Method for producing proteoglycan and proteoglycan

Abstract

To provide a method for producing proteoglycan that can produce proteoglycan in a simpler manner than conventional methods for producing proteoglycan and can produce proteoglycan in which a core protein is bound to a sugar chain.SOLUTION: The method for producing proteoglycan disclosed herein includes an extraction step of extracting an extract containing proteoglycan from an animal tissue, the proteoglycan containing a core protein and a glycan, and the core protein and the glycan being bound to each other.SELECTED DRAWING: Figure 4

Description

Technical Field

[0001] The present disclosure relates to a method for producing a proteoglycan and a proteoglycan.

Background Art

[0002] Proteoglycans are molecules that constitute the extracellular matrix together with collagen, hyaluronic acid, etc. The proteoglycan is excellent in water retention and is known to have various physiological functions such as an anti-inflammatory effect, a hyaluronic acid synthesis promoting effect, and a cell proliferation promoting effect. For this reason, various uses of proteoglycans in cosmetics, food and drink, etc. have been studied.

[0003] However, in the conventional method for producing proteoglycans, there are problems such as using substances harmful to humans and animals such as EDTA, the production process being complicated in order to remove the harmful substances, and the amount of proteoglycan obtained decreasing (Non-Patent Documents 1, 2).

Prior Art Documents

Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, the present disclosure aims to, for example, simplify the production of proteoglycan compared to conventional proteoglycan production methods, and to produce proteoglycan in which a core protein and a sugar chain are bound, and the like.

Means for Solving the Problems

[0006] In order to achieve the above object, the method for producing proteoglycan of the present disclosure (hereinafter, also referred to as "production method") includes an extraction step of extracting an extract containing proteoglycan from an animal tissue, the proteoglycan contains a core protein and a sugar chain, and the core protein and the sugar chain are bound to each other.

[0007] The composition of the present disclosure contains proteoglycan, and the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000.

Effects of the Invention

[0008] According to the present disclosure, it is possible to simplify the production of proteoglycans compared to conventional methods for producing proteoglycans, and to produce proteoglycans in which a core protein and a sugar chain are bound, and the like.

Brief Description of the Drawings

[0009]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Modes for Carrying Out the Invention

[0010] Hereinafter, the present disclosure will be specifically described with examples. Hereinafter, unless otherwise specified, each disclosure can incorporate the description of other disclosures.

[0011] <Definition> As used herein, "proteoglycan" means a molecule (glycoprotein) in which a protein (core protein) and a glycosaminoglycan (GAG, also referred to as "polysaccharide" or "sugar chain") are covalently bonded. The proteoglycan exists, for example, as an extracellular matrix in skin, organs, cartilage, and the like. The glycosaminoglycan is generally known as a sugar chain having a long-chain structure without a branched structure. The proteoglycan includes, for example, the following. · Aggrecan family (also referred to as lectican family or hyalectan family): Aggrecan, Versican, Neurocan, Brevican, etc. · Small Leucine Rich Proteoglycans (SLRPs) family: Biglycan, Decorin, Fibromodulin, Lumican, PG-Lb (Epiphycan), Keratocan, Mimecan, etc. · Proteoglycans of basement membrane: Perlecan, Agrin, Bamacan, etc. · Other proteoglycans: Testican, Biglycan, Serglycin, Syndecan, Dystroglycan, Claustrin, Glypican, Keratocan, etc. The proteoglycan can be classified into chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, heparan sulfate proteoglycan, or keratan sulfate proteoglycan, for example, according to the type of GAG bound to the protein.

[0012] The GAGs include, for example, chondroitin, chondroitin sulfate (CS), dermatan sulfate (DS, chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. The chondroitin includes an O-type sugar chain having a disaccharide structure of glucuronic acid and N-acetylgalactosamine as a main disaccharide structure, and an iO-type sugar chain having a disaccharide structure of iduronic acid and N-acetylgalactosamine as a main structure (hereinafter, also referred to as "chondroitin sulfate O" and "chondroitin sulfate iO", respectively). The chondroitin sulfate (CS) has a structure in which a sulfate group is added to a sugar chain in which a disaccharide of glucuronic acid and N-acetylgalactosamine repeats. The chondroitin sulfate includes, for example, chondroitin sulfate A (type A) having a disaccharide structure of glucuronic acid and N-acetylgalactosamine 4-sulfate as a main disaccharide structure, chondroitin sulfate C (type C) having a disaccharide structure of glucuronic acid and N-acetylgalactosamine 6-sulfate as a main disaccharide structure, and the like. The dermatan sulfate (DS) has a structure in which a sulfate group is added to a sugar chain in which a disaccharide of iduronic acid and N-acetylgalactosamine repeats. The dermatan sulfate includes, for example, chondroitin sulfate iA (iA type) having a disaccharide structure of iduronic acid and N-acetylgalactosamine 4-sulfate as a main disaccharide structure, chondroitin sulfate iC (iC type) having a disaccharide structure of iduronic acid and N-acetylgalactosamine 6-sulfate as a main disaccharide structure, and the like. Each chondroitin sulfate has, for example, a disaccharide structure shown in FIG. 1 as a main disaccharide structure. In FIG. 1, the sulfate group (sulfonyl group) is bonded to a hydrogen atom, but the present disclosure is not limited thereto, and the sulfate group of the GAG may be, for example, ionized by the elimination of a hydrogen atom or may form a salt.

[0013] In this specification, "extraction" means extracting a specific component from an object and / or the state where a specific component has been extracted from the object. The extraction can be carried out, for example, using an extractant such as a solvent. The "extraction" can be carried out, for example, by obtaining at least one extraction step. In this specification, an object containing the component extracted from the object is called an extract. Also, a liquid containing the component extracted from the object is called an extractant. The extract and the extractant may contain, for example, the proteoglycan. The extract or the extractant may contain, for example, components other than the proteoglycan derived from the animal tissue subjected to the extraction (other components). Examples of the other components include lipids, nucleic acids, proteins, etc.

[0014] In this specification, "purification" means identifying, separating, recovering from components in their natural state, the state of being identified and separated, and / or the state of being recovered from components in their natural state. The "purification" can be carried out, for example, by obtaining at least one purification step. The purification can also be referred to as isolation.

[0015] Hereinafter, the present disclosure will be described with examples, but the present disclosure is not limited to the following examples and can be arbitrarily modified and implemented. Also, each description in the present disclosure and each embodiment can be mutually incorporated unless otherwise specified. In this specification, when the expression "~" is used, it is used in the sense of including the numerical values or physical values before and after it. Also, in this specification, the expression "A and / or B" includes "only A", "only B", and "both A and B".

[0016] <Method for producing proteoglycan> In one aspect, the present disclosure provides a method for producing a proteoglycan. The production method of the present disclosure includes an extraction step of extracting an extract containing a proteoglycan from an animal tissue, the proteoglycan includes a core protein and a sugar chain, and the core protein and the sugar chain are bound.

[0017] In the extraction step, an extract containing proteoglycan is extracted from the animal tissue. Specifically, in the extraction step, the animal tissue is brought into contact with an extraction solution, so that the proteoglycan present in the animal tissue is extracted into the extraction solution. Thereby, in the extraction step, the proteoglycan can be extracted into the extraction solution. For example, by recovering the extraction solution, an extraction solution containing the proteoglycan can be recovered. In the extraction step, the contact between the animal tissue and the extraction solution can be carried out by, for example, a known contact method between a solid and a liquid. Specifically, it can be carried out by mixing the animal tissue and the extraction solution.

[0018] In the extraction of proteoglycan from the animal tissue, in order to promote the release of proteoglycan from the animal tissue, a protease or peptidase such as collagenase, and / or a sugar chain degrading enzyme such as cellulase, glucanase, chondroitinase, a metal chelating agent (chelator) such as EDTA, etc. are added and carried out. Therefore, in the obtained extract or extraction solution, the proteoglycan is extracted in a state where a part or all of the core protein and / or sugar chain of the proteoglycan is decomposed. On the other hand, in the extraction step, for example, the extraction step is carried out without decomposing the core protein and / or sugar chain constituting the proteoglycan, that is, in a state not containing or adding an exogenous degrading enzyme and / or metal chelating agent. Therefore, in the production method of the present disclosure, the proteoglycan can be extracted in a state containing the core protein and the sugar chain and in a state where the core protein and the sugar chain are bound, that is, in a state where the decomposition of the core protein and / or the sugar chain is suppressed. Further, in the production method of the present disclosure, for example, a proteoglycan having a structure closer to that of the proteoglycan in the animal tissue can be extracted. Examples of the exogenous degrading enzyme include proteases, peptidases, sugar chain degrading enzymes, chondroitinases, etc. that are not derived from the animal tissue.

[0019] The animal is not particularly limited, and examples thereof include mammalian animals such as pigs and cows (mammals); avian animals such as chickens (birds); fish such as flatfish of the family Bothidae like the common flounder, salmonids such as white salmon and Atlantic salmon, and fish such as rays (including, for example, the thornback ray). The animal is preferably a pig, a cow, a bird, a flounder, a salmon, a ray, etc.

[0020] The types of GAGs that make up the proteoglycan vary depending on the type of the animal. Therefore, in the extraction step, by adjusting the origin of the animal tissue or mixing animal tissues from different animals, the composition ratio of the disaccharide structure of GAGs that make up the extracted proteoglycan can be adjusted. When the animal is a mammal or a bird, the proteoglycan derived from the animal relatively contains more GAGs having, for example, a disaccharide structure of type A or iA as compared with proteoglycans of other animals. As a specific example, in the proteoglycan derived from the bronchial cartilage of the pig, the content ratios of the type A and iA disaccharide structures are, for example, 40 to 60% and 10 to 30%, respectively. In the proteoglycan derived from the abomasum of the cow, the content ratios of the type A and iA disaccharide structures are, for example, 5 to 25% and 30 to 60%, respectively. Also, in the proteoglycan derived from the breast cartilage of the chicken, the content ratios of the type A and iA disaccharide structures are, for example, 50 to 75% and 3 to 10%, respectively. Therefore, when extracting a proteoglycan with a higher composition ratio of proteoglycans containing GAGs having a disaccharide structure of type A or iA, as the animal, animal tissues derived from the mammals and / or the birds are used or added, whereby in the extraction step, the content ratio of GAGs having a disaccharide structure of type A or iA in the sugar chain of the obtained proteoglycan can be increased. When the animal is a fish, the proteoglycan derived from the animal relatively contains less GAGs having, for example, a disaccharide structure of type A or iA as compared with proteoglycans of other animals. As a specific example, in the proteoglycan derived from the nasal bone of the rainbow trout, the content ratios of the type A and iA disaccharide structures are, for example, 10 to 30% and 5 to 12.5%, respectively. Also, in the proteoglycan derived from the fin of the little skate, the content ratios of the type A and iA disaccharide structures are, for example, 25 to 45% and 3 to 10%, respectively. In the proteoglycan derived from the nasal cartilage of the Atlantic salmon, the content ratios of the type A and iA disaccharide structures are, for example, 16 to 26% and 2 to 13%, respectively.In the proteoglycan derived from the fin of the eel, the content ratios of the A-type and the iA-type disaccharide structures are, for example, 5 to 15% and 4 to 13%, respectively. Therefore, when extracting a proteoglycan having a lower composition ratio of a proteoglycan containing a GAG having an A-type or iA-type disaccharide structure, as the animal, an animal tissue derived from the fish is used or added, so that in the extraction step, the content ratio of the GAG having an A-type or iA-type disaccharide structure in the sugar chain of the obtained proteoglycan can be lowered.

[0021] When the animal is a fish such as a white salmon, an Atlantic salmon, a Japanese whiting, or a skate, the proteoglycan derived from the animal relatively contains more GAGs having a C-type or iC-type disaccharide structure, for example, compared to proteoglycans of other animals. As a specific example, in the proteoglycan derived from the nasal cartilage of the white salmon, the content ratios of the C-type and iC-type disaccharide structures are, for example, 45 to 65% and 0 to 13%, respectively, or 45 to 55% and 3 to 13%, respectively. Also, in the proteoglycan derived from the fin of the Japanese whiting, the content ratios of the C-type and iC-type disaccharide structures are, for example, 30 to 50% and 0 to 10%, respectively. In the proteoglycan derived from the nasal cartilage of the Atlantic salmon, the content ratios of the C-type and iC-type disaccharide structures are, for example, 42 to 52% and 1 to 12%, respectively. In the proteoglycan derived from the fin of the skate (Raja kenojei), the content ratios of the C-type and iC-type disaccharide structures are, for example, 50 to 70% and 0 to 10%, respectively. Therefore, when extracting a proteoglycan having a higher composition ratio of a proteoglycan containing a GAG having a C-type or iC-type disaccharide structure, as the animal, an animal tissue derived from the fish can be used, and in the extraction step, the content ratio of the GAG having a C-type or iC-type disaccharide structure in the sugar chain of the obtained proteoglycan can be increased. When the animal is a mammal or a bird, the proteoglycan derived from the animal relatively contains less GAGs having a C-type or iC-type disaccharide structure, for example, compared to proteoglycans of other animals. As a specific example, in the proteoglycan derived from the bronchial cartilage of the pig, the content ratios of the C-type and iC-type disaccharide structures are, for example, 10 to 30% and 1 to 5%, respectively. In the proteoglycan derived from the abomasum of the cow, the content ratios of the C-type and iC-type disaccharide structures are, for example, 10 to 30% (more preferably 10% to 25%) and 10% or less, respectively. In the proteoglycan derived from the breast cartilage of the chicken, the total content ratios of the C-type and iC-type disaccharide structures are, for example, 15 to 35% and 0 to 3%, respectively.Therefore, when extracting a proteoglycan with a lower composition ratio of proteoglycans containing GAG having a C-type or iC-type disaccharide structure, as the animal, an animal tissue derived from the mammal or bird can be used, and in the extraction step, the content ratio of GAG having a C-type or iC-type disaccharide structure in the sugar chain of the obtained proteoglycan can be lowered.

[0022] The content ratio of the disaccharide structure in the proteoglycan can be calculated by analyzing the composition of the disaccharide structure of the proteoglycan, that is, the composition of the disaccharide structure of GAG in the proteoglycan. Specifically, the method for measuring the disaccharide structure of the sugar chain of the proteoglycan can be carried out, for example, with reference to the measurement methods in References 1 and 2 below and in accordance with Examples 1(2) and (3) described later. Reference 1: Takeda Naoko et al. “Facile analysis of contents and compositions of the chondroitin sulfate / dermatan sulfate hybrid chain in shark and ray tissues.” Carbohydrate research vol. 424 (2016): 54-8. doi:10.1016 / j.carres.2016.02.006 Reference 2: Tamura, Jun-ichi et al. “Sulfation patterns and the amounts of chondroitin sulfate in the diamond squid, Thysanoteuthis rhombus.” Bioscience, biotechnology, and biochemistry vol. 73,6 (2009): 1387-91. doi:10.1271 / bbb.90037

[0023] First, prior to the measurement of the disaccharide structure, GAG is prepared from the proteoglycan. Specifically, GAG is purified from the proteoglycan to be measured according to Example 1(2) described below. Next, according to Example 1(3) described below, unsaturated disaccharides are prepared from the obtained GAG, and the content ratio of each disaccharide structure is calculated from the HPLC peak area of the obtained unsaturated disaccharides. That is, the peak area of each disaccharide structure in the total peak area of HPLC is calculated as the content ratio of each disaccharide structure.

[0024] (HPLC measurement conditions) Sample preparation: The sugar chain (50 μg) is diluted with H2O (50 μl), BSA solution (0.05 mg / 5 μl), and 250 mmol / l Tris-HCl (AcOH) buffer (10 μl) (pH 8.0), and then chondroitinase ABC (25 mU / 25 μl) or chondroitinase AC (25 mU / 25 μl) is used to digest it under the conditions of 37 °C or 30 °C for 8 hours to prepare unsaturated disaccharides. HPLC system: LC-10AD (manufactured by Shimadzu Corporation) Column: PA-03 (PA-G) column Eluent: Aqueous NaH2PO4 solution Flow rate: 1.0 ml / min

[0025] The molecular weight (peak top molecular weight) of the proteoglycan and the molecular weight (peak top molecular weight) of the GAG vary depending on the type of the animal. Therefore, in the extraction step, the molecular weights of the proteoglycan to be extracted and the GAG constituting the proteoglycan can be adjusted by adjusting the origin of the animal tissue or mixing animal tissues derived from different animals. As a specific example, in the proteoglycan derived from the rainbow trout, the molecular weight of the proteoglycan is, for example, 300,000 to 800,000, 600,000 to 750,000. In the GAG derived from the rainbow trout, the molecular weight of the GAG is, for example, 60,000 to 100,000, 70,000 to 90,000. In the proteoglycan derived from the Atlantic salmon, the molecular weight of the proteoglycan is, for example, 1,000,000 to 1,300,000, 1,100,000 to 1,250,000. In the GAG derived from the Atlantic salmon, the molecular weight of the GAG is, for example, 30,000 to 150,000, 50,000 to 80,000. In the proteoglycan derived from the black rockfish, the molecular weight of the proteoglycan is, for example, 600,000 to 1,000,000, 800,000 to 900,000. In the GAG derived from the black rockfish, the molecular weight of the GAG is, for example, 50,000 to 110,000, 70,000 to 100,000. In the GAG derived from the skate, the molecular weight of the GAG is, for example, 140,000 to 280,000, 190,000 to 230,000. In the proteoglycan derived from the chicken, the molecular weight of the proteoglycan is, for example, 500,000 to 750,000, 600,000 to 700,000. In the GAG derived from the chicken, the molecular weight of the GAG is, for example, 30,000 to 90,000, 40,000 to 70,000. In the proteoglycan derived from the pig, the molecular weight of the proteoglycan is, for example, 500,000 to 750,000, 600,000 to 700,000. In the GAG derived from the pig, the molecular weight of the GAG is, for example, 10,000 to 50,000, 20,000 to 40,000. In the proteoglycan derived from the cow, the molecular weight of the proteoglycan is, for example, 300,000 to 600,000, 400,000 to 500,000. In the GAG derived from the cow, the molecular weight of the GAG is, for example, 5,000 to 300,000, 6,000 to 250,000.

[0026] In this specification, the molecular weight of the proteoglycan and the molecular weight of the GAG may be, for example, the peak top molecular weight measured by the GPC (Gel Permeation Chromatography) method. The molecular weight of the proteoglycan can be measured using the GPC method according to Example 1(1) described below. The GPC method can be calculated, for example, by performing under the following conditions, injecting the following standard samples (molecular weight markers, pullulan) into the HPLC system individually, and obtaining a molecular weight calibration curve.

[0027] (Measurement conditions for peak top molecular weight (Mp)) HPLC system: LC-10AD (manufactured by Shimadzu Corporation) Column: TSKgel G5000-PWXL φ7.8mm×300mm (manufactured by Tosoh Corporation) Eluent: Phosphate buffer with pH 6.8 Flow rate: 0.5 ml / min Column temperature: 40 °C Detector: Differential refractive index detector (RID-10A manufactured by Shimadzu Corporation) Injection volume: 50 μl Molecular weight marker: Shodex STANDARD P-82 (pullulan) The peak top molecular weight (Mp) and weight average molecular weight (Mw) / number average molecular weight (Mn) of the molecular weight marker are as follows. STD P-800: Mp: 739,000, Mw / Mn: 1.24 STD P-400: Mp: 348,000, Mw / Mn: 1.33 STD P-200: Mp: 216,000, Mw / Mn: 1.22 STD P-100: Mp: 107,000, Mw / Mn: 1.12 STD P-50: Mp: 49,400, Mw / Mn: 1.08 STD P-20: Mp: 22,000, Mw / Mn: 1.08 STD P-10: Mp: 9,800, Mw / Mn: 1.07 STD P-5: Mp: 6,300, Mw / Mn: 1.09

[0028] The method for measuring the molecular weight of the GAG can be carried out using the GPC method, for example, according to Example 1(2) described below. The GPC method can be carried out under the following conditions, for example. The standard sample (molecular weight marker, pullulan) is individually injected into the HPLC system to obtain a molecular weight calibration curve for calculation. (Measurement conditions for the peak top molecular weight of GAG) HPLC system: LC-10AD (manufactured by Shimadzu Corporation) Sample: Dilute GAG (50 μg) with H2O (50 μg) Column: Linear connected size exclusion column (OHpak SB-G 6B 100 × 4.6 mm and SB-805 HQ 250 × 4.6 mm) Eluent: 0.1 mol / l NaNO3 Flow rate: 1 ml / min

[0029] The molecular weight of the proteoglycan and the molecular weight of the GAG may be the number average molecular weight. The number average molecular weight can be measured by the GPC method according to Example 1(1) described below, similar to the peak top molecular weight. In the GAG derived from Oncorhynchus keta, the number average molecular weight of the GAG is, for example, 20,000 to 130,000, 40,000 to 110,000. In the GAG derived from Salmo salar, the number average molecular weight of the GAG is, for example, 10,000 to 120,000, 30,000 to 100,000. In the GAG derived from Pleuronectes yokohamae, the number average molecular weight of the GAG is, for example, 30,000 to 140,000, 50,000 to 120,000. In the GAG derived from Raja porosa, the number average molecular weight of the GAG is, for example, 230,000 to 340,000, 250,000 to 320,000. In the GAG derived from Gallus gallus, the number average molecular weight of the GAG is, for example, 10,000 to 110,000, 20,000 to 80,000. In the proteoglycan derived from Sus scrofa, in the GAG derived from Sus scrofa, the number average molecular weight of the GAG is, for example, 10,000 to 60,000, 20,000 to 40,000.

[0030] The molecular weight of the proteoglycan and the molecular weight of the GAG may be weight-average molecular weights. The weight-average molecular weight can be measured by the GPC method in accordance with Example 1(1) described later, similar to the peak-top molecular weight. In the GAG derived from the ayu, the weight-average molecular weight of the GAG is, for example, 80,000 to 190,000, 100,000 to 170,000. In the GAG derived from the Atlantic salmon, the weight-average molecular weight of the GAG is, for example, 60,000 to 170,000, 90,000 to 140,000. In the GAG derived from the black rockfish, the weight-average molecular weight of the GAG is, for example, 90,000 to 200,000, 110,000 to 170,000. In the GAG derived from the skate, the number-average molecular weight of the GAG is, for example, 370,000 to 480,000, 390,000 to 460,000. In the GAG derived from the chicken, the weight-average molecular weight of the GAG is, for example, 40,000 to 150,000, 60,000 to 140,000. In the proteoglycan derived from the pig, in the GAG derived from the pig, the weight-average molecular weight of the GAG is, for example, 10,000 to 110,000, 30,000 to 90,000.

[0031] The animal tissue is, for example, a tissue containing proteoglycan. As an example, epithelial tissues such as skin; cartilage tissues such as cartilage; digestive organs; circulatory organs; respiratory organs; and placenta, etc. can be mentioned. As specific examples, the animal tissue is, for example, cartilage, fin, digestive tract, circulatory system, respiratory system, and ear, etc. The type of chondroitin sulfate constituting the proteoglycan varies depending on the type of the animal tissue. The animal tissue having many proteoglycans relatively rich in GAGs having disaccharide structures of type A and type C, compared with type iA and type iC proteoglycans respectively, is, for example, cartilage tissue. Therefore, when extracting a proteoglycan with a higher composition ratio of proteoglycan containing GAG having a disaccharide structure of type A or type C, by using the cartilage tissue as the animal tissue, in the extraction step, the content ratio of GAG having a disaccharide structure of type A or type C in the sugar chain of the obtained proteoglycan can be increased.

[0032] In addition, the animal tissue having many proteoglycans that relatively contain a large amount of GAGs having iA-type and iC-type disaccharide structures, respectively, compared with A-type and C-type proteoglycans, includes, for example, the digestive tract. Therefore, when extracting a proteoglycan having a higher composition ratio of a proteoglycan containing a GAG having an iA-type or iC-type disaccharide structure, by using the digestive tract as the animal tissue, in the extraction step, the content ratio of the GAG having an iA-type or iC-type disaccharide structure in the sugar chain of the obtained proteoglycan can be increased.

[0033] In the extraction step, examples of the extract include an aqueous guanidine hydrochloride solution, an aqueous acetic acid solution, an aqueous urea solution, and an aqueous magnesium chloride solution. The concentration of the aqueous guanidine hydrochloride solution is, for example, 3 to 5 mol / l (hereinafter also referred to as "M"). The concentration of the aqueous acetic acid solution is, for example, preferably 3 to 5 M.

[0034] The extract may contain other components, for example, within a range where the proteoglycan in a state where the core protein and the sugar chain are bound can be extracted, and / or within a range where safety can be ensured when used in vivo. Examples of the other components include sugar chain degrading enzyme inhibitors such as protease inhibitors, peptidase inhibitors, cellulase inhibitors, glucanase inhibitors, and chondroitinase inhibitors. The other components may be of one type or a combination of two or more types.

[0035] The extract is preferably configured to suppress the degradation of the core protein and / or the sugar chain in the proteoglycan. The extract does not contain, for example, exogenous sugar chain degrading enzymes such as proteases, peptidases, and cellulases, and chondroitinases. The exogenous substance means, for example, being derived from a different species from the target animal tissue. The extract preferably does not contain a molecule that catalyzes the hydrolysis of the core protein and / or the sugar chain in the proteoglycan. Examples of the molecule that catalyzes the hydrolysis include hydrochloric acid.

[0036] In the extraction step, the quantitative ratio of the animal tissue to the extraction solution is not particularly limited. The amount (volume) of the extraction solution is, for example, 1 to 10 times the amount (mass) of the animal tissue (w / v), preferably 3 to 7 times the amount (w / v). As a specific example, when the animal tissue is 1 g and 3 times the amount of the extraction solution is used, the amount of the extraction solution is, for example, 3 ml.

[0037] The time (extraction time) in the extraction step is not particularly limited as long as it is within a range capable of extracting proteoglycan in a state where the core protein and the sugar chain are bound. The extraction time is, for example, 1 day to 2 weeks, preferably about 7 days.

[0038] The temperature (extraction temperature) in the extraction step is not particularly limited as long as it is within a range capable of extracting an extract containing proteoglycan in a state where the core protein and the sugar chain are bound. The extraction temperature is, for example, 0°C to 10°C, preferably 0°C to 5°C, more preferably 4°C.

[0039] The pH (extraction pH) in the extraction step is not particularly limited as long as it is within a range capable of extracting an extract containing proteoglycan in a state where the core protein and the sugar chain are bound. The extraction pH is, for example, pH 2 to pH 9, preferably pH 6.

[0040] In the extraction step, for example, as long as it is within a range capable of extracting an extract containing proteoglycan in a state where the core protein and the sugar chain are bound, stirring may be carried out in parallel with the extraction.

[0041] The molecular weight of the proteoglycan extracted in the extraction step, the types of GAGs contained in the proteoglycan, and the molecular weight of the GAGs are not particularly limited and can be proteoglycans in a desired numerical range.

[0042] In the animal tissue, the molecular weight of the proteoglycan is, for example, usually 400,000 or more. Therefore, if the molecular weight of the proteoglycan obtained in the extraction step is 400,000 or more, preferably 600,000 or more, it can be evaluated that the proteoglycan has a core protein and a sugar chain bound thereto and is in a state where degradation of the core protein and / or the sugar chain is suppressed.

[0043] The molecular weight of the proteoglycan obtained in the extraction step is, for example, 400,000 to 1,500,000, 600,000 to 1,500,000, 400,000 to 1,200,000, or 600,000 to 1,200,000. Also, for example, the description of the molecular weight of the proteoglycan described above can be applied to the molecular weight of the proteoglycan derived from the tissues of various animals.

[0044] The molecular weight of the GAG (sugar chain) constituting the proteoglycan obtained in the extraction step is, for example, 20,000 to 250,000, 26,000 to 224,000, preferably 26,000 to 75,000, 196,000 to 224,000. Also, for example, the description of the molecular weight of the GAG constituting the proteoglycan described above can be applied to the molecular weight of the GAG constituting the proteoglycan derived from the tissues of various animals.

[0045] The GAG (sugar chain) constituting the proteoglycan may contain, for example, disaccharide structures of type A and / or type iA. In this case, in the proteoglycan obtained in the extraction step, the content ratio of the proteoglycan having the disaccharide structure of type A and / or type iA may be higher than the content ratio of other disaccharide structures. As a specific example, in the proteoglycan, the content ratio of the disaccharide structure of type A and / or type iA is, for example, 20 to 90%, preferably 30 to 70%. Further, in the proteoglycan, the ratio (A:iA) of the ratio (A) of the disaccharide structure of type A to the ratio (iA) of the disaccharide structure of type iA is, for example, 1:1 to 20:1, preferably 1.62:1 to 10.2:1. The ratio (A:iA) varies depending on the origin of the proteoglycan. When the origin of the proteoglycan is a pig's ear, the ratio (A:iA) is, for example, 0.5:1 to 4:1, 1:1 to 2:1, and preferably about 1.62:1. When the origin of the proteoglycan is a pig's bronchial cartilage, the ratio (A:iA) is, for example, 1:1 to 5:1, 2:1 to 3:1, and preferably about 2.6:1. When the origin of the proteoglycan is a pig's ear, the ratio (A:iA) is, for example, 3:1 to 4:1, and preferably about 3.52:1. When the origin of the proteoglycan is a chicken's sternal cartilage, the ratio (A:iA) is, for example, 7:1 to 13:1, 9:1 to 11:1, and preferably about 10.2:1.

[0046] The sugar chain of the proteoglycan may contain, for example, C-type and / or iC-type disaccharide structures. In this case, in the proteoglycan, the content ratio of the C-type and / or the iC-type disaccharide structure may be higher than the content ratio of other disaccharide structures. As a specific example, in the proteoglycan, the content ratio of the C-type and / or the iC-type disaccharide structure is, for example, 20 to 60%. Further, the ratio (C:iC) of the ratio (C) of the C-type disaccharide structure to the ratio (iC) of the iC-type disaccharide structure is, for example, 4:1 to 100:0, preferably 7:1 to 100:0, 7.46:1 to 100:0. The ratio (C:iC) varies depending on the origin of the proteoglycan. When the origin of the proteoglycan is the nasal cartilage of Oncorhynchus keta, the ratio (C:iC) is, for example, 5:1 to 10:1, 7:1 to 8:1, preferably about 7.46:1. When the origin of the proteoglycan is the entire fin of Raja porosa, the ratio (C:iC) is, for example, 20:1 to 30:1, 23:1 to 24:1, preferably about 23.8:1. When the origin of the proteoglycan is the base or cartilage of the fin of Raja porosa, or the fin of Galeus sauteri, the ratio (C:iC) is, for example, 90:10 to 100:0, 95:5 to 100:0.

[0047] The content ratios of the A-type, iA-type, C-type, and iC-type disaccharide structures of the GAG (sugar chain) constituting the proteoglycan obtained in the extraction step are, for example, 5 to 75%, 3 to 30%, 10 to 80%, and 0 to 13%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 600,000 to 1,500,000, 600,000 to 1,200,000. Further, the molecular weight of the GAG is, for example, 26,000 to 224,000, preferably 26,000 to 75,000, 196,000 to 224,000. In the composition of the present disclosure, the total content ratio of the A-type, iA-type, C-type, and iC-type disaccharide structures is 100% or less (the same applies hereinafter).

[0048] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that make up the proteoglycan obtained in the extraction step are, for example, 40 to 60%, 10 to 30%, 10 to 30%, and 1 to 5%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 500,000 to 750,000, 600,000 to 700,000. Also, the molecular weight of the GAG is, for example, 10,000 to 50,000, 20,000 to 40,000.

[0049] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that make up the proteoglycan obtained in the extraction step are, for example, 50 to 75%, 3 to 10%, 15 to 35%, and 0 to 3%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 500,000 to 750,000, 600,000 to 700,000. Also, the molecular weight of the GAG is, for example, 30,000 to 90,000, 40,000 to 70,000.

[0050] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that make up the proteoglycan obtained in the extraction step are, for example, 10 to 30%, 5 to 12.5%, 45 to 55%, and 3 to 13%, or 10 to 30%, 5 to 12.5%, 45 to 65%, and 0 to 13%, respectively. Also, the molecular weight of the proteoglycan is, for example, 300,000 to 800,000, 600,000 to 750,000. The molecular weight of the GAG is, for example, 60,000 to 100,000, 70,000 to 90,000.

[0051] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that make up the proteoglycan obtained in the extraction step are, for example, 25 to 45%, 3 to 10%, 30 to 50%, and 0 to 10%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 600,000 to 1,000,000, 800,000 to 900,000. Also, the molecular weight of the GAG is, for example, 50,000 to 110,000, 70,000 to 100,000.

[0052] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that constitute the proteoglycan obtained in the extraction step are, for example, 16 to 26%, 2 to 13%, 42 to 52%, and 1 to 12%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 1 million to 1.3 million, 1.1 million to 1.25 million. Also, the molecular weight of the GAG is, for example, 30,000 to 150,000, 50,000 to 80,000.

[0053] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that constitute the proteoglycan obtained in the extraction step are, for example, 5 to 15%, 4 to 13%, 50 to 70%, and 0 to 10%, respectively.

[0054] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that constitute the proteoglycan obtained in the extraction step are, for example, 5 to 25%, 30 to 60%, 10 to 30% (more preferably 10% to 25%), and 10% or less, more preferably 5 to 25%, 30 to 60%, 10% to 25%, and 10% or less. In this case, the molecular weight of the proteoglycan is, for example, 300,000 to 600,000, 400,000 to 500,000. Also, the molecular weight of the GAG is, for example, 5,000 to 300,000, 6,000 to 250,000.

[0055] After the extraction step, for example, the extract may be filtered. The filtration method of the extract is not particularly limited, and can be carried out by, for example, a known solid-liquid separation method. Specifically, it may be carried out using filtration means such as a filter, or solid-liquid separation may be carried out by centrifugation or the like. The filtration step may be carried out once or a plurality of times.

[0056] The method for producing a proteoglycan of the present disclosure may include a purification step of purifying a purified product containing the proteoglycan from the extract. The purification can be carried out, for example, by bringing the filtered extract into contact with a solvent in which the solubility of the proteoglycan is low, and precipitating or depositing the proteoglycan. Therefore, the purification step can also be referred to as, for example, a step of precipitating or depositing the proteoglycan in the extract. Examples of the solvent include organic solvents such as alcohols such as ethanol and methanol. In the purification step, for example, the precipitate or deposit may be further separated by centrifugation, and the precipitate or deposit may be recovered.

[0057] The method for producing a proteoglycan of the present disclosure may include a fractionation step of fractionating a proteoglycan that satisfies a predetermined peak top molecular weight from the purified product. The fractionation method is not particularly limited as long as it can fractionate the proteoglycan in a state where the core protein and the sugar chain are bound. The fractionation method can be carried out, for example, by a known method capable of fractionating components based on molecular weight. Specifically, it may be carried out using an ultrafiltration membrane (molecular weight fractionation membrane) or a dialysis membrane, or by silica gel column chromatography, gel filtration chromatography, ion exchange column chromatography, or the like. The fractionation molecular weight of the molecular weight fractionation membrane can be set according to, for example, the target molecular weight. The fractionation method may be carried out by centrifugation using a container having the molecular weight fractionation membrane. When performing the centrifugation, as the container, for example, a membrane Amicon Ultra centrifugal filter unit manufactured by Merck Millipore can be used.

[0058] In the fractionation step, it is preferable that the purified product is subjected to the fractionation step, for example, in a liquid state. Therefore, when the purified product obtained in the purification step is solid, it is preferable to disperse the purified product in a solvent prior to the fractionation step. The solvent is not particularly limited as long as it can dissolve the proteoglycan in a state where the core protein and the sugar chain are bound, and examples thereof include buffer solutions such as phosphate buffer solutions, purified water, and aqueous solvents such as aqueous sodium acetate solutions. The pH of the solvent is, for example, pH 4 to pH 9.

[0059] The method for producing a proteoglycan of the present disclosure may include a step of recovering a solid fraction of the proteoglycan in which the core protein and the sugar chain are bound from a fraction containing the proteoglycan. The recovery method is not particularly limited as long as it can recover the proteoglycan in a state where the core protein and the sugar chain are bound. The recovery method can be carried out, for example, by a known method capable of recovering a solid fraction of the proteoglycan from the liquid containing the proteoglycan, and specifically, it can be carried out by a drying treatment such as freeze-drying or spray drying.

[0060] The production method of the present disclosure may perform a pretreatment on the animal tissue prior to the extraction step. Examples of the pretreatment include treatment such as mincing and defatting of the animal. In the production method of the present disclosure, since the animal tissue can relatively increase the contact area with the extract and improve the extraction efficiency of the proteoglycan, it is preferably minced. The mincing can be carried out by, for example, treatments such as cutting treatment, crushing treatment, and ultrasonic treatment. Specifically, examples of the mincing include mechanical crushing using a homogenizer, crushing by ultrasonic treatment, and the like. Therefore, the production method of the present disclosure may optionally include a step of mincing the animal tissue prior to the extraction step. Further, in the production method of the present disclosure, when an unminced animal tissue is used in the extraction step, in the extraction step, after contact between the animal tissue and the extract, a mincing treatment may be performed on the obtained mixture.

[0061] <Composition> In another aspect, the present disclosure provides a composition comprising a proteoglycan. The composition of the present disclosure contains a proteoglycan. The peak top molecular weight of the proteoglycan is, for example, 400,000 to 1,200,000. According to the composition of the present disclosure, for example, the proliferation of cells, particularly skin fibroblasts, can be promoted.

[0062] The composition of the present disclosure contains, for example, a proteoglycan obtained by the method for producing the proteoglycan of the present disclosure. The composition of the present disclosure can, for example, incorporate the description of the method for producing the proteoglycan of the present disclosure.

[0063] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAG (sugar chain) constituting the proteoglycan contained in the composition of the present disclosure are, for example, 5 to 75%, 3 to 30%, 10 to 80%, and 0 to 13%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 600,000 to 1,500,000, 600,000 to 1,200,000. Also, the molecular weight of the GAG is, for example, 26,000 to 224,000, preferably 26,000 to 75,000, 196,000 to 224,000. In the composition of the present disclosure, the total content ratio of the disaccharide structures of type A, iA type, C type, and iC type is 100% or less (the same applies hereinafter).

[0064] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAG (sugar chain) constituting the proteoglycan contained in the composition of the present disclosure are, for example, 40 to 60%, 10 to 30%, 10 to 30%, and 1 to 5%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 500,000 to 750,000, 600,000 to 700,000. Also, the molecular weight of the GAG is, for example, 10,000 to 50,000, 20,000 to 40,000.

[0065] The content ratios of the A-type, iA-type, C-type, and iC-type disaccharide structures of GAG that constitute the proteoglycan contained in the composition of the present disclosure are, for example, 50 to 75%, 3 to 10%, 15 to 35%, and 0 to 3%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 500,000 to 750,000, 600,000 to 700,000. Also, the molecular weight of the GAG is, for example, 30,000 to 90,000, 40,000 to 70,000.

[0066] The content ratios of the A-type, iA-type, C-type, and iC-type disaccharide structures of GAG that constitute the proteoglycan contained in the composition of the present disclosure are, for example, 10 to 30%, 5 to 12.5%, 45 to 55%, and 3 to 13%, respectively, or 10 to 30%, 5 to 12.5%, 45 to 65%, and 0 to 13%. Also, the molecular weight of the proteoglycan is, for example, 300,000 to 800,000, 600,000 to 750,000. The molecular weight of the GAG is, for example, 60,000 to 100,000, 70,000 to 90,000.

[0067] The content ratios of the A-type, iA-type, C-type, and iC-type disaccharide structures of GAG that constitute the proteoglycan contained in the composition of the present disclosure are, for example, 25 to 45%, 3 to 10%, 30 to 50, and 0 to 10%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 600,000 to 1,000,000, 800,000 to 900,000. Also, the molecular weight of the GAG is, for example, 50,000 to 110,000, 70,000 to 100,000.

[0068] The content ratios of the A-type, iA-type, C-type, and iC-type disaccharide structures of GAG that constitute the proteoglycan contained in the composition of the present disclosure are, for example, 16 to 26%, 2 to 13%, 42 to 52%, and 1 to 12%, respectively. In this case, the molecular weight of the proteoglycan is, for example, 1,000,000 to 1,300,000, 1,100,000 to 1,250,000. Also, the molecular weight of the GAG is, for example, 30,000 to 150,000, 50,000 to 80,000.

[0069] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that constitute the proteoglycan contained in the composition of the present disclosure are, for example, 5 to 15%, 4 to 13%, 50 to 70%, and 0 to 10%, respectively. In this case, the molecular weight of the GAG is, for example, 140,000 to 280,000, 190,000 to 230,000.

[0070] The content ratios of the disaccharide structures of type A, iA type, C type, and iC type of GAGs that constitute the proteoglycan contained in the composition of the present disclosure are, for example, 5 to 25%, 30 to 60%, 10 to 30% (more preferably 10% to 25%), and 10% or less, more preferably 5 to 25%, 30 to 60%, 10% to 25%, and 10% or less, respectively. In this case, the molecular weight of the proteoglycan is, for example, 300,000 to 600,000, 400,000 to 500,000. Further, the molecular weight of the GAG is, for example, 5,000 to 300,000, 6,000 to 250,000.

[0071] In this specification, "promotion of cell proliferation" means that the proliferation of cells, particularly human-derived cells, is promoted or enhanced. The promotion of the cell proliferation can be carried out, for example, by evaluating the proliferation of the cells according to Example 1(4) described below. The cells are not particularly limited and can be any cells, for example, cells derived from skin tissue, preferably skin fibroblasts.

[0072] The composition of the present disclosure can promote the proliferation of skin fibroblasts, for example, by being used for an administration subject. The usage conditions (administration conditions) of the composition of the present disclosure are not particularly limited, and for example, the administration form, administration timing, dosage, etc. can be appropriately set according to the type of the administration subject, etc.

[0073] The composition of the present disclosure can be used, for example, in vivo also in in vitro and can also be used in

[0074] The administration subject of the composition of the present disclosure is not particularly limited. The composition of the present disclosure in vivoWhen used, the administration subject may be, for example, a human or a non-human animal excluding humans. Examples of the non-human animal include mammals such as mice, rats, rabbits, dogs, sheep, horses, cats, goats, monkeys, guinea pigs, and birds. When the composition of the present disclosure is in vitro used, the administration subject may be, for example, cells, tissues, organs, etc. Examples of the cells include cells collected from a living body, cultured cells, etc. Examples of the tissue or organ include tissues (biological tissues) or organs collected from a living body.

[0075] In the following topical skin composition (for example, a transdermal administration or a composition for application to the skin) or the following oral administration composition containing the proteoglycan of the present disclosure, the blending amount of the proteoglycan may be in a range that exhibits a promoting effect on the proliferation of the cells, particularly skin fibroblasts, that is, an effective amount.

[0076] The administration forms of the composition of the present disclosure include oral administration and parenteral administration. Examples of the parenteral administration include transdermal administration, application (contact) to the skin, etc. The application to the skin may include application to the oral mucosa, that is, application or contact to epithelial cells in the oral cavity. Further, the application to the skin may include, in addition to or instead of application to the skin surface, administration or injection into or under the skin through the skin surface. The administration or injection into the skin through the skin surface can be carried out, for example, using microneedles.

[0077] The dosage form of the composition of the present disclosure is not particularly limited and can be appropriately determined, for example, according to the administration form. Examples of the dosage form include liquid and solid. When the administration form is oral administration, examples of the dosage form include tablets, pills, capsules, granules, powders, solutions, etc.

[0078] The compositions of the present disclosure may, for example, optionally contain additives, and the additives preferably include pharmaceutically acceptable additives or pharmaceutically acceptable carriers. The additives are not particularly limited, and examples thereof include base materials, excipients, coloring agents, lubricants, binders, disintegrants, stabilizers, coating agents, preservatives, flavoring agents such as flavoring and deodorizing agents, and the like.

[0079] The excipients include, for example, sugar derivatives such as lactose, lactose hydrate, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; organic excipients such as pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium metasilicate aluminate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and inorganic excipients such as calcium sulfate. Examples of the coloring agent include, for example, yellow ferric oxide. Examples of the lubricant include metal stearates such as stearic acid, calcium stearate, and magnesium stearate; talc; polyethylene glycol; silica; and hardened vegetable oil. Examples of the flavoring and odor-correcting agent include, for example, spices such as cocoa powder, peppermint oil, aromatic powder, peppermint oil, borneol, and cinnamon powder, sweeteners, and acidulants. Examples of the binder include, for example, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, and macrogol. Examples of the disintegrant include, for example, cellulose derivatives such as carboxymethyl cellulose and carboxymethyl cellulose calcium; chemically modified starches and chemically modified celluloses such as carboxymethyl starch, sodium carboxymethyl starch, crosslinked polyvinyl pyrrolidone, and sodium starch glycolate. Examples of the stabilizer include, for example, paraoxybenzoic acid esters such as methyl paraben and propyl paraben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol; thimerosal; dehydroacetic acid; and sorbic acid. Examples of the coating agent include, for example, hypromellose, macrogols such as macrogol 6000, talc, and titanium oxide.

[0080] When the composition of the present disclosure is an oral administration composition, specific examples of the oral administration composition include, for example, beverages, foods, pharmaceuticals, quasi-drugs, and the like.

[0081] When the composition of the present disclosure is used for transdermal administration or application to the skin (hereinafter, also referred to as "external preparation for skin"), the form of the external preparation for skin can be an ampoule, capsule, powder, granule, liquid, gel, foam, emulsion, sheet, mist, spray agent, etc., according to the usage form. Examples of the usage form include pharmaceuticals (types); quasi-drugs (types); local or systemic external preparations for skin; pharmaceutical and / or cosmetic preparations applied to the scalp and hair; bath agents added to bath water for use; other preparations; etc. Examples of the local or systemic external preparations for skin include basic cosmetics such as lotion, emulsion, cream, ointment, lotion, oil, pack, etc., facial cleansers or skin cleansers such as solid soap, liquid soap, hand wash, etc., massage agents, cleansing agents, hair removers, depilatory agents, beard shaving treatment agents, aftershave lotion, pre-shave lotion, shaving cream, foundation, lipstick, blush, eyeshadow, eyeliner, mascara, etc. makeup cosmetics, perfumes, nail beauty agents, nail enamels, nail enamel removers, poultices, plasters, tape agents, sheet agents, patches, aerosol agents, gargles (such as dentifrices and mouthwashes), etc. Examples of the pharmaceutical and / or cosmetic preparations applied to the scalp and hair include shampoo agents, conditioner agents, hair treatment agents, pre-hair treatment agents, permanent solutions, hair dyes, hair styling agents, hair tonics, hair growth and nourishing agents, poultices, plasters, tape agents, sheet agents, aerosol agents, etc. Examples of the other preparations include antiperspirants or deodorants, antiperspirants, sanitary products, sanitary napkins, wet tissues, etc.

[0082] The external preparation for skin can be optionally selected and / or used in combination with the components and / or additives exemplified below within the range that does not prevent the promoting effect on the proliferation of skin fibroblasts, if necessary.

[0083] (1) Various oils and fats Avocado oil, almond oil, perilla oil, sesame oil, olive oil, orange oil, orange raffia oil, sesame oil, cocoa butter, chamomile oil, carrot oil, cucumber oil, beef tallow fatty acid, kukui nut oil, safflower oil, shea butter, liquid shea butter, soybean oil, camellia oil, corn oil, rapeseed oil, peppermint oil, castor oil, cottonseed oil, peanut oil, turtle oil, mink oil, egg yolk oil, palm oil, palm kernel oil, beeswax, coconut oil, beef tallow, lard, squalene, squalane, pristane or hydrogenated products of these oils and fats (hardened oils, etc.).

[0084] (2) Waxes Beeswax, carnauba wax, whale wax, lanolin, liquid lanolin, reduced lanolin, hard lanolin, candelilla wax, montan wax, shellac wax, rice wax, etc.

[0085] (3) Mineral oils Liquid paraffin, petrolatum, paraffin, ozokerite, ceresin, microcrystalline wax, etc.

[0086] (4) Fatty acids Lauric acid, myristic acid, palmitic acid, stearic acid, behenic acid, oleic acid, linoleic acid, linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, 12 - hydroxystearic acid, undecylenic acid, tall oil, lanolin fatty acid and other natural fatty acids, isononanoic acid, caproic acid, 2 - ethylbutanoic acid, isopentanoic acid, 2 - methylpentanoic acid, 2 - ethylhexanoic acid, isopentanoic acid and other synthetic fatty acids.

[0087] (5) Alcohols Ethanol, isopropanol, lauryl alcohol, cetyl alcohol, stearyl alcohol, oleyl alcohol, lanolin alcohol, cholesterol, phytosterol, phenoxyethanol and other natural alcohols, 2 - hexyldecanol, isostearyl alcohol, 2 - octyldodecanol and other synthetic alcohols.

[0088] (6) Polyhydric alcohols Ethylene oxide, ethylene glycol, diethylene glycol, triethylene glycol, ethylene glycol monoethyl ether, ethylene glycol monobutyl ether, diethylene glycol monomethyl ether, diethylene glycol monoethyl ether, polyethylene glycol, propylene oxide, propylene glycol, polypropylene glycol, 1,3-butylene glycol, pentyl glycol, glycerin, pentaerythritol, threitol, arabitol, xylitol, ribitol, galactitol, sorbitol, mannitol, lactitol, maltitol, etc.

[0089] (7) Esters Isopropyl myristate, isopropyl palmitate, butyl stearate, hexyl laurate, myristyl myristate, oleyl oleate, decyl oleate, octyldodecyl myristate, hexyldecyl dimethyloctanoate, cetyl lactate, myristyl lactate, diethyl phthalate, dibutyl phthalate, lanolin acetate, ethylene glycol monostearate, propylene glycol monostearate, propylene glycol dioleate, etc.

[0090] (8) Metal soaps Aluminum stearate, magnesium stearate, zinc stearate, calcium stearate, zinc palmitate, magnesium myristate, zinc laurate, zinc undecylenate, etc.

[0091] (9) Gums, sugars or water-soluble high molecular compounds Arabic gum, benzoin gum, dammar gum, guaiac resin, Irish moss, karaya gum, tragacanth gum, carob gum, quince seed, agar, casein, lactose, fructose, sucrose or its esters, trehalose or its derivatives, dextrin, gelatin, pectin, starch, carrageenan, carboxymethyl chitin or chitosan, hydroxyalkyl (C2-C4) chitin or chitosan to which alkylene (C2-C4) oxides such as ethylene oxide are added, low-molecular chitin or chitosan, chitosan salts, sulfated chitin or chitosan, phosphorylated chitin or chitosan, alginic acid or its salts, hyaluronic acid or its salts, chondroitin sulfate or its salts, heparin, ethyl cellulose, methyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, sodium carboxyethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, nitrocellulose, crystalline cellulose, polyvinyl alcohol, polyvinyl methyl ether, polyvinyl pyrrolidone, polyvinyl methacrylate, polyacrylate salts, polyalkylene oxides such as polyethylene oxide and polypropylene oxide or their cross-linked polymers, carboxyvinyl polymer, polyethyleneimine, etc.

[0092] (10) Surfactants Anionic surfactants (alkyl carboxylates, alkyl sulfonates, alkyl sulfate esters, alkyl phosphate esters), cationic surfactants (alkyl amine salts, alkyl quaternary ammonium salts), amphoteric surfactants: carboxylic acid type amphoteric surfactants (amino type, betaine type), sulfate ester type amphoteric surfactants, sulfonic acid type amphoteric surfactants, phosphate ester type amphoteric surfactants, nonionic surfactants (ether type nonionic surfactants, ether ester type nonionic surfactants, ester type nonionic surfactants, block polymer type nonionic surfactants, nitrogen-containing type nonionic surfactants), other surfactants (natural surfactants, derivatives of protein hydrolysates, polymeric surfactants, surfactants containing titanium and silicon, fluorocarbon surfactants), etc.

[0093] (11) Various vitamins Group A vitamins: retinol, retinal (vitamin A1), dehydroretinal (vitamin A2), carotenes, lycopene (provitamin A); Group B vitamins: thiamine hydrochloride, thiamine sulfate (vitamin B1), riboflavin (vitamin B2), pyridoxine (vitamin B6), cyanocobalamin (vitamin B12), folic acids, nicotinic acids, pantothenic acids, biotin, choline, inositols; Group C vitamins: ascorbic acid or its derivatives; Group D vitamins: ergocalciferol (vitamin D2), cholecalciferol (vitamin D3), dihydrotachysterol; Group E vitamins: vitamin E or its derivatives, ubiquinones; Group K vitamins: phylloquinone (vitamin K1), menaquinone (vitamin K2), menadione (vitamin K3), menadiol (vitamin K4); others, essential fatty acids (vitamin F), carnitine, ferulic acid, γ - oryzanol, orotic acid, Group P vitamins (rutin, eriocitrin, hesperidin), vitamin U, etc.

[0094] (12) Various amino acids Valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, lysine, glycine, alanine, asparagine, glutamine, serine, cysteine, cystine, tyrosine, proline, hydroxyproline, aspartic acid, glutamic acid, hydroxylysine, arginine, ornithine, histidine, etc., and their sulfates, phosphates, nitrates, citrates, or amino acid derivatives such as pyrrolidonecarboxylic acid.

[0095] (13) Additives The external preparation for skin may further contain various additives derived from animals or plants. The additives can be arbitrarily selected from various materials after performing processing routinely carried out according to, for example, the type and form of the product to be added. The processing can be, for example, a treatment of arbitrarily selecting and / or combining crushing, milling, washing, hydrolysis, fermentation, purification, pressing, extraction, fractionation, filtration, drying, powdering, granulation, dissolution, sterilization, pH adjustment, deodorization, decolorization, etc.

[0096] The solvent used for the extraction can be selected taking into consideration the purpose and type of the product to be used, or the subsequent processing. The extraction solvent is preferably one or a mixture of two or more selected from various organic solvents such as water, lower alcohols or water-containing lower alcohols such as water, methanol, ethanol, propyl alcohol, isopropyl alcohol, butanol, and isobutanol, polyhydric alcohols or water-containing polyhydric alcohols such as propylene glycol, 1,3-butylene glycol, and glycerin, acetone, and ethyl acetate. However, when the inclusion of an organic solvent is not preferable depending on the application, the extraction solvent may be water alone or ethanol, which is easy to remove after extraction, may be used alone or in any mixture with water, or may be extracted by squeezing.

[0097] When the additives derived from plant or animal raw materials are used in external preparations or cosmetics for systemic or local use, the external preparations for skin can be expected to have, for example, cosmetic effects such as protection of the skin and hair, moisturizing, improving feel and texture, imparting softness, easing irritation, relieving stress through fragrance, cell activation (preventing cell aging), suppressing inflammation, improving skin and hair quality, preventing and improving rough skin, hair growth, hair care, preventing hair loss, imparting shine, a cleansing effect, relieving fatigue, promoting blood flow, and a hot bath effect, as well as fragrance, deodorizing, thickening, preserving, buffering, and other effects.

[0098] In addition to the above, the topical skin preparation can be made into a product that is expected to have various cosmetic and medicinal effects of each of the raw material materials that are known to date, and by combining these, it is possible to enhance the effect intended by this disclosure and create a product that is expected to have multifunctional effects.

[0099] <Method of promoting cell proliferation> In another aspect, the present disclosure discloses a method for promoting cell proliferation. The method for promoting cell proliferation of the present disclosure uses the composition of the present disclosure. According to the method for promoting cell proliferation of the present disclosure, for example, it is expected to obtain the effect of promoting cell proliferation.

[0100] The method for promoting cell proliferation according to the present disclosure includes a step of using the composition according to the present disclosure for a subject. The use may be, for example, contact with the skin or the like, or administration.

[0101] In the method for promoting cell proliferation according to the present disclosure, the use step may be, for example, in vitro or in vivo performed. The subject (administration subject) and administration conditions of the method for promoting cell proliferation according to the present disclosure can, for example, refer to the description of the administration subject and administration conditions in the composition according to the present disclosure.

[0102] The cells are not particularly limited and can be any cells, for example, cells derived from skin tissue, preferably dermal fibroblasts.

[0103] <Use> The present disclosure is a composition or the use of a composition for promoting cell proliferation. The present disclosure is the use of a composition for manufacturing a composition for promoting cell proliferation.

Examples

[0104] Next, examples of the present disclosure will be described. However, the present disclosure is not limited by the following examples. Commercially available reagents were used based on their protocols unless otherwise indicated. Note that "mol / l" may also be denoted as "M".

[0105] [Example 1] Regarding the proteoglycan of the present disclosure, it was confirmed that a proteoglycan in which a core protein and a sugar chain are bound can be produced by the production method of the present disclosure.

[0106] (1) Purification of proteoglycan As raw materials, 100 - 150 g each of the nasal cartilage of white salmon, the nasal cartilage of Atlantic salmon, the laryngeal cartilage of pigs, the hyaline cartilage (sternal cartilage) of chickens, and the fins of black rockfish were prepared. To the above raw materials, 5 times the amount (W / V) of a 4M guanidine hydrochloride aqueous solution was added. After the addition, immersion extraction was carried out under the condition of 4°C for 1 week. After the extraction, filtration was performed using a 60 - mesh sieve and filter paper (3 μm). After the filtration, 3 times the amount (v / v) of ethanol was added. After the addition, it was allowed to stand under the condition of 4°C overnight. After the standing, filtration was carried out using filter paper (3 μm) to recover the precipitate. After the recovery, a phosphate buffer (pH 6.8) was added to dissolve the precipitate. After the dissolution, fractionation purification was carried out using a molecular weight fractionation membrane (MW 50,000, manufactured by Asahi Kasei Corporation). After the fractionation purification, the proteoglycan fraction was recovered. After the recovery, the proteoglycan fraction was freeze - dried. After the freeze - drying, a proteoglycan solid was obtained. Regarding the proteoglycan solid, HPLC analysis was performed under the following measurement conditions. These results are shown in Figure 2 and Table 1.

[0107] (Measurement conditions for HPLC) HPLC system: LC - 10AD Column: TSKgel G5000 - PWXL Φ7.8 mm×300 mm (manufactured by Tosoh Corporation) Eluent: Phosphate buffer at pH 6.8 Flow rate: 0.5 ml / min Column temperature: 40°C Detector: Differential refractive index detector (RID - 10A, manufactured by Shimadzu Corporation) Injection volume: 50 μl Molecular weight marker: Shodex STANDARD P - 82 (pullulan)

[0108] Figure 2 is a graph showing the results of HPLC analysis of each proteoglycan solid. In Figure 2, the vertical axis represents peak intensity (mV), and the horizontal axis represents retention time (min). As shown in Figure 2(A), the elution position of the proteoglycan in the nasal cartilage of rainbow trout was around 13.246, and the peak top molecular weight was 680,000. As shown in Figure 2(B), the elution position of the proteoglycan in the nasal cartilage of Atlantic salmon was around 12.418, and the peak top molecular weight was 1,170,000. As shown in Figure 2(C), the elution position of the proteoglycan in the laryngeal cartilage of pigs was around 13.296, and the peak top molecular weight was 640,000. As shown in Figure 2(D), the elution positions of the proteogly in the calcaneal cartilage of chickens were around 13.443 and 16.816, and the combined peak top molecular weight of the two peaks was 630,000. As shown in Figure 2(E), the elution positions of the proteoglycan in the fin of black rockfish were around 13.082 and 17.699, and the combined peak top molecular weight of the two peaks was 860,000.

[0109]

Table 1

[0110] Table 1 above is a table showing the proteoglycan purity (%) and solid content yield (%) of each proteoglycan solid. As shown in Table 1, the proteoglycan purity of the nasal cartilage of rainbow trout was 42.7%, and the solid content yield was 2.62%. As shown in Table 1, the proteoglycan purity of the nasal cartilage of Atlantic salmon was 22.5%, and the solid content yield was 2.39%. As shown in Table 1, the proteoglycan purity of the laryngeal cartilage of pigs was 101.0%, and the solid content yield was 0.26%. As shown in Table 1, the proteoglycan purity of the calcaneal cartilage of chickens was 108.8%, and the solid content yield was 1.04%. As shown in Table 1, the proteoglycan purity of the fin of black rockfish was 77.7%, and the solid content yield was 1.14%.

[0111] (2) Analysis of GAG in each proteoglycan For the proteoglycan obtained in Example 1(1) above, analysis of GAG was performed. Specifically, GAG was purified from the proteoglycan obtained in Example 1(1) above. 10 g of the proteoglycan obtained in Example 1(1) was treated with 80 ml of boiling water for 10 minutes. After the treatment, 100 ml of 0.5 mol / l boric acid buffer (pH 7.0) was added. After the addition, 0.67 g of protease N Amano G (manufactured by Amano Enzyme Inc.) was added, and then incubated at 55°C for 7 days. After the incubation, quenching was performed with 5% trichloroacetic acid. After the quenching, insolubles were filtered and precipitated with 80% ethanol containing 1.25% NaOAc (sodium acetate). After the precipitation, centrifugation was performed at 0°C, and the obtained precipitate was vacuum dried to obtain a crude sugar chain. Next, 100 mg of the crude sugar chain was diluted with 0.05 mol / l acetic acid (pH 4.0) containing 0.15 mol / l LiCl. After the dilution, it was applied to a DEAE-cellulose column (φ2.2×15 cm, manufactured by Wako Pure Chemical Industries, Ltd.). After the application, the column was washed stepwise with 160 ml of buffer containing 0.15, 0.5, 1.0, or 2.0 mol / l LiCl. After the washing, the carbazole-positive fraction was collected and dialyzed, and the sample after dialysis was desalted using a gel permeation column (LH-20, H2O, φ1.1×80 cm) to obtain GAG. Thereafter, 50 μg of the GAG was diluted with 50 μg of H2O. After the dilution, HPLC analysis was performed using a linear-linked size exclusion column (OHpak SB-G 6B 100×4.6 mm and SB-805 HQ 250×4.6 mm).

[0112] (Measurement conditions for HPLC) HPLC system: LC-10AD (manufactured by Shimadzu Corporation) Column: OHpak SB-G 6B 100×4.6 mm and SB-805 HQ 250×4.6 mm Eluent: 0.1 mol / l NaNO3 Flow rate: 1 mL / min

[0113] As a result, the peak top molecular weight (Mp) of the GAG derived from white salmon was 49,000, the number average molecular weight (Mn) was 78,000, the weight average molecular weight (Mw) was 137,000, and the polydispersity (d) was 1.75. The peak top molecular weight of the GAG derived from Atlantic salmon was 44,000, the number average molecular weight was 63,000, the weight average molecular weight was 110,000, and the polydispersity was 1.75. The peak top molecular weight derived from the flounder was 75,000, the number average molecular weight was 87,000, the weight average molecular weight was 141,000, and the polydispersity was 1.61. The peak top molecular weight of the GAG derived from chicken was 49,000, the number average molecular weight was 53,000, the weight average molecular weight was 92,000, and the polydispersity was 1.74. The peak top molecular weight of the GAG derived from pig was 26,000, the number average molecular weight was 32,000, the weight average molecular weight was 61,000, and the polydispersity was 1.94. The peak top molecular weight of the GAG derived from skate fins was 224,000, the number average molecular weight was 281,000, the weight average molecular weight was 423,000, and the polydispersity was 1.51. The peak top molecular weight and the like of each measured GAG are described in Table 2 below.

[0114]

Table 2

[0115] (3) Disaccharide structure of each proteoglycan Regarding the GAG obtained in Example 1(2) above, the disaccharide structure of each proteoglycan was analyzed. Specifically, 50 μg of the GAG obtained in Example 1(2) was diluted with 50 μl of H2O, a BSA solution (0.05 mg / 5 μl), and 10 μl of a 250 mmol / l Tris-HCl(AcOH) solution (pH 8.0). After the dilution, chondroitinase ABC (25 mU / 25 μl, manufactured by Sigma Aldrich) or chondroitinase AC (25 mU / 25 μl, manufactured by Sigma Aldrich) was used to digest the resulting diluted solution under the conditions of 37 °C or 30 °C for 8 hours. After the digestion, the unsaturated disaccharides generated by the digestion were subjected to HPLC analysis using a PA-03 (PA-G) column. In this analysis, based on the HPLC peak area of the standard unsaturated disaccharides, the content ratio (composition ratio) of each disaccharide structure was calculated. These results are shown in Table 3 below.

[0116]

Table 3

[0117] As shown in Table 3 above, the content of the disaccharide structure was as follows. · GAG derived from the nasal cartilage of rainbow trout Chondroitin sulfate O: 10%, chondroitin sulfate iO: 4.0%, chondroitin sulfate A: 21%, chondroitin sulfate iA: 7.6%, chondroitin sulfate C: 47%, chondroitin sulfate iC: 6.3% · GAG derived from the bronchial cartilage of pigs Chondroitin sulfate O: 6.1%, chondroitin sulfate iO: 0%, chondroitin sulfate A: 52%, chondroitin sulfate iA: 20%, chondroitin sulfate C: 19%, chondroitin sulfate iC: 2.1% · GAG derived from the chest cartilage of chickens Chondroitin sulfate O: 5.2%, chondroitin sulfate iO: 0%, chondroitin sulfate A: 63%, chondroitin sulfate iA: 6.2%, chondroitin sulfate C: 24%, chondroitin sulfate iC: 0.3% · GAG derived from the fin of *Scomber japonicus* Chondroitin sulfate O: 14%, chondroitin sulfate iO: 2.1%, chondroitin sulfate A: 34%, chondroitin sulfate iA: 6.8%, chondroitin sulfate C: 40%, chondroitin sulfate iC: 0% · GAG derived from the base of the fin of *Anguilla japonica* Chondroitin sulfate O: 8.8%, chondroitin sulfate iO: 2.8%, chondroitin sulfate A: 11%, chondroitin sulfate iA: 8.6%, chondroitin sulfate C: 60%, chondroitin sulfate iC: 0% It was found that mammalian or avian proteoglycans contain relatively more GAGs with A-type or iA-type disaccharide structures compared to fish proteoglycans. Also, fish proteoglycans were found to contain relatively more GAGs with C-type or iC-type disaccharide structures compared to mammalian or avian proteoglycans. From the above results, it was found that the types of GAGs constituting the proteoglycans differ depending on the type of animal from which they are derived.

[0118] (4) Cell proliferation effect of proteoglycan Regarding the proteoglycan obtained in the above Example 1(1), it was examined whether it had cell proliferation activity. Specifically, by comparing with the cell proliferation activity of chondroitin sulfate A, it was examined whether the proteoglycan obtained in the above Example 1(1) had cell proliferation activity. Human dermal fibroblasts (Normal Human Dermal Fibroblasts: NHDF, purchased from Kurabo) were cultured under the conditions of 37 °C and 5% CO2 using DMEM supplemented with 10% FBS and antibiotics. After the above culture, each proteoglycan obtained in the above Example 1(1) or chondroitin sulfate A (4SGAG) was added to 1% FBS DMEM, and the cells were cultured. The proteoglycan or 4SGAG was used at concentrations of 3.9 μg / ml, 7.8 μg / ml, 15.6 μg / ml, 31.3 μg / ml, 62.5 μg / ml, 125 μg / ml, 250 μg / ml, or 500 μg / ml. For proteoglycans from any origin, regarding 500 μg / ml, since it did not dissolve completely, a solution that was not completely dissolved was used. The above culture was carried out for 72 hours. After the above culture, the number of cells was measured using a CCK-8 assay kit (Dojindo Laboratories). Also, as a negative control, it was carried out in the same manner except that each proteoglycan obtained in the above Example 1(1) was not added. As a positive control, it was carried out in the same manner except that C-028 derived from salmon nasal cartilage was added instead of each proteoglycan obtained in the above Example 1(1). These results are shown in FIGS. 3 and 4.

[0119] Figure 3 is a graph showing the results of the cell proliferation test of chondroitin sulfate A. In Figure 3, the vertical axis indicates the relative value of the cell number, and the horizontal axis indicates the concentration of chondroitin sulfate A. The numerical values on the vertical axis in Figure 3 indicate the values of each group with the value of the negative control (NC) group set to 100. The numerical values on the vertical axis of Figure 3 are as follows. NC group: 100, 3.9 μg / ml group: 96.5, 7.8 μg / ml group: 97.0, 15.6 μg / ml group: 98.5, 31.3 μg / ml group: 97.3, 62.5 μg / ml group: 100.0, 125 μg / ml group: 98.3, 250 μg / ml group: 96.5, 500 μg / ml group: 96.4 As shown in Fig. 3, no cell proliferation effect was observed when chondroitin sulfate A was added compared to the case without addition (negative control).

[0120] Fig. 4 is a graph showing the results of the cell proliferation test of each proteoglycan obtained in Example 1(1) above. In Fig. 4, the vertical axis represents the relative value of the number of cells, and the horizontal axis represents the concentration of the proteoglycan. The numerical values on the vertical axis in Fig. 4 are shown with the value of the negative control (NC) group set to 100 for each group. The numerical values on the vertical axis in Fig. 4(A) are as follows. NC group: 100, 3.91 μg / ml C-028 group: 103.5, 7.81 μg / ml C-028 group: 106.5, 15.63 μg / ml C-028 group: 108.4, 31.25 μg / ml C-028 group: 118.1, 62.5 μg / ml C-028 group: 115.4, 125 μg / ml C-028 group: 120.8, 250 μg / ml C-028 group: 128.3, 500 μg / ml C-028 group: 130.3 The numerical values on the vertical axis in Fig. 4(B) are as follows. NC group: 100, 3.9 μg / ml salmon group: 99.3, 7.8 μg / ml salmon group: 103.5, 15.6 μg / ml salmon group: 103.6, 31.3 μg / ml salmon group: 106.4, 62.5 μg / ml salmon group: 106.6, 125 μg / ml salmon group: 106.0, 250 μg / ml salmon group: 113.4, 500 μg / ml salmon group: 120.2 The numerical values on the vertical axis in Fig. 4(C) are as follows. NC group: 100, Atlantic salmon group at 3.9 μg / ml: 99.7, Atlantic salmon group at 7.8 μg / ml: 102.9, Atlantic salmon group at 15.6 μg / ml: 105.5, Atlantic salmon group at 31.3 μg / ml: 103.7, Atlantic salmon group at 62.5 μg / ml: 104.2, Atlantic salmon group at 125 μg / ml: 103.6, Atlantic salmon group at 250 μg / ml: 92.5, Atlantic salmon group at 500 μg / ml: 79.2 The numerical values on the vertical axis of Fig. 4(D) are as follows. NC group: 100, chicken group at 3.9 μg / ml: 102.5, chicken group at 7.8 μg / ml: 107.2, chicken group at 15.6 μg / ml: 109.4, chicken group at 31.3 μg / ml: 110.8, chicken group at 62.5 μg / ml: 115.8, chicken group at 125 μg / ml: 120.0, chicken group at 250 μg / ml: 130.6, chicken group at 500 μg / ml: 137.5 The numerical values on the vertical axis of Fig. 4(E) are as follows. NC group: 100, pig group at 3.9 μg / ml: 96.9, pig group at 7.8 μg / ml: 100.1, pig group at 15.6 μg / ml: 104.8, pig group at 31.3 μg / ml: 105.7, pig group at 62.5 μg / ml: 105.2, pig group at 125 μg / ml: 110.6, pig group at 250 μg / ml: 117.0, pig group at 500 μg / ml: 121.5 The numerical values on the vertical axis of Fig. 4(F) are as follows. NC group: 100, Japanese eel group at 3.9 μg / ml: 101.3, Japanese eel group at 7.8 μg / ml: 102.3, Japanese eel group at 15.6 μg / ml: 105.1, Japanese eel group at 31.3 μg / ml: 108.8, Japanese eel group at 62.5 μg / ml: 107.8, Japanese eel group at 125 μg / ml: 112.2, Japanese eel group at 250 μg / ml: 118.8, Japanese eel group at 500 μg / ml: 124.8 As shown in Fig. 4(A), cell growth activity was observed when C-028 (positive control) was added, as compared with the case where no addition was made (negative control). As shown in Figs. 4(B) to (F), in the proteoglycans derived from the nasal cartilage of rainbow trout, the epiphyseal cartilage of chicken, the laryngeal cartilage of pig, and the fin of rockfish obtained in Example 1(1) above, a cell growth effect was observed in a proteoglycan concentration-dependent manner.

[0121] From the results of Figs. 3 and 4, no cell growth activity of dermal fibroblasts was observed with only type A chondroitin sulfate constituting the proteoglycan. In contrast, the proteoglycan in which the core protein produced by the method of the present disclosure is bound to the sugar chain showed cell growth activity of dermal fibroblasts. From the above, it was found that the proteoglycan in which the core protein produced by the method of the present disclosure is bound to the sugar chain has cell growth activity of dermal fibroblasts.

[0122] [Example 2] The composition of GAG of the proteoglycan obtained in Example 1 above was analyzed.

[0123] (1) Analysis of GAG in each proteoglycan GAG analysis was performed on various proteoglycans of Example 1(1) above. Specifically, GAG was purified from various proteoglycans purified by the same method as in Example 1(1) above by the same method as in Example 1(2), and HPLC analysis was performed. As the various proteoglycans, a proteoglycan derived from the nasal cartilage of rainbow trout (sCSPG), a proteoglycan derived from the sternal cartilage of chicken (cCSPG), or a proteoglycan derived from the bronchial cartilage of pig (pCSPG) was used.

[0124] As a result, the peak top molecular weight (Mp) of GAG (sGAG) derived from the nasal cartilage of rainbow trout was 84,000, the peak top molecular weight (Mp) of GAG (cGAG) derived from the sternal cartilage of chicken was 77,000, and the peak top molecular weight (Mp) of GAG (pGAG) derived from the bronchial cartilage of pig was 51,000.

[0125] (3) Disaccharide structure in each proteoglycan For each of the various GAGs obtained in Example 2(1) above, the disaccharide structure of each proteoglycan was analyzed. Specifically, the content ratio (composition ratio) of each disaccharide structure was calculated in the same manner as in Example 1(3). These results are shown in Table 4 below.

[0126]

Table 4

[0127] As shown in Table 4 above, the content of the disaccharide structure was as follows. · GAG (sGAG) derived from the nasal cartilage of rainbow trout by guanidine extraction Chondroitin sulfate O: 11%, chondroitin sulfate iO: 0%, chondroitin sulfate A: 27%, chondroitin sulfate iA: 0%, chondroitin sulfate C: 61%, chondroitin sulfate iC: 0% · GAG (cGAG) derived from the sternal cartilage of chicken by guanidine extraction Chondroitin sulfate O: 5.2%, chondroitin sulfate iO: 0%, chondroitin sulfate A: 63%, chondroitin sulfate iA: 6.5%, chondroitin sulfate C: 24%, chondroitin sulfate iC: 0.3% · GAG (pGAG) derived from the bronchial cartilage of pig by guanidine extraction Chondroitin sulfate O: 6.1%, chondroitin sulfate iO: 0%, chondroitin sulfate A: 52%, chondroitin sulfate iA: 20%, chondroitin sulfate C: 19%, chondroitin sulfate iC: 2.1%

[0128] [Example 3] Regarding the proteoglycan of the present disclosure, it was confirmed that a proteoglycan in which a core protein and a sugar chain are bound can be produced by the production method of the present disclosure.

[0129] (1-1) Purification of bovine-derived proteoglycan As a raw material, about 824 g of the abomasum of a cow (Holstein) was prepared. The abomasum was washed and cut into approximately 2 cm squares while removing as much fat as possible. After the cutting, four times the amount of ethanol (3288 ml) was added, and the mixture was allowed to stand for 10 minutes, and this was repeated once. Thereafter, four times the amount of ethanol (3288 ml) was added, and the mixture was allowed to stand overnight. After the standing, the ethanol was removed and air drying was performed. After the air drying, vacuum drying was performed to obtain 173 g of a defatted dried product.

[0130] (1-2) Guanidine extraction Next, for the defatted dried product, purification of proteoglycan by guanidine extraction was performed. Specifically, 87 g of the defatted dried product of Example 3(1-1) was added with 350 ml of a 4M guanidine hydrochloride aqueous solution, and immersion extraction was performed under the condition of 4 °C for 3 days. After the immersion extraction, 347 ml of a 4M guanidine hydrochloride aqueous solution was added, and further immersion extraction was performed under the condition of 4 °C for 3 days. After the immersion extraction, suction filtration and dialysis were performed. After the dialysis, lyophilization was performed to obtain 9.93 g of a proteoglycan solid (cow stomach-derived guanidine-extracted proteoglycan (dermatan sulfate proteoglycan: DSPG)).

[0131] (1-3) Acetic acid extraction In parallel with the guanidine extraction, for the defatted dried product, purification of proteoglycan by acetic acid extraction was performed. Specifically, 86 g of the defatted dried product of Example 3(1-1) was added with 344 ml of a 4 wt% acetic acid aqueous solution, and immersion extraction was performed under the condition of 4 °C for 3 days. After the immersion extraction, 344 ml of a 4 wt% acetic acid aqueous solution was added, and further immersion extraction was performed under the condition of 4 °C for 3 days. After the immersion extraction, suction filtration and dialysis were performed. After the dialysis, lyophilization was performed to obtain 0.53 g of a proteoglycan solid (cow stomach-derived acetic acid-extracted proteoglycan).

[0132] (2-1) Purification of pig-derived proteoglycan As raw materials, the stomachs of pigs (6 months old) weighing approximately 1282 g were prepared. The stomachs were washed and minced into approximately 2 cm squares while removing as much fat as possible. After mincing, four times the amount of ethanol (5182 ml) was added, and the mixture was allowed to stand for 10 minutes, and this was repeated once. Thereafter, four times the amount of ethanol (5182 ml) was added, and the mixture was allowed to stand overnight. After standing, the ethanol was removed and air drying was performed. After air drying, drying under reduced pressure was carried out to obtain 261 g of defatted dried product.

[0133] (2-2) Guanidine extraction Next, for the defatted dried product, purification of proteoglycan by guanidine extraction was performed. Specifically, 131 g of the defatted dried product of Example 3(2-1) was added to 1047 ml of a 4M guanidine hydrochloride aqueous solution, and immersion extraction was carried out under the condition of 4 °C for 6 days. After the immersion extraction, suction filtration and dialysis were performed. After dialysis, lyophilization was carried out to obtain 11.92 g of proteoglycan solid (guanidine-extracted proteoglycan derived from pig stomach).

[0134] (2-3) Acetic acid extraction In parallel with Example 3(2-2), for the defatted dried product, purification of proteoglycan by acetic acid extraction was performed. Specifically, 130 g of the defatted dried product of Example 3(2-1) was added to 1040 ml of a 4 wt% acetic acid aqueous solution, and immersion extraction was carried out under the condition of 4 °C for 6 days. After the immersion extraction, suction filtration and dialysis were performed. After dialysis, lyophilization was carried out to obtain 0.44 g of proteoglycan solid (acetic acid-extracted proteoglycan derived from pig stomach).

[0135] (3) Analysis of the disaccharide structure of each proteoglycan Analysis of the disaccharide structure was performed on the guanidine-extracted proteoglycan derived from bovine stomach, the acetic acid-extracted proteoglycan derived from bovine stomach, the guanidine-extracted proteoglycan derived from porcine stomach, and the acetic acid-extracted proteoglycan derived from porcine stomach. Specifically, digestion and HPLC analysis were performed on the guanidine-extracted proteoglycan derived from bovine stomach in Example 3(1-2), the acetic acid-extracted proteoglycan derived from bovine stomach in Example 3(1-3), the guanidine-extracted proteoglycan derived from porcine stomach in Example 3(2-2), and the acetic acid-extracted proteoglycan derived from porcine stomach in Example 3(2-3) in the same manner as in Example 1(3). The results are shown in FIG. 5.

[0136] FIG. 5 is a graph showing the results of HPLC analysis of the disaccharide structure of each proteoglycan. In FIG. 5, the vertical axis indicates the peak intensity (AU), and the horizontal axis indicates the retention time (min). FIG. 5(A) shows the results of the guanidine-extracted proteoglycan derived from bovine stomach, FIG. 5(B) shows the results of the acetic acid-extracted proteoglycan derived from bovine stomach, FIG. 5(C) shows the results of the guanidine-extracted proteoglycan derived from porcine stomach, and FIG. 5(D) shows the results of the acetic acid-extracted proteoglycan derived from porcine stomach. As shown in FIG. 5, it was found that the guanidine-extracted proteoglycan derived from bovine stomach and the guanidine-extracted proteoglycan derived from porcine stomach contain chondroitin sulfate (CS) and dermatan sulfate (DS).

[0137] (4-1) Ion exchange of guanidine-extracted proteoglycan with Tris buffer By using guanidine extraction for the purification of proteoglycan, the uronic acids (such as glucuronic acid and iduronic acid) of GAG that constitute proteoglycan are substituted with guanidine salts. Therefore, if guanidine-extracted proteoglycan is used as it is, harmful guanidine is released. For this reason, salt exchange of guanidine-extracted proteoglycan was performed with Tris buffer. Specifically, 60 ml of Tris buffer (20 mmol / l Tris-HCl, 137 mmol / l NaCl) was added to 0.2 g of the bovine stomach-derived proteoglycan of Example 3(1-2). After the addition, ultrasonic disruption was performed and left standing overnight. After the standing, centrifugation was performed to obtain a precipitate and a supernatant. The precipitate and the supernatant were each subjected to dialysis and freeze-drying.

[0138] (4-2) Salt exchange of guanidine-extracted proteoglycan with an aqueous sodium hydrogen carbonate solution In parallel with Example 3(4-1) above, salt exchange of guanidine-extracted proteoglycan was performed with an aqueous sodium hydrogen carbonate solution. Specifically, 30 ml of NaHCO3 was added to 0.1 g of the bovine stomach-derived proteoglycan of Example 3(1-2). After the addition, ultrasonic disruption was performed and left standing overnight. After the standing, centrifugation was performed to obtain a precipitate and a supernatant. The precipitate and the supernatant were each subjected to dialysis and freeze-drying.

[0139] (4-3) Analysis of the disaccharide structure of each proteoglycan Analysis of the disaccharide structure was performed on the precipitate and the supernatant after salt exchange. Specifically, using the freeze-dried products of the precipitate and the supernatant after salt exchange obtained in Example 3(4-1) and (4-2), digestion and HPLC analysis were performed in the same manner as in Example 1(3). These results are shown in Figure 6 and Table 5 below.

[0140] Figure 6 is a graph showing the results of HPLC analysis of the disaccharide structure of each proteoglycan. In Figure 6, the vertical axis indicates the peak intensity (AU), and the horizontal axis indicates the retention time (min). Figure 6(A) shows the results of the Tris buffer-treated precipitate, Figure 6(B) shows the results of the Tris buffer-treated supernatant, Figure 6(C) shows the results of the sodium bicarbonate aqueous solution-treated precipitate, and Figure 6(D) shows the results of the sodium bicarbonate aqueous solution-treated supernatant. As shown in Figure 6, it was found that the proteoglycan contained chondroitin sulfate sulfate (CS) and dermatan sulfate (DS) even after the salt exchange of guanidine.

[0141]

Table 5

[0142] The above Table 5 shows the total amount of chondroitin sulfate and dermatan sulfate (CS-DS amount) contained in the defatted dried product when converted to 100 g of defatted dried product of the fourth stomach of cattle after salt exchange. As shown in the above Table 5, the CS-DS amount of the Tris buffer-treated supernatant was 182 mg, the CS-DS amount of the sodium bicarbonate aqueous solution-treated supernatant was 190 mg, the CS-DS amount of the Tris buffer-treated precipitate was 50 mg, and the CS-DS amount of the sodium bicarbonate aqueous solution-treated precipitate was 24 mg. Also, although not shown in the above Table 4, the peak top molecular weight of the proteoglycan contained in the Tris buffer-treated supernatant was 460 kDa.

[0143] (5) Detection of decorin Next, it was examined whether decorin could be detected in the proteoglycan after the salt exchange. Specifically, for the lyophilized product of the supernatant after salt exchange obtained in Example 3 (4-1) or (4-2) above, reduction and alkylation of the proteoglycan were performed using FOCUS Protein Reduction-Alkylation (786-231, manufactured by G-Biosciences). To 500 μl of 1 mg / ml proteoglycan after salt exchange with a neutral buffer (pH 7.5), 2.5 μl of Reductant Buffer was added and stirred for 10 seconds. After the stirring, 10 ml of FOCUS Protein Reductant was added and allowed to stand under the conditions of 55°C for 1 hour. After the standing, 2.5 ml of Alkylation Buffer was added and stirred for 10 seconds. After the stirring, 0.4 mol / l iodoacetamide was added, and the sample was allowed to stand and alkylated under the conditions of room temperature (about 25°C, the same hereinafter) for 1 hour in the dark. Next, a PVDF membrane (Immobilon-FL membrane, manufactured by Merck) was treated with methanol for 1 minute to hydrophilize it. After the hydrophilization, the PVDF membrane was replaced with TBS under the conditions of 5 minutes. After the replacement, the PVDF membrane was set in a filtration blotting apparatus (manufactured by BioRad). After the setting, the alkylated sample was applied to the well and aspirated. After the aspiration, the PVDF membrane was recovered from the filtration blotting apparatus and allowed to stand and dry at room temperature. After the standing and drying, the PVDF membrane was washed with TBS containing 0.05% Tween-20 (Tris buffer, TBS-T). After the washing, the PVDF membrane was blocked using EveryBlot Blocking Buffer (manufactured by BioRad) under the conditions of 5 minutes. After the blocking, a primary antibody (anti-Decorin antibody, Cat No: 14667-1-AP, manufactured by Proteintech) diluted 1 / 1000-fold was added, and a primary reaction was performed under the conditions of 4°C for 18 hours. After the primary reaction, washing was performed 5 times for 5 minutes using TBS-T.After the washing, StarBright Blue (manufactured by BioRad) diluted 1 / 2500 times and 0.05% SDS were added, and a secondary reaction was carried out under the conditions of room temperature for 1 hour. After the secondary reaction, washing was performed 6 times for 5 minutes each using TBS-T. After the washing, the PVDF membrane was washed with ultrapure water and replaced with ultrapure water. After the replacement, fluorescence detection of the PVDF membrane was performed using a multiplex fluorescence detector (ChemiDoc Touch MP, manufactured by BioRad). These results are shown in FIG. 7.

[0144] FIG. 7 is a photograph showing the detection results of decorin. In FIG. 7, the left column shows the results of BSA, and the right column shows the results of bovine-derived proteoglycan (DSPG). As shown in FIG. 7, a signal could be detected in bovine-derived DSPG. From these results, it was found that the bovine-derived proteoglycan extracted with guanidine and salt-substituted with a neutral buffer (pH 7.5) preserved decorin as the core protein.

[0145] Although the present disclosure has been described with reference to the embodiments and examples above, the present disclosure is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0146] This application claims priority based on Japanese Patent Application No. 2022-211853 filed on December 28, 2022, and incorporates all of the disclosure thereof herein.

[0147] <Supplementary Note> Some or all of the above embodiments and examples can be described as follows, but are not limited thereto. <Method for producing proteoglycan> (Supplementary Note 1) An extraction step of extracting an extract containing proteoglycan from animal tissue is included, The proteoglycan includes a core protein and a sugar chain, A method for producing a proteoglycan in which the core protein and the sugar chain are bound. (Appendix 2) A purification step of purifying a purified product containing proteoglycan from the extract, and a fractionation step of fractionating a proteoglycan that satisfies a predetermined peak top molecular weight from the purified product. The production method according to Appendix 1. (Appendix 3) The production method according to Appendix 2, including a proteoglycan recovery step of recovering a solid fraction of proteoglycan from the fraction containing the proteoglycan. (Appendix 4) The production method according to any one of Appendices 1 to 3, wherein the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000. (Appendix 5) The production method according to any one of Appendices 1 to 4, wherein the peak top molecular weight of the sugar chain of the proteoglycan is 20,000 to 250,000. (Appendix 6) The sugar chain of the proteoglycan contains a disaccharide structure of type A and / or type iA, In the proteoglycan, the content ratio of the disaccharide structure of type A and / or type iA is higher than the content ratio of other disaccharide structures. The production method according to any one of Appendices 1 to 5. (Appendix 7) The sugar chain of the proteoglycan contains a disaccharide structure of type A and / or type iA, In the proteoglycan, the content ratio of the disaccharide structure of type A and / or type iA is 20 to 90%. The production method according to any one of Appendices 1 to 6. (Appendix 8) The sugar chain of the proteoglycan contains a disaccharide structure of type A and / or type iA, In the proteoglycan, the ratio (A:iA) of the ratio (A) of the disaccharide structure of type A to the ratio (iA) of the disaccharide structure of type iA is 1:1 to 20:1. The production method according to any one of Appendices 7. (Appendix 9) The sugar chain of the proteoglycan contains a disaccharide structure of type C and / or type iC, In the proteoglycan, the content ratio of the C-type and / or the iC-type disaccharide structure is higher than the content ratio of other disaccharide structures. The production method according to any one of Supplementary Notes 1 to 8. (Supplementary Note 10) The sugar chain of the proteoglycan contains a C-type and / or an iC-type disaccharide structure. In the proteoglycan, the content ratio of the C-type and / or the iC-type disaccharide structure is 20 to 50%. The production method according to any one of Supplementary Notes 1 to 9. (Supplementary Note 11) The sugar chain of the proteoglycan contains a C-type and / or an iC-type disaccharide structure. The ratio (C:iC) of the ratio (C) of the C-type disaccharide structure to the ratio (iC) of the iC-type disaccharide structure is 4:1 to 100:0. The production method according to any one of Supplementary Notes 1 to 10. (Supplementary Note 12) The animal includes at least one of mammals and fish. The production method according to any one of Supplementary Notes 1 to 11. (Supplementary Note 13) The animal is selected from the group consisting of pigs, cows, chickens, flounders, salmon, and sharks. The production method according to any one of Supplementary Notes 1 to 12. (Supplementary Note 14) The animal tissue is selected from the group consisting of cartilage, fins, digestive organs, circulatory organs, respiratory organs, and ears. The production method according to any one of Supplementary Notes 1 to 13. <Composition containing proteoglycan> (Supplementary Note 15) Contains proteoglycan. The peak top molecular weight of the proteoglycan is 400,000 to 1,200,000. Composition. (Supplementary Note 16) The peak top molecular weight of the sugar chain of the proteoglycan is 20,000 to 250,000. The composition according to Supplementary Note 15. (Supplementary Note 17) The sugar chain of the proteoglycan contains an A-type and / or an iA-type disaccharide structure. The composition according to Supplementary Note 15 or 16, wherein in the proteoglycan, the content ratio of the disaccharide structure of the A type and / or the iA type is higher than the content ratio of other disaccharide structures. (Supplementary Note 18) The sugar chain of the proteoglycan contains a disaccharide structure of the A type and / or the iA type, The composition according to any one of Supplementary Notes 15 to 17, wherein the content ratio of the sugar chain having a disaccharide structure of the A type and / or the iA type is 20 to 90%. (Supplementary Note 19) The sugar chain of the proteoglycan contains a disaccharide structure of the A type and / or the iA type, The composition according to any one of Supplementary Notes 15 to 18, wherein the ratio (A:iA) of the ratio (A) of the disaccharide structure of the A type to the ratio (iA) of the disaccharide structure of the iA type is 1:1 to 20:1. (Supplementary Note 20) The sugar chain of the proteoglycan contains a disaccharide structure of the C type and / or the iC type, The composition according to any one of Supplementary Notes 15 to 19, wherein in the proteoglycan, the content ratio of the disaccharide structure of the C type and / or the iC type is higher than the content ratio of other disaccharide structures. (Supplementary Note 21) The sugar chain of the proteoglycan contains a disaccharide structure of the C type and / or the iC type, The composition according to any one of Supplementary Notes 15 to 20, wherein in the proteoglycan, the content ratio of the disaccharide structure of the C type and / or the iC type is 20 to 50%. (Supplementary Note 22) The sugar chain of the proteoglycan contains a disaccharide structure of the C type and / or the iC type, The composition according to any one of Supplementary Notes 15 to 21, wherein the ratio (C:iC) of the ratio (C) of the disaccharide structure of the C type to the ratio (iC) of the disaccharide structure of the iC type is 7:1 to 100:0. (Supplementary Note 23) The composition according to any one of Supplementary Notes 15 to 22, which is derived from at least one of mammals and fish. (Supplementary Note 24) A composition according to any one of Appendices 15 to 23, derived from an animal selected from the group consisting of pigs, cows, chickens, flounders, salmon, and rays. (Appendix 25) A composition according to any one of Appendices 15 to 24, derived from a tissue selected from the group consisting of cartilage, fins, digestive organs, circulatory organs, respiratory organs, and ears. (Appendix 26) A composition according to any one of Appendices 15 to 25, for use in promoting cell proliferation. <Method for promoting cell proliferation> (Appendix 27) A method for promoting cell proliferation, which comprises using a composition containing a proteoglycan according to any one of Appendices 15 to 26. (Appendix 28) The proliferation promotion method according to Appendix 27, which comprises a step of using, for a subject, a composition containing the proteoglycan. (Appendix 29) in vitro Or in vivo The proliferation promotion method according to Appendix 26 or 27, for use therein. <Use> (Appendix 30) Use of a composition containing a proteoglycan according to any one of Appendices 15 to 26, for use in promoting cell proliferation.

Industrial applicability

[0148] As described above, according to the present disclosure, it is possible to produce a proteoglycan more simply than the conventional method for producing a proteoglycan, and to produce a proteoglycan in which a core protein and a sugar chain are bound. Therefore, the present disclosure can be said to be extremely useful, for example, in the cosmetic field and the pharmaceutical field.

Claims

1. Contains proteoglycans, The peak top molecular weight of the proteoglycan is 400,000 to 1,200,000, the sugar chain of the proteoglycan contains a C-type and / or iC-type disaccharide structure; A composition, wherein the content of the C-type and / or iC-type disaccharide structures in the proteoglycan is higher than the content of other disaccharide structures.

2. The composition according to claim 1, wherein the content of the C-type and / or iC-type disaccharide structures in the proteoglycan is 20 to 50%.

3. 3. The composition according to claim 1, wherein the ratio (C:iC) of the C-type disaccharide structures (C) to the iC-type disaccharide structures (iC) is 7:1 to 100:

0.

4. 3. The composition according to claim 1 or 2, which is derived from at least one of mammals and fish.

5. 3. The composition of claim 1 or 2, derived from an animal selected from the group consisting of pigs, cows, poultry, flounder, salmon, and rays.

6. 3. The composition of claim 1 or 2, which is derived from tissue selected from the group consisting of cartilage, fin, digestive, circulatory, respiratory, and ear tissues.

7. The composition according to claim 1 or 2 for use in promoting cell proliferation.

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