WO2023038480A1 - Composition, for preventing, treating or relieving influenza virus infection, comprising mixture of agrimonia pilosa extract and galla rhois extract as active ingredient - Google Patents

Composition, for preventing, treating or relieving influenza virus infection, comprising mixture of agrimonia pilosa extract and galla rhois extract as active ingredient Download PDF

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WO2023038480A1
WO2023038480A1 PCT/KR2022/013577 KR2022013577W WO2023038480A1 WO 2023038480 A1 WO2023038480 A1 WO 2023038480A1 KR 2022013577 W KR2022013577 W KR 2022013577W WO 2023038480 A1 WO2023038480 A1 WO 2023038480A1
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influenza virus
extract
virus infection
preventing
aprg64
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PCT/KR2022/013577
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French (fr)
Korean (ko)
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강세찬
서영진
김형건
이영근
최순호
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에이피알지 주식회사
경희대학교 산학협력단
중앙대학교 산학협력단
성균관대학교산학협력단
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Priority claimed from KR1020220113158A external-priority patent/KR20230039782A/en
Application filed by 에이피알지 주식회사, 경희대학교 산학협력단, 중앙대학교 산학협력단, 성균관대학교산학협력단 filed Critical 에이피알지 주식회사
Publication of WO2023038480A1 publication Critical patent/WO2023038480A1/en

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    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • 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)
    • A61K36/22—Anacardiaceae (Sumac family), e.g. smoketree, sumac or poison oak
    • 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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/36—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/49—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds
    • A61K8/4973—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom
    • A61K8/498—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing heterocyclic compounds with oxygen as the only hetero atom having 6-membered rings or their condensed derivatives, e.g. coumarin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/60—Sugars; Derivatives thereof
    • A61K8/602—Glycosides, e.g. rutin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
    • A61Q17/005—Antimicrobial preparations
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/20—Natural extracts
    • A23V2250/21—Plant extracts
    • A23V2250/2116—Flavonoids, isoflavones
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/30—Other Organic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a composition for preventing, treating or improving influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois .
  • Influenza commonly known as 'the flu', is an acute respiratory disease caused by the influenza virus. It is highly contagious and causes large and small group infections or pandemics around the world every year. It is a highly contagious disease that infects 10-20% of the general population within ⁇ 3 weeks.
  • influenza virus is one of the viruses that cause severe respiratory symptoms in the elderly, children, certain chronic diseases, and immunocompromised patients, leading to death in severe cases (Hien, TT et al. N. Eng. J. Med ., 350, 1179, 2004).
  • Influenza virus taxonomically belongs to Orthomyxovirus , and there are three types of A, B, and C types, and particularly, the epidemically spreading types are known as A and B types.
  • a and B types On the surface of these viruses, there are two types of surface antigens, glycoproteins, hamagglitinin (HA) and neuraminidase (NA), and eight segmented RNAs exist inside.
  • Hemagglutinin is one of the antigenic projections sprouting on the influenza virus surface. It is in the form of a trimer composed of a head and a stem, and the double head part is related to most antigenic mutations and attaches the virus by binding to the terminal sialic acid residue on the surface of the host cell and sequentially the virus enters the host cell.
  • Neuraminidase is an enzyme of a glycoprotein in the viral envelope membrane. It is a mushroom-shaped tetramer with a head and stem shape, and has an active site on the upper surface of the head. The virus replicated and multiplied in the infected cell cuts the alpha-ketosidic bond connecting the oligosaccharide portion on the cell surface and the terminal neuraminic acid residue to release the virus outside the host cell. It plays a major role in penetration into cells of the respiratory tract (a. Mark, VI Nature review 6, 987, 2007. b. Huberman, K et al. Virology 214, 294, 1995).
  • Virus surface antigens mutate within the same subtype, and new antigenic variants emerge every year.
  • the avian influenza virus which has been a problem until recently, among influenza viruses, has mutated to infect various types of birds such as chickens, turkeys, ducks and wild birds.
  • chickens are infected due to rapid propagation, more than 80% of them die, so it is a viral disease that causes the greatest damage and threat to the poultry industry worldwide, and its ripple effect is not limited to the poultry industry. It has been reported to cause disease to (Gubareva, L. V. et al. Lancet, 355, 2000).
  • Influenza is contracted by inhaling aerosols containing the virus, which are usually airborne through coughing or sneezing. In addition to this, it can be transmitted by excrement, saliva, nasal mucus, feces and blood of other objects, but most transmission is droplet infection caused by inhalation of aerosols.
  • the most obvious symptom is a sudden high fever of 38-40 ° C within 24 hours of infection, systemic symptoms such as headache, muscle pain and fatigue, and respiratory symptoms such as sore throat, cough, expectoration and rhinitis also appear. In addition, abdominal pain, vomiting and convulsions may occur rarely. Healthy people recover after showing symptoms for several days, but patients with chronic lung disease, heart disease, and immunocompromised patients may die from complications such as pneumonia, as well as encephalopathy, myelitis, Reye syndrome, myositis, Complications such as myocarditis and pericarditis may be accompanied. Children have similar symptoms as adults, but they have higher fevers, febrile seizures may occur, and otitis media, pseudomembranous laryngitis, and myalgia are more common.
  • 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 the only avian influenza (AI) treatment exclusively produced in the world. It is an antiviral drug that has a therapeutic effect by blocking the enzyme function that proliferates the virus, and it 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.
  • oseltamir has a side effect of severe vomiting
  • zanamivir has a high antiviral effect, but has low bioavailability and rapid excretion from the kidneys.
  • the present inventors completed the present invention by confirming that influenza virus infection can be effectively prevented or treated using the Seonhakcho extract and gall nut extract.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating influenza virus infection, a food composition for preventing or improving influenza virus infection, and influenza virus infection containing a mixture of an extract of Agrimonia pilosa and a galla rhois extract as an active ingredient. It is to provide a quasi-drug composition for preventing or suppressing a viral infection.
  • Another object of the present invention is a flavonoid compound represented by the following formula (1) or a triterpenoid compound represented by the following formula (2), isomers thereof, and pharmaceutically acceptable salts thereof selected from the group consisting of It is to provide a pharmaceutical composition for preventing or treating influenza virus infection and a quasi-drug composition for preventing or suppressing influenza virus infection, comprising any one or more as active ingredients:
  • R 1 is H or OH
  • R 2 is any one selected from the group consisting of ⁇ -L-rhamnopyranosyl, H, ⁇ -D-glucopyranosyl, rutinosyl, and OH;
  • R 3 is H or ⁇ -D-glucuronosyl
  • Another object of the present invention is selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and food-acceptable salts thereof. It is to provide a food composition for preventing or improving influenza virus infection comprising at least one of the active ingredients.
  • the present invention is a pharmaceutical composition for preventing or treating influenza virus infection and influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient It provides a quasi-drug composition for prevention or suppression.
  • the present invention provides a food composition for preventing or improving influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient.
  • the Seonhakcho extract or the gall nut extract may be extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 alcohol, and mixed solvents thereof, but is limited thereto It is not.
  • the Seonhakcho extract or the nut gall extract may be extracted using 30 to 95% ethanol, but is not limited thereto.
  • the mixture may be mixed so that the weight ratio of Seonhakcho extract: Nut gall extract is 10: 1 to 1: 10, but is not limited thereto.
  • influenza virus may be an influenza A type H1N1 virus, but is not limited thereto.
  • the mixture can inhibit the expression of M1 or NP gene of influenza virus, but is not limited thereto.
  • the present invention is any one selected from the group consisting of flavonoid compounds represented by the following formula (1) or triterpenoid compounds represented by the following formula (2), isomers thereof, and pharmaceutically acceptable salts thereof.
  • a pharmaceutical composition for preventing or treating influenza virus infection and a quasi-drug composition for preventing or suppressing influenza virus infection, comprising at least one as an active ingredient are provided:
  • R 1 is H or OH
  • R 2 is any one selected from the group consisting of ⁇ -L-rhamnopyranosyl, H, ⁇ -D-glucopyranosyl, rutinosyl, and OH;
  • R 3 is H or ⁇ -D-glucuronosyl
  • the quasi-drug may be at least one selected from the group consisting of disinfectant cleaner, shower foam, gargreen, wet tissue, detergent soap, hand wash, and ointment, but is not limited thereto.
  • the present invention is any one selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and food-acceptable salts thereof. It provides a food composition for preventing or improving influenza virus infection containing one or more as active ingredients.
  • the food composition may be a health functional food composition, but is not limited thereto.
  • the flavonoid compound is Afzelin, Apigenin, Apigenin 7-O- ⁇ -D-glucuronide (Apigenin 7-O- ⁇ - It may be any one or more selected from the group consisting of D-glucuronide), Astragalin, Nicotiflorin, Quercetin, Quercitrin, and Rutin, but is not limited thereto. no.
  • the triterpenoid compound may be ursolic acid, but is not limited thereto.
  • the present invention provides a method for preventing or treating influenza virus infection comprising the step of administering to a subject in need of a composition containing a mixture of the extract of Agrimonia pilosa and the extract of Galla rhois as an active ingredient do.
  • the present invention provides a method for preventing or inhibiting influenza virus infection comprising the step of using a composition comprising a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois as an active ingredient by an individual in need thereof do.
  • the present invention is any one selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically acceptable salts thereof.
  • a method for preventing or treating influenza virus infection comprising administering a composition containing one or more active ingredients to a subject in need thereof.
  • the present invention is any one selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically acceptable salts thereof.
  • a method for preventing or inhibiting influenza virus infection comprising the step of using a composition containing one or more active ingredients by an individual in need thereof.
  • the present invention provides a composition comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient for preventing, treating, improving, or inhibiting influenza virus infection.
  • the present invention provides the use of a mixture of an extract of Agrimonia pilosa and a galla rhois extract for the preparation of a preparation for preventing, treating, improving or inhibiting influenza virus infection.
  • the present invention is a group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically or food-acceptable salts thereof.
  • a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically or food-acceptable salts thereof.
  • the present invention relates to a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and It provides the use of a composition containing at least one selected from the group consisting of pharmaceutically or food-acceptable salts thereof as an active ingredient.
  • a composition for preventing, treating, or improving influenza virus infection containing a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois as an active ingredient has no side effects on the human body and is safe, while cell lesions through a virus-dependent pathway It has excellent antiviral activity according to effect, influenza virus-related gene reduction effect, etc.
  • the antiviral effect was confirmed in vivo, and the active ingredients of the mixed extract also have remarkably excellent antiviral activity, it can be usefully used as a composition for preventing, treating, or improving influenza virus infectious diseases.
  • 1 is a graph showing the results of testing the cytotoxicity of APRG64 in MDCK cell lines.
  • FIGS. 2a and 2b are photographs and graphs showing the cytopathic effect inhibitory activity of APRG64 in IAV-infected MDCK cell lines.
  • Figure 2c is a graph showing that the cytopathic effect inhibitory activity of APRG64 is by a virus-dependent pathway.
  • Figure 3a is a graph showing the inhibitory effect of influenza virus M1 and NP gene expression according to APRG64 treatment, and a photograph showing the result of Western blot experiment on the expression inhibitory effect of influenza virus M1 and NS1 protein.
  • Figure 3b is a graph showing the plaque analysis results for the influenza virus particle reduction effect according to the treatment of APRG64.
  • Figure 3c is a photograph and a graph showing the results of immunocytochemical analysis on the effect of reducing cells expressing influenza virus NP according to APRG64 treatment.
  • 4 is a diagram showing structural formulas and functional groups of 9 compounds isolated from APRG64.
  • 5a and 5b are graphs showing the effect of reducing the gene expression of M1 and NP of influenza virus when treated with 9 compounds isolated from APRG64 cancer cell lines.
  • Figures 6a and 6b are photographs and graphs showing the cytopathic effect inhibitory activity in IAV-infected MDCK cell lines by apigenin.
  • 6C is a graph showing that the activity of apigenin to inhibit the effect of apigenin on cell damage is a virus-dependent pathway.
  • Figure 7a is a graph showing the effect of suppressing the expression of influenza virus M1 and NP genes according to apigenin treatment, and a photograph showing the results of Western blot experiments on the effect of suppressing the expression of influenza virus M1 and NS1 proteins.
  • Figure 7b is a graph showing the results of plaque analysis on the effect of reducing influenza virus particles according to apigenin treatment.
  • 7c is a photograph and a graph showing the results of immunocytochemical analysis on the effect of reducing cells expressing influenza virus NP according to apigenin treatment.
  • Figure 8a is a photograph showing the results of Western blot experiments showing the activity of inhibiting influenza virus replication according to apigenin treatment.
  • 8b and 8c are graphs showing the reduction of M1 and NP gene expression of influenza virus in an experiment confirming the inhibitory activity of APRG64 and apigenin on influenza virus replication through the initial mechanism of viral replication.
  • Figure 8d is a graph showing the experimental results confirming the influenza virus replication inhibitory activity of APRG64 and apigenin through RNA polymerase activity inhibition.
  • 8e is a photograph showing the results of a Western blot experiment confirming the inhibitory activity of APRG64 and apigenin on influenza virus replication through inhibition of MAPK activity.
  • 9a is a diagram showing a schematic view of a mouse model for intranasal administration of APRG64 and apigenin.
  • 9B is a graph showing the effect of reducing IAV titers in the lungs of influenza virus-infected mice when APRG64 and apigenin were administered intranasally.
  • 10a is a diagram showing a schematic view of a mouse model for oral administration of APRG64.
  • Figure 10b is a graph showing the effect of inhibiting weight loss in mice infected with influenza virus when APRG64 was orally administered.
  • 10c is a graph showing the effect of suppressing the death of influenza virus-infected mice when APRG64 was orally administered.
  • 10d is a graph showing the effect of reducing viral RNA and infectious virus particles in the lungs of influenza virus-infected mice when APRG64 was orally administered.
  • 10e is a graph showing the reduction of IFN- ⁇ , TNF- ⁇ , and IL-6 expressions induced in influenza virus-infected mice upon oral administration of APRG64.
  • 11a to 11c show the results of influenza virus suppression experiments through molecular docking simulation using APRG64-derived Chemical Formulas 1 to 3.
  • the present invention provides a pharmaceutical composition for preventing or treating influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois as an active ingredient.
  • Seonhakcho extract may be a whole plant extract including Seonhakcho leaves, stems, roots, or both, and according to an embodiment of the present invention, it may be an Agrimonia pilosa leaf extract, but is not limited thereto don't
  • the seonhakcho can be used without limitation, such as directly harvested, cultivated, or commercially available.
  • gall extract may be a nut gall leaf, stem, branch, root, or whole plant extract including both thereof, and according to an embodiment of the present invention, galla rhois may be a leaf extract, but thereby Not limited.
  • the gall nut can be used without limitation, such as directly collected, cultivated, or commercially available.
  • Seonhakcho Agrimonia pilosa Extract and nut gall ( Galla rhois )
  • the mixture of the extract was abbreviated as APRG64.
  • extract refers to an extract obtained by the extraction treatment of the sagebrush or gall gall, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a purified product or a purified product of the extract, or a mixture thereof. Etc., the extract itself and extracts of all formulations that can be formed using the extract.
  • extract may include a crude extract, a polar solvent-soluble extract or a non-polar solvent-soluble extract, a fermented extract, and the like.
  • any lactic acid bacteria may be applied to enhance the influenza virus inhibitory effect of the Seonhakcho or Nut gall extract according to the present invention, but is not limited thereto.
  • the Seonhakcho extract or the gall nut extract may be in the form of a dried product, but is not limited thereto.
  • the method for extracting the Sunhakcho or gall nut is not particularly limited, and can be extracted according to a method commonly used in the art.
  • a method using an extraction device such as supercritical extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, or ultrasonic extraction, or a method using an adsorption resin including XAD and HP-20 may be used.
  • Non-limiting examples of the extraction method include a heating extraction method, a cold extraction method, a reflux cooling extraction method, a steam distillation method, an ultrasonic extraction method, an elution method, a compression method, and the like, which are performed alone or in combination of two or more methods. It can be.
  • the extract may be additionally subjected to a conventional fractionation process and may be purified according to a conventional purification method.
  • the extract included in the composition of the present invention may be prepared by pulverizing the primary extract extracted by the hot water extraction or solvent extraction method through an additional process such as distillation under reduced pressure and freeze drying or spray drying.
  • the primary extract can be further purified by various chromatography methods such as silica gel column chromatography, high performance liquid chromatography, and thin layer chromatography to obtain additional purified fractions.
  • the extract may include all extracts, separated compounds, fractions and purified products obtained in each step of extraction, fractionation or purification, dilution, concentration, or drying thereof.
  • the type of extraction solvent used to extract the Sunhakcho or the gallbladder is not particularly limited, and according to a conventional method known in the art for extracting an extract from natural products, that is, conventional temperature and pressure It can be extracted using conventional solvents under the conditions.
  • a solvent alcohol having 1 to 4 carbon atoms including purified water, ethanol, methanol, isopropanol, butanol, etc., and acetone, ether, benzene ), chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and the like may be used alone or in combination.
  • the Seonhakcho extract or the nut gall extract may be extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 alcohol, and mixed solvents thereof, and according to an embodiment of the present invention, ethanol It can be extracted using a solvent, but is not limited thereto.
  • methanol or ethanol aqueous solution may be used, and the methanol or ethanol aqueous solution is 30 to 95% (v / v), more specifically 40 to 60% (v / v), most specifically 50% (v / v ) may be methanol or ethanol, but is not limited thereto.
  • the ethanol when used as a solvent to extract the sunflower or the nut gall, for example, 10% to 100% ethanol, 10% to 90% ethanol, 10% to 80% ethanol, 10% to 70% ethanol, 10% to 60% Ethanol, 10% to 50% Ethanol, 20% to 90% Ethanol, 20% to 80% Ethanol, 20% to 70% Ethanol, 20% to 60% Ethanol, 20% to 50% Ethanol, 30 % to 90% Ethanol, 30% to 80% Ethanol, 30% to 70% Ethanol, 30% to 60% Ethanol, 30% to 50% Ethanol, 40% to 90% Ethanol, 40% to 80% Ethanol, 40% to 70% ethanol, 40% to 60% ethanol, 40% to 50% ethanol, 45% to 55% ethanol, or 50% ethanol, but is not limited thereto.
  • the prepared extract may then be filtered or concentrated or dried to remove the solvent, and both filtration, concentration and drying may be performed.
  • filtration may use filter paper or a vacuum filter
  • concentration may use a vacuum vacuum concentrator or vacuum rotary evaporator
  • drying may be performed by vacuum drying, vacuum drying, boiling drying, spray drying, freeze drying, and the like.
  • vacuum drying vacuum drying, boiling drying, spray drying, freeze drying, and the like.
  • the number of extractions may be carried out one or more times, but as the extraction continues, the yield of the active ingredient significantly decreases, so it may not be economical to perform the extraction repeatedly five times or more. Accordingly, the number of extractions is preferably 1 to 5 times, and more preferably 2 to 5 repeated extractions, but is not limited thereto.
  • the extraction temperature is preferably 20°C to 100°C, more preferably 20°C to 80°C, and most preferably room temperature, but is not limited thereto.
  • the extraction time is preferably one to 10 hours, but is not limited thereto.
  • the mixture may be mixed so that the weight ratio of Seonhakcho extract: gall nut extract is 10:1 to 1:10, and also, the weight ratio is 1:0.1 to 9, 1:0.1 to 8, 1:0.1 to 7, 1:0.1 to 6, 1:0.1 to 5, 1:0.1 to 4, 1:0.1 to 3, 1:0.1 to 2, 1:0.1 to 1, 1:0.1 to 0.9, 1:0.1 to 0.8, 1:0.1 to 0.7, or 1:0.1 to 0.6.
  • it may be mixed in a weight ratio of 6:4, but is not limited thereto.
  • the mixture may inhibit the expression of the M1 or NP gene of influenza virus, but is not limited thereto.
  • the present invention relates to at least one selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically acceptable salts thereof.
  • a pharmaceutical composition for preventing or treating influenza virus infection comprising as an active ingredient:
  • R 1 is H or OH
  • R 2 is any one selected from the group consisting of ⁇ -L-rhamnopyranosyl, H, ⁇ -D-glucopyranosyl, rutinosyl, and OH;
  • R 3 is H or ⁇ -D-glucuronosyl
  • the flavonoid compounds include Afzelin, Apigenin, Apigenin 7-O- ⁇ -D-glucuronide, It may be at least one selected from the group consisting of Astragalin, Nicotiflorin, Quercetin, Quercitrin, and Rutin, but is not limited thereto.
  • the triterpenoid compound may be ursolic acid, but is not limited thereto.
  • the compound represented by Compound 10 may have a molecular formula of C 41 H 32 O 28 and a molecular weight of 940.67, [(2R,3R,4S,5R,6S)-3,4,5,6-tetrakis[ (3,4,5-trihydroxybenzoyl)oxy]oxan-2-yl]methyl 3,4,5-trihydroxybenzoate may have the IUPAC name.
  • the compound represented by Compound 2 may have a molecular formula of C 30 H 48 O 3 and a molecular weight of 456.7, (1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10 -hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H It can have the IUPAC name of -picene-4a-carboxylic acid.
  • the compound represented by compound 3 may have a molecular formula of C 15 H 10 O 7 and a molecular weight of 302.2, and the IUPAC of 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one can have a name.
  • the method for obtaining the compound may be chemically synthesized by a method known in the field to which the present invention pertains, or a commercially available material may be used, but is not limited thereto.
  • the extract or mixture may include one or more selected from the group consisting of Compounds 1 to 10 as an active ingredient, standard material, or active material, but is not limited thereto.
  • the extract or mixture may contain a high dose of the compound, but is not limited thereto.
  • influenza refers to an infectious disease caused by an influenza virus of the Orthomyxoviridae family among viruses that cause cold symptoms. Compared to the common cold, systemic symptoms such as fever, muscle pain, and headache appear distinctly, and the incubation period is about 1 to 3 days. Influenza is classified into A, B, and C types, and their overall structure and composition are the same. They have a diameter of 80 to 120 nm, and are filament-shaped in the early stage of infection, but become circular in the later stage.
  • Influenza virus the viral envelope surrounding the central nucleus is largely composed of glycoproteins that can be distinguished into two types, hemagglutinin (H) and neuraminidase (N), and the nucleus is composed of viral RNA (viral RNA). RNA) and viral proteins necessary to protect and activate it. Types B and C have low susceptibility and low genetic diversity, so there are almost no subtypes, and they appear infrequently and do not become prevalent. It is again divided into several subtypes according to the reaction. In nature, there are 18 H serotypes and 11 N serotypes, and mainly H1/2/3 and N1/2 are the subtypes that cause influenza in humans. According to one embodiment of the present invention, the influenza virus may be an influenza A type H1N1 virus, but is not limited thereto.
  • the term “molecular docking simulation” means structure-based drug design by predicting the binding suitability of a small molecule ligand to an appropriate target binding site based on the association between biologically relevant molecules such as proteins, peptides, nucleic acids, carbohydrates, and lipids.
  • influenza inhibitory activity was determined based on the ability to bind to influenza virus proteins.
  • the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • the excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.
  • compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods.
  • tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta agents, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external agents are creams, gels, patches, sprays, ointments, and warning agents.
  • lotion, liniment, pasta, or cataplasma may have formulations such as the like.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, 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.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like may be used.
  • a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.
  • Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.
  • tragacantha methylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, HPMC 2910, etc. and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.
  • Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; tonicity agents such as sodium chlor
  • the suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolen (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Buytyrum Tego-G -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hyde Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neos
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin.
  • excipients for example, starch, calcium carbonate, sucrose, etc.
  • lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc.
  • 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 suspending agents.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.
  • the pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.
  • subject means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. of mammals.
  • administration means providing a given composition of the present invention to a subject by any suitable method.
  • prevention refers to any action that suppresses or delays the onset of a desired disease
  • treatment means that the desired disease and its resulting metabolic abnormality are improved or improved by administration of the pharmaceutical composition according to the present invention. All actions that are advantageously altered are meant, and “improvement” means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administration of the composition according to the present invention.
  • the present invention provides a food composition for preventing or improving influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient.
  • the present invention is any one selected from the group consisting of flavonoid compounds represented by the following formula (1) or triterpenoid compounds represented by the following formula (2), isomers thereof, and food-acceptable salts thereof. It provides a food composition for preventing or improving influenza virus infection comprising one or more as active ingredients:
  • R 1 is H or OH
  • R 2 is any one selected from the group consisting of ⁇ -L-rhamnopyranosyl, H, ⁇ -D-glucopyranosyl, rutinosyl, and OH;
  • R 3 is H or ⁇ -D-glucuronosyl
  • "food” means a natural product or processed product containing one or more nutrients, preferably means a state that can be eaten directly through a certain degree of processing process, and usually means As, it means to include all health functional foods, beverages, food additives and beverage additives.
  • the food composition may be a health functional food composition, but is not limited thereto.
  • the "functional food” is the same term as food for special health use (FoSHU), medicine processed to efficiently display bioregulatory functions in addition to nutritional supply, It means a food with high medical effect, and can be manufactured into tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or film formulations.
  • “functionality” means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions.
  • Seonhakcho of the present invention Agrimonia pilosa ) Extract and nut gall ( Galla rhois )
  • the mixture of the extract and its derived compound can be added as it is or used together with other food or food ingredients, according to conventional methods can be used
  • the mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).
  • the freshwater extract of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material.
  • the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
  • Examples of foods to which the above substances can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in a conventional sense.
  • the health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages.
  • the aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.
  • the proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.
  • the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, A carbonation agent used in carbonated beverages and the like may be contained.
  • the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.
  • the present invention provides a quasi-drug composition for preventing or suppressing influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient.
  • the present invention is any one selected from the group consisting of flavonoid compounds represented by the following formula (1) or triterpenoid compounds represented by the following formula (2), isomers thereof, and pharmaceutically acceptable salts thereof.
  • a quasi-drug composition for preventing or inhibiting influenza virus infection comprising at least one as an active ingredient:
  • R 1 is H or OH
  • R 2 is any one selected from the group consisting of ⁇ -L-rhamnopyranosyl, H, ⁇ -D-glucopyranosyl, rutinosyl, and OH;
  • R 3 is H or ⁇ -D-glucuronosyl
  • the quasi-drug is a preparation used for sterilization, insecticide, and similar purposes for the prevention of infectious diseases described in Article 2, Subparagraph 7, Item C of the Pharmaceutical Affairs Act, and is used for human or animal It may refer to repellents, repellents, preventives, pesticides, or attracting insecticides such as flies and mosquitoes used for the health of people.
  • the quasi-drug may include external skin preparations and personal hygiene products.
  • it may be a disinfectant cleanser, shower foam, gargreen, wet tissue, detergent soap, hand wash, or ointment, but is not limited thereto.
  • the quasi-drug composition according to the present invention When used as a quasi-drug additive, the composition may be added as it is or used together with other quasi-drugs or quasi-drug ingredients, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined depending on the purpose of use.
  • the quasi-drug composition of the present invention may be prepared in the form of, for example, a general emulsified formulation and a solubilized formulation.
  • a general emulsified formulation for example, it may have formulations such as emulsions, creams, ointments, sprays, oil gels, gels, oils, aerosols, and smokers such as lotions, but it may be used without limitation as long as it exhibits the pest control inducing effect of the present invention. .
  • the quasi-drug composition appropriately blends oil, water, surfactant, moisturizer, lower alcohol having 1 to 4 carbon atoms, a thickener, a chelating agent, a colorant, a preservative, or a flavoring agent, etc., which are generally formulated in quasi-drug compositions, in each dosage form, as necessary. and can be used.
  • the MDCK cell line used in the experiment was cultured under conditions of 37°C, 5% CO 2 and MEM (Minimum essential) containing 10% fetal bovine serum (FBS, Hyclone Thermo Scientific) and 1% penicillin/streptomycin (P/S, Gibco). medium, Gibco) medium.
  • FBS fetal bovine serum
  • P/S penicillin/streptomycin
  • the leaves of Agrimonia pilosa and Galla rhois were purchased from BioKorea Co., LTd (Seoul), and voucher specimens (BMRI-AP-1601, BMRI-RG-1602) were purchased from Biomedical Research Center, Kyunghee University, Yongin, Korea. deposited in A dried sample (20 kg) was extracted with 50% ethanol at 80 ⁇ 2° C. for 6 hours and then filtered. Then, it was concentrated using a rotary evaporator and freeze-dried to obtain 1.57 kg of Seonhakcho extract and 11.59 kg of nut gall extract. A mixture of Seonhakcho extract and gall nut extract was used in a ratio of 6:4, respectively, and was abbreviated as APRG64. All samples were stored at 4°C until use.
  • CPE cytopathic effect
  • IAV-induced CPE inhibitory activity of APRG64 confirmed in Experimental Example 1-2 is not a virus-independent cell death but a virus-dependent cell death effect, it was confirmed whether the same effect appears under an apoptosis inducer.
  • MDCK cells infected with IAV were treated with 200 nM of staurosporine (STS), an apoptosis inducer, and then additionally treated with 10 ⁇ g/ml APRG64 to determine cell death (cell viability).
  • STS staurosporine
  • 10 ⁇ g/ml APRG64 1% crystal violet staining was used, the images were processed with ImageJ software to quantify the cells attached to the plate, and all graphs were made based on the average of three repetitions.
  • IAV-induced CPE inhibitory activity of APRG64 confirmed in Experimental Example 1 was based on the inhibition of viral replication. To confirm this, MDCK cells infected with IAV were treated with APRG64, and RT-qPCR and Western blot experiments were performed on the production of influenza virus mRNA, protein, and infectious particles.
  • MDCK cells cultured in MEM medium were infected with IAV at a multiplicity of infection (MOI) of 0.1 for 1 hour, then treated with APRG64 (10 ⁇ g/ml), and after 6 hours, M1, which forms the structure of influenza virus, And the expression level of viral nucleoprotein (NP) was analyzed by RT-qPCR.
  • NucleoZOL Macherey-Nagel, Duren, Germany
  • ReverTraAce qPCR RT kit Toyobo, Osaka, Japan
  • a cDNA synthesis kit RevertraAce qPCR RT kit, Toyobo
  • CFX Connect real-time system Bio-Rad, Hercules, CA, USA
  • Classification (Content) direction order SEQ ID NO: 1 (M1 primer) forward 5′-AAGACCAATCCTGTCACCTCTG-3′ SEQ ID NO: 2 (M1 primer) reverse 5'-CAAAACGTCTACGCTGCAGTCC-3' SEQ ID NO: 3 (NP primer) forward 5′-CCAGATCAGTGTGCAGCCTA-3′ SEQ ID NO: 4 (NP primer) reverse 5′-CTCTGGCTTTGCACTTTCC-3′ SEQ ID NO: 5 (GAPDH primer) forward 5′-AACATCATCCCTGCTTCCAC-3′ SEQ ID NO: 6 (GAPDH primer) reverse 5′-GACCACCTGGTCCTCAGTGT-3′
  • M1 and NS1 proteins of IAV were analyzed by Western blot, and actin was used as a loading control.
  • cells were lysed using NP-40 buffer (ELPIS Biotech, Daejeon, Korea) in the presence of a protease inhibitor (Thermo Scientific).
  • Cell extracts were loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA), and then the membrane was incubated in TBST solution for blocking.
  • % BSA for 1 hour followed by incubation with primary antibodies (Abcam, Cambridge, UK) overnight at 4°C.
  • HRP horseradish peroxidase
  • APRG64 treatment was shown to strongly suppress the expression of IAV M1 (Fig. 3a, left graph) and NP (Fig. 3a, center graph) mRNA expression, and consistent with these results, M1 and non-structural protein 1 (NS1 ) was found to be substantially inhibited by APRG64 treatment (Fig. 3a, right picture).
  • infectious virus particles were titrated through plaque assay. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour and then treated with 10 ⁇ g/ml APRG64 for 36 hours, followed by 0.3% BSA and 0.5% agarose (Affymetrix, Santa Clara, CA, USA). It was incubated for 2 days with MEM containing. Cells were then fixed with 3.5% formaldehyde and removed from an agarose gel, and plaques were identified by staining the cells with a 1% crystal violet solution.
  • Immunocytochemical analysis was performed to confirm the effect of APRG64 on reducing IAV NP-expressing cells. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour, then treated with 10 ⁇ g/ml APRG64 for 6 hours, fixed with 4% formaldehyde, and then treated with 0.5% Triton X-100 (Sigma-Aldrich). permeabilized. After treatment with an anti-IAV NP antibody (Abcam), cells were incubated with Alexa Fluor 488-conjugated secondary antibody (Thermo Scientific) and DAPI (4',6-diamidino-2-phenylindole) solution (Sigma-Aldrich) was used to detect nuclei.
  • the anti-IAV activity was confirmed.
  • the anti-IAV activity was confirmed through the expression levels of M1 and viral nucleoprotein (NP), which form the structure of influenza virus, after each compound was treated with IAV-infected cells.
  • MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour, and then treated with afgeline (Compound 1, 10 ⁇ g/ml), apigenin (Compound 2, 5 ⁇ g/ml), and apigenin 7-O-glu.
  • Curonide (Compound 3, 10 ⁇ g/ml), Astragaline (Compound 4, 10 ⁇ g/ml), Nicotiflorin (Compound 5, 10 ⁇ g/ml), Quercetin (Compound 6, 2.5 ⁇ g/ml), Quercetin (Compound 6, 2.5 ⁇ g/ml)
  • Relative expression levels of M1 and NP mRNAs of IAV were measured by RT -Analyzed by qPCR. RT-qPCR analysis was performed in the same manner as in Example 2-1 except for the concentration of each compound.
  • MDCK cells were infected with IAV at an MOI of 0.1 and then treated with 0.625, 1.25, 2.50 or 5.00 ⁇ g/ml of apigenin, and after 24 hours, the cells were incubated with 1% crystal violet solution. Adherent cells were analyzed by staining. In addition, the EC 50 of apigenin was calculated by quantifying the attached cells at 24 hpi, and CPE analysis was performed in the same manner as in Experimental Example 1-2 except for this.
  • apigenin was additionally treated after treatment with the apoptosis inducer to confirm cell death.
  • MDCK cells were treated with 5 ⁇ g/ml of apigenin and then additionally treated with 200 nM of staurosporine (STS) to perform CPE analysis to confirm cell viability.
  • STS staurosporine
  • the image was processed with ImageJ software to quantify the cells attached to the plate, and the graph showed the average of three repetitions, and the experiment was performed in the same manner as in Experimental Example 1-3 except for this.
  • RT-qPCR analysis and Western blotting were performed. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour and then treated with 5 ⁇ g/ml apigenin, and after 6 hours, the expression levels of M1 and NP mRNA of IAV were analyzed by RT-qPCR, Virus M1 and NS1 protein expression levels were analyzed by Western blot. The experimental method and conditions were performed in the same manner as in Experimental Example 2-1.
  • MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour, then treated with 5 ⁇ g/ml apigenin, and plaque assay was performed at 36 hpi to measure the infectious virus titer and presented in a graph. At this time, the plaque analysis method and conditions were performed in the same manner as in Experimental Example 2-2.
  • Immunocytochemical analysis was performed to confirm the increase or decrease of IAV NP-expressing cells (IAV-NP) by apigenin. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour and then treated with 5 ⁇ g/ml apigenin for 6 hours, and then the reduction of IAV-NP expressing cells was confirmed by immunocytochemical analysis, At this time, immunocytochemical analysis was performed in the same manner as in Experimental Example 2-3.
  • apigenin is the main antiviral component of APRG64.
  • the antiviral activity of APRG64 and apigenin against other strains of IAV was investigated.
  • MDCK cells were infected with another influenza strain, A/PR/8/34 (H1N1), at an MOI of 0.1 for 1 hour, and treated with APRG64 (10 ⁇ g/ml) or apigenin (5 ⁇ g/ml), respectively.
  • APRG64 10 ⁇ g/ml
  • apigenin 5 ⁇ g/ml
  • APRG64 and apigenin were confirmed to strongly reduce viral protein expression in PR8-infected cells, respectively (FIG. 8a).
  • the anti-IAV activity of APRG64 and apigenin was similar to that of oseltamivir phosphate (Tamiflu), an antiviral neuraminidase inhibitor clinically used for the treatment of IAV infection, influenza virus It was confirmed that the treatment effect was remarkably good.
  • IAV replication begins when the virus attaches to and enters the host cell. Whether APRG64 and apigenin target this early stage of viral replication to inhibit IAV replication was analyzed.
  • MDCK cells were treated with 10 ⁇ g/ml APRG64 or 5 ⁇ g/ml apigenin for 1 hour at 4° C., respectively, and infected with IAV at an MOI of 0.1. Then, the mRNA expression levels of M1 and NP of IAV were analyzed by RT-qPCR at 6 hpi. In addition, the mRNA expression levels of M1 and NP of IAV after treatment with APRG64 or apigenin at 1, 2 or 4 hours (hpi) after infection were analyzed.
  • APRG64 and apigenin significantly inhibited the expression of M1 and NP RNA of IAV, respectively (FIG. 8b).
  • treatment with APRG64 or apigenin at 1, 2 or 4 hours post infection (hpi) also reduced the expression levels of M1 and NP RNA of IAV (Fig. 8c). According to these results, it was confirmed that APRG64 and apigenin inhibit the early stages of IAV replication including viral attachment and entry.
  • RdRP IAV RNA-dependent RNA polymerase
  • IAV M1 and NP IAV M1 and NP (+) It can be measured by the expression level of the RNA strand. Therefore, by analyzing the expression levels of (+) RNA strands of IAV M1 and NP according to APRG64 and apigenin treatment, it was confirmed whether or not IAV RdRP activity was affected. Specifically, MDCK cells were infected with IAV at 0.1 MOI for 1 hour.
  • IAV infection dramatically induced phosphorylation of ERK and SAPK, but this increase was not observed in cells treated with APRG64 or apigenin (FIG. 8e).
  • APRG64 and apigenin were intranasally administered to a mouse model to confirm antiviral effects.
  • dpi the number of infectious viral particles in the lungs was measured by plaque assay. Other than that, it was performed under the same conditions as the plaque analysis method according to Experimental Example 2-2.
  • APRG64 was orally administered to mice, it was confirmed whether the killing effect caused by IAV was suppressed. Specifically, wild-type C57BL/6 mice prepared as described above were orally administered with 0, 25 or 50 mg/kg of APRG64 for 3 days, and then infected with 1 ⁇ 10 3 pfu of IAV. In addition, APRG64 was orally administered to the mice for an additional 5 days, and mouse survival was monitored for 14 days.
  • mice were significantly protected from IAV-induced death in a dose-dependent manner (FIG. 10c). Specifically, all of the mouse group not administered with APRG64 died on day 10, whereas all of the group treated with APRG64 maintained survival for 14 days. In particular, the survival rate of mice administered with 50 mg/kg of APRG64 was 80% or more, confirming that the antiviral effect against IAV was remarkably excellent.
  • RNA, cytokines, and infectious viral particles in mouse lungs were analyzed by RT-qPCR or plaque assay.
  • wild-type C57BL/6 mice prepared for oral administration of Experimental Example 8-3 were orally treated with 50 mg/kg of APRG64 for 3 days, and then the mice were infected with 1 ⁇ 10 3 pfu of IAV, and then 5 The same amount of APRG64 was additionally treated for 10 days.
  • mice lungs were collected and viral RNA, titers and production of inflammatory cytokines were analyzed to determine the relative mRNA expression levels and infectious virus titers of M1 and NP of IAV, and the inflammatory cytokines IFN- ⁇ , TNF- ⁇ and The relative expression level of IL-6 was analyzed by RT-qPCR.
  • Classification (Content) direction order SEQ ID NO: 7 (IFN- ⁇ primer) forward 5′-GGCCATCAGCAACAACATAAGCGT-3′ SEQ ID NO: 8 (IFN- ⁇ primer) reverse 5′-TGGGTTGTTGACCTCAAACTTGGC-3′ SEQ ID NO: 9 (TNF- ⁇ primer) forward 5′-GCCTCTTCTCATTCCTGCTTG-3′ SEQ ID NO: 10 (TNF- ⁇ primer) reverse 5′-CTGATGAGAGGGAGGCCATT-3′ SEQ ID NO: 11 (IL-6 primer) forward 5′-ACGGCCTTCCCTACTTCACA-3′ SEQ ID NO: 12 (IL-6 primer) reverse 5'-CATTTCCACGATTTCCCAGA-3'
  • APRG64 can be used to treat IAV infection as a drug that can be administered intranasally and orally, which is safe and exhibits remarkably excellent effects.
  • a molecular docking simulation was performed on the influenza virus for the APRG64-derived compound to evaluate the virus inhibitory ability.
  • influenza A/H1N1 the compound continuously binds to the subunit viral RdRp (PA, PB1, PB2) that affects replication and transcription in the virus. Therefore, in the present invention, blind docking was performed for the subunit viral RdRp (PA, PB1, PB2).
  • molecular docking simulation was performed for the C-terminal domain, middle domain, and Cap binding domain.
  • Influenza virus also has a Receptor binding domain (RBD) like the spike protein of coronavirus (SARS-CoV-2). RBD; and A pocket, which plays a major role in the process of cell membrane fusion after RBD enters the cell and before exporting its RNA into the cell.
  • RBD Receptor binding domain
  • a composition for preventing, treating, or improving influenza virus infection containing a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois as an active ingredient has no side effects on the human body and is safe, while antiviral through a virus-dependent route activity is excellent.
  • antiviral effects have been confirmed in vivo, and the active ingredients of the mixed extracts also have remarkably excellent antiviral activities, so it is expected that they can be usefully used as a composition for preventing, treating, or improving influenza virus infectious diseases.
  • the industrial applicability of the present invention is recognized.

Abstract

The present invention relates to a composition, for preventing, treating or relieving influenza virus infection, comprising a mixture of an Agrimonia pilosa extract and a Galla rhois extract as an active ingredient, and does not cause side effects to a human body and thus exhibits excellent safety, and also has excellent antiviral activity due to a cell lesion effect through a virus-dependent pathway, an influenza virus-related gene reducing effect, and the like. Further, the present invention not only is confirmed with an in vivo antiviral effect but also has notably excellent antiviral activity in the active ingredient of the mixed extracts, and thus may be utilized for a composition for preventing, treating or relieving influenza virus infection.

Description

선학초 추출물과 오배자 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방, 치료 또는 개선용 조성물Composition for preventing, treating or improving influenza virus infection comprising a mixture of Seonhakcho extract and gall nut extract as an active ingredient
본 발명은 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 포함하는 인플루엔자 바이러스 감염증 예방, 치료 또는 개선용 조성물에 관한 것이다.The present invention relates to a composition for preventing, treating or improving influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois .
본 출원은 2021년 09월 08일에 출원된 한국특허출원 제10-2021-0119674호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다. This application claims priority based on Korean Patent Application No. 10-2021-0119674 filed on September 08, 2021, and all contents disclosed in the specification and drawings of the application are incorporated into this application.
또한, 본 출원은 2022년 09월 07일에 출원된 한국특허출원 제10-2022-0113158호에 기초한 우선권을 주장하며, 해당 출원의 명세서 및 도면에 개시된 모든 내용은 본 출원에 원용된다. In addition, this application claims priority based on Korean Patent Application No. 10-2022-0113158 filed on September 7, 2022, and all contents disclosed in the specification and drawings of the application are incorporated into this application.
인플루엔자(Influenza)는 흔히 ‘독감’이라 알려져 있고, 인플루엔자 바이러스(influenza virus)에 의해서 발병되는 급성 호흡기 질환으로, 전염성이 매우 강해 매년 전 세계적으로 크고 작은 집단감염이나 대유행을 일으키며, 유행이 시작되면 2~3주 이내에 통상 인구의 10~20%가 감염될 정도로 감염성이 큰 질병이다. 또한, 인플루엔자 바이러스는 고령자, 소아, 특정한 만성질환자, 면역부전 환자에게 심각한 호흡기 증상을 유발하는 바이러스 중 하나로 심각할 경우 사망에 이른다(Hien, T. T. et al. N. Eng. J. Med., 350, 1179, 2004). Influenza, commonly known as 'the flu', is an acute respiratory disease caused by the influenza virus. It is highly contagious and causes large and small group infections or pandemics around the world every year. It is a highly contagious disease that infects 10-20% of the general population within ~3 weeks. In addition, the influenza virus is one of the viruses that cause severe respiratory symptoms in the elderly, children, certain chronic diseases, and immunocompromised patients, leading to death in severe cases (Hien, TT et al. N. Eng. J. Med ., 350, 1179, 2004).
인플루엔자 바이러스는 분류학적으로 오르쏘믹소바이러스(Orthomyxovirus)에 속하며, A, B, C형의 3가지 형이 있으며 특히 유행적으로 확산되는 형은 A, B형으로 알려져 있다. 이들 바이러스 표면에는 당단백질인 적혈구 응집소(hamagglitinin, HA)와 뉴라미니다아제(neuraminidase, NA)라는 두 종류의 표면 항원이 존재하고 내부에는 8개의 분절된 RNA가 존재한다. 헤마글루티닌은 인플루엔자 바이러스 표면에 돋아나 있는 항원성 돌기중 하나이다. 이는 머리와 줄기로 구성된 트라이머(trimer) 형태이고, 이중 머리 부분은 대부분의 항원변이와 관련되어 있으며 숙주세포의 표면에 있는 말단 시알산 잔기와 결합하여 바이러스를 부착시키고 순차적으로 바이러스가 숙주세포로 침투할 수 있게 한다(Chandrasekaran, A. et al. Nature biotechnology, 26, 107, 2008). 뉴라미니다아제는 바이러스 외피 막에 있는 당단백질의 효소이다. 이는 머리와 줄기 형태를 가지는 버섯모양의 테트라머(tetramer) 형태이고, 머리 상단 표면에 활성자리가 있다. 감염된 세포 내에서 복제 및 증식된 바이러스가 세포 표면의 올리고사카라이드 부분과 말단 뉴라민산(neuraminic acid) 잔기를 연결해주는 알파-케토사이딕 본드(ketosidic bond)를 절단하여 바이러스를 숙주세포 밖으로 배출하여 호흡기 점막 세포로 침투하는 데 주요한 역할을 한다(a. Mark, V. I. Nature review 6, 987, 2007. b. Huberman, K et al. Virology 214, 294, 1995).Influenza virus taxonomically belongs to Orthomyxovirus , and there are three types of A, B, and C types, and particularly, the epidemically spreading types are known as A and B types. On the surface of these viruses, there are two types of surface antigens, glycoproteins, hamagglitinin (HA) and neuraminidase (NA), and eight segmented RNAs exist inside. Hemagglutinin is one of the antigenic projections sprouting on the influenza virus surface. It is in the form of a trimer composed of a head and a stem, and the double head part is related to most antigenic mutations and attaches the virus by binding to the terminal sialic acid residue on the surface of the host cell and sequentially the virus enters the host cell. to penetrate (Chandrasekaran, A. et al. Nature biotechnology, 26, 107, 2008). Neuraminidase is an enzyme of a glycoprotein in the viral envelope membrane. It is a mushroom-shaped tetramer with a head and stem shape, and has an active site on the upper surface of the head. The virus replicated and multiplied in the infected cell cuts the alpha-ketosidic bond connecting the oligosaccharide portion on the cell surface and the terminal neuraminic acid residue to release the virus outside the host cell. It plays a major role in penetration into cells of the respiratory tract (a. Mark, VI Nature review 6, 987, 2007. b. Huberman, K et al. Virology 214, 294, 1995).
바이러스의 표면 항원들은 동일한 아형에서 변이를 일으키고, 매년 새로운 항원 변이주가 출현한다. 특히, 인플루엔자 바이러스 중 최근까지 문제가 되는 조류 인플루엔자 바이러스는 대변이가 일어나 닭, 칠면조, 오리 및 야생조류 등 여러 종류의 조류를 감염시켰다. 빠른 전파로 인해 닭이 감염되면, 80% 이상이 폐사하므로 전 세계적으로 양계산업에 가장 큰 피해와 위협을 주는 바이러스 질환이며, 그 파급효과는 양계산업에만 한정되어 있지 않고 인체에 대한 감염으로 인하여 사람에게 질병을 일으키는 것으로 보고되고 있다(Gubareva, L. V. et al. Lancet, 355, 2000).Virus surface antigens mutate within the same subtype, and new antigenic variants emerge every year. In particular, the avian influenza virus, which has been a problem until recently, among influenza viruses, has mutated to infect various types of birds such as chickens, turkeys, ducks and wild birds. When chickens are infected due to rapid propagation, more than 80% of them die, so it is a viral disease that causes the greatest damage and threat to the poultry industry worldwide, and its ripple effect is not limited to the poultry industry. It has been reported to cause disease to (Gubareva, L. V. et al. Lancet, 355, 2000).
인플루엔자는 보통 기침이나 재채기를 통해 공기 중으로 나오는, 바이러스가 함유된 연무질을 흡입함으로써 인플루엔자에 감염된다. 이 외에도 타 개체의 배설물, 침, 콧물, 대변과 혈액으로도 전염될 수 있지만, 대부분의 전염은 연무질 흡입으로 인한 비말감염이라고 할 수 있다. Influenza is contracted by inhaling aerosols containing the virus, which are usually airborne through coughing or sneezing. In addition to this, it can be transmitted by excrement, saliva, nasal mucus, feces and blood of other objects, but most transmission is droplet infection caused by inhalation of aerosols.
가장 뚜렷한 증상은 감염 24시간 이내에 38~40℃의 갑작스러운 고열이며, 두통, 근육통 및 피로감 등의 전신 증상과 인후통, 기침, 객담 및 비염 등의 호흡기 증상도 나타난다. 또한 복통, 구토 및 경련 등이 드물게 발생할 수 있다. 건강한 사람은 수일간 증상을 보인 후 회복되지만, 만성 폐 질환, 심장 질환자 및 면역 저하자 등은 폐렴과 같은 합병증이 발생하여 사망할 수 있으며, 이외에 뇌증, 척수염(transverse myelitis), Reye 증후군, 근염, 심근염 및 심낭염 등의 합병증이 동반될 수 있다. 소아의 경우 성인에서와 비슷한 증상을 보이지만, 열이 더 높게 나고, 열성 경련이 일어날 수 있으며, 중이염, 위막성 후두염(croup) 및 근육통도 더 흔하게 발생한다.The most obvious symptom is a sudden high fever of 38-40 ° C within 24 hours of infection, systemic symptoms such as headache, muscle pain and fatigue, and respiratory symptoms such as sore throat, cough, expectoration and rhinitis also appear. In addition, abdominal pain, vomiting and convulsions may occur rarely. Healthy people recover after showing symptoms for several days, but patients with chronic lung disease, heart disease, and immunocompromised patients may die from complications such as pneumonia, as well as encephalopathy, myelitis, Reye syndrome, myositis, Complications such as myocarditis and pericarditis may be accompanied. Children have similar symptoms as adults, but they have higher fevers, febrile seizures may occur, and otitis media, pseudomembranous laryngitis, and myalgia are more common.
종래 인플루엔자의 치료에 사용되는 약물로는 오셀타미비르(상품명: 타미플루), 자나미비르(상품명: 릴렌자), 페라미비르(en:Peramivir), 아만타딘(en:Amantadine) 등이 있다. 타미플루로 불리는 오셀타미비르가 현재 H1N1 인플루엔자 A형의 치료에 주로 사용되고 있다. 타미플루는 세계에서 유일하게 독점 생산하는 조류 인플루엔자(AI) 치료제이다. 바이러스를 증식시키는 효소 기능을 막아 치료 효과를 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. 이는 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 이용률이 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 the only avian influenza (AI) treatment exclusively produced in the world. It is an antiviral drug that has a therapeutic effect by blocking the enzyme function that proliferates the virus, and it 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 low bioavailability and rapid excretion from the kidneys.
현재까지 개발된 항인플루엔자 치료제들은 부작용이 나타내는 경우가 대부분이다. 따라서, 인플루엔자의 예방 및 치료에 효과적인 항인플루엔자 조성물의 개발이 시급한 실정이다.Most of the anti-influenza treatments developed so far have side effects. Therefore, there is an urgent need to develop an anti-influenza composition effective for preventing and treating influenza.
이에 본 발명자들은 선학초 추출물 및 오배자 추출물을 사용하여 인플루엔자 바이러스 감염증을 효과적으로 예방 또는 치료할 수 있다는 것을 확인하여 본 발명을 완성하였다.Accordingly, the present inventors completed the present invention by confirming that influenza virus infection can be effectively prevented or treated using the Seonhakcho extract and gall nut extract.
본 발명자들은 인플루엔자 바이러스 감염증의 치료를 위해서 연구한 결과 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물 및 이의 유래 화합물에 뛰어난 항인플루엔자 효과가 있는 것을 확인하였는바, 이에 기초하여 본 발명을 완성하였다.As a result of research for the treatment of influenza virus infection, the present inventors have confirmed that the mixture of Agrimonia pilosa extract and Galla rhois extract and its derived compounds have an excellent anti-influenza effect, based on this the present invention completed.
이에 본 발명의 목적은 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물, 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물, 및 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating influenza virus infection, a food composition for preventing or improving influenza virus infection, and influenza virus infection containing a mixture of an extract of Agrimonia pilosa and a galla rhois extract as an active ingredient. It is to provide a quasi-drug composition for preventing or suppressing a viral infection.
본 발명의 다른 목적은 하기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 하기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물 및 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물을 제공하는 것이다:Another object of the present invention is a flavonoid compound represented by the following formula (1) or a triterpenoid compound represented by the following formula (2), isomers thereof, and pharmaceutically acceptable salts thereof selected from the group consisting of It is to provide a pharmaceutical composition for preventing or treating influenza virus infection and a quasi-drug composition for preventing or suppressing influenza virus infection, comprising any one or more as active ingredients:
[화학식 1][Formula 1]
Figure PCTKR2022013577-appb-img-000001
Figure PCTKR2022013577-appb-img-000001
상기 화학식에서, In the above formula,
R1은 H 또는 OH이고,R 1 is H or OH;
R2는 α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, 및 OH로 이루어진 군으로 선택되는 어느 하나이고 ,R 2 is any one selected from the group consisting of α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, and OH;
R3은 H 또는 β-D-glucuronosyl이고, R 3 is H or β-D-glucuronosyl;
[화학식 2][Formula 2]
Figure PCTKR2022013577-appb-img-000002
.
Figure PCTKR2022013577-appb-img-000002
.
본 발명의 또 다른 목적은 상기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 상기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 식품학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물을 제공하는 것이다.Another object of the present invention is selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and food-acceptable salts thereof. It is to provide a food composition for preventing or improving influenza virus infection comprising at least one of the active ingredients.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 본 발명이 속하는 기술분야의 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. will be.
상기와 같은 본 발명의 목적을 달성하기 위하여, 본 발명은 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물 및 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물을 제공한다. 또한, 본 발명은 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물을 제공한다.In order to achieve the object of the present invention as described above, the present invention is a pharmaceutical composition for preventing or treating influenza virus infection and influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient It provides a quasi-drug composition for prevention or suppression. In addition, the present invention provides a food composition for preventing or improving influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient.
본 발명의 일 실시예에 있어서, 상기 선학초 추출물 또는 상기 오배자 추출물은 물, C1 내지 C4의 알코올, 및 이들의 혼합용매로 이루어지는 군에서 선택된 하나 이상의 용매로 추출된 것일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the Seonhakcho extract or the gall nut extract may be extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 alcohol, and mixed solvents thereof, but is limited thereto It is not.
본 발명의 일 실시예에 있어서, 상기 선학초 추출물 또는 상기 오배자 추출물은 30 내지 95% 에탄올을 사용하여 추출된 것일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the Seonhakcho extract or the nut gall extract may be extracted using 30 to 95% ethanol, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 혼합물은 선학초 추출물 : 오배자 추출물의 중량비가 10:1 내지 1:10이 되도록 혼합될 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the mixture may be mixed so that the weight ratio of Seonhakcho extract: Nut gall extract is 10: 1 to 1: 10, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 인플루엔자 바이러스는 인플루엔자 A형 H1N1 바이러스일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the influenza virus may be an influenza A type H1N1 virus, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 혼합물은 인플루엔자 바이러스의 M1, 또는 NP 유전자의 발현을 억제시킬 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the mixture can inhibit the expression of M1 or NP gene of influenza virus, but is not limited thereto.
또한, 본 발명은 하기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 하기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물 및 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물을 제공한다:In addition, the present invention is any one selected from the group consisting of flavonoid compounds represented by the following formula (1) or triterpenoid compounds represented by the following formula (2), isomers thereof, and pharmaceutically acceptable salts thereof. A pharmaceutical composition for preventing or treating influenza virus infection and a quasi-drug composition for preventing or suppressing influenza virus infection, comprising at least one as an active ingredient are provided:
[화학식 1][Formula 1]
Figure PCTKR2022013577-appb-img-000003
Figure PCTKR2022013577-appb-img-000003
상기 화학식에서, In the above formula,
R1은 H 또는 OH이고,R 1 is H or OH;
R2는 α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, 및 OH로 이루어진 군으로 선택되는 어느 하나이고,R 2 is any one selected from the group consisting of α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, and OH;
R3은 H 또는 β-D-glucuronosyl이고,R 3 is H or β-D-glucuronosyl;
[화학식 2][Formula 2]
Figure PCTKR2022013577-appb-img-000004
.
Figure PCTKR2022013577-appb-img-000004
.
본 발명의 일 실시예에 있어서, 상기 의약외품은 소독청결제, 샤워폼, 가그린, 물티슈, 세제비누, 핸드워시, 및 연고제로 이루어지는 군에서 선택된 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the quasi-drug may be at least one selected from the group consisting of disinfectant cleaner, shower foam, gargreen, wet tissue, detergent soap, hand wash, and ointment, but is not limited thereto.
또한, 본 발명은 상기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 상기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 식품학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물을 제공한다. In addition, the present invention is any one selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and food-acceptable salts thereof. It provides a food composition for preventing or improving influenza virus infection containing one or more as active ingredients.
본 발명의 일 실시예에 있어서, 상기 식품 조성물은 건강 기능성 식품 조성물일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the food composition may be a health functional food composition, but is not limited thereto.
본 발명의 일 실시예에 있어서, 상기 플라보노이드(flavonoid) 화합물은 아프젤린(Afzelin), 아피제닌(Apigenin), 아피제닌 7-O-β-D-글루쿠로나이드(Apigenin 7-O-β-D-glucuronide), 아스트라갈린(Astragalin), 니코티플로린(Nicotiflorin), 케르세틴(Quercetin), 케르시트린(Quercitrin), 및 루틴(Rutin)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아니다. In one embodiment of the present invention, the flavonoid compound is Afzelin, Apigenin, Apigenin 7-O-β-D-glucuronide (Apigenin 7-O-β- It may be any one or more selected from the group consisting of D-glucuronide), Astragalin, Nicotiflorin, Quercetin, Quercitrin, and Rutin, but is not limited thereto. no.
본 발명의 일 실시예에 있어서, 상기 트리테르펜(triterpenoid) 화합물은 우르솔산(Ursolic acid)일 수 있으나, 이에 제한되는 것은 아니다.In one embodiment of the present invention, the triterpenoid compound may be ursolic acid, but is not limited thereto.
또한, 본 발명은 상기 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 조성물을 이를 필요로 하는 개체에 투여하는 단계를 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료방법을 제공한다.In addition, the present invention provides a method for preventing or treating influenza virus infection comprising the step of administering to a subject in need of a composition containing a mixture of the extract of Agrimonia pilosa and the extract of Galla rhois as an active ingredient do.
또한, 본 발명은 상기 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 조성물을 이를 필요로 하는 개체가 사용하는 단계를 포함하는 인플루엔자 바이러스 감염증 예방 또는 억제방법을 제공한다.In addition, the present invention provides a method for preventing or inhibiting influenza virus infection comprising the step of using a composition comprising a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois as an active ingredient by an individual in need thereof do.
또한, 본 발명은 상기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 상기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 조성물을 이를 필요로 하는 개체에 투여하는 단계를 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료방법을 제공한다.In addition, the present invention is any one selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically acceptable salts thereof. Provided is a method for preventing or treating influenza virus infection comprising administering a composition containing one or more active ingredients to a subject in need thereof.
또한, 본 발명은 상기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 상기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 조성물을 이를 필요로 하는 개체가 사용하는 단계를 포함하는 인플루엔자 바이러스 감염증 예방 또는 억제방법을 제공한다.In addition, the present invention is any one selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically acceptable salts thereof. Provided is a method for preventing or inhibiting influenza virus infection comprising the step of using a composition containing one or more active ingredients by an individual in need thereof.
또한, 본 발명은 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 조성물의 인플루엔자 바이러스 감염증 예방, 치료, 개선, 또는 억제 용도를 제공한다.In addition, the present invention provides a composition comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient for preventing, treating, improving, or inhibiting influenza virus infection.
또한, 본 발명은 인플루엔자 바이러스 감염증 예방, 치료, 개선 또는 억제용 제제의 제조를 위한 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물의 용도를 제공한다.In addition, the present invention provides the use of a mixture of an extract of Agrimonia pilosa and a galla rhois extract for the preparation of a preparation for preventing, treating, improving or inhibiting influenza virus infection.
또한, 본 발명은 상기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 상기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 또는 식품학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 조성물의 인플루엔자 바이러스 감염증 예방, 치료, 개선, 또는 억제 용도를 제공한다.In addition, the present invention is a group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically or food-acceptable salts thereof. Provides the use of preventing, treating, improving, or suppressing influenza virus infection of a composition comprising any one or more selected from as an active ingredient.
또한, 본 발명은 인플루엔자 바이러스 감염증 예방, 치료, 개선 또는 억제용 제제의 제조를 위한 상기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 또는 식품학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 조성물의 용도를 제공한다.In addition, the present invention relates to a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and It provides the use of a composition containing at least one selected from the group consisting of pharmaceutically or food-acceptable salts thereof as an active ingredient.
선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방, 치료 또는 개선용 조성물은 인체에 부작용이 없어 안전성이 우수하면서도, 바이러스 의존적인 경로를 통한 세포병변 효과, 인플루엔자 바이러스 관련 유전자 감소 효과 등에 따른 항바이러스 활성이 뛰어나다. 이에 더하여, 생체 내에서도 항바이러스 효과가 확인되었을 뿐만 아니라, 혼합 추출물의 활성 성분 역시 현저히 우수한 항바이러스 활성을 가진다는 점에서 인플루엔자 바이러스 감염 질환의 예방, 치료 또는 개선용 조성물로 유용하게 활용될 수 있다.A composition for preventing, treating, or improving influenza virus infection containing a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois as an active ingredient has no side effects on the human body and is safe, while cell lesions through a virus-dependent pathway It has excellent antiviral activity according to effect, influenza virus-related gene reduction effect, etc. In addition, in that the antiviral effect was confirmed in vivo, and the active ingredients of the mixed extract also have remarkably excellent antiviral activity, it can be usefully used as a composition for preventing, treating, or improving influenza virus infectious diseases.
도 1은 MDCK 세포주에서 APRG64의 세포독성을 시험한 결과를 나타낸 그래프이다.1 is a graph showing the results of testing the cytotoxicity of APRG64 in MDCK cell lines.
도 2a 및 도 2b는 IAV 감염된 MDCK 세포주에서 APRG64의 세포병변 효과 억제 활성을 나타낸 사진 및 그래프이다.2a and 2b are photographs and graphs showing the cytopathic effect inhibitory activity of APRG64 in IAV-infected MDCK cell lines.
도 2c는 APRG64의 세포병변 효과 억제 활성이 바이러스 의존적 경로에 의한 것임을 나타내는 그래프이다.Figure 2c is a graph showing that the cytopathic effect inhibitory activity of APRG64 is by a virus-dependent pathway.
도 3a는 APRG64 처리에 따른 인플루엔자 바이러스 M1 및 NP 유전자 발현 억제 효과를 나타낸 그래프 및 인플루엔자 바이러스 M1 및 NS1 단백질의 발현 억제 효과에 대한 웨스턴 블롯 실험 결과를 나타낸 사진이다.Figure 3a is a graph showing the inhibitory effect of influenza virus M1 and NP gene expression according to APRG64 treatment, and a photograph showing the result of Western blot experiment on the expression inhibitory effect of influenza virus M1 and NS1 protein.
도 3b는 APRG64의 처리에 따른 인플루엔자 바이러스 입자 감소 효과에 대한 플라크 분석 결과를 나타낸 그래프이다.Figure 3b is a graph showing the plaque analysis results for the influenza virus particle reduction effect according to the treatment of APRG64.
도 3c는 APRG64 처리에 따른 인플루엔자 바이러스의 NP를 발현하는 세포 감소 효과에 대한 면역세포화학 분석 결과를 나타낸 사진 및 그래프이다.Figure 3c is a photograph and a graph showing the results of immunocytochemical analysis on the effect of reducing cells expressing influenza virus NP according to APRG64 treatment.
도 4는 APRG64에서 분리된 9가지의 화합물의 구조식을 나타낸 그림 및 작용기를 나타낸 도표이다.4 is a diagram showing structural formulas and functional groups of 9 compounds isolated from APRG64.
도 5a 및 도 5b는 APRG64에서 분리된 9가지의 화합물 암 세포주에 처리 시 인플루엔자 바이러스의 M1 및 NP의 유전자 발현 감소 효과를 나타낸 그래프이다.5a and 5b are graphs showing the effect of reducing the gene expression of M1 and NP of influenza virus when treated with 9 compounds isolated from APRG64 cancer cell lines.
도 6a 및 도 6b는 아피제닌에 의한 IAV 감염된 MDCK 세포주에서의 세포병변 효과 억제 활성을 나타낸 사진 및 그래프이다.Figures 6a and 6b are photographs and graphs showing the cytopathic effect inhibitory activity in IAV-infected MDCK cell lines by apigenin.
도 6c는 아피제닌의 세포 병변 효과 억제 활성이 바이러스 의존적 경로임을 나타내는 그래프이다.6C is a graph showing that the activity of apigenin to inhibit the effect of apigenin on cell damage is a virus-dependent pathway.
도 7a는 아피제닌 처리에 따른 인플루엔자 바이러스 M1 및 NP 유전자 발현 억제 효과를 나타낸 그래프 및 인플루엔자 바이러스 M1 및 NS1 단백질의 발현 억제 효과에 대한 웨스턴 블롯 실험 결과를 나타낸 사진이다.Figure 7a is a graph showing the effect of suppressing the expression of influenza virus M1 and NP genes according to apigenin treatment, and a photograph showing the results of Western blot experiments on the effect of suppressing the expression of influenza virus M1 and NS1 proteins.
도 7b는 아피제닌 처리에 따른 인플루엔자 바이러스 입자 감소 효과에 대한 플라크 분석 결과를 나타낸 그래프이다.Figure 7b is a graph showing the results of plaque analysis on the effect of reducing influenza virus particles according to apigenin treatment.
도 7c는 아피제닌 처리에 따른 인플루엔자 바이러스의 NP를 발현하는 세포 감소 효과에 대한 면역세포화학 분석 결과를 나타낸 사진 및 그래프이다.7c is a photograph and a graph showing the results of immunocytochemical analysis on the effect of reducing cells expressing influenza virus NP according to apigenin treatment.
도 8a는 아피제닌 처리에 따른 인플루엔자 바이러스 복제 억제 활성을 나타낸 웨스턴 블롯 실험 결과를 나타낸 사진이다.Figure 8a is a photograph showing the results of Western blot experiments showing the activity of inhibiting influenza virus replication according to apigenin treatment.
도 8b 및 도 8c는 바이러스 복제의 초기 기전 타겟을 통한 APRG64 및 아피제닌의 인플루엔자 바이러스 복제 억제 활성을 확인한 실험에서 인플루엔자 바이러스의 M1 및 NP 유전자 발현 감소를 나타낸 그래프이다.8b and 8c are graphs showing the reduction of M1 and NP gene expression of influenza virus in an experiment confirming the inhibitory activity of APRG64 and apigenin on influenza virus replication through the initial mechanism of viral replication.
도 8d는 RNA 중합효소 활성 억제를 통한 APRG64 및 아피제닌의 인플루엔자 바이러스 복제 억제 활성을 확인한 실험결과를 나타낸 그래프이다.Figure 8d is a graph showing the experimental results confirming the influenza virus replication inhibitory activity of APRG64 and apigenin through RNA polymerase activity inhibition.
도 8e는 MAPK 활성 억제를 통한 APRG64 및 아피제닌의 인플루엔자 바이러스 복제 억제 활성을 확인한 웨스턴 블롯 실험 결과를 나타낸 사진이다.8e is a photograph showing the results of a Western blot experiment confirming the inhibitory activity of APRG64 and apigenin on influenza virus replication through inhibition of MAPK activity.
도 9a는 APRG64 및 아피제닌의 비강 투여 마우스 모델 모식도를 나타낸 그림이다.9a is a diagram showing a schematic view of a mouse model for intranasal administration of APRG64 and apigenin.
도 9b는 APRG64 및 아피제닌의 비강 투여시, 인플루엔자 바이러스에 감염된 마우스 폐에서의 IAV 역가 감소 효과를 나타내는 그래프이다.9B is a graph showing the effect of reducing IAV titers in the lungs of influenza virus-infected mice when APRG64 and apigenin were administered intranasally.
도 10a는 APRG64의 경구 투여 마우스 모델 모식도를 나타낸 그림이다.10a is a diagram showing a schematic view of a mouse model for oral administration of APRG64.
도 10b는 APRG64 경구 투여시, 인플루엔자 바이러스에 감염된 마우스의 체중 감소 억제 효과를 나타낸 그래프이다.Figure 10b is a graph showing the effect of inhibiting weight loss in mice infected with influenza virus when APRG64 was orally administered.
도 10c는 APRG64 경구 투여시, 인플루엔자 바이러스에 감염된 마우스의 사망 억제 효과를 나타낸 그래프이다.10c is a graph showing the effect of suppressing the death of influenza virus-infected mice when APRG64 was orally administered.
도 10d는 APRG64 경구 투여시, 인플루엔자 바이러스에 감염된 마우스 폐에서의 바이러스 RNA 및 감염성 바이러스 입자 감소 효과를 나타낸 그래프이다.10d is a graph showing the effect of reducing viral RNA and infectious virus particles in the lungs of influenza virus-infected mice when APRG64 was orally administered.
도 10e는 APRG64 경구 투여시, 인플루엔자 바이러스에 감염된 마우스에서 유도된 IFN-γ, TNF-α 및 IL-6 발현 감소를 나타낸 그래프이다.10e is a graph showing the reduction of IFN-γ, TNF-α, and IL-6 expressions induced in influenza virus-infected mice upon oral administration of APRG64.
도 11a 내지 도 11c는 APRG64 유래 화학식 1 내지 3을 이용하여 분자도킹 시뮬레이션을 통한 인플루엔자 바이러스 억제 실험의 결과를 나타낸 것이다.11a to 11c show the results of influenza virus suppression experiments through molecular docking simulation using APRG64-derived Chemical Formulas 1 to 3.
이하, 본 발명에 대해 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois as an active ingredient.
본 발명에 있어서, “선학초 추출물”은 선학초 잎, 줄기, 뿌리, 또는 이를 모두 포함한 식물 전체 추출물일 수 있으며, 본 발명의 일 실시예에 따르면 선학초(Agrimonia pilosa) 잎 추출물일 수 있으나, 이에 제한되지 않는다. 상기 선학초는 직접 채취, 재배한 것 또는 시판되는 것 등을 제한 없이 사용할 수 있다.In the present invention, "Seonhakcho extract" may be a whole plant extract including Seonhakcho leaves, stems, roots, or both, and according to an embodiment of the present invention, it may be an Agrimonia pilosa leaf extract, but is not limited thereto don't The seonhakcho can be used without limitation, such as directly harvested, cultivated, or commercially available.
본 발명에 있어서, “오배자 추출물”은 오배자 잎, 줄기, 가지, 뿌리, 또는 이를 모두 포함한 식물 전체 추출물일 수 있으며, 본 발명의 일 실시예에 따르면 오배자(Galla rhois) 잎 추출물일 수 있으나, 이에 제한되지 않는다. 상기 오배자는 직접 채취, 재배한 것 또는 시판되는 것 등을 제한 없이 사용할 수 있다. In the present invention, "nut gall extract" may be a nut gall leaf, stem, branch, root, or whole plant extract including both thereof, and according to an embodiment of the present invention, galla rhois may be a leaf extract, but thereby Not limited. The gall nut can be used without limitation, such as directly collected, cultivated, or commercially available.
본 발명에 있어서, 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물은 APRG64로 약칭하였다.In the present invention, Seonhakcho ( Agrimonia pilosa ) Extract and nut gall ( Galla rhois ) The mixture of the extract was abbreviated as APRG64.
본 발명에 있어서, “추출물”은 상기 선학초 또는 상기 오배자의 추출처리에 의하여 얻어지는 추출액, 상기 추출액의 희석액이나 농축액, 상기 추출액을 건조하여 얻어지는 건조물, 상기 추출액의 조정제물이나 정제물, 또는 이들의 혼합물 등, 추출액 자체 및 추출액을 이용하여 형성 가능한 모든 제형의 추출물을 포함한다. 또한, “추출물”에는 조 추출물, 극성용매 가용 추출물 또는 비극성 용매 가용 추출물, 발효 추출물 등이 포함될 수 있다. In the present invention, "extract" refers to an extract obtained by the extraction treatment of the sagebrush or gall gall, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, a purified product or a purified product of the extract, or a mixture thereof. Etc., the extract itself and extracts of all formulations that can be formed using the extract. In addition, the “extract” may include a crude extract, a polar solvent-soluble extract or a non-polar solvent-soluble extract, a fermented extract, and the like.
또한, 상기 발효 추출물의 제조에 있어서 본 발명에 따른 선학초 또는 오배자 추출물의 인플루엔자 바이러스 억제 효과를 증진시키기 위하여 임의의 유산균 등이 적용될 수 있으나, 이에 제한되는 것은 아니다.In addition, in the preparation of the fermented extract, any lactic acid bacteria may be applied to enhance the influenza virus inhibitory effect of the Seonhakcho or Nut gall extract according to the present invention, but is not limited thereto.
본 발명의 일 실시예에 따르면, 상기 선학초 추출물 또는 상기 오배자 추출물은 건조물 형태일 수 있으나, 이에 제한되지 않는다.According to one embodiment of the present invention, the Seonhakcho extract or the gall nut extract may be in the form of a dried product, but is not limited thereto.
본 발명의 상기 선학초 추출물 또는 상기 오배자 추출물에 있어서, 상기 선학초 또는 오배자를 추출하는 방법은 특별히 제한되지 아니하며, 당해 기술분야에서 통상적으로 사용하는 방법에 따라 추출할 수 있다. 예를 들어, 초임계추출, 아임계추출, 고온추출, 고압 추출 또는 초음파추출법 등의 추출 장치를 이용한 방법 또는 XAD 및 HP-20을 포함한 흡착 수지를 이용하는 방법 등을 이용할 수 있다. 상기 추출 방법의 비제한적인 예로는, 가열추출법, 냉침추출법, 환류냉각추출법, 수증기증류법, 초음파추출법, 용출법, 압착법 등을 들 수 있으며, 이들은 단독으로 수행되거나 2 종 이상의 방법을 병용하여 수행될 수 있다. 또한, 예를 들어 진탕 추출, 속슬렛(Soxhlet) 추출 또는 환류 추출을 이용하는 것이 바람직하나, 이에 제한되지 않는다. 또한 목적에 따라서 추출물은 추가적으로 통상의 분획 공정을 수행할 수 있으며, 통상의 정제 방법에 따라 정제될 수 있다.In the Sunhakcho extract or the nut gall extract of the present invention, the method for extracting the Sunhakcho or gall nut is not particularly limited, and can be extracted according to a method commonly used in the art. For example, a method using an extraction device such as supercritical extraction, subcritical extraction, high-temperature extraction, high-pressure extraction, or ultrasonic extraction, or a method using an adsorption resin including XAD and HP-20 may be used. Non-limiting examples of the extraction method include a heating extraction method, a cold extraction method, a reflux cooling extraction method, a steam distillation method, an ultrasonic extraction method, an elution method, a compression method, and the like, which are performed alone or in combination of two or more methods. It can be. In addition, it is preferable to use, for example, shaking extraction, Soxhlet extraction or reflux extraction, but is not limited thereto. In addition, depending on the purpose, the extract may be additionally subjected to a conventional fractionation process and may be purified according to a conventional purification method.
예를 들어, 본 발명의 조성물에 포함되는 추출물은 상기의 열수 추출 또는 용매 추출법으로 추출된 1차 추출물을 감압 증류 및 동결 건조 또는 분무 건조 등과 같은 추가 과정을 통해 분말화하여 제조할 수 있다. 또 상기 1차 추출물을 실리카겔 컬럼 크로마토그래피(silica gel column chromatography), 고성능 액체 크로마토그래피 (high performance liquid chromatography), 박층 크로마토그래피(thin layer chromatography) 등의 다양한 크로마토그래피를 통해 추가적으로 정제 분획을 얻을 수 있다. 따라서 본 발명에 있어서 추출물은 추출, 분획 또는 정제의 각 단계에서 얻어지는 모든 추출액, 분리 화합물, 분획 및 정제물, 이들의 희석, 농축, 건조물 등이 포함 될 수 있다.For example, the extract included in the composition of the present invention may be prepared by pulverizing the primary extract extracted by the hot water extraction or solvent extraction method through an additional process such as distillation under reduced pressure and freeze drying or spray drying. In addition, the primary extract can be further purified by various chromatography methods such as silica gel column chromatography, high performance liquid chromatography, and thin layer chromatography to obtain additional purified fractions. . Therefore, in the present invention, the extract may include all extracts, separated compounds, fractions and purified products obtained in each step of extraction, fractionation or purification, dilution, concentration, or drying thereof.
본 발명에서 상기 선학초 또는 상기 오배자를 추출하는 데에 사용되는 추출 용매의 종류는 특별히 제한되지 아니하며, 천연물로부터 추출물을 추출하는 당업계에 공지된 통상적인 방법에 따라, 즉, 통상적인 온도, 압력의 조건 하에서 통상적인 용매를 사용하여 추출할 수 있다. 용매로서 정제수, 에탄올(ethanol), 메탄올(methanol), 아이소프로판올(isopropanol), 부탄올(butanol), 등을 포함하는 탄소 수 1 내지 4의 알코올 및 아세톤(acetone), 에테르(ether), 벤젠(benzene), 클로로폼(chloroform), 에틸아세테이트(ethyl acetate), 메틸렌 클로라이드(methylene chloride), 헥산(hexane), 사이클로헥세인(cyclohexane) 등의 각종 용매를 단독 또는 혼합하여 사용할 수 있다. 예를 들어, 상기 선학초 추출물 또는 상기 오배자 추출물은 물, C1 내지 C4의 알코올, 및 이들의 혼합용매로 이루어지는 군에서 선택된 하나 이상의 용매로 추출될 수 있으며, 본 발명의 일 실시예에 따르면 에탄올을 용매로 사용하여 추출할 수 있으나, 이에 제한되지 않는다. 또한, 메탄올 또는 에탄올 수용액이 사용될 수 있으며, 상기 메탄올 또는 상기 에탄올 수용액은 30 내지 95%(v/v), 더욱 구체적으로 40 내지 60%(v/v), 가장 구체적으로 50%(v/v) 메탄올 또는 에탄올일 수 있지만, 이에 제한되지 않는다.In the present invention, the type of extraction solvent used to extract the Sunhakcho or the gallbladder is not particularly limited, and according to a conventional method known in the art for extracting an extract from natural products, that is, conventional temperature and pressure It can be extracted using conventional solvents under the conditions. As a solvent, alcohol having 1 to 4 carbon atoms including purified water, ethanol, methanol, isopropanol, butanol, etc., and acetone, ether, benzene ), chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and the like may be used alone or in combination. For example, the Seonhakcho extract or the nut gall extract may be extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 alcohol, and mixed solvents thereof, and according to an embodiment of the present invention, ethanol It can be extracted using a solvent, but is not limited thereto. In addition, methanol or ethanol aqueous solution may be used, and the methanol or ethanol aqueous solution is 30 to 95% (v / v), more specifically 40 to 60% (v / v), most specifically 50% (v / v ) may be methanol or ethanol, but is not limited thereto.
본 발명에 있어서, 상기 에탄올을 용매로 사용하여 선학초 또는 상기 오배자를 추출할 경우, 예컨대 10 % 내지 100 % 에탄올, 10 % 내지 90 % 에탄올, 10 % 내지 80 % 에탄올, 10 % 내지 70 % 에탄올, 10 % 내지 60 % 에탄올, 10 % 내지 50 % 에탄올, 20 % 내지 90 % 에탄올, 20 % 내지 80 % 에탄올, 20 % 내지 70 % 에탄올, 20 % 내지 60 % 에탄올, 20 % 내지 50 % 에탄올, 30 % 내지 90 % 에탄올, 30 % 내지 80 % 에탄올, 30 % 내지 70 % 에탄올, 30 % 내지 60 % 에탄올, 30 % 내지 50 % 에탄올, 40 % 내지 90 % 에탄올, 40 % 내지 80 % 에탄올, 40 % 내지 70 % 에탄올, 40 % 내지 60 % 에탄올, 40 % 내지 50 % 에탄올, 45 % 내지 55 % 에탄올, 또는 50 % 에탄올을 사용할 수 있으나, 이에 제한되지 않는다.In the present invention, when the ethanol is used as a solvent to extract the sunflower or the nut gall, for example, 10% to 100% ethanol, 10% to 90% ethanol, 10% to 80% ethanol, 10% to 70% ethanol, 10% to 60% Ethanol, 10% to 50% Ethanol, 20% to 90% Ethanol, 20% to 80% Ethanol, 20% to 70% Ethanol, 20% to 60% Ethanol, 20% to 50% Ethanol, 30 % to 90% Ethanol, 30% to 80% Ethanol, 30% to 70% Ethanol, 30% to 60% Ethanol, 30% to 50% Ethanol, 40% to 90% Ethanol, 40% to 80% Ethanol, 40% to 70% ethanol, 40% to 60% ethanol, 40% to 50% ethanol, 45% to 55% ethanol, or 50% ethanol, but is not limited thereto.
상기 제조된 추출물은 이후 여과하거나 농축 또는 건조과정을 수행하여 용매를 제거할 수 있으며, 여과, 농축 및 건조를 모두 수행할 수 있다. 예컨대, 여과는 여과지를 이용하거나 감압여과기를 이용할 수 있으며, 농축은 진공 감압 농축기 또는 진공회전증발기를 이용할 수 있고, 건조는 감압건조, 진공건조, 비등건조, 분무건조, 동결건조법 등을 수행할 수 있으나, 이에 제한되는 것은 아니다.The prepared extract may then be filtered or concentrated or dried to remove the solvent, and both filtration, concentration and drying may be performed. For example, filtration may use filter paper or a vacuum filter, concentration may use a vacuum vacuum concentrator or vacuum rotary evaporator, and drying may be performed by vacuum drying, vacuum drying, boiling drying, spray drying, freeze drying, and the like. However, it is not limited thereto.
추출 횟수는 1회 이상 실시할 수 있으나, 추출이 계속될수록 활성 성분은 수득량이 현저히 감소되므로 5회 이상 반복 실시하는 것은 경제적이지 않을 수 있다. 이에, 추출 횟수는 1 내지 5회인 것이 바람직하며, 2 내지 5회 반복 추출하는 것이 더욱 바람직하나, 이에 제한되는 것은 아니다.The number of extractions may be carried out one or more times, but as the extraction continues, the yield of the active ingredient significantly decreases, so it may not be economical to perform the extraction repeatedly five times or more. Accordingly, the number of extractions is preferably 1 to 5 times, and more preferably 2 to 5 repeated extractions, but is not limited thereto.
상기 추출 용매를 건조된 선학초 또는 오배자 분량에 1 내지 30배, 1 내지 20배, 또는 1 내지 10배 첨가하여 추출하는 것이 바람직하나, 이에 제한되는 것은 아니다. 추출 온도는 20℃ 내지 100℃인 것이 바람직하고, 20℃ 내지 80℃인 것이 더욱 바람직하고, 실온인 것이 가장 바람직하나, 이에 제한되는 것은 아니다. 또한, 상기 추출 시간은 1 내지 10시간인 것이 바람직하나, 이에 제한되지 않는다.It is preferable to perform extraction by adding the extraction solvent 1 to 30 times, 1 to 20 times, or 1 to 10 times to the amount of dried Seonhakcho or gall nut, but is not limited thereto. The extraction temperature is preferably 20°C to 100°C, more preferably 20°C to 80°C, and most preferably room temperature, but is not limited thereto. In addition, the extraction time is preferably one to 10 hours, but is not limited thereto.
본 발명에 있어서, 상기 혼합물은 선학초 추출물 : 오배자 추출물의 중량비가 10:1 내지 1:10이 되도록 혼합될 수 있으며, 또한, 중량비가 1:0.1 내지 9, 1:0.1 내지 8, 1:0.1 내지 7, 1:0.1 내지 6, 1:0.1 내지 5, 1:0.1 내지 4, 1:0.1 내지 3, 1:0.1 내지 2, 1:0.1 내지 1, 1:0.1 내지 0.9, 1:0.1 내지 0.8, 1:0.1 내지 0.7, 또는 1:0.1 내지 0.6이 되도록 혼합될 수 있다. 본 발명의 일 실시예에 따르면, 6:4의 중량비로 혼합될 수 있으나, 이에 제한되지 않는다.In the present invention, the mixture may be mixed so that the weight ratio of Seonhakcho extract: gall nut extract is 10:1 to 1:10, and also, the weight ratio is 1:0.1 to 9, 1:0.1 to 8, 1:0.1 to 7, 1:0.1 to 6, 1:0.1 to 5, 1:0.1 to 4, 1:0.1 to 3, 1:0.1 to 2, 1:0.1 to 1, 1:0.1 to 0.9, 1:0.1 to 0.8, 1:0.1 to 0.7, or 1:0.1 to 0.6. According to one embodiment of the present invention, it may be mixed in a weight ratio of 6:4, but is not limited thereto.
또한, 상기 혼합물은 인플루엔자 바이러스의 M1 또는 NP 유전자의 발현을 억제시킬 수 있으나, 이에 제한되지 않는다.In addition, the mixture may inhibit the expression of the M1 or NP gene of influenza virus, but is not limited thereto.
본 발명은 하기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물을 제공한다:The present invention relates to at least one selected from the group consisting of a flavonoid compound represented by Formula 1 or a triterpenoid compound represented by Formula 2, isomers thereof, and pharmaceutically acceptable salts thereof. Provides a pharmaceutical composition for preventing or treating influenza virus infection comprising as an active ingredient:
[화학식 1][Formula 1]
Figure PCTKR2022013577-appb-img-000005
Figure PCTKR2022013577-appb-img-000005
상기 화학식에서, In the above formula,
R1은 H 또는 OH이고,R 1 is H or OH;
R2는 α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, 및 OH로 이루어진 군으로 선택되는 어느 하나이고,R 2 is any one selected from the group consisting of α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, and OH;
R3은 H 또는 β-D-glucuronosyl이고, R 3 is H or β-D-glucuronosyl;
[화학식 2][Formula 2]
Figure PCTKR2022013577-appb-img-000006
.
Figure PCTKR2022013577-appb-img-000006
.
본 발명에서, 상기 플라보노이드(flavonoid) 화합물은 아프젤린(Afzelin), 아피제닌(Apigenin), 아피제닌 7-O-β-D-글루쿠로나이드(Apigenin 7-O-β-D-glucuronide), 아스트라갈린(Astragalin), 니코티플로린(Nicotiflorin), 케르세틴(Quercetin), 케르시트린(Quercitrin), 및 루틴(Rutin)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the flavonoid compounds include Afzelin, Apigenin, Apigenin 7-O-β-D-glucuronide, It may be at least one selected from the group consisting of Astragalin, Nicotiflorin, Quercetin, Quercitrin, and Rutin, but is not limited thereto.
본 발명에서, 상기 트리테르펜(triterpenoid) 화합물은 우르솔산(Ursolic acid)일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the triterpenoid compound may be ursolic acid, but is not limited thereto.
본 발명에서, 화합물 10으로 표시되는 화합물은 분자식은 C41H32O28, 분자량은 940.67일 수 있으며, [(2R,3R,4S,5R,6S)-3,4,5,6-tetrakis[(3,4,5-trihydroxybenzoyl)oxy]oxan-2-yl]methyl 3,4,5-trihydroxybenzoate의 IUPAC 네임을 가질 수 있다.In the present invention, the compound represented by Compound 10 may have a molecular formula of C 41 H 32 O 28 and a molecular weight of 940.67, [(2R,3R,4S,5R,6S)-3,4,5,6-tetrakis[ (3,4,5-trihydroxybenzoyl)oxy]oxan-2-yl] methyl 3,4,5-trihydroxybenzoate may have the IUPAC name.
본 발명에서, 화합물 2로 표시되는 화합물은 분자식은 C30H48O3, 분자량은 456.7일 수 있으며, (1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10-hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H-picene-4a-carboxylic acid의 IUPAC 네임을 가질 수 있다.In the present invention, the compound represented by Compound 2 may have a molecular formula of C 30 H 48 O 3 and a molecular weight of 456.7, (1S,2R,4aS,6aR,6aS,6bR,8aR,10S,12aR,14bS)-10 -hydroxy-1,2,6a,6b,9,9,12a-heptamethyl-2,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydro-1H It can have the IUPAC name of -picene-4a-carboxylic acid.
본 발명에서, 화합물 3으로 표시되는 화합물은 분자식은 C15H10O7, 분자량은 302.2일 수 있으며, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one의 IUPAC 네임을 가질 수 있다.In the present invention, the compound represented by compound 3 may have a molecular formula of C 15 H 10 O 7 and a molecular weight of 302.2, and the IUPAC of 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxychromen-4-one can have a name.
또한, 상기 화합물의 획득 방법은 본 발명이 속한 분야에서 공지된 방법으로 화학적으로 합성하거나, 시판되는 물질을 사용할 수 있으나, 이에 제한되지 않는다. 상기 추출물 또는 혼합물에 유효성분, 표준물질 또는 활성물질로 화합물 1 내지 10으로 이루어지는 군에서 선택된 하나 이상이 포함될 수 있으나, 이에 제한되지 않는다. 또한, 상기 추출물 또는 혼합물은 상기 화합물을 고용량 함유할 수 있으나, 이에 제한되지 않는다.In addition, the method for obtaining the compound may be chemically synthesized by a method known in the field to which the present invention pertains, or a commercially available material may be used, but is not limited thereto. The extract or mixture may include one or more selected from the group consisting of Compounds 1 to 10 as an active ingredient, standard material, or active material, but is not limited thereto. In addition, the extract or mixture may contain a high dose of the compound, but is not limited thereto.
본 발명에서 용어, “인플루엔자(influenza)”는 감기 증세를 일으키는 바이러스 중 오소믹소바이러스과의 인플루엔자 바이러스가 유발하는 감염성 질환을 일컫는다. 일반 감기에 비해 발열, 근육통, 두통 등의 전신적인 증상이 뚜렷하게 나타나며 잠복기는 1~3일 정도이다. 인플루엔자는 A, B, C형으로 구분되며 이들의 전체적 구조 및 조성이 동일하며, 직경 80~120nm의 크기로 감염 초기에는 필라멘트 모양이지만, 후기에는 원형이 된다. 인플루엔자 바이러스는 중앙의 핵을 둘러싸고 있는 바이러스성 외피는 크게 헤마글루티닌(H)과 뉴라미니다아제(N) 두 종류로 구별할 수 있는 당단백질로 구성되어있으며, 핵은 바이러스성 RNA(viral RNA)와 이를 보호하고 활성화하는데 필요한 바이러스성 단백질(viral protein)을 포함한다. B형과 C형의 경우 감수성이 낮고 유전학적 다양성이 떨어져 아형이 거의 존재하지 않으며, 적은 빈도로 출현하여 유행이 잘 일어나지 않는 반면, A형은 헤마글루티닌과 뉴라미니다제에 대한 항체의 반응 여부에 따라 다시 여러 개의 아형으로 나눠지며, 자연계에는 H혈청형이 18개, N혈청형이 11개 존재하며 주로 H1/2/3 및 N1/2가 인간에게 독감을 일으키는 아형이다. 본 발명의 일 실시예에 따르면 상기 인플루엔자 바이러스는 인플루엔자 A형 H1N1 바이러스일 수 있으나, 이에 제한되지 않는다.In the present invention, the term "influenza" refers to an infectious disease caused by an influenza virus of the Orthomyxoviridae family among viruses that cause cold symptoms. Compared to the common cold, systemic symptoms such as fever, muscle pain, and headache appear distinctly, and the incubation period is about 1 to 3 days. Influenza is classified into A, B, and C types, and their overall structure and composition are the same. They have a diameter of 80 to 120 nm, and are filament-shaped in the early stage of infection, but become circular in the later stage. Influenza virus, the viral envelope surrounding the central nucleus is largely composed of glycoproteins that can be distinguished into two types, hemagglutinin (H) and neuraminidase (N), and the nucleus is composed of viral RNA (viral RNA). RNA) and viral proteins necessary to protect and activate it. Types B and C have low susceptibility and low genetic diversity, so there are almost no subtypes, and they appear infrequently and do not become prevalent. It is again divided into several subtypes according to the reaction. In nature, there are 18 H serotypes and 11 N serotypes, and mainly H1/2/3 and N1/2 are the subtypes that cause influenza in humans. According to one embodiment of the present invention, the influenza virus may be an influenza A type H1N1 virus, but is not limited thereto.
본 발명에서 사용된 용어 “분자 도킹 시뮬레이션”이란 단백질, 펩타이드, 핵산, 탄수화물, 지질과 같은 생물학적으로 관련된 분자간의 연관성을 토대로 적절한 표적 결합부위에 대한 소분자 리간드의 결합 적합성을 예측하여, 구조 기반 약물설계에서 많이 사용되는 방법으로 본 발명에선 인플루엔자 바이러스 단백질과의 결합능을 바탕으로 인플루엔자 저해 활성여부를 판단하였다. As used herein, the term “molecular docking simulation” means structure-based drug design by predicting the binding suitability of a small molecule ligand to an appropriate target binding site based on the association between biologically relevant molecules such as proteins, peptides, nucleic acids, carbohydrates, and lipids. As a method widely used in the present invention, influenza inhibitory activity was determined based on the ability to bind to influenza virus proteins.
본 발명에 따른 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 상기 부형제는 예를 들어, 희석제, 결합제, 붕해제, 활택제, 흡착제, 보습제, 필름-코팅 물질, 및 제어방출첨가제로 이루어진 군으로부터 선택된 하나 이상일 수 있다. The pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions. The excipient may be, for example, one or more selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a moisturizer, a film-coating material, and a controlled release additive.
본 발명에 따른 약학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 서방형 과립제, 장용과립제, 액제, 점안제, 엘실릭제, 유제, 현탁액제, 주정제, 트로키제, 방향수제, 리모나아데제, 정제, 서방형정제, 장용정제, 설하정, 경질캅셀제, 연질캅셀제, 서방캅셀제, 장용캅셀제, 환제, 틴크제, 연조엑스제, 건조엑스제, 유동엑스제, 주사제, 캡슐제, 관류액, 경고제, 로션제, 파스타제, 분무제, 흡입제, 패취제, 멸균주사용액, 또는에어로졸 등의 외용제 등의 형태로 제형화하여 사용될 수 있으며, 상기 외용제는 크림, 젤, 패치, 분무제, 연고제, 경고제, 로션제, 리니멘트제, 파스타제 또는 카타플라스마제 등의 제형을 가질 수 있다. The pharmaceutical compositions according to the present invention are powders, granules, sustained-release granules, enteric granules, solutions, eye drops, elsilic agents, emulsions, suspensions, spirits, troches, perfumes, and limonadese, respectively, according to conventional methods. , tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusate, It can be formulated and used in the form of external preparations such as warning agents, lotions, pasta agents, sprays, inhalants, patches, sterile injection solutions, or aerosols, and the external agents are creams, gels, patches, sprays, ointments, and warning agents. , lotion, liniment, pasta, or cataplasma may have formulations such as the like.
본 발명에 따른 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, 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.
제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
본 발명에 따른 정제, 산제, 과립제, 캡슐제, 환제, 트로키제의 첨가제로 옥수수전분, 감자전분, 밀전분, 유당, 백당, 포도당, 과당, 디-만니톨, 침강탄산칼슘, 합성규산알루미늄, 인산일수소칼슘, 황산칼슘, 염화나트륨, 탄산수소나트륨, 정제 라놀린, 미결정셀룰로오스, 덱스트린, 알긴산나트륨, 메칠셀룰로오스, 카르복시메칠셀룰로오스나트륨, 카올린, 요소, 콜로이드성실리카겔, 히드록시프로필스타치, 히드록시프로필메칠셀룰로오스(HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, 프로필렌글리콜, 카제인, 젖산칼슘, 프리모젤 등 부형제; 젤라틴, 아라비아고무, 에탄올, 한천가루, 초산프탈산셀룰로오스, 카르복시메칠셀룰로오스, 카르복시메칠셀룰로오스칼슘, 포도당, 정제수, 카제인나트륨, 글리세린, 스테아린산, 카르복시메칠셀룰로오스나트륨, 메칠셀룰로오스나트륨, 메칠셀룰로오스, 미결정셀룰로오스, 덱스트린, 히드록시셀룰로오스, 히드록시프로필스타치, 히드록시메칠셀룰로오스, 정제쉘락, 전분호, 히드록시프로필셀룰로오스, 히드록시프로필메칠셀룰로오스, 폴리비닐알코올, 폴리비닐피롤리돈 등의 결합제가 사용될 수 있으며, 히드록시프로필메칠셀룰로오스, 옥수수전분, 한천가루, 메칠셀룰로오스, 벤토나이트, 히드록시프로필스타치, 카르복시메칠셀룰로오스나트륨, 알긴산나트륨, 카르복시메칠셀룰로오스칼슘, 구연산칼슘, 라우릴황산나트륨, 무수규산, 1-히드록시프로필셀룰로오스, 덱스트란, 이온교환수지, 초산폴리비닐, 포름알데히드처리 카제인 및 젤라틴, 알긴산, 아밀로오스, 구아르고무(Guar gum), 중조, 폴리비닐피롤리돈, 인산칼슘, 겔화전분, 아라비아고무, 아밀로펙틴, 펙틴, 폴리인산나트륨, 에칠셀룰로오스, 백당, 규산마그네슘알루미늄, 디-소르비톨액, 경질무수규산 등 붕해제; 스테아린산칼슘, 스테아린산마그네슘, 스테아린산, 수소화식물유(Hydrogenated vegetable oil), 탈크, 석송자, 카올린, 바셀린, 스테아린산나트륨, 카카오지, 살리실산나트륨, 살리실산마그네슘, 폴리에칠렌글리콜 4000, 6000, 유동파라핀, 수소첨가대두유(Lubri wax), 스테아린산알루미늄, 스테아린산아연, 라우릴황산나트륨, 산화마그네슘, 마크로골(Macrogol), 합성규산알루미늄, 무수규산, 고급지방산, 고급알코올, 실리콘유, 파라핀유, 폴리에칠렌글리콜지방산에테르, 전분, 염화나트륨, 초산나트륨, 올레인산나트륨, dl-로이신, 경질무수규산 등의 활택제가 사용될 수 있다.Corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, di-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, phosphoric acid as additives for tablets, powders, granules, capsules, pills, and troches according to the present invention Calcium monohydrogen, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethyl Excipients such as cellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate acetate, carboxymethyl cellulose, calcium carboxymethyl cellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethyl cellulose, sodium methyl cellulose, methyl cellulose, microcrystalline cellulose, dextrin Binders such as hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch arc, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, and polyvinylpyrrolidone may be used, Hydroxypropyl Methyl Cellulose, Corn Starch, Agar Powder, Methyl Cellulose, Bentonite, Hydroxypropyl Starch, Sodium Carboxymethyl Cellulose, Sodium Alginate, Calcium Carboxymethyl Cellulose, Calcium Citrate, Sodium Lauryl Sulfate, Silicic Anhydride, 1-Hydroxy Propyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, disintegrants such as amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid; Calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopod, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acid, higher alcohol, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, Lubricants such as sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid may be used.
본 발명에 따른 액제의 첨가제로는 물, 묽은 염산, 묽은 황산, 구연산나트륨, 모노스테아린산슈크로스류, 폴리옥시에칠렌소르비톨지방산에스텔류(트윈에스텔), 폴리옥시에칠렌모노알킬에텔류, 라놀린에텔류, 라놀린에스텔류, 초산, 염산, 암모니아수, 탄산암모늄, 수산화칼륨, 수산화나트륨, 프롤아민, 폴리비닐피롤리돈, 에칠셀룰로오스, 카르복시메칠셀룰로오스나트륨 등이 사용될 수 있다.Additives for the liquid formulation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, and the like may be used.
본 발명에 따른 시럽제에는 백당의 용액, 다른 당류 혹은 감미제 등이 사용될 수 있으며, 필요에 따라 방향제, 착색제, 보존제, 안정제, 현탁화제, 유화제, 점조제 등이 사용될 수 있다.In the syrup according to the present invention, a solution of white sugar, other sugars, or a sweetener may be used, and aromatics, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickeners, etc. may be used as necessary.
본 발명에 따른 유제에는 정제수가 사용될 수 있으며, 필요에 따라 유화제, 보존제, 안정제, 방향제 등이 사용될 수 있다.Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.
본 발명에 따른 현탁제에는 아카시아, 트라가칸타, 메칠셀룰로오스, 카르복시메칠셀룰로오스, 카르복시메칠셀룰로오스나트륨, 미결정셀룰로오스, 알긴산나트륨, 히드록시프로필메칠셀룰로오스, HPMC 1828, HPMC 2906, HPMC 2910 등 현탁화제가 사용될 수 있으며, 필요에 따라 계면활성제, 보존제, 안정제, 착색제, 방향제가 사용될 수 있다.Acacia, tragacantha, methylcellulose, carboxymethylcellulose, carboxymethylcellulose sodium, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, HPMC 2910, etc. and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.
본 발명에 따른 주사제에는 주사용 증류수, 0.9%염화나트륨주사액, 링겔주사액, 덱스트로스주사액, 덱스트로스+염화나트륨주사액, 피이지(PEG), 락테이티드 링겔주사액, 에탄올, 프로필렌글리콜, 비휘발성유-참기름, 면실유, 낙화생유, 콩기름, 옥수수기름, 올레인산에칠, 미리스트산 이소프로필, 안식향산벤젠과 같은 용제; 안식향산나트륨, 살리실산나트륨, 초산나트륨, 요소, 우레탄, 모노에칠아세트아마이드, 부타졸리딘, 프로필렌글리콜, 트윈류, 니정틴산아미드, 헥사민, 디메칠아세트아마이드와 같은 용해보조제; 약산 및 그 염(초산과 초산나트륨), 약염기 및 그 염(암모니아 및 초산암모니움), 유기화합물, 단백질, 알부민, 펩톤, 검류와 같은 완충제; 염화나트륨과 같은 등장화제; 중아황산나트륨(NaHSO3)이산화탄소가스, 메타중아황산나트륨(Na2S2O5), 아황산나트륨(Na2SO3),질소가스(N2),에칠렌디아민테트라초산과 같은 안정제; 소디움비설파이드 0.1%, 소디움포름알데히드 설폭실레이트, 치오우레아, 에칠렌디아민테트라초산디나트륨, 아세톤소디움비설파이트와 같은 황산화제; 벤질알코올, 클로로부탄올, 염산프로카인, 포도당, 글루콘산칼슘과 같은 무통화제; 시엠시나트륨, 알긴산나트륨, 트윈 80, 모노스테아린산알루미늄과 같은 현탁화제를 포함할 수 있다.Injections according to the present invention include distilled water for injection, 0.9% sodium chloride injection, IV injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated IV injection, ethanol, propylene glycol, non-volatile oil-sesame oil , solvents such as cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, twins, nijuntinamide, hexamine, and dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; tonicity agents such as sodium chloride; Stabilizers such as sodium bisulfite (NaHSO 3 ) carbon dioxide gas, sodium metabisulfite (Na 2 S 2 O 5 ), sodium sulfite (Na 2 SO 3 ), nitrogen gas (N 2 ), ethylenediaminetetraacetic acid; Sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, ethylenediamine disodium tetraacetate, acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; Suspending agents such as Siemesis sodium, sodium alginate, Tween 80, aluminum monostearate may be included.
본 발명에 따른 좌제에는 카카오지, 라놀린, 위텝솔, 폴리에틸렌글리콜, 글리세로젤라틴, 메칠셀룰로오스, 카르복시메칠셀룰로오스, 스테아린산과 올레인산의 혼합물, 수바날(Subanal), 면실유, 낙화생유, 야자유, 카카오버터+콜레스테롤, 레시틴, 라네트왁스, 모노스테아린산글리세롤, 트윈 또는 스판, 임하우젠(Imhausen), 모놀렌(모노스테아린산프로필렌글리콜), 글리세린, 아뎁스솔리두스(Adeps solidus), 부티룸 태고-G(Buytyrum Tego-G), 세베스파마 16 (Cebes Pharma 16), 헥사라이드베이스 95, 코토마(Cotomar), 히드록코테 SP, S-70-XXA, S-70-XX75(S-70-XX95), 히드록코테(Hydrokote) 25, 히드록코테 711, 이드로포스탈 (Idropostal), 마사에스트라리움(Massa estrarium, A, AS, B, C, D, E, I, T), 마사-MF, 마수폴, 마수폴-15, 네오수포스탈-엔, 파라마운드-B, 수포시로(OSI, OSIX, A, B, C, D, H, L), 좌제기제 IV 타입 (AB, B, A, BC, BBG, E, BGF, C, D, 299), 수포스탈 (N, Es), 웨코비 (W, R, S, M ,Fs), 테제스터 트리글리세라이드 기제 (TG-95, MA, 57)와 같은 기제가 사용될 수 있다.The suppository according to the present invention includes cacao butter, lanolin, witapsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lannet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolen (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Buytyrum Tego-G -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxycote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hyde Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecobi (W, R, S, M, Fs), testosterone triglyceride base (TG-95, MA, 57) and The same mechanism can be used.
경구 투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 추출물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, sucrose, etc. ) or by mixing lactose and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
경구 투여를 위한 액상제제로는 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, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. Liquid preparations for oral administration include suspensions, solutions for oral administration, 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. there is. 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 suspending agents.
본 발명에 따른 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서, “약학적으로 유효한 양”은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level is the type of patient's disease, severity, activity of the drug, It may be determined according to factors including sensitivity to the drug, administration time, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.
본 발명에 따른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 본 발명이 속하는 기술분야에 통상의 기술자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention belongs.
본 발명의 약학적 조성물은 개체에게 다양한 경로로 투여될 수 있다. 투여의 모든 방식은 예상될 수 있는데, 예를 들면, 경구 복용, 피하 주사, 복강 투여, 정맥 주사, 근육 주사, 척수 주위 공간(경막내) 주사, 설하 투여, 볼점막 투여, 직장 내 삽입, 질 내 삽입, 안구 투여, 귀 투여, 비강 투여, 흡입, 입 또는 코를 통한 분무, 피부 투여, 경피 투여 등에 따라 투여될 수 있다.The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration can be envisaged, eg oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intrathecal) injection, sublingual administration, buccal administration, intrarectal insertion, vaginal It can be administered by intraoral insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like.
본 발명의 약학적 조성물은 치료할 질환, 투여 경로, 환자의 연령, 성별, 체중 및 질환의 중등도 등의 여러 관련 인자와 함께 활성성분인 약물의 종류에 따라 결정된다.The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient together with various related factors such as the disease to be treated, the route of administration, the age, sex, weight and severity of the disease of the patient.
본 발명에서 “개체”란 질병의 치료를 필요로 하는 대상을 의미하고, 보다 구체적으로는 인간 또는 비-인간인 영장류, 생쥐(mouse), 쥐(rat), 개, 고양이, 말, 및 소 등의 포유류를 의미한다.In the present invention, "subject" means a subject in need of treatment of a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, cow, etc. of mammals.
본 발명에서 “투여”란 임의의 적절한 방법으로 개체에게 소정의 본 발명의 조성물을 제공하는 것을 의미한다. 본 발명에서 “예방”이란 목적하는 질환의 발병을 억제하거나 지연시키는 모든 행위를 의미하고, “치료”란 본 발명에 따른 약학적 조성물의 투여에 의해 목적하는 질환과 그에 따른 대사 이상 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미하며, “개선”이란 본 발명에 따른 조성물의 투여에 의해 목적하는 질환과 관련된 파라미터, 예를 들면 증상의 정도를 감소시키는 모든 행위를 의미한다.In the present invention, "administration" means providing a given composition of the present invention to a subject by any suitable method. In the present invention, “prevention” refers to any action that suppresses or delays the onset of a desired disease, and “treatment” means that the desired disease and its resulting metabolic abnormality are improved or improved by administration of the pharmaceutical composition according to the present invention. All actions that are advantageously altered are meant, and "improvement" means any action that reduces a parameter related to a target disease, for example, the severity of a symptom, by administration of the composition according to the present invention.
본 발명은 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물을 제공한다.The present invention provides a food composition for preventing or improving influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient.
또한, 본 발명은 하기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 하기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 식품학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물을 제공한다:In addition, the present invention is any one selected from the group consisting of flavonoid compounds represented by the following formula (1) or triterpenoid compounds represented by the following formula (2), isomers thereof, and food-acceptable salts thereof. It provides a food composition for preventing or improving influenza virus infection comprising one or more as active ingredients:
[화학식 1][Formula 1]
Figure PCTKR2022013577-appb-img-000007
Figure PCTKR2022013577-appb-img-000007
상기 화학식에서, In the above formula,
R1은 H 또는 OH이고,R 1 is H or OH;
R2는 α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, 및 OH로 이루어진 군으로 선택되는 어느 하나이고,R 2 is any one selected from the group consisting of α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, and OH;
R3은 H 또는 β-D-glucuronosyl이고, R 3 is H or β-D-glucuronosyl;
[화학식 2][Formula 2]
Figure PCTKR2022013577-appb-img-000008
.
Figure PCTKR2022013577-appb-img-000008
.
본 발명에 있어서, “식품”이란 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 건강기능성 식품, 음료, 식품 첨가제 및 음료 첨가제 등을 모두 포함하는 의미이다. In the present invention, "food" means a natural product or processed product containing one or more nutrients, preferably means a state that can be eaten directly through a certain degree of processing process, and usually means As, it means to include all health functional foods, beverages, food additives and beverage additives.
본 발명에 있어서, 상기 식품 조성물은 건강기능성 식품 조성물일 수 있으나, 이에 제한되지 않는다.In the present invention, the food composition may be a health functional food composition, but is not limited thereto.
본 발명에 있어서, 상기 “건강기능성 식품(functional food)”이란, 특정보건용 식품(food for special health use, FoSHU)과 동일한 용어로, 영양 공급 외에도 생체조절기능이 효율적으로 나타나도록 가공된 의학, 의료효과가 높은 식품을 의미하며, 정제, 캡슐제, 환제, 과립제, 분말, 액제, 편상(flake), 페이스트, 시럽제, 겔, 젤리, 바(bar), 또는 필름 제형으로 제조될 수 있다. 여기서 “기능성”이라 함은 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적 작용 등과 같은 보건용도에 유용한 효과를 얻는 것을 의미한다.In the present invention, the "functional food" is the same term as food for special health use (FoSHU), medicine processed to efficiently display bioregulatory functions in addition to nutritional supply, It means a food with high medical effect, and can be manufactured into tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or film formulations. Here, “functionality” means obtaining useful effects for health purposes, such as adjusting nutrients for the structure and function of the human body or physiological functions.
본 발명의 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물 및 이의 유래 화합물을 식품 첨가물로 사용할 경우, 이를 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합양은 사용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시 본 발명의 담팔수 추출물은 원료에 대하여 15 중량% 이하, 또는 10 중량% 이하의 양으로 첨가될 수 있다. 그러나, 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.Seonhakcho of the present invention ( Agrimonia pilosa ) Extract and nut gall ( Galla rhois ) When the mixture of the extract and its derived compound is used as a food additive, it can be added as it is or used together with other food or food ingredients, according to conventional methods can be used The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment). In general, when preparing food or beverage, the freshwater extract of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알콜 음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강기능식품을 모두 포함한다.There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, tea, drinks, There are alcoholic beverages and vitamin complexes, and includes all health functional foods in a conventional sense.
본 발명에 따른 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당 및 과당과 같은 모노사카라이드, 말토오스 및 수크로오스와 같은 디사카라이드, 덱스트린 및 시클로덱스트린과 같은 폴리사카라이드, 및 자일리톨, 소르비톨 및 에리트리톨 등의 당알콜이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 mL당 일반적으로 약 0.01-0.20g, 또는 약 0.04-0.10g 이다.The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional components, like conventional beverages. The aforementioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.01-0.20 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, A carbonation agent used in carbonated beverages and the like may be contained. In addition, the composition of the present invention may contain fruit flesh for preparing natural fruit juice, fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The ratio of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.
또한, 본 발명은 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물을 제공한다.In addition, the present invention provides a quasi-drug composition for preventing or suppressing influenza virus infection comprising a mixture of an extract of Agrimonia pilosa and an extract of galla rhois as an active ingredient.
또한, 본 발명은 하기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 하기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물을 제공한다:In addition, the present invention is any one selected from the group consisting of flavonoid compounds represented by the following formula (1) or triterpenoid compounds represented by the following formula (2), isomers thereof, and pharmaceutically acceptable salts thereof. Provided is a quasi-drug composition for preventing or inhibiting influenza virus infection comprising at least one as an active ingredient:
[화학식 1][Formula 1]
Figure PCTKR2022013577-appb-img-000009
Figure PCTKR2022013577-appb-img-000009
상기 화학식에서, In the above formula,
R1은 H 또는 OH이고,R 1 is H or OH;
R2는 α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, 및 OH로 이루어진 군으로 선택되는 어느 하나이고,R 2 is any one selected from the group consisting of α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, and OH;
R3은 H 또는 β-D-glucuronosyl이고, R 3 is H or β-D-glucuronosyl;
[화학식 2][Formula 2]
Figure PCTKR2022013577-appb-img-000010
.
Figure PCTKR2022013577-appb-img-000010
.
본 발명에 따른 “인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물”에 있어서, 의약외품은 약사법 제2조 제7호 다목에 기재된 감염병 예방을 위하여 살균ㆍ살충 및 이와 유사한 용도로 사용되는 제제로서, 사람 또는 동물의 보건을 위해 사용되는 파리, 모기 등의 기피제, 구제제, 방지제, 방제제 또는 유인살충제를 의미할 수 있다.In the “quasi-drug composition for preventing or inhibiting influenza virus infection” according to the present invention, the quasi-drug is a preparation used for sterilization, insecticide, and similar purposes for the prevention of infectious diseases described in Article 2, Subparagraph 7, Item C of the Pharmaceutical Affairs Act, and is used for human or animal It may refer to repellents, repellents, preventives, pesticides, or attracting insecticides such as flies and mosquitoes used for the health of people.
또한, 상기 의약외품은 피부외용제 및 개인위생용품을 포함할 수 있다. 예를 들어, 소독청결제, 샤워폼, 가그린, 물티슈, 세제비누, 핸드워시, 또는 연고제일 수 있으나 이에 제한되지는 않는다.In addition, the quasi-drug may include external skin preparations and personal hygiene products. For example, it may be a disinfectant cleanser, shower foam, gargreen, wet tissue, detergent soap, hand wash, or ointment, but is not limited thereto.
본 발명에 따른 상기 의약외품 조성물을 의약외품 첨가물로 사용할 경우, 상기 조성물을 그대로 첨가하거나 다른 의약외품 또는 의약외품 성분과 함께 사용 할 수 있고, 통상적인 방법에 따라 적절하게 사용할 수 있다. 유효성분의 혼합량은 사용 목적에 따라 적합하게 결정될 수 있다.When the quasi-drug composition according to the present invention is used as a quasi-drug additive, the composition may be added as it is or used together with other quasi-drugs or quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use.
본 발명의 의약외품 조성물은 일 예로, 일반적인 유화 제형 및 가용화 제형의 형태로 제조된 것일 수 있다. 예를 들어, 로션 등과 같은 유액, 크림, 연고, 스프레이, 오일젤, 젤, 오일, 에어로졸, 연막제와 같은 제형을 가질 수 있으나, 본 발명의 해충 방제 유도 효과를 나타내는 것이라면 제한되지 않고 사용할 수 있다. 또한 상기 의약외품 조성물은 각각의 제형에 일반적으로 의약외품 조성물에 배합되는 유분, 물, 계면활성제, 보습제, 탄소수 1 내지 4의 저급 알코올, 증점제, 킬레이트제, 색소, 방부제 또는 향료 등을 필요에 따라 적절히 배합하여 사용할 수 있다.The quasi-drug composition of the present invention may be prepared in the form of, for example, a general emulsified formulation and a solubilized formulation. For example, it may have formulations such as emulsions, creams, ointments, sprays, oil gels, gels, oils, aerosols, and smokers such as lotions, but it may be used without limitation as long as it exhibits the pest control inducing effect of the present invention. . In addition, the quasi-drug composition appropriately blends oil, water, surfactant, moisturizer, lower alcohol having 1 to 4 carbon atoms, a thickener, a chelating agent, a colorant, a preservative, or a flavoring agent, etc., which are generally formulated in quasi-drug compositions, in each dosage form, as necessary. and can be used.
본 발명에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다. The terms used in the present invention have been selected from general terms that are currently widely used as much as possible while considering the functions in the present invention, but they may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technologies, and the like. In addition, in a specific case, there is also a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, not simply the name of the term.
이하, 본 발명의 이해를 돕기 위하여 바람직한 실시예를 제시한다. 그러나 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐, 하기 실시예에 의해 본 발명의 내용이 한정되는 것은 아니다.Hereinafter, a preferred embodiment is presented to aid understanding of the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.
실시예 1. 세포배양Example 1. Cell culture
실험에 사용된 MDCK 세포주는 37℃, 5% CO2의 조건으로 10% 소 태아 혈청(FBS, Hyclone Thermo Scientific) 및 1% 페니실린/스트렙토마이신(P/S, Gibco)이 포함된 MEM(Minimum essential medium, Gibco) 배지에서 배양하였다. The MDCK cell line used in the experiment was cultured under conditions of 37°C, 5% CO 2 and MEM (Minimum essential) containing 10% fetal bovine serum (FBS, Hyclone Thermo Scientific) and 1% penicillin/streptomycin (P/S, Gibco). medium, Gibco) medium.
실시예 2. 추출물의 제조Example 2. Preparation of extracts
선학초(Agrimonia pilosa)와 오배자(Galla rhois)의 잎은 BioKorea Co., LTd(Seoul)에서 구입하고, 바우처 표본(BMRI-AP-1601, BMRI-RG-1602)은 한국 용인 경희대학교 바이오메디컬연구센터에 기탁하였다. 건조된 샘플(20kg)을 80±2℃에서 6시간 동안 50% 에탄올로 추출한 다음 여과하였다. 그 후, 회전 증발기를 사용하여 농축시키고 동결 건조하여 선학초 추출물 1.57kg 및 오배자 추출물 11.59kg을 얻었다. 선학초 추출물과 오배자 추출물의 혼합물은 각각 6:4의 비율로 혼합하여 사용하였으며, APRG64로 약칭하였다. 모든 샘플은 사용 전까지 4℃에서 보관하였다. The leaves of Agrimonia pilosa and Galla rhois were purchased from BioKorea Co., LTd (Seoul), and voucher specimens (BMRI-AP-1601, BMRI-RG-1602) were purchased from Biomedical Research Center, Kyunghee University, Yongin, Korea. deposited in A dried sample (20 kg) was extracted with 50% ethanol at 80±2° C. for 6 hours and then filtered. Then, it was concentrated using a rotary evaporator and freeze-dried to obtain 1.57 kg of Seonhakcho extract and 11.59 kg of nut gall extract. A mixture of Seonhakcho extract and gall nut extract was used in a ratio of 6:4, respectively, and was abbreviated as APRG64. All samples were stored at 4°C until use.
실험예 1. IAV 유도 세포병변 효과에 대한 APRG64의 억제 활성 확인Experimental Example 1. Confirmation of inhibitory activity of APRG64 on IAV-induced cytopathic effect
IAV에 대한 항바이러스 활성을 조사하기 위하여 CPE(세포병변 효과, cytopathic effect) 분석을 실시하였다. 구체적으로, MDCK 세포를 0.1의 감염 다중도(MOI)에서 1시간 동안 IAV(A/California/07/2009)에 감염시킨 후, 37℃, CO2 조건의 인큐베이터에서 24시간 동안 배양하였다. APRG64를 농도별(0, 2.5, 5, 및 10㎍/㎖)로 24시간 동안 처리한 후, 세포독성 및 CPE에 대한 효과를 확인하였다. 이 때, 부착된 세포의 백분율을 분석하기 위해 세포를 1% 크리스탈 바이올렛 용액으로 염색하여 세포의 형태학적 변화 여부를 관찰하고, 부착된 세포의 백분율을 그래프로 나타내었다.To investigate the antiviral activity against IAV, CPE (cytopathic effect) assay was performed. Specifically, MDCK cells were infected with IAV (A/California/07/2009) for 1 hour at a multiplicity of infection (MOI) of 0.1, and then cultured for 24 hours in an incubator at 37° C. under CO 2 conditions. After treatment with APRG64 at different concentrations (0, 2.5, 5, and 10 μg/ml) for 24 hours, the effect on cytotoxicity and CPE was confirmed. At this time, in order to analyze the percentage of attached cells, cells were stained with 1% crystal violet solution to observe cell morphological changes, and the percentage of attached cells was graphed.
실험예 1-1. APRG64의 세포 독성의 확인Experimental Example 1-1. Confirmation of cytotoxicity of APRG64
그 결과, 전 구간의 농도에 대하여 APRG64는 세포 독성을 나타내지 않는 것으로 확인되었다(도 1).As a result, it was confirmed that APRG64 did not exhibit cytotoxicity for all concentrations (FIG. 1).
실험예 1-2. APRG64의 IAV 유도 CPE 억제 활성 확인Experimental Example 1-2. Confirmation of IAV-induced CPE inhibitory activity of APRG64
그 결과, 도 2a 및 도 2b에 나타난 바와 같이 APRG64 무처리 시 MDCK 세포에서 강력한 CPE가 관찰되었지만, APRG64 처리한 경우에는 용량 의존적 방식으로 IAV 유도 CPE를 유의하게 억제하는 것으로 확인되었으며, 이때의 50% 유효 농도(EC50)는 6.274㎍/㎖로 확인되었다.As a result, as shown in Figures 2a and 2b, strong CPE was observed in MDCK cells when APRG64 was not treated, but when APRG64 was treated, it was confirmed that IAV-induced CPE was significantly inhibited in a dose-dependent manner, at which time 50% The effective concentration (EC 50 ) was found to be 6.274 μg/ml.
실험예 1-3. APRG64의 바이러스 의존적 경로를 통한 항바이러스 효과 확인Experimental Example 1-3. Confirmation of antiviral effect through virus-dependent pathway of APRG64
실험예 1-2에서 확인된 APRG64의 IAV 유도 CPE 억제 활성이 바이러스 비의존적 세포 사멸이 아닌 바이러스 의존적 세포 사멸 효과임을 명확히 하기 위하여 세포사멸사 유도제 하에서도 동일한 효과가 나타나는지를 확인하였다. 구체적으로, IAV에 감염된 MDCK 세포에 세포자멸사 유도제인 스타우로스포린(staurosporine, STS)을 200nM 처리한 후 10㎍/㎖ APRG64를 추가적으로 처리하여 세포 사멸 여부(세포 생존율)를 확인하였다. 이 때 1% 크리스탈 바이올렛 염색을 사용하였고, ImageJ 소프트웨어로 이미지를 처리하여 플레이트에 부착된 세포를 정량화하였으며, 모든 그래프는 3회 반복의 평균을 기반으로 제작되었다.In order to clarify that the IAV-induced CPE inhibitory activity of APRG64 confirmed in Experimental Example 1-2 is not a virus-independent cell death but a virus-dependent cell death effect, it was confirmed whether the same effect appears under an apoptosis inducer. Specifically, MDCK cells infected with IAV were treated with 200 nM of staurosporine (STS), an apoptosis inducer, and then additionally treated with 10 μg/ml APRG64 to determine cell death (cell viability). At this time, 1% crystal violet staining was used, the images were processed with ImageJ software to quantify the cells attached to the plate, and all graphs were made based on the average of three repetitions.
그 결과, 실험예 1-2의 결과와는 달리 APRG64 추가군은 STS 유도 세포 사멸을 예방하지 못했다는 점에서(도 2c), APRG64가 바이러스 의존적인 경로를 통해 IAV 감염으로부터 유도된 CPE를 억제하여 MDCK 세포를 보호하는 것이 명확히 확인되었다.As a result, unlike the results of Experimental Example 1-2, in that the APRG64 additional group did not prevent STS-induced cell death (FIG. 2c), APRG64 inhibited CPE induced from IAV infection through a virus-dependent pathway. It was clearly confirmed to protect MDCK cells.
실험예 2. APRG64의 IAV 복제 억제에 따른 항바이러스 효과 확인Experimental Example 2. Confirmation of antiviral effect according to inhibition of IAV replication by APRG64
실험예 2-1. APRG64의 IAV 복제 억제 활성 확인Experimental Example 2-1. Confirmation of IAV replication inhibitory activity of APRG64
실험예 1에서 확인된 APRG64의 IAV 유도 CPE 억제 활성 효과가 바이러스 복제 억제에 기반한 것인지 여부를 확인하였다. 이를 확인하기 위하여 IAV에 감염된 MDCK 세포에 APRG64로 처리한 후 인플루엔자 바이러스 mRNA, 단백질 및 감염 입자의 생산에 대해 RT-qPCR 및 웨스턴 블롯 실험을 수행하였다.It was confirmed whether the IAV-induced CPE inhibitory activity of APRG64 confirmed in Experimental Example 1 was based on the inhibition of viral replication. To confirm this, MDCK cells infected with IAV were treated with APRG64, and RT-qPCR and Western blot experiments were performed on the production of influenza virus mRNA, protein, and infectious particles.
먼저, MEM배지에서 배양된 MDCK 세포를 0.1의 감염 다중도(MOI)에서 1시간 동안 IAV로 감염시킨 다음 APRG64(10㎍/㎖)로 처리하였고, 6시간 후, 인플루엔자 바이러스의 구조를 형성하는 M1 및 바이러스 핵단백질(Nucleoprotein, NP)의 발현 수준을 RT-qPCR로 분석하였다. 이 때, 세포에서 총 RNA를 추출하기 위해 제조업체의 지침에 따라 NucleoZOL(Macherey-Nagel, Duren, Germany)을 사용하였고, 나노그램의 RNA를 사용하여 ReverTraAce qPCR RT 키트(Toyobo, Osaka, Japan)를 사용하여 cDNA를 합성하였다. 분리된 RNA는 Nanodrop을 통해 정량한 후 cDNA 합성 키트(RevertraAce qPCR RT kit, Toyobo)를 이용해 cDNA로 합성하였으며, CFX Connect real-time system(Bio-Rad, Hercules, CA, USA)을 이용해 real-time PCR를 실시하였다. 프라이머 반응 조건은 95℃에서 10분 동안 전보온 시켰으며, 95℃에서 15초, 60℃에서 1분으로 총 40 cycle을 반복하였다. First, MDCK cells cultured in MEM medium were infected with IAV at a multiplicity of infection (MOI) of 0.1 for 1 hour, then treated with APRG64 (10 μg/ml), and after 6 hours, M1, which forms the structure of influenza virus, And the expression level of viral nucleoprotein (NP) was analyzed by RT-qPCR. At this time, NucleoZOL (Macherey-Nagel, Duren, Germany) was used according to the manufacturer's instructions to extract total RNA from cells, and ReverTraAce qPCR RT kit (Toyobo, Osaka, Japan) was used using nanograms of RNA. Thus, cDNA was synthesized. The isolated RNA was quantified using Nanodrop and then synthesized into cDNA using a cDNA synthesis kit (RevertraAce qPCR RT kit, Toyobo), and real-time using a CFX Connect real-time system (Bio-Rad, Hercules, CA, USA). PCR was performed. Primer reaction conditions were prewarmed at 95 ° C for 10 minutes, and a total of 40 cycles were repeated at 95 ° C for 15 seconds and 60 ° C for 1 minute.
구분(내용)Classification (Content) 방향direction 서열order
서열번호 1(M1 primer)SEQ ID NO: 1 (M1 primer) forwardforward 5'-AAGACCAATCCTGTCACCTCTG-3'5′-AAGACCAATCCTGTCACCTCTG-3′
서열번호 2(M1 primer)SEQ ID NO: 2 (M1 primer) reversereverse 5'-CAAAACGTCTACGCTGCAGTCC-3'5'-CAAAACGTCTACGCTGCAGTCC-3'
서열번호 3(NP primer)SEQ ID NO: 3 (NP primer) forwardforward 5'-CCAGATCAGTGTGCAGCCTA-3'5′-CCAGATCAGTGTGCAGCCTA-3′
서열번호 4(NP primer)SEQ ID NO: 4 (NP primer) reversereverse 5'-CTCTGGCTTTGCACTTTCC-3'5′-CTCTGGCTTTGCACTTTCC-3′
서열번호 5(GAPDH primer)SEQ ID NO: 5 (GAPDH primer) forwardforward 5'-AACATCATCCCTGCTTCCAC-3'5′-AACATCATCCCTGCTTCCAC-3′
서열번호 6(GAPDH primer)SEQ ID NO: 6 (GAPDH primer) reversereverse 5'-GACCACCTGGTCCTCAGTGT-3'5′-GACCACCTGGTCCTCAGTGT-3′
또한, IAV의 M1 및 NS1 단백질의 발현 수준을 웨스턴 블롯으로 분석하였으며, 액틴은 로딩 컨트롤로 사용되었다. 구체적으로, 프로테아제 억제제(Thermo Scientific)의 존재하에 NP-40 완충액(ELPIS Biotech, 대전, 한국)을 사용하여 세포를 용해시켰다. 세포 추출물을 나트륨 도데실 설페이트-폴리아크릴아미드 겔 전기영동(SDS-PAGE) 겔에 로딩한 다음 니트로셀룰로오스 멤브레인(GE Healthcare, Chicago, IL, USA)으로 옮긴 후, 차단을 위해 멤브레인을 TBST 용액에서 2% BSA와 함께 1시간 동안 인큐베이션한 다음 4℃에서 밤새 1차 항체(Abcam, Cambridge, UK)와 함께 인큐베이션하였다. 세포막은 실온에서 1시간 동안 호스라디시 퍼옥시다아제(horseradish peroxidase, HRP; Cell Signaling Technology, Beverly, MA, USA)와 접합된 2차 항체로 배양되었으며, Super Signal West Pico PLUS 화학발광기질(Thermo Scientific)을 사용하여 얼룩진 단백질을 검출하였다.In addition, the expression levels of M1 and NS1 proteins of IAV were analyzed by Western blot, and actin was used as a loading control. Specifically, cells were lysed using NP-40 buffer (ELPIS Biotech, Daejeon, Korea) in the presence of a protease inhibitor (Thermo Scientific). Cell extracts were loaded on a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, USA), and then the membrane was incubated in TBST solution for blocking. % BSA for 1 hour followed by incubation with primary antibodies (Abcam, Cambridge, UK) overnight at 4°C. Cell membranes were incubated with a secondary antibody conjugated to horseradish peroxidase (HRP; Cell Signaling Technology, Beverly, MA, USA) for 1 hour at room temperature, followed by Super Signal West Pico PLUS chemiluminescent substrate (Thermo Scientific). was used to detect stained proteins.
그 결과, APRG64 처리는 IAV M1(도 3a, 왼쪽 그래프) 및 NP(도 3a, 중앙 그래프) mRNA의 발현을 강력하게 억제하는 것으로 나타났고, 이러한 결과와 일치하게, M1 및 비구조 단백질 1(NS1)을 포함한 바이러스 단백질의 발현은 APRG64 처리에 의해 실질적으로 억제되는 것으로 확인되었다(도 3a, 오른쪽 사진).As a result, APRG64 treatment was shown to strongly suppress the expression of IAV M1 (Fig. 3a, left graph) and NP (Fig. 3a, center graph) mRNA expression, and consistent with these results, M1 and non-structural protein 1 (NS1 ) was found to be substantially inhibited by APRG64 treatment (Fig. 3a, right picture).
실험예 2-2. APRG64에 의한 IAV 감염성 바이러스 입자 감소 효과 확인Experimental Example 2-2. Confirmation of IAV infectious virus particle reduction effect by APRG64
APRG64에 의한 IAV 감염성 바이러스 입자 감소 효과를 확인하기 위하여 플라크 분석을 통해 감염성 바이러스 입자를 적정하였다. 구체적으로, MDCK 세포를 0.1의 MOI에서 1시간 동안 IAV로 감염시킨 다음 10㎍/㎖의 APRG64를 36시간 동안 처리한 후, 0.3% BSA 및 0.5% 아가로스(Affymetrix, Santa Clara, CA, USA)를 함유하는 MEM으로 2일 동안 인큐베이션하였다. 그 후 세포를 3.5% 포름알데히드로 고정하고 아가로스 겔에서 제거하였고, 플라크는 1% 크리스탈 바이올렛 용액으로 세포를 염색하여 확인하였다.In order to confirm the effect of reducing IAV infectious virus particles by APRG64, infectious virus particles were titrated through plaque assay. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour and then treated with 10 μg/ml APRG64 for 36 hours, followed by 0.3% BSA and 0.5% agarose (Affymetrix, Santa Clara, CA, USA). It was incubated for 2 days with MEM containing. Cells were then fixed with 3.5% formaldehyde and removed from an agarose gel, and plaques were identified by staining the cells with a 1% crystal violet solution.
그 결과, APRG64 처리는 감염성 바이러스 입자의 생성을 유의하게 감소시키는 것으로 확인되었으며, 이는 실험예 3-1의 결과와 일치하는 것이다(도 3b).As a result, it was confirmed that APRG64 treatment significantly reduced the production of infectious virus particles, which was consistent with the results of Experimental Example 3-1 (FIG. 3b).
실험예 2-3. APRG64에 의한 IAV NP 발현 세포의 감소 효과 확인Experimental Example 2-3. Confirmation of the reduction effect of IAV NP expressing cells by APRG64
APRG64에 의한 IAV NP 발현 세포의 감소 효과를 확인하기 위하여 면역세포화학 분석을 수행하였다. 구체적으로, MDCK 세포를 0.1의 MOI에서 1시간 동안 IAV로 감염시킨 다음 6시간 동안 10㎍/㎖의 APRG64를 처리하였고, 4% 포름알데히드로 고정한 후 0.5% Triton X-100(Sigma-Aldrich)으로 투과화시켰다. 항-IAV NP 항체(Abcam)로 처리한 후, 세포를 Alexa Fluor 488-접합 이차 항체(Thermo Scientific)와 함께 인큐베이션하였으며, DAPI(4',6-diamidino-2-phenylindole) 용액(Sigma-Aldrich)을 사용하여 핵을 검출하였다. 세포 이미지는 N-SIM S Super Resolution Microscope(Nikon, Tokyo, Japan)로 캡처하고 N-SIM 이미지 획득 소프트웨어 브라우저(Nikon)로 분석하였다. 이에 따라, 바이러스성 NP 단백질(녹색)과 핵(DAPI+, 파란색)은 면역세포화학 분석에 의해 시각화되었으며, 대표적인 이미지(왼쪽 패널) 및 DAPI+ 세포에 대한 NP+ 세포의 백분율이 표시되었다. 모든 그래프는 3회 반복의 평균을 나타낸다.Immunocytochemical analysis was performed to confirm the effect of APRG64 on reducing IAV NP-expressing cells. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour, then treated with 10 μg/ml APRG64 for 6 hours, fixed with 4% formaldehyde, and then treated with 0.5% Triton X-100 (Sigma-Aldrich). permeabilized. After treatment with an anti-IAV NP antibody (Abcam), cells were incubated with Alexa Fluor 488-conjugated secondary antibody (Thermo Scientific) and DAPI (4',6-diamidino-2-phenylindole) solution (Sigma-Aldrich) was used to detect nuclei. Cell images were captured with an N-SIM S Super Resolution Microscope (Nikon, Tokyo, Japan) and analyzed with the N-SIM image acquisition software browser (Nikon). Correspondingly, viral NP protein (green) and nuclei (DAPI + , blue) were visualized by immunocytochemical analysis, and representative images (left panel) and percentage of NP + cells relative to DAPI + cells are shown. All graphs represent the average of three replicates.
그 결과, IAV NP-발현 세포의 수는 APRG64에 의해 유의하게 감소되는 것으로 나타났고(도 3c), 이에 따르면 APRG64가 강력한 항-IAV 활성을 가진다는 것이 강력하게 뒷받침되었다.As a result, it was found that the number of IAV NP-expressing cells was significantly reduced by APRG64 (FIG. 3c), which strongly supported that APRG64 has strong anti-IAV activity.
실시예 3. APRG64의 항바이러스 성분 식별Example 3. Identification of antiviral components of APRG64
실험예 1 및 2에 따른 APRG64의 항바이러스 효과를 나타내는 구체적인 성분을 확인하기 위하여 활성 성분 분리실험을 수행하였다. 먼저, 농축 추출물(APRG64)을 EtOAc, n-BuOH 및 H2O 분획으로 분할하였다. TLC 플레이트에서 주요 플라보노이드 반점이 관찰되었기 때문에 EtOAc 분획(APRG64-E)을 사용하여 활성 대사산물을 분리하였으며, c.c.로 구분된 분수 SiO2, ODS 및 Sephadex LH-20을 사용하여 8개의 플라보노이드(flavonoids)(1-8)와 1개의 트리테르페노이드(triterpenoid)(9)를 생성하였다(표 2 및 도 4). In order to confirm specific components exhibiting the antiviral effect of APRG64 according to Experimental Examples 1 and 2, an active component separation experiment was performed. First, the concentrated extract (APRG64) was partitioned into EtOAc, n-BuOH and H 2 O fractions. Since major flavonoid spots were observed on the TLC plate, the active metabolite was isolated using the EtOAc fraction (APRG64-E), and the fractions SiO 2 , ODS and Sephadex LH-20 separated by cc were used to identify 8 flavonoids. (1-8) and one triterpenoid (9) were produced (Table 2 and Figure 4).
구체적으로, 실시예 2에서 제조된 APRG64 추출물(2kg)을 H2O(1.5L)에 현탁시킨 후 에틸 아세테이트(ethyl acetate, EtOAc, 1.5L×3) 및 n-부탄올(n-BuOH, 1.5L×3)로 순차적으로 추출하였다. 각 층을 감압 농축하여 EtOAc(APRG64-E, 178g), n-BuOH(APRG64-B, 328g) 및 H2O(APRG64-W, 494g) 분획을 얻었다. Specifically, after suspending the APRG64 extract (2kg) prepared in Example 2 in H 2 O (1.5L), ethyl acetate (ethyl acetate, EtOAc, 1.5L×3) and n-butanol (n-BuOH, 1.5L) × 3) were sequentially extracted. Each layer was concentrated under reduced pressure to obtain EtOAc (APRG64-E, 178 g), n-BuOH (APRG64-B, 328 g), and H 2 O (APRG64-W, 494 g) fractions.
APRG64-E 분획(178g)에 SiO2 c.c.(φ12×20㎝) 및 클로로포름-메탄올(CHCl3-MeOH, 30:1→20:1→10:1→3:1; 각각 18L)로 용출하여 25개의 분획(APRG64-E-1~APRG64-E-13)이 생성되었다. 25 _ Two fractions (APRG64-E-1 to APRG64-E-13) were generated.
분획 APRG64-E-13[609.2㎎, 용리 부피/총 부피(Ve/Vt) 0.224-0.230]을 ODS c.c.(φ3.5×5.0㎝) 및 아세톤-H2O(1:1→3:1; 둘 다 10L)로 용리하여 17개의 분획(APRG64-E-13-1~APRG64-E-13-17)이 생성되었다.Fraction APRG64-E-13 [609.2 mg, elution volume/total volume (Ve/Vt) 0.224-0.230] was separated by ODS cc (φ3.5×5.0 cm) and acetone-H 2 O (1:1→3:1; Both were eluted with 10 L), resulting in 17 fractions (APRG64-E-13-1 to APRG64-E-13-17).
분획 APRG64-E-13-14(150.9㎎, Ve/Vt=0.315-0.444)을 ODS c.c.(φ2.5×5.0㎝) 및 MeOH-H2O (3:1; 6L)로 용출하여 궁극적으로 12개의 분획(APRG64-E-13-14-1~APRG64-E-13-14-12)이 생성되었고, 상기 12개의 분획에는 정제된 화합물 2[아피게닌, APRG64-E-13-14-7, 10.7㎎, Ve/Vt 0.306-0.456, TLC(SiO2 F254) 머무름 계수(retention factor, Rf) 0.46, CHCl3-MeOH = 20:1] 및 화합물 9[우르솔릭 산, APRG64-E-13-14-10, 44.4㎎, Ve/Vt 0.621-0.765, TLC(SiO2 F254) Rf 0.56, CHCl3-MeOH = 20:1]가 포함되었다. Fraction APRG64-E-13-14 (150.9 mg, Ve/Vt = 0.315-0.444) was eluted with ODS cc (φ2.5×5.0 cm) and MeOH-H 2 O (3:1; 6L), ultimately reaching 12 Two fractions (APRG64-E-13-14-1 to APRG64-E-13-14-12) were produced, and the 12 fractions contained purified compounds 2 [apigenin, APRG64-E-13-14-7, 10.7 mg, Ve/Vt 0.306-0.456, TLC (SiO 2 F254) retention factor (Rf) 0.46, CHCl 3 -MeOH = 20:1] and compound 9 [ursolic acid, APRG64-E-13-14 -10, 44.4mg, Ve/Vt 0.621-0.765, TLC(SiO 2 F254) Rf 0.56, CHCl 3 -MeOH = 20:1].
분획 APRG64-E-23[7.9g, Ve/Vt 0.683-0.720]을 ODS c.c.(φ7.0×7.0㎝) 및 아세톤-H2O(1:3→1:2; 둘 다 30L)로 용리하여 12개의 분획(APRG64-E-23-1 to APRG64-E-23-12)이 생성되었고, 상기 12개의 분획에는 정제된 화합물 7[퀘르시트린, APRG64-E-23-5, 684.5㎎, Ve/Vt 0.423-0.660, TLC(SiO2 F254) Rf 0.70, CHCl3-MeOH = 2:1]이 포함되었다.Fraction APRG64-E-23 [7.9 g, Ve/Vt 0.683-0.720] was eluted with ODS cc (φ7.0×7.0 cm) and acetone-H 2 O (1:3→1:2; both 30 L). Twelve fractions (APRG64-E-23-1 to APRG64-E-23-12) were generated, and the purified compound 7 [quercitrin, APRG64-E-23-5, 684.5 mg, Ve/ Vt 0.423-0.660, TLC (SiO 2 F254) Rf 0.70, CHCl 3 -MeOH = 2:1].
분획 APRG64-E-23-3(648.3㎎, Ve/Vt=0.015-0.023)을 ODS c.c.(φ3.0×5.0㎝) 및 MeOH-H2O(1:2; 8L)로 용출하여 궁극적으로 11개의 분획(APRG64-E-23-3-1~APRG64-E-23-3-11)이 생성되었고, 상기 11개의 분획에는 정제된 화합물 6[퀘르세틴, APRG64-E-23-3-9, 24.1㎎, Ve/Vt 0.756-0.795, TLC(SiO2 F254) Rf 0.42, CHCl3-MeOH = 20:1]이 포함되었다.Fraction APRG64-E-23-3 (648.3 mg, Ve/Vt = 0.015-0.023) was eluted with ODS cc (φ3.0×5.0 cm) and MeOH-H 2 O (1:2; 8 L), ultimately reaching 11 Two fractions (APRG64-E-23-3-1 to APRG64-E-23-3-11) were generated, and the 11 fractions contained purified compounds 6 [quercetin, APRG64-E-23-3-9, 24.1 mg, Ve/Vt 0.756-0.795, TLC(SiO 2 F254) Rf 0.42, CHCl 3 -MeOH = 20:1].
분획 APRG64-E-23-6(258.3㎎, Ve/Vt=0.111-0.153)에 SiO2 c.c.(φ3.0×15㎝) 및 CHCl3-MeOH-H2O(20:3:1→10:3:1; 둘 다 6L)로 용리하여 17개의 분획(APRG64-E-23-6-1~APRG64- E-23-6-17)이 생성되었다. Fraction APRG64-E-23-6 (258.3 mg, Ve/Vt = 0.111-0.153) was added with 2 cc of SiO (φ3.0×15 cm) and CHCl3-MeOH-H 2 O (20:3:1→10:3 :1; both 6L), resulting in 17 fractions (APRG64-E-23-6-1 to APRG64-E-23-6-17).
분획 APRG64-E-23-6-3(81.4㎎, Ve/Vt=0.078-0.140)을 ODS c.c.(φ1.5×8.0㎝) 및 MeOH-H2O(2:3; 2L)로 용리하여 궁극적으로 8개의 분획(APRG64-E-23-6-3-1~APRG64-E-23-6-3-8)이 생성되었고, 상기 8개의 분획에는 정제된 화합물 1[afzelin, APRG64-E-23-6-3-6, 6.7㎎, Ve/Vt 0.538-0.629, TLC(SiO2 F254) Rf 0.54, CHCl3-MeOH=20:1]이 포함되었다.Fraction APRG64-E-23-6-3 (81.4 mg, Ve/Vt = 0.078-0.140) was eluted with ODS cc (φ1.5×8.0 cm) and MeOH-H 2 O (2:3; 2 L) to obtain the final 8 fractions (APRG64-E-23-6-3-1 to APRG64-E-23-6-3-8) were generated, and the 8 fractions contained purified compound 1 [afzelin, APRG64-E-23 -6-3-6, 6.7mg, Ve/Vt 0.538-0.629, TLC(SiO 2 F254) Rf 0.54, CHCl 3 -MeOH=20:1] were included.
분획 APRG64-E-23-6-3-4(31.8㎎, Ve/Vt=0.156-0.241)에 Sephadex LH-20 c.c.(φ1.5×50㎝) 및 100% MeOH(1.5L)로 용리하여 궁극적으로 6개의 분획(APRG64-E-23-6-3-4-1~APRG64-E-23-6-3-4-6이 생성되었고, 상기 6개의 분획에는 정제된 화합물 4[아스트라갈린, APRG64-E-23-6-3-4-2, 26.5㎎, Ve/Vt 0.502-0.742, TLC(SiO2 F254) Rf 0.50, CHCl3-MeOH-H2O=7:3:1]가 포함되었다. Fraction APRG64-E-23-6-3-4 (31.8 mg, Ve/Vt = 0.156-0.241) was eluted with Sephadex LH-20 cc (φ1.5 × 50 cm) and 100% MeOH (1.5 L) to obtain the final 6 fractions (APRG64-E-23-6-3-4-1 to APRG64-E-23-6-3-4-6 were generated, and the 6 fractions contained purified compound 4 [Astragalin, APRG64 -E-23-6-3-4-2, 26.5 mg, Ve/Vt 0.502-0.742, TLC(SiO 2 F254) Rf 0.50, CHCl3-MeOH-H 2 O=7:3:1].
분획 APRG64-E-23-8(2.4g, Ve/Vt=0.253-0.423)에 SiO2 c.c.(φ4.0×15㎝) CHCl3-MeOH-H2O(20:3:1→10:3:1→7:3:1→6:4:1; 각각 6L)로 용출하여 최종적으로 24개의 분획(APRG64-E-23-8-1~APRG64-E-23-8-24)이 생성되었고, 상기 24개의 분획에는 정제된 화합물 3[아피게닌 7-O-β-D-글루쿠로나이드, APRG64-E-23-8-3, 175.3㎎, Ve/Vt 0.056-0.209, TLC(SiO2 F254) Rf 0.52, CHCl3-MeOH-H2O=12:3:1], 화합물 5[니코티플로린, APRG64-E-23-8-5, 22.9㎎, Ve/Vt 239 0.377-0.501, TLC(SiO2 F254) Rf 0.34, CHCl3-MeOH-H2O=12:3:1], 및 화합물 8[루틴, APRG64-E-23-8-16, 7.9㎎, Ve/Vt 0.760- 0.809, TLC(SiO2 F254) Rf 0.61, CH2Cl3-MeOH-H2O = 6:4:1]이 포함되었다.Fraction APRG64-E-23-8 (2.4 g, Ve/Vt = 0.253-0.423) SiO 2 cc (φ4.0 × 15 cm) CHCl 3 -MeOH-H 2 O (20:3:1→10:3 :1→7:3:1→6:4:1; 6L each) to finally generate 24 fractions (APRG64-E-23-8-1 to APRG64-E-23-8-24) , The 24 fractions contained purified compound 3 [apigenin 7-O-β-D-glucuronide, APRG64-E-23-8-3, 175.3 mg, Ve/Vt 0.056-0.209, TLC (SiO 2 F254) Rf 0.52, CHCl 3 -MeOH-H 2 O=12:3:1], compound 5 [nicotiflorin, APRG64-E-23-8-5, 22.9mg, Ve/Vt 239 0.377-0.501, TLC (SiO 2 F254) Rf 0.34, CHCl 3 -MeOH-H 2 O=12:3:1], and compound 8 [rutin, APRG64-E-23-8-16, 7.9 mg, Ve/Vt 0.760- 0.809, TLC(SiO 2 F254) Rf 0.61, CH 2 Cl 3 -MeOH-H 2 O = 6:4:1].
구분division 이름name 특징 characteristic
화합물 1compound 1 아프젤린
(Afzelin)Afgeline
(Afzelin) 		황색 무정형 분말(MeOH); 양성 FAB/MS m/z 433 [M+H]+; IR(KBr, v) 3,400, 1,660, 1,605 및 1,500㎝-1 yellow amorphous powder (MeOH); Positive FAB/MS m/z 433 [M+H] + ; IR (KBr, v) 3,400, 1,660, 1,605 and 1,500 cm -1
화합물 2compound 2 아피제닌
(Apigenin) apigenin
(Apigenin) 		황색 무정형 분말(MeOH); 양성 FAB/MS m/z 271 [M+H]+; IR(KBr, v) 3,420, 2,935, 1,645 및 1,605㎝-1 yellow amorphous powder (MeOH); Positive FAB/MS m/z 271 [M+H] + ; IR (KBr, v) 3,420, 2,935, 1,645 and 1,605 cm -1
화합물 3compound 3 아피제닌 7-O-β-D-글루쿠로나이드
(Apigenin 7-O-β-D-glucuronide )Apigenin 7-O-β-D-glucuronide
(Apigenin 7-O-β-D-glucuronide) 		황색 무정형 분말(MeOH); 양성 FAB/MS m/z 469 [M+Na]+; IR(KBr, v) 3,455, 1,645, 1,510 및 1,365㎝-1 yellow amorphous powder (MeOH); Positive FAB/MS m/z 469 [M+Na] + ; IR (KBr, v) 3,455, 1,645, 1,510 and 1,365 cm -1
화합물 4compound 4 아스트라갈린
(Astragalin )astragalin
(Astragalin) 		황색 무정형 분말(MeOH); 양성 FAB/MS m/z 471 [M+Na]+; IR(KBr, v) 3,350, 2,930, 2,365, 1,655 및 1,610㎝-1 yellow amorphous powder (MeOH); Positive FAB/MS m/z 471 [M+Na] + ; IR (KBr, v) 3,350, 2,930, 2,365, 1,655 and 1,610 cm -1
화합물 5compound 5 니코티플로린
(Nicotiflorin)Nicotiflorin
(Nicotiflorin) 		황색 무정형 분말(MeOH); 양성 FAB/MS m/z 639 [M+Na]+; IR(KBr, v) 3,365, 2,940, 2,360, 1,655, 1,600 및 1,515㎝-1 yellow amorphous powder (MeOH); Positive FAB/MS m/z 639 [M+Na] + ; IR (KBr, v) 3,365, 2,940, 2,360, 1,655, 1,600 and 1,515 cm -1
화합물 6compound 6 케르세틴
(Quercetin)quercetin
(Quercetin) 		황색 무정형 분말(MeOH); 양성 FAB/MS m/z 303 [M+H]+; IR(KBr, v) 3,350, 1,680 및 1,615㎝-1 yellow amorphous powder (MeOH); Positive FAB/MS m/z 303 [M+H] + ; IR (KBr, v) 3,350, 1,680 and 1,615 cm -1
화합물 7compound 7 케르시트린
(Quercitrin)Quercitrin
(Quercitrin) 		황색 무정형 분말(MeOH); 양성 FAB/MS m/z 449 [M+H]+; IR(KBr, v) 3,358, 1,659, 1,610 및 1,500㎝-1 yellow amorphous powder (MeOH); positive FAB/MS m/z 449 [M+H] + ; IR (KBr, v) 3,358, 1,659, 1,610 and 1,500 cm -1
화합물 8compound 8 루틴
(Rutin)Routine
(Rutin) 		황색 비정질 분말(MeOH); 양성 FAB/MS m/z 611 [M+H]+; IR(KBr, v) 3405, 3,930, 1,660 및 1,565㎝-1 yellow amorphous powder (MeOH); Positive FAB/MS m/z 611 [M+H] + ; IR (KBr, v) 3405, 3,930, 1,660 and 1,565 cm -1
화합물 9compound 9 우르솔산
(Ursolic acid)Ursolic acid
(Ursolic acid) 		백색 무정형 분말(MeOH); 양성 FAB/MS m/z 457 [M+H]+; IR(KBr, v) 3,400, 1,732 및 1,680㎝-1 white amorphous powder (MeOH); positive FAB/MS m/z 457 [M+H] + ; IR (KBr, v) 3,400, 1,732 and 1,680 cm -1
실험예 3. APRG64으로부터 분리된 9가지 화합물의 항바이러스 활성 확인Experimental Example 3. Confirmation of antiviral activity of 9 compounds isolated from APRG64
실시예 3에 따라 APRG64에서 분리된 9가지의 성분 중 항바이러스 효과를 나타내는 성분을 식별하기 위하여 항 IAV 활성을 확인하였다. 항 IAV 활성은 실험예 2와 동일하게 IAV로 감염된 세포에 각 화합물을 처리한 후 인플루엔자 바이러스의 구조를 형성하는 M1 및 바이러스 핵단백질(Nucleoprotein, NP)의 발현양을 통해 확인하였다. 구체적으로, MDCK 세포를 0.1의 MOI에서 1시간 동안 IAV로 감염시킨 후, 아프젤린(화합물 1, 10㎍/㎖), 아피제닌(화합물 2, 5㎍/㎖), 아피제닌 7-O-글루쿠로나이드(화합물 3, 10㎍/㎖), 아스트라갈린(화합물 4, 10㎍/㎖), 니코티플로린(화합물 5, 10㎍/㎖), 케르세틴(화합물 6, 2.5㎍/㎖), 케르시트린(화합물 7, 10㎍/㎖), 루틴 (화합물 8, 5㎍/㎖) 및 우르솔산(화합물 9, 10㎍/㎖)를 각각 처리한 후 IAV의 M1 및 NP mRNA의 상대적인 발현 수준을 RT-qPCR로 분석하였다. 각 화합물의 농도를 제외하고는 실시예 2-1과 동일한 방법으로 RT-qPCR 분석을 실시하였다.In order to identify a component exhibiting an antiviral effect among the nine components isolated from APRG64 according to Example 3, the anti-IAV activity was confirmed. As in Experimental Example 2, the anti-IAV activity was confirmed through the expression levels of M1 and viral nucleoprotein (NP), which form the structure of influenza virus, after each compound was treated with IAV-infected cells. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour, and then treated with afgeline ( Compound 1, 10 μg/ml), apigenin ( Compound 2, 5 μg/ml), and apigenin 7-O-glu. Curonide (Compound 3, 10μg/ml), Astragaline (Compound 4, 10μg/ml), Nicotiflorin (Compound 5, 10μg/ml), Quercetin (Compound 6, 2.5μg/ml), Quercetin (Compound 6, 2.5μg/ml) Relative expression levels of M1 and NP mRNAs of IAV were measured by RT -Analyzed by qPCR. RT-qPCR analysis was performed in the same manner as in Example 2-1 except for the concentration of each compound.
그 결과, 9개의 화합물 중 루틴(화합물 8)을 제외한 8개(아프젤린, 아피제닌, 아피제닌 7-O-글루쿠로나이드, 아스트라갈린, 니코티플로린, 케르세틴, 케르시트린 및 우르솔산)의 화합물이 IAV의 M1 및 NP mRNA 발현을 유의하게 감소시키는 것으로 확인되었으며(각각 도 5a 및 도 5b), 특히, 아피제닌(화합물 2)의 항-IAV 활성이 가장 우수한 것으로 확인되었다.As a result, 8 of the 9 compounds (apgeline, apigenin, apigenin 7-O-glucuronide, astragaline, nicotiflorin, quercetin, quercitrin and ursolic acid) excluding rutin (compound 8) It was confirmed that the compound significantly reduced the expression of M1 and NP mRNA of IAV (FIG. 5a and FIG. 5b, respectively), and in particular, apigenin (compound 2) showed the highest anti-IAV activity.
실험예 4. IAV 유도 CPE에 대한 아피제닌의 우수한 억제 활성 확인Experimental Example 4. Confirmation of excellent inhibitory activity of apigenin on IAV-induced CPE
실험예 3에서 가장 항바이러스 효과가 우수한 것으로 확인된 아피제닌(화합물 2)의 항바이러스 효과를 추가적으로 확인하기 위하여, IAV에 의해 유도된 CPE 억제 활성 및 바이러스 의존적 경로를 통한 항바이러스 효과인지 여부를 분석하였다.In order to additionally confirm the antiviral effect of apigenin (Compound 2), which was confirmed to have the best antiviral effect in Experimental Example 3, CPE inhibition activity induced by IAV and antiviral effect through a virus-dependent pathway were analyzed. did
실험예 4-1. 아피제닌의 우수한 IAV 유도 CPE 억제 활성 효과확인Experimental Example 4-1. Confirmation of excellent IAV-induced CPE inhibitory activity of apigenin
CPE 억제 활성을 확인하기 위하여, MDCK 세포를 0.1의 MOI에서 IAV로 감염시킨 다음, 0.625, 1.25, 2.50 또는 5.00㎍/㎖의 아피제닌을 처리하였고, 24시간 후, 세포를 1% 크리스탈 바이올렛 용액으로 염색하여 부착된 세포를 분석하였다. 또한, 24 hpi에서 부착된 세포를 정량화하여 아피제닌의 EC50을 계산하였으며, 이 외에는 실험예 1-2과 동일한 방법으로 CPE 분석을 실시하였다.To confirm the CPE inhibitory activity, MDCK cells were infected with IAV at an MOI of 0.1 and then treated with 0.625, 1.25, 2.50 or 5.00 μg/ml of apigenin, and after 24 hours, the cells were incubated with 1% crystal violet solution. Adherent cells were analyzed by staining. In addition, the EC 50 of apigenin was calculated by quantifying the attached cells at 24 hpi, and CPE analysis was performed in the same manner as in Experimental Example 1-2 except for this.
그 결과, IAV에 의해 유발된 CPE가 아피제닌의 용량 의존적으로 현저히 감소하는 것으로 확인되었고(도 6a), 이 때 EC50은 1.438㎍/㎖로 측정되었으며(도 6b), 이와 같은 결과는 실험예 1-2와 상응하는 것이다.As a result, it was confirmed that the CPE induced by IAV significantly decreased in a dose-dependent manner of apigenin (FIG. 6a), and at this time, the EC 50 was measured as 1.438 μg/ml (FIG. 6b). It corresponds to 1-2.
실험예 4-2. 아피제닌의 바이러스 의존적 경로를 통한 항바이러스 효과 확인Experimental Example 4-2. Confirmation of antiviral effect through virus-dependent pathway of apigenin
실험예 4-1에 따른 CPE 억제 활성이 바이러스 의존적 경로에 기반한 것인지 확인하기 위하여 세포사멸사 유도제 처리 후 아피제닌을 추가적으로 처리하여 세포 사멸 여부를 확인하였다. 구체적으로, MDCK 세포에 5㎍/㎖의 아피제닌를 처리한 다음 200nM의 스타우로스포린(STS)을 추가적으로 처리하여 CPE 분석을 수행하여 세포 생존력을 확인하였다. 이 때, ImageJ 소프트웨어로 이미지를 처리하여 플레이트에 부착된 세포를 정량화였으며, 그래프는 3회 반복의 평균을 나타내었으며, 이 외에는 실험예 1-3과 동일한 방법으로 실험을 수행하였다.In order to confirm whether the CPE inhibitory activity according to Experimental Example 4-1 was based on the virus-dependent pathway, apigenin was additionally treated after treatment with the apoptosis inducer to confirm cell death. Specifically, MDCK cells were treated with 5 μg/ml of apigenin and then additionally treated with 200 nM of staurosporine (STS) to perform CPE analysis to confirm cell viability. At this time, the image was processed with ImageJ software to quantify the cells attached to the plate, and the graph showed the average of three repetitions, and the experiment was performed in the same manner as in Experimental Example 1-3 except for this.
그 결과, 아피제닌을 처리하였음에도 불구하고 STS에 의한 바이러스 감염-독립적 세포사멸로부터 세포를 보호하지 못하는 것으로 확인되었고(도 6c), 이는 실험예 1-3에서 APRG64 추가군은 STS 유도 세포 사멸을 예방하지 못하였다는 결과와 일치하는 것이다(도 2c).As a result, it was confirmed that cells were not protected from virus infection-independent apoptosis by STS despite treatment with apigenin (FIG. 6c), indicating that in Experimental Examples 1-3, the APRG64 addition group prevented STS-induced cell death. This is consistent with the result of not being able to (FIG. 2c).
실험예 5. 아피제닌의 IAV 복제 억제에 따른 우수한 항바이러스 효과 확인Experimental Example 5. Confirmation of excellent antiviral effect according to inhibition of IAV replication by apigenin
실험예 5-1. 아피제닌의 IAV 복제 억제 활성 확인Experimental Example 5-1. Confirmation of IAV replication inhibitory activity of apigenin
아피제닌의 IAV 복제 억제 활성을 확인하기 위하여 RT-qPCR 분석 및 웨스턴 블롯 실험을 수행하였다. 구체적으로, MDCK 세포를 0.1의 MOI에서 1시간 동안 IAV로 감염시킨 다음 5㎍/㎖의 아피제닌을 처리하였고, 6시간 후, IAV의 M1 및 NP mRNA의 발현 수준을 RT-qPCR로 분석하였고, 바이러스 M1 및 NS1 단백질 발현 수준을 웨스턴 블롯으로 분석하였다. 실험 방법 및 조건은 실험예 2-1과 동일하게 수행하였다.In order to confirm the IAV replication inhibitory activity of apigenin, RT-qPCR analysis and Western blotting were performed. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour and then treated with 5 μg/ml apigenin, and after 6 hours, the expression levels of M1 and NP mRNA of IAV were analyzed by RT-qPCR, Virus M1 and NS1 protein expression levels were analyzed by Western blot. The experimental method and conditions were performed in the same manner as in Experimental Example 2-1.
그 결과, 아피제닌 처리는 IAV의 M1 및 NP mRNA(도 7a, 왼쪽 및 중앙 그래프) 및 단백질(도 7a, 오른쪽 사진)의 발현을 크게 감소시키는 것으로 확인되었고, 이는 실험예 2-1에서 확인된 APRG64의 IAV 복제 억제 활성과 일치하는 것이다.As a result, it was confirmed that apigenin treatment greatly reduced the expression of M1 and NP mRNA (Fig. 7a, left and center graphs) and protein (Fig. 7a, right picture) of IAV, which was confirmed in Experimental Example 2-1. This is consistent with the inhibitory activity of APRG64 on IAV replication.
실험예 5-2. 아피제닌에 의한 IAV 감염성 바이러스 입자 감소 효과 확인Experimental Example 5-2. Confirmation of IAV infectious virus particle reduction effect by apigenin
MDCK 세포를 0.1의 MOI에서 1시간 동안 IAV로 감염시킨 다음 5㎍/㎖의 아피제닌을 처리하였고, 36 hpi에서 플라크 분석을 수행하여 감염성 바이러스 역가를 측정하여 그래프로 나타내었다. 이 때, 플라크 분석 방법 및 조건은 실험예 2-2와 동일하게 수행하였다.MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour, then treated with 5 μg/ml apigenin, and plaque assay was performed at 36 hpi to measure the infectious virus titer and presented in a graph. At this time, the plaque analysis method and conditions were performed in the same manner as in Experimental Example 2-2.
그 결과, 아피제닌 처리에 의해 감염성 바이러스 입자의 생성이 현저히 감소되는 것으로 확인되었으며(도 7b), 이는 실험예 2-2에서 확인된 APRG64의 IAV 바이러스 입자 감소 효과와 일치한다.As a result, it was confirmed that the production of infectious virus particles was significantly reduced by apigenin treatment (FIG. 7b), which is consistent with the effect of APRG64 on reducing IAV virus particles confirmed in Experimental Example 2-2.
실험예 5-3. 아피제닌에 의한 IAV NP 발현 세포의 감소 효과 확인Experimental Example 5-3. Confirmation of the reduction effect of IAV NP expressing cells by apigenin
아피제닌에 의한 IAV의 NP를 발현하는 세포(IAV-NP) 증감 여부를 확인하기 위하여 면역세포화학 분석을 실시하였다. 구체적으로, MDCK 세포를 0.1의 MOI에서 1시간 동안 IAV로 감염시킨 다음 6시간 동안 5㎍/㎖의 아피제닌을 처리한 후, IAV-NP 발현 세포의 감소 여부를 면역세포화학 분석으로 확인하였으며, 이 때, 면역세포화학 분석은 실험예 2-3와 동일하게 수행하였다.Immunocytochemical analysis was performed to confirm the increase or decrease of IAV NP-expressing cells (IAV-NP) by apigenin. Specifically, MDCK cells were infected with IAV at an MOI of 0.1 for 1 hour and then treated with 5 μg/ml apigenin for 6 hours, and then the reduction of IAV-NP expressing cells was confirmed by immunocytochemical analysis, At this time, immunocytochemical analysis was performed in the same manner as in Experimental Example 2-3.
그 결과, 아피제닌을 처리한 경우 IAV NP 세포가 거의 검출되지 않아(도 7c) 강력한 항바이러스 활성이 확인되었으며, 이는 실험예 2-3에서 확인된 APRG64의 IAV 바이러스 입자 감소 효과와 일치하는 것이다. As a result, when apigenin was treated, almost no IAV NP cells were detected (FIG. 7c), confirming strong antiviral activity, which is consistent with the effect of APRG64 on reducing IAV virus particles confirmed in Experimental Examples 2-3.
이와 같은 결과를 통하여 아피제닌이 APRG64의 주요 항바이러스 성분임이 확인되었다.Through these results, it was confirmed that apigenin is the main antiviral component of APRG64.
실험예 6. APRG64 및 아피제닌의 IAV 복제 억제 활성Experimental Example 6. IAV replication inhibitory activity of APRG64 and apigenin
실험예 6-1. 다른 IAV 균주에 대한 APRG64 및 아피제닌의 항바이러스 활성 확인Experimental Example 6-1. Confirmation of antiviral activity of APRG64 and apigenin against other IAV strains
APRG64와 아피제닌이 IAV의 다른 균주에 대해서도 항바이러스 활성을 갖는지 여부를 조사하였다. 이를 위해 MDCK 세포를 다른 인플루엔자 균주인 A/PR/8/34(H1N1)에 0.1의 MOI에서 1시간 동안 감염시키고 APRG64(10㎍/㎖) 또는 아피게닌(5㎍/㎖)을 각각 처리하였다. 6시간 후, IAV의 M1 및 NP 발현 수준을 웨스턴 블롯으로 분석하였으며, 웨스턴 블롯 분석은 실험예 2-1과 동일한 방법으로 수행하였다.The antiviral activity of APRG64 and apigenin against other strains of IAV was investigated. To this end, MDCK cells were infected with another influenza strain, A/PR/8/34 (H1N1), at an MOI of 0.1 for 1 hour, and treated with APRG64 (10 μg/ml) or apigenin (5 μg/ml), respectively. After 6 hours, the expression levels of M1 and NP of IAV were analyzed by Western blot, and Western blot analysis was performed in the same manner as in Experimental Example 2-1.
그 결과, 인플루엔자 A/California/07/2009 균주에 대한 항바이러스 효과와 유사하게, APRG64 및 아피제닌은 각각 PR8-감염 세포에서 바이러스 단백질 발현을 강하게 감소시키는 것으로 확인되었다(도 8a). 특히, APRG64 및 아피제닌의 항-IAV 활성이 IAV 감염 치료를 위해 임상적으로 사용되는 항바이러스성 뉴라미니다제 억제제(neuraminidase inhibitor)인 oseltamivir phosphate (Tamiflu)와 유사하게 나타났다는 점에서, 인플루엔자 바이러스 치료 효과가 현저히 우수한 것으로 확인되었다.As a result, similar to the antiviral effects against the influenza A/California/07/2009 strain, APRG64 and apigenin were confirmed to strongly reduce viral protein expression in PR8-infected cells, respectively (FIG. 8a). In particular, in that the anti-IAV activity of APRG64 and apigenin was similar to that of oseltamivir phosphate (Tamiflu), an antiviral neuraminidase inhibitor clinically used for the treatment of IAV infection, influenza virus It was confirmed that the treatment effect was remarkably good.
실험예 6-2. 바이러스 복제 초기 단계 억제에 따른 APRG64 및 아피제닌의 IAV 복제 억제 활성 확인Experimental Example 6-2. Confirmation of IAV replication inhibitory activity of APRG64 and apigenin according to inhibition of early viral replication
IAV 복제는 바이러스가 숙주 세포에 부착하여 세포 내로 유입하는 것으로 시작되는데, APRG64 및 아피제닌이 이러한 바이러스 복제의 초기 단계를 타겟하여 IAV 복제 억제 활성을 나타내는지 여부를 분석하였다. 먼저, 각 물질의 바이러스 부착에 대한 효과를 확인하기 위하여 MDCK 세포를 4℃에서 1시간 동안 10㎍/㎖의 APRG64 또는 5㎍/㎖의 아피제닌을 각각 처리하여 0.1의 MOI에서 IAV로 감염시켰다. 그 후, IAV의 M1 및 NP의 mRNA 발현 수준을 6 hpi에서 RT-qPCR에 의해 분석하였다. 또한, 감염 후 1, 2 또는 4시간(hpi)에서 APRG64 또는 아피제닌으로 처리한 후의 IAV의 M1 및 NP의 mRNA 발현 수준을 분석하였다.IAV replication begins when the virus attaches to and enters the host cell. Whether APRG64 and apigenin target this early stage of viral replication to inhibit IAV replication was analyzed. First, in order to confirm the effect of each substance on virus attachment, MDCK cells were treated with 10 μg/ml APRG64 or 5 μg/ml apigenin for 1 hour at 4° C., respectively, and infected with IAV at an MOI of 0.1. Then, the mRNA expression levels of M1 and NP of IAV were analyzed by RT-qPCR at 6 hpi. In addition, the mRNA expression levels of M1 and NP of IAV after treatment with APRG64 or apigenin at 1, 2 or 4 hours (hpi) after infection were analyzed.
그 결과, APRG64와 아피제닌은 IAV의 M1 및 NP RNA의 발현을 각각 유의하게 억제시켰다(도 8b). 또한, 감염 후 1, 2 또는 4시간(hpi)에서 APRG64 또는 아피제닌으로 처리하면 IAV의 M1 및 NP RNA 발현 수준 역시 감소하였다(도 8c). 이와 같은 결과에 따라 APRG64 및 아피제닌이 바이러스 부착 및 진입을 포함하는 IAV 복제의 초기 단계를 억제하는 것이 확인되었다.As a result, APRG64 and apigenin significantly inhibited the expression of M1 and NP RNA of IAV, respectively (FIG. 8b). In addition, treatment with APRG64 or apigenin at 1, 2 or 4 hours post infection (hpi) also reduced the expression levels of M1 and NP RNA of IAV (Fig. 8c). According to these results, it was confirmed that APRG64 and apigenin inhibit the early stages of IAV replication including viral attachment and entry.
실험예 6-3. RNA 중합효소 활성 억제에 따른 APRG64 및 아피제닌의 IAV 복제 억제 활성 확인Experimental Example 6-3. Confirmation of IAV replication inhibitory activity of APRG64 and apigenin according to RNA polymerase activity inhibition
바이러스 음성(-) RNA 가닥을 양성(+) RNA 가닥으로 전사하는 IAV RNA-의존성 RNA 중합효소(RdRP)의 활성은 IAV 복제에 중요한 역할을 수행하는데, RdRP 활성은 IAV M1 및 NP의 (+) RNA 가닥의 발현 수준에 의해 측정될 수 있다. 따라서, APRG64 및 아피제닌 처리에 따른 IAV M1 및 NP의 (+) RNA 가닥의 발현 수준을 분석하여 IAV RdRP 활성에 영향을 미치는지 여부를 확인하였다. 구체적으로, MDCK 세포를 0.1 MOI에서 1시간 동안 IAV로 감염시켰다. 5 hpi에서 바이러스 M1 및 NP RNA의 (+) 가닥이 급격히 증가했기 때문에 5 hpi에서 세포에 APRG64(10㎍/㎖) 또는 아피게닌(5㎍/㎖)을 각각 처리하고 IAV M1 및 NP의 발현 수준을 측정하였다.The activity of IAV RNA-dependent RNA polymerase (RdRP), which transcribes viral negative (-) RNA strands to positive (+) RNA strands, plays an important role in IAV replication, and RdRP activity plays a critical role in IAV M1 and NP (+) It can be measured by the expression level of the RNA strand. Therefore, by analyzing the expression levels of (+) RNA strands of IAV M1 and NP according to APRG64 and apigenin treatment, it was confirmed whether or not IAV RdRP activity was affected. Specifically, MDCK cells were infected with IAV at 0.1 MOI for 1 hour. Since the (+) strands of viral M1 and NP RNAs increased rapidly at 5 hpi, cells were treated with APRG64 (10 μg/ml) or apigenin (5 μg/ml), respectively, and the expression levels of IAV M1 and NP were measured. was measured.
그 결과, APRG64 및 아피제닌은 (+) 가닥 M1 및 NP의 RNA의 합성을 각각 유의하게 억제하여 IAV RdRP 활성을 억제하는 것으로 확인되었다(도 8d).As a result, it was confirmed that APRG64 and apigenin inhibit IAV RdRP activity by significantly inhibiting the synthesis of (+) strand M1 and NP RNA, respectively (FIG. 8d).
실험예 6-4. MAPK 활성 억제에 따른 APRG64 및 아피제닌의 IAV 복제 억제 활성 확인Experimental Example 6-4. Confirmation of IAV replication inhibitory activity of APRG64 and apigenin according to the inhibition of MAPK activity
APRG64 및 아피제닌이 IAV 복제에 필요한 것으로 알려진 MAPK(mitogen-activated protein kinase) 경로의 활성화에 영향을 미치는지 알아보기 위해 웨스턴 블롯 분석을 수행하였다. 구체적으로, MDCK 세포를 0.1 MOI에서 1시간 동안 IAV로 감염시킨 다음 APRG64(10㎍/㎖) 또는 아피게닌(5㎍/㎖)으로 처리하였고, 6 hpi에서 ERK(세포외 신호 조절 단백질 키나아제), p-ERK, SAPK(스트레스 활성화 단백질 키나아제), p-SAPK 및 액틴의 발현 수준을 웨스턴 블롯으로 측정하였으며, 모든 그래프는 3회 반복의 평균을 나타낸다.Western blot analysis was performed to determine whether APRG64 and apigenin affect the activation of the mitogen-activated protein kinase (MAPK) pathway, which is known to be required for IAV replication. Specifically, MDCK cells were infected with IAV at 0.1 MOI for 1 hour, then treated with APRG64 (10 μg/ml) or apigenin (5 μg/ml), and at 6 hpi, ERK (extracellular signal-regulated protein kinase), The expression levels of p-ERK, SAPK (stress activated protein kinase), p-SAPK and actin were measured by Western blotting, and all graphs represent the average of three replicates.
그 결과, IAV 감염은 ERK 및 SAPK의 인산화를 극적으로 유도하였으나, 이러한 증가는 APRG64 또는 아피제닌이 처리된 세포에서는 관찰되지 않았다(도 8e). As a result, IAV infection dramatically induced phosphorylation of ERK and SAPK, but this increase was not observed in cells treated with APRG64 or apigenin (FIG. 8e).
종합하면, 이러한 결과는 APRG64 및 아피제닌이 바이러스 부착, 진입, RdRP 활성화 및 MAPK 신호 전달 경로를 타겟하여 IAV 복제의 여러 단계를 억제한다는 것이 확인되었다.Taken together, these results confirm that APRG64 and apigenin inhibit multiple steps of IAV replication by targeting viral attachment, entry, RdRP activation and MAPK signaling pathways.
실험예 7. 마우스 모델에서 비강 투여에 따른 APRG64 및 아피제닌의 우수한 바이러스 효과 확인Experimental Example 7. Confirmation of excellent viral effects of APRG64 and apigenin according to intranasal administration in mouse models
실험예 2 내지 6의 in vitro 효과가 생체 내에서도 유지되는지 여부를 확인하기 위하여, 마우스 모델에 APRG64 및 아피제닌을 비강 투여하여 항바이러스 효과를 확인하였다. 구체적으로, 야생형 C57BL/6 마우스를 1×103 pfu의 IAV로 감염시킨 후 10분, 3시간 및 6hpi에서 마우스를 PBS(용매 대조군), 0.5㎎/㎏의 APRG64 또는 0.25㎎/㎏의 아피제닌(n = 5)으로 비강 내 처리하였다(도 9a). 감염 후 5일(dpi)에, 플라크 분석을 통해 폐의 감염성 바이러스 입자의 수를 측정하였다. 이외에는 실험예 2-2에 따른 플라크 분석 방법과 동일한 조건으로 수행하였다.In order to confirm whether the in vitro effects of Experimental Examples 2 to 6 are maintained in vivo, APRG64 and apigenin were intranasally administered to a mouse model to confirm antiviral effects. Specifically, wild-type C57BL/6 mice were infected with 1×10 3 pfu of IAV, and at 10 minutes, 3 hours, and 6 hpi, mice were treated with PBS (solvent control), APRG64 at 0.5 mg/kg or apigenin at 0.25 mg/kg. (n = 5) was treated intranasally (Fig. 9a). At 5 days post infection (dpi), the number of infectious viral particles in the lungs was measured by plaque assay. Other than that, it was performed under the same conditions as the plaque analysis method according to Experimental Example 2-2.
그 결과, APRG64 및 아피제닌은 폐에서 IAV 역가를 유의하게 감소시키는 것으로 확인되었다는 점에서(도 9b), APRG64 및 아피제닌의 시험관 내 항바이러스 효과는 생체 내에서도 우수한 것으로 확인되었다.As a result, it was confirmed that APRG64 and apigenin significantly reduced the IAV titer in the lung (FIG. 9b), and thus, the in vitro antiviral effects of APRG64 and apigenin were also excellent in vivo.
실험예 8. 마우스 모델에서의 APRG64 경구 투여에 따른 우수한 바이러스 효과 확인Experimental Example 8. Confirmation of excellent viral effect according to oral administration of APRG64 in mouse model
실험예 8-1. APRG64의 경구투여에 따른 마우스 체중 감소 여부 확인Experimental Example 8-1. Confirmation of mouse weight loss following oral administration of APRG64
6주령 C57BL/6 수컷 마우스를 사용하였고, 투여된 바이러스의 경우 안전성이 확보되어 있으나, 다른 설치류의 감염을 우려하여 네거티브(negative) 사육 시스템을 사용해 다른 동물과의 접촉을 차단하였다. 바이러스의 분리, 동정, 역가 계산 및 투여는 그 안전성을 확보하기 위하여 준비된 특수 클린벤치(clean bench)에서 실시하였으며, 바이러스 소독을 위한 특수 소독제를 이용하여 실험 전후 소독을 실시하였다. 체중에 직접적인 영향을 줄 수 있는 사료 급여 관리는 매일 사료의 양을 판단하여 급여함으로써 급식에 제한이 발생하지 않도록 하였다. 조명은 12시간 주기로 교체하였고, 물과 사료를 자유롭게 먹도록 하였다. 기타 동물 사육과 관련되는 사항은 중앙대학교 동물실험 윤리위원회(National Association of Laboratory Animal Care) 규정에 따라 동물실험을 수행하였다.Six-week-old C57BL/6 male mice were used, and the safety of the administered virus was secured, but contact with other animals was blocked using a negative breeding system due to concerns about infection of other rodents. Isolation, identification, titer calculation, and administration of the virus were performed on a special clean bench prepared to ensure its safety, and disinfection was performed before and after the experiment using a special disinfectant for virus disinfection. For the management of feed feeding, which can directly affect the body weight, the amount of feed was judged and fed every day so that there was no restriction in feeding. Lighting was changed every 12 hours, and water and food were freely available. Other matters related to animal breeding were performed in accordance with Chung-Ang University Animal Experiment Ethics Committee (National Association of Laboratory Animal Care) regulations.
상기 방법에 따라 준비된 마우스를 마취한 뒤 IAV를 1×103 pfu로 비강을 통해 감염시켰으며, APRG64(0.5㎎/㎏)을 IAV 감염 3일 전부터 감염 5일 후까지 총 8일 동안 경구투여하였다(도 10a). APRG64의 경구투여에 따른 항인플루엔자 효능을 평가하기 위해서 8일 동안의 APRG 경구 투여에 따른 마우스 체중 변화를 관찰하였다.After anesthetizing the mice prepared according to the above method, IAV was infected with 1×10 3 pfu through the nasal cavity, and APRG64 (0.5 mg/kg) was orally administered from 3 days before IAV infection to 5 days after infection for a total of 8 days. (Fig. 10a). In order to evaluate the anti-influenza effect of oral administration of APRG64, changes in body weight of mice following oral administration of APRG for 8 days were observed.
그 결과, 대조군에서는 IAV 감염 후부터 체중의 감소가 나타났지만, APRG64 경구 투여군에서는 대조군에 대비 체중 감소가 억제되는 것이 확인되었다(도 10b).As a result, in the control group, weight loss was observed after IAV infection, but in the APRG64 orally administered group, it was confirmed that weight loss was suppressed compared to the control group (FIG. 10b).
실험예 8-2. APRG64의 경구투여에 따른 마우스 사망 억제 효과 여부 확인Experimental Example 8-2. Confirmation of inhibitory effect on mouse death by oral administration of APRG64
동물 실험은 한국파스퇴르연구소 동물관리위원회에서 승인한 프로토콜(승인번호: IPK-21,003)에 따라 수행되었고, 6~7주 된 C57BL/6 수컷 마우스를 실험에 사용하으며, 경구 투여를 위해 마우스를 하루에 한 번 200㎕의 증류수 함유 APRG64(50㎎/㎏)로 위내 처리하였다. Animal experiments were carried out according to the protocol approved by the Animal Care Committee of the Institut Pasteur Korea (approval number: IPK-21,003), and 6-7 week old C57BL/6 male mice were used for the experiment. Intragastric treatment was performed once with 200 µl of distilled water-containing APRG64 (50 mg/kg).
APRG64를 마우스에 경구 투여하였을 때 IAV에 의한 사망 효과가 억제되는지를 확인하였다. 구체적으로, 상기와 같이 준비된 야생형 C57BL/6 마우스에 0, 25 또는 50㎎/㎏의 APRG64를 3일 동안 경구 투여한 후 1×103 pfu의 IAV로 감염시켰다. 또한, 마우스에 추가로 5일 동안 APRG64를 경구 투여하여 마우스 생존을 14일 동안 모니터링하였다.When APRG64 was orally administered to mice, it was confirmed whether the killing effect caused by IAV was suppressed. Specifically, wild-type C57BL/6 mice prepared as described above were orally administered with 0, 25 or 50 mg/kg of APRG64 for 3 days, and then infected with 1×10 3 pfu of IAV. In addition, APRG64 was orally administered to the mice for an additional 5 days, and mouse survival was monitored for 14 days.
그 결과, 마우스는 용량 의존적 방식으로 IAV에 의한 사망으로부터 유의하게 보호되는 것으로 확인되었다(도 10c). 구체적으로, APRG64를 투여하지 않은 마우스군은 10일에 모두 사망한 반면, APRG64 처리군은 14일 동안 모두 생존을 유지하였다. 특히, 50㎎/㎏의 APRG64를 투여한 마우스의 생존률은 80% 이상으로 나타나, IAV에 대한 항바이러스 효과가 현저히 우수한 것으로 확인되었다.As a result, it was confirmed that mice were significantly protected from IAV-induced death in a dose-dependent manner (FIG. 10c). Specifically, all of the mouse group not administered with APRG64 died on day 10, whereas all of the group treated with APRG64 maintained survival for 14 days. In particular, the survival rate of mice administered with 50 mg/kg of APRG64 was 80% or more, confirming that the antiviral effect against IAV was remarkably excellent.
실험예 8-4. APRG64의 경구투여에 따른 감염성 바이러스 입자 감소 효과 확인Experimental Example 8-4. Confirmation of the effect of reducing infectious virus particles according to oral administration of APRG64
APRG64의 경구투여에 따른 감염성 바이러스 입자 감소 효과 확인하기 위하여 마우스 폐의 바이러스 RNA, 사이토카인 및 감염성 바이러스 입자를 RT-qPCR 또는 플라크 분석으로 분석하였다. 구체적으로, 실험예 8-3의 경구 투여를 위해 준비된 야생형 C57BL/6 마우스에 50 mg/kg의 APRG64를 3일 동안 경구 처리한 다음, 마우스를 1×103 pfu의 IAV로 감염시킨 후, 5일 동안 추가적으로 동일한 양의 APRG64를 처리하였다. 5dpi에서 마우스 폐를 채취하여 바이러스 RNA, 역가 및 염증성 사이토카인의 생산을 분석하여 IAV의 M1 및 NP의 상대적 mRNA 발현 수준 및 감염성 바이러스 역가를 확인하였으며, 염증성 사이토카인인 IFN-γ, TNF-α 및 IL-6의 상대적 발현 수준을 RT-qPCR로 분석하였다.In order to confirm the effect of reducing infectious viral particles by oral administration of APRG64, viral RNA, cytokines, and infectious viral particles in mouse lungs were analyzed by RT-qPCR or plaque assay. Specifically, wild-type C57BL/6 mice prepared for oral administration of Experimental Example 8-3 were orally treated with 50 mg/kg of APRG64 for 3 days, and then the mice were infected with 1×10 3 pfu of IAV, and then 5 The same amount of APRG64 was additionally treated for 10 days. At 5 dpi, mouse lungs were collected and viral RNA, titers and production of inflammatory cytokines were analyzed to determine the relative mRNA expression levels and infectious virus titers of M1 and NP of IAV, and the inflammatory cytokines IFN-γ, TNF-α and The relative expression level of IL-6 was analyzed by RT-qPCR.
구분(내용)Classification (Content) 방향direction 서열order
서열번호 7(IFN-γ primer)SEQ ID NO: 7 (IFN-γ primer) forwardforward 5'-GGCCATCAGCAACAACATAAGCGT-3'5′-GGCCATCAGCAACAACATAAGCGT-3′
서열번호 8(IFN-γ primer)SEQ ID NO: 8 (IFN-γ primer) reversereverse 5'-TGGGTTGTTGACCTCAAACTTGGC-3'5′-TGGGTTGTTGACCTCAAACTTGGC-3′
서열번호 9(TNF-α primer)SEQ ID NO: 9 (TNF-α primer) forwardforward 5'-GCCTCTTCTCATTCCTGCTTG-3'5′-GCCTCTTCTCATTCCTGCTTG-3′
서열번호 10(TNF-α primer)SEQ ID NO: 10 (TNF-α primer) reversereverse 5'-CTGATGAGAGGGAGGCCATT-3'5′-CTGATGAGAGGGAGGCCATT-3′
서열번호 11(IL-6 primer)SEQ ID NO: 11 (IL-6 primer) forwardforward 5'-ACGGCCTTCCCTACTTCACA-3'5′-ACGGCCTTCCCTACTTCACA-3′
서열번호 12(IL-6 primer)SEQ ID NO: 12 (IL-6 primer) reversereverse 5'-CATTTCCACGATTTCCCAGA-3'5'-CATTTCCACGATTTCCCAGA-3'
그 결과, 바이러스 RNA(도 10d 왼쪽 및 중앙 그래프) 및 감염성 바이러스 입자(도 10d 오른쪽 그래프)는 5dpi에서 처리되지 않은 마우스의 폐와 비교하였을 때 APRG64 처리 마우스의 폐에서 유의하게 감소되는 것으로 나타났다. 또한, IAV 감염에 따라 유도된 IFN-γ, TNF-α 및 IL-6과 같은 전염증성 사이토카인의 발현은 APRG64 경구 투여에 의해 유의하게 억제되는 것으로 확인되었다(도 10e). 이와 같은 결과는 APRG64가 지질다당류(LPS)로 자극된 비장세포에서 전염증성 사이토카인의 발현을 감소시킨다는 시험관 내 사이토카인 발현 결과와 일치하였다.As a result, viral RNA (Fig. 10d left and center graphs) and infectious viral particles (Fig. 10d right graph) were significantly reduced in the lungs of APRG64-treated mice compared to the lungs of untreated mice at 5 dpi. In addition, it was confirmed that the expression of proinflammatory cytokines such as IFN-γ, TNF-α and IL-6 induced by IAV infection was significantly suppressed by oral administration of APRG64 ( FIG. 10e ). These results were consistent with the in vitro cytokine expression results that APRG64 reduced the expression of pro-inflammatory cytokines in lipopolysaccharide (LPS)-stimulated splenocytes.
상기의 실험에 따라 APRG64가 안전하면서도 현저히 우수한 효과를 나타내는 비강 및 경구 투여 가능한 약물로서 IAV 감염을 치료하는 데 사용될 수 있음이 종합적으로 시사되는 것이다.According to the above experiments, it is comprehensively suggested that APRG64 can be used to treat IAV infection as a drug that can be administered intranasally and orally, which is safe and exhibits remarkably excellent effects.
실험예 9. APRG64 유래 화합물의 분자도킹 시뮬레이션을 통한 인플루엔자 바이러스 억제능 평가Experimental Example 9. Influenza virus inhibitory ability evaluation through molecular docking simulation of APRG64-derived compounds
APRG64 유래 화합물에 대하여 인플루엔자 바이러스에 대해 분자도킹 시뮬레이션을 수행하여 바이러스 억제능을 평가하였다. A molecular docking simulation was performed on the influenza virus for the APRG64-derived compound to evaluate the virus inhibitory ability.
구체적으로, 인플루엔자 A/H1N1에 대하여 분자 모델링을 진행하였으며, 상기 화합물은 해당 바이러스에서 복제 및 전사에 영향을 주는 subunit viral RdRp (PA, PB1, PB2)과 연속적으로 결합한다. 따라서 본 발명에서는, subunit viral RdRp (PA, PB1, PB2)에 대해 blind docking으로 진행하였다. 특히, PB2의 경우 C-terminal domain, Middle domain 및 Cap binding domain에 대해 분자도킹 시뮬레이션을 수행하였다. 또한 인플루엔자 바이러스는 코로나바이러스 (SARS-CoV-2)의 스파이크 단백질과 마찬가지로 Receptor binding domain (RBD)이 존재한다. RBD; 및 RBD가 cell 내에 들어간 후, 갖고 있던 RNA를 세포 내에 내보내기 전에 세포막이 융합되는 과정에서 주요한 역할을 하는 곳인 A pocket;에 대하여 PDB 파일을 확보하여 분자도킹 시뮬레이션을 수행하였다. 도킹 결과는 Kcal/mol 단위의 결합 친화도의 형태로 얻어졌고, 도킹 과정은 별도의 10개의 컴퓨터 시스템에서 10번 되풀이하여 수행하였다. 결합 모드는 Discovery Studio 4.0을 이용하여 시각화하였고, 분자의 상호작용을 확인하기 위해 2차원 또는 3차원 이미지를 이용하였다. 그 결과는 도 11a 내지 도 11c에 나타내었다. 또한, 화합물의 도킹 후에 계산된 평균 결합 에너지 값을 표 4에 나타내었다.Specifically, molecular modeling was performed for influenza A/H1N1, and the compound continuously binds to the subunit viral RdRp (PA, PB1, PB2) that affects replication and transcription in the virus. Therefore, in the present invention, blind docking was performed for the subunit viral RdRp (PA, PB1, PB2). In particular, in the case of PB2, molecular docking simulation was performed for the C-terminal domain, middle domain, and Cap binding domain. Influenza virus also has a Receptor binding domain (RBD) like the spike protein of coronavirus (SARS-CoV-2). RBD; and A pocket, which plays a major role in the process of cell membrane fusion after RBD enters the cell and before exporting its RNA into the cell. PDB files were obtained and molecular docking simulation was performed. The docking result was obtained in the form of binding affinity in Kcal/mol unit, and the docking process was repeated 10 times on 10 separate computer systems. The binding mode was visualized using Discovery Studio 4.0, and 2D or 3D images were used to confirm molecular interactions. The results are shown in Figures 11a to 11c. Also, the average binding energy values calculated after docking of the compounds are shown in Table 4.
도 11a 내지 도 11c 및 표 4에 나타난 바와 같이, 3종의 APRG64 유래 화합물에서 인플루엔자 바이러스의 3차원 구조 기반 결합(docking)을 통하여 높은 인실리코 결합능을 보이는 것을 확인하였다. 그 중에서 화합물 9인 우르솔산이 가장 높은 억제능을 보이는 것을 확인하였다.As shown in FIGS. 11a to 11c and Table 4, it was confirmed that the three APRG64-derived compounds showed high in silico binding ability through docking based on the three-dimensional structure of influenza virus. Among them, it was confirmed that compound 9, ursolic acid, showed the highest inhibitory activity.
CompoundsCompounds Average Binding Affinity (Kcal/mol)Average Binding Affinity (Kcal/mol)
PAPA PB1PB1 PB2PB2 RBDRBD A pocketA pocket
C-terminal domainC-terminal domain Middle domainMiddle domain Cap binding domainCap binding domain
화합물 10compound 10 -8.93-8.93 -1.68-1.68 -5.55-5.55 -0.47-0.47 -3.86-3.86 -5.26-5.26 -5.57-5.57
화합물 9compound 9 -10.71-10.71 -6.47-6.47 -11.70-11.70 -8.55-8.55 -8.60-8.60 -10.33-10.33 -8.07-8.07
화합물 6compound 6 -6.01-6.01 -4.24-4.24 -6.08-6.08 -5.20-5.20 -6.52-6.52 -6.43-6.43 -9.45-9.45
상기 진술한 본 발명의 설명은 예시를 위한 것이며, 본 발명이 속하는 기술분야의 통상의 지식을 가진 자는 본 발명의 기술적 사상이나 필수적인 특징을 변경하지 않고서 다른 구체적인 형태로 쉽게 변형이 가능하다는 것을 이해할 수 있을 것이다. 그러므로 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적이 아닌 것으로 이해해야만 한다.The description of the present invention described above is for illustrative purposes, and those skilled in the art can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. There will be. Therefore, the embodiments described above should be understood as illustrative in all respects and not limiting.
선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방, 치료 또는 개선용 조성물은 인체에 부작용이 없어 안전성이 우수하면서도, 바이러스 의존적인 경로를 통한 항바이러스 활성이 뛰어나다. 이에 더하여, 생체 내에서도 항바이러스 효과가 확인되었을 뿐만 아니라, 혼합 추출물의 활성 성분 역시 현저히 우수한 항바이러스 활성을 가진다는 점에서 인플루엔자 바이러스 감염 질환의 예방, 치료 또는 개선용 조성물로 유용하게 활용될 수 있을 것으로 기대되는 바 본 발명의 산업상 이용가능성이 인정된다.A composition for preventing, treating, or improving influenza virus infection containing a mixture of an extract of Agrimonia pilosa and an extract of Galla rhois as an active ingredient has no side effects on the human body and is safe, while antiviral through a virus-dependent route activity is excellent. In addition, antiviral effects have been confirmed in vivo, and the active ingredients of the mixed extracts also have remarkably excellent antiviral activities, so it is expected that they can be usefully used as a composition for preventing, treating, or improving influenza virus infectious diseases. As expected, the industrial applicability of the present invention is recognized.

Claims (18)

  1. 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물.Seonhakcho ( Agrimonia pilosa ) Extract and nut gall ( Galla rhois ) A pharmaceutical composition for preventing or treating influenza virus infection comprising a mixture of an extract as an active ingredient.
  2. 제1항에 있어서, According to claim 1,
    상기 선학초 추출물 또는 상기 오배자 추출물은 물, C1 내지 C4의 알코올, 및 이들의 혼합용매로 이루어지는 군에서 선택된 하나 이상의 용매로 추출되는 것인 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물.The Seonhakcho extract or the nut gall extract is extracted with one or more solvents selected from the group consisting of water, C 1 to C 4 alcohol, and mixed solvents thereof. A pharmaceutical composition for preventing or treating influenza virus infection.
  3. 제1항에 있어서,According to claim 1,
    상기 선학초 추출물 또는 상기 오배자 추출물은 30 내지 95% 에탄올을 사용하여 추출된 것인 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물.The Seonhakcho extract or the nut gall extract is a pharmaceutical composition for preventing or treating influenza virus infection that is extracted using 30 to 95% ethanol.
  4. 제1항에 있어서, According to claim 1,
    상기 혼합물은 선학초 추출물 : 오배자 추출물의 중량비가 10:1 내지 1:10이 되도록 혼합되는 것인 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물.The mixture is a pharmaceutical composition for preventing or treating influenza virus infection that is mixed so that the weight ratio of the extract of Seonhakcho: the extract of gall nut is 10: 1 to 1: 10.
  5. 제1항에 있어서,According to claim 1,
    상기 인플루엔자 바이러스는 인플루엔자 A형 H1N1 바이러스인 것인 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물.The influenza virus is a pharmaceutical composition for preventing or treating influenza virus infection, which is an influenza A type H1N1 virus.
  6. 제1항에 있어서,According to claim 1,
    상기 혼합물은 인플루엔자 바이러스의 M1 또는 NP 유전자의 발현을 억제시키는 것인 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating influenza virus infection, wherein the mixture inhibits the expression of the M1 or NP gene of influenza virus.
  7. 하기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 하기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물:At least one active ingredient selected from the group consisting of a flavonoid compound represented by Formula 1 below, a triterpenoid compound represented by Formula 2 below, isomers thereof, and pharmaceutically acceptable salts thereof. A pharmaceutical composition for preventing or treating influenza virus infection comprising:
    [화학식 1][Formula 1]
    Figure PCTKR2022013577-appb-img-000011
    Figure PCTKR2022013577-appb-img-000011
    상기 화학식에서, In the above formula,
    R1은 H 또는 OH이고,R 1 is H or OH;
    R2는 α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, 및 OH로 이루어진 군으로 선택되는 어느 하나이고,R 2 is any one selected from the group consisting of α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, and OH;
    R3은 H 또는 β-D-glucuronosyl이고, R 3 is H or β-D-glucuronosyl;
    [화학식 2][Formula 2]
    Figure PCTKR2022013577-appb-img-000012
    .
    Figure PCTKR2022013577-appb-img-000012
    .
  8. 제7항에 있어서,According to claim 7,
    상기 플라보노이드(flavonoid) 화합물은 아프젤린(Afzelin), 아피제닌(Apigenin), 아피제닌 7-O-β-D-글루쿠로나이드(Apigenin 7-O-β-D-glucuronide), 아스트라갈린(Astragalin), 니코티플로린(Nicotiflorin), 케르세틴(Quercetin), 케르시트린(Quercitrin), 및 루틴(Rutin)으로 이루어진 군으로부터 선택되는 어느 하나 이상인 것인 인플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물.The flavonoid compounds include Afzelin, Apigenin, Apigenin 7-O-β-D-glucuronide, Astragalin ), nicotiflorin, quercetin, quercitrin, and rutin, and any one or more selected from the group consisting of influenza virus infection prevention or treatment pharmaceutical composition.
  9. 제7항에 있어서, According to claim 7,
    상기 트리테르펜(triterpenoid) 화합물은 우르솔산(Ursolic acid)인 것인 플루엔자 바이러스 감염증 예방 또는 치료용 약학적 조성물.The triterpenoid compound is ursolic acid (Ursolic acid), a pharmaceutical composition for preventing or treating influenza virus infection.
  10. 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물.A food composition for preventing or improving influenza virus infection, comprising a mixture of an extract of Seonhakcho ( Agrimonia pilosa ) and an extract of galla rhois ( Galla rhois ) as an active ingredient.
  11. 하기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 하기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 식품학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물: At least one active ingredient selected from the group consisting of a flavonoid compound represented by the following formula (1) or a triterpenoid compound represented by the following formula (2), isomers thereof, and food-acceptable salts thereof Influenza virus infection prevention or improvement food composition comprising:
    [화학식 1][Formula 1]
    Figure PCTKR2022013577-appb-img-000013
    Figure PCTKR2022013577-appb-img-000013
    상기 화학식에서, In the above formula,
    R1은 H 또는 OH이고,R 1 is H or OH;
    R2는 α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, 및 OH로 이루어진 군으로 선택되는 어느 하나이고,R 2 is any one selected from the group consisting of α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, and OH;
    R3은 H 또는 β-D-glucuronosyl이고, R 3 is H or β-D-glucuronosyl;
    [화학식 2][Formula 2]
    Figure PCTKR2022013577-appb-img-000014
    .
    Figure PCTKR2022013577-appb-img-000014
    .
  12. 제10항 또는 제11항에 있어서, According to claim 10 or 11,
    상기 식품 조성물은 건강 기능성 식품 조성물인 것인 인플루엔자 바이러스 감염증 예방 또는 개선용 식품 조성물.The food composition is a food composition for preventing or improving influenza virus infection that is a health functional food composition.
  13. 선학초(Agrimonia pilosa) 추출물과 오배자(Galla rhois) 추출물의 혼합물을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물.Seonhakcho ( Agrimonia pilosa ) Extract and nut gall ( Galla rhois ) A quasi-drug composition for preventing or inhibiting influenza virus infection, comprising a mixture of an extract as an active ingredient.
  14. 하기 화학식 1로 표시되는 플라보노이드(flavonoid) 화합물 또는 하기 화학식 2로 표시되는 트리테르펜(triterpenoid) 화합물, 이들의 이성질체, 및 이들의 약학적으로 허용 가능한 염으로 이루어지는 군으로부터 선택되는 어느 하나 이상을 유효성분으로 포함하는 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물:At least one active ingredient selected from the group consisting of a flavonoid compound represented by Formula 1 below, a triterpenoid compound represented by Formula 2 below, isomers thereof, and pharmaceutically acceptable salts thereof. A quasi-drug composition for preventing or inhibiting influenza virus infection comprising:
    [화학식 1][Formula 1]
    Figure PCTKR2022013577-appb-img-000015
    Figure PCTKR2022013577-appb-img-000015
    상기 화학식에서, In the above formula,
    R1은 H 또는 OH이고,R 1 is H or OH;
    R2는 α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, 및 OH로 이루어진 군으로 선택되는 어느 하나이고,R 2 is any one selected from the group consisting of α-L-rhamnopyranosyl, H, β-D-glucopyranosyl, rutinosyl, and OH;
    R3은 H 또는 β-D-glucuronosyl이고, R 3 is H or β-D-glucuronosyl;
    [화학식 2][Formula 2]
    Figure PCTKR2022013577-appb-img-000016
    .
    Figure PCTKR2022013577-appb-img-000016
    .
  15. 제13항 또는 제14항에 있어서, The method of claim 13 or 14,
    상기 의약외품은 소독청결제, 샤워폼, 가그린, 물티슈, 세제비누, 핸드워시, 및 연고제로 이루어지는 군에서 선택된 하나 이상인 것인 인플루엔자 바이러스 감염증 예방 또는 억제용 의약외품 조성물.The quasi-drug is a quasi-drug composition for preventing or inhibiting influenza virus infection, wherein the quasi-drug is at least one selected from the group consisting of disinfectant cleaner, shower foam, gargreen, wet tissue, detergent soap, hand wash, and ointment.
  16. 제1항 또는 제7항의 조성물을 이를 필요로 하는 개체에 투여하는 단계를 포함하는 인플루엔자 바이러스 감염증 예방, 치료 또는 억제 방법.A method for preventing, treating or inhibiting influenza virus infection comprising administering the composition of claim 1 or 7 to a subject in need thereof.
  17. 제1항 또는 제7항의 조성물의 인플루엔자 바이러스 감염증 예방, 치료, 개선, 또는 억제 용도.Use of the composition of claim 1 or 7 for prevention, treatment, improvement, or inhibition of influenza virus infection.
  18. 인플루엔자 바이러스 감염증 예방, 치료, 개선 또는 억제용 제제의 제조를 위한 제1항 또는 제7항의 조성물의 용도.Use of the composition of claim 1 or 7 for the manufacture of an agent for preventing, treating, ameliorating or inhibiting influenza virus infection.
PCT/KR2022/013577 2021-09-08 2022-09-08 Composition, for preventing, treating or relieving influenza virus infection, comprising mixture of agrimonia pilosa extract and galla rhois extract as active ingredient WO2023038480A1 (en)

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