Agents and methods for activating NRF2

Abstract

We provide a technology that can activate NRF2. [Solution] An NRF2 activator comprising one or more chondroitin sulfate oligosaccharides selected from (a) to (c) below as an active ingredient: (a) a disaccharide having a chondroitin sulfate structure (a disaccharide of CS), (b) a trisaccharide having two N-acetyl-D-galactosamine residues and a chondroitin sulfate structure (a trisaccharide of CS having two GalNac residues), (c) a tetrasaccharide having a chondroitin sulfate structure (a tetrasaccharide of CS). According to the present invention, NRF2 can be activated. This is expected to promote homeostatic reactions in the body, such as antioxidant reactions, anti-inflammatory reactions, and detoxification reactions, and contribute to maintaining and improving health. It is also expected to contribute to the prevention and improvement of various unhealthy conditions and diseases caused by oxidative stress, inflammation, and toxic chemicals.

Description

Technical Field

[0001] The present invention relates to an agent and method for activating NRF2 using chondroitin sulfate oligosaccharide as an active ingredient, and the use of chondroitin sulfate oligosaccharide for producing an activator of NRF2.

Background Art

[0002] NRF2 (Nuclear factor erythroid 2-related factor 2), also called NFE2L2, is a protein with a calculated molecular weight of about 67,000 and is a transcriptional activator that promotes the transcription of target genes. Its target genes are enzymes and the like responsible for maintaining homeostasis against many environmental or endogenous abnormalities and toxic chemical substances such as oxidative stress and inflammation, and many of them are known.

[0003] Therefore, activation of NRF2 improves antioxidant, anti-inflammatory, and detoxification reactions in the living body, and is considered to contribute to the prevention and improvement of various unhealthy states and chronic diseases (such as neurodegenerative diseases, cardiovascular diseases, metabolic diseases, etc.) caused by oxidative stress, inflammation, and toxic chemical substances. For example, a plurality of substances having an NRF2 activation effect have been approved as therapeutic agents for specific diseases or are undergoing clinical trials. For example, dimethyl fumarate, an NRF2 activator, has been approved as a therapeutic agent for relapsing-remitting multiple sclerosis, and ursodiol has been approved as a therapeutic agent for primary biliary cirrhosis. In addition, clinical trials of bardoxolone methyl have been conducted as a therapeutic agent for chronic kidney disease, Alport syndrome, and pulmonary hypertension, and clinical trials of RTA-408 (Omaveloxone) have been conducted as a therapeutic agent for Friedreich's ataxia, ocular inflammation, and pain after ophthalmic surgery. RTA-408 has also been reported to be effective in improving regenerative ability in diabetic wound healing in preclinical trials. In addition, clinical trials of ursodiol have been conducted as a therapeutic agent for primary biliary cirrhosis (Non-Patent Document 1).

[0004] On the other hand, chondroitin sulfate (CS) is a type of glycosaminoglycan that has a structure in which sulfate groups are attached to a long sugar chain consisting of a disaccharide repeat structure of uronic acid and N-acetyl-D-galactosamine (GalNAc). Due to the presence of numerous sulfate groups, it is strongly negatively charged and has excellent water retention and elastic properties. In the body, it is widely distributed not only in cartilage but also in connective tissue such as skin, brain and all other tissues, and interacts with cell growth factors and extracellular matrix components to control various cellular activities such as cell adhesion, migration, proliferation, differentiation, and morphogenesis. In particular, it is known to play an important role in the cushioning effect in cartilage. The present inventors have succeeded in developing a technology to produce chondroitin sulfate oligosaccharide (CS oligosaccharide) with excellent solubility and absorption into the body by hydrolyzing CS under high temperature and high pressure conditions to reduce its molecular weight (Patent Document 1). [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Patent No. 6146733 [Non-patent literature]

[0006] [Non-Patent Document 1] Natalia Robledinos-Anton et al., Review Article Activators and Inhibitors of NRF2: A Review of Their Potential for Clinical Development, Oxidative Medicine and Cellular Longevity Volume 2019, Article ID 9372182, 20 pages, Published 14 July 2019, https: / / doi.org / 10.1155 / 2019 / 937218 [Overview of the project] [Problems that the invention aims to solve]

[0007] As described above, NRF2 is a transcription factor that plays an important role in maintaining homeostasis in living organisms, including antioxidant, anti-inflammatory, and detoxification reactions. It is believed that activating NRF2 can contribute to maintaining and improving health, as well as preventing and improving various unhealthy conditions and diseases caused by oxidative stress, inflammation, and toxic chemicals. Therefore, the present invention aims to provide a technology that can activate NRF2. [Means for solving the problem]

[0008] The inventors diligently conducted research to explore novel applications for CS oligosaccharides and discovered that CS disaccharides, tetrasaccharides, and CS trisaccharides containing two GalNAc residues exhibit high NRF2 activation activity. Based on these findings, the inventors completed the following inventions.

[0009] (1) The NRF2 activator according to the present invention (sometimes referred to as "this agent") comprises one or more CS oligosaccharides selected from (a) to (c) below as an active ingredient; (a) Disaccharides having a chondroitin sulfate structure (disaccharides of CS), (b) A trisaccharide having two N-acetyl-D-galactosamine residues and a chondroitin sulfate structure (a trisaccharide of CS having two GalNAc residues), (c) A tetrasaccharide having a chondroitin sulfate structure (a tetrasaccharide of CS).

[0010] (2) This agent may also be used to promote the expression of NRF2 target genes. That is, the present invention also provides an NRF2 target gene expression promoter comprising one or more CS oligosaccharides selected from (a) to (c) above as an active ingredient.

[0011] (3) In the present invention, the NRF2 target gene may be a gene having an antioxidant response element (5'-TGAC / GNNNGC-3') in its promoter region or a gene encoding thioredoxin reductase 1.

[0012] (4) This agent may also be used as an antioxidant. That is, the present invention also provides an antioxidant comprising one or more CS oligosaccharides selected from (a) to (c) above as an active ingredient.

[0013] (5) This drug may also be used as an anti-inflammatory agent. That is, the present invention also provides an anti-inflammatory agent comprising one or more CS oligosaccharides selected from (a) to (c) above as an active ingredient.

[0014] (6) A method for activating NRF2 according to the present invention comprises the step of activating NRF2 in a human or animal by administering one or more CS oligosaccharides selected from (a) to (c) above to the human or animal.

[0015] (7) The use according to the present invention is the use of one or more CS oligosaccharides selected from (a) to (c) above for the production of an NRF2 activator.

[0016] From the viewpoint of industrial applicability, the present invention may be implemented in ways other than medical procedures. [Effects of the Invention]

[0017] According to the present invention, NRF2 can be activated. This is expected to promote homeostatic reactions in the body, such as antioxidant reactions, anti-inflammatory reactions, and detoxification reactions, thereby contributing to the maintenance and improvement of health. Furthermore, it is expected to contribute to the prevention and improvement of various unhealthy conditions and diseases caused by oxidative stress, inflammation, and toxic chemicals. The present invention can be used in all applications where activating NRF2 is meaningful. [Brief explanation of the drawing]

[0018] [Figure 1] This is a structural formula showing the disaccharide CS oligosaccharide (CS2). The left side shows the CS2 structure with a saturated non-reducing end, and the right side shows the CS2 structure with a double bond between the C4-C5 non-reducing ends. [Figure 2]It is a structural formula showing a trisaccharide CS oligosaccharide (CS3GG) having two GalNAc residues. On the left is CS3GG with a saturated non-reducing end structure, and on the right is CS3GG with a double bond at C4-C5 of the non-reducing end. [Figure 3] It is a diagram schematically showing a CS hydrolysis apparatus used in the examples. [Figure 4] It is a bar graph showing the expression level of the HMOX1 gene in EA.hy926 cells cultured in the absence (no addition group) or presence (CS addition group) of a test substance. [Figure 5] (I) is a bar graph showing the expression level of the HMOX1 gene in HepG2 cells cultured in the absence (no addition group) or presence (CS addition group) of a test substance. (II) is a bar graph showing the expression level of the same gene in NRK-52E cells. [Figure 6] It is a bar graph showing the expression level of the HMOX1 gene in L929 cells cultured in the absence (no addition group) or presence (CS addition group) of a test substance. (I) shows the results obtained with n = 6 using CS disaccharide and high molecular weight CS as test substances, and (II) shows the results obtained with n = 4 using CS disaccharide and CS trisaccharide GG as test substances. [Figure 7] It is a bar graph showing the expression level of the GCLM gene in NRK-52E cells cultured in the absence (no addition group) or presence (CS addition group) of a test substance. [Figure 8] It is a bar graph showing the expression level of the GCLC gene in NRK-52E cells cultured in the absence (no addition group) or presence (CS addition group) of a test substance. [Figure 9] It is a bar graph showing the expression level of the GSTA2 gene in HepG2 cells cultured in the absence (no addition group) or presence (CS addition group) of a test substance. [Figure 10] It is a bar graph showing the expression level of the NQO1 gene in L929 cells cultured in the absence (no addition group) or presence (CS addition group) of a test substance. [Figure 11]This bar graph shows the results of measuring the expression-promoting activity of test substances against the promoter region of the GSTA2 gene using a luciferase reporter assay. (I) shows the expression-promoting activity when CS2 sugar is used as the test substance, (II) shows the activity when CS4 sugar is used as the test substance, and (III) shows the activity when high molecular weight CS is used as the test substance. [Figure 12] (I) is a schematic diagram showing a test method for evaluating the permeability of a test substance to human three-dimensional cultured epidermis. (II) is a bar graph showing the amount of permeation when high molecular weight CS, CS4 sugar, and CS2 sugar were used as test substances in the test.

[0019] The present invention will be described in detail below.

[0020] An NRF2 activator refers to a composition that has the effect of activating NRF2, or a composition used for the purpose of bringing about such an effect in a living organism.

[0021] Activating NRF2 means promoting the expression of genes that are transcriptionally induced by NRF2 (NRF2 target genes) in any cell, tissue, or organ, either in vivo or of biological origin.

[0022] To promote the expression of an NRF2 target gene means to increase the transcription level of the gene or the activity or protein level of the protein encoded by the gene (hereinafter sometimes referred to as "transcription level, etc.") compared to when the present invention is not used. In other words, promoting the expression of an NRF2 target gene includes reducing the degree of decrease in the transcription level, etc., even if it decreases or does not change, compared to when the present invention is not used.

[0023] Many NRF2 target genes have an antioxidant response element (ARE: 5'-TGAC / GNNNGC-3') in their transcriptional regulatory region (promoter region). NRF2 is known to form a heterodimer with MAF (small muscle aponeurosis fibromatosis) and bind to the ARE sequence, thereby inducing its transcription. Therefore, in the present invention, NRF2 target genes may also have an ARE sequence in their promoter region. Currently, about 250 human genes containing an ARE sequence are known (Non-Patent Literature 1). More specifically, examples of ARE sequence-containing genes include, for example, the protein-coding genes shown in Table 1 (Azhwar Raghunatha et al., Antioxidantresponseelements: Discovery, classes, regulation and potential applications, Redox Biology 17 (2018), pp. 297-314). [Table 1]

[0024] Among these, for example, glutathione S-transferase (GST) exists in cytoplasmic and membrane-bound forms, which are encoded by two different supergene families. GST has the function of detoxifying electrophilic compounds such as carcinogens, drugs, toxic chemicals, and oxidative stress products through glutathione conjugation. Based on its primary structure and substrate specificity, GST is classified into many classes such as α, μ, π, σ, θ, δ, ε, Ω, ζ, λ, and T (Akira Hiratsuka, "Recent Advances in Hepatic Drug Metabolism 5. Glutathione S-Transferase," Liver, Vol. 42, No. 6, pp. 303-304). GSTA2 is subunit 2 of GST belonging to the α(A) class, and the human GSTA2 gene is disclosed in NCBI Reference Sequence:NC_000006.12(REGION:52750087..52763475) (total length 13389 bp, SEQ ID NO: 21).

[0025] Furthermore, heme oxygenase, for example, is an enzyme involved in heme metabolism, cleaving heme to form biliverdin. The formed biliverdin is converted to bilirubin by biliverdin reductase. Heme oxygenase exists as two isozymes: inductive heme oxygenase-1 and constitutive heme oxygenase-2. Heme oxygenase 1 (HMOX1) has cytoprotective effects that protect cells from oxidative stress damage and also has anti-inflammatory effects (Akihiro Taniguchi, Review article: Heme oxygenase and biological defense mechanisms; Paradigm shift in anti-inflammatory therapy, Journal of the Japanese Society for Clinical Immunology, Vol. 30, No. 1, 2007, pp. 11-21). The human HMOX1 gene is disclosed in NCBI Reference Sequence: NC_000022.11 (REGION: 35381096..35394207) (13112 bp, SEQ ID NO: 22).

[0026] Furthermore, glutamate-cysteine ​​ligase, for example, is an enzyme composed of two subunits: a heavy-chain catalytic subunit (GCLC) and a light-chain regulatory subunit (GCLM). This enzyme, also known as γ-glutamylcysteine ​​synthase (GCS), plays a central role in maintaining glutathione levels in the body by catalyzing the rate-limiting step in the glutathione synthesis reaction. The glutathione synthesized by this enzyme primarily acts as a "defense substance for cell survival," responsible for removing reactive oxygen species and oxidative stress, and eliminating foreign substances (such as drugs), as well as participating in a variety of reactions including intracellular signal transduction and hormonal action. The human GCLM gene is disclosed in NCBI Reference Sequence: NC_000001.11 (REGION: complement(93885199..93909430)) (24232 bp total length, SEQ ID NO: 23). The human GCLC gene is disclosed in NCBI Reference Sequence:NC_000006.12(REGION: complement(53497341..53545101)) (total length 47761 bp, SEQ ID NO: 24).

[0027] Furthermore, NRF2 target genes are not limited to ARE sequence-containing genes; for example, they may be genes encoding thioredoxin reductase (TXNRD).

[0028] Thioredoxin reductase is an enzyme that uses thioredoxin and NADP+ as substrates to produce thioredoxin disulfide, NADPH, and H+ (thioredoxin + NADP(+) ⇔ thioredoxin disulfide + NADPH + H(+)). Thioredoxin (Trx) is an antioxidant enzyme with an SH group possessing redox activity within its molecule. It is a multifunctional protein that exhibits protective effects against oxidative stress and reactive oxygen species, as well as being involved in intracellular signal transduction. The thioredoxin system, composed of thioredoxin, thioredoxin reductase, and NADPH, is one of the major antioxidant mechanisms in cells and is universally present from archaea to humans. Reduced thioredoxin binds to oxidized target proteins, reducing the disulfide bonds (SS) of the target proteins to thiol groups (-SH), while the thiol group of thioredoxin itself is oxidized. Oxidized thioredoxin is reduced back to its reduced form by the action of thioredoxin reductase in the presence of NADPH (Nikken Zail Co., Ltd., Japan Institute for Aging Control, Home > Product Information Redox / Anti-aging > Thioredoxin-1 Measurement Kit, [online], [Searched November 27, 2024], Internet).<https: / / www.jaica.com / products_redox_thioredoxin1_kit_pc.html> Mammals have three types of thioredoxin reductase isozymes, and of these, it is known that only TXNRD1 expression is induced when cells are exposed to various stresses. The human TXNRD1 gene is disclosed in NCBI Reference Sequence: NC_000012.12 (REGION: 104215779..104350307) (total length 134529 bp, SEQ ID NO: 25).

[0029] As described above, activating NRF2 can promote the expression of genes encoding various proteins that have antioxidant, anti-inflammatory, and waste-elimination effects. Therefore, the active ingredient of the present invention can be used to prevent or reduce oxidation in living organisms, prevent or reduce inflammation in living organisms, and promote the detoxification or elimination of substances that are unnecessary for living organisms. In other words, the active ingredient of the present invention can be used as an antioxidant or an anti-inflammatory agent.

[0030] NRF2 activation can be confirmed by methods known to those skilled in the art. For example, as shown in the examples described later, one method is to confirm the mRNA expression level of the NRF2 target gene by quantitative PCR or microarray. If the mRNA expression level increases, it can be said that NRF2 has been activated. Specifically, for example, a test substance is added to the culture medium of cells derived from a tissue known to express the NRF2 target gene, and quantitative PCR is performed using a primer specific to the gene. A primer specific to the NRF2 target gene can be designed based on the nucleotide sequence of the known NRF2 target gene mentioned above.

[0031] Furthermore, NRF2 activation can also be confirmed by a reporter assay, for example, in which a vector linking the transcriptional regulatory region of an NRF2 target gene to a reporter gene (e.g., luciferase) is introduced into cells containing NRF2. If the expression level of the reporter gene (e.g., the luminescence signal mediated by luciferase) increases, it can be said that NRF2 has been activated.

[0032] A chondroitin sulfate structure is a structure in which a sulfate group is attached to a sugar chain consisting of a disaccharide formed by the linkage of uronic acid (such as D-glucuronic acid or iduronic acid) and GalNAc, or a structure in which uronic acid and GalNAc are repeated alternately.

[0033] CS oligosaccharides are those that have a chondroitin sulfate structure but have fewer constituent sugars (sugar residues) than chondroitin sulfate. In this invention, chondroitin sulfate may be referred to as "high molecular weight CS" or "high molecular weight CS" in contrast to CS oligosaccharides. Generally, the number of sugars in high molecular weight CS is said to be in the range of approximately 60 to 500 sugars, and the molecular weight is in the range of approximately 15,000 to 124,000.

[0034] The present invention uses one or more CS oligosaccharides selected from (a) to (c) below (sometimes referred to as "this CS oligosaccharide") as an active ingredient; (a) Disaccharides having a chondroitin sulfate structure (disaccharides of CS), (b) A trisaccharide having two N-acetyl-D-galactosamine residues and a chondroitin sulfate structure (a trisaccharide of CS having two GalNAc residues), (c) A tetrasaccharide having a chondroitin sulfate structure (a tetrasaccharide of CS).

[0035] Examples of disaccharide unit structures in CS include the glucuronic acid (GlcA)-GalNAc4-sulfate structure (so-called chondroitin sulfate A structure), the GlcA-GalNAc6-sulfate structure (so-called chondroitin sulfate C structure), the iduronic acid-GalNAc4-sulfate structure (so-called chondroitin sulfate B structure, also called dermatan sulfate), the GlcA2-sulfate-GalNAc6-sulfate structure (so-called chondroitin sulfate D structure), the GlcA-GalNAc4,6-sulfate structure (so-called chondroitin sulfate E structure), the GlcA2-sulfate-GalNAc4,6-sulfate structure (so-called chondroitin sulfate T structure), and the O structure, which lacks sulfate group bonding. Even within a single polymer CS, the sulfate group bonding is heterogeneous, and therefore, in many cases, a single polymer CS is thought to possess multiple structures.

[0036] This CS oligosaccharide can be obtained by breaking down high-molecular-weight CS to reduce its molecular weight. In other words, the chondroitin sulfate structure of this CS oligosaccharide may be any of the following: structure A, structure C, structure D, structure E, structure O, structure T, or a mixture thereof.

[0037] While high molecular weight chondroitin sulfate oligosaccharides are not absorbed in the intestinal tract when taken orally, this chondroitin sulfate oligosaccharide is low molecular weight, so it has high permeability through the intestinal wall and can be absorbed directly into the body from the intestinal tract when taken orally (International application PCT / JP2021 / 3414, Japanese Patent No. 6146733: paragraphs

[0081] -

[0086] , [Figure 18], [Figure 19]). This chondroitin sulfate oligosaccharide absorbed into the body is thought to migrate into the bloodstream (Hiroko Mizuta et al, Quantification of orally administered chondroitin sulfate oligosaccharides in human plasma and urine, Glycobiology, Volume 33, Issue 9, September 2023, Pages 755-763, https: / / doi.org / 10.1093 / glycob / cwad054), reach various tissues, and exert NRF2 activating effects.

[0038] Examples of methods for reducing the molecular weight of CS include hydrolysis under high temperature and high pressure conditions (175°C ≤ temperature T ≤ 220°C and 5 MPa ≤ pressure P ≤ 25 MPa) (Japanese Patent No. 6146733), hydrolysis with an acid such as hydrochloric acid (Cifonelli, Carbohydrate Res., Vol. 2, pp. 150-161, 1966), desorption or hydrolysis using enzymes such as chondroitinase ABC, chondroitinase ACII, testicular hyaluronidase, and CS-degrading enzyme (Japanese Patent Publication No. 9-168384), and maintaining an aqueous solution of CS with a pH of 2.5 to 12.0 under hydrothermal conditions of 100 to less than 160°C for 5 to less than 20 minutes (Japanese Patent Publication No. 2010-77256).

[0039] Of the above methods, the hydrolysis method allows for obtaining CS oligosaccharides with a saturated structure at the non-reducing end (saturated type). Here, saturated type refers to a structure in which the sugar residue located at the non-reducing end of the oligosaccharide does not have a double bond, and unsaturated type refers to a structure in which the sugar residue has a double bond. Figure 1 shows the saturated and unsaturated structures of a disaccharide CS oligosaccharide (CS2). In the saturated type, there is no double bond between the carbon at position 4 and the carbon at position 5 (C4-C5) of the uronic acid residue located at the non-reducing end of CS2. Figure 2 shows the saturated and unsaturated structures of a trisaccharide CS oligosaccharide having two GalNAc residues (CS3GG). In the saturated type, there is no double bond between the carbon at position 4 and the carbon at position 5 (C4-C5) of the GalNAc residue located at the non-reducing end of CS3GG. Saturated CS oligosaccharides, which do not have a double bond between C4 and C5, are less likely to be assimilated by intestinal bacteria and have been reported to be able to be transferred into the bloodstream while maintaining their chondroitin sulfate structure (International application PCT / JP2021 / 3414; Example 3, etc.). Therefore, saturated CS oligosaccharides are considered to be more easily absorbed by the body.

[0040] Furthermore, saturated CS oligosaccharides do not absorb ultraviolet light at a wavelength of 240 nm, while unsaturated CS oligosaccharides do absorb light at this wavelength. Therefore, by subjecting a sample to high-performance liquid chromatography (HPLC) and detecting it at a wavelength of 240 nm with a UV detector, it is possible to determine whether it is saturated or unsaturated CS oligosaccharide based on whether or not a peak is detected.

[0041] CS oligosaccharides with a desired number of sugar residues, such as disaccharides or trisaccharides, can be obtained by methods known to those skilled in the art. For example, a mixture of CS oligosaccharides with different numbers of sugar residues can be used as raw materials, and by performing gel filtration HPLC under the following conditions, oligosaccharides with the desired number of sugar residues, such as only disaccharides or only trisaccharides, can be fractionated and used. <Conditions for gel filtration HPLC> Column: Superdex Increase 30 / 100 Mobile phase: 50 mM ammonium bicarbonate (NH4HCO3) aqueous solution Flow rate: 0.5mL / min Detector: RI detector

[0042] When obtaining this CS oligosaccharide by decomposing CS, commercially available raw material CS extracted from animal cartilage, etc., may also be used. The composition ratio of disaccharide units in CS contained in animal cartilage (the ratio of disaccharide units of a specific structure to the total number of disaccharide units contained in CS) is known to vary depending on the source animal. Chondroitin sulfate derived from the cartilage of cartilaginous fish such as rays, sharks, chimaeras, and whale sharks is mainly composed of chondroitin sulfate C, but also contains chondroitin sulfates A, D, E, and O. On the other hand, chondroitin sulfate derived from the cartilage of mammals such as cows, whales, rabbits, sheep, and pigs, and birds such as chickens, is mainly composed of chondroitin sulfate A, but also contains chondroitin sulfates C and O. Chondroitin sulfate derived from the cartilage of mollusks such as squid is mainly composed of chondroitin sulfate E, but also contains chondroitin sulfates A, C, and O. The disaccharide unit composition ratio of chondroitin sulfate in cartilaginous fish is, for example, chondroitin sulfate C:chondroitin sulfate D = 50-70:1-10, and it also contains chondroitin sulfate O, A, E, etc.

[0043] The disaccharide unit composition ratio of CS can be quantified by treating CS with chondroitinase to produce unsaturated disaccharides, separating them by HPLC, and detecting the peaks corresponding to each disaccharide unit (A, C, etc.) of CS (see JHFA Product Explanation Manual: Chondroitin Sulfate Foods, published August 20, 2015, Japan Health & Nutrition Food Association, pp. 13-18). Furthermore, the origin of CS can be determined by "animal-derived DNA testing" or "animal-derived protein testing" based on the DNA or proteins remaining in CS (for example, see Japan Food Research Laboratories, http: / / www.jfrl.or.jp / item / allergens / post-62.html).

[0044] As described above, this CS oligosaccharide can be obtained by decomposing high-molecular-weight CS to reduce its molecular weight, or it can be obtained by chemical synthesis, or a commercially available product (for example, "nano-type chondroitin (Marukyo Biofoods)") may be used.

[0045] This CS oligosaccharide can be used by administering it to a living organism, whether human or animal. Examples of administration methods include oral, subcutaneous, intravenous, intramuscular, intrathecal, sublingual, oral mucosal, rectal, vaginal, eye drops, ear drops, nasal, oral inhalation, spray inhalation, and transdermal.

[0046] More specific administration methods can be appropriately determined depending on the form of the product using this CS oligosaccharide. For example, for topical products such as cosmetics, quasi-drugs, and pharmaceuticals, administration methods include applying, patching, or spraying the CS oligosaccharide (or a product containing it) onto the skin. Alternatively, the CS oligosaccharide can be incorporated into skin cleansers or bath products, and the CS oligosaccharide can be absorbed into the body through the skin or mucous membranes by using such products.

[0047] Furthermore, in the case of products intended for internal use (for example, pharmaceuticals, food additives, supplements, enteral nutrition formulas, nutritional foods, infant formulas, and other ordinary foods and beverages), the CS oligosaccharide can be incorporated into the product and administered orally or enterally.

[0048] Since this CS oligosaccharide exhibits an activating effect on NRF2, it can be used to produce NRF2 activators. Furthermore, since this CS oligosaccharide exhibits the effect of promoting the expression of NRF2 target genes, it can be used to manufacture NRF2 target gene expression promoters. Furthermore, this CS oligosaccharide can be said to exert antioxidant effects because it promotes the expression of genes that encode proteins that exhibit antioxidant activity. Therefore, this CS oligosaccharide can be used to manufacture antioxidants. Furthermore, this CS oligosaccharide can be said to exert anti-inflammatory effects because it promotes the expression of genes that encode proteins that exert anti-inflammatory effects. Therefore, this CS oligosaccharide can be used to manufacture anti-inflammatory agents.

[0049] This product can be in various forms, including those consisting solely of the active ingredient, CS oligosaccharide, as well as in combination with appropriate excipients, carriers, and other materials, such as in confectionery, beverages, processed foods, health foods, infant foods, and other everyday foods; in animal feed, pet food, and beverages for livestock, racehorses, and companion animals; in pharmaceuticals, quasi-drugs, supplements, food additives, feed additives, and cosmetics. These can be manufactured by methods known to those skilled in the art. As shown in Example 4 described later, the 2-4 sugars of CS exhibit excellent skin permeability. Therefore, this CS oligosaccharide can be suitably used, for example, as a compounding material in cosmetics that prevent or improve skin damage and maintain skin health.

[0050] The content of this CS oligosaccharide in food and beverages (food compositions) varies depending on the form of the food or beverage, but can be, for example, 0.001 to 99% by mass, 0.01 to 80% by mass, or 1 to 80% by mass (measured by HPLC) based on dry mass. Since animal feed, etc., is almost the same as food and beverages except that the target is animals, the descriptions of food and beverages in this specification can also be applied to animal feed, etc.

[0051] The content of this CS oligosaccharide in pharmaceuticals, quasi-drugs, supplements, food additives, and feed additives varies depending on the dosage form, but can be, for example, 0.001 to 90% by mass, 0.01 to 85% by mass, or 0.1 to 80% by mass (measured by HPLC method) based on dry mass.

[0052] Furthermore, the content of this CS oligosaccharide in cosmetics varies depending on the dosage form, but can be, for example, 0.0001 to 80% by mass, 0.001 to 60% by mass, or 0.01 to 50% by mass (measured by HPLC method) based on dry mass.

[0053] The recommended daily dose (intake) of this CS oligosaccharide can be appropriately determined depending on the product form of the agent according to the present invention, the age, weight, sex of the recipient, the method of administration, and based on non-clinical or clinical test results. Specifically, examples of recommended daily doses of this CS oligosaccharide include 10 mg / day or more, 20 mg / day or more, 30 mg / day or more, 40 mg / day or more, 50 mg / day or more, 60 mg / day or more, 70 mg / day or more, 80 mg / day or more, 90 mg / day or more, 100 mg / day or more, and 2000 mg / day or less.

[0054] The duration of administration of this CS oligosaccharide can be set as appropriate, but it is preferable to take it repeatedly over a long period of time. Specific examples of administration periods include, for example, 1 day or more, 3 days or more, 1 week or more, 2 weeks or more, 3 weeks or more, 4 weeks or more, 5 weeks or more, 6 weeks or more, 7 weeks or more, or 8 weeks or more.

[0055] The present invention will be described below based on various examples. The technical scope of the present invention is not limited to the features shown in these examples. [Examples]

[0056] <Testing Method> This embodiment was carried out by the following method unless otherwise specified. (1) Test substance The test substances used were disaccharides (CS2), trisaccharides (CS3-sugar GG, CS3-sugar UU), tetrasaccharides (CS4), pentasaccharides (CS5-sugar GG, CS5-sugar UU), hexasaccharides (CS6-sugar), octasaccharides (CS8-sugar), and decasaccharides (CS10-sugar), as well as high molecular weight CS. Here, "CS3-sugar GG" refers to a trisaccharide of CS with GalNAc at both ends, such as "GalNAc-GlcA-GalNAc". "CS3-sugar UU" refers to a trisaccharide of CS with uronic acid at both ends, such as "GlcA-GalNAc-GlcA". The same applies to "CS5-sugar GG" and "CS5-sugar UU". These test substances were prepared as follows.

[0057] (1-1) Preparation of polymer CS 280 kg of ray cartilage was placed in an oblique-axis kneader, and 300 g of papain was added while stirring. The mixture was reacted at 55°C for 3 hours. After inactivation by maintaining the temperature at 92°C for 10 minutes, the mixture was passed through a wire mesh for coarse filtration, and the filtrate was collected. The filtrate was cooled to 50°C, 10 kg of diatomaceous earth was added as a filter aid and stirred well. The mixture was then filtered using a pressurized filter to obtain a pale yellow, clear filtrate. The filtrate was subjected to a filter equipped with an ultrafiltration membrane with a molecular weight cutoff of 13000 and dialyzed for 12 hours with water added as needed. The internal liquid (dialysis retained solution) was collected, heat-sterilized at 92°C, and then spray-dried using a spray dryer to obtain 10.2 kg of white powder, which was used as crude purified chondroitin sulfate.

[0058] 10 g of crudely purified chondroitin sulfate was dissolved in 500 mL of deionized water, 5 g of activated carbon was added, and the mixture was stirred overnight at 4°C. Next, diatomaceous earth was added as a filter aid, and the mixture was filtered for clarity, and the filtrate was collected. Ethanol was added to a final concentration of 70 (v / v)%, the mixture was stirred well, and the mixture was left to stand overnight at 4°C. The white precipitate was collected by filtering through a glass filter and washed with cooled 70 (v / v) ethanol. This was dissolved in 400 mL of deionized water, ethanol was added to a final concentration of 80 (v / v)%, the mixture was stirred well, and the mixture was left to stand overnight at 4°C. The white precipitate was collected by filtering through a glass filter and washed with cooled 80 (v / v) ethanol. This was dissolved in 400 mL of deionized water, ethanol was added to a final concentration of 90 (v / v)%, the mixture was stirred well, and the mixture was left to stand overnight at 4°C. The white precipitate was collected by filtering through a glass filter and washed separately with cooled 90 (v / v) ethanol. This mixture was thoroughly stirred in ethanol, and the resulting white precipitate was collected by filtering through a glass filter. It was then dried in a desiccator to obtain 3.8 g of purified chondroitin sulfate white powder. This was then used as high molecular weight chondroitin sulfate.

[0059] This example aims to evaluate the differences in functionality based on the molecular weight of CS. Therefore, in order to remove low-molecular-weight fragments of CS (CS oligosaccharides) that may be contained in the high-molecular-weight CS obtained in the above procedure, the fraction was further subjected to liquid chromatography, and a fraction with a narrow peak width and a large peak height that did not contain CS oligosaccharides was isolated. The weight-average molecular weight of this fraction was approximately 8900 or approximately 70000.

[0060] (1-2) Preparation of CS2-10 sugars Crudely purified chondroitin sulfate was hydrolyzed under high temperature and high pressure to obtain the oligosaccharide chondroitin sulfate. Specifically, first, an oligosaccharide production apparatus (Figure 3) described in Japanese Patent Publication No. 6146733 was prepared. That is, as shown in Figure 3, the apparatus consists of a water container for distilled water, a high-pressure pump A (Millflow controlled capacity pump M150 pulseless C24-Z3, Nikkiso), a heater (electric heater), a sensor A, a raw material container for raw materials, a high-pressure pump B (Millflow controlled capacity pump M150 pulseless C23-X1, Nikkiso), a mixing T-tube, and a reaction section (stainless steel 316 piping with a lumen diameter of 0.5 mm (internal space volume of 46,800 mm³) 3 ~191000mm 3 The system consists of a water bath, a sensor B, a sensor C, a back pressure valve (High Pressure / Back Pressure 26-1762-66-314, TESCOM), and a product container for holding the reaction products. The water container, raw material container, and product container are all connected by stainless steel piping. The inlet temperature of the reaction section is monitored by sensor B, and the outlet temperature by sensor C. The pressure is monitored by sensor D.

[0061] 10 kg of crudely purified chondroitin sulfate was dissolved in 500 L of water and adjusted to Brix 2.0 to prepare the raw material solution. Degassed distilled water was placed in a water container and continuously pumped using high-pressure pump A, then heated with a heater. The raw material solution was placed in a raw material container and continuously pumped using high-pressure pump B. The heated distilled water and the raw material solution at room temperature were mixed in a mixing T-tube at the inlet of the reaction section, and hydrolysis was carried out in the reaction section by reacting the water with the chondroitin sulfate contained in the raw material solution. The reaction conditions were a reaction section temperature of 175-220°C, a reaction section pressure of 5-25 MPa, and a reaction time of 8.8 seconds. Subsequently, the reaction was quickly terminated by directly cooling the stainless steel piping in a water bath. After that, the pressure in the stainless steel piping was reduced using a back pressure valve, and the reaction product was collected in a product container. The flow rate of the distilled water was set to at least three times the flow rate of the raw material solution.

[0062] The reaction product was subjected to a filtration apparatus equipped with an ultrafiltration membrane with a molecular weight cutoff of 450, and ultrafiltration was carried out while adding ion-exchanged water to the internal retaining liquid as appropriate several times. Finally, the internal retaining liquid, which had been concentrated and purified to a Brix of about 3-4, was recovered and spray-dried with a spray dryer to obtain CS oligosaccharide powder. The CS oligosaccharide obtained thereby mainly contains oligosaccharides with 2 to 12 constituent sugars (Japanese Patent Publication No. 6146733: paragraphs

[0057] ,

[0060] , [Figure 6], [Figure 7]).

[0063] Next, the CS oligosaccharide powder was dissolved in deionized water, adsorbed by anion exchange chromatography, and then eluted by NaCl gradient elution to obtain the disaccharide fraction, the trisaccharide fractions CS3-sugar GG and CS3-sugar UU, the tetrasaccharide fraction, the pentasaccharide fractions CS5-sugar GG and CS5-sugar UU, the hexasaccharide fraction, the octasaccharide fraction, the decasaccharide fraction, and the dodecsaccharide fraction. Each of the separated fractions was desalted by electrodialysis, concentrated in an evaporator, and purified by gel filtration chromatography (*1). After desalting again by electrodialysis, impurities were removed by activated carbon treatment. This was concentrated in an evaporator, filtered through a 0.22 μm membrane filter, and then freeze-dried to obtain CS2-sugar, CS3-sugar GG, CS3-sugar UU, CS4-sugar, CS5-sugar GG, CS5-sugar UU, CS6-sugar, CS8-sugar, and CS10-sugar powders. The purity of each sample was confirmed by HPLC (*2), and the molecular weight was confirmed by liquid chromatography / mass spectrometry (LCMS) (*3).

[0064] *1: The conditions for gel filtration chromatography are as follows. Column capacity: 11.5L Filler: Biogel P-6 Gel (Bio Rad) Mobile phase: 1M NaCL aqueous solution Flow rate: 60mL / min Elution fraction: The elution peak is separated into fractions (1 fraction = 50 mL). Each fraction is analyzed by HPLC, and fractions with a purity of 95% or higher are mixed and recovered.

[0065] *2: The HPLC conditions are as follows. Column: Superdex Increase 30 / 100 Mobile phase: 50 mM ammonium bicarbonate (NH4HCO3) aqueous solution Flow rate: 0.5mL / min Detector: Evaporative light scattering detector (ELSD)

[0066] *3: The conditions for LCMS are as follows. Column: Acclaim SEC300 Mobile phase: 25 mM ammonium bicarbonate (NH4HCO3) aqueous solution Flow rate: 0.2mL / min Detector: Thermo Scientific TSQ Fortis Plus

[0067] Furthermore, the test substances prepared in (1-2) above (CS2 sugar, CS3 sugar GG, CS3 sugar UU, CS4 sugar, CS5 sugar GG, CS5 sugar UU, CS6 sugar, CS8 sugar, and CS10 sugar) are obtained by hydrolysis of CS and therefore have a structure in which the non-reducing end is saturated. In addition, since the CS in this example is made from ray cartilage, it contains 60% or more chondroitin sulfate C.

[0068] (2) Cultured cells The following types of cells were used. Human-derived vascular endothelial cells EA.hy926 (American Type Culture Collection) Normal rat kidney-derived epithelial cell-like cells NRK-52E (National Institute of Biomedical Innovation, Health and Nutrition, JCRB Cell Bank) Human liver cancer-derived hepatocytes HepG2 (Japan CRB Cell Bank, National Institute of Biomedical Innovation, Health and Nutrition) Mouse-derived fibroblast-like cells L929 (Cell Materials Development Laboratory, BioResource Research Center, RIKEN)

[0069] (2) Culture medium The following culture media were used for each cell type. EA.hy926 cells were cultured in DMEM (4.5 g / L glucose) medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Biosera), and further supplemented with antibiotics (Antibiotic Antimycotic Solution (100×)) (GIBCO). NRK-52E cells were cultured in DMEM medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Biosera), and further supplemented with MEM non-essential amino acid 100-fold concentrate (Nacalai Tesque) and antibiotic (Antibiotic Antimycotic Solution (100×)) (GIBCO). HepG2 cells were cultured in DMEM medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Biosera), and further supplemented with antibiotics (Antibiotic Antimycotic Solution (100×)) (GIBCO). L929 cells were cultured in RPMI1640 medium (Nacalai Tesque) supplemented with 10% fetal bovine serum (Biosera), and further supplemented with antibiotics (Antibiotic Antimycotic Solution (100×)) (GIBCO).

[0070] <Example 1> Gene expression analysis using microarrays (1) Cell culture in the presence of the test substance In Example 1, CS2 sugar was used as the test substance. NRK-52E cells were cultured in a 12-well plate (CORNIG) using culture medium at a rate of 0.75 × 10⁶ 5 Seeds were seeded to a concentration of cells / mL, and each well was divided into a test substance group and a control group. After overnight incubation, the test substance group's culture medium was changed to one containing the test substance at a concentration of 1 mg / mL. The control group's culture medium was changed to one without the test substance. After the culture medium change, the cells were incubated for a further 24 or 48 hours.

[0071] (2) DNA microarrays After removing the culture medium from the cultured cells, total RNA was extracted using ISOGENII (Nippon Gene) according to the instructions and dissolved in distilled water. Using this total RNA as a template, reverse transcription and biotin labeling were performed using the GeneChip® WT PLUS Reagent Kit (Thermo Fisher Scientific) to obtain biotin-labeled cDNA. The biotin-labeled cDNA was hybridized to the DNA microarray "Applied Biosystems Clariom S" (Thermo Fisher Scientific), and the fluorescence intensity was measured using the GeneChip Scanner 3000 7G system (Thermo Fisher Scientific). The measurement data was analyzed using the software "Transcriptome Analysis Console" (Thermo Fisher Scientific), and the average value of 2 to 6 wells (n=2 to 6) for each group was calculated. The fluorescence intensity of the group treated with the test substance was expressed as an expression ratio with the fluorescence intensity of the control group set to 1. That is, an expression ratio greater than 1 indicates that the group treated with the test substance has a higher amount of RNA of the gene (higher expression level of the gene) than the control group. Table 2 shows representative NRF2 target genes with an expression ratio greater than 1.3. [Table 2]

[0072] As shown in Table 2, the expression ratios were as follows: NAD(P)H quinone reductase (NQO1) gene increased 49.0 times, aldokereductase family 1 member B1 (AKR1B1) gene increased 1.6 times, cytochrome P450 1A1 (CYP1A1) gene increased 1.7 times, heme oxygenase 1 (HMOX1) gene increased 28.5 times, γ-glutamylcysteine ​​synthase regulatory subunit (GCLM) gene increased 2.5 times, sequestosome-1 (SQSTM1) gene increased 1.3 times, γ-glutamylcysteine ​​synthase catalytic subunit (GCLC) gene increased 1.8 times, and thioredoxin reductase 1 (TXNRD1) gene increased 2.9 times. Specifically, the expression levels of the genes NQO1, AKR1B1, CYP1A1, HMOX1, GCLM, CBR3, SQSTM1, GCLC, and TXNRD1 were higher in the group treated with the test substance than in the control group. This result clearly shows that the expression of numerous NRF2 target genes was enhanced in cells cultured with CS2 sugar. Therefore, it was revealed that CS2 sugar can promote the expression of NRF2 target genes.

[0073] <Example 2> Gene expression analysis by RT-PCR (1) Administration of test substance to cultured cells In this second example, the test substances used included CS2 sugar, CS3 sugar GG, CS3 sugar UU, CS4 sugar, CS5 sugar GG, CS5 sugar UU, CS6 sugar, CS8 sugar, CS10 sugar, and high molecular weight CS (Mw8900), as well as N-acetyl-D-galactosamine 6-sulfate (GalNAc6s) and disaccharides formed by the bonding of D-glucuronic acid and N-acetyl-D-galactosamine that do not contain a sulfate group (2mer0s).

[0074] Various cultured cells (EA.hy926, HepG2, NRK-52E, L929) were cultured in medium at a rate of 0.75 × 10⁶ 5After diluting to cells / mL, 1 mL was seeded into each well of a 12-well plate (CORNIG) and incubated in a CO2 incubator at 5% CO2 and 37°C for 24 hours. Subsequently, the test substance was added to the culture medium to a concentration of 1 mg / mL to create the CS-added group. A control group without the test substance was also established and designated as the no-addition group. Each group consisted of 4 or 6 wells (n=4 or 6). These were incubated for a further 24 hours under the same conditions.

[0075] (2) Analysis of gene expression levels by quantitative PCR After culturing was complete, 0.4 mL of the RNA extraction reagent "ISOGENII" (Nippon Gene) was added to each well to lyse the cells. Total RNA was extracted from the lysed cell solution according to the reagent's instructions. cDNA was synthesized from this total RNA by reverse transcription using the PrimeScript™ RT reagent Kit (Takara Bio) according to the intercalator protocol. 80 μL of ultrapure water was added to 10 μL of the reaction solution to prepare the cDNA solution.

[0076] Quantitative PCR was performed using TB Green® Fast qPCR Mix (Takara Bio) according to the attached instructions. The target genes for expression level detection were the HMOX1 gene, GCLM gene, GCLC gene, the genes encoding glutathione-S-transferase α2 (GSTA2) and NQO1, and the GAPDH gene (internal standard gene) encoding glyceraldehyde 3-phosphate dehydrogenase.

[0077] The total volume of the reaction mixture was 10 μL, and 4 μL of cDNA solution was used as the template. The primers used were those shown in SEQ ID NOs: 1-16 below. PCR reaction and detection were performed using the LightCycler® 96 system (Roche). The reaction conditions were 40 cycles of shuttle PCR at 94°C for 5 seconds and 60°C for 10 seconds. [Primers for human HMOX1 gene amplification; EA.hy926 cells, HepG2 cells] Forward primer: 5'-CCAGGCAGAGAATGCTGAGTTC-3' (SEQ ID NO: 1) Reverse primer: 5'-AAGACTGGGCTCTCCTTGTTGC-3' (SEQ ID NO: 2) 《Primers for amplification of the rat HMOX1 gene; NRK-52E cells》 Forward primer: 5'-AGCATGTCCCAGGATTTGTC-3' (SEQ ID NO: 3) Reverse primer: 5'-TCACCAGCTTAAAGCCTTCC-3' (SEQ ID NO: 4) [Primers for human GCLM gene amplification; EA.hy926 cells, HepG2 cells] Forward primer: 5'-TCTTGCCTCCTGCTGTGTGATG-3' (SEQ ID NO: 5) Reverse primer: 5'-TTGGAAACTTGCTTCAGAAAGCAG-3' (SEQ ID NO: 6) 《Primers for amplifying the rat GCLM gene; NRK-52E cells》 Forward primer: 5'-GCCACCAGATTTGACTGCCTTT-3' (SEQ ID NO: 7) Reverse primer: 5'-CAGGGATGCTTTCTTGAAGAGCTT-3' (SEQ ID NO: 8) 《Primers for rat GCLC gene amplification; NRK-52E cells》 Forward primer: 5'-GCTTTCTCCTACCTGTTTCTTG-3' (SEQ ID NO: 9) Reverse primer: 5'-TGGCAGAGTTCAGTTCCG-3' (SEQ ID NO: 10) Primers for human GSTA2 gene amplification; HepG2 cells Forward primer: 5'-CTGCCCTTTAGTCAACCTGAGG-3' (SEQ ID NO: 11) Reverse primer: 5'-ACAAGGTAGTCTTGTCCGTGGC-3' (SEQ ID NO: 12) Primers for amplifying the mouse NQO1 gene; L929 cells Forward primer: 5'-GCCGAACACAAGAAGCTGGAAG-3' (SEQ ID NO: 13) Reverse primer: 5'-GGCAAATCTCTGCTACGAGCACT-3' (SEQ ID NO: 14) [Primers for human GAPDH gene amplification; EA.hy926 cells, HepG2 cells] Forward primer: 5'-TGATTCTACCCACGGCAAGT-3' (SEQ ID NO: 15) Reverse primer: 5'-AGCATCACCCCATTTGATGT-3' (SEQ ID NO: 16) 《Primers for amplifying the rat GAPDH gene; NRK-52E cells》 Forward primer: 5'-TGATTCTACCCACGGCAAGT-3' (SEQ ID NO: 17) Reverse primer: 5'-AGCATCACCCCATTTGATGT-3' (SEQ ID NO: 18)

[0078] The expression levels of the HMOX1, GCLM, GCLC, GSTA2, and NQO1 genes were calculated as a ratio (relative ratio) to the expression level of the GAPDH gene using the following formula 1. Formula 1: Relative ratio = 2^"Cq value of each gene (HMOX1, GCLM, GCLC, or GSTA2)" / 2^"Cq value of the GAPDH gene"

[0079] For each test section, the mean of the relative ratio (n=4 or 6) was calculated, and a test of the test sections was performed. The statistical test method used was one-way analysis of variance (Dunnett test, with the alternative hypothesis being that the unadded group ≠ each CS-added group). The significance level was set at P<0.05 (*: P<0.05, **: P<0.01). Standard deviations are shown as error bars in the figures.

[0080] The quantitative results of HMOX1 gene expression levels are shown in Figures 4-6. As shown in these figures, the relative ratio of HMOX1 gene expression levels was significantly higher in the CS2 sugar-added group compared to the unadded group in all EA.hy926, HepG2, NRK-52E, and L929 cell groups. Furthermore, the same relative ratio was significantly higher in the CS3 sugar-added group compared to the unadded group, as shown in EA.hy926 cells (Figure 4), HepG2 cells (Figure 5(I)), and L929 cells (Figure 6(II)). Similarly, the same relative ratio was significantly higher in the CS4 sugar-added group compared to the unadded group, as shown in HepG2 cells (Figure 5(I)). In other words, HMOX1 gene expression was enhanced in cells cultured in the presence of CS2 sugar, CS3 sugar-added group (GG), or CS4 sugar.

[0081] Figure 7 shows the quantitative results of GCLM gene expression levels. As shown in the figure, the relative ratio of GCLM gene expression levels was significantly higher in NRK-52E cells when CS2 sugar was used compared to when it was not used. In other words, GCLM gene expression was enhanced in cells cultured in the presence of CS2 sugar.

[0082] Figure 8 shows the quantitative results of GCLC gene expression levels. As shown in the figure, the relative ratio of GCLC gene expression levels was significantly higher in NRK-52E cells when CS2 sugar was used compared to when it was not used. In other words, GCLC gene expression was enhanced in cells cultured in the presence of CS2 sugar.

[0083] Figure 9 shows the quantitative results of GSTA2 gene expression levels. As shown in the figure, the relative ratio of GSTA2 gene expression levels was significantly higher in HepG2 cells when CS2 sugar or CS3 sugar GG was used compared to the control group. In other words, GSTA2 gene expression was enhanced in cells cultured in the presence of CS2 sugar or CS3 sugar GG.

[0084] Figure 10 shows the quantitative results of NQO1 gene expression levels. As shown in the figure, the relative ratio of NQO1 gene expression levels was significantly higher in L929 cells when CS2 sugar was added compared to the control group. In other words, NQO1 gene expression was enhanced in cells cultured in the presence of CS2 sugar.

[0085] As described above, the expression of multiple NRF2 target genes was enhanced in several types of cells cultured with CS2 sugar, CS3 sugar GG, or CS4 sugar. These results demonstrate that CS2 sugar, CS3 sugar GG, and CS4 sugar can promote the expression of NRF2 target genes.

[0086] On the other hand, as shown in Figures 5-10, when CS3 sugar UU, CS5 sugar GG, CS5 sugar UU, CS6 sugar, CS8 sugar, CS10 sugar, GalNAc6s, or 2mer0s were used, no significant increase in the gene expression of HMOX1, GCLM, GCLC, and GSTA2 was observed. From this, it became clear that the structure of a composition that exhibits an NRF2 activating effect is preferably a disaccharide, trisaccharide, or tetrasaccharide composed of N-acetyl-D-galactosamine and uronic acid, has a sulfate group, and if it is a trisaccharide, has two GalNAc residues (GalNAc at both ends).

[0087] <Example 3> Gene expression analysis by reporter assay (1) Plasmid A plasmid containing the GSTA2 promoter region and the firefly luciferase gene (SEQ ID NO: 19) was prepared and used as the reporter plasmid. In addition, a plasmid "pGL4.73hRluc-SV40" (SEQ ID NO: 20) was prepared by ligating the constitutive expression promoter SV40 upstream of the sea urchin luciferase gene, and this was used as the internal standard plasmid. The reporter plasmid and internal standard plasmid were mixed in a ratio of 1.9:0.1 (by weight) and dissolved in Opti-MEM to a concentration of 10 μg / mL (total DNA amount) to prepare the plasmid solution.

[0088] (2) Introduction of plasmids into cells HepG2 cells 1.0 × 10 6 Cells were seeded in 100 mm dishes to a cell / dish ratio and cultured for 1 day. 1 / 43 volume of the gene transfer reagent "X-tremeGENE HP" (Roche) was added to the plasmid solution to create the transfection solution, which was allowed to stand for 15 minutes. 1000 μL of the transfection solution was added to each dish and cultured for 6 hours to introduce the reporter plasmid and internal standard plasmid into HepG2 cells.

[0089] (3) Cell culture in the presence of the test substance HepG2 cells into which plasmids have been introduced were dispersed using trypsin and placed in a 96-well plate in a 2.0 × 10⁶ arrangement. 4 The cells were reseeded to a cell / well concentration, and the medium was replaced with DMEM medium without phenol red. After 1 hour, the medium was replaced with DMEM medium (without phenol red) containing the test substances (CS2 sugar, CS4 sugar, high molecular weight CS (weight-average molecular weight 8900), t-BHQ) at each test concentration. The test concentrations for CS2 sugar were 0.2, 0.4, 1.0, 2.0, and 5.0 mg / mL. The test concentrations for CS4 sugar were 0.4, 1.0, 2.0, 5.0, 10.0, and 25.0 mg / mL. The test concentrations for high molecular weight CS were 0.4, 1.0, 2.0, 5.0, and 10.0 mg / mL. The test concentration for tert-butylhydroquinone (t-BHQ) (positive control) was 20.0 μM. For the negative control, DMEM medium (without phenol red) containing ultrapure water at a final concentration of 10 (w / w)% was used. The cells were then incubated in a CO2 incubator for 48 hours.

[0090] (4) Measurement of luminescence and calculation of expression-promoting activity After culturing, the cells were washed with phosphate-buffered saline and lysed using a dual luciferase assay system (Promega). Subsequently, substrate solution was added, and the luminescence levels of firefly luciferase and sea urchin luciferase were measured using the plate reader "AB-2350 Phelios" (ATTO). Based on the measurement results, the "luciferase expression-promoting activity" was calculated using equations 2 and 3 below. The above procedure was performed using 3 wells per sample (including positive and negative controls), and the average value of the 3 wells was used as the test result. The expression-promoting activity is shown in Figure 11. Equation 2: Luminescence correction value = Luminescence due to firefly luciferase / Luminescence due to sea urchin luciferase Equation 3: Luciferase expression-promoting activity = Corrected luminescence value in sample with test substance / Corrected luminescence value in negative control

[0091] As shown in Figure 11(I), when CS2 sugar was used as the test substance, the expression-promoting activity increased in proportion to the added concentration, reaching 6.2 times that of the negative control at 5.0 mg / mL. Similarly, as shown in Figure 11(II), when CS4 sugar was used as the test substance, the expression-promoting activity also increased in proportion to the added concentration, reaching 10.9 times that of the negative control at 25.0 mg / mL, which was almost equivalent to the value of the positive control t-BHQ. On the other hand, as shown in Figure 11(III), when high molecular weight CS was used as the test substance, no significant increase in expression-promoting activity was observed at any of the added concentrations from 0.4 to 10.0 mg / mL.

[0092] Specifically, in cells cultured in the presence of CS2 or CS4 sugars, the expression of luciferase linked to the promoter region of the GSTA2 gene was enhanced as the concentration of these test substances increased. This result revealed that CS2 and CS4 sugars can promote the expression of NRF2 target genes.

[0093] <Example 4> Evaluation of skin permeability Using "EPI-MODEL12" (Japan Tissue Engineering Co., Ltd.), a human three-dimensional cultured epidermis formed by culturing and layering normal human skin cells (hereinafter referred to as the epidermal model), the skin permeability of test substances (CS2 sugar, CS4 sugar, high molecular weight CS (Mw70000)) was evaluated according to the attached instruction manual. Specifically, 1 mL of the culture medium provided with the product was dispensed into each well of a 12-well plate, and the EPI-MODEL12, still in its cup, was placed on top of the medium and incubated at 37°C and 5% CO2 for 1 hour to perform pre-culture. Subsequently, 1 mL of degassed HBSS (Hank equilibrium salt solution) was dispensed into each well of another 12-well plate as the receiver solution. The pre-cultured EPI-MODEL12, still in its cup, was placed on top of the solution and incubated at 32°C and 5% CO2 for 30 minutes. Next, HBSS containing 1 mg / mL of each test substance was added to the cup of EPI-MODEL12, and the mixture was incubated at 32°C and 5% CO2 for 24 hours with stirring at 20 rpm. After that, 1 mL of the receiver solution was taken, and the content of the test substance (the amount of test substance that permeated the epidermal model) was quantified by HPLC analysis, and this was defined as the permeation amount of the test substance.

[0094] In HPLC analysis, CS2 and CS4 sugars in the receiver solution were labeled with 2-AB (2-aminobenzamide) according to a standard method, and then their fluorescence intensity was measured by HPLC with a fluorescence detector. High molecular weight CS in the receiver solution was converted to CS2 sugar using CS-degrading enzyme, and then its fluorescence intensity was measured by HPLC in the same manner. A calibration curve was created based on the fluorescence intensity of samples containing known amounts of CS2 or CS4 sugars, and the concentration of each test substance (persistence of test substance, ng / mL / 24hr) was calculated by applying the fluorescence intensity measurements of the receiver solution. The results are shown in Figure 12.

[0095] As shown in Figure 12, the amount of the test substance permeated was significantly greater for both CS4 sugars and CS2 sugars compared to high molecular weight CS. This result revealed that CS2-CS4 sugars have high skin permeability and can penetrate into the body even when administered transdermally.

Claims

1. An activator of NRF2 (Nuclear factor erythroid 2-related factor 2), comprising one or more chondroitin sulfate oligosaccharides selected from (a) to (c) below as the active ingredient; (a) Disaccharide having a chondroitin sulfate structure, (b) A trisaccharide having two N-acetyl-D-galactosamine residues and a chondroitin sulfate structure, (c) A tetrasaccharide having a chondroitin sulfate structure.

2. The agent according to claim 1, which is an expression promoter for NRF2 target genes.

3. The agent according to claim 2, wherein the NRF2 target gene is a gene having an antioxidant response element (5'-TGAC / GNNNGC-3') in its promoter region or a gene encoding thioredoxin reductase 1.

4. The agent according to claim 1, which is an antioxidant.

5. The agent according to claim 1, which is an anti-inflammatory agent.

6. A method for activating NRF2 (Nuclear factor erythroid 2-related factor 2), comprising the step of activating NRF2 in a human or animal by administering one or more chondroitin sulfate oligosaccharides selected from (a) to (c) below to the human or animal; (a) Disaccharide having a chondroitin sulfate structure, (b) A trisaccharide having two N-acetyl-D-galactosamine residues and a chondroitin sulfate structure, (c) A tetrasaccharide having a chondroitin sulfate structure.

7. Use of one or more chondroitin sulfate oligosaccharides selected from (a) to (c) below to produce an activator of NRF2 (Nuclear factor erythroid 2-related factor 2); (a) Disaccharide having a chondroitin sulfate structure, (b) A trisaccharide having two N-acetyl-D-galactosamine residues and a chondroitin sulfate structure, (c) A tetrasaccharide having a chondroitin sulfate structure.

Images