WO2020175974A2 - 활성산소 제거를 위한 프러시안 블루/키토산 나노입자 복합체 및 이의 용도 - Google Patents

활성산소 제거를 위한 프러시안 블루/키토산 나노입자 복합체 및 이의 용도 Download PDF

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WO2020175974A2
WO2020175974A2 PCT/KR2020/095009 KR2020095009W WO2020175974A2 WO 2020175974 A2 WO2020175974 A2 WO 2020175974A2 KR 2020095009 W KR2020095009 W KR 2020095009W WO 2020175974 A2 WO2020175974 A2 WO 2020175974A2
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Prior art keywords
prussian blue
disease
chitosan
nanoparticle complex
active oxygen
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PCT/KR2020/095009
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English (en)
French (fr)
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WO2020175974A3 (ko
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최원일
오혜련
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한국세라믹기술원
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Publication of WO2020175974A2 publication Critical patent/WO2020175974A2/ko
Publication of WO2020175974A3 publication Critical patent/WO2020175974A3/ko

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6939—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being a polysaccharide, e.g. starch, chitosan, chitin, cellulose or pectin
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • 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
    • A61K8/58—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing atoms other than carbon, hydrogen, halogen, oxygen, nitrogen, sulfur or phosphorus
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • 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
    • A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
    • A61K8/73—Polysaccharides
    • A61K8/736—Chitin; Chitosan; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • 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
    • A23V2200/20—Ingredients acting on or related to the structure
    • A23V2200/25—Nanoparticles, nanostructures
    • 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
    • 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
    • A23V2250/50—Polysaccharides, gums
    • A23V2250/51—Polysaccharide
    • A23V2250/511—Chitin, chitosan
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/41—Particular ingredients further characterized by their size
    • A61K2800/413—Nanosized, i.e. having sizes below 100 nm
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/52—Stabilizers
    • A61K2800/522—Antioxidants; Radical scavengers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/57—Compounds covalently linked to a(n inert) carrier molecule, e.g. conjugates, pro-fragrances

Definitions

  • the present invention relates to the Prussian blue/chitosan nanoparticle complex for removing active oxygen and its use.
  • the Prussian blue/chitosan nanoparticle complex according to the present invention has excellent active oxygen removal ability and antibacterial properties, is stable, and is bioactive. Because of its excellent suitability, it can be effectively used in the development of antioxidants, pharmaceutical compositions, quasi-drug compositions, cosmetic compositions or food compositions.
  • Active oxygen that exist in nature exist in the form of substances that have unpaired electrons and react quickly with the main change compound.
  • the active oxygen generated in the human body has an oxidation/reduction action and an oxidation/reduction effect. It is known to be produced as a result of the metabolism of substances (1993, Drugs and Aging, 3, 60-80).
  • active oxygen generated in foods induces oxidation, which lowers the quality, and when ingested, it is known to be produced in the human body. It causes various diseases (2008, Carbohydrate polymers, 74, 953-956).
  • the generation of free radicals in food mainly causes rancidity by chain reaction with unsaturated fats present in foods, reducing the flavor of food.
  • the active oxygen (free radicals, free radicals) generated through various metabolisms and aging in the human body are usually balanced by the active oxygen-removing enzyme present in the body, but external factors such as fine dust and ultraviolet rays, and tumors It is abnormally increased due to internal factors such as inflammation and cardiovascular disease. This excessively produced active oxygen
  • active oxygen is a nucleic acid or
  • antioxidant vitamins retinol, tocophenol, etc.
  • gold nanoparticles platinum, vanadium, and cerium oxide nanoparticles.
  • Prussian blue nanoparticles are known as peroxidase (POD), By showing the functions of catalase (CAT), superoxide dismutase (SOD), etc.), the possibility to effectively remove active oxygen and to safely show antioxidant effects in the body was raised.
  • CAT catalase
  • SOD superoxide dismutase
  • Prussian blue nanoparticles have been approved by the US Food and Drug Administration (US FDA), have the ability to remove radioactive substances and are highly safe in vivo without cytotoxicity. Also, near-infrared (NIR) wavelength Because of its strong absorbance, Prussian blue nanoparticles themselves have low stability in the body and can easily cause aggregation, as well as photo-specific ablation agents, photoacoustic imaging contrast agents, and MRI contrast agents for cancer treatment. In that, organic substances (polyvinylpyrrolidone (PVP), polylactic acid (PLA),
  • This invention aims to provide an antioxidant containing Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the present invention also uses Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the present invention also uses Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the present invention also uses Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the present invention seeks to provide an antioxidant comprising a Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the chitosan nanoparticles have a molecular weight of 1 to 501 ⁇ .
  • the chitosan nanoparticles have a molecular weight of 101 ⁇ .
  • the Prussian blue/chitosan nanoparticle complex has a size of 1 to 500 11111. According to the second embodiment,
  • the present invention is to provide a pharmaceutical composition for preventing or treating diseases caused by excessive production of active oxygen, comprising a Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the molecular weight of the chitosan nanoparticles is characterized in that the molecular weight is 1 to 5 ⁇ .
  • the molecular weight of the chitosan nanoparticles is characterized by being 101 ⁇ .
  • the size of the nanoparticle complex is from 1 to 500 11111.
  • diseases caused by the overproduction of the active oxygen are stroke, Parkinson's disease, Alzheimer's disease, aging, heart disease, ischemia, arteriosclerosis, skin disease, inflammation, rheumatism, autoimmunity. It is characterized by disease, hyperlipidemia, liver disease, diabetes, cancer, chronic ulcer, burn or wound.
  • the pharmaceutical composition according to the present invention can be administered to a patient by a general oral or parenteral administration method, and may be in any form of solid or liquid.
  • the pharmaceutical composition according to the present invention may further contain one or more pharmaceutically acceptable liquid or solid carriers.
  • diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • the solid preparations may be tablets, pills, powders, granules, capsules, pellets, granules, or powders, and these solid preparations contain carriers, excipients and/or diluents in the complex.
  • lactose sucrose (Yeri, dextrose ((16X(X086)), 2020/175974 1»(:1 ⁇ 1 ⁇ 2020/095009 mannitol, malitol, sorbitol, xylitol, erythritol, starch, acacia rubber (acacia rubber),
  • the liquid formulation may be a solution, suspension or emulsion, and may contain various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to the commonly used simple diluent, liquid paraffin, etc.
  • a suspending agent it can be prepared by dispersing the finely divided active ingredient in a viscous substance such as natural or synthetic gum, resin, methyl cellulose, sodium carboxymethyl cellulose and a known suspending agent.
  • a viscous substance such as natural or synthetic gum, resin, methyl cellulose, sodium carboxymethyl cellulose and a known suspending agent.
  • Zeros include injections, drops, infusions, ointments, sprays, suspensions, emulsions, and suppositories.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations and suppositories.
  • Non-aqueous solvents and suspensions include
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used.
  • suppositories witepsol
  • Macrogol, cacao butter, laurin, glycerogelatin, etc. can be used.
  • the present invention is to provide a quasi-drug composition for preventing or treating diseases caused by excessive production of active oxygen, comprising a Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the chitosan nanoparticles have a molecular weight of 1 to 50 kDa.
  • the chitosan nanoparticles have a molecular weight of 10 kDa.
  • the size of the nanoparticle complex is characterized by being 1 to 500 nm.
  • diseases caused by the excess production of active oxygen are stroke, Parkinson's disease, Alzheimer's disease, aging, heart disease, ischemia, arteriosclerosis, skin disease, inflammation, rheumatism, autoimmune It is characterized by disease, hyperlipidemia, liver disease, diabetes, cancer, chronic ulcer, burn or wound.
  • the quasi-drug composition is sterilized.
  • It is characterized by being a cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.
  • the present invention is intended to provide a cosmetic composition for preventing or improving symptoms or diseases caused by active oxygen, comprising a Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the symptoms or diseases caused by the active oxygen are skin aging, wrinkles, skin pigmentation, atopy, acne, psoriasis, or eczema.
  • the molecular weight of the chitosan nanoparticles is characterized in that the molecular weight is 1 to 5 ⁇ .
  • the chitosan nanoparticles have a molecular weight of 101 ⁇ .
  • the size of the Prussian blue/chitosan nanoparticle composite is 1 to 500 11111.
  • the cosmetic may be prepared in the form of ampoules, creams, lotions, lotions, essences or packs.
  • dextrin ( ⁇ !) for easy preservation and handling.
  • the present invention seeks to provide a food composition containing the Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the molecular weight of the chitosan nanoparticles is characterized in that the molecular weight is 1 to 5 ⁇ .
  • the molecular weight of the chitosan nanoparticles is
  • the size of the Prussian blue/chitosan nanoparticle complex is 1 to 500 11111.
  • the above foods are meat, sausage, bread, chocolate, candy, snacks, sweets, pizza, ramen, other noodles, rice cakes, dairy products including ice cream, various soups, beverages, It is characterized by being a tea, drink, alcoholic beverage or vitamin complex.
  • the food composition includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, fectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal resection, stabilizers, Preservatives, glycerin, alcohol, may contain carbon used in carbonated beverages.
  • the composition of the present invention may contain the flesh for the manufacture of natural fruit juice, fruit or vegetable beverages. It can be used in combination or in combination.
  • the Prussian blue/chitosan nanoparticle complex according to the present invention has excellent active oxygen removal ability and antibacterial properties, is stable, and has excellent biocompatibility, so it is effective for the development of antioxidants, pharmaceutical compositions, quasi-drug compositions, cosmetic compositions or food compositions. It is expected to be used.
  • Figure 1 is the production of Prussian blue/chitosan nanoparticles (: NP) according to the present invention
  • FIG. 3 shows the results of evaluation of stability in the third distilled water of Wamo Xiannan NPs (Fig.
  • the present invention aims to provide an antioxidant comprising a Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the present invention also uses Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the present invention also uses Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the present invention also uses Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • the present invention also uses Prussian blue/chitosan nanoparticle complex as an active ingredient.
  • PB/Chi NPs PB/Chi NPs ⁇ r solution volume of acetone (99%, Sigma aldrich), and then freeze-dried for 3 days.
  • the sizes of PB/Chi NPs coated with chitosan having different molecular weights from PB NPs were analyzed using a Zetasizer (Nano-ZS, Malvern) equipment.
  • PB NPs without chitosan coating were produced in a size of about 30 nm.
  • the size increased from 30 nm to 60 nm, and partial precipitation of chitosan in the case of 50 kDa and WOkDa Occurred (Fig. 2). Therefore, it was confirmed that the chitosan types capable of stably producing PB/Chi NP among chitosan libraries ranged from 3 kDa to 20 kDa.
  • PB NP was partially precipitated after 3 weeks and PB/Chi3k was partially precipitated after 4 weeks.
  • PB/ChiWk NP did not change in size for 4 weeks, and PB/Chi20k NP did not precipitate, but the nanoparticle size gradually increased over 4 weeks. Therefore, PB/ChiWk NP was the optimal group with the best stability in aqueous solution. On the other hand, PB NP and PB/Chi3k NP were also immediately precipitated when dispersed in PBS, so that the size change of the nanoparticles was analyzed with PB/ChilOk NP and PB/Chi20k NP.
  • antioxidant power was calculated according to the following formula. As a result, it was confirmed that the antioxidant power of itself (0 2 ⁇ -active oxygen removal ability) was about 20%, while the antioxidant power was significantly increased when echitosan (3, 10, 2 ⁇ ) was coated. Among them, PB It was confirmed that the antioxidant power of /ChiWk NP was the most excellent (Fig. 5).
  • the active oxygen removal capacity was calculated according to the following consciousness.
  • the cells were cultured in an environment of 37 O C and 5% CO 2 for 12 hours.
  • Cells were treated with different concentrations of PB/ChilOk NP solution (1000, 100, 50, 20, 10 or g/ml) for 24 hours.
  • PB/ChilOk is excellent in biocompatibility because it does not cause cytotoxicity even at a high concentration of lmg/ml (Fig. 7).
  • Cells (NIH 3T3) were dispensed with 1 ⁇ 10 4 cells per well in a 96-well plate and grown for 12 hours at 37°C, 5% CO 2. Cells were grown in lmg/ml chitosan (10kDa) and PB. Each of the /ChilOk NP solutions was treated, and H 2 0 2 (5 ⁇ iM) or LPS (5ng/ml) was treated, followed by incubation for 8 hours. After washing the cells with PBS, H 2 DCFDA (1 child) was added and incubated for 90 minutes. Fluorescence (ex/450nm, em/525nm) was immediately determined to analyze the intracellular active oxygen radical.

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Abstract

본 발명은 활성산소 제거를 위한 프러시안 블루/키토산 나노입자 복합체 및 이의 용도에 관한 것이다. 본 발명에 따른 프러시안 블루/키토산 나노입자 복합체는 활성산소제거 능력 및 항균성이 뛰어나고 안정적이며 생체적합성이 우수하므로, 항산화제, 약제학적 조성물, 의약외품 조성물, 화장료 조성물 또는 식품 조성물의 개발에 효과적으로 사용될 수 있다.

Description

2020/175974 1»（：1^1{2020/095009 명세서
발명의명칭:활성산소제거를위한프러시안블루/키토산
나노입자복합체및이의용도
기술분야
[1] 본발명은활성산소제거를위한프러시안블루/키토산나노입자복합체및 이의용도에관한것이다.본발명에따른프러시안블루/키토산나노입자 복합체는활성산소제거능력및항균성이뛰어나고안정적이며생체적합성이 우수하므로,항산화제 ,약제학적조성물,의약외품조성물,화장료조성물또는 식품조성물의개발에효과적으로사용될수있다.
四
배경기술
[3] 자연계에존재하는활성산소 (자유라디칼, free radicals)는짝지어지지않은 전자를가지는물질의형태로존재하여주변화합물과빠르게반응하는 물질이다.인체에서발생하는활성산소는산화/환원작용과물질의대사작용의 결과로생성되는것으로알려져있다 (1993, Drugs and Aging, 3, 60-80).또한, 식품에서발생된활성산소는산화작용을유발하여품질을저하시키고이를 섭취하면인체내에서각종질병을유발한다 (2008, Carbohydrate polymers, 74, 953-956).식품에서활성산소의발생은주로식품내에존재하는불포화지방과 연쇄반응하여산패를유발하여식품의풍미 (Flavor)를저하시켜식품의가치를 하락시키며일단활성산소로인한산화작용이일어나면지속적으로불포화 지방산을산화시켜연쇄반응을유발해서산화에의한산패를지속시킨다.천연 식품에존재하는비타민 C와 E,그리고다양한페놀화합물과같은항산화 물질들은식품에존재하는활성산소의억제를유발한다 (2009, Food Chemistry,
114, 881-888).이러한활성산소제거기능을하는물질들의섭취는체내에서 노화나소화혹은대사를통해생산되는활성산소를제거하는기능을한다.
[4] 인체내에서각종대사와노화를통해발생하는활성산소 (자유라디칼, free radicals)는체내에존재하는활성산소제거효소에의해보통균형을이루고 있지만미세먼지나자외선등의외부요인및종양이나염증,심혈관질환등의 내부요인에의해비이상적으로증가한다.이과잉생성된활성산소는
단백질 (Proteins),지방 (lipids)과반응하여노화 (Aging)를촉진시키고 (2002, Archives of Biochemistry and Biophysics, 379, 354-9),빈혈,당뇨또는혈관수죽성 심장질환같은만성질환을유발한다. (2007, Biochemistry(Moscow), 2, 809-827) 또한활성산소 (프리라디칼, free radicals)는핵산 (Nucleic acid)이나
염색체 (Chromosome)의산화를유발하여생명에치명적인각종암을발생시키는 주요요인중의하나로알려져 있다. (2003, Biol.Pharm. Bull. 26, 1129-1134). 이러한이유로체내에서과잉발생하는활성산소의제거는노화를비롯한각종 2020/175974 1»（：1^1{2020/095009 만성질병과생명에치명적인암의 예방의필수불가결한것이다.
[5]
[6] 효과적으로활성산소들을제거할수있는물질들은이미많이개발되어왔다. 대표적인예로는항산화제인비타민계열 (retinol, tocophenol등),금나노입자, 플래티넘,바나듐，산화세륨나노입자들이있다.최근에는프러시안블루 나노입자 (Prussian blue nanoparticle)들이항산화효소 (peroxidase (POD), catalase (CAT), superoxide dismutase (SOD)등)의기능을보여줌으로써활성산소들을 효과적으로제거하고생체내에서안전하게항산화효능을보여줄수있는 가능성이제기됐다.
[7] 프러시안블루나노입자들은미국식약처 (US FDA)의승인을받은물질로 방사능물질들을제거할수있는기능을가지고있으며세포독성이전혀없는 생체내안전성이높은물질이다.또한근적외선 (NIR)파장에서강한흡광도를 보임으로써암치료를위한 photo比 lermal ablation agent와 photoacoustic imaging contrast agent, MRI조영제로조명되고있다.하지만프러시안블루나노입자 자체는생체내안정성이낮아쉽게뭉침현상 (aggregation)이발생할수있어서, 유기물질들을 (polyvinylpyrrolidone (PVP), polylactic acid(PLA),
polydiallyldime比 lylammonium chloride(PDDA)등) template로첨가해구조적인 안정성을향상시키는연구들이많이진행되고있다.
[8]
발명의상세한설명
기술적과제
[9] 이에본발명자들은과학기술정보통신부기본연구지원사업지원하에
(과제고유번호: 2018R1D1A1B07043620,과제명 :효과적인종양진단및치료가 동시에가능한신규페로신기반테라그노시스나노입자개발)프러시안블루 나노입자들의안정성을향상시킬수있을뿐아니라항산화효능을증가시킬수 있는키토산을 template로사용하여프러시안블루/키토산나노입자 (Prussian blue/chitosan nanoparticle, PB/Chi NP)복합체를제조하고,이의활성산소제거 효과를확인함으로써본발명을완성하였다.본발명에따른프러시안 블루/키토산나노입자복합체의제조방법에대한모식도를도 1에나타내었다.
[1이 본발명은프러시안블루/키토산나노입자복합체를유효성분으로포함하는 항산화제를제공하는것을목적으로한다.
[11] 본발명은또한프러시안블루/키토산나노입자복합체를유효성분으로
포함하는약제학적조성물을제공하는것을목적으로한다.
[12] 본발명은또한프러시안블루/키토산나노입자복합체를유효성분으로
포함하는의약외품조성물을제공하는것을목적으로한다.
[13] 본발명은또한프러시안블루/키토산나노입자복합체를유효성분으로
포함하는화장료조성물을제공하는것을목적으로한다. 2020/175974 1»（：1/10公020/095009 본발명은또한프러시안블루/키토산나노입자복합체를유효성분으로 포함하는식품조성물을제공하는것을목적으로한다. 과제해결수단
제 1구현예에따르면,
본발명은프러시안블루/키토산나노입자복합체를유효성분으로포함하는 항산화제를제공하고자한다.
8] 본발명에따른항산화제에 있어서,상기키토산나노입자의분자량은 1내지 501止^인것을특징으로한다.
본발명에따른항산화제에 있어서,상기키토산나노입자의분자량은 101止 인 것을특징으로한다.
본발명에따른항산화제에 있어서 ,상기프러시안블루/키토산나노입자 복합체의크기는 1내지 500 11111인것을특징으로한다. 제 2구현예에따르면,
본발명은프러시안블루/키토산나노입자복합체를유효성분으로포함하는 활성산소과잉생성으로인해유발되는질환의 예방또는치료용약제학적 조성물을제공하고자한다.
본발명에따른약제학적조성물에있어서,상기키토산나노입자의분자량은 1 내지 5아止^인것을특징으로한다.
본발명에따른약제학적조성물에있어서,상기키토산나노입자의분자량은 101止^인것을특징으로한다.
본발명에따른약제학적조성물에있어서,상기프러시안블루/키토산
]]]] ]] ]]]] ]]] 나노입자복합체의크기는 1내지 500 11111인것을특징으로한다.
2479047356561
22222 2221 본발명에따른약제학적조성물에있어서,상기활성산소과잉생성으로인해 유발되는질환은뇌졸중,파킨슨병,알츠하이머병,노화,심장질환,허혈, 동맥경화,피부질환,염증,류마티스,자가면역질환,고지혈증,간질환,당뇨병 , 암,만성궤양,화상또는창상인것을특징으로한다.
[281 본발명에따른약제학적조성물은일반적인경구또는비경구투여방법으로 환자에게투여될수있으며 ,고체또는액체형태어떠한형태로도가능하다. 또한,본발명에따른약제학적조성물은약제학적으로허용가능한 1종이상의 액체또는고체담체를더포함할수있다.또한보통사용하는충진제,증량제, 결합제,습윤제,붕해제,계면활성제등의희석제또는부형제를사용하여 조제될수있다.상기고체형태의제제에는정제 ,환제 ,산제，과립제,캡슐제 , 펠렛제 ,세립제또는분제일수있으며 ,이러한고형제제는상기복합체에담체 , 부형제및/또는희석제를첨가하여조제할수있다.상기담체,부형제및/또는 희석제로는락토오스 수크로오스 ( 예리,덱스트로오스 ((16X(X086), 2020/175974 1»（：1^1{2020/095009 만니톨 (mannitol),말리톨 (malitol),소르비톨 (sorbitol),자일리톨 (xylitol), 에리트리톨 (eri仕 iritol),전분 (starch),아카시아고무 (acacia rubber),
알지네이트 (alginate),젤라틴 (gelatin),칼슘포스페이트 (calcium phosphate),칼슘 실리케이트 (calcium silicate),셀룰로오스 (cellulose),쓸리비닐피롤리돈 (polyvinyl pyrrolidone),마그네슘스테아레이트 (magnesium stearate)및광물유등이 있다. 상기 액체형태의 제제는용액,현탁액또는유탁액일수있으며,흔히사용되는 단순희석제인물,리퀴드파라핀이외에 여러가지부형제,예를들면습윤제, 감미제,방향제,보존제등이포함될수있다.경구용으로적당한수성
현탁제로는미분된활성성분을천연또는합성검,수지,메틸셀룰로오스 (methyl cellulose),소디움카르복시메틸셀룰로오스 (Sodium carboxymethyl cellulose)및 공지의 현탁제와같은점성물질에분산시켜제조될수있다.비경구투여제로는 주사제,점적제,수액,연고,스프레이제,현탁제,유제,좌제등을들수있다. 비경구투여를위한제제에는멸균된수용액,비수성용제 ,현탁제，유제, 동결건조제제및좌제가포함된다.비수성용제,현탁제로는
프로필렌글리콜 (propylene glycol),쓸리에틸렌글리콜 (polyethylene glycol), 올리브오일과같은식물성오일,에틸올레이트 (ethyl oleate)와같은주사가능한 에스테르등이사용될수있다.좌제의 기제로는위텝솔 (witepsol),
마크로골 (macrogol),카카오지,라우린지,글리세로젤라틴 (glycerogelatin)등이 사용될수있다.
[29]
[3이 제 3구현예에따르면,
[31] 본발명은프러시안블루/키토산나노입자복합체를유효성분으로포함하는 활성산소과잉생성으로인해유발되는질환의 예방또는치료용의약외품 조성물을제공하고자한다.
[32] 본발명에 따른의약외품조성물에 있어서 ,상기 키토산나노입자의분자량은 1 내지 50kDa인것을특징으로한다.
[33] 본발명에 따른의약외품조성물에 있어서 ,상기 키토산나노입자의분자량은 10kDa인것을특징으로한다.
[34] 본발명에 따른의약외품조성물에 있어서 ,상기프러시안블루/키토산
나노입자복합체의크기는 1내지 500 nm인것을특징으로한다.
[35] 본발명에 따른의약외품조성물에 있어서 ,상기 활성산소과잉생성으로인해 유발되는질환은뇌졸중,파킨슨병,알츠하이머병,노화,심장질환,허혈, 동맥경화,피부질환,염증,류마티스,자가면역질환,고지혈증,간질환,당뇨병, 암,만성궤양,화상또는창상인것을특징으로한다.
[36] 본발명에 따른의약외품조성물에 있어서 ,상기 의약외품조성물은소독
청결제,샤워폼,가그린,물티슈,세제비누,핸드워시,가습기충진제,마스크, 연고제또는필터충진제인것을특징으로한다.
[37] 2020/175974 1»（：1^1{2020/095009
[38] 제 4구현예에따르면,
[39] 본발명은프러시안블루/키토산나노입자복합체를유효성분으로포함하는 활성산소로유발되는증상또는질환의 예방또는개선용화장료조성물을 제공하고자한다.
[4이 본발명에 따른화장료조성물에 있어서 ,상기 활성산소로유발되는증상또는 질환은피부노화,주름생성,피부색소침착,아토피,여드름,건선또는습진인 것을특징으로한다.
[41] 본발명에 따른화장료조성물에 있어서 ,상기 키토산나노입자의분자량은 1 내지 5아止^인것을특징으로한다.
[42] 본발명에 따른화장료조성물에 있어서 ,상기 키토산나노입자의분자량은 101止^인것을특징으로한다.
[43] 본발명에 따른화장료조성물에 있어서 ,상기프러시안블루/키토산나노입자 복합체의크기는 1내지 500 11111인것을특징으로한다.
[44] 본발명에 따른화장료조성물에 있어서,상기 화장료는엠플,크림,로션, 화장수,에센스또는팩의 형태로제조될수있다.또한,보존이나취급을 용이하게하기위하여 덱스트린（（^ !!）,사이클로덱스트린仁） 0（16 11）등의 통상제제화에사용되는캐리어 ,그밖의 임의의조제를부가하여도좋다.
[45]
[46] 제 5구현예에따르면,
[47] 본발명은프러시안블루/키토산나노입자복합체를유효성분으로포함하는 식품조성물을제공하고자한다.
[48] 본발명에 따른식품조성물에 있어서 ,상기 키토산나노입자의분자량은 1 내지 5아止^인것을특징으로한다.
[49] 본발명에 따른식품조성물에 있어서 ,상기 키토산나노입자의분자량은
101止^인것을특징으로한다.
[5이 본발명에 따른식품조성물에 있어서 ,상기프러시안블루/키토산나노입자 복합체의크기는 1내지 500 11111인것을특징으로한다.
[51] 본발명에 따른식품조성물에 있어서,상기식품은육류,소세지,빵,쵸코렛, 캔디류,스넥류,과자류,피자,라면,기타면류,낌류,아이스크림류를포함한 낙농제품,각종스프,음료수,차,드링크제,알콜음료또는비타민복합제인 것을특징으로한다.
[52] 본발명에 따른식품조성물에 있어서 ,상기식품조성물은여러가지 영양제 , 비타민,전해질,풍미제,착색제,펙트산및그의 염,알긴산및그의 염,유기산, 보호성콜로이드증 절제，안정화제，방부제，글리세린,알콜,탄산 음료에사용되는탄 함유할수있다.그밖에본발명의조성물은 천연과일쥬스,과일 또는야채음료의제조를위한과육을함유할수 있다.이러한성분은
Figure imgf000007_0001
로또는조합하여사용할수있다.
[53] 2020/175974 1»（：1^1{2020/095009 발명의효과
[54] 본발명에 따른프러시안블루/키토산나노입자복합체는활성산소제거 능력 및항균성이뛰어나고안정적이며 생체적합성이우수하므로,항산화제 , 약제학적조성물,의약외품조성물,화장료조성물또는식품조성물의 개발에 효과적으로사용될수있을것으로기대된다.
[55]
도면의간단한설명
[56] 도 1은본발명에 따른프러시안블루/키토산나노입자 （： NP）의제조
방법에 대한모식도를나타낸다.
Figure imgf000008_0001
크기를분석한결과를나타낸다（도 2에서부분적으로침전된나노입자그룹은
’▲’로표시하였다）
[58] 도 3은 와모 셌난 NPs의 3차증류수내안정성 평가결과를나타낸다（도
3에서부분적으로침전된나노입자의그룹은’▲’로표시하였다.）
[59] 내안정성 평가결과를나타낸다
[6이
Figure imgf000008_0002
항산화력（0 2· -활성산소제거 능력）을 평가한결과를나타낸다.
[61]
Figure imgf000008_0003
항산화력（0比활성산소제거능력）을 평가한결과를나타낸다.
[62] 세포독성 결과를나타낸다.
[63]
Figure imgf000008_0004
활성산소라디칼제거능평가결과를나타낸다.
[64]
발명의실시를위한최선의형태
[65] 본발명은프러시안블루/키토산나노입자복합체를유효성분으로포함하는 항산화제를제공하는것을목적으로한다.
[66] 본발명은또한프러시안블루/키토산나노입자복합체를유효성분으로
포함하는약제학적조성물을제공하는것을목적으로한다.
[67] 본발명은또한프러시안블루/키토산나노입자복합체를유효성분으로
포함하는의약외품조성물을제공하는것을목적으로한다.
[68] 본발명은또한프러시안블루/키토산나노입자복합체를유효성분으로
포함하는화장료조성물을제공하는것을목적으로한다.
[69] 본발명은또한프러시안블루/키토산나노입자복합체를유효성분으로
포함하는식품조성물을제공하는것을목적으로한다.
이
발명의실시를위한형태
1] 이하,발명의 이해를돕기위해다양한실시예를제시한다.하기실시예는
발명을보다쉽게 이해하기위하여 제공되는것일뿐발명의보호범위가하기 2020/175974 1»（：1^1{2020/095009 실시예에한정되는것은아니다.
2]
3] ＜실시예＞
[74] 심시예 1.프러시아블루/키토산나노입자 (PB/Chi NP)의제조
[75] 키토산 (150mg)을 3ml의 3차증류수 (deionized water)에녹여준뒤 potassium
ferricyanide(8.5mg, 5mM) lml첨가하여상온에서 30분동안 450rpm으로 stirring하며반응시켰다. 550rpm으로 stirring하고있는반응용액에 iron chloride tetrahydrate(5mg, 5mM) lml을천천히 dropwise해준뒤 1시간동안반응시켰다. 키토산의분자량별 (DAC90; 3, 10, 20, 50, 100 kDa)로위의방법을반복해 복합체를제조하였다.반응중생성되는 KC1을제거하기위해 PB NP는 ultrafiltration(Amicon Ultra- 15 filter)을진행하고 PB/Chi NPs^r용액부피 4배의 acetone(99%, Sigma aldrich)으로침전시킨뒤, 3일간동결건조하였다. PB NP와 분자량이다른키토산이코팅된 PB/Chi NPs의크기를 Zetasizer(Nano-ZS, Malvern)장비를이용하여분석하였다.
[76] 그결과,키토산이코팅되지않은 PB NP는 30nm정도의크기로제조되었으며 , 분자량이높은키토산이코팅될수록크기가 30nm에서 60nm까지증가하면서 키토산의분자량이 50kDa및 WOkDa인경우부분적인침전이발생하였다 (도 2). 따라서,키토산라이브러리중 PB/Chi NP를안정적으로제조할수있는키토산 종류는 3kDa에서 20kDa까지인것으로확인되었다.
7]
8] ＜실험예＞
[79] 심험예 1.프러시아블루/키토산나노입자의아정성평가
[8이 동결건조된상기실시예 1에서제조된프러시안블루/키토산나노입자를 3차 증류수및 phosphate-buffered saline (PBS)에분산후 37°C, WOrpm에서안정성 평가를진행하였다.이때,상기실시예 1에서불안정하게제조된것으로확인된 PB/Chi50k NP와 PB/Chil00k NP는제외하였다.시간별로 3차증류수에서나노 입자의안정성을확인하기위해서 0일, 1일, 3일, 7일, 2주, 3주, 4주에나노 입자의크기및색상변화를분석하였다.
[81] 그결과, PB NP는 3주뒤부분적으로침전되었고 PB/Chi3k는 4주뒤
부분적으로침전되어서안정성이떨어지는것으로나타났다. PB/ChiWk NP는 4주동안크기변화가없었고 PB/Chi20k NP는침전되진않았지만나노입자 크기가 4주에걸쳐점차증가하는것으로나타났다.따라서,수용액내의 안정성이가장뛰어난최적화그룹은 PB/ChiWk NP임이확인되었다 (도 3).한편, PB NP와 PB/Chi3k NP는 PBS에분산시켰을때도바로침전되어서 PB/ChilOk NP와 PB/Chi20k NP로만나노입자의크기변화를분석하였다. PB/ChilOk NP의 경우 2주동안크기변화가거의없었지만 PB/Chi20k NP의경우 2주뒤에나노 입자의크기가확연히증가하였다.따라서 , PB/ChilOk NP가가장안정적으로 제조될수있음이확인되었다 (도 4) 2020/175974 1»(：1/10公020/095009
심험예 2.프러시아를루/키토산나노입자의활성산소제거능평가
2.1 DPPH assay
키토산, PB NP와 PB/Chi NPs (10mg/ml)를 1ml의 3차증류수에녹여준뒤 96-well plate에 150 \xl분주하였다. DPPH용액 (2mg, 0.5mM)을 methanol 10ml°1] 제조한뒤나노입자용액을분주한 wells에 150 를분주하였다.상온에서 30분간어둠속에서반응시켜준후 absorbance分 =517nm)를측정하였다. DPPH 없이 methanol에서의 PB 와 PB/Chi 요의 absorbance도즉정하여 표의
background absorbance를배제시키고활성산소제거능을평가하였다.한편, ascorbic acidQOmg/ml)를 positive contr이로사용하였다.즉정된 absorbance를 바탕으로하기의식에따라항산화력을계산하였다.
Figure imgf000010_0001
그결과, 자체의항산화력 (0 2· -활성산소제거능력)은약 20%인반면, 에키토산 (3, 10, 2此 )을코팅한경우항산화력이현저히증가하는 것으로확인되었다.그중에서, PB/ChiWk NP의항산화력이가장우수한것으로 확인되었다 (도 5).
[89]
[9이 2.2 Hydroxyl radical assay
[91] 키토산, PB NP와 PB/ChilOk NPs용액 (3mg/ml)을준비하였다. 15ml tube에
0.1ml EDTA(O.lmM), 0.1ml FeCB(O.lmM), 0.1ml H 20 2(1 mM), 0.1ml
2-deoxy-D-ribose(3.75 mM), lml나노입자, 0.5ml phosphate buffer(20 mM)와 0.1ml
] ] ] ] ] ]
24 7358 ascorbic acid(O.lmM)를넣어주었다.하나는 H 20 2와나노입자대신 3차
9888888
rL rL rL rL992451
증류수를,또다른하나는나노입자대신 3차증류수를넣어 control과 blank를 준비하였다.그후, 1시간동안 37OC, lOOrpm에서반응시켰다.그다음, 15 ml NaOH(0.05 M)에녹인 2-thiobarbituric acid(0.15g, TBA)와 15ml 3차증류수에녹인 trichloroacetic acid(0.3g, TCA)를 lml씩반응용액에첨가하고 85°C의 water bath에서 20분간중탕하였다.무색이던반응용액이분홍색으로변하면 96-well plate에 300[xl씩분주하고 absorbance (X=535nm)를즉정하였다.즉정된
absorbance를바탕으로하기의식에따라활성산소제거능력을계산하였다.
[93]
Figure imgf000010_0002
그결과, PB NP,키토산과 PB/Chi NP그룹모두항산화력 (OH·활성산소제거 능력)이 positive control인 ascorbic acid (약 25%)보다높은것으로확인되었다. PB 2020/175974 1»（：1^1{2020/095009
NP의 경우약 30%의 항산화력을나타낸반면, PB 에 키토산 Chi3k, ChilOk및 Chi20k을코팅한경우모두 95%이상의항산화력이 있는것으로확인되었다 (도
6).
[96]
[97] 심험예 3.프러시아블루/키토산나노입자의세포독성 평가
[98] 세포 (Raw 264.7 macrophage)를 96-well plate에 한 well당세포수 1X10 4씩
분주하고 12시간동안 37OC, 5% C0 2환경에서 배양하였다.세포에다른농도의 PB/ChilOk NP용액 (1000, 100, 50, 20, 10나 g/ml)을처리하고 24시간
incubation하였다.세포생존율은 cck-8분석법을통해 absorbance (X=450nm)에서 측정하였다.
[99] 그결과, PB/ChilOk 는고농도인 lmg/ml에서도세포독성을일으키지 않기 때문에 생체적합성이우수한물질임이 입증되었다 (도 7).
0이
[101] 심험예 4.프러시아블루/키토산나노입자의세포내활성산소라디칼제거능 평가
[102] 세포 (NIH 3T3)를 96-well plate에 한 well당세포수 1X10 4씩분주하고 12시간 동안 37°C, 5% CO 2환경에서키웠다.세포에 lmg/ml의 키토산 (10kDa)및 PB/ChilOk NP용액을각각처리하고 H 20 2 (5^iM)혹은 LPS (5ng/ml)를처리후 8시간 incubation하였다. PBS로세포를 washing해준뒤, H 2DCFDA (1아 )를 첨가하고 90분 incubation하였다.세포내활성산소라디칼을분석하기위해 형광도 (ex/450nm, em/525nm)를즉정하였다.
[103] 그결과,키토산과 PB/ChilOk 를처리했을때각각 10과 30%의
활성산소라디칼이 제거능이 있음이 확인되었다.따라서 PB와키토산복합체가 세포를 oxidative damage로부터보호해줄수있음이 입증되었다 (도 8).
[104]
[105] 이상으로본발명내용의특정한부분을상세히기술하였는바,당업계의
통상의지식을가진자에게 있어서 이러한구체적 기술은단지 바람직한 실시태양일뿐이며,이에의해본발명의 범위가제한되는것이아닌점은 명백할것이다.따라서,본발명의실질적인범위는첨부된청구항들과그것들의 등가물에 의하여정의된다고할것이다.

Claims

2020/175974 1»（：1/10公020/095009 청구범위
[청구항 1] 프러시안블루/키토산나노입자복합체를유효성분으로포함하는
항산화제.
[청구항 2] 제 1항에 있어서,
상기키토산나노입자의분자량은 1내지 50吐¾인것을특징으로하는 것인,항산화제.
[청구항 3] 제 1항에 있어서,
상기프러시안블루/키토산나노입자복합체의크기는 1내지 500 11111인 것을특징으로하는것인,항산화제.
[청구항 4] 프러시안블루/키토산나노입자복합체를유효성분으로포함하는
활성산소과잉생성으로인해유발되는질환의 예방또는치료용 약제학적조성물로서,
상기활성산소과잉생성으로인해유발되는질환은뇌졸중,파킨슨병, 알츠하이머병,노화,심장질환,허혈,동맥경화,피부질환,염증,류마티스, 자가면역질환,고지혈증,간질환,당뇨병 ,암,만성궤양,화상또는창상인 것을특징인것을특징으로하는것인,약제학적조성물.
[청구항 5] 프러시안블루/키토산나노입자복합체를유효성분으로포함하는
활성산소과잉생성으로인해유발되는질환의 예방또는치료용 의약외품조성물로서 ,
상기활성산소과잉생성으로인해유발되는질환은뇌졸중,파킨슨병, 알츠하이머병,노화,심장질환,허혈,동맥경화,피부질환,염증,류마티스, 자가면역질환,고지혈증,간질환,당뇨병 ,암,만성궤양,화상또는창상인 것을특징인것을특징으로하는것인,의약외품조성물.
[청구항 6] 제 5항에 있어서,
상기의약외품조성물은소독청결제 ,샤워폼,가그린,물티슈,세제비누, 핸드워시,가습기충진제,마스크,연고제또는필터충진제인것을 특징으로하는것인,의약외품조성물.
[청구항 7] 프러시안블루/키토산나노입자복합체를유효성분으로포함하는
활성산소로유발되는증상또는질환의 예방또는개선용화장료 조성물로서,
상기활성산소로유발되는증상또는질환은피부노화,주름생성, 피부색소침착,아토피,여드름,건선또는습진인것을특징으로하는 것인,화장료조성물.
[청구항 8] 제 7항에 있어서,
상기화장료는엠플,크림,로션,화장수,에센스또는팩인것을특징으로 하는것인,화장료조성물.
[청구항 9] 프러시안블루/키토산나노입자복합체를유효성분으로포함하는식품 2020/175974 1»（：1/10公020/095009 조성물.
[청구항 10] 제 9항에 있어서,
상기식품은육류,소세지,빵,쵸코렛,캔디류,스넥류,과자류,피자,라면, 기타면류,낌류,아이스크림류를포함한낙농제품,각종스프,음료수,차, 드링크제,알콜음료또는비타민복합제를특징으로하는것인,식품 조성물.
PCT/KR2020/095009 2019-02-26 2020-02-25 활성산소 제거를 위한 프러시안 블루/키토산 나노입자 복합체 및 이의 용도 WO2020175974A2 (ko)

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