KR20160064991A - A pharmaceutical or food composition containing the fermentative products of elm-tree converted biologically by Basidiomycota hyphae or lysate of Basidiomycota hyphae with anti-allergenic effect - Google Patents
A pharmaceutical or food composition containing the fermentative products of elm-tree converted biologically by Basidiomycota hyphae or lysate of Basidiomycota hyphae with anti-allergenic effect Download PDFInfo
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- KR20160064991A KR20160064991A KR1020150165206A KR20150165206A KR20160064991A KR 20160064991 A KR20160064991 A KR 20160064991A KR 1020150165206 A KR1020150165206 A KR 1020150165206A KR 20150165206 A KR20150165206 A KR 20150165206A KR 20160064991 A KR20160064991 A KR 20160064991A
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- elm
- fermented
- mycelium
- antiallergic
- allergic
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- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
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Abstract
The present invention relates to a therapeutic pharmaceutical composition or food composition having an antiallergic ability including fermented microorganisms (mycelium mycelium) or further biologically converted biologically through enzymatic treatment, and the present invention relates to mycelial fermentation and enzymatic treatment The elm fermented biosynthesized by the biosynthesis method has a greater effect of antiallergic substance compared to the non fermented elm.
Description
The present invention relates to a therapeutic pharmaceutical composition or food composition having antiallergic ability including fermented microorganisms (mycelium mycelium) or biologically converted biologically through enzymatic treatment.
Allergic diseases are so widespread that about 25% of all people suffer from one or two allergies. The prevalence of allergic rhinitis in developed countries has increased by 10% every year for the past 30 years. In recent years, the statistics of allergic patients in urban areas show that 15 ~ 20% of rhinitis.
Common treatments for allergic diseases include avoidance, drug, surgery and immunotherapy. Avoidance is the most basic and important treatment method to avoid causative antigens. It is a very important method for the management of allergic diseases, but there is a difficulty in completely avoiding causative agents that cause immune reactions. Drug therapy has shown the greatest progress in the treatment of allergic diseases, but current drug therapy is a treatment that inhibits the secretion of chemical messengers. In addition, steroids, antihistamines, sympathomimetics, theophylline, etc. have been used in the selection of therapeutic agents for 20 years or more, with the exception of leukotriene modifiers. Although there is a growing concern about the importance of anti-inflammatory drugs such as steroids for persistent allergic diseases such as chronic asthma, this treatment strategy is not aimed at primary prevention or cure of allergic diseases. Operative therapy includes surgery for cirrhosis, cryosurgery, diathermy, neurosurgery, and laser ablation. However, since the pathology of allergic diseases is not corrected by surgery, , The symptoms often recur. Immunotherapy is a method to induce a change of immune response in the body by continuously and regularly administering the causative antigen when the antigen is revealed through various inspection methods, and as a method to reduce the susceptibility to the causative substance.
As described above, since most of the allergic diseases have no special therapeutic agent or preventative agent other than the symptomatic treatment, there is a need for development of raw materials and products for health functional foods derived from natural materials. Up to now, technologies such as an immunomodulating composition (KR 0757220) and a health promotion food composition (KR 2005-0101134) using an elm extract as an active ingredient have been developed, but fermentation of microorganisms (mycelium mycelium) The development of technologies for the treatment and prevention of allergic diseases containing the biologically transformed elm fermented product as an active ingredient has been insufficient.
Accordingly, the present invention relates to a therapeutic pharmaceutical composition or food composition having antiallergic ability including a fermented elm fermented biotransformed into a bacterium mycelium or a bacterium thereof and the effect thereof.
Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems occurring in the prior art, and it is an object of the present invention to provide a food composition having an antiallergic function comprising fermented Mycelium of basidiomycetes or further biologically- will be. However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.
Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.
Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
In one embodiment of the present invention, the basidiomycetes are fungi, which are cells of the fungus as a result of the sexual reproduction of the fungus as a result of the sexual reproduction. There are many things known as mushrooms which are parasitic to other organisms because they can not make their own nutrients. Typical examples are mushroom, mushroom, shiitake, mushroom, mushroom, and mushroom. On the freshly corrugated side of adult mushrooms, a lot of volcanic ash is formed. Most of the basidiomycetes mushrooms live in secondary life in the fluid of higher plants. In particular, they decompose cellulose and lignin to play an important role in the biological circulation of the material. In addition, they form mycorrhiza in the roots of trees, There are many. There are other kinds that are important as edible bacillus or should be cautious with poisoning.
In one embodiment of the present invention, mycelium is a nutritive cell of a filamentous fungus. Functional differentiation such as nutritional function or physiological function can also be seen. Morphologically, the presence or absence of the bulkhead is used for classification, and it develops in germination tube of spore and elongates by tip growth. The group of branched mycelium is called mycelium. Depending on the physiological function, it is distinguished into basal hypha that grows on the surface or inside of the pouch or host, and parasitic bacteria which grows in the air. Parasitoids depend on the transfer of nutrients from basal hyphae, some of which differentiate into spore forming organs and the like. If there are organic matter and suitable environmental conditions such as appropriate humidity and temperature, various mycelial tissues are formed. The mycelial membrane of mycelium of fungus is simple, but it can be seen that it forms a braid connection. In the fungi, it branches into several branches and is entangled like a thread. It does not have chlorophyll and does not have photosynthesis. It attaches to the host organism and absorbs nutrients. In the case of the mushroom-bearing bacterium, the mycelium germinated in the spore has one nucleus, which when fused with another mycelium has two nuclei in one cell. Mycelium with one nucleus is called mycelium, and mycelium with two nuclei is called secondary mycelium. The cell wall of the mycelium is generally composed of chitin or hemicellulose, which is sometimes combined.
In the present specification, the mycelia of mycelia can be selected from mycelium of mushroom, chaga mushroom, shiitake mushroom, mushroom mushroom, mushroom mushroom, The mycelia of these mycelia were collected from the KCCM, KCTC, KCLB, KACC, ATCC, And the like.
In one embodiment of the present invention, elm is a plant of Ulmus rosberry elm including limm (Ulmus parvifolia Jacg.), Which is a large tree that leaves in winter. The whole length grows about 20 ~ 30m and grows at the base of the temperate northern part. Leaves are alternate, inverted ovate, pointed end, with double sawtooth on edge. The front side is full color and the back side is light green color. There are rough hairs on the back, and it is stiff. In April, before the leaves are blossomed, small pale flowers bloom in the axilla. The fruit is round in June, but it is round and flat with wings around it. The bark is dark gray brown and cracks irregularly vertically. The roots of elm and elm are called rhizome, stem skin is dermis, and leaves are leaf. The roots of the root are taken in the fall, the stem is taken in the spring and dried in the windy shade. Elm trees are known to have gastroenteritis, enteritis, bronchitis, rhinitis, insomnia, spots and freckles. Gastrointestinal diseases, enteritis, bronchitis, rhinitis, dried roots or stem shells, dried dalyeoseo drink. Leaves are collected in spring and dried in the windy shade. In insomnia, they drink on dalyeoseo.
In the present specification, the elm or elm powder is composed of all or part of the elm, and the elm part may be at least one selected from the group consisting of elm roots, root hair, stem, branches, leaves, , But is not limited thereto.
In one embodiment of the present invention, bioconversion refers to the conversion of a substance added using a physiological function of an organism into a chemically modified form.
In one embodiment of the present invention, fermentation refers to a process in which a microorganism decomposes an organic substance using an enzyme contained therein. Fermentation is a phenomenon in which organic matter changes into a simple compound by enzymatic action and releases free energy. In general, microorganisms decompose organic matter and accumulate metabolites. That is, fermentation of alcohol by anaerobic digestion of sugars by anaerobic digestion of yeast, fermentation of lactic acid by lactic acid bacteria decomposing sugar nonoxiously into lactic acid is a typical fermentation, but now, A phenomenon in which alcohol is oxidized to acetic acid by use of oxygen, and a phenomenon in which mold oxidizes glucose to gluconic acid by using oxygen in the air are also referred to as acetic acid fermentation and gluconic acid fermentation. In addition, these anaerobic fermentation can be divided into anaerobic fermentation and aerobic fermentation (oxidation fermentation), and other anaerobic fermentation fermentations include glycerol fermentation, acetone butanol fermentation, 2, 3-butylene glycol fermentation, butyric acid fermentation, Fermentation, and methane fermentation. Examples of aerobic fermentation include citric acid fermentation, itaconic acid fermentation, yeast acid fermentation, 2-keto glutaric acid fermentation, oxalic acid fermentation, fumaric acid fermentation and sorbose fermentation. In addition, production of amino acids, vitamins and antibiotics by microorganisms is also called glutamic acid fermentation, riboflavin fermentation, penicillin fermentation and the like, which is not a good title. The name of the fermentation is usually called the product, but sometimes the name of the substrate is given by cellulosic fermentation, pectin fermentation and so on.
In one embodiment of the present invention, an allergic disease is a disease caused by allergies. Allergy refers to an antigen-antibody reaction that occurs when an IgE antibody is produced against a substance of interest (antigen or allergen), and then the same antigen enters the body. The major diseases are bronchial asthma, allergic rhinitis, urticaria, leukemia, drug allergy, seropositivity, and various types of diseases depending on the type of allergen or tissues causing allergic reactions. Diagnosis of allergic diseases refers to the progress of the family or patient until that time, the time of onset, the living environment, and the contents of the meal consumed. In some cases, it may be necessary to remove the suspicious allergen and observe the improvement of the symptoms, and to induce allergens. Other skin reactions include scratch tests or skin reactions. Therapeutic methods include elimination of allergens, desensitization therapy, and drug therapy to suppress allergic reactions. As a typical allergic disease of the respiratory system, allergic rhinitis shows hypersensitivity to the nasal mucosa of a specific substance. After the nasal mucosa is exposed to the causative substance (allergen), all parts of the nasal mucosa IgE antibody-mediated inflammatory cells are involved in inflammatory reactions caused by various mediators secreted by these cells. It is characterized by three main symptoms: continuous seizure sneezing, clear runny nose, and nasal congestion. Allergic rhinitis can be suspected when two or more of these three symptoms are present. In addition to the characteristic symptoms, it may be accompanied by symptoms such as itching around the nose, headache, loss of smell, and complications such as otitis media, sinusitis and pharyngitis.
In one embodiment of the invention, IgE is an antibody protein, also called immunoglobulin E. It is often called an "allergic antibody" because it plays an important role in allergic reactions. If you are allergic to certain substances (allergens), the immune system mistakenly thinks that harmful substances are harmful to your body. When you are exposed to this particular substance, the immune system begins to produce IgE to protect your body. IgE antibodies remain in the body and then come into contact with allergens again, causing allergic reactions. As a result, people with allergies have elevated serum IgE levels, and IgE has specificity for each allergen.
In one embodiment of the present invention, histamine is a substance produced by the action of histidine decarboxylase, which is an amino acid, histidine. It is mainly stored in mast cells and is released when the cells are damaged due to wound, bacterial infection, Histamine released plays an important role in immune function by expanding the blood vessels and increasing the permeability of the blood vessels, allowing white blood cells and various plasma proteins to move quickly to the site where the bacteria have entered. The excessive secretion of histamine due to excessive immune responses may cause allergic reactions and anaphylaxis. Besides these immune functions, it is one of powerful active agents that regulate the movement of the uterus during pregnancy and secrete gastric juice. It also causes an increase in blood flow due to vasodilation.
In an embodiment of the present invention, the pathogenesis of allergy is described in detail. Allergy refers to a symptom in which an exaggerated immune response to an exogenous substance is exerted. The allergic disease is mainly caused by a Th2 cell mediated immune response Lt; / RTI > Acute and mild allergic diseases such as seasonal allergic rhinitis are characterized by increased allergen-specific IgE antibodies, IgE-dependent activation of mast cells, and invasion of activated Th2 cells and eosinophils into the mucosal surface.
In relation to the onset of allergy, ① allergen-inducing antigen enters our body to activate antigen-specific Th2 cells, ② cytokine secreted by activated Th2 cells (IL-4, etc.) When class switching occurs and IgE antibody production is induced and secreted, our body becomes sensitive to allergens. At this time, when the allergen is reintroduced, the IgE antibody mediated reaction occurs and allergic symptoms appear. That is, when the IgE antibody binds to the IgE receptor of the mast cell and the mast cell is sensitized, the mast cell is activated when the allergic antigen is re-introduced to the mast cell surface, and the degranulation is immediately induced and the intracellular granule such as histamine And inflammatory cytokines, are secreted out of the cell. Allergic reactions are triggered by several chemical allergen mediators secreted. Allergic reactions can be divided into early reaction and late reaction. The initial reaction occurs within a few minutes and secretes mediators of already-produced mediators such as histamine, β-hexosaminidase, and serotonin and newly synthesized lipid components from mast cells And the late response occurs within a few hours. IL-4, IL-5, and IL-13, which are typical cytokines of Th2 immune response, are synthesized and secreted to continue the inflammation reaction. Mediators and cytokines of lipid components, newly produced by mast cells, are the major factors causing allergic acute and chronic inflammatory responses. (4) Eosinophil infiltration and induction of eosinophils in peripheral tissues by cytokines (IL-5, etc.) secreted from cells and mast cells induces eosinophil hyperplasia, which is called eosinophilic allergy. In addition, IL-13 induced hypersensitivity by inducing hypersensitivity. Eosinophilia is a direct cause of induction of allergic hypersensitivity reaction.
In the case of an allergic disease caused by an excessive immune response, it is necessary to control the aggressive immune response. Inhibition of the Th2 immune response by inducing the Th1 immune response and the Treg immune response, (IL-4, IL-5, and IL-13), (3) reduce eosinophils and mast cells in allergic inflammation sites, and Immunomodulating treatments such as reducing hypersensitivity suggest new possibilities for the treatment of allergic diseases.
In one embodiment of the present invention, sepsis refers to a condition in which a microorganism is infected and a serious inflammatory reaction occurs in the whole body. The number of breaths increased more than 24 times per minute (ventilation), heart rate more than 90 times per minute (tachycardia), increase in leukocyte count in blood test, or significant decrease in respiratory rate If you have symptoms that are more than a branch, it is called systemic inflammatory response syndrome (SIRS). This systemic inflammatory response syndrome is called sepsis when it is caused by microbial infection. The source of the infection can be any organs of the body. Pneumonia, pyelonephritis, meningitis, cellulitis, infective endocarditis, peritonitis, pressure ulcer, cholecystitis, and cholangitis may cause sepsis. If such infection occurs, the causative microorganism may invade into the blood and cause sepsis. However, even if microorganisms do not penetrate into the blood, systemic sepsis may occur due to the inflammation reaction of some body parts and the production of inflammatory substances. Early symptoms of sepsis may include respiratory arrest, neurological disturbances such as loss of locomotion (time, place, cognitive ability) or delirium. The skin may appear visually opaque due to lowering of blood pressure and lowering of the amount of blood supplied to the distal end of the body. Bacteremia (a symptom of germs circulating in the blood) can cause germs to move around the bloodstream, to position themselves in certain parts of the body and cause pathological changes in those areas. As the skin changes specific to the causative bacteria, it may help to diagnose the cause of sepsis. Symptoms of the digestive system include nausea, vomiting, diarrhea and intestinal paralysis, and severe gastrointestinal bleeding may also occur.
In one embodiment of the present invention, the pharmaceutical composition means a composition to be administered for a specific purpose. For the purpose of the present invention, the pharmaceutical composition of the present invention is used for enhancing the survival rate or inhibiting the inflammatory active substance in the sepsis-induced group, and comprises a composition for this purpose and a pharmaceutically acceptable carrier, excipient or diluent . The pharmaceutical composition according to the present invention comprises 0.1 to 100% by weight of the active ingredient corresponding to the pharmaceutical composition of the present invention, based on the total weight of the composition. 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 one embodiment of the present invention, the food composition is variously used as a food composition for improving allergic diseases. The food composition containing the composition of the present invention as an active ingredient may be used in various foods such as beverages, Tea, vitamin complex, powder, granule, tablet, capsule, confection, rice cakes, bread and the like. Since the food composition of the present invention is composed of a natural food and its fermented product which have little toxicity and side effects, it can be safely used for prolonged use even for prophylactic purposes. When the composition of the present invention is contained in the food composition, the amount thereof may be added in a proportion of 0.1 to 100% of the total weight. Here, when the food composition is prepared in a beverage form, there are no particular limitations other than those containing the food composition at the indicated ratios and may contain various flavors or natural carbohydrates such as ordinary beverages as an additional ingredient. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and sugar sugars such as polysaccharide, dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol can do. Examples of the flavors include natural flavors (such as tau martin, stevia extract (for example, rebaudioside A and glycyrrhizin), and synthetic flavors (for example, saccharin and aspartame). The food composition of the present invention can be used as a food composition containing various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, Stabilizers, antiseptics, glycerin, alcohols, carbonating agents used in carbonated beverages, etc. These components may be used independently or in combination. The ratio of these additives is not so important, Is generally selected in the range of 0.1 to 100 parts by weight per part.
In one embodiment of the present invention, the cosmetic composition is a composition for improving an allergic skin condition or an allergic skin disease. The cosmetic composition comprising the composition of the present invention as an active ingredient can be used as a skin lotion, a nutritional lotion, a nutrition essence, a massage cream, a cosmetic bath additive, a body lotion, a body milk, a bath oil, a baby oil, Skin lotions, skin creams, sunscreen cosmetics, cleansing milks, hair removal products, cosmetics, face and body lotions, face and body creams, skin whitening creams, hand lotions, hair lotions, (Non-medical use), cream soap, facial wash, hair rinse, make-up soap, tooth whitening gel, toothpaste, etc. . ≪ / RTI > To this end, the compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used in the preparation of cosmetic compositions. The carrier, excipient or diluent which may further be added to the cosmetic composition of the present invention include purified water, oil, wax, fatty acid, fatty acid alcohol, fatty acid ester, surfactant, humectant, thickener, antioxidant, Buffers, lower alcohols, and the like, but are not limited thereto. Further, if necessary, it may contain a whitening agent, a moisturizing agent, a vitamin, an ultraviolet screening agent, a perfume, a dye, an antibiotic, an antibacterial agent, and an antifungal agent. As the oil, hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil and avocado oil may be used. Examples of the wax include wax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, Can be used. Examples of the fatty acid include stearic acid, linoleic acid, linolenic acid and oleic acid. The fatty acid alcohols include cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol and hexadecanol As the fatty acid ester, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used. As the surfactant, a cationic surfactant, an anionic surfactant and a nonionic surfactant known in the art can be used, and a surfactant derived from a natural material is preferably used. In addition, it may contain a hygroscopic agent, a thickening agent, an antioxidant and the like widely known in the field of cosmetics, and the kind and amount thereof are well known in the art.
In one embodiment of the present invention, there is provided a method for culturing an elm in a liquid culture medium comprising (a) culturing the elm in a liquid phase, (b) culturing the organism fermented by inoculating mycelium with mycelia in a liquid medium, (C) a biotransformation product of the fermentation and enzyme treatment which has improved antiallergic efficacy from the fermentation product produced by the biotransformation fermentation process of the step (b) through the biotransformation enzyme treatment process The present invention provides a method of producing fermented products having increased antiallergic activity, comprising the steps of: (a) cultivating the elm in the step (a), wherein the elm powder obtained by powdering the elm is treated with a hydrolytic enzyme Wherein the enzymatic treatment is carried out using a cellulolytic enzyme capable of degrading the cell wall component and a cellulolytic enzyme capable of degrading cell wall components, Wherein the fibrinolytic enzyme is selected from the group consisting of cellulase, hemi-cellulase, pectinase and glucanase, wherein the fibrinolytic enzyme has an increased antiallergic efficacy Wherein the mycelia of the bacterium of step (b) are selected from the group consisting of mycelia of mushrooms selected from the group consisting of mushroom, mushroom, mushroom, mushroom, mushroom, mushroom and mushroom Wherein the bioconversion product of step (c) inhibits secretion of IgE in IgE-secreting B cells and inhibits the secretion of histamine in mast cells. Wherein the biological conversion product of step (c) is selected from the group consisting of TNF-a, IL-6 , IL-10 and IFN-β, wherein the bioconversion product of step (c) is used for the production of TNF-α, which is an indicator of systemic inflammation in sepsis, alpha, IL-1 [beta], and nitric oxide in the culture medium.
In another embodiment of the present invention, there is provided a fermented product having increased antiallergic activity by fermenting culture broth by inoculating mycelium hyphae into elm, wherein the allergen provides an allergic rhinitis-enhanced fermented product, The antiallergic effect reduces the production of IgE in allergic symptoms and inhibits the production of IL-2, IL-10, IL-12 and gamma-interferon in addition to inhibiting the production of IL-4, IL-5 and IL- Wherein the antiallergic effect is reduced in a concentration of eotaxin and VCAM-1 in the bronchoalveolar lavage fluid in allergic symptoms, and the anti-allergic effect is reduced in the bronchial alveolar lavage fluid, (Eicosanoids), LTC4, and prostaglandin D2.
In another embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of allergy comprising an fermented product having increased antiallergic potency obtained by fermenting culture broth by inoculating mycelium hyphae in an elm, wherein the allergen is an allergic rhinitis To provide a pharmaceutical composition.
In another embodiment of the present invention, there is provided a pharmaceutical composition for the treatment of inflammation comprising fermented product having increased antiallergic activity, which is cultured and fermented by inoculating mycelium hyphae in an elm, wherein the inflammation treatment is an inflammation treatment A pharmaceutical composition is provided.
In another embodiment of the present invention, there is provided an antiallergenic food composition comprising an fermented product having increased antiallergic activity, which is fermented by inoculation with mycelium hyphae in an elm.
In another embodiment of the present invention, there is provided a cosmetic composition for anti-allergy comprising a fermented product having increased antiallergic potency, which is fermented by culture inoculated with basidiomycetes hyphae in elm.
Hereinafter, the present invention will be described in detail.
The fermented elm fermented by microorganisms (mycelia of mycelia) or biologically converted by further enzymatic treatment contains a large amount of antiallergenic substance compared to the non-fermented elm extract (elm). Therefore, It is anticipated that it will have the effect of reducing the incidence of allergy.
FIG. 1 is a schematic view showing a cultivation medium of an elm, a bioconversion process, and a fermentation powder obtained according to an embodiment of the present invention.
FIG. 2 is a graph showing the activity of inhibiting IgE antibody production according to the concentration of the raw material and the fermented product of each elm of the present invention according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of inhibiting manganese degranulation inhibition activity according to the concentration of elm material and fermented material according to an embodiment of the present invention.
FIG. 4 is a graph showing histamine release inhibitory activity according to the concentration of the elm material and the fermented product, according to an embodiment of the present invention.
FIG. 5 is a graph showing the results of cytotoxicity measurement according to the concentration of elm material and fermented material according to an embodiment of the present invention.
FIG. 6 is a graph showing the results of evaluating the TNF-α-expressing ability according to the concentration of the elm material and the fermented product according to an embodiment of the present invention.
FIG. 7 is a graph showing the results of evaluating the IL-6 expression-producing ability according to the concentration of elm ash and fermented product according to an embodiment of the present invention.
FIG. 8 is a graph showing the results of evaluating the ability of IL-10 to express according to the concentration of elm material and fermented product according to an embodiment of the present invention.
FIG. 9 is a graph showing the results of IFN-.beta. Expression assay according to the concentration of elm material and fermented product according to an embodiment of the present invention.
10 is a diagram illustrating a method of producing an experimental animal model of OVA-sensitization (allergic rhinitis / asthma) mouse according to an embodiment of the present invention.
11 is a graph showing the results of measurement of IgE concentration in BALF and blood of an OVA-sensitized animal model according to an embodiment of the present invention.
FIG. 12 is a graph showing the results of measurement of concentrations of representative cytokines of Th1, Th2, and Treg immunoreactions in bronchoalveolar lavage fluid (BALF) of an OVA-sensitized animal model according to an embodiment of the present invention.
FIG. 13 is a graph showing the results of measurement of concentrations of representative cytokines of Th1, Th2, and Treg immunoreactions in the blood of an OVA-sensitized animal model according to an embodiment of the present invention.
FIG. 14 is a graph showing the results of confirming the effect of BALF on the number of immune cells such as eosinophils in an OVA-sensitized animal model according to an embodiment of the present invention. FIG.
FIG. 15 is a graph showing the results of confirming the inhibitory effect of inflammation-related substances in bronchoalveolar lavage fluid (BALF) of an OVA-sensitized animal model according to an embodiment of the present invention.
16 is a graph showing mortality change according to dose of elm fermented product in an animal model induced by sepsis according to an embodiment of the present invention.
Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
Example 1. Bioconversion process of fermentation and enzyme treatment
Example 1-1. Elm pretreatment
For the pretreatment of the purchased elm from the raw material to the pulverized powder, mold contamination degree of the raw material state was measured, and water washing work was performed to remove foreign matters and contaminants from the natural raw material. (1) air washing step to remove foreign matters and mold spores, (2) water washing step to remove residual pesticides, (3) alcohol (Et-OH) to remove contaminants such as microorganisms, Washing of the raw materials was carried out through the washing step. After washing and cutting, the raw materials were dried in a hot air drier capable of mass drying, and subjected to pulverization of each of the natural raw materials through crude pulverization and pulverization.
Example 1-2. Obtain fermented powder of elm
Elm trees which removed foreign and fungal contamination were subjected to enzymatic treatment and heat treatment sterilization process for liquid culture medium. The enzymes are hydrolyzed by cellulosic and hemi-cellulosic hydrolytic enzymes capable of decomposing bark, muscle, root, and leaf, and hydrolytic enzymes such as pectinase, glucosidase and amylase hydrolytic enzymes . After the enzyme was added, the cells were incubated at 60 ° C for 1 hour and sterilized at a high temperature for 30 minutes to culture the elm.
Then, the mycelium of mycelia cultivated separately in the culture (fermentation) medium was added. When the concentration of the residual carbon source is depleted to a certain concentration or less under the condition of inoculating 10% (v / v) culture of mycelium mycelium culture at 28 ~ 30 ° C and pH 4.5 ~ 7, And cultured for 7 to 10 days in a food process. The above mycelium of mycelia may be selected from mycelia such as mushroom, chaga mushroom, shiitake mushroom, mushroom mushroom, mushroom mushroom, snow mushroom mushroom, and skirting mushroom, which are medicinal and edible bacillus species, Ryu Myung-sa is a member of the Korean Society for Microbiology (KCCM), Korea Biotechnology Research Institute (KCTC), Korea Cell Line Bank (KCLB), Korea Agricultural Microbiology Resource Center (KACC) It is possible to use the strain in an institution.
The enzymatic treatment of the biotransformation process for the above fermented product is carried out by adding various enzymes such as cellulose decomposing enzyme, cellulase, hemicellulase, pectinase and glucanase after hot water extraction to an appropriate amount (The optimum amount indicated in the product manual of each enzyme preparation). Examples of the enzymes include commercially available enzymes such as a cellulase (a compound derived from Aspergillus niger), a filtrate (a compound derived from Filtrase (Tricoderma reesei)), a rapidase (a compound derived from a fibrinolytic enzyme, Viscozyme (Complex containing Aspergillus niger) and Sumizyme (a complex containing Aspergillus niger-derived pectinase) was added at the recommended concentration (10% v / v) given in the product manual of each enzyme preparation, The enzyme / substrate reaction was performed by rotating at 250 rpm for 1 to 3 hours.
The elm fermented by the biotransformation process was sterilized by enzyme inactivation process at 90 ℃ for 1 hour and then lyophilized and powdered. The supernatant of the elm fermented product having the above immunoactivity was dissolved in 10 times of water and centrifuged at 10,000 rpm for 30 minutes to remove insoluble residues. The supernatant was lyophilized to obtain a water-soluble powder, or 5-fold ethanol Was added to induce precipitation at 4 캜 for 24 hours or more, and the precipitate was lyophilized to obtain a powder of a polymeric polysaccharide fraction having immunological activity. The above culture broth culture, bioconversion process and fermentation powder obtaining process of the elm described above are shown in Fig.
Example 2. Biotransduced Elm Fermented Evaluation of in vitro efficacy
Example 2-1. In B cells IgE Evaluation of Antibody Production Inhibitory Activity
The U266B1 cell line (human B cell) was purchased from the World Cell Line Bank (ATCC) to confirm the IgE production inhibitory effect of the biotransfected elm fermented product. The cells were incubated at 37 ° C in 5% CO 2 and 15% And incubated in RPMI-1640 medium supplemented with heat-inactivated fetal bovine serum (FBS) and 100 units of penicillin and 100 μg / ml streptomycin. After inoculation the U266B1 cells to 1 x 10 6 cells / well density in a 96 well culture plate and cultured overnight. After incubation, B cells were stimulated by treatment with 10 μg / ml of LPS and 5 ng / ml of IL-4. With the stimulation, the elm and the fermented material were treated at a concentration of 1, 10, and 100 μg / ml, respectively, for 72 hours. After incubation, the supernatant was collected and the amount of IgE secreted from U266B1 cells was measured using a Sandwich ELISA Assay (BETHYL, Montgomery, TX) kit. The elm was purchased from Yeongcheon (CM) in Gyeongbuk province and Hoengseong (HS) in Gangwon province. Both the raw material and the fermented product were found to have a concentration - dependent inhibitory activity against IgE production. The inhibitory activity of CM and elm elsewhere in Kangwon province was 25.3% and 66.9%, respectively, and that of HS elicia was 27.2% and 66.2%, respectively. But the differences between the two countries were not observed. It was found that the inhibitory activity of IgE production was significantly increased in the fermented product compared to the elm. Especially, the IgE antibody production inhibitory activity was higher at a concentration of 1 ㎍ / ㎖ of the fermented product at a concentration of 100 ㎍ / It is. The results are shown in Fig.
Example 2-2. In mast cells Degranulation Evaluation of inhibitory activity
Beta-hexyl sosami is a kinase (β-hexosaminidase) inhibition were obtained for (mast cells of rat origin) RBL-2H3 cell line for the activity evaluation at the ATCC, 37 ℃, 5
In the beta - hexosaminidase assay, no significant difference in the inhibitory activity of the raw materials and the fermented product was observed in the respective raw materials and the fermented product according to the source of the stimulants. A23187 showed 61% inhibitory activity at the concentration of 100 ㎍ / ㎖ in the fresh elm of Hongcheon (JM) in Gangwon province. The inhibitory activity was 100% in the elm biotransformed fermentation products from Youngwon (SD) and Yeongwon (HG) ㎍ / ㎖ concentration, the highest inhibitory activity was 61.2%, but it did not show any significant difference from other elm trees.
In the case of stimulation with DNP-BSA, 58.9% at 100 ㎍ / ㎖ concentration and 59.9% at 100 ㎍ / ㎖ concentration in elm fermented from JC (YC), Chungcheongbuk-do province But there was no difference between the countries of origin. In addition, the activity difference between the raw material and the fermented product did not appear to be significant, and it was found that the increase in the activity of inhibiting beta - hexosaminidase release by fermentation / enzyme - bioconversion was not significantly affected. The results are shown in Fig.
Example 2-3. Evaluation of histamine release inhibitory activity in mast cells
The RBL-2H3 cell line for evaluation of histamine release inhibitory activity was cultured in the same manner as in Example 2-2. The cells were sensitized with A23187, and then treated with 1, 10, and 100 μg / ml concentrations of HS elongate ash and fermented products. To measure histamine, cultured RBL-2H3 cells were treated with 0.25% trypsin and the cells were harvested and suspended in thyloid buffer (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl2, 1.1 mM MgCl2, 11.9 mM NaHCO3, 0.4 mM NaH2PO4, 5.6 mM glucose, pH 7.2) at a concentration of 1 × 10 6 cells / well, and then dispensed into a 24-well culture dish. Subsequently, the cells were treated with an appropriate concentration of material and incubated for 15 minutes. Then, 10 μM of A23187 knife? The cells were treated with calcium ionophore and cultured again for 20 minutes. After culturing, the reaction was terminated by treatment at 4 ° C for 10 minutes, and the supernatant was recovered. To 1 ml of the recovered supernatant was added 0.2 ml of 1 N NaOH and 0.1 ml of 1% OPT (o-phthalaldehyde), and the mixture was allowed to stand at room temperature for 5 minutes. The reaction was terminated by treatment with 0.2 ml of 1 N HCl and then measured using a fluorescence meter (Molecular Devices, Sunnyvale, Calif.) At excitation wavelength of 360 nm and emission wavelength of 450 nm.
Histamine release inhibitory activity was measured by using HS elongate and fermented products in Kangwondo, Korea. As a result, the inhibitory activity of the raw material was 31.6% and the fermented product was 39.9% at the concentration of 100 ㎍ / ㎖, And the increase of the activity was slightly observed by the biological conversion process. The results are shown in Fig.
Example 2-4. Cytotoxicity Assessment
The RBL-2H3 cell line cultured by the method of Example 2-2 was dispensed into a 96-well culture dish at a concentration of 1 x 10 4 cells / well, and then the elm material and the fermented material were mixed at 1, 10, and 100 μg / And cultured for 48 hours. After the incubation, the supernatant was removed and 200 μl of a 0.5 mg / ml MTT reagent (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) diluted in the medium was treated and cultured again for 3 hours Respectively. After incubation, the supernatant was removed and 200 μl of DMSO was added to dissolve the intracellular precipitate. The absorbance at 570 nm was measured using a microplate reader (Bio-Rad, Hercules, Calif.).
In the RBL-2H3 mast cell line, the cytotoxicity of HS elongates was not observed at 100 ㎍ / ㎖ of both the raw material and the fermented product. The results are shown in Fig.
Example 2-5. In macrophages TNF Immune response using the expression rate of Activation ability evaluation
In order to evaluate the activation of congenital immune responses of elm fermented products, culture and assay analysis of macrophages (macrophages) were carried out using 5% DMEM medium. The macrophages (macrophages) used for the immunoactivity measurement were RAW 264.7 cells, and the subcultures were subcultured in 0.5 ml each at a concentration of 1 × 10 5 cells / ml in each well of a 24-well culture dish. After culturing for 24 hours, the cell culture was confirmed with a microscope, and the medium was completely removed and then washed 2-3 times with the medium without serum. Each well was filled with 0.5 ml of 5% FBS DMEM medium supplemented with a sample. The concentration of the sample was 1/10, starting at a concentration of 100 μg / ml, and adjusted to 1, 10, and 100 μg / Respectively. In addition, LPS in a final concentration of 1 / / ml was treated with a positive control (200 / / ml stock). 24 wells treated with the sample were incubated for 8-24 hours, and 400 μl of the culture solution of each well was taken. 400 μl of the culture solution was centrifuged at 12,000 rpm for 5 minutes, and the supernatant was used for assay analysis.
The expression of TNF-α in the macrophages was examined in order to confirm the innate immune response activation efficiency of the elm and the fermented product. Experiments were conducted by treating the elm material and the fermented product at a concentration of 10 and 100 μg / ml. The culture supernatant, which had been treated for 18 hours after the sample was treated, was analyzed with an assay of TNF-α (R & D system, MTA00B) according to the manufacturer's protocol.
As a result, it was confirmed that the expression of TNF-α was higher in elm fermented product than that of elm. The results are shown in Fig.
Example 2-6. Immune response using the expression rate of IL-6 in macrophages Activation ability evaluation
The expression of IL-6 in macrophages cultured in the same manner as in Example 2-5 was examined in order to confirm the innate immune response activation efficiency of the elm and the fermented product. Experiments were conducted by treating the elm material and the fermented product at a concentration of 10 and 100 μg / ml. The culture supernatant, which had been treated for 18 hours after the sample was treated, was analyzed with an assay of IL-6 (R & D system, M6000B) according to the manufacturer's protocol.
The results showed that IL-6 was almost not expressed in the group treated with elm, but the level of IL-6 was significantly increased in the group treated with the elm fermented product. The results are shown in Fig.
Example 2-7. Immune response using the expression rate of IL-10 in macrophages Activation ability evaluation
The expression of IL-10 in macrophages cultured in the same manner as in Example 2-5 was examined in order to confirm the innate immune response activation efficiency of the elm and the fermented product. Experiments were conducted by treating the elm material and the fermented product at a concentration of 10 and 100 μg / ml. The culture supernatant, which was treated for 18 hours after the sample was treated, was analyzed by IL-10 (R & D system, M1000) assay according to the manufacturer's protocol.
As a result, it was confirmed that IL-10 expression was higher in elm fermented product than in elm. The results are shown in Fig.
Example 2-8. In macrophages IFN Immune response using the expression rate of? Activation ability evaluation
The expression of IFN-β was examined in macrophages cultured in the same manner as in Example 2-5 in order to confirm the effect of activating the innate immune response of the elm material and the fermented material. And 100 占 퐂 / ml. The culture supernatant, which had been treated for 18 hours after the sample treatment, was taken and assayed according to the protocol of the manufacturer using an IFN-β (R & D system, 42400) assay.
The results showed that the expression of IFN-β was higher in elm fermented than in elm. The results are shown in Fig.
IFN-β is a cytokine that induces a Th1 immune response and is an immunomodulatory cytokine that inhibits the Th1 and Th17 immune responses while inducing a Th1 immune response. Allergy is known to occur as the Th2 immune response increases. The elm fermented product is expected to inhibit the expression of the cytokine associated with the Th2 immune response by inducing the expression of INF-β associated with the Th1 immune response.
Example 3. In the allergic rhinitis model elm Fermented In the body (in vivo ) Efficacy evaluation
Example 3-1. Allergic rhinitis Establishment of induction experimental animal model
An animal model of OVA-sensitization (allergic rhinitis / asthma) mice was established to evaluate the antiallergic activity of the elm fermented product. Egg white albumin (ovalbumin) was used as an antigen and alumina was used as a reinforcement agent. On the first day of experiment and 14 days, albumin albumin / alumina was sensitized by two intraperitoneal injections. After 2 weeks of elapsed time, allergic reaction was induced by inhalation of albumin albumin for 30 days for 30 days from 30 days. The evaluation of the elm fermented product was carried out by two ovalbumin / alumina abdominal injections on the day of the experiment and on the 14th day. From
Example 3-2. BALF And blood IgE produce Control ability evaluation
As shown in FIG. 10, OVA-sensitized (allergic rhinitis / asthma) experimental animal model prepared by the method of Example 3-1 induces an allergic reaction by inhalation of albumin albumin daily for 5 days from the 30th day of animal model production. In addition, the elm fermented product was administered daily from the 15th day until the end of the experiment, after sensitization with two albumin / alumina abdominal injections on the day of the experiment and on the 14th day, and mice were sacrificed on the 35th day, ) And blood were collected. The amount of immunoglobulin E (IgE) in the recovered BALF was 43.8% in the elm and 83.1% in the fermented product, indicating that the inhibitory activity against IgE production was significantly increased. Immunoglobulin E in the blood was also inhibited by 25.8% in the elm and 72.1% in the fermented product. The results are shown in Fig.
Example 3-3. At BALF Cytokine generation Control ability evaluation
As a result of inhibition of IgE production in OVA-sensitized (allergic rhinitis / asthmatic) experimental animal models due to the administration of the elm fermented product, in order to investigate the degree of activation of Th2 cells regulating the proliferation and activity of B cells, (BALF), the amount of representative cytokines of Th1 and Treg immunoreactivity, together with representative cytokines of Th2 immune response, were measured using an ELISA method. As a result, the production of IL-4, IL-5 and IL-13, which are representative cytokines of Th2 immune response, in the group fed with the elm fermented product was significantly suppressed compared to the control group (OVA only) And the control group was also inhibited compared to the control group. On the other hand, the production of IL-10, a representative cytokine of Treg immunoreactivity, with IL-2 and IL-12, which are representative cytokines of Th1 immune response, was observed in the group fed with the elm fermented product as well as the control group Compared to the control group. The results are shown in Fig.
Example 3-4. Evaluation of cytokine concentration in blood
The concentrations of representative cytokines of Th1, Th2, and Treg immunoreactivity according to administration of the elm fermented product in the serum of OVA-sensitized (allergic rhinitis / asthma) experimental animal models were measured using the ELISA method as in Example 3-3 Respectively. As a result, IL-10, which is a representative cytokine of Treg immunoreactivity, is produced by elm fermentation (BALF) together with IL-2 and IL-12, which are typical cytokines of Th1 immune response, In the control group and the control group, as compared with the control group and the control group. Gamma-interferon (Interferon-γ or IFN-γ), which has been shown to inhibit eosinophil infiltration in allergic inflammatory sites, is a representative cytokine secreted by Th1 cells. In the group fed with fermented elm, And the intake of raw materials was also higher than that of the control group. On the other hand, the production of IL-4, IL-5 and IL-13, which are representative cytokines of Th2 immune response, in the group fed with the elm fermented product was decreased compared to the control group (OVA only) , And the consumption of raw materials was also decreased compared to the control group. The results are shown in Fig.
Example 3-5. At BALF Eosinophilia inhibition and On cell number Check the effect
(BALF) of the experimental animal model of OVA-sensitization (allergic rhinitis / asthma) due to the administration of the elm fermented product to inhibit eosinophilia and cell number. Experimental results showed that the number of immune cells in the BALF was significantly reduced in the group fed with the fermented elm compared to the group fed the raw material. In particular, the number of cells in eosinophils and lymphocytes was significantly reduced. The results are shown in Fig.
Example 3-6. At BALF Evaluation of the inhibitory effect of inflammation-related substances
The concentrations of eosinophils and eicosanoids, an inflammation mediator, were determined in mouse bronchoalveolar lavage fluid (BALF) of OVA-sensitized (allergic rhinitis / asthma) experimental animal models by administration of elm fermented product. The results showed that eotaxin and VCAM-1 levels were decreased in the group fed with the elm fermented product, and eosinophil counts were decreased compared to the group consuming the OVA-sensitized group and elm. In addition, it was confirmed that the production of inflammatory mediators such as Eicosanoids, LTC4 and prostaglandin D2 was also inhibited in mouse BALF. The results are shown in Fig.
Example 4. Elm in the sepsis model Fermented In the body (in vivo ) Safety evaluation
Example 4-1. Establishment of an animal model for induction of sepsis
Based on the in vivo safety evaluation results of elm fermented products, the limit dose and route of administration of fermented elm in vivo were evaluated. The mice were fed with fermented elm at a concentration of 1, 10 and 100 mg / kg for 2 weeks by intraperitoneal injection. Lethal dose of LPS (20 ㎍ / kg) and galactosamine (700 mg / kg) Sepsis was induced.
Example 4-2. Comparison of lethal dose by concentration and route of administration
As a result of examining the mortality rate of mice up to 60 hours after the induction of sepsis in the experimental animal model according to the method of Example 4-1, 2 mice in the experimental group administered with 1 mg / kg of the elm fermented product in 10 mice per experiment group, Five mice in the 10 mg / kg ip group and 4 mice in the 100 mg / kg ip group were found to survive. In other words, as the dose was increased to 1, 10, and 100 ㎎ / ㎏, the survival rate was improved and it was confirmed that the elm fermented material is a very safe immune modulating material. The results are shown in Fig.
Example 4-3. Systemic inflammatory response Indicators( TNF -α, IL- 1β , Nitric oxide) evaluation
As shown in Example 4-2, the mortality rate of sepsis at the LPS lethal dose was decreased due to the administration of the elm fermented product, so as to confirm the inflammation inhibitory effect of the developmental material in the systemic inflammatory reaction. The elm fermented at 10 mg / kg was administered orally for 2 weeks according to the determined dosage and route of administration, and the sub-lethal dose of LPS (5 μg / kg) and
As a result of the experiment, it was confirmed that the mouse treated with the fermented substance of the elm inhibited TNF-α production by 40% or more as compared with the control. The production of IL-1β, which is a typical inflammatory cytokine, also showed a high production inhibitory activity of 45% or more, confirming that the elm fermented product has an inflammatory response inhibitory activity. The amount of nitric oxide in serum, another indicator of systemic inflammatory reaction caused by LPS, was also measured to show a high production inhibitory activity of 55% or more. The results are shown in Table 1.
Claims (18)
(b) performing a bioconversion fermentation process in which a mycelium of mycelium is inoculated in a liquid medium of a culture medium of step (a) to effect culture fermentation; And
(c) producing a bioconversion product of the fermentation and enzyme treatment in which the antiallergic effect is improved through the bioconversion enzyme treatment process from the fermentation product produced by the bioconversion fermentation process in the step (b). A method for producing a fermented product with increased allergic potency.
In the step (a) of the step of culturing the elm, the elm powder powdered with elm is treated with a hydrolyzing enzyme and then sterilized to make a liquid elm medium. Way.
Wherein said enzymatic treatment is by enzymatic degradation of cellulolytic enzymes and amylases which are capable of decomposing cell wall components.
Wherein said fibrinolytic enzyme is selected from the group consisting of cellulase, hemi-cellulase, pectinase and glucanase.
Wherein the mycelium of step (b) comprises mycelium of bacterium selected from the group consisting of a mushroom, a mushroom, a shiitake, a mushroom, a mushroom, a mushroom and a mushroom, ≪ / RTI >
Wherein the bioconversion product of step (c) inhibits the secretion of IgE in IgE-secreting B cells and inhibits the secretion of histamine in mast cells.
Wherein the bioconversion product of step (c) promotes the expression of TNF-a, IL-6, IL-10 and IFN-? In macrophages.
The bioconversion product of step (c) may be a method for producing a fermented product having increased anti-inflammatory activity, which decreases the concentration of TNF-α, IL-1β and nitric oxide in serum, which is an indicator of systemic inflammation in sepsis symptoms
Wherein the allergen is an allergic rhinitis, the fermentation product having increased antiallergic potency.
The antiallergic effect reduces the production of IgE in allergic symptoms and inhibits the production of IL-2, IL-10, IL-12 and gamma-interferon in addition to inhibiting the production of IL-4, IL-5 and IL- Wherein the fermentation product has an increased antiallergic activity.
Wherein the antiallergic effect inhibits the decrease in the concentration of Eotaxin and VCAM-1 in bronchial alveolar lavage fluid and the production of inflammatory mediators Eicosanoids, LTC4 and prostaglandin D2 in allergic symptoms. Fermented product with increased allergic efficacy.
Wherein the allergic is allergic rhinitis.
Wherein said inflammation treatment is a sepsis treatment.
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KR20200043296A (en) * | 2018-10-17 | 2020-04-27 | (주)에스티알바이오텍 | A pharmaceutical composition for the treatment of colorectal disease comprising the fermentative products of Kalopanax septemlobus as an active ingredient |
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KR20210057378A (en) * | 2019-11-12 | 2021-05-21 | 주식회사 네이처텍 | A hyperlipemia improvement health functional food composition containing hot water extract of Ulmus macrocarpa Hance and Moringa as an active ingredient and its preparing method |
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