KR20180046021A - Composition including neoagarooligosaccharide for preventing, improving or treating inflammatory disease - Google Patents

Abstract

The present invention relates to a composition comprising a neoagarooligosaccharide mixture as an effective component for a novel use. The effective component of the composition according to the present invention, a neoagarooligosaccharide mixture and the like decrease the expression of various inflammatory cytokines such as TNF-α and IL-6 to effectively suppress inflammatory responses. In addition, the effective component of the composition according to the present invention, a neoagarooligosaccharide mixture and the like suppress the differentiation of T cells into Th1 cells, Th2 cells, and Th17 cells and increase the differentiation of T cells into Treg cells, thereby effectively regulating immune reactions. Accordingly, the composition according to the present invention may be used as a drug or functional food for preventing, alleviating or treating various inflammatory diseases.

Description

[0001] The present invention relates to a composition for preventing, ameliorating or treating an inflammatory disease including neoagarol oligosaccharide,

The present invention relates to a composition for preventing, improving or treating an inflammatory disease, wherein the composition for preventing, improving or treating an inflammatory disease of the present invention is characterized by effectively suppressing the expression of an inflammatory cytokine or modulating an inflammatory response to an appropriate level .

Inflammation is a normal and protective in vivo defense manifestation that is localized to physical trauma, harmful chemicals, microbial infections, or tissue damage caused by stimuli in vivo metabolites. These inflammations are triggered by various chemical mediators produced from damaged tissues and migrating cells, and these chemical mediators are known to vary according to the type of inflammation process. In normal cases, the organism neutralizes or eliminates the cause of inflammation through the inflammation reaction, regenerates the upper tissue and regenerates the normal structure and function, but if not, the disease state such as chronic inflammation also proceeds. In addition, when the inflammation is improperly induced by an autoimmune reaction such as a harmless substance such as pollen, asthma or rheumatoid arthritis, the defense reaction itself rather damages the tissue, and therefore, a prophylactic or therapeutic agent for inflammatory diseases is required. Although most inflammatory diseases can be observed in almost all clinical diseases, some of these inflammatory diseases are bacterial diseases that can be cured by administration of antibiotics, but most of them are due to tissue damage due to autoimmune reaction, so there is no specific treatment It is known as an incurable disease. The most common agents for the prophylaxis or treatment of inflammatory diseases are largely classified into those for the prevention or treatment of steroidal and non-steroidal inflammatory diseases, and most of them are synthetic agents which are effective ingredients for the prophylactic or therapeutic agents for inflammatory diseases The compound has many disadvantages in addition to main action and often accompanies various side effects. That is, drugs suitable for the prevention or treatment of non-steroidal inflammatory diseases (NSAIDs) suitable for the treatment of inflammatory diseases are used to alleviate inflammation, steroid agents are used if inflammation is severe or NSAIDs are not effective, Or surgery. In this case, the NSAIDs used in the present invention mainly inhibit cyclooxygenase (COX) and inhibit the production of prostaglandins involved in the inflammatory reaction, thereby exhibiting an action for preventing or treating inflammatory diseases. However, Kidney dysfunction, and the like, resulting in difficulty in long-term use. In order to solve the side effects of such synthetic compounds, development and research of materials having inflammation-regulating activity derived from natural materials have been actively conducted.

Agar is a representative algae-derived polysaccharide widely used for food additives, medicines, cosmetics, livestock feed and industrial raw materials for a long time. In Korea, agar is a relatively abundant fishery resource with annual production of about 2,000 ~ 5,000 tons Lt; / RTI > However, in actual use, only a part of the total production amount is processed as a simple raw material and used as cheap raw materials, and the rest of the total amount is neglected. Therefore, there is a great demand for research on the development of new applications of agar and the improvement of added value.

Agar is composed mostly of polysaccharides except for small amounts of protein, ash and fat. The polysaccharides constituting the agar include agarose, a neutral polysaccharide, and agaropectin, an acidic polysaccharide. Agarose is an agarobiose in which D-galactose and 3,6-anhydro-L-galactose are combined in a β-1,4 form, , And agarobiose has a straight-chain structure in which α-1,3 bonds are repeatedly connected to each other, and gelation power is strong. On the other hand, agaropectin, like agarose, has agarobiose as a unit, but contains an acidic group such as a sulfate group and the like and has a weak gelling power.

Among them, agarose is degraded into neoagarobiose via neoagarotetraose by β-agarase acting on β-1,4 bond, and then α-1 Galactose and 3,6-anhydro-L-galactose by α-agarase, which acts on the β-3 linkage. In addition, the agarose is degraded to a neoagarobiose by dilute acid or alpha-agarase. In general, neoagarooligosaccharides are obtained by hydrolyzing agar or agarose with beta-agarase, such as neoagarobiose, neoagarotetraose, neoagarohexaose ( neoagarohexaose, neoagarooctaose, and the like. In addition, agarooligosaccharides can be obtained by hydrolyzing agar or agarose with a dilute acid or alpha-agarase, such as Aeoagarobiose, Agarotetraose, Agarohexaose, , Agarooctaose, and the like. The neoagarooligosaccharide has 3,6-anhydro-L-galactose as a non-reducing end, while the agarooligosaccharide has 3-anhydro-L-galactose as a non- , And these structural differences cause different physiological activities.

On the other hand, the actinomycetes Streptomyces series collar (Streptomyces coelicolor A3 (2) is known to produce agarase degrading agar in the form of extracellular (secreted out of cells) (Stanier et al., 1942, J. Bacteriol .; Hodgson and Chater, 1981, J. Gen. Microbiol.), The agarase is encoded by the DagA gene. The DagA gene is a beta-agarase gene whose function is the only known in actinomycetes, and thus plays an important role in the study of agarase production through actinomycetes. In particular, Streptomyces sericola is the most widely used strain in the molecular biology of actinomycetes. In 2002, the sequence of chromosomal DNA was analyzed by the Sanger center in England (Bantley et al., 2002, Nature).

Regarding the preparation or use of neo-agarooligosaccharides, Korea Patent No. 10-0794593 discloses a method of producing Thalassomonas sp. SL-5 (Thalassomonas sp. SL-5) having agar resolving ability, KCCM 10790P, Discloses a method for producing at least one neoagarooligosaccharide selected from the group consisting of neoagarobiose, neoagarotetraose and neoagarohexaose using agarase. Korean Patent Publication No. 10-1072503 also discloses that a strain of the genus Escherichia coli SL-12 KCCM 10945P (Glaciecola sp. SL-12 KCCM 10945P) having an agar-resolving ability and a β-agarase (β- agarase is used to produce at least one neoagarooligosaccharide selected from the group consisting of neoagarobiose, neoagarotetraose and neoagarohexaose. Korean Patent No. 10-1303839 discloses beta-agarase isolated from a strain of Pseudoalteromonas sp., And a method of using neoagarotetraose and neoagarohexaose A method of producing at least one neoagarooligosaccharide selected from the group consisting of Korean Patent Registration No. 10-1295659 discloses beta-agarase isolated from Saccharophagus sp. Strain and neoagarotetraose and neoagarohexaose using the same. ) In the presence of a reducing agent. Korean Patent Registration No. 10-1212106 discloses that beta-agarase isolated from a Saccharophagus sp. Strain is selected from the group consisting of agar, neoagarotetraose, and neoagarohexose. A method of producing neoagarobiose by reacting with a substrate or more is disclosed. Korean Patent Registration No. 10-1206006 also discloses that the flameoovirga sp. Mbrc-1 KCCM 11151P strain having the agarase-degrading activity, mbrc-1 strain mbrc-1, and the beta-agarase (β -agarase is reacted with agar to produce at least one neoagarooligosaccharide selected from the group consisting of neoagarobiose, neoagarotetraose and neoagarohexaose. Korean Patent Registration No. 10-1302655 discloses a neoagarotetraose which is characterized by reacting agarase derived from Streptomyces coelicolor with agarose or agar. (neoagarotetraose) and neoagarohexaose are disclosed. Korean Patent Registration No. 10-1190078 discloses a nucleotide sequence of SEQ ID NO: 7 comprising a promoter of a trypsin gene (sprT) derived from Streptomyces griseus and a coding region of a signal peptide. A DNA fragment displayed; And a DNA fragment represented by the nucleotide sequence of SEQ ID NO: 2 from which a signal peptide code has been removed from a Streptomyces coelicolor-derived beta-agarase gene (dagA), wherein the DNA fragment is capable of transforming a prokaryote A beta-agarase recombinant expression vector and a method for producing beta-agarase using the recombinant expression vector. Korean Patent Laid-Open Publication No. 10-2014-0060045 also discloses an enzymatic production method of neoagarobiose or neoagarotetraose using a novel beta-agarase-producing gene. Korean Patent Laid-Open Publication No. 10-2009-0044987 discloses a skin whitening composition comprising neoagarotetraose as an active ingredient. As described above, the prior art is mainly concerned with a novel strain comprising a beta-agarase as a gene, a method of producing a beta-agarase through a novel strain or a recombinant strain, or a method of producing beta-agarase from agar, And a variety of physiological functions of neoagarooligosaccharides prepared by using neoagarooligosaccharides, in particular beta-agarase, have hardly been studied.

The present invention has been made under the conventional technical background, and it is an object of the present invention to provide a novel pharmaceutical use of neoagarooligosaccharides.

In one aspect, the present invention provides a composition comprising a neoagarooligosaccharide mixture as an active ingredient, wherein the neoagarooligosaccharide mixture is selected from the group consisting of neoagarobiose, neoagarotetraose, And neoagarohexaose. The present invention also provides a composition for preventing, ameliorating or treating an inflammatory disease comprising neoagarohexaose.

In order to solve the above-mentioned object, another example of the present invention relates to a method for producing a microorganism having a substrate selected from agar or agarose and a substrate selected from the group consisting of Streptomyces The present invention relates to a composition comprising an enzyme reaction product with DagA derived from coelicolor or a purified product thereof as an active ingredient, wherein the enzyme reaction product or a purified product thereof is a neoagarobiose, neoagarotetraose ) And neoagarohexaose, which is characterized in that it comprises a compound of formula (I) and a compound of formula (I).

Neoagarooligosaccharide mixture, which is an active ingredient of the composition according to the present invention, can effectively suppress the inflammatory reaction by decreasing the expression of various inflammatory cytokines such as TNF-α, IL-6 and the like. In addition, the neoagarooligosaccharide mixture, which is an active ingredient of the composition according to the present invention, inhibits the differentiation of T cells into Th1 cells, Th2 cells, and Th17 cells and effectively regulates the immune response by increasing the differentiation of T cells into Treg cells . Accordingly, the composition according to the present invention can be used as a medicine or a functional food for preventing, improving or treating various inflammatory diseases.

Figure 1 is a cleavage map of the pUWL201pw vector used for cloning the DagA gene in the present invention.
Fig. 2 is a cleavage map of a recombinant vector into which the DagA gene is introduced into the pUWL201pw vector.
3 is a graph showing the results of HPLC-ELSD analysis of the partially purified DagA enzyme reaction product obtained in Example 3 of the present invention.
Figure 4 shows the effect of neoagaroligosaccharide mixture (NAO) on the expression of IL-6, an inflammatory cytokine, in synovial cells of patients with arthritis.

Hereinafter, terms used in the present invention will be described.

As used herein, the terms "pharmaceutically acceptable" and "pharmaceutically acceptable" are intended to mean not significantly irritating the organism and not interfering with the biological activity and properties of the administered active substance.

As used herein, the term "prophylactic " means any act that inhibits the symptoms of a particular disease or delays the progress of the disease upon administration of the composition of the present invention.

The term "improvement" as used in the present invention means all actions that at least reduce the degree of symptom associated with the condition being treated.

The term "treatment" as used herein refers to any action that improves or alleviates the symptoms of a particular disease upon administration of the composition of the present invention.

The term "administering" as used herein is meant to provide any desired composition of the invention to an individual by any suitable method. The term " individual " means any animal such as a human, a monkey, a dog, a goat, a pig, or a mouse having a disease in which symptoms of a specific disease can be improved by administering the composition of the present invention.

The term " pharmaceutically effective amount " as used herein means an amount sufficient to treat a disease at a reasonable benefit or risk rate applicable to medical treatment, including the type of disease, severity, activity of the drug, The time of administration, the route and rate of excretion of the drug, the duration of the treatment, factors including drugs used simultaneously and other factors well known in the medical arts.

Hereinafter, the present invention will be described in detail.

The composition for preventing, improving or treating an inflammatory disease according to one embodiment of the present invention comprises a neoagarooligosaccharide mixture as an active ingredient. The neo-agarooligosaccharide mixture includes neoagarobiose, neoagarotetraose and neoagarohexaose. The neoagaroligosaccharide mixture may contain 1 to 10% by weight of neoagarobiose, 55 to 90% by weight of neoagarotetraose based on the total weight of the neoagarooligosaccharide mixture, 75 to 75% by weight and neoagarohexaose to 20 to 40% by weight, preferably 2 to 4% by weight of neoagarobiose, 65 to 70% by weight of neoagarotetraose, And 25 to 30% by weight of neoagarohexaose. The neoagarooligosaccharide mixture may also be an enzyme reaction product of a substrate selected from agar or agarose with DagA, a beta-agarase derived from Streptomyces coelicolor , or a purified product thereof . A method of obtaining a neoagarooligosaccharide mixture through an enzyme reaction between a substrate and DagA is described in detail later.

The composition for preventing, improving or treating an inflammatory disease according to another embodiment of the present invention comprises an enzyme of a substrate selected from Agar or Agarose and DagA which is a beta-agarase derived from Streptomyces coelicolor A reaction product or a purified product thereof as an active ingredient. The enzyme reaction product or the purified product includes neoagarobiose, neoagarotetraose and neoagarohexaose. In addition, the enzyme reaction product or the purified product may contain 1 to 10% by weight of neoagarobiose, 55 to 75% by weight of neoagarotetraose based on the total weight of neoagarooligosaccharide, It is preferable that it contains 20 to 40% by weight of neoagarohexaose, 2 to 4% by weight of neoagarobiose, 65 to 70% by weight of neoagarotetraose, and neoagarohexaose ) By weight, more preferably 25 to 30% by weight. In addition, the enzyme reaction is preferably carried out at a temperature of 35 to 45 ° C and a pH of 6 to 8. In addition, the enzyme reaction may be carried out by adding DagA to a 0.5 to 5% (w / v) agar or agarose solution at a concentration of 2 to 250 unit / ml, preferably 10 to 150 unit / ml, more preferably 10 To 100 units / ml. At this time, 3.9 ml of a 50 mM PBS solution (pH 7) in which agarose was dissolved at a concentration of 0.2% (w / v) was reacted at 40 ° C for 5 minutes (reaction solution: 4 ml) (Prepared by dissolving 6.5 g of dinitrosalicylic acid, 325 ml of 2M NaOH and 45 ml of glycerol in 1 liter of distilled water), boiled for 10 minutes, cooled and measured for absorbance at 540 nm. Absorbance at 540 nm 0.001. ≪ / RTI >

Hereinafter, a method for obtaining an active ingredient of the composition according to the present invention through an enzyme reaction between a substrate and DagA will be described. In the present invention, DagA derived from Streptomyces coelicolor means a protein having an amino acid sequence of between 31 and 309 of SEQ ID NO: 2, and is a protein having an amino acid sequence selected from the group consisting of Streptomyces coelicolor , Such as by incorporating a labeling amino acid or by altering the amino acid sequence for heterologous expression in order to make the purification more favorable, within the scope of not altering to completely different or losing agarase activity, Proteins. When DagA is originally translated from the beta-agarase gene of Streptomyces sericola, it has 309 amino acids of SEQ ID NO: 2 and is produced with a molecular weight of about 35 kDa, and the N-terminal 30 amino acid signal peptide is cleaved Lt; / RTI > (approximately 32 kDa). DagA of Streptomyces sericola The gene may be represented by the nucleotide sequence of SEQ ID NO: 1. SEQ ID NO: 1 is the nucleotide sequence of the gene present in the genome of Streptomyces sericola A3 (2) and is named "SCO3471" on NCBI (US National Bioinformation Center) database. As confirmed by in vitro experiments, the transcription of the DagA gene is regulated by four or five other promoters that are recognized by at least three other holoenzymes of the RNA polymerase. DagA Transcriptional phase analysis of the gene revealed that the transcription of DagA was initiated at the 32nd, 77th, 125th and 220th bases of the coding sequence.

To produce DagA used in the present invention through culturing of the original DagA producing strain of Streptomyces series of color, but two kinds of strains of Streptomyces Libby residence in order to increase the production efficiency (Streptomyces RTI ID = 0.0 > lividans ). < / RTI > DagA can be produced by inserting the DagA gene into a stactobacterium vector to prepare a recombinant vector, transforming the streptomyces ribidus with a recombinant vector, and culturing the transformant. In this case, the recombinant vector is preferably constructed such that the transcription of the DagA gene can be regulated by the actinomycete-derived promoter. Since there are various promoters such as sgtR promoter (sgtRp), ermE promoter (ermEp), and tipA promoter (tipAp), actinomycetes -derived promoters can be selected and used when preparing recombinant vectors. Several kinds of vectors constructed so that transcription can be regulated by these promoters have been developed. When SCO3471 is cloned into this vector, a recombinant vector having a structure in which transcription is regulated by actinomycetes-derived promoter can be produced. The transformant can be prepared by transforming the host strain with a recombinant vector in which the DagA gene is cloned. Since a variety of transformation methods are available depending on the host strain, an appropriate method can be selected and used. For example, when a Streptomyces ribidus is used as a host strain, a transformation method based on PEG (polyethylene glycol) can be used. A DagA producing strain such as a transformant can be produced in a liquid medium to produce DagA, and a culture solution can be obtained, and a high purity DagA can be produced using a conventional protein purification method such as an ultrafiltration method. At this time, if agar or agarose is included in the liquid medium, DagA can be produced more efficiently.

When the DagA thus produced is added to a substrate solution such as agar or agarose and reacted at a specific temperature and pH, an enzyme reaction product used as an active ingredient in the composition according to the present invention can be obtained. Further, the enzyme reaction product can be partially purified by ultrafiltration or passed through gel filtration chromatography to obtain a purified product of the enzyme reaction product.

The neoagarooligosaccharide mixture, which is an active ingredient of the composition according to the present invention, reduces the expression of various inflammatory cytokines such as TNF- ?, IL-6 and the like, effectively inhibits the inflammatory reaction and inhibits the Th1, Th2, Can be used as a material for preventing, ameliorating, or treating various inflammatory diseases because it can suppress the differentiation into Th17 cells and increase the differentiation of T cells into Treg cells, thereby effectively controlling the immune response. Inflammatory diseases in which the composition according to the present invention can be applied include inflammatory diseases such as inflammatory skin diseases, allergic diseases, inflammatory bowel diseases such as Crohn's desease and ulcerative colitis, peritonitis, osteomyelitis, meningitis, meningitis, encephalitis, Chronic bronchitis, acute bronchiolitis, chronic bronchitis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, lager syndrome, psoriatic arthropathies, intestinal disease, spondyloarthropathies, acute bronchitis, chronic bronchitis, Osteoarthritis, rheumatoid arthritis, arthritis associated with vasculitis, arthritis associated with vasculitis, arthritis associated with arthritis, arthritis, arthritis, arthritis, arthritis, arthritis, arthritis, , Nodular polyarteritis, hypersensitivity vasculitis, Lou Gehrig's granulomatosis, rheumatoid multiple myalgia, Arthritic arthritis, arthritic arthritis, calcium calcination arthropathy, caustic gout, non-articular rheumatism, bursitis, hay fever, tornaditis (tennis elbow), charco and joint, hemarthrosic, Hyperglycemia, hypogammaglobulinemia, familial mediterranean fever, Behrth's disease, systemic inflammation, hyperparathyroidism, hyperlipoproteinemia, hyperparathyroidism, hypertrophic osteoarthritis, hypertrophic osteoarthritis, surukilosis, surcoilosis, hemochromatosis, Acute respiratory distress syndrome, chronic obstructive pulmonary disease, acute lung injury, and bronchopulmonary dysplasia. The present invention also relates to the use of a compound of formula And broncho-pulmonary dysplasia. ≪ RTI ID = 0.0 > The allergic disease is any one selected from the group consisting of allergic skin diseases, atopic dermatitis, bronchial asthma, allergic rhinitis, allergic conjunctivitis, allergic otitis media, urticaria, asthma and anaphylactic shock . The composition according to the present invention may be formulated into a pharmaceutical composition, a food additive, a food composition (particularly a functional food) or a feed additive depending on the intended use or aspect, and the content of the active ingredient in the composition may also be a specific form, And may be adjusted in various ranges depending on purposes or aspects.

When the composition for preventing, improving or treating an inflammatory disease according to the present invention is embodied in a pharmaceutical composition, the content of the active ingredient in the pharmaceutical composition is not limited to a great extent, and may be, for example, 0.1 to 99% by weight, Preferably 0.5 to 50% by weight, more preferably 1 to 30% by weight. In addition, the pharmaceutical composition of the present invention may further comprise, in addition to the active ingredient, an additive such as a pharmaceutically acceptable carrier, excipient or diluent. Examples of carriers, excipients and diluents that can be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition of the present invention may contain at least one known active ingredient having neoagarooligosaccharide besides the effect of suppressing the inflammatory reaction or modulating the immune response. The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration by a conventional method, and can be formulated into a pharmaceutical composition such as a filler, an extender, a binder, a wetting agent, a disintegrant, Diluents or excipients. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, Sucrose, Lactose, Gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate talc may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used. Further, it can be suitably formulated according to each disease or ingredient, using appropriate methods in the art or by the method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA. The pharmaceutical composition of the present invention may be administered orally or parenterally to a mammal including a human according to a desired method. Examples of the parenteral administration method include external dermal application, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, Intravenous injection or intra-thoracic injection. The dosage of the pharmaceutical composition of the present invention is not limited as long as it is a pharmacologically effective amount and is not limited as long as it depends on the body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, Varies. The typical daily dose of the pharmaceutical composition of the present invention is not particularly limited, and is preferably 0.1 to 1000 mg / kg, more preferably 1 to 500 mg / kg, based on, for example, And may be administered once or several times a day.

When the composition for preventing, improving or treating an inflammatory disease according to the present invention is embodied in a food composition, the content of the active ingredient in the food composition is not limited to a great extent and is preferably 0.01 to 50% by weight, May be 0.1 to 25% by weight, more preferably 0.5 to 10% by weight. The food composition of the present invention may be in the form of a pill, a powder, a granule, an infusion, a tablet, a capsule, or a liquid preparation. Examples of the food include meat, sausage, bread, chocolate, candy, snack, Other noodles, gums, dairy products including ice cream, various soups, drinks, tea, functional water, drinks, alcoholic beverages and vitamin complexes. The food composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient in addition to the active ingredient. In addition, the food composition of the present invention can be used as a food composition containing various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, , A carbonating agent used in carbonated drinks, and the like. In addition, the food composition of the present invention may contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Natural flavors such as tau Martin and stevia extract, and synthetic flavors such as saccharin and aspartame may be used as the flavor.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

Example 1: Production of DagA enzyme

Polymerase chain reaction (PCR) was performed using the chromosomal DNA of Streptomyces sericola A3 (2) as a template and using the following primers and Ex-Taq (TAKARA) polymerase. As a result, amplified DagA A fragment of the gene fragment (a signal peptide of DagA and a fragment of 947 bp in length encoded with the completed peptide, corresponding to a part of the sequence of the primer connected to both ends of the nucleotide sequence of SEQ ID NO: 1) was obtained.

Primers used for amplification of the DagA gene fragment

Asm-F (Forward Primer): 5'-GACATATGGTGGTCAACCGACGTGATC-3 '(NdeI) (SEQ ID NO: 3)

Asm-R (Reverse Primer): 5'-GGTGGATCCCTACACGGCCTGATACG-3 '(BamHI) (SEQ ID NO: 4)

Then, the DagA gene was cloned into the pUWL201pw vector of FIG. 1 using the restriction enzyme site (NdeI / BamHI) of the amplified DagA gene fragment to prepare the recombinant vector of FIG. In the recombinant vector of Fig. 2, transcription of the DagA gene is regulated by the ermE promoter. Then, the recombinant strain producing the recombinant strain by transforming a Streptomyces Libby residence (Streptomyces lividans) TK24 strains, and a recombinant vector of Figure 2 was used for the production of DagA.

Specifically, the recombinant strain was inoculated into an R2YE liquid medium containing agar of 0.3% (w / v) and pre-cultured for about 60 hours at 28 ° C and 120 rpm. After the pre-culture, the cultivation was carried out for about 60 hours. The culture solution obtained through the present culture was centrifuged to remove cells, and the supernatant was separated and purified by filtration through an ultrafiltration membrane (5 kDa cut-off membrane). The enzyme concentrate which did not pass through the filtration membrane was freeze-dried to solidify it, and the solidified DagA enzyme was stored and used in the subsequent experiments.

Example 2: Measurement of agarase activity of DagA enzyme

The agarase activity of the DagA enzyme obtained in Example 1 was measured by the method of reducing equivalence (DNS method). 100 μl of the DagA enzyme solution prepared by dissolving the DagA enzyme obtained in Example 1 in PBS (phosphate buffered saline) at a concentration of 10 mg / ml and 50 ml of 50 mM PBS solution in which the agarose was dissolved at a concentration of 0.2% (w / v) (manufactured by dissolving 6.5 g of dinitrosalicylic acid, 325 ml of 2M NaOH and 45 ml of glycerol in 1 l of distilled water) was added to the reaction mixture, followed by addition of 10 ml of 10 The mixture was boiled for minutes, then allowed to cool, and the absorbance was measured at 540 nm. 1 U (Unit) of the enzyme was defined as an activity having an absorbance at 540 nm of 0.001.

Example 3: Preparation of DagA Enzyme Reaction Products

1 L of a 20 mM Tris-HCl solution in which agar was dissolved at a concentration of 1.5% (w / v) was prepared, heated at 100 캜 for about 10 minutes, and then cooled to 40 캜. To this was added a 125 U / Was treated to 20,000 U based on the amount of the whole sample and reacted for about 24 hours. Then, the enzyme reaction product was centrifuged to remove the undegraded agar, the supernatant was recovered, and the enzyme reaction product was confirmed by TLC (thin layer chromatography). The recovered supernatant was partially purified by filtration through an ultrafiltration membrane (5KDa cut-off membrane). The DagA enzyme reaction product passed through the filter membrane was lyophilized and solidified. The solidified DagA enzyme reaction product was stored and used in the subsequent experiments. The above procedure was repeated to obtain partially purified enzyme reaction products corresponding to 4 batches.

Example  4 : HPLC - ELSD  Through analysis DagA  Of the enzyme reaction product Neoagarooligosaccharide  Confirming composition

The neo-agarooligosaccharide composition of the enzyme reaction product obtained in Example 3 was confirmed by HPLC-ELSD analysis. HPLC-ELSD analysis conditions are as follows.

* Column: NH ₂ P-50 4E multimode column (250 mm x 4.6 mm)

* Mobile phase: a mixed solution of acetonitrile and water (acetonitrile: water in a volume ratio of 65:35

FIG. 3 is a graph showing the results of HPLC-ELSD analysis of the partially purified DagA enzyme reaction product obtained in Example 3. FIG. The four peak graphs shown in Figure 3 correspond to the partially purified enzyme reaction products obtained in each of the 4 batches. In addition, the area (%) in the table in Fig. 3 represents the area of the peak corresponding to each neoagaroligosaccharide as a percentage of the total peak area and can be interpreted in the same sense as% by weight in the art. As shown in FIG. 3, the DagA enzymatic reaction products are mainly composed of neoagarobiose (DP2), neoagarotetraose (DP4), and neoagarohexaose neoagarohexaose, hereinafter referred to as " DP6 "). The content of neoagarooligosaccharide in the DagA enzyme reaction product was found to be about 65 5% by weight based on the total weight of solids (including components not detected in the HPLC-ELSD assay) The above content can be selected in various ranges. For example, the content of neoagarooligosaccharide in the DagA enzyme reaction product may be 65 ± 20% by weight based on the total weight of the solids. The content of DP2, DP4 and DP6 in the DagA enzyme reaction product is 2 to 4% by weight, 65 to 70% by weight and 25 to 30% by weight, respectively, based on the total weight of the neoagarooligosaccharide containing DP2, DP4 and DP6 %. However, depending on the enzyme reaction conditions, the above content can be selected in various ranges. For example, the content of DP2, DP4, and DP6 in the DagA enzyme reaction product is 1 to 10 wt%, 55 to 75 wt%, and 20 to 20 wt%, respectively, based on the total weight of the neoagarooligosaccharide containing DP2, DP4, 40% by weight.

Example 5 Purification of DagA Enzyme Reaction Products Using Gel Filtration Chromatography

The partially purified enzyme reaction product obtained in Example 3 was purified by gel permeation chromatography (GPC; BioGel P-2 gel, Biorad, Cat. No. 150-4115) And purified and separated into neoagarohexaose (DP6), neoagarotetraose (DP4) and neoagarobiose (DP2). The purified products were then lyophilized to solidify, and the solidified DagA enzyme reaction purified product was stored and used in subsequent experiments. The purity of the purified products, DP2, DP4 and DP6, was found to be about 85% (w / w) by TLC and HPLC.

Example  6: Neoagarooligosaccharide  Test for improving arthritis efficacy of a mixture

(1) Experimental method

During arthroplasty, articular tissue was isolated and treated with collagenase to isolate synovial cells. Synovial cells from patients with isolated arthritis were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium containing 10% FBS (Fetal Bovine Serum) at a concentration of 10%. In this experiment, The cells were inoculated in a 6-well plate at a dose of 3 × 10 ⁵ per well and stimulated with IL-17, an inflammatory cytokine. To examine the effect of the neoagarooligosaccharide mixture, (Hereinafter, referred to as 'NAO') obtained in Example 1. The expression level of IL-6, an inflammatory cytokine, was analyzed by real-time PCR. 6 are shown in Table 1 below.

Gene type Forward primer sequence Reverse primer sequence IL-6 CCC ACA CAG ACA GCC ACTCA GGT TGT TTT CTG CCA CTG CC β-actin GAA ATC GTG CGT GAC ATC AAA G TGT AGT TTC ATG GAT GCC ACA

In real-time PCR analysis, nucleic acid was labeled using SYBR Green I (Roche Diagnostic, Mannheim, Germany) and fluorescence intensity was measured using a Light Cycler instrument (Roche Diagnostic).

(2) Experimental results

Figure 4 shows the effect of neoagaroligosaccharide mixture (NAO) on the expression of IL-6, an inflammatory cytokine, in synovial cells of patients with arthritis. As shown in FIG. 4, treatment of IL-17, which is an inflammatory cytokine, in synovial cells of patients with arthritis increases IL-6 activity, which is an inflammatory cytokine, but neoagarol oligosaccharide mixture (partially purified DagA enzymatic reaction product, NAO) decreased IL-6 expression in a concentration-dependent manner.

Example  7: Neoagarooligosaccharide  Test for confirming the effectiveness of the mixture for improving colitis

(1) Experimental method

Six-week-old male mice (C57BL / 6, 18-22 g) were purchased from Orient Bio. All mice were maintained under controlled environmental conditions with a humidity of 50 ± 10% and a temperature of 20 to 22 ° C. The lights were turned on for 12 hours and then turned off for 12 hours. Feeds were prepared using a standard laboratory feed (Samyang, Korea) and the drinking water was freely consumed. In all the experiments, 6 dogs were used. Acute colitis was induced with 2,4,6-trinitrobenzenesulfonic acid (TNBS) in one of the experimental animals as the normal group and the other animals in the experimental group. Specifically, an experimental animal was lightly anesthetized with ether, and then a solution of 2.5 g of NBS (2,4,6-Trinitrobenzene sulfonic acid) solution in 50% ethanol was injected into an intestine 0.1 ml each was injected vertically and kept for 30 seconds to induce inflammation. On the other hand, 0.1 ml of physiological saline was orally administered to the normal group. After that, the drug sample is dissolved in physiological saline for 3 days once a day from the next day. Oral administration is carried out at a predetermined dose, and the animal is choked by carbon dioxide the next day after the end of the sample administration, and the colon from the cecum to the anus Respectively. At this time, partially purified DagA enzyme reaction product (hereinafter referred to as "NAO") obtained in Example 3 and mesalazine as a commercial colitis therapeutic were used as drug samples.

(2) Experimental results

100 ㎎ of the extracted colon tissue from the model animals was homogenized by adding 250 ㎕ of RIPA buffer containing protease inhibitor cocktail. Then, the supernatant was centrifuged at 4 ° C and 13000 rpm for 15 minutes. The supernatant was stored at -80 ° C, and the amount of IL-6 and TNF-alpha corresponding to proinflammatory cytokine and anti-inflammatory cytokine anti-inflammatory cytokine (IL-10) expression was measured using 96-well ELISA plate kits (Pierce Biotechology, Inc., Rockford, IL, USA). Table 2 below summarizes the results of the response indicator analysis when neoagarol oligosaccharide mixture was administered to a model animal in which acute colitis was induced by TNBS.

Experiment Group Concentration of inflammatory markers in colon tissues (pg / mg) TNBS processing Administered drug sample Single dose of drug sample (mg / kg) TNF-a IL-6 IL-10 - vehicle - 13.5 20.6 18.1 + vehicle - 106.7 84.5 7.9 + NAO One 56.4 44.7 11.1 + NAO 10 51.1 39.3 15.7 + NAO 50 37.6 29.5 17.8 + Mesalazine 50 40.2 32.7 14.6

As shown in Table 2 above, the neoagarooligosaccharide mixture (the partially purified DagA enzyme reaction product obtained in Example 3, NAO) showed a colitis improving effect similar to that of mesalazine, a commercial therapeutic agent.

Example  8 : Neoagarooligosaccharide  Test for confirming the modulation of the immune response of the mixture

(1) Experimental method

The spleen of C56BL / 6J mice was isolated and appropriately pulverized, suspended in RPMI 1640 containing 10% FCS, and CD4 T cells were isolated using CD4 T cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany). The separated CD4 T cells were divided into 5 × 10 ⁵ cells per well in a 12-well plate. Anti-CD3, anti-CD28, IL-2, and IL-12 were induced to induce the differentiation of T cells into Th1 cells, Anti-CD3, anti-CD28, IL-6, and TGF-β in order to induce the differentiation of T cells into T17 cells, and IL-2 and IL- CD3 and anti-CD28, and the cells were cultured for 4 days in a predetermined amount in the respective partially purified DagA enzyme reaction products (hereinafter referred to as "NAO") obtained in Example 3.

(2) Experimental results

After the cultivation of CD4 T cells was completed, the ability of T cells to differentiate into Th1 cells, Th2 cells, Th17 cells and Treg cells was measured. Specifically, cells of the culture were stained with anti-FoxP3 or anti-IL-17A antibody, and Th1 cells and Th2 cells were cultured using a FACS (C6 Flow Cytometer System, San Jose, CA, USA) The distribution of the cells, Th17 cells and Treg cells was analyzed, and the results are shown in Table 13 below. As shown in the following Table 3, the neoagarooligosaccharide mixture (the partially purified DagA enzyme reaction product obtained in Example 3, NAO) inhibited the differentiation of T cells into Th1 cells, Th2 cells and Th17 cells in a concentration-dependent manner T cells to Treg cells, suggesting that the neoagarooligosaccharide mixture can harmonically regulate the immune or inflammatory response.

Spleen T cell treatment drug and treatment concentration Rate of differentiation inhibition Differentiation increase rate Th1 cells Th2 cells Th17 cells Treg cells No treatment - - - - NAO: 10 占 퐂 / ml + ++ + + NAO: 100 占 퐂 / ml ++ ++ ++ ++ NAO: 500 占 퐂 / ml +++ +++ +++ +

* Inhibition rate: -, <10%; +, 10-30%; ++, 30-60%; +++, > 60%

* Growth rate: -, <10%; +, 10-50%; ++, 50-100%; +++, > 100%

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the scope of protection of the present invention is not limited to the specific embodiments but should be construed as including all embodiments belonging to the claims attached hereto.

&Lt; 110 > DyneBio Inc. <120> Composition including neoagarooligosaccharide for preventing,          improving or treating inflammatory disease &Lt; 130 > DP-16-772 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 930 <212> DNA <213> DagA gene derived from Streptomyces coelicolor A3 (2) <400> 1 gtggtcaacc gacgtgatct catcaagtgg agtgccgtcg cactcggagc gggtgcgggg 60 ctcgcgggtc ccgcacccgc cgctcatgcc gcagacctcg aatgggaaca gtaccccgtg 120 ccggccgccc ctggcggaaa caggtcctgg cagcttctcc ccagccattc ggacgacttc 180 aactacaccg gcaagcctca aaccttcagg ggcagatggc tggaccagca caaggatggc 240 tggtcgggcc cggccaacag cctctacagtg gcgcgccatt cctgggtggc tgacggaaat 300 ctcatcgtcg agggccgcag ggcgccggac gggagggtct actgcggcta cgtgacctcc 360 cgcaccccag tcgagtaccc tctctatacc gaagtactca tgcgtgtgag cgggctgaag 420 ctctcatcga atttctggct cctgagcaga gacgacgtca acgagattga cgtgatcgaa 480 tgctacggca acgagtcatt gcacggaaag cacatgaaca ccgcctacca catattccag 540 cggaacccct tcactgaact ggcgagaagc cagaaggggt atttcgcaga tgggagctac 600 gggtacaatg gtgagactgg gcaggtgttt ggggacggcg ccgggcaacc tcttcttcgg 660 aatggattcc accgctacgg cgtgcactgg ataagcgcca ccgaattcga tttctacttc 720 aacggcaggt tggtgcgccg gctgaaccgg tcgaacgacc tcagggaccc ccggagccgg 780 ttcttcgacc agccaatgca tctgatcctc aacaccgaga gtcatcagtg gcgcgtcgac 840 cgaggtatcg aacccacgga cgcggaactc gcagacccca gcatcaacaa catctactac 900 cgctgggtca ggacgtatca ggccgtgtag 930 <210> 2 <211> 309 <212> PRT <213> DagA enzyme derived from Streptomyces coelicolor A3 (2) <400> 2 Met Val Asn Arg Arg Asp Leu Ile Lys Trp Ser Ala Val Ala Leu Gly   1 5 10 15 Ala Gly Ala Gly Leu Ala Gly Ala Pro Ala Ala Ala His Ala Ala Asp              20 25 30 Leu Glu Trp Glu Gln Tyr Pro Val Pro Ala Ala Pro Gly Gly Asn Arg          35 40 45 Ser Trp Gln Leu Leu Pro Ser Ser Ser Asp Asp Phe Asn Tyr Thr Gly      50 55 60 Lys Pro Gln Thr Phe Arg Gly Arg Trp Leu Asp Gln His Lys Asp Gly  65 70 75 80 Trp Ser Gly Pro Ala Asn Ser Leu Tyr Ser Ala Arg His Ser Trp Val                  85 90 95 Ala Asp Gly Asn Leu Ile Val Glu Gly Arg Arg Ala Pro Asp Gly Arg             100 105 110 Val Tyr Cys Gly Tyr Val Thr Ser Arg Thr Pro Val Glu Tyr Pro Leu         115 120 125 Tyr Thr Glu Val Leu Met Arg Val Ser Gly Leu Lys Leu Ser Ser Asn     130 135 140 Phe Trp Leu Leu Ser Arg Asp Asp Val Asn Glu Ile Asp Val Ile Glu 145 150 155 160 Cys Tyr Gly Asn Glu Ser Leu His Gly Lys His Met Asn Thr Ala Tyr                 165 170 175 His Ile Phe Gln Arg Asn Pro Phe Thr Glu Leu Ala Arg Ser Gln Lys             180 185 190 Gly Tyr Phe Ala Asp Gly Ser Tyr Gly Tyr Asn Gly Glu Thr Gly Gln         195 200 205 Val Phe Gly Asp Gly Ala Gly Gln Pro Leu Leu Arg Asn Gly Phe His     210 215 220 Arg Tyr Gly Val His Trp Ile Ser Ala Thr Glu Phe Asp Phe Tyr Phe 225 230 235 240 Asn Gly Arg Leu Val Arg Arg Leu Asn Arg Ser Asn Asp Leu Arg Asp                 245 250 255 Pro Arg Ser Phe Phe Asp Gln Pro Met His Leu Ile Leu Asn Thr             260 265 270 Glu Ser His Gln Trp Arg Val Asp Arg Gly Ile Glu Pro Thr Asp Ala         275 280 285 Glu Leu Ala Asp Pro Ser Ile Asn Asn Ile Tyr Tyr Arg Trp Val Arg     290 295 300 Thr Tyr Gln Ala Val 305 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for cloning DagA (Asm-F) <400> 3 gacatatggt ggtcaaccga cgtgatc 27 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for cloning DagA (Asm-R) <400> 4 ggtggatccc tacacggcct gatacg 26

Claims (10)

A composition comprising a neoagarooligosaccharide mixture as an active ingredient,
Wherein the neoagarooligosaccharide mixture comprises neoagarobiose, neoagarotetraose and neoagarohexaose. 9. The pharmaceutical composition according to claim 1, wherein the neoagarooligosaccharide mixture comprises neoagarobiose, neoagarotetraose and neoagarohexaose.
The method according to claim 1, wherein the inflammatory disease is inflammatory bowel disease, Crohn's disease and ulcerative colitis, peritonitis, osteomyelitis, meningitis, meningitis, encephalitis, pancreatitis, traumatic shock, Inflammatory bowel disease, chronic bronchitis, chronic bronchitis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, intestinal disease, spondyloarthropathies, juvenile asthma, cystic fibrosis, stroke, acute bronchitis, acute bronchiolitis, Inflammatory arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, platelet arthritis, Lyme disease, arthritis associated with ' vasculitis syndrome &apos;, nodular polyarteritis, hypersensitivity Vasculitis, Lou Gehrig's granulomatosis, rheumatoid polyposis myalgia, arthritic cell arteritis, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and / or prophylaxis of a disease or condition selected from the group consisting of psoriasis, psoriatic gout, non-arthritic rheumatism, bursitis, hay fever, tennis elbow, charco and joint, hemarthrosic, Hypothyroidism, hypogammaglobulinemia, familial mediterranean fever, Beckhard disease, systemic lupus erythematosus, recurrent fever, psoriasis, multiple sclerosis, multiple sclerosis, hemorrhoids, sickle cell disease and other hemochromatosis, Chronic obstructive pulmonary disease, acute lung injury, and broncho-pulmonary dysplasia, which are associated with a number of diseases, such as atherosclerosis, septicemia, septic shock, multiorgan dysfunction syndrome, acute respiratory distress syndrome, And the like. The allergic disease is any one selected from the group consisting of allergic skin diseases, atopic dermatitis, bronchial asthma, allergic rhinitis, allergic conjunctivitis, allergic otitis media, urticaria, asthma and anaphylactic shock Or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of inflammatory diseases.
The method of claim 1, wherein the neoagarooligosaccharide mixture comprises 1 to 10% by weight neoagarobiose, 55 to 75% by weight neoagarotetraose based on the total weight of the neoagarooligosaccharide mixture, And 20 to 40% by weight of neoagarohexaose. The pharmaceutical composition for preventing or treating inflammatory diseases according to claim 1,
A composition comprising an enzyme reaction product of a substrate selected from agar or agarose and DagA as a beta-agarase derived from Streptomyces coelicolor or a purified product thereof as an active ingredient,
Wherein the enzyme reaction product or the purified product thereof comprises neoagarobiose, neoagarotetraose and neoagarohexaose. 9. The pharmaceutical composition according to claim 1, wherein the enzyme reaction product or the purified product thereof comprises neoagarobiose, neoagarotetraose and neoagarohexaose.
5. The method of claim 4, wherein the inflammatory disease is selected from inflammatory bowel disease such as inflammatory skin disease, allergic disease, Crohn's desease and ulcerative colitis, peritonitis, osteomyelitis, meningitis, meningitis, encephalitis, pancreatitis, Inflammatory bowel disease, chronic bronchitis, chronic bronchitis, osteoarthritis, gout, spondyloarthropathies, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, intestinal disease, spondyloarthropathies, juvenile asthma, cystic fibrosis, stroke, acute bronchitis, acute bronchiolitis, Inflammatory arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, platelet arthritis, Lyme disease, arthritis associated with the vasculitis syndrome, nodular polyarteritis nervosa, irritable bowel syndrome, irritable bowel syndrome, Vasculitis, Lou Gehrig's granulomatosis, rheumatoid polyposis myalgia, arthritic cell arteritis, The present invention relates to the use of a compound of formula (I) or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment and / or prophylaxis of a disease or condition selected from the group consisting of psoriasis, psoriatic gout, non-arthritic rheumatism, bursitis, hay fever, tennis elbow, charco and joint, hemarthrosic, Hypothyroidism, hypogammaglobulinemia, familial mediterranean fever, Beckhard disease, systemic lupus erythematosus, recurrent fever, psoriasis, multiple sclerosis, multiple sclerosis, hemorrhoids, sickle cell disease and other hemochromatosis, Chronic obstructive pulmonary disease, acute lung injury, and broncho-pulmonary dysplasia, which are associated with a number of diseases, such as atherosclerosis, septicemia, septic shock, multiorgan dysfunction syndrome, acute respiratory distress syndrome, And the like. The allergic disease is any one selected from the group consisting of allergic skin diseases, atopic dermatitis, bronchial asthma, allergic rhinitis, allergic conjunctivitis, allergic otitis media, urticaria, asthma and anaphylactic shock Or a pharmaceutically acceptable salt thereof, for the prophylaxis or treatment of inflammatory diseases.
5. The method according to claim 4, wherein the enzyme reaction product or the purified product thereof comprises 1 to 10% by weight of neoagarobiose, 55 to 75% by weight of neoagarotetraose based on the total weight of neoagarooligosaccharide, And 20 to 40% by weight of neoagarohexaose, based on the total weight of the pharmaceutical composition.
A composition comprising a neoagarooligosaccharide mixture as an active ingredient,
Wherein the neoagarooligosaccharide mixture comprises neoagarobiose, neoagarotetraose and neoagarohexaose. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
8. The method of claim 7, wherein the neoagarooligosaccharide mixture comprises 1 to 10% by weight neoagarobiose, 55 to 75% neoagarotetraose based on the total weight of the neoagarooligosaccharide mixture, And 20 to 40% by weight of neoagarohexaose. The composition for preventing or improving inflammatory diseases according to claim 1,
A composition comprising an enzyme reaction product of a substrate selected from agar or agarose and DagA as a beta-agarase derived from Streptomyces coelicolor or a purified product thereof as an active ingredient,
Wherein the enzyme reaction product or the purified product thereof comprises neoagarobiose, neoagarotetraose, and neoagarohexaose. &Lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt;
10. The method of claim 9, wherein the enzyme reaction product or the purified product thereof comprises 1 to 10% by weight of neoagarobiose, 55 to 75% by weight of neoagarotetraose based on the total weight of neoagarooligosaccharide, And 20 to 40% by weight of neoagarohexaose, based on the total weight of the food composition.

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