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WO2023038240A1 - Pharmaceutical composition for preventing or treating diseases caused by influenza a virus and enhancing immunity comprising castanea crenata sieb - Google Patents

Pharmaceutical composition for preventing or treating diseases caused by influenza a virus and enhancing immunity comprising castanea crenata sieb Download PDF

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Publication number
WO2023038240A1
WO2023038240A1 PCT/KR2022/008646 KR2022008646W WO2023038240A1 WO 2023038240 A1 WO2023038240 A1 WO 2023038240A1 KR 2022008646 W KR2022008646 W KR 2022008646W WO 2023038240 A1 WO2023038240 A1 WO 2023038240A1
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WIPO (PCT)
Prior art keywords
influenza
virus
chestnut honey
honey
immune function
Prior art date
Application number
PCT/KR2022/008646
Other languages
French (fr)
Korean (ko)
Inventor
김세건
한상미
우순옥
김효영
최홍민
이혜진
문효정
최장기
권은빈
김영수
정환석
Original Assignee
대한민국(농촌진흥청장)
한국 한의학 연구원
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Publication date
Priority claimed from KR1020210120737A external-priority patent/KR20230037778A/en
Priority claimed from KR1020210120738A external-priority patent/KR20230037779A/en
Application filed by 대한민국(농촌진흥청장), 한국 한의학 연구원 filed Critical 대한민국(농촌진흥청장)
Publication of WO2023038240A1 publication Critical patent/WO2023038240A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/63—Arthropods
    • A61K35/64—Insects, e.g. bees, wasps or fleas
    • A61K35/644—Beeswax; Propolis; Royal jelly; Honey
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/49—Fagaceae (Beech family), e.g. oak or chestnut
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention confirms the antiviral activity effect and immune function enhancing effect of chestnut honey on influenza A virus, and various compositions for preventing, improving or treating diseases caused by influenza A virus including chestnut honey, or , It relates to a composition for various uses for enhancing immune function.
  • Immunity is a body's defense against external factors, and the human body's immune system is maintained by interactions between various immune cells such as monocytes, neutrophils, and phagocytes. Among them, macrophages phagocytose foreign antigens, and inflammatory factors such as nitric oxide (NO), TNF- ⁇ (tumor necrosis factor- ⁇ ), IL-1 ⁇ (Interleukin-1 ⁇ ), and IL-6, which are immune regulators.
  • NO nitric oxide
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-1 ⁇ Interleukin-1 ⁇
  • IL-6 interleukin-6
  • Cytokines pro-inflammatory cytokine
  • innate immunity such as natural killer cells and dendritic cells
  • foreign antigens and delivering them to T cells to activate and It is known to contribute to the activation of adaptive immunity by regulating differentiation.
  • influenza virus is an RNA enveloped virus and infects humans and animals, causing respiratory complications with high morbidity and mortality.
  • Influenza viruses basically consist of an internal nucleocapsid or core of RNA associated with a nucleoprotein surrounded by a viral envelope with a lipid bilayer structure and external glycoproteins.
  • the inner layer of the viral envelope is mainly composed of matrix proteins, and the outer layer is mostly composed of host-derived lipid substances.
  • Influenza virus contains two surface antigens, the glycoproteins neuraminidase (NA) and hemagglutinin (HA), which appear as spikes 10 to 12 nm long on the surface of the particle. . It is these surface proteins, in particular hemagglutinin, that determine the antigenic specificity of influenza subtypes.
  • NA glycoproteins neuraminidase
  • HA hemagglutinin
  • influenza A virus In the case of influenza A virus, it is classified according to the serological characteristics of HA and NA subtypes, and 18 HA subtypes (H1 - H18) and 11 NA subtypes (N1 - N11) have been reported.
  • Influenza virus causes a serious global disease that kills 25 - 500,000 people worldwide each year, infects 10 - 40% of children, and causes economic loss to countries. After the Spanish flu pandemic in 1918, the Asian flu in 1957, and the Hong Kong flu in 1968, influenza pandemics appeared every few decades. died. Due to the possibility of a highly contagious new mutant outbreak in the southern hemisphere, pandemic measures are being taken, but social repercussions and concerns are not diminishing.
  • Tamiflu oseltamivir
  • zanamivir trade name: Relenza
  • peramivir en: Peramivir
  • amantadine en: Amantadine
  • Oseltamivir also called Tamiflu
  • Tamiflu is an antiviral drug that has a therapeutic effect by blocking the function of an enzyme that proliferates the virus, and is most effective when taken within 48 hours of the onset of symptoms.
  • the main treatment effects are reduction of flu symptoms exacerbation, reduction of secondary complications such as bronchitis or pneumonia, and reduction of incubation period of flu.
  • Zanamivir is also known as Zanamivir and the trade name is Relenza. It acts as a neuraminidase inhibitor and is used for the treatment of influenza types A and B.
  • oseltamir has a side effect of severe vomiting, and zanamivir has a high antiviral effect, but has a low bioavailability and rapid excretion from the kidney.
  • chestnut honey While studying the efficacy of chestnut honey, the present inventors found that chestnut honey induces the expression of factors related to innate immunity, shows an antiviral effect against influenza A virus, and phosphorylates molecules related to activation of interferon signaling pathway. The present invention was completed by confirming that it induces.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by influenza A virus.
  • an object of the present invention is to provide a food composition for preventing or improving diseases caused by influenza A virus.
  • Another object of the present invention is to provide a health functional food composition for preventing or improving diseases caused by influenza A virus.
  • Another object of the present invention is to provide a feed additive composition for preventing or improving diseases caused by influenza A virus.
  • Another object of the present invention is to provide a method for treating diseases caused by influenza A virus.
  • Another object of the present invention is to provide a pharmaceutical composition for enhancing immune function.
  • Another object of the present invention is to provide a food composition for enhancing immune function.
  • Another object of the present invention is to provide a health functional food composition for enhancing immune function.
  • Another object of the present invention is to provide a composition for feed additives for enhancing immune function.
  • Another object of the present invention is to provide a method for enhancing immune function.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases caused by influenza A virus containing chestnut honey.
  • the present invention provides a food composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
  • the present invention provides a health functional food composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
  • the present invention provides a feed additive composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
  • the present invention provides a method for treating a disease caused by influenza A virus comprising the step of administering chestnut honey to a subject.
  • the present invention provides a pharmaceutical composition for enhancing immune function containing chestnut honey.
  • the present invention provides a food composition for enhancing immune function containing chestnut honey.
  • the present invention provides a health functional food composition for enhancing immune function containing chestnut honey.
  • the present invention provides a feed additive composition for enhancing immune function containing chestnut honey.
  • the present invention provides a method for enhancing immune function comprising administering chestnut honey to a subject.
  • the present invention confirms the antiviral effect and innate immunity enhancement effect of chestnut honey against influenza A virus.
  • chestnut honey is an expression of influenza A virus protein and the expression of inflammatory cytokines increased by its infection and NF-kB It was confirmed that it reduced the activity of influenza A virus, significantly reduced the mortality rate of animal models infected with influenza A virus, and suppressed weight loss caused by viral infection.
  • phosphorylation of factors involved in the expression of innate immunity-related factors and the activation of immune-related signaling pathways were induced when administered to normal cells, and chestnut honey was used for various purposes for preventing, improving or treating diseases caused by influenza A virus. It can be usefully used as a supplement, or it can be usefully used for various purposes for enhancing immune function.
  • Figure 1a is a result of confirming intracellular fluorescence of influenza A virus infected with chestnut honey.
  • Figure 1b is a result of confirming the effect of chestnut honey on influenza A virus infection, quantifying intracellular fluorescence of 1a (A) and confirming the effect of chestnut honey on influenza A virus protein expression (B).
  • significance was confirmed by performing Two-way ANOVA with Tukey's post hoc, *** means P ⁇ 0.001 value compared to the control result value, ## and ### mean P ⁇ 0.01 and P ⁇ 0.001 values, respectively, compared to the result values of the virus infected group (vehicle).
  • Figure 2 is a result of confirming the effect of chestnut honey on the expression or activity of inflammatory factors caused by influenza A virus infection.
  • A is the result of confirming TNF- ⁇ expression
  • C in FIG. 2 is the result of confirming the NF-kB activity.
  • significance was confirmed by performing two-way ANOVA accompanied by Tukey's post hoc, and ** and *** were respectively P ⁇ 0.1, P ⁇ 0.001 value, and #, ## and ### mean P ⁇ 0.05, P ⁇ 0.01, P ⁇ 0.001 values, respectively, compared to the result value of the virus infected group (vehicle).
  • Figure 3 is a result of confirming the effect of chestnut honey on the expression of cytokines related to the enhancement of immune function.
  • A is the result of confirming the expression of TNF- ⁇
  • B of FIG. 2 is the result of confirming the expression of IL-6.
  • the significance was confirmed by performing a two-way ANOVA with Tukey's post hoc, and *, **, and *** are P ⁇ 0.5, respectively, compared to the control result value. P ⁇ 0.01, P ⁇ 0.001 means the value.
  • Figure 4 is the result of confirming the effect of chestnut honey on the phosphorylation of molecules related to the activation of the interferon signaling pathway.
  • Figure 5 is the result of confirming the death rate (A) and weight change (B) after administering chestnut honey to an animal model infected with influenza virus.
  • the present invention provides a pharmaceutical composition for preventing or treating diseases caused by influenza A virus, including chestnut honey.
  • the present invention provides a pharmaceutical composition for enhancing immune function containing chestnut honey.
  • the "chestnut honey” is a honey with a chestnut flower as a source of honey, has a dark brown or black color, and is characterized by a bitter taste compared to other honey.
  • the chestnut honey of the present invention is chestnut honey itself, an extract extracted with a solvent known in the art, a diluted solution diluted with a solvent known in the art, a concentrate concentrated by a known method, a dried product dried by a known method, or a known method It can be provided as a manufactured purified product.
  • the known concentration method is not limited thereto, but may be concentrated using, for example, precipitation concentration, evaporation concentration, vacuum concentration, ultrafiltration, reverse osmosis, or centrifugation.
  • the known drying method is not limited thereto, but may be dried using, for example, freeze drying, spray drying or hot air drying.
  • a powdering process may be further included as needed.
  • the purified product may be prepared by a known filtration method, but is not limited thereto, and may be prepared by, for example, filtration using a filter net or microfilter, centrifugation, or separatory funnel.
  • the chestnut honey showed an effect of reducing the expression of influenza virus proteins in a concentration-dependent manner in cells infected with influenza A virus.
  • the chestnut honey is any selected from the group consisting of TNF- (tumor necrosis factor- ⁇ ) and IL-6 (Interleukin-6), which are inflammatory cytokines increased by influenza A virus infection It showed the effect of reducing the expression of one and the activity of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells).
  • TNF- tumor necrosis factor- ⁇
  • IL-6 Interleukin-6
  • chestnut honey can be usefully utilized in various fields for the prevention, improvement, or treatment of diseases caused by influenza A virus infection
  • influenza A virus of the present invention is preferably an H1N1 strain influenza A virus.
  • the disease caused by the influenza A virus may be at least one selected from the group consisting of bird flu, influenza, cold, sore throat, bronchitis and pneumonia, but is not limited thereto.
  • tumor necrosis factor- ⁇ TNF- ⁇
  • interleukin-6 interleukin-6
  • the TNF- ⁇ is a cytokine mainly secreted by activated macrophages, and as a cell signaling protein related to an inflammatory response, it is a cytokine that induces an acute phase response.
  • TNF- ⁇ is mainly secreted by activated macrophages, and is also produced by various types of cells such as CD4 lymphocytes, NK cells, neutrophils, and mast cells.
  • a major role of TNF- ⁇ is the regulation of the activity and function of immune cells.
  • it is also known as an endogenous pyrogen, and it is known to induce fever, mediate cell death and inflammatory response, and perform functions to suppress tumor formation and viral replication.
  • the IL-6 also induces fever and stimulates the liver to induce the production of acute phase proteins such as CRP (Creactive protein).
  • CRP reactive protein
  • chestnut honey is TANK-binding enzyme 1 (TANK-binding kinase 1, TBK1) and signal transducer-transcription activator 1 (Signal transducer) involved in the activation of the interferon signaling pathway and activator of transcription 1, STAT1) phosphorylation was induced.
  • TANK-binding enzyme 1 TANK-binding kinase 1, TBK1
  • Signal transducer-transcription activator 1 Signal transducer-transcription activator 1
  • the interferon is a natural protein made in the immune cells of vertebrates, activates innate immune cells such as NK cells and macrophages, and increases the expression of the major histocompatibility complex (MHC) to macrophages or dendritic cells improve the antigen presenting function of In addition, it activates cell immunity and also acts on cell death.
  • MHC major histocompatibility complex
  • the interferon is classified into type 1 interferon, type 2 interferon and type 3 interferon.
  • the type 1 interferon activates intracellular antimicrobial mechanisms and affects the development of innate and adaptive immune responses.
  • Canonical type 1 interferon signaling activates the Janus Kinase (JAK) - signal transducer and activator of transcription (STAT) pathway, leading to transcription of interferon-stimulated genes (ISGs).
  • JNK Janus Kinase
  • STAT activator of transcription
  • ISGs interferon-stimulated genes
  • the TBK1 phosphorylates STING, a protein that plays a key role in the innate immune response to DNA in the cytoplasm
  • IRF-3 a transcription factor.
  • IRF-3 phosphorylated by TBK1 regulates the expression of type I interferons after dimerization and translocation to the nucleus.
  • chestnut honey since chestnut honey induces the expression and phosphorylation of factors that play an important role in enhancing immune function, it can be used to enhance the immune function of individuals with reduced or suppressed immune function.
  • the lowering or suppression of the immune function may be caused by an immune-related disease such as acquired immunodeficiency syndrome or by a reason such as radiation treatment.
  • the pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. there is.
  • the pharmaceutical composition of the present invention may additionally include any one or more selected from pharmaceutically acceptable carriers, excipients and diluents.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the chestnut honey of the present invention, for example, starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin, etc.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions.
  • As a base for the suppository witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
  • the dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 0.1 mg/kg/day to 1000 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.
  • the pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection.
  • the pharmaceutical composition for preventing or treating a disease caused by influenza A virus infection has already been verified for safety and is known to have antiviral activity against influenza A virus in order to increase the effect of preventing or treating a disease, in addition to the active ingredient. It may further include any compound or natural extract.
  • composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of diseases caused by influenza A virus.
  • the pharmaceutical composition for enhancing immune function may further include, in addition to the active ingredient, any compound or natural extract whose safety has already been verified and is known to have an immune function enhancing activity for enhancing immune function. .
  • composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers to enhance immune function.
  • the present invention provides a food composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
  • the present invention provides a health functional food composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
  • the present invention provides a food composition for enhancing immune function containing chestnut honey.
  • the present invention provides a health functional food composition for enhancing immune function containing chestnut honey.
  • the food composition according to the present invention includes all types of functional food, nutritional supplements, health food and food additives.
  • the term "health functional food” refers to a food manufactured and processed by using a specific ingredient as a raw material or by extraction, concentration, purification, mixing, etc. It refers to food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. by the above components, and performs functions related to disease prevention or health recovery. say what you can do
  • Food compositions of this type can be prepared in various forms according to conventional methods known in the art.
  • the type of food composition may be formulated in any one form selected from the group consisting of powders, tablets, capsules, pills and liquids according to conventional methods known in the art, but is not limited thereto. It can be prepared in various forms using methods known in the art.
  • the chestnut honey of the present invention may be granulated, encapsulated, and powdered to be ingested or prepared in the form of tea, juice, and drink to be consumed.
  • the chestnut honey of the present invention can be prepared in the form of a composition by mixing it with a known substance or active ingredient known to have an antiviral effect against influenza A virus.
  • functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled food, jam, marmalade, etc.), fish, meat, and their processed foods (e.g., ham, sausage corned beef, etc.) , breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort It can be prepared by adding the chestnut honey of the present invention to food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).
  • fruits and their processed foods e.g., canned fruit, bottled food, jam, marmalade, etc.
  • fish, meat, and their processed foods e.g., ham, sausage corned beef, etc.
  • breads and noodles e.g. udon,
  • the preferred content of the chestnut honey of the present invention in the food composition of the present invention is not limited thereto, but may be, for example, 0.01 to 80% by weight of the final food, preferably 0.01 to 50% of the final food. weight percent.
  • the chestnut honey of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.
  • the present invention provides a feed additive composition for preventing or improving diseases caused by influenza A virus, including chestnut honey.
  • the present invention provides a composition for feed additives for enhancing immune function containing chestnut honey.
  • feed of the present invention refers to any natural or artificial oral diet, one meal meal, etc., or a component of the one meal meal eaten by animals, and the feed containing the composition for feed addition according to the present invention as an active ingredient is It can be prepared with various types of feed known in the art, preferably, concentrated feed, roughage and / or special feed may be included, but is not limited thereto.
  • composition for feed additive may mean “feed additive”, which alleviates symptoms of disease in animals, supplements nutrients and prevents weight loss, enhances the digestibility of fiber in feed, improves milk quality, and breeds. It includes substances added to feed for the purpose of various effects, such as preventing disorders, improving conception rates, and preventing high-temperature stress in the summer.
  • the composition for feed additive of the present invention corresponds to supplementary feed under the Feed Management Act, and is a mineral preparation such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, and a trace mineral preparation such as zinc, copper, cobalt, and selenium.
  • vitamins such as kerotene, vitamins A, D, E, nicotinic acid, and vitamin B complex, protected amino acids such as methionine and lysine, protected fatty acids such as calcium salts of fatty acids, probiotics (lactic acid bacteria), yeast cultures, and fungal fermentation products. Probiotics, yeasts, and the like may be further included.
  • the enriched feed includes seed fruits including grains such as wheat, oats and corn, bran including rice bran, bran, barley bran, etc. as a by-product obtained by refining grains, soybeans, fluids, sesame seeds, linseed, coco Fish meal, which is a by-product obtained from oil extraction of palms, residual starch, which is the main component of starch residue, which is the remainder after removing starch from sweet potatoes, potatoes, etc., fish meal, fish residue, and fresh liquid obtained from fish Animal feed such as fish soluble, meat meal, blood meal, feather meal, skim milk powder, dried whey, which is the balance when manufacturing cheese from milk and casein from skim milk, yeast, chlorella , algae, but not limited thereto.
  • seed fruits including grains such as wheat, oats and corn, bran including rice bran, bran, barley bran, etc.
  • bran including rice bran, bran, barley bran, etc.
  • by-product obtained by refining grains soybeans
  • Forages among the feed include raw grass feed such as wild grass, grass, green cutting, turnip for feed, beet for feed, root vegetables such as Lutherbearer, a kind of turnip, raw grass, green crops, grain Silage, which is a stored feed made by lactic acid fermentation by filling the back into a silo, wild grass, hay dried by cutting grass, straw of breeding crops, and leaves of legumes, but is not limited thereto.
  • Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea or its derivative, diureide isobutane, and supplements for ingredients that are likely to be insufficient when only natural feed ingredients are mixed, or formulated feeds to improve the storability of feeds.
  • feed additives and dietary supplements which are substances added in small amounts, but are not limited thereto.
  • the feed composition according to the present invention can be prepared by adding chestnut honey in an appropriate effective concentration range according to various feed manufacturing methods known in the art.
  • composition for feed additives according to the present invention can be applied without limitation to an individual who aims to prevent influenza A virus, alleviate or treat symptoms infected with the virus, or enhance immune function.
  • it can be applied to any object such as non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds and fish.
  • the present invention provides a method for treating a disease caused by influenza A virus comprising administering chestnut honey to a subject.
  • the subject is preferably a mammal, including a human, and is a patient in need of treatment for a disease caused by influenza A virus infection, a patient in need of treatment for a disease caused by influenza A virus infection, and a disease treatment caused by influenza A virus infection.
  • a mammal including a human
  • the chestnut honey of the present invention can be treated in combination with drugs or treatment methods for the treatment of diseases caused by influenza A virus infection.
  • the chestnut honey of the present invention can be treated simultaneously or sequentially with other drugs or treatment methods for the treatment of diseases caused by influenza A virus infection.
  • the present invention provides a method for enhancing immune function comprising administering chestnut honey to a subject.
  • the subject is preferably a mammal, including a human, and is a patient in need of prevention, improvement or treatment of symptoms and diseases caused by reduced immune function, a patient who needs treatment for symptoms and diseases caused by reduced immune function, and immune function It includes all patients who are undergoing treatment for symptoms and diseases caused by lowered immune function, patients who have been treated for symptoms and diseases caused by lowered immune function, and patients who need to receive treatment for symptoms and diseases caused by lowered immune function. Patients who have undergone surgical operations for the treatment of symptoms and diseases may also be included.
  • the chestnut honey of the present invention can be treated in combination with drugs or treatment methods for the treatment of symptoms and diseases caused by reduced immune function.
  • the chestnut honey of the present invention can be treated simultaneously or sequentially with other drugs or treatment methods for the treatment of symptoms and diseases caused by reduced immune function.
  • RAW 264.7 cells were cultured in a 24-well plate, and then cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Fetal bovine serum) and 1% penicillin/streptomycin at 1.25, 2.5, and 5 Alternatively, chestnut honey diluted in distilled water at a concentration of 10 mg/mL (purchased from an open market) was treated and cultured for 18 hours.
  • influenza A/PR8/34-GFP 10 MOI
  • DMEM fetal calf serum
  • proteins were separated and electrophoresis was performed on an 8-12% acrylamide gel with the same protein amount using the Bradford method. Afterwards, the protein was transferred with PVDF (Polyvinylidene fluoride), and the non-specific binding of the protein was blocked with bovine serum albumin (BSA), and influenza A virus proteins HA (Hemagglutinin), PB1, PA, and M1 from genetex were used. , The target protein was detected using a secondary antibody (IgG-HRP from santa cruz) specifically binding to the M2 antibody.
  • PVDF Polyvinylidene fluoride
  • CON is a negative control cell group not treated with anything
  • vehicle is an A/PR8/34-GFP virus infection group
  • IFN- ⁇ is a positive control group
  • A/PR8 /34-GFP virus infection 1,000 Units/ml IFN- ⁇ treatment group.
  • 1.25 is the A/PR8/34-GFP virus infection and 1.25 mg/mL chestnut honey treatment group
  • 2.5 is the A/PR8/34-GFP virus infection and 2.5 mg/mL chestnut honey treatment group
  • 5 is the A/PR8/34 -GFP virus infection and 5 mg / mL chestnut honey treatment group
  • 10 A / PR8 / 34-GFP virus infection and 10 mg / mL chestnut honey treatment group.
  • CON is a negative control cell group without any treatment
  • vehicle is A/PR8/34-GFP is an influenza A virus infection group
  • 1.25 is A /PR8/34-GFP virus infection and 2.5 mg/mL chestnut honey treatment group
  • 2.5 was A/PR8/34-GFP virus infection and 2.5 mg/mL chestnut honey treatment group
  • 5 was A/PR8/34-GFP virus infection And 5 mg / mL chestnut honey treatment group
  • NF-kB activity of NF-kB was confirmed using immunofluorescence (IF). After blocking non-specific binding of the fixed cells using BSA, the specific binding of the primary antibody, CST's NF-kB, and the secondary antibody, Thermo's alexa fluor-568, was confirmed through a fluorescence microscope. Through this, the effect of chestnut honey on the activity of NF-kB, which is increased during influenza A virus infection, was confirmed and shown in C in FIG. 2.
  • IF immunofluorescence
  • Chestnut honey was treated with cells to confirm the effect on immune function-related factors.
  • chestnut honey diluted in distilled water at a concentration of 1.25, 2.5, 5, or 10 mg/mL in a medium (DMEM) supplemented with 10% FBS (Fetal Bovine Serum), 1% penicillin, and 1% streptomycin (purchased from an open market) and 100 ng/mL of LPS (Lipopolysaccharide), respectively.
  • DMEM fetal bovine Serum
  • penicillin Fetal Bovine Serum
  • streptomycin purchased from an open market
  • LPS Lipopolysaccharide
  • RAW 264.7 cells were treated with 100 ng/ml of LPS (control) or 5 mg/ml of chestnut honey, cultured, and the cells of each experimental group were recovered at 0, 6, 12, and 24 hours. Then, it was dissolved and Western blotting was performed. At this time, as a negative control group, cells from the untreated experimental group were also collected at the same time intervals and Western blotting was performed.
  • 6-week-old BALB/c female mice were randomly assigned to each of 4 groups, 10 mice each. Chestnut honey dissolved in sterile distilled water was orally administered once daily for 2 weeks at concentrations of 300 mg/kg and 600 mg/kg. During the 2 weeks of administration, the virus-infected group (Negative) was not administered with chestnut honey and sterile distilled water, and the virus-infected group (Virus control) was administered with sterile distilled water. After infection with influenza A virus (A/PR8/34-GFP) at a concentration of 5LD50 (5-fold half-lethal dose) through the nasal cavity, the virus-infected group and the chestnut honey-administered group were measured for body weight and survival rate every day for 10 days. A weight loss of more than 25% of the initial body weight before infection was regarded as death by sacrifice from an ethical point of view.
  • a weight loss of more than 25% of the initial body weight before infection was regarded as death by sacrifice from an ethical point of view.
  • the 600 mg / kg chestnut honey administration group showed a survival rate of 60% compared to the virus infection group (Virus control). Through this, it was confirmed that the administration of chestnut honey significantly reduced the mortality caused by influenza A virus.
  • the virus control group showed significant weight loss due to influenza A virus infection, while the chestnut honey administration group confirmed that weight loss due to influenza A virus infection was significantly suppressed.
  • chestnut honey reduces the expression of influenza A virus proteins and the increased expression of inflammatory cytokines and the activity of NF-kB by infection, significantly reduces mortality in animal models infected with influenza A virus, and is effective against viral infection. It has been confirmed that it has the effect of inducing phosphorylation of factors involved in the expression of innate immunity-related factors and the activation of immune-related signaling pathways, thereby preventing diseases caused by influenza A virus, It can be usefully used for various purposes for improvement or treatment, and can be usefully used for various purposes for enhancing immune function.

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Abstract

The present invention has confirmed that Castanea crenata Sieb reduces the expression of an influenza A virus protein, the expression of inflammatory cytokines increased by infection therewith, and the activity of NF-kB, remarkably reduces the mortality of an animal model infected with influenza A virus, suppresses weight loss caused by viral infection, and has the effect of inducing the expression of innate immunity-related factors and the phosphorylation of factors involved in the activation of immune-related signaling pathways. Therefore, Castanea crenata Sieb can have various useful applications for the prevention, alleviation or treatment of diseases caused by influenza A virus, and can have various useful applications for enhancing immune function.

Description

밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 치료 및 면역증진용 약학적 조성물Pharmaceutical composition for preventing or treating diseases caused by influenza A virus and enhancing immunity containing chestnut honey
본 발명은 밤꿀의 인플루엔자 A 바이러스(influenza A virus)에 대한 항바이러스 활성 효과 및 면역기능 증진 효과를 확인한 것으로, 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방, 개선 또는 치료를 위한 다양한 용도의 조성물 또는, 면역기능 증진을 위한 다양한 용도의 조성물에 관한 것이다.The present invention confirms the antiviral activity effect and immune function enhancing effect of chestnut honey on influenza A virus, and various compositions for preventing, improving or treating diseases caused by influenza A virus including chestnut honey, or , It relates to a composition for various uses for enhancing immune function.
면역이란 외부 인자에 대한 생체의 방어 수단으로 인체의 면역계는 단핵구(monocyte), 호중구(neutrophile) 및 포식세포(phagocyte) 등과 같은 다양한 면역세포들간의 상호작용에 의하여 그 체계가 유지된다. 그 중 대식세포 (macrophage)는 외부 항원을 포식하고, 면역 조절인자인 NO(nitric oxide)와 TNF-α(tumor necrosis factor-α), IL-1β(Interleukin-1β), IL-6와 같은 염증성 사이토카인 (pro-inflammatory cytokine)을 분비하여 자신의 포식작용을 증가시키거나 자연살해세포, 수지상세포 등의 선천면역의 활성을 유도하는 한편 외부 항원을 인식한 후 T 세포에 전달하여 세포의 활성화와 분화를 조절함으로써 적응 면역의 활성에도 기여하는 것으로 알려져 있다.Immunity is a body's defense against external factors, and the human body's immune system is maintained by interactions between various immune cells such as monocytes, neutrophils, and phagocytes. Among them, macrophages phagocytose foreign antigens, and inflammatory factors such as nitric oxide (NO), TNF-α (tumor necrosis factor-α), IL-1β (Interleukin-1β), and IL-6, which are immune regulators. Cytokines (pro-inflammatory cytokine) are secreted to increase their own phagocytosis or induce activation of innate immunity such as natural killer cells and dendritic cells, while recognizing foreign antigens and delivering them to T cells to activate and It is known to contribute to the activation of adaptive immunity by regulating differentiation.
따라서 인체의 면역기능 저하는 결국 감염 및 질병의 발생뿐만 아니라 종양의 발생 및 암환자의 회복을 지연시키는 요인이 되기도 한다. 이러한 관점에서 면역자극은 체내의 면역 방어체계 향상을 위한 중요한 전략으로 대두되고 있으며 최근 이러한 요구에 부응하여 면역 잠재력을 향상시킬 수 있는 천연물 및 식이소재 개발에 관심이 집중되고 있다.Therefore, the decrease in the immune function of the human body eventually becomes a factor that delays the occurrence of infections and diseases as well as the occurrence of tumors and recovery of cancer patients. From this point of view, immunostimulation has emerged as an important strategy for improving the body's immune defense system, and attention has recently been focused on the development of natural products and dietary materials that can improve immune potential in response to this demand.
한편, 인플루엔자 바이러스(Influenza virus)는 RNA 외피 (enveloped) 바이러스이며 사람과 동물을 감염시켜 높은 이환율과 사망률을 나타내는 호흡기 합병증을 유발한다. 인플루엔자 바이러스는 기본적으로 지질 이층 구조 및 외부 당단백질을 갖는 바이러스 외피에 의해 둘러싸인 핵단백질과 결합된 RNA의 내부 뉴클레오캡시드 또는 코어로 구성된다. 바이러스 외피의 내층은 주로 기질 단백질로 구성되며, 외층은 대부분 숙주 유래된 지질 물질로 구성된다. 인플루엔자 바이러스는 두 개의 표면 항원인 당단백질 뉴라미니다제(neuraminidase, NA) 및 헤마글루티닌(hemagglutinin, HA)을 포함하며, 이들은 입자의 표면에서 10 내지 12 nm 길이의 스파이크(spike)로서 나타난다. 인플루엔자 아형(subtype)의 항원 특이성을 결정하는 것이 바로 이러한 표면 단백질, 특히 헤마글루티닌이다.Meanwhile, influenza virus is an RNA enveloped virus and infects humans and animals, causing respiratory complications with high morbidity and mortality. Influenza viruses basically consist of an internal nucleocapsid or core of RNA associated with a nucleoprotein surrounded by a viral envelope with a lipid bilayer structure and external glycoproteins. The inner layer of the viral envelope is mainly composed of matrix proteins, and the outer layer is mostly composed of host-derived lipid substances. Influenza virus contains two surface antigens, the glycoproteins neuraminidase (NA) and hemagglutinin (HA), which appear as spikes 10 to 12 nm long on the surface of the particle. . It is these surface proteins, in particular hemagglutinin, that determine the antigenic specificity of influenza subtypes.
A형 인플루엔자 바이러스(Influenza A virus)의 경우, HA 및 NA 아형의 혈청학 특성에 따라 분류되며, 18개의 HA 아형 (H1 - H18) 및 11개의 NA 아형 (N1 - N11)이 보고되었다.In the case of influenza A virus, it is classified according to the serological characteristics of HA and NA subtypes, and 18 HA subtypes (H1 - H18) and 11 NA subtypes (N1 - N11) have been reported.
인플루엔자 바이러스는 세계적으로 매년 25 - 50만 명의 목숨을 빼앗고 소아의 10 - 40%가 감염되고 있으며, 국가 경제적 손실을 야기하는 심각한 글로벌 질환을 유발한다. 1918년 스페인 독감 대유행 이후, 1957년 아시아 독감, 1968년 홍콩 독감 등 수십 년 주기로 인플루엔자 대유행이 나타났으며, 2009년 H1N1 인플루엔자 바이러스(2009pdm)의 출현으로 세계적으로 27만명이 감염되었고, 3200명 이상이 사망하였다. 남반구에서 높은 전염성의 새로운 변이주 창궐 가능성 때문에 대유행 대책을 세우고 있음에도 사회적 파장과 우려는 감소하지 않고 있다.Influenza virus causes a serious global disease that kills 25 - 500,000 people worldwide each year, infects 10 - 40% of children, and causes economic loss to countries. After the Spanish flu pandemic in 1918, the Asian flu in 1957, and the Hong Kong flu in 1968, influenza pandemics appeared every few decades. died. Due to the possibility of a highly contagious new mutant outbreak in the southern hemisphere, pandemic measures are being taken, but social repercussions and concerns are not diminishing.
종래 인플루엔자의 치료에 사용되는 약물로는 오셀타미비르(상품명: 타미플루), 자나미비르(상품명: 릴렌자), 페라미비르(en:Peramivir), 아만타딘(en:Amantadine) 등이 있다. 타미플루로 불려지는 오셀타미비르가 현재 Ｈ１Ｎ１ 인플루엔자 A형의 치료에 주로 사용되고 있다. 타미플루는 바이러스를 증식시키는 효소 기능을 막아 치료효과를 내는 항바이러스제이며, 증상이 발생한 뒤 48시간 안에 복용해야 효과가 크다. 주요 치료 효과는 독감 증세의 악화 감소, 기관지염이나 폐렴 등 2차 합병증 발생 감소, 독감 잠복 기간의 감소 등이다. 인플루엔자 A와 B의 치료제로도 쓰인다. 자나미비르는 자나미비어(Zanamivir)로도 불리며 상품명은 릴렌자(Relenza)이다. 이는 뉴라미니다아제 저해제로 작용하여 인플루엔자 A 및 B형의 치료에 사용되고 있다. 그러나, 오셀타미르는 심각한 구토증세가 나타나는 부작용이 있으며 자나미비르는 항바이러스 효과는 높지만 생체 이용율이 낮고 신장에서의 배출이 빠르다는 단점이 있다.Conventional drugs used for the treatment of influenza include oseltamivir (trade name: Tamiflu), zanamivir (trade name: Relenza), peramivir (en: Peramivir), amantadine (en: Amantadine), and the like. Oseltamivir, also called Tamiflu, is currently used primarily for the treatment of H1N1 influenza type A. Tamiflu is an antiviral drug that has a therapeutic effect by blocking the function of an enzyme that proliferates the virus, and is most effective when taken within 48 hours of the onset of symptoms. The main treatment effects are reduction of flu symptoms exacerbation, reduction of secondary complications such as bronchitis or pneumonia, and reduction of incubation period of flu. It is also used as a treatment for influenza A and B. Zanamivir is also known as Zanamivir and the trade name is Relenza. It acts as a neuraminidase inhibitor and is used for the treatment of influenza types A and B. However, oseltamir has a side effect of severe vomiting, and zanamivir has a high antiviral effect, but has a low bioavailability and rapid excretion from the kidney.
이렇듯 현재까지 개발된 항인플루엔자 치료제들은 다양한 부작용에 대한 문제점을 가지고 있기 때문에 인플루엔자의 예방 및 치료에 효과적인 항인플루엔자 조성물의 개발이 시급한 실정이다.As such, since anti-influenza therapeutics developed to date have problems with various side effects, there is an urgent need to develop an anti-influenza composition effective for preventing and treating influenza.
본 발명자들은 밤꿀의 효능에 대하여 연구하던 중, 밤꿀이 선천면역 관련 인자의 발현을 유도하고, 인플루엔자 A 바이러스에 대한 항바이러스 효과를 보이며, 인터페론(Interferon) 신호전달 경로의 활성화와 연관있는 분자의 인산화를 유도하는 것을 확인하여 본 발명을 완성하였다. While studying the efficacy of chestnut honey, the present inventors found that chestnut honey induces the expression of factors related to innate immunity, shows an antiviral effect against influenza A virus, and phosphorylates molecules related to activation of interferon signaling pathway. The present invention was completed by confirming that it induces.
따라서 본 발명의 목적은 인플루엔자 A 바이러스에 의한 질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by influenza A virus.
따라서 본 발명의 목적은 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선용 식품 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a food composition for preventing or improving diseases caused by influenza A virus.
본 발명의 다른 목적은 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선용 건강기능식품 조성물을 제공하는 것이다. Another object of the present invention is to provide a health functional food composition for preventing or improving diseases caused by influenza A virus.
본 발명의 또 다른 목적은 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선을 위한 사료첨가용 조성물을 제공하는 것이다. Another object of the present invention is to provide a feed additive composition for preventing or improving diseases caused by influenza A virus.
본 발명의 또 다른 목적은 인플루엔자 A 바이러스에 의한 질환을 치료하는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating diseases caused by influenza A virus.
본 발명의 또 다른 목적은 면역기능 증진용 약학적 조성물을 제공하는 것이다. Another object of the present invention is to provide a pharmaceutical composition for enhancing immune function.
본 발명의 또 다른 목적은 면역기능 증진용 식품 조성물을 제공하는 것이다. Another object of the present invention is to provide a food composition for enhancing immune function.
본 발명의 또 다른 목적은 면역기능 증진용 건강기능식품 조성물을 제공하는 것이다. Another object of the present invention is to provide a health functional food composition for enhancing immune function.
본 발명의 또 다른 목적은 면역기능 증진을 위한 사료첨가용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for feed additives for enhancing immune function.
본 발명의 또 다른 목적은 면역기능을 증진시키는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for enhancing immune function.
상기 목적을 달성하기 위하여, 본 발명은 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating diseases caused by influenza A virus containing chestnut honey.
또한 상기 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선용 식품 조성물을 제공한다.In addition, in order to achieve the above other object, the present invention provides a food composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, in order to achieve the above another object, the present invention provides a health functional food composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선을 위한 사료첨가용 조성물을 제공한다.In addition, in order to achieve the above another object, the present invention provides a feed additive composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 개체에 투여하는 단계를 포함하는 인플루엔자 A 바이러스에 의한 질환을 치료하는 방법을 제공한다.In addition, in order to achieve the above another object, the present invention provides a method for treating a disease caused by influenza A virus comprising the step of administering chestnut honey to a subject.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 포함하는 면역기능 증진용 약학적 조성물을 제공한다.In addition, in order to achieve the above another object, the present invention provides a pharmaceutical composition for enhancing immune function containing chestnut honey.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 포함하는 면역기능 증진용 식품 조성물을 제공한다.In addition, in order to achieve the above another object, the present invention provides a food composition for enhancing immune function containing chestnut honey.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 포함하는 면역기능 증진용 건강기능식품 조성물을 제공한다.In addition, in order to achieve the above another object, the present invention provides a health functional food composition for enhancing immune function containing chestnut honey.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 포함하는 면역기능 증진을 위한 사료첨가용 조성물을 제공한다.In addition, in order to achieve the above another object, the present invention provides a feed additive composition for enhancing immune function containing chestnut honey.
또한 상기 또 다른 목적을 달성하기 위하여, 본 발명은 밤꿀을 개체에 투여하는 단계를 포함하는 면역기능을 증진시키는 방법을 제공한다.In addition, in order to achieve the above another object, the present invention provides a method for enhancing immune function comprising administering chestnut honey to a subject.
본 발명은 밤꿀의 인플루엔자 A 바이러스에 대한 항바이러스 효과와 선천 면역 증진 효과를 확인한 것으로, 본 발명에 따르면 밤꿀은 인플루엔자 A 바이러스 단백질의 발현 및 이의 감염에 의해 증가된 염증성 사이토카인의 발현과 NF-kB의 활성을 감소시키며 인플루엔자 A 바이러스를 감염시킨 동물 모델의 사망률을 현저히 감소시키고, 바이러스 감염에 의한 체중 감소를 억제함을 확인하였다. 더불어 정상 세포에 투여시 선천면역 관련 인자의 발현 및 면역 관련 신호전달 경로의 활성화에 관여하는 인자의 인산화를 유도함을 확인한 바, 밤꿀을 인플루엔자 A 바이러스에 의한 질환의 예방, 개선 또는 치료를 위한 다양한 용도로 유용하게 활용할 수 있고 또는, 면역기능 증진을 위한 다양한 용도로 유용하게 활용할 수 있다.The present invention confirms the antiviral effect and innate immunity enhancement effect of chestnut honey against influenza A virus. According to the present invention, chestnut honey is an expression of influenza A virus protein and the expression of inflammatory cytokines increased by its infection and NF-kB It was confirmed that it reduced the activity of influenza A virus, significantly reduced the mortality rate of animal models infected with influenza A virus, and suppressed weight loss caused by viral infection. In addition, it was confirmed that phosphorylation of factors involved in the expression of innate immunity-related factors and the activation of immune-related signaling pathways were induced when administered to normal cells, and chestnut honey was used for various purposes for preventing, improving or treating diseases caused by influenza A virus. It can be usefully used as a supplement, or it can be usefully used for various purposes for enhancing immune function.
도 1a는 밤꿀이 처리된 인플루엔자 A 바이러스가 감염된 세포내 형광을 확인한 결과이다.Figure 1a is a result of confirming intracellular fluorescence of influenza A virus infected with chestnut honey.
도 1b는 밤꿀의 인플루엔자 A 바이러스의 감염에 미치는 영향을 확인한 결과로, 상기 1a의 세포내 형광을 정량한 결과(A)와 밤꿀이 인플루엔자 A 바이러스 단백질 발현에 미치는 영향을 확인한 결과(B)이다. 상기 도 1b에 있어서, Tukey’s post hoc를 수반하는 이원분산분석(Two-way ANOVA)를 수행하여 유의성을 확인하였고, ***은 대조군(control) 결과 값 대비 P<0.001 값을 의미하고, ## 및 ###은 바이러스 감염군(vehicle) 결과 값 대비 각각 P<0.01, P<0.001 값을 의미한다.Figure 1b is a result of confirming the effect of chestnut honey on influenza A virus infection, quantifying intracellular fluorescence of 1a (A) and confirming the effect of chestnut honey on influenza A virus protein expression (B). In FIG. 1B, significance was confirmed by performing Two-way ANOVA with Tukey's post hoc, *** means P<0.001 value compared to the control result value, ## and ### mean P<0.01 and P<0.001 values, respectively, compared to the result values of the virus infected group (vehicle).
도 2는 밤꿀의 인플루엔자 A 바이러스 감염에 의한 염증성 인자의 발현 또는 활성에 미치는 영향을 확인한 결과로, 도 2 중 A는 TNF-α 발현을 확인한 결과이고, 도 2 중 B는 IL-6의 발현을 확인한 결과이며, 도 2 중 C는 NF-kB 활성을 확인한 결과이다. 상기 도 2 중 A 및 B에 있어서, Tukey’s post hoc를 수반하는 이원분산분석(Two-way ANOVA)를 수행하여 유의성을 확인하였고, ** 및 ***은 대조군(control) 결과 값 대비 각각 P<0.1, P<0.001 값을 의미하고, #, ## 및 ###은 바이러스 감염군(vehicle) 결과 값 대비 각각 P<0.05, P<0.01, P<0.001 값을 의미한다.Figure 2 is a result of confirming the effect of chestnut honey on the expression or activity of inflammatory factors caused by influenza A virus infection. In FIG. 2, A is the result of confirming TNF-α expression, and in FIG. This is the confirmation result, and C in FIG. 2 is the result of confirming the NF-kB activity. In A and B of FIG. 2, significance was confirmed by performing two-way ANOVA accompanied by Tukey's post hoc, and ** and *** were respectively P< 0.1, P<0.001 value, and #, ## and ### mean P<0.05, P<0.01, P<0.001 values, respectively, compared to the result value of the virus infected group (vehicle).
도 3은 밤꿀의 면역기능 증진 관련 사이토카인의 발현에 미치는 영향을 확인한 결과로, 도 3 중 A는 TNF-α 발현을 확인한 결과이고, 도 2 중 B는 IL-6의 발현을 확인한 결과이다. 상기 도 3에 있어서, Tukey’s post hoc를 수반하는 이원분산분석(Two-way ANOVA)를 수행하여 유의성을 확인하였고, *, ** 및 ***은 대조군(control) 결과 값 대비 각각 P<0.5, P<0.01, P<0.001 값을 의미한다.Figure 3 is a result of confirming the effect of chestnut honey on the expression of cytokines related to the enhancement of immune function. In FIG. 3, A is the result of confirming the expression of TNF-α, and B of FIG. 2 is the result of confirming the expression of IL-6. In FIG. 3, the significance was confirmed by performing a two-way ANOVA with Tukey's post hoc, and *, **, and *** are P<0.5, respectively, compared to the control result value. P<0.01, P<0.001 means the value.
도 4는 밤꿀이 인터페론 신호전달 경로의 활성화와 관련있는 분자들의 인산화에 미치는 영향을 확인한 결과이다.Figure 4 is the result of confirming the effect of chestnut honey on the phosphorylation of molecules related to the activation of the interferon signaling pathway.
도 5는 인플루엔자 바이러스에 감염된 동물 모델에 밤꿀을 투여한 후, 이의 사망률(A) 및 체중 변화(B)를 확인한 결과이다.Figure 5 is the result of confirming the death rate (A) and weight change (B) after administering chestnut honey to an animal model infected with influenza virus.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 밤꿀을 포함하는 인플루엔자 A 바이러스(Influenza A virus)에 의한 질환 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating diseases caused by influenza A virus, including chestnut honey.
또한 본 발명은 밤꿀을 포함하는 면역기능 증진용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for enhancing immune function containing chestnut honey.
본 발명에 있어서, 상기 “밤꿀”은 밤나무꽃을 밀원으로 하는 꿀로 짙은 갈색 또는 검은 색을 띄며, 다른 꿀에 비해 쓴 맛이 나는 것이 특징이다. 현재까지 밤꿀의 다양한 효능에 대하여 보고되었지만 본 발명과 같이 인플루엔자 A 바이러스에 대한 항바이러스 효과에 대해서는 보고된 바 없다.In the present invention, the "chestnut honey" is a honey with a chestnut flower as a source of honey, has a dark brown or black color, and is characterized by a bitter taste compared to other honey. Although various effects of chestnut honey have been reported so far, there has been no report on the antiviral effect against influenza A virus as in the present invention.
본 발명의 밤꿀은 밤꿀 자체, 당분야에 알려진 용매로 추출한 추출물, 당분야에 알려진 용매로 희석한 희석액, 공지의 방법에 의하여 농축된 농축액, 공지의 방법에 의하여 건조된 건조물 또는 공지의 방법에 의하여 제조된 정제물로서 제공될 수 있다.The chestnut honey of the present invention is chestnut honey itself, an extract extracted with a solvent known in the art, a diluted solution diluted with a solvent known in the art, a concentrate concentrated by a known method, a dried product dried by a known method, or a known method It can be provided as a manufactured purified product.
상기 공지의 농축 방법은 이에 제한되지는 않으나, 예를 들어 침전농축, 증발농축, 감압농축, 한외여과법, 역삼투법 또는 원심분리법을 이용하여 농축할 수 있다.The known concentration method is not limited thereto, but may be concentrated using, for example, precipitation concentration, evaporation concentration, vacuum concentration, ultrafiltration, reverse osmosis, or centrifugation.
상기 공지의 건조 방법은 이에 제한되지는 않으나, 예를 들어 동결 건조, 분무 건조 또는 열풍건조를 이용하여 건조할 수 있다. 또한 필요에 따라 분말화 과정을 추가적으로 더 포함할 수 있다.The known drying method is not limited thereto, but may be dried using, for example, freeze drying, spray drying or hot air drying. In addition, a powdering process may be further included as needed.
상기 정제물은 공지의 여과 방법에 의하여 제조될 수 있으며 이에 제한되지는 않으나, 예를 들어 여과망 또는 마이크로필터를 이용한 여과, 원심분리 또는 분액깔때기를 이용하여 제조될 수 있다. The purified product may be prepared by a known filtration method, but is not limited thereto, and may be prepared by, for example, filtration using a filter net or microfilter, centrifugation, or separatory funnel.
본 발명의 일실시예에 따르면, 상기 밤꿀은 인플루엔자 A 바이러스에 감염된 세포에 있어서, 농도의존적으로 인플루엔자 바이러스 단백질의 발현을 감소시키는 효과를 보였다.According to one embodiment of the present invention, the chestnut honey showed an effect of reducing the expression of influenza virus proteins in a concentration-dependent manner in cells infected with influenza A virus.
또한 본 발명의 또다른 실시예에 따르면, 상기 밤꿀은 인플루엔자 A 바이러스 감염에 의해 증가된 염증성 사이토카인인 TNF-(tumor necrosis factor-α) 및 IL-6(Interleukin-6)으로 이루어진 군에서 선택된 어느 하나의 발현과, NF-kB(nuclear factor kappa-light-chain-enhancer of activated B cells)의 활성을 감소시키는 효과를 보였다.In addition, according to another embodiment of the present invention, the chestnut honey is any selected from the group consisting of TNF- (tumor necrosis factor-α) and IL-6 (Interleukin-6), which are inflammatory cytokines increased by influenza A virus infection It showed the effect of reducing the expression of one and the activity of NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells).
또한 본 발명의 또 다른 실시예에 따르면, H1N1 계통 인플루엔자 A 바이러스를 감염시킨 동물 모델에 밤꿀을 2주동안 투여한 경우, 마우스의 사망률이 현저히 감소되며, 바이러스 감염에 의한 체중 감소가 억제됨을 확인하였다.In addition, according to another embodiment of the present invention, when chestnut honey was administered to an animal model infected with H1N1 strain influenza A virus for 2 weeks, it was confirmed that the mortality rate of mice was significantly reduced and weight loss caused by viral infection was suppressed. .
따라서 밤꿀은 인플루엔자 A 바이러스 감염에 의한 질환의 예방, 개선 또는 치료를 위한 다양한 분야에서 유용하게 활용할 수 있고, 본 발명의 인플루엔자 A 바이러스는 바람직하게는 H1N1 계통 인플루엔자 A 바이러스 일 수 있다. Therefore, chestnut honey can be usefully utilized in various fields for the prevention, improvement, or treatment of diseases caused by influenza A virus infection, and the influenza A virus of the present invention is preferably an H1N1 strain influenza A virus.
본 발명에 있어서, 상기 인플루엔자 A 바이러스에 의한 질환은 조류 독감, 독감, 감기, 인후염, 기관지염 및 폐렴으로 구성되는 군으로부터 선택된 1종 이상인 것일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the disease caused by the influenza A virus may be at least one selected from the group consisting of bird flu, influenza, cold, sore throat, bronchitis and pneumonia, but is not limited thereto.
더불어 본 발명의 또다른 실시예에 따르면 상기 밤꿀을 정상세포에 처리시 농도의존적으로 종양괴사인자-α(tumor necrosis factor-α, TNF-α) 및 인터루킨-6(interleukin-6, IL-6) 발현을 유도하는 효과를 보였다.In addition, according to another embodiment of the present invention, tumor necrosis factor-α (TNF-α) and interleukin-6 (interleukin-6, IL-6) concentration-dependently when the chestnut honey is treated with normal cells showed an effect of inducing expression.
상기 TNF-α는 주로 활성화된 대식세포에 의해 분비되는 사이토카인이며, 염증반응과 관련된 세포 신호전달 단백질로서, 급성기반응을 유도하는 사이토카인이다. TNF-α는 주로 활성화된 대식세포가 분비하며, CD4 림프구, NK 세포, 호중구, 그리고 비만세포 등과 같은 다양한 종류의 세포에서도 생산된다. TNF-α의 주요 역할은 면역 세포의 활성 및 기능 조절이다. 더불어 내인성발열물질로도 알려져 있으며 발열을 유도하고 세포사멸과 염증반응을 매개하여, 종양의 생성과 바이러스 복제를 억제하는 기능을 수행하는 것으로 알려져 있다.The TNF-α is a cytokine mainly secreted by activated macrophages, and as a cell signaling protein related to an inflammatory response, it is a cytokine that induces an acute phase response. TNF-α is mainly secreted by activated macrophages, and is also produced by various types of cells such as CD4 lymphocytes, NK cells, neutrophils, and mast cells. A major role of TNF-α is the regulation of the activity and function of immune cells. In addition, it is also known as an endogenous pyrogen, and it is known to induce fever, mediate cell death and inflammatory response, and perform functions to suppress tumor formation and viral replication.
또한 상기 IL-6도 발열을 유도하며, 간을 자극하여 CRP(Creactive protein)와 같은 급성상단백질의 생성을 유도한다. 더불어 B-세포를 활성화시키고 항체생산을 증가시켜 항원 특이적 면역반응을 촉진하는 중요한 사이토카인이다.In addition, the IL-6 also induces fever and stimulates the liver to induce the production of acute phase proteins such as CRP (Creactive protein). In addition, it is an important cytokine that promotes antigen-specific immune responses by activating B-cells and increasing antibody production.
또한 본 발명의 또다른 실시예에 따르면 밤꿀은 인터페론(Interferon) 신호전달 경로의 활성화에 관여하는 TANK-결합 효소 1(TANK-binding kinase 1, TBK1) 및 신호변환자-전사활성자 1(Signal transducer and activator of transcription 1, STAT1)의 인산화를 유도하였다.In addition, according to another embodiment of the present invention, chestnut honey is TANK-binding enzyme 1 (TANK-binding kinase 1, TBK1) and signal transducer-transcription activator 1 (Signal transducer) involved in the activation of the interferon signaling pathway and activator of transcription 1, STAT1) phosphorylation was induced.
상기 인터페론은 척추동물의 면역 세포에서 만들어지는 자연 단백질로서, NK세포와 대식세포 등의 선천면역 세포들을 활성화시키며, 주조직 적합성 복합체(major histocompatibility complex, MHC)의 발현을 증대시켜 대식세포나 수지상세포의 항원제시 기능을 향상시킨다. 더불어 세포면역을 활성화시키며, 세포사멸 작용도 한다.The interferon is a natural protein made in the immune cells of vertebrates, activates innate immune cells such as NK cells and macrophages, and increases the expression of the major histocompatibility complex (MHC) to macrophages or dendritic cells improve the antigen presenting function of In addition, it activates cell immunity and also acts on cell death.
상기 인터페론은 제 1형 인터페론, 제 2형 인터페론 및 제 3형 인터페론으로 분류되는데 상기 제 1형 인터페론은 세포내의 항균성 기작들을 활성화하고 선천성 (innate), 후천성 면역반응(adaptive immune response)의 발달에 영향을 준다. 표준 1형 인터페론 시그널링은 JAK(Janus Kinase) - 신호 변환기 및 전사 활성제(signal transducer and activator of transcription, STAT) 경로를 활성화하여, 인터페론 유도형 유전자(IFN-stimulated genes, ISGs)의 전사(transcription)를 유도한다. 상기 TBK1은 세포질 내 DNA에 대한 선천성 면역 반응에서 핵심적인 역할을 하는 단백질인 STING 및 전사인자인 IRF-3를 인산화시킨다. TBK1에 의해서 인산화된 IRF-3는 이합체화 및 핵으로 전이된 후 제 1형 인터페론의 발현을 조절하게 된다. The interferon is classified into type 1 interferon, type 2 interferon and type 3 interferon. The type 1 interferon activates intracellular antimicrobial mechanisms and affects the development of innate and adaptive immune responses. give Canonical type 1 interferon signaling activates the Janus Kinase (JAK) - signal transducer and activator of transcription (STAT) pathway, leading to transcription of interferon-stimulated genes (ISGs). induce The TBK1 phosphorylates STING, a protein that plays a key role in the innate immune response to DNA in the cytoplasm, and IRF-3, a transcription factor. IRF-3 phosphorylated by TBK1 regulates the expression of type I interferons after dimerization and translocation to the nucleus.
따라서 밤꿀은 면역기능 증진에 중요한 역할을 하는 인자들의 발현 및 인산화를 유도하는 바, 면역기능이 저하 또는 억제된 개체의 면역기능을 증진시키기 위하여 활용될 수 있다. 상기 면역기능이 저하 또는 억제는 후천성 면역결핍증(Acquired immunodeficiency syndrome)과 같은 면역관련 질환에 의해서, 또는 방사선 치료 등과 같은 이유에 의하여 야기될 수 있다.Therefore, since chestnut honey induces the expression and phosphorylation of factors that play an important role in enhancing immune function, it can be used to enhance the immune function of individuals with reduced or suppressed immune function. The lowering or suppression of the immune function may be caused by an immune-related disease such as acquired immunodeficiency syndrome or by a reason such as radiation treatment.
본 발명의 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 더불어 본 발명의 약학적 조성물은 약학적으로 허용되는 담체, 부형제 및 희석제로 이루어진 선택된 어느 하나 이상을 추가적으로 포함할 수 있다. 상기 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 밤꿀에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스 또는 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜, 폴리에틸렌글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다. The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. there is. In addition, the pharmaceutical composition of the present invention may additionally include any one or more selected from pharmaceutically acceptable carriers, excipients and diluents. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the chestnut honey of the present invention, for example, starch, calcium carbonate, sucrose or lactose, It is prepared by mixing gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions for oral use, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
본 발명의 약학적 조성물의 투여량은 치료받을 대상의 연령, 성별, 체중과, 치료할 특정 질환 또는 병리 상태, 질환 또는 병리 상태의 심각도, 투여경로 및 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 투여량 결정은 당업자의 수준 내에 있으며, 일반적으로 투여량은 0.01㎎/㎏/일 내지 대략 2000㎎/㎏/일의 범위이다. 더 바람직한 투여량은 0.1㎎/㎏/일 내지 1000㎎/㎏/일이다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Determination of dosage based on these factors is within the level of those skilled in the art, and generally dosages range from 0.01 mg/kg/day to approximately 2000 mg/kg/day. A more preferred dosage is 0.1 mg/kg/day to 1000 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The dosage is not intended to limit the scope of the present invention in any way.
본 발명의 약학적 조성물은 쥐, 가축, 인간 등의 포유동물에 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내 주사에 의해 투여될 수 있다. The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, and humans through various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection.
본 발명에 있어서, 인플루엔자 A 바이러스 감염에 의한 질환 예방 또는 치료용 약학적 조성물은 유효성분 이외에, 질환의 예방 또는 치료 효과 상승을 위하여 이미 안전성이 검증되고 인플루엔자 A 바이러스에 항바이러스 활성을 갖는 것으로 공지된 임의의 화합물이나 천연 추출물을 추가로 포함할 수 있다.In the present invention, the pharmaceutical composition for preventing or treating a disease caused by influenza A virus infection has already been verified for safety and is known to have antiviral activity against influenza A virus in order to increase the effect of preventing or treating a disease, in addition to the active ingredient. It may further include any compound or natural extract.
더불어 본 발명의 약학적 조성물은 인플루엔자 A 바이러스에 의한 질환 예방 또는 치료를 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of diseases caused by influenza A virus.
또한 본 발명에 있어서, 면역기능 증진용 약학적 조성물은 유효성분 이외에, 면역기능 증진을 위하여 이미 안전성이 검증되고 면역기능 증진 활성을 갖는 것으로 공지된 임의의 화합물이나 천연 추출물을 추가로 포함할 수 있다.In addition, in the present invention, the pharmaceutical composition for enhancing immune function may further include, in addition to the active ingredient, any compound or natural extract whose safety has already been verified and is known to have an immune function enhancing activity for enhancing immune function. .
더불어 본 발명의 약학적 조성물은 면역기능 증진을 위하여 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.In addition, the pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers to enhance immune function.
또한 본 발명은 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
더불어 본 발명은 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving diseases caused by influenza A virus containing chestnut honey.
또한 본 발명은 밤꿀을 포함하는 면역기능 증진용 식품 조성물을 제공한다.In addition, the present invention provides a food composition for enhancing immune function containing chestnut honey.
더불어 본 발명은 밤꿀을 포함하는 면역기능 증진용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for enhancing immune function containing chestnut honey.
본 발명에 따른 식품 조성물은 기능성 식품(functional food), 영양 보조제(nutritional supplement), 건강식품(health food) 및 식품 첨가제(food additives) 등의 모든 형태를 포함한다. The food composition according to the present invention includes all types of functional food, nutritional supplements, health food and food additives.
본 발명에 있어서 용어, "건강기능식품"이란 건강보조의 목적으로 특정성분을 원료로 하거나 식품 원료에 들어있는 특정성분을 추출, 농축, 정제, 혼합 등의 방법으로 제조, 가공한 식품을 말하며, 상기 성분에 의해 생체 방어, 생체리듬의 조절, 질병의 방지와 회복 등 생체조절기능을 생체에 대하여 충분히 발휘할 수 있도록 설계되고 가공된 식품을 말하는 것으로서, 질병의 예방 또는 건강의 회복 등과 관련된 기능을 수행할 수 있는 것을 말한다.In the present invention, the term "health functional food" refers to a food manufactured and processed by using a specific ingredient as a raw material or by extraction, concentration, purification, mixing, etc. It refers to food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. by the above components, and performs functions related to disease prevention or health recovery. say what you can do
상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. 상기 유형의 식품 조성물은 당업계에 공지된 통상적인 방법에 따라 분말제, 정제, 캡슐제, 환제 및 액제로 이루어진 군에서 선택된 어느 하나의 형태로 제형화 될 수 있으나, 이에 제한되는 것은 아니다. 당 업계에 공지된 방법을 이용하여 다양한 형태로 제조할 수 있다.Food compositions of this type can be prepared in various forms according to conventional methods known in the art. The type of food composition may be formulated in any one form selected from the group consisting of powders, tablets, capsules, pills and liquids according to conventional methods known in the art, but is not limited thereto. It can be prepared in various forms using methods known in the art.
예를 들면, 건강식품으로는 본 발명의 밤꿀 자체를 과립화, 캡슐화 및 분말화하여 섭취하거나 차, 쥬스 및 드링크의 형태로 제조하여 음용하도록 할 수 있다. 또한, 본 발명의 밤꿀을 인플루엔자 A 바이러스에 대하여 항바이러스 효과를 가진다고 알려진 공지의 물질 또는 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.For example, as a health food, the chestnut honey of the present invention may be granulated, encapsulated, and powdered to be ingested or prepared in the form of tea, juice, and drink to be consumed. In addition, the chestnut honey of the present invention can be prepared in the form of a composition by mixing it with a known substance or active ingredient known to have an antiviral effect against influenza A virus.
또한, 기능성 식품으로는 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마멀레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면,스파게티, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 밤꿀을 첨가하여 제조할 수 있다.In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g., canned fruit, bottled food, jam, marmalade, etc.), fish, meat, and their processed foods (e.g., ham, sausage corned beef, etc.) , breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine, vegetable protein, retort It can be prepared by adding the chestnut honey of the present invention to food, frozen food, various seasonings (eg, soybean paste, soy sauce, sauce, etc.).
본 발명의 식품 조성물 중 상기 본 발명의 밤꿀의 바람직한 함유량으로는 이에 한정되지 않지만 예를 들어 최종적으로 제조된 식품 중 0.01 내지 80 중량%일 수 있으며, 바람직하게는 최종적으로 제조된 식품 중 0.01 내지 50 중량%일 수 있다.The preferred content of the chestnut honey of the present invention in the food composition of the present invention is not limited thereto, but may be, for example, 0.01 to 80% by weight of the final food, preferably 0.01 to 50% of the final food. weight percent.
또한, 본 발명의 밤꿀을 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다.In addition, in order to use the chestnut honey of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.
또한 본 발명은 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선을 위한 사료첨가용 조성물을 제공한다.In addition, the present invention provides a feed additive composition for preventing or improving diseases caused by influenza A virus, including chestnut honey.
더불어 본 발명은 밤꿀을 포함하는 면역기능 증진을 위한 사료첨가용 조성물을 제공한다.In addition, the present invention provides a composition for feed additives for enhancing immune function containing chestnut honey.
본 발명의 용어 "사료"란, 동물이 먹는 임의의 천연 또는 인공 구정식, 한끼식 등 또는 상기 한끼식의 성분을 의미하며, 본 발명에 따른 사료 첨가용 조성물을 유효성분으로 포함하는 사료는 당업계에 공지된 다양한 형태의 사료로 제조가능하며, 바람직하게는 농후 사료, 조사료 및/또는 특수사료가 포함될 수 있으나, 이로 제한되는 것은 아니다.The term "feed" of the present invention refers to any natural or artificial oral diet, one meal meal, etc., or a component of the one meal meal eaten by animals, and the feed containing the composition for feed addition according to the present invention as an active ingredient is It can be prepared with various types of feed known in the art, preferably, concentrated feed, roughage and / or special feed may be included, but is not limited thereto.
본 발명에서 용어, “사료첨가용 조성물”은 “사료첨가제”를 의미할 수 있으며, 이는 동물에 있어 질병 증상의 완화, 영양소 보충 및 체중감소 예방, 사료 내 섬유소의 소화 이용성 증진, 유질개선, 번식장애 예방 및 수태율 향상, 하절기 고온 스트레스 예방 등 다양한 효과를 목적으로 사료에 첨가하는 물질을 포함한다.In the present invention, the term "composition for feed additive" may mean "feed additive", which alleviates symptoms of disease in animals, supplements nutrients and prevents weight loss, enhances the digestibility of fiber in feed, improves milk quality, and breeds. It includes substances added to feed for the purpose of various effects, such as preventing disorders, improving conception rates, and preventing high-temperature stress in the summer.
본 발명의 사료첨가용 조성물은 사료관리법상의 보조사료에 해당하며, 탄산수소나트륨, 벤토나이트(bentonite), 산화마그네슘, 복합광물질 등의 광물질제제, 아연, 구리, 코발트, 셀레늄 등의 미량 광물질인 미네랄제제, 케로틴, 비타민 A D, E, 니코틴산, 비타민 B 복합체 등의 비타민제, 메티오닌, 라이신 등의 보호아미노산제, 지방산 칼슘염 등의 보호지방산제, 생균제(유산균제), 효모배양물, 곰팡이 발효물 등의 생균, 효모제 등이 추가로 포함될 수 있다.The composition for feed additive of the present invention corresponds to supplementary feed under the Feed Management Act, and is a mineral preparation such as sodium bicarbonate, bentonite, magnesium oxide, and complex minerals, and a trace mineral preparation such as zinc, copper, cobalt, and selenium. , vitamins such as kerotene, vitamins A, D, E, nicotinic acid, and vitamin B complex, protected amino acids such as methionine and lysine, protected fatty acids such as calcium salts of fatty acids, probiotics (lactic acid bacteria), yeast cultures, and fungal fermentation products. Probiotics, yeasts, and the like may be further included.
상기 사료 중 농후사료로는 밀, 귀리, 옥수수 등의 곡류를 포함하는 종자열매류, 곡물을 정제하고 얻는 부산물로서 쌀겨, 밀기울, 보릿겨 등을 포함하는 겨류, 콩, 유체, 깨, 아마인, 코코야자 등을 채유하고 얻는 부산물인 깻묵류와 고구마, 감자 등에서 녹말을 뺀 나머지인 녹말찌꺼기의 주성분인 잔존녹말질류 등의 찌꺼기류, 어분, 물고기찌꺼기, 어류에서 얻은 신선한 액상물을 농축시킨 것인 피시솔루블(fish soluble), 육분(肉粉), 혈분, 우모분, 탈지분유, 우유에서 치즈, 탈지유에서 카제인을 제조할 때의 잔액인 훼이(whey)를 건조한 건조훼이 등의 동물질사료, 효모, 클로렐라, 해조류가 있으나 이에 제한되지 않는다.Among the feeds, the enriched feed includes seed fruits including grains such as wheat, oats and corn, bran including rice bran, bran, barley bran, etc. as a by-product obtained by refining grains, soybeans, fluids, sesame seeds, linseed, coco Fish meal, which is a by-product obtained from oil extraction of palms, residual starch, which is the main component of starch residue, which is the remainder after removing starch from sweet potatoes, potatoes, etc., fish meal, fish residue, and fresh liquid obtained from fish Animal feed such as fish soluble, meat meal, blood meal, feather meal, skim milk powder, dried whey, which is the balance when manufacturing cheese from milk and casein from skim milk, yeast, chlorella , algae, but not limited thereto.
상기 사료 중 조사료로는 야초, 목초, 풋베기 등의 생초(生草)사료, 사료용 순무, 사료용 비트, 순무의 일종인 루터베어거 등의 뿌리채소류, 생초, 풋베기작물, 곡실(穀實) 등을 사일로에 채워 놓고 젖산발효시킨 저장사료인 사일리지(silage), 야초, 목초를 베어 건조시킨 건초, 종축용(種畜用) 작물의 짚, 콩과 식물의 나뭇잎이 있으며, 이에 제한되지 않는다. 특수사료에는 굴껍데기, 암염 등의 미네랄 사료, 요소나 그 유도체인 디우레이드이소부탄 등의 요소사료, 천연사료원료만을 배합했을 때 부족하기 쉬운 성분을 보충하거나, 사료의 저장성을 높이기 위해서 배합사료에 미량으로 첨가하는 물질인 사료첨가물, 식이보조제가 있으나 이에 제한되지 않는다.Forages among the feed include raw grass feed such as wild grass, grass, green cutting, turnip for feed, beet for feed, root vegetables such as Lutherbearer, a kind of turnip, raw grass, green crops, grain Silage, which is a stored feed made by lactic acid fermentation by filling the back into a silo, wild grass, hay dried by cutting grass, straw of breeding crops, and leaves of legumes, but is not limited thereto. Special feeds include mineral feeds such as oyster shells and rock salt, urea feeds such as urea or its derivative, diureide isobutane, and supplements for ingredients that are likely to be insufficient when only natural feed ingredients are mixed, or formulated feeds to improve the storability of feeds. There are feed additives and dietary supplements, which are substances added in small amounts, but are not limited thereto.
본 발명에 따른 상기 사료첨가용 조성물은 당업계에 공지된 다양한 사료 제조방법에 따라 적절한 유효 농도 범위에서 밤꿀을 첨가하여 제조 가능하다.The feed composition according to the present invention can be prepared by adding chestnut honey in an appropriate effective concentration range according to various feed manufacturing methods known in the art.
또한 본 발명에 따른 사료첨가용 조성물은 인플루엔자 A 바이러스 예방 또는, 상기 바이러스에 감염되어 증상의 완화 또는 치료를 목적으로 하는 개체 또는 면역기능 증진을 목적으로 하는 개체라면 제한없이 적용가능하다. 예를 들면, 원숭이, 개, 고양이, 토끼, 모르모트, 랫트, 마우스, 소, 양, 돼지, 염소 등과 같은 비인간 동물, 조류 및 어류 등 어느 개체에도 적용이 가능하다.In addition, the composition for feed additives according to the present invention can be applied without limitation to an individual who aims to prevent influenza A virus, alleviate or treat symptoms infected with the virus, or enhance immune function. For example, it can be applied to any object such as non-human animals such as monkeys, dogs, cats, rabbits, guinea pigs, rats, mice, cows, sheep, pigs, goats, birds and fish.
또한 본 발명은 밤꿀을 개체에 투여하는 단계를 포함하는 인플루엔자 A 바이러스에 의한 질환을 치료하는 방법을 제공한다.In addition, the present invention provides a method for treating a disease caused by influenza A virus comprising administering chestnut honey to a subject.
상기 개체는 인간을 포함한 포유류인 것이 바람직하며, 인플루엔자 A 바이러스 감염에 의한 질환의 치료를 필요로 하는 환자로 인플루엔자 A 바이러스 감염에 의한 질환의 치료를 필요로 하는 환자, 인플루엔자 A 바이러스 감염에 의한 질환 치료 중인 환자, 인플루엔자 A 바이러스 감염에 의한 질환 치료를 받은 적이 있는 환자, 인플루엔자 A 바이러스 감염에 의한 질환 치료를 받을 필요가 있는 환자를 모두 포함하며, 인플루엔자 A 바이러스 감염에 의한 질환 치료를 위하여 외과적 수술을 시행한 환자 또한 포함될 수 있다.The subject is preferably a mammal, including a human, and is a patient in need of treatment for a disease caused by influenza A virus infection, a patient in need of treatment for a disease caused by influenza A virus infection, and a disease treatment caused by influenza A virus infection This includes all patients who are under treatment, patients who have been treated for diseases caused by influenza A virus infection, and patients who need to be treated for diseases caused by influenza A virus infection, and who undergo surgical operation to treat diseases caused by influenza A virus infection. Patients may also be included.
또한 본 발명의 밤꿀은 기존의 인플루엔자 A 바이러스 감염에 의한 질환 치료를 위한 약물 또는 치료방법과 병용하여 처리될 수 있다. 본 발명의 밤꿀을 병용 처리하는 경우, 이는 인플루엔자 A 바이러스 감염에 의한 질환 치료를 위한 다른 약물 또는 치료방법과 동시에 또는 순차적으로 처리될 수 있다. In addition, the chestnut honey of the present invention can be treated in combination with drugs or treatment methods for the treatment of diseases caused by influenza A virus infection. When the chestnut honey of the present invention is treated in combination, it can be treated simultaneously or sequentially with other drugs or treatment methods for the treatment of diseases caused by influenza A virus infection.
더불어 본 발명은 밤꿀을 개체에 투여하는 단계를 포함하는 면역기능을 증진시키는 방법을 제공한다.In addition, the present invention provides a method for enhancing immune function comprising administering chestnut honey to a subject.
상기 개체는 인간을 포함한 포유류인 것이 바람직하며, 면역기능 저하에 의한 증상 및 질환의 예방, 개선 또는 치료를 필요로 하는 환자로 면역기능 저하에 의한 증상 및 질환의 치료를 필요로 하는 환자, 면역기능 저하에 의한 증상 및 질환 치료 중인 환자, 면역기능 저하에 의한 증상 및 질환 치료를 받은 적이 있는 환자, 면역기능 저하에 의한 증상 및 질환 치료를 받을 필요가 있는 환자를 모두 포함하며, 면역기능 저하에 의한 증상 및 질환 치료를 위하여 외과적 수술을 시행한 환자 또한 포함될 수 있다.The subject is preferably a mammal, including a human, and is a patient in need of prevention, improvement or treatment of symptoms and diseases caused by reduced immune function, a patient who needs treatment for symptoms and diseases caused by reduced immune function, and immune function It includes all patients who are undergoing treatment for symptoms and diseases caused by lowered immune function, patients who have been treated for symptoms and diseases caused by lowered immune function, and patients who need to receive treatment for symptoms and diseases caused by lowered immune function. Patients who have undergone surgical operations for the treatment of symptoms and diseases may also be included.
또한 본 발명의 밤꿀은 기존의 면역기능 저하에 의한 증상 및 질환 치료를 위한 약물 또는 치료방법과 병용하여 처리될 수 있다. 본 발명의 밤꿀을 병용 처리하는 경우, 이는 면역기능 저하에 의한 증상 및 질환 치료를 위한 다른 약물 또는 치료방법과 동시에 또는 순차적으로 처리될 수 있다. In addition, the chestnut honey of the present invention can be treated in combination with drugs or treatment methods for the treatment of symptoms and diseases caused by reduced immune function. When the chestnut honey of the present invention is treated in combination, it can be treated simultaneously or sequentially with other drugs or treatment methods for the treatment of symptoms and diseases caused by reduced immune function.
상술한 본 발명의 내용은 상호 모순되지 않는 한, 서로 동일하게 적용되며, 당해 기술분야의 통상의 기술자가 적절한 변경을 가해 실시하는 것 또한 본 발명의 범주에 포함된다.The contents of the present invention described above are equally applied to each other unless contradictory to each other, and implementation by adding appropriate changes by a person skilled in the art is also included in the scope of the present invention.
이하 본 발명을 실시예를 통해 상세하게 설명하나 본 발명의 범위가 하기 실시예로만 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited only to the following examples.
실시예 1. 밤꿀의 인플루엔자 A 바이러스에 대한 항바이러스 효과 확인Example 1. Confirmation of antiviral effect of chestnut honey against influenza A virus
H1N1 계통 인플루엔자 A 바이러스인 인플루엔자 A 바이러스(Influenza virus; A/PR8/34)에 대한 밤꿀의 항바이러스 활성 분석을 수행하였다. The antiviral activity of chestnut honey against influenza A virus (A/PR8/34), which is an H1N1 strain influenza A virus, was analyzed.
먼저 24-웰 플레이트에 RAW 264.7 세포를 배양한 후 10% FBS(Fetal bovine serum)와 1% 페니실린(penicillin)/스트렙토마이신(streptomycin)이 첨가된 DMEM(Dulbecco's modified Eagle's medium)에 1.25, 2.5, 5 또는 10 mg/mL의 농도로 증류수에 희석한 밤꿀(오픈마켓에서 구입)을 처리하여 18 시간 배양하였다. First, RAW 264.7 cells were cultured in a 24-well plate, and then cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS (Fetal bovine serum) and 1% penicillin/streptomycin at 1.25, 2.5, and 5 Alternatively, chestnut honey diluted in distilled water at a concentration of 10 mg/mL (purchased from an open market) was treated and cultured for 18 hours.
배양이 완료된 후 1% 페니실린/스트렙토마이신이 첨가된 DMEM하에서 상기 세포를 인플루엔자A/PR8/34-GFP(10 MOI)로 감염시켰다. 감염 2시간 후 감염액(상층액)을 제거하고 PBS(phosphate buffer saline)로 세척한 후 10% FBS와 1% 페니실린/스트렙토마이신이 첨가된 DMEM를 넣어주고 24시간동안 배양하였다. 이때 양성대조군으로 1,000Units/ml의 IFN-β(interferon-β)를 처리하였다. 그런 후 인플루엔자 A 바이러스 단백질의 발현 수준을 확인하기 위해 웨스턴블롯(western blot)분석을 수행하였다. 먼저 세포를 회수한 후 단백질을 분리하고 브래드포드(bradford)법을 이용하여 동일한 단백질 양으로 8-12% 아크릴아마이드 겔(acrylamide gel)에 전기영동을 진행하였다. 이 후 PVDF(Polyvinylidene fluoride)로 단백질을 트랜스퍼(transfer)한 후 소혈청 알부민(bovine serum albumin, BSA)로 단백질의 비특이적 결합을 차단하고 genetex사의 인플루엔자 A 바이러스 단백질 HA(Hemagglutinin), PB1, PA, M1, M2항체와 특이적으로 결합하는 2차 항체(santa cruz사의 IgG-HRP)를 이용하여 표적 단백질을 검출하였다. After the culture was completed, the cells were infected with influenza A/PR8/34-GFP (10 MOI) in DMEM supplemented with 1% penicillin/streptomycin. After 2 hours of infection, the infection solution (supernatant) was removed, washed with PBS (phosphate buffer saline), and DMEM supplemented with 10% FBS and 1% penicillin/streptomycin was added and cultured for 24 hours. At this time, as a positive control group, 1,000 Units/ml of IFN-β (interferon-β) was treated. Then, western blot analysis was performed to confirm the expression level of influenza A virus protein. First, after recovering the cells, proteins were separated and electrophoresis was performed on an 8-12% acrylamide gel with the same protein amount using the Bradford method. Afterwards, the protein was transferred with PVDF (Polyvinylidene fluoride), and the non-specific binding of the protein was blocked with bovine serum albumin (BSA), and influenza A virus proteins HA (Hemagglutinin), PB1, PA, and M1 from genetex were used. , The target protein was detected using a secondary antibody (IgG-HRP from santa cruz) specifically binding to the M2 antibody.
그 결과는 도 1a 및 도 1b에 나타내었다. 상기 도 1a 및 도 1b에 있어서, CON은 음성 대조군으로 아무것도 처리하지 않은 세포군이고, 베히클(vehicle)은 A/PR8/34-GFP 바이러스 감염군이며, IFN-β는 양성 대조군으로, A/PR8/34-GFP 바이러스 감염 및 1,000Units/㎖의 IFN-β 처리군이다. 또한 1.25는 A/PR8/34-GFP 바이러스 감염 및 1.25 mg/mL 밤꿀 처리군이고, 2.5는 A/PR8/34-GFP 바이러스 감염 및 2.5 mg/mL 밤꿀 처리군이며, 5는 A/PR8/34-GFP 바이러스 감염 및 5 mg/mL 밤꿀 처리군이고, 10은 A/PR8/34-GFP 바이러스 감염 및 10 mg/mL 밤꿀 처리군이다.The results are shown in Figures 1a and 1b. In FIGS. 1A and 1B, CON is a negative control cell group not treated with anything, vehicle is an A/PR8/34-GFP virus infection group, and IFN-β is a positive control group, A/PR8 /34-GFP virus infection and 1,000 Units/ml IFN-β treatment group. In addition, 1.25 is the A/PR8/34-GFP virus infection and 1.25 mg/mL chestnut honey treatment group, 2.5 is the A/PR8/34-GFP virus infection and 2.5 mg/mL chestnut honey treatment group, and 5 is the A/PR8/34 -GFP virus infection and 5 mg / mL chestnut honey treatment group, 10 A / PR8 / 34-GFP virus infection and 10 mg / mL chestnut honey treatment group.
도 1a 및 도 1b(A)에 나타낸 바와 같이, 밤꿀은 농도 의존적으로 인플루엔자 A 바이러스 감염에 의한 GFP 형광을 감소시켰다. 더불어 도 1b(B)에 나타낸 바와 같이, 인플루엔자 A 바이러스 단백질인 HA, PB1, PA, M1 및 M2의 발현 또한 밤꿀 처리에 의해 농도 의존적으로 감소됨을 확인하였다.As shown in Figures 1a and 1b (A), chestnut honey reduced GFP fluorescence caused by influenza A virus infection in a concentration-dependent manner. In addition, as shown in Figure 1b (B), it was confirmed that the expression of influenza A virus proteins HA, PB1, PA, M1 and M2 was also reduced in a concentration-dependent manner by treatment with chestnut honey.
실시예 2. 밤꿀의 인플루엔자 A 바이러스 감염에 따른 염증성 인자에 미치는 영향 확인Example 2. Confirmation of the effect of chestnut honey on inflammatory factors following influenza A virus infection
더불어 밤꿀이 인플루엔자 A 바이러스 감염에 따른 세포내 염증성 사이토카인 및 NF-kB 활성에 미치는 영향을 확인하였다. In addition, the effect of chestnut honey on intracellular inflammatory cytokines and NF-kB activity following influenza A virus infection was confirmed.
이를 위하여 상기 실시예 1과 동일한 방법으로 다양한 농도의 밤꿀을 처리한 RAW 264.7 세포를 인플루엔자A/PR8/34-GFP로 감염시켰다. 16시간 배양이 끝난 후, 각 웰의 상층액을 수거하여 1,500 rpm으로 5분간 원심분리한 후, TNF-α(tumor necrosis factor-α)와 IL-6(Interleukin-6)의 생성정도를 ELISA(enzyme-linked immunosorbent assay) 기법을 이용하여 측정하였다. 그 결과를 도 2에 나타내었으며, 상기 도 2 중 A 및 B에 있어서 CON은 음성 대조군으로 아무것도 처리하지 않은 세포군이고, vehicle은 A/PR8/34-GFP는 인플루엔자 A 바이러스 감염군이며, 1.25는 A/PR8/34-GFP 바이러스 감염 및 2.5 mg/mL 밤꿀 처리군이고, 2.5는 A/PR8/34-GFP 바이러스 감염 및 2.5 mg/mL 밤꿀 처리군이며, 5는 A/PR8/34-GFP 바이러스 감염 및 5 mg/mL 밤꿀 처리군이고, 10은 A/PR8/34-GFP 바이러스 감염 및 10 mg/mL 밤꿀 처리군이다.To this end, in the same manner as in Example 1, RAW 264.7 cells treated with various concentrations of chestnut honey were infected with influenza A/PR8/34-GFP. After 16 hours of incubation, the supernatant from each well was collected and centrifuged at 1,500 rpm for 5 minutes. It was measured using enzyme-linked immunosorbent assay) technique. The results are shown in FIG. 2, and in A and B of FIG. 2, CON is a negative control cell group without any treatment, vehicle is A/PR8/34-GFP is an influenza A virus infection group, and 1.25 is A /PR8/34-GFP virus infection and 2.5 mg/mL chestnut honey treatment group, 2.5 was A/PR8/34-GFP virus infection and 2.5 mg/mL chestnut honey treatment group, 5 was A/PR8/34-GFP virus infection And 5 mg / mL chestnut honey treatment group, 10 A / PR8 / 34-GFP virus infection and 10 mg / mL chestnut honey treatment group.
또한 NF-kB의 활성은 면역형광법(immunofluorecsence, IF)을 이용하여 확인하였다. 고정된 세포의 비특이적 결합을 BSA를 이용하여 차단한 후 1차 항체인 CST사의 NF-kB와 2차 항체인 thermo사의 alexa fluor-568의 특이적 결합을 형광현미경을 통해 확인하였다. 이를 통해 인플루엔자 A 바이러스 감염시 증가되는 NF-kB의 활성에 밤꿀이 미치는 영향을 확인하여 도 2 중 C에 나타내었다. In addition, the activity of NF-kB was confirmed using immunofluorescence (IF). After blocking non-specific binding of the fixed cells using BSA, the specific binding of the primary antibody, CST's NF-kB, and the secondary antibody, Thermo's alexa fluor-568, was confirmed through a fluorescence microscope. Through this, the effect of chestnut honey on the activity of NF-kB, which is increased during influenza A virus infection, was confirmed and shown in C in FIG. 2.
도 2에 나타낸 바와 같이, 밤꿀은 농도의존적으로 인플루엔자 A 바이러스 감염에 의하여 증가된 염증성 사이토카인인 TNF-α 및 IL-6의 발현을 감소시켰다. 더불어 NF-kB의 활성도 감소시킨 바, 인플루엔자 A 바이러스 감염에 대하여 농도의존적인 치료 효과를 보임을 확인하였다.As shown in Figure 2, chestnut honey reduced the expression of TNF-α and IL-6, which are inflammatory cytokines increased by influenza A virus infection, in a concentration-dependent manner. In addition, since the activity of NF-kB was also reduced, it was confirmed that a concentration-dependent therapeutic effect on influenza A virus infection was shown.
실시예 3. 밤꿀의 면역 관련 사이토카인 발현 유도 효과 확인Example 3. Immune-related cytokine expression induction effect confirmation of chestnut honey
밤꿀을 세포에 처리하여 면역기능 관련 인자에 미치는 영향을 확인하였다. 먼저 10% FBS(Fetal Bovine Serum)와 1% 페니실린(penicillin), 1% 스트렙토마이신(streptomycin)이 첨가된 배지(DMEM)에 1.25, 2.5, 5 또는 10 mg/mL의 농도로 증류수에 희석한 밤꿀(오픈마켓에서 구입)과 100 ng/mL의 LPS(Lipopolysaccharide)를 각각 처리하였다. 이를 24-웰 플레이트에서 배양한 RAW 264.7 세포에 분주하고 18 시간동안 배양하였다. 배양이 끝난 후, 각 웰의 배지를 수거하여 1500rpm으로 5분간 원심분리한 후, TNF-α와 IL-6의 생성 정도를 ELISA 기법을 이용하여 측정하였다. Chestnut honey was treated with cells to confirm the effect on immune function-related factors. First, chestnut honey diluted in distilled water at a concentration of 1.25, 2.5, 5, or 10 mg/mL in a medium (DMEM) supplemented with 10% FBS (Fetal Bovine Serum), 1% penicillin, and 1% streptomycin (purchased from an open market) and 100 ng/mL of LPS (Lipopolysaccharide), respectively. This was seeded into RAW 264.7 cells cultured in a 24-well plate and cultured for 18 hours. After the incubation, the medium of each well was collected and centrifuged at 1500 rpm for 5 minutes, and then the levels of TNF-α and IL-6 production were measured using ELISA technique.
그 결과 도 3에 나타낸 바와 같이, 10mg/ml의 밤꿀을 처리한 실험군의 경우 음성대조군(CON)에 비해 TNF-α의 발현이 9배 증가하였다(도 3 중 A). 또한 10mg/ml의 밤꿀을 처리한 실험군은 IL-6의 발현도 음성대조군(CON)에 비해 11배 증가하였다. As a result, as shown in Figure 3, in the case of the experimental group treated with 10 mg / ml of chestnut honey, the expression of TNF-α increased 9 times compared to the negative control group (CON) (A in Figure 3). In addition, the experimental group treated with 10 mg/ml of chestnut honey increased the expression of IL-6 11 times compared to the negative control group (CON).
실시예 4. 밤꿀의 인터페론 신호전달 경로의 활성화와 관련있는 분자의 인산화 유도 효과 확인Example 4. Confirmation of the phosphorylation-inducing effect of molecules related to the activation of the interferon signaling pathway of chestnut honey
더불어 밤꿀이 인터페론 신호전달 경로의 활성화에 관여하는 분자의 인산화에 미치는 영향을 확인하였다. In addition, the effect of chestnut honey on the phosphorylation of molecules involved in the activation of the interferon signaling pathway was confirmed.
이를 위하여 RAW 264.7 세포에 100 ng/ml의 LPS(대조군) 또는 5mg/ml의 밤꿀을 처리하고 배양하며 0, 6, 12, 24시간별로 각 실험군의 세포를 회수하였다. 그런후 이를 용해하여 웨스턴블롯을 수행하였다. 이때 음성대조군으로 무처리 실험군의 세포도 동일 시간별로 회수하여 웨스턴블롯을 수행하였다.To this end, RAW 264.7 cells were treated with 100 ng/ml of LPS (control) or 5 mg/ml of chestnut honey, cultured, and the cells of each experimental group were recovered at 0, 6, 12, and 24 hours. Then, it was dissolved and Western blotting was performed. At this time, as a negative control group, cells from the untreated experimental group were also collected at the same time intervals and Western blotting was performed.
웨스턴 블롯은 bradford법을 이용하여 동일한 단백질 양으로 8-12% 아크릴아마이드 겔에 전기영동을 진행한 후 폴리비닐리딘다이플루(polyvinyliden difluoride, PVDF)막으로 단백질 트랜스퍼(transfer)하였다. 이 후 소혈청알부민 (bovine serum albumin, BSA)으로 단백질의 비특이적 결합을 차단한 후 1차(CST사의 p-SATA1, p-TBK) 및 2차 항체(santa cruz사의 IgG-HRP)와 반응시키고 화학발광(chemiluminescence)반응으로 표적 단백질을 검출하였다. For Western blotting, electrophoresis was performed on an 8-12% acrylamide gel with the same amount of protein using the bradford method, followed by protein transfer to a polyvinyliden difluoride (PVDF) membrane. Afterwards, the non-specific binding of proteins was blocked with bovine serum albumin (BSA), followed by reaction with primary (CST's p-SATA1, p-TBK) and secondary antibodies (Santa Cruz's IgG-HRP) and chemical The target protein was detected by a chemiluminescence reaction.
그 결과 도 4에 나타낸 바와 같이, 밤꿀을 처리한 실험군에서 처리한 지 24시간이 경과되었을때 인터페론 신호전달 경로의 활성화에 관여하는 TBK1 및 STAT1 분자의 인산화가 현저히 증가된 것을 확인하였다.As a result, as shown in Figure 4, it was confirmed that the phosphorylation of TBK1 and STAT1 molecules involved in the activation of the interferon signaling pathway was significantly increased when 24 hours had passed since the treatment in the experimental group treated with chestnut honey.
실시예 5. 밤꿀의 바이러스 감염 동물모델에서의 항바이러스 효능 확인Example 5. Confirmation of antiviral efficacy in virus infection animal models of chestnut honey
6주령 BALB/c 암컷 마우스를 4군 각 10마리씩 무작위 배정하였다. 멸균 증류수로 녹인 밤꿀을 300 mg/kg와 600 mg/kg 농도로 2주 동안 매일 1회 경구 투여하였다. 투여를 진행하는 2주 동안 바이러스 미감염군(Negative)은 밤꿀 및 멸균 증류수를 투여하지 않았고 바이러스만 감염시킨 군(Virus control)은 멸균 증류수를 투여하였다. 바이러스 감염군과 밤꿀 투여군에 인플루엔자 A 바이러스(A/PR8/34-GFP)를 5LD50(5배의 반수치사량)농도로 비강을 통해 감염시킨 후 10일 동안 매일 체중 및 생존율을 측정하였다. 감염 전의 개시 체중 25% 이상 체중감소는 윤리적인 관점에서 희생시켜 사망으로 간주하였다.6-week-old BALB/c female mice were randomly assigned to each of 4 groups, 10 mice each. Chestnut honey dissolved in sterile distilled water was orally administered once daily for 2 weeks at concentrations of 300 mg/kg and 600 mg/kg. During the 2 weeks of administration, the virus-infected group (Negative) was not administered with chestnut honey and sterile distilled water, and the virus-infected group (Virus control) was administered with sterile distilled water. After infection with influenza A virus (A/PR8/34-GFP) at a concentration of 5LD50 (5-fold half-lethal dose) through the nasal cavity, the virus-infected group and the chestnut honey-administered group were measured for body weight and survival rate every day for 10 days. A weight loss of more than 25% of the initial body weight before infection was regarded as death by sacrifice from an ethical point of view.
그 결과, 도 5 중 A에 나타낸 바와 같이, 600 mg/kg의 밤꿀 투여군은 바이러스 감염군(Virus control)에 비해 60%의 생존율을 보였다. 이를 통해 밤꿀의 투여가 인플루엔자 A 바이러스에 의한 사망률을 현저히 감소시킴을 확인하였다.As a result, as shown in A in Figure 5, the 600 mg / kg chestnut honey administration group showed a survival rate of 60% compared to the virus infection group (Virus control). Through this, it was confirmed that the administration of chestnut honey significantly reduced the mortality caused by influenza A virus.
더불어 도 5 중 B에 나타낸 바와 같이, 바이러스 감염군(Virus control)은 인플루엔자 A 바이러스 감염에 의해 현저한 체중 감소를 보인 반면, 밤꿀 투여군은 인플루엔자 A 바이러스 감염에 의한 체중 감소가 현저히 억제되었음을 확인하였다. In addition, as shown in B in FIG. 5, the virus control group showed significant weight loss due to influenza A virus infection, while the chestnut honey administration group confirmed that weight loss due to influenza A virus infection was significantly suppressed.
종합적으로 밤꿀은 인플루엔자 A 바이러스 단백질의 발현 및 이의 감염에 의해 증가된 염증성 사이토카인의 발현과 NF-kB의 활성을 감소시키며, 인플루엔자 A 바이러스를 감염시킨 동물 모델의 사망률을 현저히 감소시키고, 바이러스 감염에 의한 체중 감소를 억제하며, 선천면역 관련 인자의 발현 및 면역 관련 신호전달 경로의 활성화에 관여하는 인자의 인산화를 유도하는 효과를 보유하고 있음이 확인된 바, 이를 인플루엔자 A 바이러스에 의한 질환의 예방, 개선 또는 치료를 위한 다양한 용도로 유용하게 활용할 수 있고, 면역기능 증진을 위한 다양한 용도로 유용하게 활용할 수 있다.Overall, chestnut honey reduces the expression of influenza A virus proteins and the increased expression of inflammatory cytokines and the activity of NF-kB by infection, significantly reduces mortality in animal models infected with influenza A virus, and is effective against viral infection. It has been confirmed that it has the effect of inducing phosphorylation of factors involved in the expression of innate immunity-related factors and the activation of immune-related signaling pathways, thereby preventing diseases caused by influenza A virus, It can be usefully used for various purposes for improvement or treatment, and can be usefully used for various purposes for enhancing immune function.

Claims (14)

  1. 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating diseases caused by influenza A virus containing chestnut honey.
  2. 제 1항에 있어서,According to claim 1,
    상기 인플루엔자 A 바이러스는 H1N1 계통 인플루엔자 A 바이러스인 것을 특징으로 하는, 약학적 조성물.The pharmaceutical composition, characterized in that the influenza A virus is an H1N1 strain influenza A virus.
  3. 제 1항에 있어서,According to claim 1,
    상기 인플루엔자 A 바이러스에 의한 질환은 조류 독감, 독감, 감기, 인후염, 기관지염 및 폐렴으로 이루어진 군으로부터 선택된 1종 이상인 것을 특징으로 하는, 약학적 조성물.The disease caused by the influenza A virus is characterized in that at least one selected from the group consisting of bird flu, flu, cold, sore throat, bronchitis and pneumonia, pharmaceutical composition.
  4. 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선용 식품 조성물.A food composition for preventing or improving disease caused by influenza A virus containing chestnut honey.
  5. 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선용 건강기능식품 조성물.Health functional food composition for preventing or improving disease caused by influenza A virus containing chestnut honey.
  6. 밤꿀을 포함하는 인플루엔자 A 바이러스에 의한 질환 예방 또는 개선을 위한 사료첨가용 조성물.Feed additive composition for preventing or improving disease caused by influenza A virus containing chestnut honey.
  7. 밤꿀을 개체에 투여하는 단계를 포함하는 인플루엔자 A 바이러스에 의한 질환을 치료하는 방법.A method of treating a disease caused by influenza A virus comprising administering chestnut honey to a subject.
  8. 밤꿀을 포함하는 면역기능 증진용 약학적 조성물.Pharmaceutical composition for enhancing immune function containing chestnut honey.
  9. 제 8항에 있어서,According to claim 8,
    상기 밤꿀은 종양괴사인자-α(tumor necrosis factor-α, TNF-α), 인터루킨-6(interleukin-6, IL-6) 및 인터페론-베타(interferon-beta, INF-β)로 이루어진 군에서 선택된 어느 하나 이상의 발현을 증가시키는 것을 특징으로 하는, 약학적 조성물.The chestnut honey is selected from the group consisting of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interferon-beta (INF-β) A pharmaceutical composition characterized by increasing the expression of any one or more.
  10. 제 8항에 있어서,According to claim 8,
    상기 밤꿀은 TANK-결합 효소 1(TANK-binding kinase 1, TBK1), JNK(c-Jun N-terminal kinases) 및 신호변환자-전사활성자 1/2(Signal transducer and activator of transcription 1/2, STAT1/2)로 이루어진 군에서 선택된 어느 하나 이상의 인산화를 증가시키는 것을 특징으로 하는, 약학적 조성물.The chestnut honey is TANK-binding enzyme 1 (TANK-binding kinase 1, TBK1), JNK (c-Jun N-terminal kinases) and signal transducer-activator of transcription 1/2 (Signal transducer and activator of transcription 1/2, STAT1/2) characterized in that to increase the phosphorylation of any one or more selected from the group consisting of, a pharmaceutical composition.
  11. 밤꿀을 포함하는 면역기능 증진용 식품 조성물.Food composition for enhancing immune function containing chestnut honey.
  12. 밤꿀을 포함하는 면역기능 증진용 건강기능식품 조성물.Health functional food composition for enhancing immune function containing chestnut honey.
  13. 밤꿀을 포함하는 면역기능 증진을 위한 사료첨가용 조성물.Feed additive composition for enhancing immune function containing chestnut honey.
  14. 밤꿀을 개체에 투여하는 단계를 포함하는 면역기능을 증진시키는 방법. A method for enhancing immune function comprising administering chestnut honey to a subject.
PCT/KR2022/008646 2021-09-10 2022-06-17 Pharmaceutical composition for preventing or treating diseases caused by influenza a virus and enhancing immunity comprising castanea crenata sieb WO2023038240A1 (en)

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KR1020210120738A KR20230037779A (en) 2021-09-10 2021-09-10 Pharmaceutical composition for preventing or treating of diseases caused by influenza A virus comprising chestnut honey as an active ingredient
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Citations (4)

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Publication number Priority date Publication date Assignee Title
KR100295389B1 (en) * 1998-05-22 2001-09-17 신민규 Antiviral pharmaceutical composition useful for prevention and treatment of a cold caused by influenza a virus and a functional food
KR20110049618A (en) * 2009-11-05 2011-05-12 한국 한의학 연구원 Composition for prevention or treatment of disease originated from influenza virus
KR20200024185A (en) * 2018-04-21 2020-03-06 류형준 Pharmaceutical and food composition for treating or alleviating influenza a
KR20200140166A (en) * 2019-06-05 2020-12-15 이재원 miscellaneous honey health supplements containing lacquer and the making method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100295389B1 (en) * 1998-05-22 2001-09-17 신민규 Antiviral pharmaceutical composition useful for prevention and treatment of a cold caused by influenza a virus and a functional food
KR20110049618A (en) * 2009-11-05 2011-05-12 한국 한의학 연구원 Composition for prevention or treatment of disease originated from influenza virus
KR20200024185A (en) * 2018-04-21 2020-03-06 류형준 Pharmaceutical and food composition for treating or alleviating influenza a
KR20200140166A (en) * 2019-06-05 2020-12-15 이재원 miscellaneous honey health supplements containing lacquer and the making method

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CIANCIOSI DANILA; FORBES-HERNANDEZ TAMARA Y.; ALVAREZ-SUAREZ JOSÉ M.; ANSARY JOHURA; QUINZI DENISE; AMICI ADOLFO; NAVARRO-HORTAL M: "Anti-inflammatory activities of Italian Chestnut and Eucalyptus honeys on murine RAW 264.7 macrophages", JOURNAL OF FUNCTIONAL FOODS, ELSEVIER BV, NL, vol. 87, 15 September 2021 (2021-09-15), NL , XP086892934, ISSN: 1756-4646, DOI: 10.1016/j.jff.2021.104752 *

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