KR102584002B1 - A composition for treating inflammation comprising the fermentative products of Ecklonia cava - Google Patents

Abstract

The present invention includes the steps of (a) powdering Ecklonia cava and converting it into a liquid culture medium; (b) performing a biotransformation fermentation process of culturing and fermenting by inoculating Basidiomycete mycelia into the Ecklonia cava liquid medium after converting the culture medium in step (a); (c) performing a bioconversion enzyme treatment process of treating each fermented product produced by the bioconversion fermentation process in step (b) with a cellulolytic enzyme; and (d) providing a method for producing a composition for treating inflammation comprising the fermented Ecklonia cava produced in step (c).

Description

A composition for treating inflammation comprising the fermentative products of Ecklonia cava}

The present invention relates to a composition for treating or improving inflammation containing fermented Ecklonia cava as an active ingredient.

As the concentration and duration of fine dust (particulate matter, PM) increases, interest in the effects of fine dust on the human body is increasing. Among them, ultrafine dust (PM2.5) refers to fine dust with an aerodynamic diameter of 2.5 μm or less, and is a secondary air pollutant produced by reacting in the atmosphere with primary pollutants emitted directly from air pollution sources ( NO3-, SO4-, NH4-, polyacromatic hydrocarbon (PAH), quinone, etc.) are the main substances. According to the World Health Organization (2013), long-term exposure to PM10 (fine dust with an aerodynamic diameter of 10 μm or less) increases respiratory tract-related diseases and mortality, but PM2.5 is a stronger risk factor. It was reported that it works. In particular, it has been reported that fine dust deposited in the body induces oxidative stress and inflammatory responses and causes acute worsening of respiratory and circulatory diseases. Therefore, it is believed that research is needed to suppress oxidative stress and inflammatory responses in the human body that can be induced by fine dust.

Previously, the use of Ecklonia cava extract as an active ingredient for anti-inflammatory and inflammatory neurodegenerative diseases has been disclosed (see Korean Patent Publication No. 10-20180080896). However, inflammation can be caused by physical trauma, harmful chemicals, bacteria, and mold. , It is only a local body's defense response that appears in response to a pathological condition caused by infection by a virus or irritant substances in the body's metabolites. On the other hand, the cause of allergic rhinitis and asthma can be said to be a common cause of hypersensitivity to a specific substance, an allergy (antigen). The phenomenon of breathing difficulties due to allergic inflammation of the bronchial tubes is called asthma, and it is called asthma. Allergic rhinitis is a disease in which the normal function of the nose is lost due to allergies and inflammation, causing the four major symptoms of sneezing, runny nose, itching, and nasal congestion. As treatments for relieving rhinitis and asthma, products using oral agents, inhalers, tulobuterol asthma patches, and aroma essential oils as an alternative therapy are being marketed. Inhalation preparations have fewer side effects compared to oral preparations, but they are difficult to apply to infants and children or unconscious patients, and it is difficult to receive prescriptions for prescription drugs in emergency situations. Additionally, when nebulizers are used, etc. Due to frequent inhalation, the mist may irritate the nasal mucosa, causing sneezing, or cause additional inflammation of the nasal mucosa due to air pressure during inhalation.

All of the prior technologies have the problem of not being able to provide an appropriate treatment effect depending on the age of the rhinitis or asthma patient, the patient's condition, treatment time, surrounding environment, patient's sleep or activity status, etc. Therefore, compositions for treating or alleviating respiratory inflammation such as rhinitis still have a very urgent problem with a lot of room for development.

Therefore, the present invention was completed to solve the above problems.

The present invention was devised to solve the problems of the prior art as described above, and its purpose is to treat allergic rhinitis and asthma by fermenting Ecklonia cava. However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

DETAILED DESCRIPTION OF THE INVENTION Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details, such as specific forms, compositions, and processes, are set forth in order to provide a thorough understanding of the invention. However, certain embodiments may be practiced without one or more of these specific details or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Accordingly, the phrases “in one embodiment” or “an embodiment” appearing in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in any suitable way in one or more embodiments.

Unless there is a special definition within the present invention, all scientific and technical terms used in this specification have the same meaning as commonly understood by a person skilled in the art in the technical field to which the present invention pertains.

In the present invention, Gamtae is a seaweed of brown algae that is important as food for abalone and is found in the deep sea. It is a perennial seaweed of the Kelp family of the Kelp order, a brown algae that inhabits the coast of Jeju Island. It is mainly distributed in Jeju Island and the southern coast of Korea. The cylindrical stem is 1 to 2 meters long and is brown. At the end of the stem is a single, flat middle leaf shaped like a piece of double leaf. The middle leaf is brown, about 1m long, and has pinnate small leaves on both sides. When dried, it takes on a black color. Young plants that appear in spring have stems about 5 to 10 cm long and middle leaves about 20 to 30 cm long. In addition, Ecklonia cava is a type of brown algae belonging to the seaweed family of the kelp order. It is distributed in temperate coastal areas such as the Korean Peninsula and Japan, and is abundantly produced in Jeju Island in Korea. Ecklonia cava contains bioactive substances such as peptides, polysaccharides, carotenoids, fucoidan, and phlorotannins. In particular, phlorotannin, a major bioactive substance mainly found in brown algae; Phlorotannins (eckol, dieckol, 6,6′-bieckol, and eckstolonol, etc.) are used as substitutes for chemical compounds in various industrial fields such as health functional foods and functional cosmetics due to their excellent antioxidant, anti-inflammatory, anti-wrinkle formation, and hair growth promoting effects. It is attracting attention.

In the present invention, bioconversion refers to inducing a structural change in an added substance through biological methods and converting it into a chemically modified form, leading to the creation of a new ingredient.

In the present invention, fermentation refers to a process in which microorganisms decompose organic matter using their own enzymes. Fermentation is a phenomenon in which organic matter changes into a simple compound through enzyme action and releases free energy, but generally refers to a phenomenon in which microorganisms accumulate metabolites through the decomposition process of organic matter. In other words, alcohol fermentation in which yeast anaerobically decomposes sugar into ethyl alcohol and carbon dioxide, and lactic acid fermentation in which lactic acid bacteria anaerobically decompose sugar into lactic acid are typical fermentations, but currently, acetic acid bacteria consume oxygen in the air. The phenomenon of oxidizing alcohol to acetic acid and the phenomenon of mold oxidizing glucose to gluconic acid using oxygen in the air are also called acetic acid fermentation and gluconic acid fermentation, respectively. These are also discussed by dividing them into anaerobic fermentation and aerobic fermentation (oxidative fermentation). Anaerobic fermentations other than those mentioned above include glycerol fermentation, acetone fermentation, butanol fermentation, 2, 3-butylene glycol fermentation, and butyric acid fermentation. , propionic acid fermentation, methane fermentation, etc., and aerobic fermentation includes citric acid fermentation, itaconic acid fermentation, succinic acid fermentation, 2-ketoglutaric acid fermentation, oxalic acid fermentation, fumaric acid fermentation, and sorbose fermentation. In addition, the production of amino acids, vitamins, and antibiotics by microorganisms is also called, for example, glutamic acid fermentation, riboflavin fermentation, and penicillin fermentation, which are not very good names. In principle, the name of the fermentation action is called the product, but sometimes the name of the substrate is given, such as cellulose fermentation or pectin fermentation.

In the present invention, the term 'allergic rhinitis' refers to the tendency to abnormally produce IgE in response to foreign substances entering the body from the outside. In other words, cases where IgE-mediated hypersensitivity reactions manifest clinically as symptoms are called 'allergic diseases', and traditionally allergic rhinitis, asthma, allergic conjunctivitis, atopic dermatitis, and urticaria are classified as such diseases. Allergic diseases correspond to IgE-dependent (IgE-mediated) allergic diseases. Allergic rhinitis is characterized by mucosal inflammation and hyperreactivity, very similar to allergic asthma. Allergens that cause rhinitis are inhalable, just like asthma. Typically, outdoor allergens, plant pollen, and mold cause seasonal rhinitis, while indoor allergens such as dust mites, cockroaches, and pets cause perennial symptoms. The pathogenesis of allergic rhinitis at the cellular level is the interaction between the Fab portion of specific IgE attached to the surface of both mast cells and basophils, and antigens (allergens), as in allergic asthma. The interaction between antigen and IgE causes the release of preformed inflammatory mediators such as histamine and tryptase, as well as prostaglandin D2 and leukotrienes, which are released through de novo synthesis. bring about These substances cause acute symptoms such as severe local itching, sneezing, nasal discharge, and runny nose. Histamine is probably the most important mediator in terms of the acute clinical manifestations of allergic rhinitis, as it is the only inflammatory mediator responsible for the full spectrum of rhinitis symptoms. As in the lower respiratory tract, allergen exposure to the nasal mucosa not only causes an acute (early) reaction, but also a late reaction characterized by recurrence of symptoms such as nasal congestion, a secondary release reaction of inflammatory media, and an increase in eosinophils, basophils, monocytes, and neutrophils in nasal fluid. bring about

In the present invention, asthma is an inflammatory disease characterized by hyperresponsiveness of the bronchial tubes, which can lead to life-threatening airway disorders and cause difficulty in breathing or breathing (especially when exhaling). It may cause symptoms such as chest tightness. Things that can trigger an asthma attack include sudden changes in temperature or humidity, allergies, upper respiratory infections, strenuous exercise, stress, and heavy smoking.

TSLP is mainly produced in keratinocytes of the skin and epithelial cells of the respiratory tract and nasal mucosa and stimulates immature myeloid dendritic cells (mDC), which use the heterodimeric TSLP receptor to transform the immature dendritic cells into mature dendritic cells. manifests. TSLP-activated dendritic cells differentiate naive CD4+ T cells into a Th2 phenotype and promote the proliferation of Th2 memory cells by expressing the costimulatory molecule OX40 ligand along with the Th2 chemotactic cytokines TARC (CCL17) and MDC (CCL22). In addition, TSLP inhibits the IL-12 p40 receptor expressed on dendritic cells, suppresses the Th1 immune response, and further promotes the Th2 immune response. TSLP activates the transcription of the IL-4 gene in Th2 cells, promotes the production of IL-13 in mast cells, and worsens the allergic inflammatory response by increasing the response of basophils and infiltrating eosinophils. Therefore, TSLP plays a key role in the onset of atopic dermatitis, allergic rhinitis, and asthma. TSLP is expressed in epithelial cells of patients with atopic dermatitis, allergic rhinitis, and asthma. Additionally, overexpression of TSLP in skin keratinocytes in mice amplifies the inflammatory response to sensitized inhalant antigens.

In the present invention, a pharmaceutical composition refers to a composition administered for a specific purpose. The composition for this purpose may include a pharmaceutically acceptable carrier, excipient, or diluent. The pharmaceutical composition according to the present invention contains 0.1 to 100% by weight of an active ingredient corresponding to the pharmaceutical composition of the present invention based on the total weight of the composition. Carriers, excipients, and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, and calcium silicate. , cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

In addition, the dosage of the composition according to the present invention may vary depending on the patient's condition, body weight, disease state, drug form, administration route and period, but may be appropriately selected by those skilled in the art. However, for desirable pharmacological effects, it is preferable to administer the extract or compound according to the present invention at 0.0001 to 100 mg/kg per day. The frequency of administration may be once a day, or it may be divided into several doses. However, this dosage does not limit or limit the scope of the present invention.

In the present invention, the food composition refers to a food composition that is used in various ways to improve a specific purpose. The food composition containing the composition of the present invention as an active ingredient is used in various foods, such as beverages, gum, tea, and vitamin complexes. , can be manufactured in the form of powder, granules, tablets, capsules, cookies, rice cakes, bread, etc. Since the food composition of the present invention has almost no toxicity and side effects, it can be used safely even when taken for a long period of time for preventive purposes. When the composition of the present invention is included in a food composition, it can be added in an amount of 0.1 to 100% of the total weight. Here, when the food composition is manufactured in the form of a beverage, there are no particular limitations other than containing the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates can be contained as additional ingredients like ordinary beverages. That is, natural carbohydrates include common sugars such as monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, dextrin, and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavoring agent include natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). In addition, the present invention The food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, It may contain stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These components can be used independently or in combination. The ratio of these additives is not so important, but 100 weight of the composition of the present invention It is generally selected in the range of 0.1 to 100 parts by weight per part.

In the present invention, a cosmetic composition is a composition for improving inflammation and atopic skin conditions or inflammation and atopic skin diseases. Cosmetic compositions containing the composition of the present invention as an active ingredient include lotion, nutritional lotion, nutritional essence, massage cream, beauty bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, and sunscreen. Screen lotion, sunscreen cream, suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilator {cosmetic}, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, Cosmetic cream, jasmine oil, bath soap, liquid soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicated soap (non-medical use), cream soap, facial wash, hair rinse, toilet soap, teeth whitening gel, toothpaste, etc. It can be manufactured in the form of. To this end, the composition of the present invention may further include an appropriate carrier, excipient, or diluent commonly used in the production of cosmetic compositions. Carriers, excipients, or diluents that can be further added to the cosmetic composition of the present invention include purified water, oil, wax, fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, and chelates. These include, but are not limited to, rating agents, buffering agents, lower alcohols, etc. Additionally, if necessary, it may contain whitening agents, moisturizers, vitamins, sunscreen, perfume, dyes, antibiotics, antibacterial agents, and antifungal agents. Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil can be used as the oil, and waxes include beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin. It can be used. Stearic acid, linoleic acid, linolenic acid, and oleic acid can be used as fatty acids, and cetyl alcohol, octyldodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol can be used as fatty alcohols. And as fatty acid esters, isopropyl myristate, isopropyl palmitate, and butyl stearate can be used. As surfactants, cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred whenever possible. In addition, it may contain moisture absorbents, thickeners, antioxidants, etc., which are widely known in the cosmetics field, and their types and amounts are known in the art.

In the present invention, the cellulase family of hydrolytic enzymes may include cellulase, hemicellulase, pectinase, or glucanase that can decompose tree bark, root bark, roots, leaves, etc. Commercially available cellulase preparations include, for example, Cellulase T "Amano", Cellulase A "Amano" (manufactured by Amano Enzyme Inc.), Tricerase KSM, Multifact A40, and Cellulase GC220 (hereinafter Genen). [manufactured by Core Kyowa Co., Ltd.], Cellulase GODO-TCL, Cellulase GODO TCD-H, Besserex, Cellulase GODO-ACD [manufactured by GODO SHUSEI CO., LTD.], Cellulase [ manufactured by Toyobo Co., Ltd.], Cellizer, Cellulase XL-522 [manufactured by Nagase ChemteX Corporation], Cellsoft, Denimax [manufactured by Novozymes. (manufactured by Novozymes A/S)], Cellulosine AC40, Cellulosine AL, Cellulosine T2 [manufactured by HBI Enzymes Inc.], Cellulase "Onozuka" 3S, Cellulase Y-NC [manufactured by Yakult Pharmaceutical Industry Co., Ltd.], Sumitim AC, Sumitim C [manufactured by SHINNIHON CHEMICALS Corporation], Entilon CM , Entilon MCH, Bioheat (manufactured by Rakuto Kasei Industrial CO., LTD.), etc.

According to one embodiment of the present invention, the present invention is a method for preventing and improving inflammation, comprising as an active ingredient a fermented product obtained by culturing Ecklonia cava into a liquid culture medium, inoculating it with Basidiomycete hyphae, culturing and fermenting it, and then treating it with a cellulolytic enzyme. Alternatively, a therapeutic composition is provided.

In the present invention, the inflammation may be allergic rhinitis or asthma.

In the present invention, the culture medium may be made by pulverizing Ecklonia cava, treating the Ecklonia cava powder in a liquid phase with a hydrolytic enzyme as needed, and then sterilizing it to make an Ecklonia cava medium.

In the present invention, the hydrolytic enzyme may be at least one of the cellulase family of hydrolytic enzymes.

The composition provided by the present invention may be used in the form of a pharmaceutical composition, cosmetic composition, or food composition, but is not limited thereto.

According to another embodiment of the present invention, (a) cultivating Ecklonia cava into a liquid culture medium; (b) performing a biotransformation fermentation process of culturing and fermenting by inoculating the basidiomycete hyphae into the Ecklonia cava culture medium obtained in step (a); and (c) performing a bioconversion enzyme treatment process of treating a fibrinolytic enzyme from the fermented product produced by the bioconversion fermentation process in step (b). A composition for preventing, improving, or treating inflammation. A manufacturing method is provided.

In the present invention, the cultivation medium of Ecklonia cava in step (a) may be made by pulverizing Ecklonia cava and treating the Ecklonia cava powder with a hydrolytic enzyme in a liquid phase to make Ecklonia cava medium. Here, after treatment with the hydrolytic enzyme, sterilization or sterilization may be performed as needed, but is not limited to this.

In the present invention, the hydrolytic enzyme in step (a) may be at least one hydrolytic enzyme of the cellulase series.

In the present invention, the basidiomycete hyphae of step (b) may be composed of basidiomycete hyphae selected from the group consisting of shiitake mushrooms, sage mushrooms, chaga mushrooms, maitake mushrooms, wood ear mushrooms, snowflake mushrooms, and skirt mushrooms. In the present invention, the basidiomycete hyphae is 2% (v/v) to 18% (v/v), preferably 5% (v/v) to 15% (v/v), more preferably 7% ( v/v) to 13% (v/v), but is not limited thereto.

In addition, in the present invention, the culture fermentation in step (b) is performed at a temperature of 28 to 30 ℃ and pH 4.5 to 7, and the concentrated Ecklonia cava medium is added when the concentration of the residual carbon source is depleted below a certain concentration. It is preferable to culture for 7 to 10 days in a fed-batch process, but it is not limited to this.

In the present invention, the fibrinolytic enzyme in step (c) may be cellulase alone or a combination of one or more selected from the group consisting of cellulase, hemicellulase, pectinase, and glucanase. Specific examples include fibrinolytic enzyme. Commercially available enzymes include the cellulolytic enzyme Viscozyme (complex derived from Aspergillus), cellulase (Cellulase: derived from Aspergillus niger), Filtrase (complex derived from Tricoderma reesei), and Rapidase (complex derived from Aspergillus niger). derived complex) and Sumizyme (complex containing pectinase derived from Aspergillus niger) can be used, but is not limited thereto. In the present invention, the fibrinolytic enzyme may be added in an amount of 0.01% (v/v) to 1% (v/v), preferably 0.01% (v/v) to 0.1% (v/v). It is not limited to this.

In the present invention, the bioconversion enzyme treatment process in step (c) may be performed under temperature conditions of 50 to 60° C. with stirring for 1 to 3 hours, but is not limited thereto.

In the present invention, the Ecklonia cava bioconversion product in step (d) is IL-4, IL-5, IL-13, TSLP (Thymic stromal lymphopoietin), or IL at the site of allergic rhinitis or asthma lesions. -31, increase the expression level of IFN-γ, increase the expression level of IL-2, IL-12 or IFN-γ in the spleen, or increase the expression level of TNF-α, It may decrease the amount of IL-1β, IL-6, or IgE or increase the amount of galectin-9.

According to another embodiment of the present invention, a food composition for preventing or improving inflammation, comprising as an active ingredient a fermented product obtained by culturing Ecklonia cava into a liquid culture medium, inoculating Basidiomycete hyphae, culturing and fermenting, and then treating it with a cellulolytic enzyme. provides.

In the present invention, the inflammation may be allergic rhinitis or asthma.

According to another embodiment of the present invention, a cosmetic composition for preventing or improving inflammation, comprising as an active ingredient the fermented product obtained by culturing Ecklonia cava into a liquid culture medium, inoculating it with Basidiomycete mycelia, culturing and fermenting it, and then treating it with a fibrinolytic enzyme. provides.

In the present invention, the inflammation may be allergic rhinitis or asthma.

The fermented Ecklonia cava product provided in the present invention contains more substances for improving or treating allergic rhinitis and asthma compared to unfermented Ecklonia cava raw material and Ecklonia extract, so its use may be effective in reducing allergic rhinitis or asthma. It is expected that

Figure 1 is a schematic diagram showing the process of an experiment using an animal model of allergic rhinitis and asthma, according to an embodiment of the present invention.
Figure 2 shows the results of examining the effects of Ecklonia cava raw material, Ecklonia cava fermented product, and Ecklonia cava alcohol extract fraction in an animal model of allergic rhinitis and asthma induced by OVA, according to an embodiment of the present invention.
Figure 3 shows the results of administering raw Ecklonia cava, fermented Ecklonia cava, and Ecklonia cava alcohol extract fraction in an animal model of allergic rhinitis and asthma induced by OVA, according to an embodiment of the present invention, and measuring the TLSP concentration in lesion tissue.
Figure 4 shows the immunoglobulin concentration in bronchoalveolar lavage fluid (BALF) and serum after applying Ecklonia cava raw material, Ecklonia cava fermented product, and Ecklonia cava alcohol extract fraction in an animal model of allergic rhinitis and asthma induced by OVA, according to an embodiment of the present invention. This is the result of measurement.
Figure 5 shows the results of measuring Th2 cytokines in bronchoalveolar lavage fluid after applying Ecklonia cava raw material, Ecklonia cava fermented product, and Ecklonia cava alcohol extract fraction in an animal model of allergic rhinitis and asthma induced by OVA, according to an embodiment of the present invention.
Figure 6 shows the results of measuring Th1 cytokines in the blood after applying Ecklonia cava raw material, Ecklonia cava fermented product, and Ecklonia cava alcohol extract fraction in an animal model of allergic rhinitis and asthma induced by OVA, according to an embodiment of the present invention.
Figure 7 shows measurement results by cell type after applying Ecklonia cava raw material, Ecklonia cava fermented product, and Ecklonia cava alcohol extract fraction in an animal model of allergic rhinitis and asthma induced by OVA, according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example 1. Bioconversion process of fermentation and enzyme treatment

Example 1-1. Pre-processing of gamtae and harvesting of gamtae

In order to pre-process the purchased gamtae raw material to obtain fermented gamtae product, the degree of mold contamination of the raw material was measured, and then the raw material of gamtae was washed to remove foreign substances and contaminants. The washing process was performed in three stages: ① air washing step to remove foreign substances and mold spores, ② water washing step to remove heavy metals, etc., and ③ alcohol (Et-OH) washing step to remove contamination such as microorganisms. Washing of raw materials was performed through the steps. After completing the washing process, the raw materials were dried and then subjected to a grinding process to obtain Ecklonia cava.

Example 1-2. Obtaining fermented Ecklonia cava powder

Ecklonia cava from which foreign substances and mold contamination were removed was subjected to enzyme treatment and heat sterilization after adding water to convert it into a liquid culture medium. A hydrolytic enzyme of the cellulase series was used, and after adding the enzyme, it was reacted at 60°C for 1 hour and sterilized at high temperature for 30 minutes to perform culture (fermentation) of Ecklonia cava into a medium. At this time, commercially available enzyme products can be used, and in this case, the test was conducted at the appropriate amount and under appropriate conditions (pH, temperature) suggested in the product instructions.

Afterwards, shiitake mushroom hyphae among the separately cultured basidiomycete hyphae were added to the culture (fermentation) medium. Basidiomycete mycelial seed culture was inoculated at 10% (v/v) and cultured under conditions of 28-30°C and pH 4.5-7. In addition, it can be cultured for 7 to 10 days through a fed-batch culture process in which concentrated liquid Ecklonia cava medium is added when the concentration of the remaining carbon source is depleted below a certain concentration. The above basidiomycete mycelium can be used by selecting any one from the medicinal and edible basidiomycetes, such as shiitake mushroom, saenghang mushroom, chaga mushroom, maitake mushroom, wood ear mushroom, snowflake mushroom, and skirt mushroom. is preferable, and the effect was confirmed to be the same. In addition, these Basidiomycete hyphae are collected from the Korea Center for Microbial Conservation (KCCM), Korea Research Institute of Bioscience and Biotechnology Life Resources Center (KCTC), Korea Cell Line Bank (KCLB), Korea Agricultural Microbial Resource Center (KACC), and the American Standard Strain Culture Collection Center ( Strains can be purchased and used from organizations such as ATCC).

For the enzyme treatment of the bioconversion process for the fermented product, cellulase, one of the cellulolytic enzymes, was used. At this time, cellulase can be used alone, or commercially available enzyme products containing various enzymes such as cellulase, hemicellulase, pectinase, and glucanase can be used. You can. In this case, it was carried out by adding an appropriate amount (the optimal amount presented in the product instructions for each enzyme). Enzymes that are sold commercially include the fibrinolytic enzyme Laminex (complex derived from Tricoderma reesei), Viscozyme (complex derived from Aspergillus), Cellulase (complex derived from Aspergillus niger), and Filtrase (complex derived from Aspergillus niger). One or more of the fibrinolytic enzymes can be selected and used, such as Rapidase (a complex derived from Tricoderma reesei), Rapidase (a complex derived from Aspergillus niger), and Sumizyme (a complex containing pectinase derived from Aspergillus niger). It was confirmed that NEX is preferable and that the effect is the same. The recommended concentration (0.05%) suggested in the product description of the Laminex enzyme was added, and the enzyme/substrate reaction was performed by rotating at 250 rpm for 1 to 3 hours at 50 to 60°C.

The fermented Ecklonia cava product produced through the bioconversion process was subjected to an enzyme inactivation process and sterilization process at 90°C for 1 hour, then freeze-dried and powdered.

Example 2. Evaluation of the efficacy of fermented Ecklonia cava in allergic rhinitis/asthma model

Experimental animals .

The mice used in the experiment were 5-week-old female BALB/c mice purchased from Orient Bio (Seongnam, Korea), and were raised with sufficient water and feed while maintaining the room temperature at 22 ± 2°C until used in the experiment. .

Animal testing to evaluate allergic rhinitis/asthma efficacy.

To induce allergic rhinitis and asthma, mice were randomly separated into 10 mice per group, and after one week of adaptation diet, they were stimulated by intraperitoneal injection of OVA (Ovalbumin) and aluminum hydroxide three times at one-week intervals. Starting 16 days after the start of stimulation, the developed material was administered dietaryly according to the daily intake. Afterwards, individual material dietary administration was carried out for 10 days (days 16 to 25 from the start of stimulation), and from the 11th day (day 25 from the start of stimulation) OVA stimulation and individual material dietary extract destimulation were repeated for 5 days, and on the 6th day of repeated stimulation ( Mice were sacrificed (30 days after stimulation began), BALF (bronchoalveolar lavage fluid) was extracted, and blood was collected from the heart to evaluate the effects of fermented Ecklonia cabbage on suppressing allergic rhinitis and asthma. The progress of the animal experiment is shown in Figure 1.

ELISA.

Blood collected from the heart was treated at 4°C for 30 minutes to induce blood coagulation, and then centrifuged at 2,000g for 30 minutes to separate serum. The amount of immunoglobulin in the separated serum was measured using a mouse immunoglobulin ELISA kit (Abcam, Cambridge, Cambridgeshire, England). BALF (bronchoalveolar lavage fluid) was placed in TNT lysis buffer (10mM Tris-HCl, 150mM NaCl, 0.05% Tween 20, pH 8.0), disrupted using a homogenizer, and then centrifuged at 13,000rpm for 30 minutes to obtain the supernatant. recovered. The amount of TSLP (Thymic Stromal Lymphopoietin) in the supernatant was measured using the mouse TSLP quantikine ELISA kit (R&D systems, Minneapolis, MN).

Result 1. Evaluation of anti-allergic efficacy in OVA-induced allergic rhinitis/asthma mouse model

An increase in infiltrating cells in the lesion tissue of mice suffering from allergic rhinitis and asthma was confirmed. It was confirmed that the infiltration of immune cells increased significantly compared to normal mice due to the induction of allergic rhinitis. In the case of mice administered Ecklonia mackerel and Ecklonia root extract fraction, a slight inhibitory effect was observed, but in fermented Ecklonia cava, the infiltrating cells were significantly reduced compared to the control group (OVA) and compared to the Ecklonia cava root and alcohol extract fraction, showing the most outstanding effect. It was shown to have inhibitory activity. The results are shown in Figure 2.

Result 2. TLSP measurement in lesion tissue in OVA-induced allergic rhinitis/asthma mouse model

The amount of TSLP in the bronchoalveolar lavage fluid (BALF) of mice with allergic rhinitis and asthma was measured to confirm the effect of fermented Ecklonia cabbage on inhibiting TSLP expression. After sacrificing mice, the amount of TSLP in BALF (bronchoalveolar lavage fluid) was measured using ELISA. As a result of inducing allergic rhinitis or asthma, the expression level of TSLP in BALF increased by about 70% compared to the normal control group. The expression level of TSLP increased by OVA decreased by about 10% and 21% compared to the increase in Ecklonia cava raw material and Ecklonia cava alcohol extract fractions, confirming a slight inhibitory effect. On the other hand, it was confirmed that fermented Ecklonia cabbage had significantly improved inhibitory activity compared to the raw product by drastically reducing it to about 70%.

In allergic rhinitis and asthma, TSLP produced by keratinocytes has been reported to induce chronic Th2 inflammatory response by activating dendritic cells. Therefore, it is expected that administration of fermented Ecklonia cabbage can alleviate allergic rhinitis or asthma by controlling chronic inflammatory responses by suppressing the production of TSLP. The results are shown in Figure 3.

Result 3. Investigation to evaluate anti-allergic efficacy in OVA-induced allergic rhinitis/asthma mouse model: evaluation of immunoglobulins in BALF (bronchoalveolar lavage fluid) and serum

The amount of immunoglobulins in the BALF and serum of mice with allergic rhinitis and asthma was measured to confirm the inhibitory effect of fermented Ecklonia cabbage on immunoglobulin production. After sacrificing the mouse, the amount of IgE present in BALF (bronchoalveolar lavage fluid), blood was collected from the heart, serum was separated, and the amount of immunoglobulins (IgG, IgG1, IgG2a) present in the serum were measured using ELISA method. As a result, allergic rhinitis or As asthma was induced, compared to the control group, which was a normal tissue, the production of IgE in BALF and IgG, IgG1, and IgG2a in serum increased significantly by about 18-fold, 28-fold, 18-fold, and 5-fold, respectively. IgE in BALF increased by OVA was about 25% and 27% when applying Ecklonia cava raw material and Ecklonia cava alcohol extract fraction compared to the control group treated only with OVA, and IgG in serum increased by OVA was about 18% and 23%, respectively. IgG1 decreased by approximately 18% and 24%, and IgG2a decreased by approximately 16% and 14%, respectively.

On the other hand, in fermented Ecklonia cabbage, IgE production in BALF decreased by about 60%, IgG in serum decreased by about 50%, IgG1 decreased by about 56%, and IgG2a decreased by about 29%, showing a dramatically improved inhibitory activity compared to raw Ecklonia cabbage. Confirmed. In addition, it was confirmed that the effect of suppressing immunoglobulin activity in fermented Ecklonia cabbage was much more excellent than that of the commercially available product (PLAG).

Considering the above results, fermented Ecklonia cava is evaluated to be able to effectively suppress allergic rhinitis and asthma. The results are shown in Figure 4.

Result 4. Investigation of Th2 cytokine regulation effect in OVA-induced allergic rhinitis/asthma mouse model: suppression of enhanced Th2 immune response

The amount of Th2 cytokines in the bronchoalveolar lavage fluid of mice with allergic rhinitis and asthma was measured to confirm the effect of the developed material on regulating Th2 immune responses. As a result of confirming the amount of IL-4, IL-5, and IL-13 in bronchoalveolar lavage fluid by ELISA method, the expression of cytokines in OVA-induced allergic rhinitis and asthma mouse model was approximately 8 to 14 times higher than that of normal mice. It was confirmed that it does. In the case of mice treated with Ecklonia cava root extract and Ecklonia cava alcohol extract fractions, IL-4, IL-5, and IL-13 were all decreased by 9% and 14%, respectively, compared to the OVA-induced allergic rhinitis and asthma mouse model (control group). %, 19% and 13%, 21%, and 15%, respectively, a very weak slight inhibitory effect was observed. On the other hand, the inhibitory activity of IL-4, IL-5, and IL-13 in fermented Ecklonia cabbage was significantly improved by 39%, 41%, and 50%, and 33%, 31%, and 28%, respectively, compared to the control (OVA) and Ecklonia root product. showed. Additionally, it showed much higher inhibitory activity than PLAG. The results are shown in Figure 5.

Result 5. Investigation of Th1 cell cytokine regulation effect in OVA-induced allergic rhinitis/asthma mouse model: recovery of decreased Th1 immune response

The amount of Th1 cytokines in the blood of OVA-induced allergic rhinitis and asthma mouse models was measured to confirm the effect of the developed material on regulating Th1 immune responses. As a result of confirming the expression levels of IL-2, IL-12, and IL-10 among Th1 cytokines using the ELISA method, the expression levels of IL-2, IL-12, and IL-10 in the blood increased as allergic rhinitis and asthma were induced. It was confirmed that it was significantly reduced. The cytokine recovery effect was very minimal at 13%, 37%, 15%, and 10%, 31%, and 11%, respectively, in the Ecklonia cava raw material and Ecklonia cava alcohol extract fractions, whereas in mice administered Ecklonia cava fermented product, the control effect (OVA ), it was confirmed that the expression levels of IL-2, IL-12, and IL-10 were dramatically improved, recovering by about 51%, 64%, and 60%, respectively. It also showed much higher improvement than PLAG. Considering the above results, fermented Ecklonia cava is evaluated to be able to effectively suppress allergic rhinitis or asthma through recovery of the Th1 immune response. The results are shown in Figure 6.

Result 6. Investigation of anti-allergy control efficacy in cells isolated from OVA-induced allergic rhinitis/asthma mouse model: recovery of enhanced immune response

The amount of immune cells isolated from mice suffering from allergic rhinitis and asthma was measured to confirm the effect of the developed material on regulating immune responses. As allergic rhinitis and asthma were induced, it was confirmed that the number of Lymphocyte, Neutrophil, Macrophage, and Eosinophil cells increased by about 12-fold, 11-fold, 5-fold, and 49-fold, respectively, compared to immune cells isolated from normal mice. In the case of the number of immune cells measured after treatment with Ecklonia cava raw material and Ecklonia cava alcohol extract fractions, all decreased by 12%, 13%, 17%, 13%, and 14%, 19%, and 8%, respectively, compared to the control group (OVA). , a slight inhibitory effect was observed at 9%. On the other hand, in fermented Ecklonia cava, the number of Lymphocyte, Neutrophil, Macrophage, and Eosinophil cells was confirmed to be reduced to approximately 42%, 29%, 20%, and 25% higher than the control group (OVA). In addition, it was confirmed that the inhibitory ability was greatly improved compared to the raw Ecklonia cava and Ecklonia cava alcohol extract fractions. And it showed much improved inhibitory activity than PLAG. The results are shown in Figure 7.

Fermented Ecklonia cava, described in detail in the present invention, showed a much more improved effect than Ecklonia cava root, Ecklonia cava extract, and PLAG in improving typical allergic rhinitis and asthma. It is known that when allergic rhinitis or asthma is induced, the expression of immunoglobulins, especially IgE, at the lesion site and in the serum increases significantly. In the present invention, as allergic rhinitis and asthma were induced, it was confirmed that the production of IgE in the lesion area and IgG, IgG1, and IgG2a in the serum increased significantly by about 18 times, 28 times, 18 times, and 4.2 times, respectively, compared to the control group, which was a normal tissue. did. Accordingly, when Ecklonia cava raw material and Ecklonia cava alcohol extract fraction were treated, IgE production decreased by 25% and 21%, respectively. Of note, it was confirmed that the fermented Ecklonia cava product described in the present invention had the effect of dramatically reducing the production of IgE in BALF by about 60%.

Additionally, in allergic rhinitis and asthma, TSLP has been reported to induce chronic Th2 inflammatory response by activating dendritic cells. In the present invention, when fermented Ecklonia cava was administered, the inhibitory activity against TSLP was about 70%, which confirmed that it had excellent inhibitory activity. Therefore, it is expected that it will be able to alleviate allergic rhinitis or asthma by improving the chronic inflammatory response with a significantly improved effect than the 5% and 10% inhibitory effects shown by the Ecklonia cava root and the Ecklonia cava alcohol extract fraction, respectively.

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (12)

(a) Powdering Ecklonia cava into liquid culture medium;
(b) performing a bioconversion fermentation process of culturing and fermenting the liquid Ecklonia cava grown in the culture medium of step (a) with shiitake mushroom mycelia;
(c) performing a bioconversion enzyme treatment process of treating each fermented product produced by the bioconversion fermentation process in step (b) with a cellulolytic enzyme; and
(d) A method for producing a composition for treating inflammation comprising the fermented Ecklonia cava produced in step (c),
The method wherein the inflammation is allergic rhinitis or asthma.
According to clause 1,
The method of converting the Ecklonia cava culture medium in step (a) is to powder the Ecklonia cava, treat the Ecklonia cava powder in a liquid phase with a hydrolytic enzyme, and then sterilize it to make a liquid Ecklonia cava medium.
According to clause 2,
The method wherein the hydrolytic enzyme is a hydrolytic enzyme of the cellulase family.
delete According to clause 1,
The method wherein the cellulolytic enzyme in step (C) is cellulase alone or a combination of one or more selected from the group consisting of cellulase, hemicellulase, pectinase, or glucanase.
According to clause 1,
The fermented Ecklonia cava product in step (d) suppresses the secretion of IgE and secretes TSLP, which plays a role in the onset of allergic rhinitis or asthma, along with Th2 cytokines such as IL-4, IL-5, or IL-13. How to suppress.
A pharmaceutical composition for preventing or treating inflammation comprising fermented Ecklonia cava by inoculating shiitake mushroom mycelium into Ecklonia cava,
A pharmaceutical composition wherein the inflammation is allergic rhinitis or asthma.
delete A food composition for preventing or improving inflammation comprising fermented Ecklonia cava by inoculating shiitake mushroom mycelium into Ecklonia cava,
A food composition wherein the inflammation is allergic rhinitis or asthma.
delete A cosmetic composition for preventing or improving inflammation containing fermented Ecklonia cava by inoculating shiitake mushroom mycelium into Ecklonia cava,
A cosmetic composition wherein the inflammation is allergic rhinitis or asthma. delete