detail. In this specification, "~" for a numerical range represents a range from the lower limit value or more to the upper limit value or less, including both end values, unless otherwise specified. Also, when a numerical range is indicated, the upper limit value and the lower limit value can be appropriately combined, and the numerical ranges formed by appropriate combinations are also disclosed.

[0016] The raw material used in this invention is cartilage derived from fish. There are no particular restrictions on the type of fish or the part of the cartilage tissue used; for example, salmon nasal cartilage (ice head), shark cartilage, ray cartilage, squid cartilage, etc. Salmon nasal cartilage (ice head) is particularly preferred because it has a high proteoglycan content and is readily available at low cost as it is a part that is usually discarded in the fisheries processing industry. For example, in salmon roe processing and fillet processing, a large amount of heads are discarded from the salmon after they are landed, so these can be obtained, and the nasal cartilage can be extracted from these heads and used.

[0017] In any non-limited embodiment of the present invention, it is preferable to use raw cartilage derived from fish as the extraction raw material. This makes it easier to extract proteoglycans in a state closer to their in vivo state by using fresh raw materials. In this specification, "raw cartilage" refers to raw materials that have not undergone a heating history of 30°C or higher and have not undergone a freeze-thaw process. In general, raw materials that have undergone a heating history of 30°C or higher are prone to denaturation and decomposition of biomolecules such as proteins, making it difficult to extract them in a form close to their natural state. Furthermore, in order to avoid the growth of bacteria, it is preferable to receive the cartilage from a partner seafood processing company, etc., without delay after it has been caught.

[0018] When using fish cartilage as an extraction raw material, processing such as mincing it using a meat grinder, meat chopper, or homogenizer, or preparing dried or freeze-dried fish cartilage and then grinding it using a pulverizer, mill, or muscoloader, can increase the contact area between the raw material and the extraction solvent, thereby improving extraction efficiency. When the extraction raw material is in the form of a pulverized product, it is preferable to grind it to a particle size such that approximately 90% or more of the total mass passes through a 30-mesh pass (mesh opening: 500 μm), and more preferably to a particle size such that approximately 90% or more of the total mass passes through a 60-mesh pass (mesh opening: 250 μm). Alternatively, it is preferable to prepare the pulverized product so that approximately 90% or more of the total mass passes through a screen with a diameter of 0.3 mm or more and 0.75 mm or less.

[0019] When preparing freeze-dried fish cartilage as an extraction material, freezing can be carried out by means of a freezing device commonly known to those skilled in the art, or by simply placing it in a freezer. There are no particular restrictions on the freezing temperature, and it is appropriate to freeze the material until it reaches a temperature of -40°C to -10°C. However, to freeze it more completely and avoid even partial or temporary freeze-thawing, it is more preferable to freeze it until it reaches a temperature of -40°C to -30°C. Drying can usually be carried out by means of a vacuum freeze-dryer commonly known to those skilled in the art. The freeze-drying settings are typically a shelf temperature of -40°C to 50°C and a vacuum level of 0.1 Pa to 2000 Pa.

[0020] In any non-limited embodiment of the present invention, the freezing method described above is preferably carried out under slow freezing conditions. Hereinafter, "slow freezing" means that in the freezing process of the raw material, the raw material is frozen for a predetermined period of time at a temperature range of -5°C or higher and below 0°C, which is the ice crystal formation temperature range, thereby allowing the ice crystals in the raw material to grow and enlarge sufficiently. As a result, the cartilage tissue is more sufficiently broken down, and consequently, proteoglycans in a form closer to that of nature can be made into a material that is even easier to utilize. Specifically, slow freezing can be carried out by using a freezing device with set temperature change conditions, or by leaving the raw material in a freezer set to appropriate temperature conditions, for example, by freezing the raw material at a temperature range of -5°C or higher and below 0°C for 30 minutes or more.

[0021] Figure 1 shows one embodiment of a method for producing a hyaluronic acid-complex proteoglycan-containing composition according to the present invention. As shown in Figure 1, this embodiment includes an extraction solvent addition step (indicated as S1 in Figure 1) in which fish cartilage is used as the extraction raw material and an alkaline aqueous solution is added to the extraction raw material as the extraction solvent; an extraction process step (indicated as S2 in Figure 1) in which components contained in the extraction raw material to which the extraction solvent has been added are transferred to the extraction solvent; and an extraction termination step (indicated as S3 in Figure 1) in which the treatment with the extraction solvent is stopped. The hyaluronic acid-complex proteoglycan-containing composition is obtained as the target product.

[0022] Examples of alkaline agents that serve as solutes in alkaline aqueous solutions used as extraction solvents include alkali metal hydroxides such as sodium hydroxide (NaOH) and potassium hydroxide (KOH), and alkaline earth metal hydroxides such as magnesium hydroxide (Mg(OH)2) and calcium hydroxide (Ca(OH)2). However, they are not limited to these, and any alkaline agent that generally provides alkalinity or basicity is acceptable.

[0023] The pH of the alkaline aqueous solution used as the extraction solvent is preferably 10 to 13, more preferably 11 to 12.8, and particularly preferably 11.4 to 12.4. In this case, the concentration range of the alkaline agent that serves as the solute in the alkaline aqueous solution may be 0.0001 to 0.1 M (mol / L), 0.001 to 0.064 M (mol / L), 0.0025 to 0.025 M (mol / L), etc. Typically, for example, when sodium hydroxide is used as the alkaline agent, its concentration range may be 0.0004 to 0.4 w / v%, 0.004 to 0.25 w / v%, 0.01 to 0.1 w / v%, etc.

[0024] In any non-limited embodiment of the present invention, the extraction solvent may contain a water-soluble inorganic salt as a solute. This allows for more effective extraction of proteoglycans (PG) and hyaluronic acid (HA) from the raw materials by appropriately increasing the ionic strength of the extraction solvent. Examples of water-soluble inorganic salts for increasing the ionic strength of the extraction solvent include sodium chloride, potassium chloride, sodium sulfate, and ammonium sulfate, but are not limited to these; any salt that is generally water-soluble and functions as an ion donor is acceptable. In this case, the concentration range of the water-soluble inorganic salt may be 0.001 to 1 M (mol / L), 0.01 to 0.7 M (mol / L), 0.05 to 0.5 M (mol / L), etc. Typically, for example, when sodium chloride is used as the water-soluble inorganic salt for increasing the ionic strength of the extraction solvent, the concentration range may be 0.01 to 5 w / v%, 0.1 to 4 w / v%, 0.3 to 3 w / v%, etc.

[0025] In the manufacturing method according to the present invention, the extraction solvent addition step involves adding an extraction solvent to the raw material for extraction. In this case, there are no particular restrictions on the amount of extraction solvent to be added. Typically, for example, the amount of extraction solvent may be 0.5 to 500 times the weight of the raw material for extraction, in the range of 1 to 250 times, in the range of 2.5 to 200 times, in the range of 5 to 150 times, or in the range of 10 to 100 times.

[0026] In the manufacturing method according to the present invention, the extraction process involves transferring the components contained in the extraction raw material from the extraction solvent to the extraction solvent. In this case, there are no particular restrictions on the extraction conditions. Typically, for example, the extraction temperature is preferably 10°C or lower, more preferably 0 to 10°C, even more preferably 1 to 9°C, and particularly preferably 2 to 8°C. The extraction time is preferably 0.01 to 2 hours, more preferably 0.05 to 1.75 hours, even more preferably 1 to 1.5 hours, and particularly preferably 0.5 to 1.25 hours.

[0027] In the manufacturing method according to the present invention, the extraction termination step involves stopping the treatment with the extraction solvent. Here, in this specification, "stopping the treatment with the extraction solvent" means that in the extraction treatment step, a process is carried out to transfer the components contained in the extraction raw material to the extraction solvent, but the state is brought to a state in which the extraction solvent does not substantially affect the extraction raw material. More specifically, as shown in the examples described later, in the process of extracting proteoglycans (PG) using an alkaline aqueous solution as the extraction solvent, if the process is prolonged, dissociation or decomposition of hyaluronic acid (HA) may occur, making it difficult to extract both together. However, by bringing the state in which the extraction solvent does not substantially affect the extraction raw material, such problems can be avoided.

[0028] In the extraction termination step described above, the extraction process is performed to transfer components contained in the raw material to the extraction solvent, and the extraction solvent is brought to a state where it does not substantially affect the raw material. To achieve this, the effects of extraction, elution, dissolution, and decomposition of the extract by the alkaline aqueous solution used as the extraction solvent must be stopped. The means for doing so are not particularly limited, but typically, the above objective can be achieved by, for example, (1) adjusting the pH of the extraction solvent added to the raw material, and / or (2) removing the raw material from the extraction solvent by solid-liquid separation. More specifically, the pH of the extraction solvent can be adjusted by neutralization, dilution, etc. In addition, the raw material can be separated from the extraction solvent by solid-liquid separation by centrifugation, membrane treatment, etc.

[0029] In any non-limited embodiment of the present invention, the prepared composition obtained after the extraction termination step may have its proteoglycan content increased by means well known to those skilled in the art. That is, the prepared composition contains low molecular weight salts, proteins, and other impurities besides the target substance, and is a crude composition containing these. However, by subjecting it to conventional purification methods such as clarification filtration using diatomaceous earth or activated carbon, or ultrafiltration, these impurities can be removed, and a purified composition with a highly increased proteoglycan content can be obtained.

[0030] After the process described above, the extract may be dried by means of a vacuum dryer, spray dryer, or other means commonly known to those skilled in the art, or the dried product may be further crushed, pulverized, or otherwise dried into a powder. In the form of a dried product, since the moisture has been removed, similar to the freeze-dried product described above, spoilage is prevented and storage stability is excellent. During drying, excipients such as dextrin, crystalline cellulose, silica, etc., may be added in a formulation.

[0031] For powdering, the same methods as for the freeze-dried cartilage described above can be used, utilizing pulverizers, mills, muscoloaders, and other means commonly known to those skilled in the art. The particle size after powdering is preferably such that approximately 90% or more of the total material passes through a 30-mesh filter (mesh opening: 500 μm), and more preferably approximately 90% or more of the total material passes through a 60-mesh filter (mesh opening: 250 μm). Alternatively, it is preferable that approximately 90% or more of the total material passes through a screen with a diameter of 0.3 mm to 0.75 mm.

[0032] By the method described above, a hyaluronic acid complex-type proteoglycan can be obtained that also contains hyaluronic acid (HA), which forms a non-covalent complex in vivo. A hyaluronic acid complex-type proteoglycan-containing composition can then be obtained.

[0033] Generally, methods for measuring the amount of proteoglycans contained in a material include HPLC (High Performance Liquid Chromatography), enzymatic decomposition, capillary electrophoresis, and mass spectrometry. Additionally, the carbazole sulfate method (Galambos method), which measures the amount of glucuronic acid derived from glycosaminoglycans and converts that value, is also known.

[0034] Furthermore, methods for measuring the molecular weight of proteoglycans include gel filtration chromatography (size exclusion chromatography), SDS-PAGE, agarose gel electrophoresis, ultracentrifugation using a centrifuge, absorbance measurement (UV-Vis absorption spectroscopy), small-angle X-ray scattering (SAXS), and viscosity measurement. For more accurate molecular weight measurement of biomolecules, methods such as mass spectrometry (mass spectroscopy) and structural analysis using static light scattering with multi-angle light scattering detectors are known.

[0035] On the other hand, methods for measuring hyaluronic acid content include, for example, HPLC, ion exchange chromatography, capillary electrophoresis, enzymatic degradation, and ELISA (enzyme-linked immunosorbent assay). In recent years, assay systems using competitive ELISA, which utilize hyaluronic acid-binding proteins that specifically recognize high molecular weight hyaluronic acid, have also become available (product name "Hyaluronic Acid Measurement Kit," manufactured by PG Research Co., Ltd.). This method allows for more accurate measurement of high molecular weight hyaluronic acid.

[0036] In the hyaluronic acid complex proteoglycan-containing composition obtained by the present invention, the proteoglycan content per unit of solid content is preferably 0.1 to 12% by mass, more preferably 0.2 to 11% by mass, and even more preferably 0.3 to 10% by mass, as measured by the carbazole sulfuric acid method and based on the amount of glucuronic acid derived from glycosaminoglycans per unit of solid content.

[0037] In the hyaluronic acid complex proteoglycan-containing composition obtained by the present invention, the hyaluronic acid content per unit of solid content is preferably 0.01 to 2% by mass, more preferably 0.05 to 1% by mass, and even more preferably 0.1 to 0.5% by mass, as measured by a method using a hyaluronic acid-binding protein that specifically binds to hyaluronic acid.

[0038] The hyaluronic acid complex proteoglycan-containing composition obtained by the present invention preferably has an average molecular weight (Mw) of 2.65 million Da or more, more preferably 3 to 4 million Da, and even more preferably 3.2 to 4 million Da, as measured by the SEC-MALS method (size exclusion chromatography-multi-angle light scattering method).

[0039] In any non-limited embodiment of the present invention, it is preferable that the amount of hyaluronic acid (HA) is above a certain level relative to the amount of proteoglycan (PG) in order to standardize the quality of the resulting hyaluronic acid complex-type proteoglycan-containing composition. The measurement of this amount is not limited, but it is preferable to use the carbazole sulfuric acid method, which measures the amount of glucuronic acid derived from glycosaminoglycans and converts the amount of proteoglycan from that value, and the measurement method using a hyaluronic acid-binding protein that specifically binds to hyaluronic acid. Furthermore, for the hyaluronic acid complex proteoglycans contained in the resulting composition, it is preferable that the mass ratio of (1) the amount of glucuronic acid derived from glycosaminoglycans per unit solid content, measured by the carbazole sulfuric acid method, and (2) the amount of hyaluronic acid per unit solid content, measured by a measurement method using hyaluronic acid-binding protein that specifically binds to hyaluronic acid, is 50:1 to 2:1, more preferably 30:1 to 2.5:1, and particularly preferably 25:1 to 3:1.

[0040] Here, it is generally known that the chondroitin sulfate content (mass%) ([CS]) of proteoglycans is determined from the glucuronic acid content (mass%) ([Gluc]) derived from glycosaminoglycans measured by the carbazole sulfate method described above using the following formula. • [CS] = [Gluc] × 2.593 (In the above formula, "2.593" is the conversion factor for chondroitin sulfate, which has a disaccharide composed mainly of N-acetyl-D-galactosamine and D-glucuronic acid as its repeating constituent sugar.)

[0041] As described above, the hyaluronic acid complex proteoglycan-containing composition obtained by the present invention may have its proteoglycan content increased by means of means well known to those skilled in the art. In the form of the crude composition after preparation, the above [CS] equivalent amount may be preferably 10 to 60% by mass, more preferably 20 to 55% by mass, and even more preferably 30 to 50% by mass. Furthermore, in the form of a purified composition in which the proteoglycan content has been highly increased by clarification filtration or ultrafiltration, the above [CS] equivalent amount may be preferably 50 to 100% by mass, more preferably 60 to 97% by mass, and even more preferably 70 to 95% by mass.

[0042] The hyaluronic acid complex-type proteoglycan-containing composition obtained by the present invention may further contain vitamin C, imidazole peptide, collagen peptide, salmon ovary membrane peptide, β-hydroxy-β-methylbutyrate (HMB), etc., in addition to the hyaluronic acid complex-type proteoglycan. Such a hyaluronic acid complex-type proteoglycan-containing composition can be used for, for example, cosmetics, health foods and supplements, pharmaceuticals, quasi-drugs, etc., and can be particularly suitable as a raw material for these products. Furthermore, it can be used not only for humans but also for animals such as pets. [Examples]

[0043] The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited in any way by these examples.

[0044] <Testing Method> In the following preparation and test examples, the molecular weight distribution of proteoglycans was determined by the SEC-MALS method (size exclusion chromatography-multi-angle light scattering) (analysis software "ASTRA", Wyatt Technology), the amount of glucuronic acid derived from glycosaminoglycans necessary for estimating the amount of proteoglycan recovered was determined by the carbazole sulfate method, and the amount of hyaluronic acid (HA) was determined by a competitive ELISA method using hyaluronic acid-binding protein (HABP).

[0045] (Analysis conditions for the SEC-MALS method) • HPLC: HITACHI Chromaster series • Detector 1: RI detector • Detector 2: Multi-angle light scattering detector: DAWNHELEOS II (manufactured by Wyatt Technology) • Columns: OHpak SB-806M HQ (Exclusion limit: 20,000,000), OHpak SB-804 HQ (Exclusion limit: 1,000,000) ·Mobile phase: 0.2M-NaCl solution ·Flow rate: 0.5mL / min ·Temperature: 40℃ dn / dc value: 0.14

[0046] (Carbazole sulfuric acid method) This method involves detecting glucuronic acid, a component of glycosaminoglycans, by color development with carbazole reagent and measuring the concentration using a spectrophotometer. Specifically, in this test example, 10 equivalents of concentrated sulfuric acid were added to 1 equivalent volume of the sample, cooled with ice water, and after cooling with ice, carbazole reagent solution was added. The mixture was then heated in a boiling bath for approximately 10 minutes, cooled again with ice water, and the absorbance at 530 nm was measured. On the other hand, a calibration curve using a glucuronic acid standard solution was prepared in advance using the same method, and the uronic acid concentration in the sample was determined by applying it to this curve.

[0047] (Hyaluronic acid quantitative analysis) A competitive assay using hyaluronic acid-binding protein (HABP) is known as a method for quantifying high molecular weight hyaluronic acid. Specifically, in this test example, a commercially available kit (product name "Hyaluronic Acid Measurement Kit," manufactured by PG Research Co., Ltd.) was used to quantify the hyaluronic acid as follows.

[0048] A 96-well plate treated with hyaluronic acid (HA) coating solution was used. 200 μL of blocking solution was added to each well, and the plate was allowed to stand at room temperature for 30 minutes to block the mixture. After washing with washing solution (300 μL / well), 50 μL / well of appropriately diluted sample was added, followed by 50 μL / well of the same volume of biotin-labeled hyaluronic acid-binding protein (Biotin-HABP). The plate was gently agitated and allowed to stand at room temperature for 1 hour to allow the reaction to occur. Subsequently, the plate was washed four times with washing solution, and then 100 μL / well of HRP-labeled avidin (HRP-avidin), a biotin-binding protein, was added. The plate was allowed to stand at room temperature for 1 hour. After washing four more times with washing solution, 100 μL / well of substrate solution was added, and the plate was allowed to react at room temperature for 20-30 minutes under light-shielding conditions to allow color development. Finally, 100 μL / well of stop solution was added to stop the reaction, and the absorbance at 450 nm was measured using a plate reader. On the other hand, a calibration curve using hyaluronic acid standard solutions was prepared in advance using the same method, and the hyaluronic acid concentration in the sample was determined by applying it to this curve.

[0049] [Preparation Example 1] (Product 1) 100g of salmon nasal cartilage was minced using a meat chopper to obtain a paste-like surimi. To this surimi, 1200mL of an alkaline aqueous solution with a pH of 12.1, containing sodium hydroxide at a concentration of 0.0125M (mol / L) (0.05w / v%) and sodium chloride at a concentration of 0.171M (mol / L) (1w / v%), was added as an extraction solvent, and the mixture was stirred at 4°C for 0.5 hours. The resulting solution was separated into solid and liquid portions by centrifugation to obtain the supernatant. Hydrochloric acid was added to the supernatant to neutralize it to a pH of 7, and the neutralized solution was separated into solid and liquid portions again by centrifugation to obtain the supernatant. The supernatant was clarified by diatomaceous earth filtration, and then desalted to remove low molecular weight components using an ultrafiltration membrane (molecular weight cutoff: 10-100kDa).

[0050] The obtained samples were analyzed using the SEC-MALS method, a molecular weight distribution analysis method based on the measurement of absolute molecular weight using a multi-angle light scattering detector. The molecular weight distribution shown in Table 1 below was obtained. The average molecular weight (Mw) was 3.62 million Da.

[0051] [Table 1]

[0052] Furthermore, when the hyaluronic acid content in the sample was confirmed using a competitive ELISA method with hyaluronic acid-binding protein (HABP), it was found to be 0.33% by mass in the solid content.

[0053] Furthermore, the amount of glucuronic acid derived from glycosaminoglycans contained in the sample was confirmed to be 1.071% by mass in the solid content using the carbazole sulfuric acid method.

[0054] Furthermore, the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was 30.8, when the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was denoted as "[Gluc]" and the mass percentage of hyaluronic acid per unit of solid content in the sample was denoted as "[HA]". ·{([HA] / ([Gluc])}×100

[0055] [Preparation Example 2] (Product 2) The sample was prepared in the same manner as in Preparation Example 1, except that instead of the alkaline aqueous solution used in Preparation Example 1, an alkaline aqueous solution with a sodium hydroxide concentration of 0.02 M (mol / L) (0.08 w / v%) and a sodium chloride concentration of 0.0856 M (mol / L) (0.5 w / v%) with a pH of 12.3 was used as the extraction solvent, and the extraction temperature was changed to 8°C and the stirring time to 0.3 hours.

[0056] The obtained samples were analyzed by the SEC-MALS method in the same manner as in Preparation Example 1, and the molecular weight distribution shown in Table 2 below was obtained. The average molecular weight (Mw) was 3,461,000 Da.

[0057] [Table 2]

[0058] Furthermore, when the hyaluronic acid content in the sample was confirmed using a competitive ELISA method with hyaluronic acid-binding protein (HABP), it was found to be 0.19% by mass in the solid content.

[0059] Furthermore, the amount of glucuronic acid derived from glycosaminoglycans contained in the sample was confirmed to be 0.491% by mass in the solid content using the carbazole sulfuric acid method.

[0060] Furthermore, the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was 38.7, when the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was denoted as "[Gluc]" and the mass percentage of hyaluronic acid per unit of solid content in the sample was denoted as "[HA]". ·{([HA] / ([Gluc])}×100

[0061] [Preparation Example 3] (Product 3) The sample was prepared in the same manner as in Preparation Example 1, except that instead of the alkaline aqueous solution used in Preparation Example 1, an alkaline aqueous solution with a sodium hydroxide concentration of 0.0075 M (mol / L) (0.03 w / v%) and a sodium chloride concentration of 0.342 M (mol / L) (2 w / v%) with a pH of 11.88 was used as the extraction solvent, and the extraction temperature was changed to 2°C and the stirring time to 0.1 hours.

[0062] The obtained samples were analyzed by the SEC-MALS method in the same manner as in Preparation Example 1, and the molecular weight distribution shown in Table 3 below was obtained. The average molecular weight (Mw) was 3,442,000 Da.

[0063] [Table 3]

[0064] Furthermore, when the hyaluronic acid content in the sample was confirmed using a competitive ELISA method with hyaluronic acid-binding protein (HABP), it was found to be 0.075% by mass in the solid content.

[0065] Furthermore, the amount of glucuronic acid derived from glycosaminoglycans contained in the sample was confirmed to be 0.199% by mass in the solid content using the carbazole sulfuric acid method.

[0066] Furthermore, the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was 37.6, when the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was denoted as "[Gluc]" and the mass percentage of hyaluronic acid per unit of solid content in the sample was denoted as "[HA]". ·{([HA] / ([Gluc])}×100

[0067] [Preparation Example 4] (Comparative Product 1) As the extraction solvent, instead of the alkaline aqueous solution used in Preparation Example 1, an alkaline aqueous solution with a sodium hydroxide concentration of 0.02 M (mol / L) (0.08 w / v%) and no added sodium chloride was used, with a pH of 12.3. The sample was prepared in the same manner as in Preparation Example 1, except that the extraction temperature was changed to 10°C and the stirring time to 12 hours.

[0068] The obtained samples were analyzed by SEC-MALS in the same manner as in Preparation Example 1, and the molecular weight distribution shown in Table 4 below was obtained. The average molecular weight (Mw) was 2,142,000 Da.

[0069] [Table 4]

[0070] Furthermore, when the hyaluronic acid content in the sample was confirmed using a competitive ELISA method with hyaluronic acid-binding protein (HABP), it was found to be 0.06% by mass in the solid content.

[0071] Furthermore, the amount of glucuronic acid derived from glycosaminoglycans in the sample was confirmed to be 15.04% by mass in the solid content using the carbazole sulfuric acid method.

[0072] Furthermore, the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was 0.4, when the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was denoted as "[Gluc]" and the mass percentage of hyaluronic acid per unit of solid content in the sample was denoted as "[HA]". ·{([HA] / ([Gluc])}×100

[0073] [Preparation Example 5] (Comparative Product 2) The sample was prepared in the same manner as in Preparation Example 1, except that instead of the alkaline aqueous solution used in Preparation Example 1, an alkaline aqueous solution with a sodium hydroxide concentration of 0.125 M (mol / L) (0.5 w / v%) and no added sodium chloride was used as the extraction solvent, and the extraction temperature was changed to 4°C and the stirring time to 18 hours.

[0074] The obtained samples were analyzed by SEC-MALS in the same manner as in Preparation Example 1, and the molecular weight distribution shown in Table 4 below was obtained. The average molecular weight (Mw) was 450,000 Da.

[0075] [Table 5]

[0076] Furthermore, when the hyaluronic acid content in the sample was confirmed using a competitive ELISA method with hyaluronic acid-binding protein (HABP), it was found to be 0% by mass (not detected) in the solid content.

[0077] Furthermore, the amount of glucuronic acid derived from glycosaminoglycans in the sample was confirmed to be 16.19% by mass in the solid content using the carbazole sulfuric acid method.

[0078] Furthermore, the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was 0, when the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was denoted as "[Gluc]" and the mass percentage of hyaluronic acid per unit of solid content in the sample was denoted as "[HA]". ·{([HA] / ([Gluc])}×100

[0079] [Preparation Example 6] (Comparative Product 3) The sample was prepared in the same manner as in Preparation Example 1, except that instead of the alkaline aqueous solution used in Preparation Example 1, an alkaline aqueous solution with a sodium hydroxide concentration of 0.05 M (mol / L) (0.2 w / v%) and no added sodium chloride was used as the extraction solvent, and the extraction temperature was changed to 9°C and the stirring time to 18 hours.

[0080] The obtained samples were analyzed by SEC-MALS in the same manner as in Preparation Example 1, and the molecular weight distribution shown in Table 6 below was obtained. The average molecular weight (Mw) was 2,616,000 Da.

[0081] [Table 6]

[0082] Furthermore, when the hyaluronic acid content in the sample was confirmed using a competitive ELISA method with hyaluronic acid-binding protein (HABP), it was found to be 0.05% by mass in the solid content.

[0083] Furthermore, the amount of glucuronic acid derived from glycosaminoglycans in the sample was confirmed to be 16.19% by mass in the solid content using the carbazole sulfuric acid method.

[0084] Furthermore, the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was 0.3, when the mass percentage of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample was denoted as "[Gluc]" and the mass percentage of hyaluronic acid per unit of solid content in the sample was denoted as "[HA]". ·{([HA] / ([Gluc])}×100

[0085] <Test Example 1> The relationship between extraction time and the amount of proteoglycan and hyaluronic acid recovered was investigated. To this end, 100 g of salmon nasal cartilage was minced using a meat chopper device in the same manner as in the preparation example described above to obtain a paste-like surimi. To this surimi, 1200 mL of an alkaline aqueous solution with a pH of 12.3 and a sodium hydroxide concentration of 0.01875 M (mol / L) (0.075 w / v%) was added as the extraction solvent, and the mixture was stirred at 7°C for a predetermined time.

[0086] After adding the extraction solvent, samples were taken from the sample at various time intervals. Hyaluronic acid was quantified using a competitive ELISA method with hyaluronic acid-binding protein (HABP) in the same manner as in the preparation example described above, and proteoglycans were quantified using the carbazole sulfuric acid method, as glucuronic acid derived from glycosaminoglycans. For normalization, the content (mass%) per solid content of the sample was calculated.

[0087] The results obtained are shown in Figures 2 to 5.

[0088] As shown in Figures 2 and 3, the hyaluronic acid content (mass%) per unit of solid matter in the sample increased within 0.5 hours after the addition of the extraction solvent, and decreased thereafter. On the other hand, the glucuronic acid content (mass%) derived from glycosaminoglycans per unit of solid matter in the sample increased over 3 hours after the addition of the extraction solvent, and did not fluctuate significantly thereafter.

[0089] Figures 4 and 5 are charts showing the results from Figures 2 and 3 converted to percentages expressed by the following formula, where "[Gluc]" represents the mass %) content of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample, and "[HA]" represents the mass %) content of hyaluronic acid per unit of solid content in the sample. ·{([HA] / ([Gluc])}×100

[0090] As shown in Figures 4 and 5, the proportion of hyaluronic acid in the solid content of the sample reached a maximum around 0.3 hours after the addition of the extraction solvent, and after that time, the proportion decreased rapidly.

[0091] <Test Example 2> The relationship between the concentration of sodium chloride in the extraction solvent and the amount of proteoglycans and hyaluronic acid recovered was investigated. To this end, the concentration of sodium chloride in the alkaline aqueous solution used as the extraction solvent was varied, and the rest of the test method was the same as in Test Example 1. After adding the extraction solvent, a portion of the sample was sampled at various elapsed time points, and the glucuronic acid and hyaluronic acid derived from glycosaminoglycans were quantified and normalized as the content (mass%) per solid content of the sample.

[0092] The results obtained are shown in Figures 6 to 11.

[0093] As shown in Figures 6 and 7, increasing the concentration of sodium chloride in the alkaline aqueous solution used as the extraction solvent resulted in an increase in the recovery amount of proteoglycans represented by glucuronic acid derived from glycosaminoglycans. Furthermore, this increase in sodium chloride concentration increased to 1 w / v%, but there was no further increase even when the sodium chloride concentration was increased further.

[0094] As shown in Figures 8 and 9, increasing the concentration of sodium chloride in the alkaline aqueous solution used as the extraction solvent resulted in an increase in the amount of hyaluronic acid recovered. Furthermore, this increase in hyaluronic acid recovery increased as the sodium chloride concentration was raised to 1 w / v%, but there was no further increase even when the sodium chloride concentration was increased further.

[0095] Figures 10 and 11 are charts showing the results from Figures 6-9 converted to percentages expressed by the following formula, where "[Gluc]" represents the mass %) content of glucuronic acid derived from glycosaminoglycans per unit of solid content in the sample, and "[HA]" represents the mass %) content of hyaluronic acid per unit of solid content in the sample. ·{([HA] / ([Gluc])}×100

[0096] As shown in Figures 10 and 11, the proportion of hyaluronic acid in the solid content of the sample reached a maximum around 0.3 hours after the addition of the extraction solvent, and after that time, the proportion decreased rapidly.

[0097] Based on the results of Preparation Examples 1-6 and Test Examples 1 and 2, it was concluded that to efficiently obtain the target hyaluronic acid complex proteoglycan, it is useful to add the alkaline aqueous solution used as the extraction solvent and then stop the extraction process at a predetermined point.

Claims

1. A method for producing a hyaluronic acid-complex proteoglycan-containing composition using fish cartilage as an extractant, The extraction solvent addition step involves adding an alkaline aqueous solution to the extraction raw material, which has a pH of 10 or higher and a pH of 13 or lower, and is prepared by adding a water-soluble inorganic salt that increases the ionic strength of the extraction solvent. An extraction process is performed to transfer components contained in the extraction material from the extraction material to which the extraction solvent has been added to the extraction solvent. The system includes an extraction termination step, which involves performing an extraction termination step to stop the process with the extraction solvent, The extraction termination step includes (1) a process of adjusting the pH of the extraction solvent added to the extraction raw material, and / or (2) a process of removing the extraction raw material from the extraction solvent by solid-liquid separation. A method for producing the hyaluronic acid complex-type proteoglycan-containing composition, wherein the extraction time in the extraction process is 0.05 hours or more and 0.5 hours or less.

2. A method for producing a hyaluronic acid complex-type proteoglycan-containing composition according to claim 1, wherein the extraction temperature in the extraction process is 10°C or lower.

3. A method for producing a hyaluronic acid complex-type proteoglycan-containing composition according to claim 2, wherein the extraction temperature is 0°C or higher and 4°C or lower.

4. The method for producing the hyaluronic acid complex proteoglycan-containing composition according to any one of claims 1 to 3, wherein the mass ratio of the hyaluronic acid complex proteoglycan contained in the composition is 50:1 to 2:1, wherein (1) the amount of glucuronic acid derived from glycosaminoglycans measured by the carbazole sulfuric acid method per unit of solid content and (2) the amount of hyaluronic acid measured by a measurement method using a hyaluronic acid-binding protein that specifically binds to hyaluronic acid.

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