WO2021080357A1 - Antibiotic silver nanoparticles using tomato extract, and method for producing same - Google Patents

Antibiotic silver nanoparticles using tomato extract, and method for producing same Download PDF

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Publication number
WO2021080357A1
WO2021080357A1 PCT/KR2020/014523 KR2020014523W WO2021080357A1 WO 2021080357 A1 WO2021080357 A1 WO 2021080357A1 KR 2020014523 W KR2020014523 W KR 2020014523W WO 2021080357 A1 WO2021080357 A1 WO 2021080357A1
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Prior art keywords
silver nanoparticles
tomato extract
tomatoes
present
tomato
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PCT/KR2020/014523
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French (fr)
Korean (ko)
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김성욱
최정수
이민우
신운철
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고려대학교 산학협력단
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Priority claimed from KR1020200137373A external-priority patent/KR20210048428A/en
Publication of WO2021080357A1 publication Critical patent/WO2021080357A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/26—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests in coated particulate form
    • A01N25/28—Microcapsules or nanocapsules
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N59/00—Biocides, pest repellants or attractants, or plant growth regulators containing elements or inorganic compounds
    • A01N59/16—Heavy metals; Compounds thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
    • A01N65/08—Magnoliopsida [dicotyledons]
    • A01N65/38—Solanaceae [Potato family], e.g. nightshade, tomato, tobacco or chilli pepper

Definitions

  • the present invention relates to an antimicrobial silver nanoparticle using a tomato extract, a method for preparing the same, and a use of the antimicrobial silver nanoparticle.
  • Nanoparticles have various functions depending on the metal type, size, and shape of the particles.
  • silver nanoparticles are known to have biocidal activities including antibacterial, antifungal, antiviral and anticancer activities.
  • biocidal activities including antibacterial, antifungal, antiviral and anticancer activities.
  • research into potential therapeutic biomolecules for silver nanoparticles is being actively conducted, and as the tendency to consider economic and environmental benefits becomes more pronounced, an efficient synthesis method that does not contain harmful chemicals and pollutants has been developed.
  • an increasing number of studies to be done is a chemical synthesis method using sodium borohydride (NaBH4), and sodium borohydride reduces Ag + ions to Ag 0 in an aqueous silver nitrate solution.
  • NaBH4 sodium borohydride
  • this method has a problem that is not safe because it requires the use of harmful reagents in the synthesis process and a problem of environmental pollution occurs. Moreover, there is a problem in that the silver nanoparticles produced by chemical synthesis have low biocompatibility and very high toxicity, and thus are not suitable for nanomedical applications.
  • an object of the present invention is to provide a method for producing antimicrobial silver nanoparticles using tomato extract, comprising adding and stirring a tomato extract to a silver nitrate solution at room temperature.
  • Another object of the present invention is to provide antimicrobial silver nanoparticles using the tomato extract prepared by the method of the present invention.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing and treating candidiasis, comprising antimicrobial silver nanoparticles using the tomato extract of the present invention.
  • Another object of the present invention is to provide a composition for external application for skin for improving candidiasis, including antimicrobial silver nanoparticles using the tomato extract of the present invention.
  • Another object of the present invention is to provide a composition for inhibiting or removing the formation of a biofilm formed by Candida bacteria, including antimicrobial silver nanoparticles using the tomato extract of the present invention.
  • the present invention provides a method for producing antimicrobial silver nanoparticles using a tomato extract, comprising the step of adding and stirring a tomato extract to a silver nitrate solution at room temperature.
  • the tomato extract is obtained by washing the surface of the tomato and homogenizing the fruit portion from which the skin has been removed to obtain tomato juice, and then centrifuging the tomato juice to obtain a supernatant, followed by filtration It may be a tomato extract obtained by.
  • the silver nitrate solution may be a solution in which the concentration of silver nitrate is 3 to 7 mM.
  • the tomatoes are Heirloom Tomatoes, Beefsteak Tomatoes, Plum Tomatoes, Yellow Tomatoes, Cherry Tomatoes, Green Tomatoes (Green Tomatoes), Campari Tomatoes (Campari Tomatoes), Cherry Tomatoes (Pear Tomatoes), Brandywine Tomatoes (Brandywine Tomatoes) and Cherokee Purple Tomatoes (Cherokee Purple Tomatoes) may be selected from the group consisting of.
  • the present invention provides antimicrobial silver nanoparticles using the tomato extract prepared by the method of the present invention.
  • the silver nanoparticles may have antibacterial activity against Candida bacteria.
  • the silver nanoparticles may have an average particle size of about 100 nm in diameter.
  • the antimicrobial activity may be one having antimicrobial activity through inhibition of growth, inhibition of engraftment, or inhibition of biofilm formation against Candida bacteria.
  • the Candida bacteria are Candida albicans, Candidan tropicalis, Candida glabrata, Candida glabrata, C. krusei, and Candida parafid. It may be selected from the group consisting of C. parapsilosis.
  • the present invention provides a pharmaceutical composition for preventing and treating candidiasis, comprising antimicrobial silver nanoparticles using the tomato extract of the present invention.
  • the composition is Candida albicans (C. albicans), Candida tropicalis (Candidan tropicalis), Candida glabrata (Candida glabrata), Candida Crusei (C. krusei) and Candida parapicillo It may have antifungal activity against Candida bacteria selected from the group consisting of cis (C. parapsilosis).
  • the present invention provides a composition for external application for skin for improving candidiasis, including antimicrobial silver nanoparticles using tomato extract.
  • the present invention provides a composition for inhibiting or removing the formation of a biofilm formed by Candida bacteria, including antimicrobial silver nanoparticles using the tomato extract of the present invention.
  • the antimicrobial silver nanoparticles using the tomato extract prepared by the method of the present invention are characterized by excellent growth inhibitory activity against Candida bacteria.
  • the silver nanoparticles using the tomato extract of the present invention adhere to the surface of the Candida bacteria It can induce the formation of pores and inhibits normal growth and has excellent antibacterial activity against Candida bacteria. Therefore, the antimicrobial silver nanoparticles using the tomato of the present invention have an effect that can be widely used not only in the field of antibacterial, antifungal and anti-biofilm, but also in the field of nanomedicine.
  • FIG. 1 is a photograph showing a color of a silver nitrate solution, a tomato extract, and a mixed solution of a silver nitrate solution and a tomato extract in the process of synthesizing silver nanoparticles according to an embodiment of the present invention.
  • FIG. 2 shows the result of analyzing the synthesis of silver nanoparticles according to the reaction time for a mixture of a silver nitrate solution and a tomato extract using a UV-Vis spectrum in an embodiment of the present invention.
  • Figure 3 shows a scanning electron microscope observation photograph and EDXM spectrum analysis results of the silver nanoparticles using the tomato extract prepared in the present invention.
  • Figure 4 is a graph showing the particle size distribution by synthesis time (a: 24 hours, b: 48 hours, c: 72 hours) for the silver nanoparticles using the tomato extract prepared in the present invention.
  • FIG. 6 shows the antimicrobial activity of silver nanoparticles against Candida bacteria using the tomato extract of the present invention, and is a result of analyzing the degree of inhibition of growth of Candida bacteria by concentration of silver nanoparticle treatment.
  • FIG. 8 shows SEM observation photos of Candida bacteria according to treatment with silver nanoparticles using the tomato extract of the present invention, (a) control C. albicans (3000x magnification), (b) 64 ⁇ g/mL in C. albicans bacteria Of the group treated with silver nanoparticles (3,000X magnification), (c) the group treated with 64 ⁇ g/mL silver nanoparticles in C. albicans (20,000 ⁇ magnification), (d) the control C. parasilopsis (3000X magnification) , (e) C. parasilopsis bacteria treated with 64 ⁇ g/mL silver nanoparticles (3000x magnification), (f) C.
  • the present invention is characterized in providing a new method for producing silver nanoparticles that are safe and excellent in antibacterial activity without the use of harmful reagents that have been a problem in the conventional silver nanoparticle production process.
  • tomatoes which are natural materials
  • antimicrobial silver nanoparticles using tomato extract comprising the step of adding and stirring a tomato extract to a silver nitrate solution at room temperature. It is characterized by providing a manufacturing method of.
  • the present invention confirmed that while studying a natural plant material capable of eco-friendly production of antimicrobial silver nanoparticles, when using tomato extract, it was possible to manufacture silver nanoparticles with excellent antibacterial properties while being stable and without the production of harmful by-products.
  • the tomato extract can serve as a reductant agent and a capping agent in the synthesis of antimicrobial silver nanoparticles.
  • the surface of the tomato is washed with deionized water and the skin is removed.
  • the tomato juice is obtained by homogenizing the fruit part (pulp) of the tomato from which the skin has been removed.
  • the fruit portion of the tomato from which the skin has been removed may be used to make juice in a finely cut form.
  • the homogenization can be juiced through a blender device or the like.
  • the tomato extract is added dropwise to the silver nitrate solution and slowly stirred to prepare the antimicrobial silver nanoparticles of the present invention.
  • the silver nitrate (AgNO 3 ) solution may be a solution containing silver nitrate in a concentration of 3 mM to 7 mM. If a solution of less than 3mM concentration is used, there is a problem that silver nanoparticles are not formed properly, whereas if a solution exceeding 7mM concentration is used, impurities are generated. Therefore, it is preferable to use a silver nitrate solution in the above concentration range, and in one embodiment of the present invention, a silver nitrate solution having a concentration of 5 mM was used.
  • the mixed reaction of the tomato extract and the silver nitrate solution may be used so that the volume ratio of the tomato extract to the silver nitrate solution is 1:2 to 1:6.
  • the volume ratio is out of the range, there is a problem that the antimicrobial silver nanoparticles according to the present invention are not prepared or other impurities are formed.
  • the reaction was performed in a volume ratio of 1:4.
  • the reaction to produce silver nitrate by mixing and stirring according to the dropwise addition of the tomato extract to the silver nitrate solution proceeds for 15 to 20 minutes at room temperature, and the stirring may be performed at a speed of 120 to 150 rpm, which is a mild stirring condition.
  • the stirring may be performed at a speed of 120 to 150 rpm, which is a mild stirring condition.
  • Ag + ions are reduced to Ag 0 ions by treatment of the tomato extract.
  • the silver nanoparticles generated in the mixed solution by the reaction of the tomato extract and the silver nitrate solution can be obtained as a precipitated pellet by centrifuging the mixed solution, and the pellet is washed several times with deionized water.
  • the antimicrobial silver nanoparticles of the invention can be obtained.
  • varieties of tomatoes that can be used in the present invention are not limited thereto, but Heirloom Tomatoes, Beefsteak Tomatoes, Plum Tomatoes, Yellow Tomatoes, and Cherry Tomatoes ( Cherry Tomatoes, Green Tomatoes, Campari Tomatoes, Pear Tomatoes, Brandywine Tomatoes or Cherokee Purple Tomatoes.
  • silver nanoparticles prepared using a UV-Vis spectrophotometer, a scanning electron microscope (SEM), dynamic light scattering (DLS), and Fourier transform-infrared spectroscopy (FT-IR) The characteristics of were analyzed.
  • SEM scanning electron microscope
  • DLS dynamic light scattering
  • FT-IR Fourier transform-infrared spectroscopy
  • the silver nanoparticles using the tomato extract prepared in the present invention mainly have a spherical shape, and the particle size is an average of about 100 nm in diameter without aggregation.
  • tomato extract played a role as a reducing agent and capping agent involved in synthesis and dispersion in the process of synthesizing silver nanoparticles. It was found that the substance was contained.
  • the antimicrobial activity against silver nanoparticles using the tomato extract prepared by the method of the present invention was analyzed.
  • the minimum inhibitory concentration (MIC) according to the silver nanoparticle treatment of the present invention was measured for various Candida bacteria.
  • MIC minimum inhibitory concentration
  • Biofilm is also called a biofilm, and refers to a state in which microorganisms produce polysaccharides around cells and aggregate with neighboring microorganisms through this to form a film on the surface of a solid or living body. It has a structural feature consisting of microbial secretions. Microorganisms secrete enzymes that produce polysaccharides to the outside of cells to form water-soluble or insoluble polysaccharides from sugar substances existing in the external environment, and when such insoluble polysaccharides are continuously accumulated, a thin biofilm in the form of a film is formed. The biofilm formed in this way helps coagulation between microorganisms and, as a result, can cause various diseases due to the biofilm of harmful bacteria.
  • the silver nanoparticles using the tomato extract of the present invention have the activity of inhibiting the formation of a biofilm formed by Candida bacteria, and the silver nanoparticles of the present invention form a large hole in the film of the biofilm. It was found that it was possible to induce apoptosis of harmful bacteria by enabling the formation of additional pores. In addition, it was found that the silver nanoparticles of the present invention bind to the surface of harmful bacteria to inhibit normal growth and cause abnormalities in cell membranes, thereby having excellent antibacterial activity against harmful bacteria.
  • the present invention can provide antimicrobial silver nanoparticles using the tomato extract prepared by the method of the present invention.
  • the silver nanoparticles prepared by the method of the present invention have a particle size of about 100 nm on average and have a spherical shape.
  • the antimicrobial silver nanoparticles using the tomato extract of the present invention may have excellent antimicrobial activity, preferably may have antibacterial activity against Candida, and the Candida is not limited thereto, but Candida is Candida. Albicans (C. albicans), Candidan tropicalis (Candidan tropicalis), Candida glabrata (Candida glabrata), Candida cruse (C. krusei) and Candida parapsilosis (C. parapsilosis) to be selected from the group consisting of. I can.
  • Candida is Candida. Albicans (C. albicans), Candidan tropicalis (Candidan tropicalis), Candida glabrata (Candida glabrata), Candida cruse (C. krusei) and Candida parapsilosis (C. parapsilosis) to be selected from the group consisting of. I can.
  • the antimicrobial activity of the silver nanoparticles using the tomato extract of the present invention against Candida bacteria has antibacterial activity by acting through inhibition of growth, engraftment, or inhibition of biofilm formation against Candida bacteria.
  • the present invention can provide a pharmaceutical composition for the prevention and treatment of candidiasis, including antimicrobial silver nanoparticles using tomato extract.
  • Candidiasis is a disease caused by infection of parts or parts of the body by Candida bacteria (fungi or fungi).Candida bacteria often cause infections confined to the superficial layer of the skin or mucous membrane, and inflammation of the oropharynx or esophagus, including thrush, Symptoms such as vulvitis, vaginitis, and peritonitis appear.
  • Candida proliferates in the oropharynx or esophagus the mouth feels uncomfortable, tastes poor, and can cause pain when chewing or passing food.
  • Candidal vulvovaginitis is the most common superficial candidiasis, and symptoms such as itching of the vulva, itching, vaginal pain, pain during intercourse, and white lumpy vaginal discharge appear.
  • Candida bacteria invade various organs such as the kidney, heart, liver, brain, and eyes, causing pathological changes and fever, It can be accompanied by general physical symptoms that can occur in common infectious diseases, such as chills.
  • Candida bacteria causing candidiasis are not limited thereto, but candida albicans, Candidan tropicalis, Candida glabrata, and C. krusei ) And Candida parapsilosis (C. parapsilosis) may be selected from the group consisting of.
  • prevention refers to any action that suppresses or delays the onset of candidiasis by administration of the pharmaceutical composition of the present invention, and “treatment” is already induced by the administration of the pharmaceutical composition of the present invention. It refers to any action that improves or benefits the symptoms of candidiasis.
  • Antimicrobial silver nanoparticles using the tomato extract according to the present invention may be appropriately formulated together with a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colors and fragrances, etc., when administered orally.
  • the injection is a physiological saline solution.
  • aqueous solvents such as Ringel's solution, vegetable oils, higher fatty acid esters (e.g., oleic acid ethyl, etc.) and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.).
  • a buffering agent such as compressed air, nitrogen, carbon dioxide, or hydrocarbon-based low boiling point solvent can be conveniently delivered from a pressurized pack or nebulizer in the form of an aerosol spray.
  • the formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier as described above.
  • it when administered orally, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, and in the case of injections, it may be prepared in the form of unit dosage ampoules or multiple dosage forms.
  • the route of administration of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue.
  • administration means introducing a predetermined substance to a patient by any suitable method, and is formulated for human use and administered by various routes.
  • the pharmaceutical composition of the present invention may be administered by a parenteral route, such as intravascular, intravenous, intraarterial, intramuscular or subcutaneous, orally, nasal, rectal, transdermal, or by inhalation via aerosol.
  • a parenteral route such as intravascular, intravenous, intraarterial, intramuscular or subcutaneous, orally, nasal, rectal, transdermal, or by inhalation via aerosol.
  • it may be administered as a bolus or may be slowly injected, but it is preferably administered by intramuscular or subcutaneous injection.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to exhibit an antimicrobial effect against Candida and an amount that does not cause side effects or serious or excessive immune reactions, and the effective dose level is to be treated.
  • the pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
  • the pharmaceutical composition according to the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method.
  • Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are prepared by mixing at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like.
  • Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
  • the present invention can provide a composition for external application for skin for improving candidiasis, including antimicrobial silver nanoparticles using tomato extract.
  • the present invention can provide a composition for inhibiting the production of a biofilm formed by Candida bacteria, including antimicrobial silver nanoparticles using a tomato extract.
  • the antimicrobial silver nanoparticles prepared by the method of the present invention can ultimately cause apoptosis of Candida bacteria by inhibiting the production of the biofilm by forming holes in the film of the biofilm formed by Candida bacteria.
  • the silver nanoparticles containing the tomato extract prepared by the method of the present invention has excellent antibacterial activity and can be used as an antibacterial composition, and in particular, can be used as an antibacterial composition against Candida bacteria.
  • Silver nitrate (AgNO 3 ) used for the synthesis of silver nanoparticles was purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and used.
  • the tomato extract (10 mL) prepared in ⁇ 1-1> was added dropwise over 20 minutes to 40 mL of silver nitrate solution (AgNO 3 , 5 mM) and incubated at room temperature under gentle stirring (120 rpm).
  • AgNO 3 silver nitrate solution
  • AgNPs silver nanoparticles
  • a mixture of silver nitrate and tomato extract containing silver nanoparticles was centrifuged at 13,000 rpm for 15 minutes. Then, the pellets of the silver nanoparticles were washed three times with deionized water to remove impurities. Finally, freeze-drying was performed to obtain a powder of silver nanoparticles (AgNPs) using the purified tomato extract of the present invention, and then dissolved in deionized water and stored as a stock solution, and then used in the following examples.
  • the UV-Vis spectrophotometer is an analysis method used to confirm the synthesis of nanoparticles.To confirm the yield of particle synthesis over time, a mixture of tomato extract and silver nitrate was measured every 3 minutes in the absorbance range of 300 to 800 nm. , The measurement was performed until a change in absorbance was no longer detected.
  • image analysis using a scanning electron microscope is a method of analyzing the morphological characteristics of the size and shape of nanoparticles.
  • a solution of colloidal silver nanoparticles using the tomato extract prepared in the present invention is buried on a cover glass. After drying overnight, the cover glass was placed over a copper stub and fixed with carbon tape.
  • SEM images were acquired at 10KV acceleration voltage with a JEOL, Ltd (Tokyo, Japan) instrument (JSM-6701F), and EDAX analysis was performed simultaneously with the instrument included in the SEM to confirm the presence of elemental silver.
  • FT-IR Fourier transform-infrared spectroscopy
  • the color change is caused by the reduction of Ag+ ions, and is an index confirming the synthesis of silver nanoparticles (AgNP).
  • AgNP silver nanoparticles
  • the mixture of the silver nitrate solution and the tomato extract gradually changed from colorless to yellow by reaction, and then changed to reddish brown after about 10 minutes, confirming that silver nanoparticles were synthesized (FIG. 1).
  • the control group was found to be colorless.
  • SPR surface plasmon resonance
  • the SRP pattern depends on the characteristics of each metal particle, such as the size, shape, dielectric properties of the medium used for synthesis, and the bonding interactions between the nanoparticles.
  • ⁇ max in the mixture of the silver nitrate solution and tomato extract of the present invention was observed at 445 nm, and the intensity of the SPR band increased with the reaction time, confirming that silver nanoparticles were synthesized (FIG. 2).
  • the maximum absorbance was recorded at 12 minutes, thereby confirming that the reaction was terminated.
  • the present inventors found that the tomato extract played a role as a reducing agent and capping agent involved in synthesis and dispersion in the process of synthesizing silver nanoparticles, and through the lower result of FIG. 2, in the EDAX spectrum It was confirmed that the strong signal of the silver atom was confirmed, and it was also found that silver nanoparticles were synthesized by the method of the present invention. In addition, signals of carbon, oxygen, sulfur, and potassium, which are organic substances present in the tomato extract, were confirmed.
  • the size distribution and colloidal stability of the silver nanoparticles of the present invention were analyzed for 72 hours through dynamic light scattering analysis.
  • the z-average value was found to be 99.16 nm (3a) at 24 hours, 100.2 nm (3b) at 48 hours, and 108.5 nm (3c) at 72 hours, which This means that the size remained almost constant over 48 hours.
  • PDI polydispersity index
  • the apparent absorption peaks of the tomato extract were observed at 3296.71, 2137.28, 1635.83 and 1063.18 cm -1.
  • the peak at 3296.71 cm -1 corresponds to the stretching vibration of the -OH bond in alcohol and phenol
  • the weak intensity peak at 2137.28 cm -1 is -C ⁇ C- bond in the alkynyl groups. It is due to the expansion and contraction of the vibration.
  • the peak at 1063.18 cm -1 can be estimated to indicate CO elongation vibration in esters and ethers.
  • C. albicans (ATCC 90028), C. parasilopsis (ATCC 90018) and C. glabrata (ATCC 90030) corresponding to Candida bacteria as harmful bacteria of the present invention
  • the minimum inhibitory concentration (MIC) of silver nanoparticles was measured.
  • a dilution solution obtained by continuously diluting the silver nanoparticles prepared using the tomato extract according to the present invention twice was prepared at a final concentration in the range of 2 to 64 ⁇ g/mL, and added to a 96-well flat bottom microtiter plate.
  • Biofilms are known to be the main cause of persistent infections in the human body or infections related to implanted medical devices because microbes are surrounded by various foreign substances and can be formed on a living body or inanimate surface. Accordingly, the present inventors conducted an experiment to confirm whether the silver nanoparticles of the present invention have an inhibitory effect on the existing biofilm (pre-existing biofilm).
  • the Candida biofilm was fixed on the surface of a 96-well plate at a temperature of 37° C. for 24 hours. After washing with a PBS solution to remove suspended cells, the wells were filled with fresh RPMI 1640 medium and treated with the silver nanoparticles of the present invention having the same concentration as used in the MIC test of Example ⁇ 3-1>, and then 37 It was further incubated for 24 hours at a temperature of °C. Thereafter, the biofilm attached to the bottom of the plate was suspended by pipetting, and absorbance was measured at 620 nm using a microplate reader device. At this time, the concentration of silver nanoparticles whose turbidity was reduced to 50% or less was regarded as the minimum inhibitory concentration (MIC) for biofilm formation.
  • MIC minimum inhibitory concentration
  • the silver nanoparticles using the tomato extract of the present invention have the activity of inhibiting the biofilm against all Candida bacteria used in the experiment.
  • the MIC values of C. albicans and C. glabrata were found to be 32 ⁇ g/mL, and the MIC of C. parasilopsis was 8 ⁇ g/mL.
  • C. parasilopsis has the highest sensitivity to the silver nanoparticles of the present invention.
  • the present inventors analyzed the morphological image using a scanning electron microscope to confirm the effect of the silver nanoparticles prepared using the tomato extract of the present invention on biofilm inhibition.
  • the suspension of the Candida strain was adjusted to 0.5 McFarland in RPMI 1640 medium and inoculated into a 6-well plate containing a custom-made 20 mm x 20 mm polystyrene cover slip.
  • Polystyrene coverslips were pre-incubated at 37°C for 2 hours to allow Candida strains to adhere, and after pre-cultivation, the coverslips were gently washed with PBS to remove non-adherent cells, and test wells of 64 ⁇ g/mL of the present invention was filled with a medium containing silver nanoparticles, and then incubated at 37° C. for 48 hours to form a biofilm.
  • the control well was used as a group treated with fresh RPMI 1640 medium that does not contain the silver nanoparticles of the present invention.
  • the biofilm on the polystyrene cover slip was washed with PBS and fixed overnight using 2.5% glutaraldehyde using PBS buffer.
  • coverslips were dehydrated using ethanol (70, 80, 90, and 100%) having different ethanol contents and dried overnight in a dryer.
  • the sample was then coated with platinum using an automatic magnetron sputter coater system and observed with a scanning electron microscope. A representative image of the effect of silver nanoparticles on the entire sample surface was confirmed.
  • FIG. 8 shows SEM micrographs of Candida species before and after treatment with the silver nanoparticles of the present invention for 48 hours.
  • Cells of Candida species are irreversibly attached to the substrate or interface to form a biofilm in the microbial community, which acts as a major cause of Candida pathogenicity.
  • This physiological structure makes Candida species resistant to various antimicrobial agents.
  • Candida species are known to have three types of yeast, pseudo-hyphae, and hyphae as a cytological form.
  • C. glabrata does not exhibit heterogeneous morphology and only forms yeast morphology, C.
  • C. albicans forms yeast and pseudohyphae morphologies, often called giant cells
  • C. albicans forms yeast, pseudohyphae, and hyphae morphologies, often called giant cells.
  • the control C. albicans (8a) showed a typical and smooth morphology, and yeast and hyphae morphology were found in the biofilm.
  • the silver nanoparticles of the present invention treated C. albicans (8b), no hyphae was observed, and only single or budding yeasts with rimose membranes were observed.
  • the image (8c) confirming the C. albicans treated with the silver nanoparticles of the present invention at high magnification, it was found that the silver nanoparticles adhered and aggregated on the surface of the nearby yeast (black arrow in the image).
  • the control biofilm (8d) was mainly composed of yeast and similar hyphae with a regular shape, and when the silver nanoparticles of the present invention were treated (8e), it had a solid film. Yeast was present throughout the sample, but no similar hyphae was observed.
  • the image (8f) magnified 20,000 times the silver nanoparticles of the present invention were found to be located on the surface of wrinkled abnormal yeast and similar hyphae (black arrows in the image).
  • C. glabrata As a result of scanning electron microscopy analysis of C. glabrata, it was found that the biofilm (8g) of the control C. glabrata consisted only of blastoconidia, which are spherical or elliptical, which is significantly smaller than that of C. albicans and C. parasilopsis.
  • C. glabrata (8h) treated with silver nanoparticles of the present invention it has an abnormal shape, and some pores and broken membranes were observed in yeast. 8i observed at 20,000 x magnification, it could be observed that silver nanoparticles of the present invention (black arrows in the image) adhered around the perforated membrane of C. glabrata observed in C. albicans and C. parasilopsis. .
  • the present inventors believe that the silver nanoparticles using the tomato extract of the present invention can interfere with the membrane potential and affect the interaction of the membrane structure with respect to harmful bacteria or fungi, in particular, it is possible to form a large hole in the membrane. In addition, it was found that the formation of additional pores could ultimately lead to apoptosis of harmful bacteria or fungi. In addition, it was found that the silver nanoparticles of the present invention bind to the surface of harmful bacteria or fungi to inhibit normal growth, cause abnormalities in cell membranes, and inhibit the formation of biofilms, thereby having excellent antibacterial activity.

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Abstract

The present invention relates to antibiotic silver nanoparticles using a tomato extract, and a method for producing same, and, particularly, to: a method for producing antibiotic silver nanoparticles using a tomato extract, comprising a step for adding a tomato extract to a silver nitrate solution at room temperature and mixing same; antibiotic silver nanoparticles, using a tomato extract, produced by the method; a pharmaceutical composition, for preventing and treating candidiasis, comprising the silver nanoparticles; a candidiasis-alleviating composition to be used externally on the skin; and a composition for removing or inhibiting formation of biofilm due to Candida fungus. In the present invention, it has been confirmed that the tomato extract can act as a reductant and a capping agent in the process of synthesizing the silver nanoparticles, and the silver nanoparticles using a tomato extract of the present invention has an excellent growth inhibiting activity against Candida fungus and has excellent antibiotic activity by attaching to the surface of Candida fungus to induce formation of pores on the film and by inhibiting normal growth. Therefore, the silver nanoparticles using a tomato extract of the present invention can be widely utilized in antifungal and antibiotic films and in the field of nanomedicine for treating candidiasis.

Description

토마토 추출물을 이용한 항균성 은 나노입자 및 이의 제조방법Antimicrobial silver nanoparticles using tomato extract and method for preparing the same
본 발명은 토마토 추출물을 이용한 항균성 은 나노입자, 이의 제조방법 및 상기 항균성 은 나노입자의 용도에 관한 것이다.The present invention relates to an antimicrobial silver nanoparticle using a tomato extract, a method for preparing the same, and a use of the antimicrobial silver nanoparticle.
나노입자의 합성은 오랫동안 연구되고 있으며, 나노의학 분야를 발전시키기 위해 나노입자를 적용하기 위한 다양한 연구가 진행 중에 있다. 나노입자는 입자의 금속 유형, 크기 및 모양에 따라 다양한 기능을 가지며, 특히 은 나노입자는 항균, 항진균, 항 바이러스 및 항암 활성을 포함하는 살균(biocidal) 활성이 있는 것으로 알려져 있다. 이러한 이유로 은 나노입자는 잠재적으로 치료용 생체분자로의 연구가 활발히 진행되고 있으며 경제적, 환경적인 편익을 고려하는 경향이 더욱 뚜렷해 짐에 따라 유해 화학물질 및 오염물질을 포함하지 않는 효율적인 합성방법을 개발하고자 하는 연구가 증가하고 있다. 현재 가장 널리 사용되는 은 나노입자의 제조는 나트륨 보로하이드라이드(NaBH4)를 사용하는 화학 합성방법으로, 나트륨 보로 하이드라이드는 질산은 수용액에서 Ag + 이온을 Ag0로 환원시킨다.Synthesis of nanoparticles has been studied for a long time, and various studies are underway to apply nanoparticles to advance the field of nanomedicine. Nanoparticles have various functions depending on the metal type, size, and shape of the particles. In particular, silver nanoparticles are known to have biocidal activities including antibacterial, antifungal, antiviral and anticancer activities. For this reason, research into potential therapeutic biomolecules for silver nanoparticles is being actively conducted, and as the tendency to consider economic and environmental benefits becomes more pronounced, an efficient synthesis method that does not contain harmful chemicals and pollutants has been developed. There is an increasing number of studies to be done. Currently, the most widely used preparation of silver nanoparticles is a chemical synthesis method using sodium borohydride (NaBH4), and sodium borohydride reduces Ag + ions to Ag 0 in an aqueous silver nitrate solution.
그러나 이러한 방법은 합성과정에서 유해한 시약을 사용해야 하고 환경오염의 문제가 발생하기 때문에 안전하지 못한 문제점이 있다. 더욱이, 화학적 합성에 의해 생성된 은 나노입자는 생체 적합성이 낮고 독성이 매우 높아 나노의학 응용 분야에 적합하지 않다는 문제점이 있다.However, this method has a problem that is not safe because it requires the use of harmful reagents in the synthesis process and a problem of environmental pollution occurs. Moreover, there is a problem in that the silver nanoparticles produced by chemical synthesis have low biocompatibility and very high toxicity, and thus are not suitable for nanomedical applications.
이에 최근에는 천연식물을 은 나노입자의 제조에 사용하고자 하는 노력이 진행되고 있는데, 이는 식물 추출물에는 다당류와 식물성 화학물질이 포함되어 있으며, 다당류의 하이드록시 기(Hydroxyl groups) 및 헤미아세탈 환원 말단(hemiacetal reducing ends)은 환원제 및 캡핑제로서 나노입자 합성에 중요한 역할을 할 수 있으며, 식물화학 물질은 Ag+의 감소와 Ag0의 안정성에 기여할 수 있기 때문이다. 식물 추출물을 은 나노입자의 개발에 활용한 종래 기술로는 치자 종자 추출물을 이용한 은 나노입자 제조방법이 개발된 바 있고(대한민국 등록특허 10-2091166), 닥풀 추출물을 포함하는 은 나노입자 분산제의 기술이 개발된 바 있다(대한민국 공개특허 10-2019-0019657).In recent years, efforts have been made to use natural plants for the production of silver nanoparticles, which include polysaccharides and phytochemicals in plant extracts, and hydroxy groups of polysaccharides and hemiacetal reducing ends ( Hemiacetal reducing ends) can play an important role in the synthesis of nanoparticles as a reducing agent and capping agent, and phytochemicals can contribute to the reduction of Ag + and the stability of Ag 0. As a conventional technique using plant extracts for the development of silver nanoparticles, a method for producing silver nanoparticles using gardenia seed extract has been developed (Korean Patent Registration No. 10-2091166), and a technology of a silver nanoparticle dispersant including a Duckweed extract. This has been developed (Republic of Korea Patent Publication 10-2019-0019657).
그러나 아직까지 천연식물을 은 나노입자의 제조에 사용하려는 연구가 미비한 실정이며, 천연식물 대상으로 토마토를 사용한 예가 없다.However, studies to use natural plants for the production of silver nanoparticles are still insufficient, and there is no example of using tomatoes as natural plants.
따라서 본 발명의 목적은 상온에서 토마토 추출물을 질산은 용액에 첨가하고 교반하는 단계를 포함하는, 토마토 추출물을 이용한 항균성 은 나노입자의 제조방법을 제공하는 것이다.Accordingly, an object of the present invention is to provide a method for producing antimicrobial silver nanoparticles using tomato extract, comprising adding and stirring a tomato extract to a silver nitrate solution at room temperature.
본 발명의 다른 목적은 본 발명의 방법으로 제조된 토마토 추출물을 이용한 항균성 은 나노입자를 제공하는 것이다.Another object of the present invention is to provide antimicrobial silver nanoparticles using the tomato extract prepared by the method of the present invention.
본 발명의 또 다른 목적은 본 발명의 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다증 예방 및 치료용 약학 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing and treating candidiasis, comprising antimicrobial silver nanoparticles using the tomato extract of the present invention.
본 발명의 또 다른 목적은 본 발명의 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다증 개선용 피부 외용제 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for external application for skin for improving candidiasis, including antimicrobial silver nanoparticles using the tomato extract of the present invention.
본 발명의 또 다른 목적은 본 발명의 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다 균이 형성하는 바이오필름의 생성억제 또는 제거용 조성물을 제공하는 것이다.Another object of the present invention is to provide a composition for inhibiting or removing the formation of a biofilm formed by Candida bacteria, including antimicrobial silver nanoparticles using the tomato extract of the present invention.
본 발명은 상온에서 토마토 추출물을 질산은 용액에 첨가하고 교반하는 단계를 포함하는, 토마토 추출물을 이용한 항균성 은 나노입자의 제조방법을 제공한다. The present invention provides a method for producing antimicrobial silver nanoparticles using a tomato extract, comprising the step of adding and stirring a tomato extract to a silver nitrate solution at room temperature.
본 발명의 일실시예에 있어서, 상기 토마토 추출물은, 토마토의 표면을 세척하고 껍질을 제거한 열매 부위를 균질화시켜 토마토 즙을 수득한 다음, 상기 토마토 즙을 원심 분리하여 상층액을 수득한 후, 여과시켜 얻은 토마토 추출물일 수 있다.In one embodiment of the present invention, the tomato extract is obtained by washing the surface of the tomato and homogenizing the fruit portion from which the skin has been removed to obtain tomato juice, and then centrifuging the tomato juice to obtain a supernatant, followed by filtration It may be a tomato extract obtained by.
본 발명의 일실시예에 있어서, 상기 질산은 용액은 질산은의 농도가 3~7mM로 포함된 용액일 수 있다.In one embodiment of the present invention, the silver nitrate solution may be a solution in which the concentration of silver nitrate is 3 to 7 mM.
본 발명의 일실시예에 있어서, 상기 토마토는 에어룸 토마토(Heirloom Tomatoes), 비프스테이크 토마토(Beefsteak Tomatoes), 플럼 토마토(Plum Tomatoes), 노란 토마토(Yellow Tomatoes), 체리 토마토(Cherry Tomatoes), 그린 토마토(Green Tomatoes), 캄파리 토마토(Campari Tomatoes), 방울 토마토(Pear Tomatoes), 브랜드와인 토마토(Brandywine Tomatoes) 및 체로키퍼플 토마토(Cherokee Purple Tomatoes)로 이루어진 군 중에서 선택되는 것일 수 있다.In one embodiment of the present invention, the tomatoes are Heirloom Tomatoes, Beefsteak Tomatoes, Plum Tomatoes, Yellow Tomatoes, Cherry Tomatoes, Green Tomatoes (Green Tomatoes), Campari Tomatoes (Campari Tomatoes), Cherry Tomatoes (Pear Tomatoes), Brandywine Tomatoes (Brandywine Tomatoes) and Cherokee Purple Tomatoes (Cherokee Purple Tomatoes) may be selected from the group consisting of.
또한 본 발명은 본 발명의 방법으로 제조된 토마토 추출물을 이용한 항균성 은 나노입자를 제공한다.In addition, the present invention provides antimicrobial silver nanoparticles using the tomato extract prepared by the method of the present invention.
본 발명의 일실시예에 있어서, 상기 은 나노입자는 칸디다 균에 대한 항균활성을 갖는 것일 수 있다.In one embodiment of the present invention, the silver nanoparticles may have antibacterial activity against Candida bacteria.
본 발명의 일실시예에 있어서, 상기 은 나노입자는 입자의 크기가 평균적으로 약 100nm의 직경을 갖는 것일 수 있다.In one embodiment of the present invention, the silver nanoparticles may have an average particle size of about 100 nm in diameter.
본 발명의 일실시예에 있어서, 상기 항균활성은 칸디다 균에 대한 생장억제, 생착억제 또는 바이오필름 형성억제를 통해 항균활성을 갖는 것일 수 있다.In one embodiment of the present invention, the antimicrobial activity may be one having antimicrobial activity through inhibition of growth, inhibition of engraftment, or inhibition of biofilm formation against Candida bacteria.
본 발명의 일실시예에 있어서, 상기 칸디다 균은 칸디다 알비칸스(C. albicans), 칸디다 트로피칼리스(Candidan tropicalis), 칸디다 글라브라타(Candida glabrata), 칸디다 크루제(C.krusei) 및 칸디다 파라피실로시스(C. parapsilosis)으로 이루어진 군 중에서 선택되는 것일 수 있다.In one embodiment of the present invention, the Candida bacteria are Candida albicans, Candidan tropicalis, Candida glabrata, Candida glabrata, C. krusei, and Candida parafid. It may be selected from the group consisting of C. parapsilosis.
또한 본 발명은 본 발명의 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다증 예방 및 치료용 약학 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing and treating candidiasis, comprising antimicrobial silver nanoparticles using the tomato extract of the present invention.
본 발명의 일실시예에 있어서, 상기 조성물은 칸디다 알비칸스(C. albicans), 칸디다 트로피칼리스(Candidan tropicalis), 칸디다 글라브라타(Candida glabrata), 칸디다 크루제(C.krusei) 및 칸디다 파라피실로시스(C. parapsilosis)으로 이루어진 군 중에서 선택되는 칸디다 균에 대한 항진균 활성을 갖는 것일 수 있다.In one embodiment of the invention, the composition is Candida albicans (C. albicans), Candida tropicalis (Candidan tropicalis), Candida glabrata (Candida glabrata), Candida Crusei (C. krusei) and Candida parapicillo It may have antifungal activity against Candida bacteria selected from the group consisting of cis (C. parapsilosis).
또한 본 발명은 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다증 개선용 피부 외용제 조성물을 제공한다.In addition, the present invention provides a composition for external application for skin for improving candidiasis, including antimicrobial silver nanoparticles using tomato extract.
또한 본 발명은 본 발명의 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다 균이 형성하는 바이오필름의 생성억제 또는 제거용 조성물을 제공한다.In addition, the present invention provides a composition for inhibiting or removing the formation of a biofilm formed by Candida bacteria, including antimicrobial silver nanoparticles using the tomato extract of the present invention.
본 발명의 방법으로 제조된 토마토 추출물을 이용한 항균성 은 나노입자는 칸디다 균에 대한 우수한 성장 억제 활성이 있는 특징이 있는데, 특히 본 발명의 토마토 추출물을 이용한 은 나노입자는 칸디다 균의 표면에 부착하여 막에 기공 형성을 유도할 수 있고 정상적인 성장을 저해하여 칸디다 균에 대한 우수한 항균 활성을 갖는다. 따라서 본 발명의 토마토를 이용한 항균성 은 나노입자는 항균, 항진균 및 항바이오필름 분야뿐만 아니라 나노의학 분야에도 널리 활용할 수 있는 효과가 있다.The antimicrobial silver nanoparticles using the tomato extract prepared by the method of the present invention are characterized by excellent growth inhibitory activity against Candida bacteria. In particular, the silver nanoparticles using the tomato extract of the present invention adhere to the surface of the Candida bacteria It can induce the formation of pores and inhibits normal growth and has excellent antibacterial activity against Candida bacteria. Therefore, the antimicrobial silver nanoparticles using the tomato of the present invention have an effect that can be widely used not only in the field of antibacterial, antifungal and anti-biofilm, but also in the field of nanomedicine.
도 1은 본 발명의 일실시예에 따른 은 나노입자 합성과정에서 질산은 용액, 토마토 추출물 및 질산은 용액과 토마토 추출물의 혼합용액에 대한 색상을 확인한 사진을 나타낸 것이다.1 is a photograph showing a color of a silver nitrate solution, a tomato extract, and a mixed solution of a silver nitrate solution and a tomato extract in the process of synthesizing silver nanoparticles according to an embodiment of the present invention.
도 2는 본 발명의 일실시예에서 질산은 용액과 토마토 추출물의 혼합물에 대한 반응 시간에 따른 은 나노입자의 합성을 UV-Vis 스펙트럼으로 분석한 결과를 나타낸 것이다.FIG. 2 shows the result of analyzing the synthesis of silver nanoparticles according to the reaction time for a mixture of a silver nitrate solution and a tomato extract using a UV-Vis spectrum in an embodiment of the present invention.
도 3은 본 발명에서 제조한 토마토 추출물을 이용한 은 나노입자에 대한 주사 전자현미경 관찰사진 및 EDXM 스펙트럼 분석 결과를 나타낸 것이다.Figure 3 shows a scanning electron microscope observation photograph and EDXM spectrum analysis results of the silver nanoparticles using the tomato extract prepared in the present invention.
도 4는 본 발명에서 제조한 토마토 추출물을 이용한 은 나노입자에 대한 합성 시간별(a: 24시간, b: 48시간, c:72시간) 입자 크기 분포도를 그래프로 나타낸 것이다.Figure 4 is a graph showing the particle size distribution by synthesis time (a: 24 hours, b: 48 hours, c: 72 hours) for the silver nanoparticles using the tomato extract prepared in the present invention.
도 5는 토마토 추출물(파란색) 및 본 발명에서 합성된 은 나노입자(검은색)에 대한 FT-IR 스펙트럼에 대한 결과를 나타낸 것이다.5 shows the results of the FT-IR spectrum for the tomato extract (blue) and silver nanoparticles (black) synthesized in the present invention.
도 6은 본 발명의 토마토 추출물을 이용한 은 나노입자의 칸디다균에 대한 항균활성을 나타낸 것으로, 은 나노입자 처리 농도별 칸디다균의 성장억제 정도를 분석한 결과이다.6 shows the antimicrobial activity of silver nanoparticles against Candida bacteria using the tomato extract of the present invention, and is a result of analyzing the degree of inhibition of growth of Candida bacteria by concentration of silver nanoparticle treatment.
도 7은 본 발명의 토마토 추출물을 이용한 은 나노입자의 기형성된 바이오필름에 대한 억제 활성을 분석한 결과이다.7 is a result of analyzing the inhibitory activity of the silver nanoparticles on the preformed biofilm using the tomato extract of the present invention.
도 8은 본 발명의 토마토 추출물을 이용한 은 나노입자 처리에 따른 칸디다균에 대한 SEM 관찰사진을 나타낸 것으로, (a) 대조군 C. albicans(3000x 배율), (b) C. albicans균에 64μg/mL의 은 나노입자를 처리한 군(3,000X 배율), (c) C. albicans균에 64μg/mL의 은 나노입자를 처리한 군(20,000x 배율), (d) 대조군 C. parasilopsis(3000x 배율), (e) C. parasilopsis균에 64μg/mL의 은 나노입자를 처리한 군(3000x 배율), (f) C. parasilopsis균에 64μg/mL의 은 나노입자를 처리한 군(20,000x 배율), (g) 대조군 C. albicans (3000x 배율), (h) C. glabrata균에 64μg/mL의 은 나노입자를 처리한 군(3,000x 배율), (i) C. glabrata균에 64μg/mL의 은 나노입자를 처리한 군(20,000x magnification)을 나타낸 것이며, 여기서 본 발명의 은 나노입자는 검은색 화살표로 나타내었고, (j) 본 발명의 은 나노입자를 나타낸 것이다(20,000x magnification).8 shows SEM observation photos of Candida bacteria according to treatment with silver nanoparticles using the tomato extract of the present invention, (a) control C. albicans (3000x magnification), (b) 64 μg/mL in C. albicans bacteria Of the group treated with silver nanoparticles (3,000X magnification), (c) the group treated with 64 μg/mL silver nanoparticles in C. albicans (20,000× magnification), (d) the control C. parasilopsis (3000X magnification) , (e) C. parasilopsis bacteria treated with 64μg/mL silver nanoparticles (3000x magnification), (f) C. parasilopsis bacteria treated with 64μg/mL silver nanoparticles (20,000x magnification), (g) Control C. albicans (3000x magnification), (h) C. glabrata bacteria treated with 64 μg/mL silver nanoparticles (3,000x magnification), (i) C. glabrata bacteria with 64 μg/mL silver The group treated with the nanoparticles (20,000x magnification) is shown, where the silver nanoparticles of the present invention are indicated by black arrows, and (j) the silver nanoparticles of the present invention are shown (20,000x magnification).
본 발명은 종래 은 나노입자의 제조과정에서 문제가 되었던 유해한 시약을 사용하지 않고도 안전하며 항균 활성이 우수한 새로운 은 나노입자의 제조방법을 제공함에 특징이 있다.The present invention is characterized in providing a new method for producing silver nanoparticles that are safe and excellent in antibacterial activity without the use of harmful reagents that have been a problem in the conventional silver nanoparticle production process.
구체적으로 본 발명은 천연의 소재인 토마토를 은 나노입자의 제조에 사용할 수 있음을 규명함에 따라, 상온에서 토마토 추출물을 질산은 용액에 첨가하고 교반하는 단계를 포함하는, 토마토 추출물을 이용한 항균성 은 나노입자의 제조방법을 제공함에 특징이 있다.Specifically, as the present invention found out that tomatoes, which are natural materials, can be used for the production of silver nanoparticles, antimicrobial silver nanoparticles using tomato extract comprising the step of adding and stirring a tomato extract to a silver nitrate solution at room temperature. It is characterized by providing a manufacturing method of.
보통 항균물질로서의 사용을 위한 은 나노입자의 종래합성은, 나노 크기의 입자로 성장하는 새로운 핵에 원자를 자기 조립함으로써 화학적 및 생물학적 방법을 사용하여 합성할 수 있고, 이때 환원제와 캡핑제가 사용되는데, 환원제로는 Sodium borohydride(NaBH4), sodium citrate, ascorbate, elemental hydrogen, Tollen’s reagent. N, N-dimethyl formamide(DMF) 및 poly (ethylene glycol) block copolymer와 같은 다양한 유기 및 무기 환원제가 은 이온(Ag+) 수용액 또는 비수용액의 환원을 위해 사용된다. 또한 캡핑제는 나노 입자의 크기 안정화를 위해 사용된다. 그러나 이러한 방법의 가장 큰 장점 중 하나는 짧은 시간 내에 다량의 나노 입자를 합성할 수 있다는 점이다. 이러한 합성 과정에 사용된 환원제 및 캡핑제와 같은 화학 물질은 독성물질이며 친환경적이지 않은 부산물을 발생시킨다는 문제점이 있다. 따라서 환경 친화적인 공정을 통한 은 나노입자의 개발이 시급한 실정이었다.Conventional synthesis of silver nanoparticles for use as antimicrobial substances can be synthesized using chemical and biological methods by self-assembling atoms in a new nucleus that grows into nano-sized particles. As a reducing agent, sodium borohydride (NaBH 4 ), sodium citrate, ascorbate, elemental hydrogen, Tollen's reagent. Various organic and inorganic reducing agents such as N, N-dimethyl formamide (DMF) and poly (ethylene glycol) block copolymer are used for the reduction of silver ion (Ag+) aqueous or non-aqueous solutions. In addition, the capping agent is used to stabilize the size of the nanoparticles. However, one of the biggest advantages of this method is that a large amount of nanoparticles can be synthesized within a short time. Chemical substances such as reducing agents and capping agents used in the synthesis process are toxic and have a problem in that they generate byproducts that are not environmentally friendly. Therefore, it was urgent to develop silver nanoparticles through environmentally friendly processes.
이러한 점에서 본 발명은 항균성 은 나노입자의 친환경적 제조가 가능한 천연 식물소재를 연구하던 중, 토마토 추출물을 사용할 경우, 안정적이면서 유해 부산물의 생산없이 항균성이 우수한 은 나노입자의 제조가 가능함을 확인하였고, 특히 본 발명에서는 토마토 추출물이 항균성 은 나노입자의 합성에서 환원제(reductant agent) 및 캡핑제(capping agent)로서의 역할을 할 수 있음을 확인하였다.In this respect, the present invention confirmed that while studying a natural plant material capable of eco-friendly production of antimicrobial silver nanoparticles, when using tomato extract, it was possible to manufacture silver nanoparticles with excellent antibacterial properties while being stable and without the production of harmful by-products. In particular, in the present invention, it was confirmed that the tomato extract can serve as a reductant agent and a capping agent in the synthesis of antimicrobial silver nanoparticles.
하기에서는 본 발명에서 제공하는 토마토 추출물을 이용한 항균성 은 나노입자의 제조과정에 대해 상세하게 설명한다.Hereinafter, the preparation process of the antimicrobial silver nanoparticles using the tomato extract provided by the present invention will be described in detail.
먼저 깨끗한 토마토를 선별한 후, 토마토의 표면을 탈이온수로 세척하고 껍질을 제거한다. 껍질이 제거된 토마토의 열매 부위(과육)를 균질화시켜 토마토 즙을 수득한다. 이때 상기 껍질이 제거된 토마토의 열매 부위는 잘게 절단한 형태로 즙을 만드는데 사용할 수 있다. 또한 상기 균질화는 블렌더 기기 등을 통해 착즙할 수 있다.First, after selecting a clean tomato, the surface of the tomato is washed with deionized water and the skin is removed. The tomato juice is obtained by homogenizing the fruit part (pulp) of the tomato from which the skin has been removed. At this time, the fruit portion of the tomato from which the skin has been removed may be used to make juice in a finely cut form. In addition, the homogenization can be juiced through a blender device or the like.
토마토 즙을 얻으면, 상기 즙을 원심분리하여 상층액을 수득함으로써 분순물을 제거하며, 수득한 상층액은 여과지를 통해 여과시켜 은 나노입자의 제조에 사용할 토마토 추출물을 수득한다. When the tomato juice is obtained, impurities are removed by centrifuging the juice to obtain a supernatant, and the obtained supernatant is filtered through a filter paper to obtain a tomato extract to be used in the manufacture of silver nanoparticles.
상기와 같이 토마토 추출물의 준비가 완료되면, 상기 토마토 추출물을 질산은 용액에 적가하고 천천히 교반하는 단계를 수행하여 본 발명의 항균성 은 나노입자를 제조한다.When the preparation of the tomato extract is completed as described above, the tomato extract is added dropwise to the silver nitrate solution and slowly stirred to prepare the antimicrobial silver nanoparticles of the present invention.
이때 상기 질산은(AgNO3) 용액은 질산은이 3mM ~ 7mM의 농도로 포함된 용액을 사용할 수 있다. 3mM 농도 미만의 용액을 사용하게 되면 은 나노입자가 제대로 형성되지 못하는 문제점이 생기며, 반면 7mM 농도를 초과하는 용액을 사용하게 되면 불순물이 생성되는 문제점이 발생한다. 따라서 상기 농도 범위의 질산은 용액을 사용하는 것이 바람직하며, 본 발명의 일실시예에서는 5mM 농도의 질산은 용액을 사용하였다.In this case, the silver nitrate (AgNO 3 ) solution may be a solution containing silver nitrate in a concentration of 3 mM to 7 mM. If a solution of less than 3mM concentration is used, there is a problem that silver nanoparticles are not formed properly, whereas if a solution exceeding 7mM concentration is used, impurities are generated. Therefore, it is preferable to use a silver nitrate solution in the above concentration range, and in one embodiment of the present invention, a silver nitrate solution having a concentration of 5 mM was used.
또한, 상기 토마토 추출물과 질산은 용액의 혼합반응은 토마토 추출물 대 질산은 용액의 부피비가 1:2~1:6이 되도록 사용할 수 있다. 이 또한 상기 부피비의 범위를 벗어날 경우, 본 발명에 따른 항균성 은 나노입자가 제조되지 않거나, 다른 불순물이 형성되는 문제점이 발생한다. 본 발명의 일실시예에서는 1:4의 부피비로 반응시켰다.In addition, the mixed reaction of the tomato extract and the silver nitrate solution may be used so that the volume ratio of the tomato extract to the silver nitrate solution is 1:2 to 1:6. In addition, when the volume ratio is out of the range, there is a problem that the antimicrobial silver nanoparticles according to the present invention are not prepared or other impurities are formed. In an example of the present invention, the reaction was performed in a volume ratio of 1:4.
상기 토마토 추출물의 질산은 용액으로의 적가에 따른 혼합 및 교반에 의한 질산은 생성반응은 상온에서 15~20 분 동안 진행되며 상기 교반은 온화한 교반조건인 120~150rpm의 속도에서 수행할 수 있다. 이때, 상기 질산은 용액에 토마토 추출물을 첨가하는 과정에서 토마토 추출물의 처리에 의해 Ag+ 이온이 Ag0 이온으로 환원된다. The reaction to produce silver nitrate by mixing and stirring according to the dropwise addition of the tomato extract to the silver nitrate solution proceeds for 15 to 20 minutes at room temperature, and the stirring may be performed at a speed of 120 to 150 rpm, which is a mild stirring condition. At this time, in the process of adding the tomato extract to the silver nitrate solution, Ag + ions are reduced to Ag 0 ions by treatment of the tomato extract.
토마토 추출물 및 질산은 용액과의 반응으로 혼합용액 내에 생성된 은 나노입자는 상기 반응이 완료된 혼합용액을 원심분리하여 침전된 펠렛으로 수득할 수 있고, 상기 펠렛은 탈이온수로 여러 차례 세척을 수행함으로써 본 발명의 항균성 은 나노입자를 수득할 수 있다.The silver nanoparticles generated in the mixed solution by the reaction of the tomato extract and the silver nitrate solution can be obtained as a precipitated pellet by centrifuging the mixed solution, and the pellet is washed several times with deionized water. The antimicrobial silver nanoparticles of the invention can be obtained.
또한 본 발명에서 사용할 수 있는 토마토의 품종으로는 이에 제한되는지 않으나, 에어룸 토마토(Heirloom Tomatoes), 비프스테이크 토마토(Beefsteak Tomatoes), 플럼 토마토(Plum Tomatoes), 노란 토마토(Yellow Tomatoes), 체리 토마토(Cherry Tomatoes), 그린 토마토(Green Tomatoes), 캄파리 토마토(Campari Tomatoes), 방울 토마토(Pear Tomatoes), 브랜드와인 토마토(Brandywine Tomatoes) 또는 체로키퍼플 토마토(Cherokee Purple Tomatoes)를 사용할 수 있다.In addition, the varieties of tomatoes that can be used in the present invention are not limited thereto, but Heirloom Tomatoes, Beefsteak Tomatoes, Plum Tomatoes, Yellow Tomatoes, and Cherry Tomatoes ( Cherry Tomatoes, Green Tomatoes, Campari Tomatoes, Pear Tomatoes, Brandywine Tomatoes or Cherokee Purple Tomatoes.
본 발명의 일실시예에서는, UV-Vis 분광 광도계, 주사 전자현미경(SEM), 동적 광산란(Dynamic light scattering (DLS)) 및 푸리에 변환-적외선 분광법(FT-IR)을 이용하여 제조된 은 나노입자의 특성을 분석하였다.In an embodiment of the present invention, silver nanoparticles prepared using a UV-Vis spectrophotometer, a scanning electron microscope (SEM), dynamic light scattering (DLS), and Fourier transform-infrared spectroscopy (FT-IR) The characteristics of were analyzed.
분석 결과, 질산은 용액과 토마토 추출물의 혼합물에서의 λmax는 445nm에서 관찰되었고, SPR 밴드의 강도는 반응시간에 따라 증가하는 것으로 나타나 은 나노입자가 합성되었음을 확인하였다.As a result of the analysis, λmax in the mixture of silver nitrate solution and tomato extract was observed at 445 nm, and the intensity of the SPR band increased with the reaction time, confirming that silver nanoparticles were synthesized.
또한, 본 발명에서 제조된 토마토 추출물을 이용한 은 나노입자는 주로 구형의 형태를 가지며, 입자 크기는 응집 없이 직경이 평균 약 100 nm인 것으로 나타났다.In addition, it was found that the silver nanoparticles using the tomato extract prepared in the present invention mainly have a spherical shape, and the particle size is an average of about 100 nm in diameter without aggregation.
또한, 본 발명의 은 나노입자의 합성에 관여하는 토마토 추출물의 작용기 분석결과, 토마토 추출물에 함유된 다양한 성분, 즉 알코올, 페놀 및 플라보노이드에 포함된 작용기들은 본 발명의 은 나노입자의 감소 및 안정화에 관여하는 캡핑제 역할을 한다는 것을 알 수 있었고, 토마토 추출물에 존재하는 유기 물질인 탄소, 산소, 황 및 칼륨의 신호를 확인할 수 있었다.In addition, as a result of functional group analysis of the tomato extract involved in the synthesis of the silver nanoparticles of the present invention, various components contained in the tomato extract, that is, the functional groups contained in alcohol, phenol and flavonoids, contribute to the reduction and stabilization of the silver nanoparticles of the present invention. It was found that it acts as a capping agent involved, and signals of carbon, oxygen, sulfur, and potassium, which are organic substances present in tomato extract, were confirmed.
이러한 결과를 통해, 본 발명자들은 토마토 추출물이 은 나노입자를 합성하는 과정에서 합성 및 분산에 관여하는 환원제 및 캡핑제로서의 역할을 하였음을 알 수 있었으며, 본 발명의 은 나노입자에 토마토 추출물 유래 유기 물질이 포함되어 있음을 알 수 있었다.Through these results, the present inventors found that tomato extract played a role as a reducing agent and capping agent involved in synthesis and dispersion in the process of synthesizing silver nanoparticles. It was found that the substance was contained.
나아가 본 발명의 다른 일실시예에서는, 본 발명의 방법으로 제조된 토마토 추출물을 이용한 은 나노입자에 대한 항균 활성을 분석하였다.Further, in another embodiment of the present invention, the antimicrobial activity against silver nanoparticles using the tomato extract prepared by the method of the present invention was analyzed.
이를 위해, 다양한 칸디다균들을 대상으로 본 발명의 은 나노입자 처리에 따른 최소억제 농도(MIC)를 측정하였는데, 그 결과, 실험에 사용한 모든 칸디다 종의 유해균들은 본 발명의 은 나노입자에 의해 성장이 저해되는 것으로 나타났다.To this end, the minimum inhibitory concentration (MIC) according to the silver nanoparticle treatment of the present invention was measured for various Candida bacteria. As a result, all harmful bacteria of Candida species used in the experiment were grown by the silver nanoparticles of the present invention. It has been shown to be inhibited.
또 다른 실험에서는 본 발명의 은 나노입자가 바이오필름에 대한 억제활성이 있는지를 확인하였는데, 그 결과 실험에 사용된 모든 칸디다균들의 바이오필름 형성을 억제하는 활성이 있음을 확인하였다.In another experiment, it was confirmed whether the silver nanoparticles of the present invention have inhibitory activity against the biofilm, and as a result, it was confirmed that the silver nanoparticles of the present invention have the activity of inhibiting biofilm formation of all Candida bacteria used in the experiment.
바이오필름(biofilm)은 생물막이라고도 불리우며, 미생물이 세포 주위에 다당체를 생산하고 이것을 매개로 인접한 미생물과 응집하여 고체나 생체 표면에 막(film)을 형성하고 있는 상태를 의미하며, 상호 연결된 미생물 세포층 및 미생물 분비물로 이루어진 구조적 특징을 가진다. 미생물은 다당류를 생성하는 효소를 세포 외부로 분비하여 외부 환경에 존재하는 당물질로부터 수용성 또는 불용성 다당류를 형성하며, 이러한 불용성 다당류가 지속적으로 축적되면 필름 형태의 얇은 바이오필름이 형성된다. 이렇게 형성된 바이오필름은 미생물 사이의 응집에 도움을 주어 결과적으로 유해균의 바이오필름에 의해 다양한 질환을 유발할 수 있다.Biofilm is also called a biofilm, and refers to a state in which microorganisms produce polysaccharides around cells and aggregate with neighboring microorganisms through this to form a film on the surface of a solid or living body. It has a structural feature consisting of microbial secretions. Microorganisms secrete enzymes that produce polysaccharides to the outside of cells to form water-soluble or insoluble polysaccharides from sugar substances existing in the external environment, and when such insoluble polysaccharides are continuously accumulated, a thin biofilm in the form of a film is formed. The biofilm formed in this way helps coagulation between microorganisms and, as a result, can cause various diseases due to the biofilm of harmful bacteria.
한편, 본 발명의 토마토 추출물을 이용한 은 나노입자는 칸디다균들에 의해 형성되는 바이오필름 형성을 억제하는 활성이 있음을 확인하였고, 또한 본 발명의 은 나노입자는 바이오필름의 막에 큰 구멍을 형성하게 하며 추가의 기공 형성도 가능하게 하여 유해균의 세포사멸을 유도할 수 있음을 알 수 있었다. 뿐만 아니라 본 발명의 은 나노입자는 유해균의 표면에 결합하여 정상적인 성장을 억제하고 세포막에 이상을 유발하여 유해균에 대한 우수한 항균 활성을 갖는다는 것을 알 수 있었다.On the other hand, it was confirmed that the silver nanoparticles using the tomato extract of the present invention have the activity of inhibiting the formation of a biofilm formed by Candida bacteria, and the silver nanoparticles of the present invention form a large hole in the film of the biofilm. It was found that it was possible to induce apoptosis of harmful bacteria by enabling the formation of additional pores. In addition, it was found that the silver nanoparticles of the present invention bind to the surface of harmful bacteria to inhibit normal growth and cause abnormalities in cell membranes, thereby having excellent antibacterial activity against harmful bacteria.
그러므로 본 발명은 본 발명의 방법으로 제조된 토마토 추출물을 이용한 항균성 은 나노입자를 제공할 수 있다.Therefore, the present invention can provide antimicrobial silver nanoparticles using the tomato extract prepared by the method of the present invention.
본 발명의 방법으로 제조된 은 나노입자는 입자의 크기가 평균적으로 약 100 nm의 직경을 가지며 구형의 형태를 갖는다.The silver nanoparticles prepared by the method of the present invention have a particle size of about 100 nm on average and have a spherical shape.
또한 본 발명의 토마토 추출물을 이용한 항균성 은 나노입자는 우수한 항균활성을 가질 수 있는데, 바람직하게는 칸디다 균에 대한 항균활성을 가질 수 있으며, 상기 칸디다 균으로는 이에 제한되지는 않으나, 칸디다 균은 칸디다 알비칸스(C. albicans), 칸디다 트로피칼리스(Candidan tropicalis), 칸디다 글라브라타(Candida glabrata), 칸디다 크루제(C.krusei) 및 칸디다 파라피실로시스(C. parapsilosis)으로 이루어진 군 중에서 선택되는 것일 수 있다.In addition, the antimicrobial silver nanoparticles using the tomato extract of the present invention may have excellent antimicrobial activity, preferably may have antibacterial activity against Candida, and the Candida is not limited thereto, but Candida is Candida. Albicans (C. albicans), Candidan tropicalis (Candidan tropicalis), Candida glabrata (Candida glabrata), Candida cruse (C. krusei) and Candida parapsilosis (C. parapsilosis) to be selected from the group consisting of. I can.
본 발명의 토마토 추출물을 이용한 은 나노입자의 칸디다 균에 대한 항균활성은 칸디다 균에 대한 생장억제, 생착억제 또는 바이오필름 형성억제를 통한 작용으로 항균활성을 갖는다.The antimicrobial activity of the silver nanoparticles using the tomato extract of the present invention against Candida bacteria has antibacterial activity by acting through inhibition of growth, engraftment, or inhibition of biofilm formation against Candida bacteria.
나아가 본 발명은 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다증(candidasis)의 예방 및 치료용 약학 조성물을 제공할 수 있다.Furthermore, the present invention can provide a pharmaceutical composition for the prevention and treatment of candidiasis, including antimicrobial silver nanoparticles using tomato extract.
칸디다증은 칸디다 균(진균 또는 곰팡이)에 의해 신체의 일부 또는 여러 부위가 감염되어 발생하는 질환으로, 칸디다 균은 흔히 피부나 점막의 표층에 국한된 감염증을 일으키며, 아구창을 포함하여 구인두나 식도의 염증, 외음부염, 질염, 손발톱 주위염 등의 증상이 나타난다. 구인두나 식도에 칸디다가 증식하게 될 경우, 입안이 불편하고 맛을 잘 느끼지 못하며 음식을 씹거나 넘길 때 통증이 나타날 수 있다. 칸디다성 외음질염은 가장 흔한 표재성 칸디다증으로, 외음부의 소양감, 따가움, 질 통증, 성교 시의 통증 및 흰 색의 덩어리진 질 분비물 등의 증상이 나타난다.Candidiasis is a disease caused by infection of parts or parts of the body by Candida bacteria (fungi or fungi).Candida bacteria often cause infections confined to the superficial layer of the skin or mucous membrane, and inflammation of the oropharynx or esophagus, including thrush, Symptoms such as vulvitis, vaginitis, and peritonitis appear. When Candida proliferates in the oropharynx or esophagus, the mouth feels uncomfortable, tastes poor, and can cause pain when chewing or passing food. Candidal vulvovaginitis is the most common superficial candidiasis, and symptoms such as itching of the vulva, itching, vaginal pain, pain during intercourse, and white lumpy vaginal discharge appear.
또한, 혈류를 통해 균이 신체 여러 부위로 퍼지는 침습성 칸디다증은 주로 호중구 감소증 환자에게서 발생하며, 이 경우 칸디다균이 신장, 심장, 간, 뇌, 안구 등 다양한 장기에 침범하여 병적인 변화를 일으키며 발열, 오한 등 일반적인 감염 질환에서 나타날 수 있는 전반적인 신체 증상이 동반될 수 있다.In addition, invasive candidiasis, in which bacteria spread to various parts of the body through the bloodstream, mainly occurs in patients with neutropenia.In this case, Candida bacteria invade various organs such as the kidney, heart, liver, brain, and eyes, causing pathological changes and fever, It can be accompanied by general physical symptoms that can occur in common infectious diseases, such as chills.
본 발명에서 상기 칸디다증을 유발시키는 칸디다균으로는 이에 제한되지는 않으나, 칸디다 알비칸스(C. albicans), 칸디다 트로피칼리스(Candidan tropicalis), 칸디다 글라브라타(Candida glabrata), 칸디다 크루제(C.krusei) 및 칸디다 파라피실로시스(C. parapsilosis)으로 이루어진 군 중에서 선택되는 것일 수 있다.In the present invention, the Candida bacteria causing candidiasis are not limited thereto, but candida albicans, Candidan tropicalis, Candida glabrata, and C. krusei ) And Candida parapsilosis (C. parapsilosis) may be selected from the group consisting of.
본 발명에서 용어 "예방"이란, 본 발명의 약학적 조성물의 투여로 칸디다증의 발병을 억제 또는 지연시키는 모든 행위를 의미하며, "치료"란, 본 발명의 약학적 조성물의 투여로 인해 이미 유발된 칸디다증의 증세가 호전되거나 이롭게 되는 모든 행위를 의미한다. In the present invention, the term "prevention" refers to any action that suppresses or delays the onset of candidiasis by administration of the pharmaceutical composition of the present invention, and "treatment" is already induced by the administration of the pharmaceutical composition of the present invention. It refers to any action that improves or benefits the symptoms of candidiasis.
본 발명에 따른 토마토 추출물을 이용한 항균성 은 나노입자는 약제학적으로 허용되는 담체와 함께 적절하게 제제화될 수 있다. 약제학적으로 허용되는 담체는 경구 투여시에는 결합제, 활택제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소 및 향료 등을 사용할 수 있으며, 주사제의 경우에는 주사제는 생리식염액 및 링겔액 등의 수성용제, 식물유, 고급 지방산 에스텔(예, 올레인산에칠 등) 및 알코올류(예, 에탄올, 벤질알코올, 프로필렌글리콜, 글리세린 등) 등의 비수성 용제 등을 이용하여 제조할 수 있고, 완충제, 보존제, 무통화제, 가용화제, 등장화제 및 안정화제 등을 혼합하여 사용할 수 있다. 분사제의 경우에는 적합한 분사제, 예컨대, 압축공기, 질소, 이산화탄소, 또는 탄화수소 기반 낮은 끓는점 용매 등을 사용하여 가압팩 또는 분무기로부터 에어로졸 스프레이 형태로 편리하게 전달될 수 있다.Antimicrobial silver nanoparticles using the tomato extract according to the present invention may be appropriately formulated together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colors and fragrances, etc., when administered orally.In the case of injections, the injection is a physiological saline solution. And aqueous solvents such as Ringel's solution, vegetable oils, higher fatty acid esters (e.g., oleic acid ethyl, etc.) and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). In addition, a buffering agent, a preservative, a painless agent, a solubilizing agent, an isotonic agent, and a stabilizer may be mixed and used. In the case of a propellant, a suitable propellant such as compressed air, nitrogen, carbon dioxide, or hydrocarbon-based low boiling point solvent can be conveniently delivered from a pressurized pack or nebulizer in the form of an aerosol spray.
본 발명의 약제학적 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릴시르, 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다.The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above. For example, when administered orally, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like, and in the case of injections, it may be prepared in the form of unit dosage ampoules or multiple dosage forms.
상기 약학적 조성물의 투여 경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다.The route of administration of the pharmaceutical composition may be administered through any general route as long as it can reach the target tissue.
본 발명의 용어 "투여"란, 어떠한 적절한 방법으로 환자에게 소정의 물질을 도입하는 것을 의미하며, 인체용으로 제형화되어 다양한 경로로 투여된다. 본 발명의 약학적 조성물은 비경구 경로, 예컨대 혈관내, 정맥내, 동맥내, 근육내 또는 피하 등의 경로로 투여될 수 있고, 경구, 비강, 직장, 경피 또는 에어로졸을 통한 흡입 경로로 투여될 수도 있으며, 볼루스(bolus)로 투여하거나 또는 서서히 주입할 수도 있으나, 바람직하게는 근육내 또는 피하 주사로 투여되는 것이 바람직하다.The term "administration" of the present invention means introducing a predetermined substance to a patient by any suitable method, and is formulated for human use and administered by various routes. The pharmaceutical composition of the present invention may be administered by a parenteral route, such as intravascular, intravenous, intraarterial, intramuscular or subcutaneous, orally, nasal, rectal, transdermal, or by inhalation via aerosol. Alternatively, it may be administered as a bolus or may be slowly injected, but it is preferably administered by intramuscular or subcutaneous injection.
본 발명의 약학적 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명의 용어 "약제학적으로 유효한 양"이란, 칸디다 균에 대한 항균 효과를 나타낼 수 있을 정도의 충분한 양과 부작용이나 심각한 또는 과도한 면역반응을 일으키지 않을 정도의 양을 의미하며, 유효 용량 수준은 치료될 장애, 장애의 중증도, 특정 화합물의 활성, 투여 경로, 제거 속도, 치료 지속 기간, 대상체의 연령, 체중, 성별, 식습관, 일반적인 건강상태, 및 의약 업계 및 의학 분야에 공지된 인자를 비롯한 다양한 인자들에 따라 달라질 것이다. "치료상 유효량" 결정시 고려되는 다양한 일반적인 사항들은 당업자에게 공지되어 있으며, 예를 들어 문헌 [Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990] 및 [Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990]에 기재되어 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to exhibit an antimicrobial effect against Candida and an amount that does not cause side effects or serious or excessive immune reactions, and the effective dose level is to be treated. Disorder, severity of the disorder, activity of a particular compound, route of administration, rate of elimination, duration of treatment, age, weight, sex, diet, general health status of the subject, and various factors, including factors known in the pharmaceutical and medical arts. It will depend on. Various general considerations to be taken into account when determining a "therapeutically effective amount" are known to those of skill in the art, for example Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990 ] And Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990.
또한, 본 발명의 약학적 조성물은 약학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다. 본 발명의 약학적 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토오스, 덱스트로오스, 수크로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀룰로오스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다.In addition, the pharmaceutical composition of the present invention may further include suitable carriers, excipients, and diluents commonly used in the preparation of pharmaceutical compositions. Carriers, excipients and diluents that may be included in the pharmaceutical composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
본 발명에 따른 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 외용제, 좌제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있다. 당해 기술분야에 알려진 적합한 제제는 문헌(Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA)에 개시되어 있는 것을 사용하는 것이 바람직하다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트, 수크로오스, 락토오스, 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조 제제, 좌제가 포함된다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.The pharmaceutical composition according to the present invention may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. I can. Suitable formulations known in the art are preferably those disclosed in Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are prepared by mixing at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, and suppositories. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.
또한 본 발명은 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다증 개선용 피부 외용제 조성물을 제공할 수 있다.In addition, the present invention can provide a composition for external application for skin for improving candidiasis, including antimicrobial silver nanoparticles using tomato extract.
나아가 본 발명은 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다 균이 형성하는 바이오필름의 생성억제용 조성물을 제공할 수 있다.Furthermore, the present invention can provide a composition for inhibiting the production of a biofilm formed by Candida bacteria, including antimicrobial silver nanoparticles using a tomato extract.
본 발명의 방법으로 제조된 항균성 은 나노입자는 칸디다 균에 의해 형성되는 바이오필름의 막에 구멍을 형성하게 하여 바이오필름의 생성을 억제함으로 통해 궁극적으로 칸디다 균의 세포사멸을 유발시킬 수 있다. The antimicrobial silver nanoparticles prepared by the method of the present invention can ultimately cause apoptosis of Candida bacteria by inhibiting the production of the biofilm by forming holes in the film of the biofilm formed by Candida bacteria.
그러므로 본 발명의 방법에 의해 제조된 토마토 추출물을 함유한 은 나노입자는 우수한 항균활성이 있어 항균용 조성물로 사용될 수 있으며, 특히 칸디다 균에 대한 항균용 조성물로 사용될 수 있다.Therefore, the silver nanoparticles containing the tomato extract prepared by the method of the present invention has excellent antibacterial activity and can be used as an antibacterial composition, and in particular, can be used as an antibacterial composition against Candida bacteria.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. These examples are for explaining the present invention more specifically, and the scope of the present invention is not limited to these examples.
<실시예 1><Example 1>
토마토 추출물을 이용한 항균성 은 나노입자의 제조 Preparation of antimicrobial silver nanoparticles using tomato extract
<1-1> 토마토 추출물의 제조<1-1> Preparation of tomato extract
신선한 보통 토마토(Lycopersicon esculentum var. commune)를 시장에서 구입한 후, 탈 이온수를 이용하여 토마토의 표면을 깨끗이 세척하였다. 과육을 얻기 위해 껍질을 벗기고 열매를 잘게 썰었다. 이후, 토마토 열매 부위를 균질화시켜 토마토 즙을 수득하였으며, 상기 토마토 즙을 8000 rpm에서 15 분 동안 원심분리하여 과일 찌꺼기를 제거하고 상층액을 수득한 다음, 이를 Whatman No.1 여과지를 사용하여 여과하였다. 여과된 수용성 토마토 추출물을 0.25㎛ 주사기 필터를 이용하여 1 회 더 여과시켜 잔류 오염물을 제거하였고, 여과액은 나노입자의 합성에 이용될 때까지 4℃에서 보관하였다.After purchasing fresh ordinary tomatoes (Lycopersicon esculentum var. commune) from the market, the surface of the tomatoes was cleaned with deionized water. Peeled and chopped the fruit to obtain the flesh. Thereafter, the tomato fruit portion was homogenized to obtain tomato juice, and the tomato juice was centrifuged at 8000 rpm for 15 minutes to remove fruit residue and a supernatant was obtained, which was then filtered using Whatman No. 1 filter paper. . The filtered water-soluble tomato extract was filtered once more using a 0.25 μm syringe filter to remove residual contaminants, and the filtrate was stored at 4° C. until used for synthesis of nanoparticles.
<1-2> 토마토 추출물을 이용한 은 나노입자의 제조<1-2> Preparation of silver nanoparticles using tomato extract
은 나노입자의 합성에 사용된 질산은(AgNO3)은 Sigma-Aldrich(미국 미주리 주 세인트 루이스)에서 구입하여 사용하였다. 상기 <1-1>에서 제조한 토마토 추출물(10 mL)을 40 mL의 질산은 용액(AgNO3, 5 mM)에 20분에 걸쳐 적가하면서 온화한 교반(120rpm) 하에 실온에서 배양하였다. 이때 대조군으로는 질산은 용액에 토마토 추출물을 첨가하지 않은 군을 사용하였다. 이후, 은 나노입자(AgNPs)의 농도를 결정하기 위해, 은 나노 입자가 포함된 질산은과 토마토 추출물의 혼합물을 13,000rpm에서 15 분 동안 원심 분리하였다. 그런 뒤, 은 나노입자의 펠렛을 탈 이온수로 3회 세척하여 불순물을 제거하였다. 마지막으로 동결건조를 수행하여 정제된 본 발명의 토마토 추출물을 이용한 은 나노입자(AgNPs)의 분말을 수득한 후, 이를 탈 이온수에 녹여 저장액으로 보관한 후, 하기의 실시예에 사용하였다. Silver nitrate (AgNO 3 ) used for the synthesis of silver nanoparticles was purchased from Sigma-Aldrich (St. Louis, Missouri, USA) and used. The tomato extract (10 mL) prepared in <1-1> was added dropwise over 20 minutes to 40 mL of silver nitrate solution (AgNO 3 , 5 mM) and incubated at room temperature under gentle stirring (120 rpm). At this time, as a control group, a group in which the tomato extract was not added to the silver nitrate solution was used. Thereafter, in order to determine the concentration of silver nanoparticles (AgNPs), a mixture of silver nitrate and tomato extract containing silver nanoparticles was centrifuged at 13,000 rpm for 15 minutes. Then, the pellets of the silver nanoparticles were washed three times with deionized water to remove impurities. Finally, freeze-drying was performed to obtain a powder of silver nanoparticles (AgNPs) using the purified tomato extract of the present invention, and then dissolved in deionized water and stored as a stock solution, and then used in the following examples.
<실시예 2><Example 2>
본 발명의 토마토 추출물을 이용하여 제조한 은 나노입자의 특성분석Characterization of silver nanoparticles prepared using the tomato extract of the present invention
상기 실시예 1에서 제조한 본 발명의 토마토 추출물을 이용한 은 나노입자에 대한 특성 분석을 다음과 같이 수행하였다.Characterization of the silver nanoparticles using the tomato extract of the present invention prepared in Example 1 was performed as follows.
UV-Vis 분광 광도계는 나노입자의 합성을 확인하는데 사용되는 분석방법으로, 시간에 따른 입자 합성의 수율을 확인하기 위해 토마토 추출물과 질산은 혼합물을 3 분마다 300~800nm의 흡광도 범위에서 흡광도를 측정하였으며, 상기 측정은 흡광도의 변화가 더 이상 감지되지 않을 때까지 수행하였다.The UV-Vis spectrophotometer is an analysis method used to confirm the synthesis of nanoparticles.To confirm the yield of particle synthesis over time, a mixture of tomato extract and silver nitrate was measured every 3 minutes in the absorbance range of 300 to 800 nm. , The measurement was performed until a change in absorbance was no longer detected.
또한, 주사 전자현미경(SEM)을 이용한 이미지 분석은 나노입자의 크기 및 모양에 대한 형태학적 특성을 분석하는 방법으로, 본 발명에서 제조된 토마토 추출물을 이용한 콜로이드의 은 나노입자 용액을 커버 유리에 묻혀 밤새도록 건조시킨 후, 커버 유리를 구리 스터브 위에 놓고 카본 테이프로 고정시켰다. SEM 이미지는 JEOL, Ltd (일본 도쿄) 기기 (JSM-6701F)로 10KV 가속 전압에서 획득하였고, SEM에 포함된 기기와 동시에 EDAX 분석을 수행하여 원소 은의 존재를 확인하였다. In addition, image analysis using a scanning electron microscope (SEM) is a method of analyzing the morphological characteristics of the size and shape of nanoparticles. A solution of colloidal silver nanoparticles using the tomato extract prepared in the present invention is buried on a cover glass. After drying overnight, the cover glass was placed over a copper stub and fixed with carbon tape. SEM images were acquired at 10KV acceleration voltage with a JEOL, Ltd (Tokyo, Japan) instrument (JSM-6701F), and EDAX analysis was performed simultaneously with the instrument included in the SEM to confirm the presence of elemental silver.
입자 크기 분포의 측정은 25 ℃에서 동적 광산란(DLS, Zetasizer nano S90 시스템, Malvern, UK)을 사용하여 분석하였고, 은 나노입자의 합성에 기여한 생체 분자의 작용기를 조사하기 위해 푸리에 변환-적외선 분광법 (FT-IR)을 사용하였다. FT-IR (Spectrum 100, Perkin Elmer Corporation, Norwalk, CT, USA) 측정은 4000 ~ 400cm-1 사이에서 수행하였다.The measurement of the particle size distribution was analyzed using dynamic light scattering (DLS, Zetasizer nano S90 system, Malvern, UK) at 25°C, and Fourier transform-infrared spectroscopy (Fourier transform-infrared spectroscopy) to investigate the functional groups of biomolecules that contributed to the synthesis of silver nanoparticles. FT-IR) was used. FT-IR (Spectrum 100, Perkin Elmer Corporation, Norwalk, CT, USA) measurement was performed between 4000 ~ 400cm -1.
<2-1> UV-Vis 분광 광도계 분석결과<2-1> UV-Vis spectrophotometer analysis result
색 변화는 Ag+ 이온의 감소에 의해 발생하며 은 나노입자(AgNP)의 합성을 확인하는 지표가 된다. UV-Vis 분광 광도계 분석 결과, 질산은 용액과 토마토 추출물의 혼합물은 반응에 의해 점차적으로 무색에서 노란색으로 바뀌었다가 약 10 분 후 적갈색으로 바뀌어 은 나노입자가 합성되었음을 확인할 수 있었다(도 1). 반면, 대조군은 무색인 것으로 나타났다. 또한, 질산은 용액과 토마토 추출물의 혼합물을 UV-Vis 분광 광도법으로 분석한 결과, 표면 플라즈몬 공명(SPR)이라고 하는 특정한 피크 패턴이 나타나는 것을 확인할 수 있었다. SRP 패턴은 각각의 금속 입자의 특징, 예컨대 크기, 모양, 합성에 사용되는 매체의 유전 특성 및 나노입자 간 결합 상호작용에 따라 달라진다. 본 발명의 질산은 용액과 토마토 추출물의 혼합물에서의 λmax는 445nm에서 관찰되었고, SPR 밴드의 강도는 반응시간에 따라 증가하여 은 나노입자가 합성되었음을 확인하였다(도 2). 또한 최대 흡광도는 12분에 기록되었고 이로써 반응이 종료되었음을 확인하였다. The color change is caused by the reduction of Ag+ ions, and is an index confirming the synthesis of silver nanoparticles (AgNP). As a result of UV-Vis spectrophotometer analysis, the mixture of the silver nitrate solution and the tomato extract gradually changed from colorless to yellow by reaction, and then changed to reddish brown after about 10 minutes, confirming that silver nanoparticles were synthesized (FIG. 1). On the other hand, the control group was found to be colorless. In addition, as a result of analyzing the mixture of silver nitrate solution and tomato extract by UV-Vis spectrophotometry, it was confirmed that a specific peak pattern called surface plasmon resonance (SPR) appeared. The SRP pattern depends on the characteristics of each metal particle, such as the size, shape, dielectric properties of the medium used for synthesis, and the bonding interactions between the nanoparticles. Λmax in the mixture of the silver nitrate solution and tomato extract of the present invention was observed at 445 nm, and the intensity of the SPR band increased with the reaction time, confirming that silver nanoparticles were synthesized (FIG. 2). In addition, the maximum absorbance was recorded at 12 minutes, thereby confirming that the reaction was terminated.
<2-2> 주사 전자현미경(SEM) 분석결과<2-2> Scanning electron microscope (SEM) analysis result
또한 본 발명에서 제조된 토마토 추출물을 이용한 은 나노입자에 대한 주사 전자현미경 관찰 결과, 도 2에 나타낸 바와 같이 본 발명의 방법에 의해 은 나노입자가 합성되었음을 SEM 이미지를 통해 확인하였다. 구체적으로, 본 발명에 의해 제조된 토마토 추출물을 이용한 은 나노입자의 모양은 주로 구형이며, 입자 크기는 응집 없이 직경이 약 100nm의 범위인 것으로 나타났다.In addition, as a result of observation with a scanning electron microscope for silver nanoparticles using the tomato extract prepared in the present invention, it was confirmed through SEM images that silver nanoparticles were synthesized by the method of the present invention as shown in FIG. 2. Specifically, it was found that the shape of the silver nanoparticles using the tomato extract prepared by the present invention is mainly spherical, and the particle size is in the range of about 100 nm in diameter without aggregation.
이러한 결과를 통해, 본 발명자들은 토마토 추출물이 은 나노입자를 합성하는 과정에서 합성 및 분산에 관여하는 환원제 및 캡핑제로서의 역할을 하였음을 알 수 있었고, 도 2의 하단 결과를 통해, EDAX 스펙트럼에서 은 원자의 강한신호를 확인하였으며, 이 또한 본 발명의 방법으로 은 나노입자가 합성되었음을 알 수 있었다. 또한 토마토 추출물에 존재하는 유기 물질인 탄소, 산소, 황 및 칼륨의 신호를 확인할 수 있었다. Through these results, the present inventors found that the tomato extract played a role as a reducing agent and capping agent involved in synthesis and dispersion in the process of synthesizing silver nanoparticles, and through the lower result of FIG. 2, in the EDAX spectrum It was confirmed that the strong signal of the silver atom was confirmed, and it was also found that silver nanoparticles were synthesized by the method of the present invention. In addition, signals of carbon, oxygen, sulfur, and potassium, which are organic substances present in the tomato extract, were confirmed.
<2-3> 동적 광산란(Dynamic light scattering (DLS)) 분석결과<2-3> Dynamic light scattering (DLS) analysis result
동적 광산란 분석을 통해 본 발명의 은 나노입자의 크기 분포와 콜로이드 안정성을 72 시간 동안 분석하였다. 그 결과, 도 4에 나타낸 바와 같이, z- 평균값은 24 시간에서 99.16nm(3a)인 것으로 나타났고, 48 시간에서 100.2nm(3b), 72 시간 에서 108.5nm(3c)로 확인되었으며, 이는 입자 크기가 48 시간 동안 거의 일정하게 유지되었음을 의미한다. 또한, 48 시간에서 0.232의 낮은 다분산 지수(PDI)가 확인되어 좁은 입자 크기 분포가 관찰되었다.The size distribution and colloidal stability of the silver nanoparticles of the present invention were analyzed for 72 hours through dynamic light scattering analysis. As a result, as shown in FIG. 4, the z-average value was found to be 99.16 nm (3a) at 24 hours, 100.2 nm (3b) at 48 hours, and 108.5 nm (3c) at 72 hours, which This means that the size remained almost constant over 48 hours. In addition, a low polydispersity index (PDI) of 0.232 was observed at 48 hours, and a narrow particle size distribution was observed.
<2-4> 푸리에 변환-적외선 분광법(FT-IR) 분석결과<2-4> Fourier transform-infrared spectroscopy (FT-IR) analysis result
FT-IR 분석을 통해 본 발명의 은 나노입자 합성에 관여하는 토마토 추출물의 작용기를 확인하였다. Ag + 이온을 Ag0로 변환하는 주요 요인은 FT-IR 스펙트럼에 표시되어진다. 본 발명에 따른 토마토 추출물 및 질산은 용액의 혼합용액에 대한 FT-IR 분석결과와 토마토 추출물만을 사용한 FT-IR 분석결과를 비교하였고 그 결과를 도 5에 나타내었다. Through FT-IR analysis, the functional groups of the tomato extract involved in the synthesis of silver nanoparticles of the present invention were confirmed. The main factor converting Ag + ions to Ag 0 is indicated in the FT-IR spectrum. The FT-IR analysis results of the mixed solution of the tomato extract and the silver nitrate solution according to the present invention and the FT-IR analysis results using only the tomato extract were compared, and the results are shown in FIG. 5.
구체적으로 도 5에 나타낸 바와 같이, 토마토 추출물의 뚜렷한 흡수 피크는 3296.71, 2137.28, 1635.83 및 1063.18 cm-1에서 관찰되었다. 여기서 3296.71 cm-1에서의 피크는 알코올과 페놀에서 -OH 결합의 신축 진동(stretching vibration)에 해당하며, 2137.28 cm-1에서 약한 강도 피크는 알키닐 그룹(alkynyl groups)에서 -C≡C- 결합의 신축 진동에 의한 것이다. 또한, 1635.83 cm-1에서의 피크는 플라보노이드 및 테르페노이드와 관련된 C = O 스트레칭 진동에 의한 피크이며, 1063.18 cm-1에서의 피크는 에스테르 및 에테르에서 C-O 신장 진동을 나타내는 것으로 추정할 수 있다. 또한, -OH 및 C = O 결합은 Ag +에서 Ag0 로의 환원과 관련이 있으며, 알코올, 페놀 및 플라보노이드에 포함된 작용기는 은 나노입자의 감소 및 안정화에 관여하는 캡핑제 역할을 한다는 것을 알 수 있다.Specifically, as shown in Figure 5, the apparent absorption peaks of the tomato extract were observed at 3296.71, 2137.28, 1635.83 and 1063.18 cm -1. Here, the peak at 3296.71 cm -1 corresponds to the stretching vibration of the -OH bond in alcohol and phenol, and the weak intensity peak at 2137.28 cm -1 is -C≡C- bond in the alkynyl groups. It is due to the expansion and contraction of the vibration. In addition, the peak at 1635.83 cm -1 is a peak due to C = O stretching vibrations associated with flavonoids and terpenoids, and the peak at 1063.18 cm -1 can be estimated to indicate CO elongation vibration in esters and ethers. In addition, -OH and C = O bonds are related to the reduction of Ag + to Ag 0 , and the functional groups contained in alcohols, phenols and flavonoids act as capping agents involved in the reduction and stabilization of silver nanoparticles. have.
<실시예 3><Example 3>
본 발명의 토마토 추출물을 이용한 은 나노입자의 항균 및 항바이오필름 활성 분석Analysis of antibacterial and anti-biofilm activity of silver nanoparticles using tomato extract of the present invention
<3-1> 본 발명의 토마토 추출물을 이용한 은 나노입자의 최소억제 농도(MIC) 분석<3-1> Analysis of the minimum inhibitory concentration (MIC) of silver nanoparticles using the tomato extract of the present invention
본 발명의 은 나노입자에 대한 항균활성 분석을 위해, 유해균으로 칸디다균에 해당하는 C. albicans (ATCC 90028), C. parasilopsis (ATCC 90018) 및 C. glabrata (ATCC 90030) 균에 대한 본 발명의 은 나노입자의 최소억제 농도(MIC)를 측정하였다. 이를 위해 본 발명에 따른 토마토 추출물을 이용하여 제조된 은 나노 입자를 연속적으로 2 배 희석한 희석액을 2 ~ 64 μg/mL 범위의 최종 농도로 준비하고, 96 웰 평평한 바닥 마이크로 타이터 플레이트에 첨가한 후, 0.5 McFarland (1~5 x 106 CFU/mL)로 조정된 칸다다 균의 현탁액을 0.5~2.5 x 103 CFU/mL의 최종 밀도로 RPMI 1640 배지에 현탁시키고, 희석된 은 나노입자를 포함하는 테스트 웰에 첨가하였다. 이후 모든 플레이트는 37 ℃에서 48 시간 동안 정치 배양하였다. 이후, 흡광도 측정에 있어서 침전된 세포의 영향을 제거하기 위해, 피펫팅을 통해 각 웰의 세포를 재현탁 한 다음, 620 nm에서의 칸디다균의 성장확인을 마이크로 플레이트 리더 (SpectraMax 190, Molecular Devices, Downingtown, PA, USA)를 사용하여 흡광도를 측정하였다. 이때 대조군에 비해 흡광도가 50 % 미만으로 감소한 최저 농도를 MIC로 간주하여 분석하였다.In order to analyze the antimicrobial activity of the silver nanoparticles of the present invention, C. albicans (ATCC 90028), C. parasilopsis (ATCC 90018) and C. glabrata (ATCC 90030) corresponding to Candida bacteria as harmful bacteria of the present invention The minimum inhibitory concentration (MIC) of silver nanoparticles was measured. To this end, a dilution solution obtained by continuously diluting the silver nanoparticles prepared using the tomato extract according to the present invention twice was prepared at a final concentration in the range of 2 to 64 μg/mL, and added to a 96-well flat bottom microtiter plate. Then, a suspension of Candada bacteria adjusted to 0.5 McFarland (1-5 x 10 6 CFU/mL) was suspended in RPMI 1640 medium at a final density of 0.5-2.5 x 10 3 CFU/mL, and the diluted silver nanoparticles were It was added to the containing test well. Thereafter, all plates were incubated at 37° C. for 48 hours. Thereafter, in order to remove the influence of the precipitated cells in the absorbance measurement, the cells in each well were resuspended through pipetting, and then the growth of Candida at 620 nm was confirmed with a microplate reader (SpectraMax 190, Molecular Devices, Downingtown, PA, USA) was used to measure absorbance. At this time, the lowest concentration at which the absorbance decreased by less than 50% compared to the control was considered as MIC and analyzed.
그 결과, 테스트 한 모든 칸디다 종의 유해균은 본 발명의 은 나노입자에 의해 성장이 저해되는 것으로 나타났으며, 은 나노입자를 8 μg/mL 처리한 경우, 실험에 사용한 C. albicans (ATCC 90028), C. parasilopsis (ATCC 90018) 및 C. glabrata (ATCC 90030) 균의 성장이 대조군에 비해 50%로 저해되는 것을 확인하였으며, 특히 대조군에 비해 8㎍/mL 이상의 양으로 본 발명의 은 나노 입자를 처리한 군에서는 이들 유해균의 성장이 유의하게 억제되었다(도 6).As a result, it was found that the growth of harmful bacteria of all Candida species tested was inhibited by the silver nanoparticles of the present invention. When the silver nanoparticles were treated with 8 μg/mL, C. albicans used in the experiment (ATCC 90028) It was confirmed that the growth of C. parasilopsis (ATCC 90018) and C. glabrata (ATCC 90030) bacteria was inhibited by 50% compared to the control, in particular, the silver nanoparticles of the present invention in an amount of 8 μg/mL or more compared to the control group. In the treated group, the growth of these harmful bacteria was significantly suppressed (FIG. 6).
<3-2> 기형성된 바이오필름에 대한 본 발명에 따른 은 나노입자의 억제활성 분석<3-2> Analysis of inhibitory activity of silver nanoparticles according to the present invention on preformed biofilm
바이오필름(biofilm)은 미생물들이 여러 외부 물질로 둘러싸인 구조로 생체 또는 무생물 표면에 형성될 수 있어 인체 내 지속 감염이나 삽입 의료 기구 관련 감염의 주원인이 되는 것으로 알려져 있다. 이에 본 발명자들은 기존 바이오필름(pre-existing biofilm)에 대하여 본 발명의 은 나노입자가 이에 대한 억제 효과가 있는지를 확인하기 위한 실험을 수행하였다.Biofilms are known to be the main cause of persistent infections in the human body or infections related to implanted medical devices because microbes are surrounded by various foreign substances and can be formed on a living body or inanimate surface. Accordingly, the present inventors conducted an experiment to confirm whether the silver nanoparticles of the present invention have an inhibitory effect on the existing biofilm (pre-existing biofilm).
이를 위해, 칸디다 바이오필름을 96웰 플레이트 표면에 37℃의 온도에서 24시간 동안 고정시켰다. 이후 PBS 용액으로 세척하여 부유 세포를 제거하였고, 상기 웰에 신선한 RPMI 1640 배지로 채우고 상기 실시예 <3-1>의 MIC 테스트에 사용한 것과 동일한 농도의 본 발명의 은 나노입자를 처리한 다음, 37℃의 온도에서 24시간 동안 추가 배양하였다. 이후 플레이트 바닥에 부착된 바이오필림을 피펫 팅으로 현탁하고 마이크로 플레이트 리더 기기를 사용하여 620 nm에서 흡광도를 측정하였다. 이때 탁도가 50% 이하로 감소되는 은 나노입자의 농도를 바이오필름 형성에 대한 최소저해농도(MIC)로 간주하였다.To this end, the Candida biofilm was fixed on the surface of a 96-well plate at a temperature of 37° C. for 24 hours. After washing with a PBS solution to remove suspended cells, the wells were filled with fresh RPMI 1640 medium and treated with the silver nanoparticles of the present invention having the same concentration as used in the MIC test of Example <3-1>, and then 37 It was further incubated for 24 hours at a temperature of °C. Thereafter, the biofilm attached to the bottom of the plate was suspended by pipetting, and absorbance was measured at 620 nm using a microplate reader device. At this time, the concentration of silver nanoparticles whose turbidity was reduced to 50% or less was regarded as the minimum inhibitory concentration (MIC) for biofilm formation.
분석 결과, 도 7에 나타낸 바와 같이, 본 발명의 토마토 추출물을 이용한 은 나노입자는 실험에 사용한 모든 칸디다 균들에 대하여 바이오필름을 억제하는 활성이 있는 것으로 나타났다. 구체적으로 C. albicans 및 C. glabrata의 MIC 값은 32 μg/mL인 것으로 나타났고, C. parasilopsis의 MIC는 8 μg/mL로 나타났다. 이를 통해 C. parasilopsis가 본 발명의 은 나노입자에 대해 가장 높은 감수성을 갖는다는 것을 알 수 있었다.As a result of the analysis, as shown in FIG. 7, it was found that the silver nanoparticles using the tomato extract of the present invention have the activity of inhibiting the biofilm against all Candida bacteria used in the experiment. Specifically, the MIC values of C. albicans and C. glabrata were found to be 32 μg/mL, and the MIC of C. parasilopsis was 8 μg/mL. Through this, it was found that C. parasilopsis has the highest sensitivity to the silver nanoparticles of the present invention.
<3-3> 본 발명의 은 나노입자가 처리된 칸디다 바이오필름의 형태학적 분석<3-3> Morphological analysis of Candida biofilm treated with silver nanoparticles of the present invention
나아가 본 발명자들은 본 발명의 토마토 추출물을 이용하여 제조된 은 나노 입자가 바이오필름 억제에 미치는 영향을 확인하기 위해 주사 전자현미경을 이용한 형태학적 이미지를 분석하였다. 이를 위해 칸디다 균주의 현탁액을 RPMI 1640 배지에서 0.5 McFarland로 조정하고 주문 제작된 20mm x 20mm 폴리스티렌 커버 슬립을 포함하는 6 웰 플레이트에 접종하였다. 폴리스티렌 커버 슬립을 37 ℃에서 2 시간 동안 사전 배양하여 칸디다 균주가 부착이 되도록 하였고, 사전 배양 후, 커버 슬립을 PBS로 부드럽게 세척하여 비 부착성 세포를 제거하고 테스트 웰을 64 μg/mL의 본 발명의 은 나노입자가 포함된 배지로 채운 다음, 37℃에서 48 시간 동안 배양하여 바이오필름이 형성되도록 하였다. 이때 대조군 웰은 본 발명의 은 나노입자를 포함하지 않은 신선한 RPMI 1640 배지로만 처리한 군을 사용하였다. 이후 바이오필름이 형성된 다음, 폴리스티렌 커버 슬립 상의 바이오필름을 PBS로 세척하고 PBS 버퍼를 이용한 2.5 % 글루타르알데히드를 이용하여 밤새도록 고정시켰다. 이후 에탄올 함량이 각기 다른 에탄올(70, 80, 90 및 100 %)을 사용하여 커버 슬립을 탈수하고 건조기에서 밤새도록 건조시켰다. 이후 자동 마그네트론 스퍼터 코팅기 시스템을 사용하여 샘플을 백금으로 코팅하고 주사 전자 현미경으로 관찰하였다. 전체 샘플 표면에 대한 은 나노 입자의 효과에 대한 대표적인 이미지를 확인하였다.Furthermore, the present inventors analyzed the morphological image using a scanning electron microscope to confirm the effect of the silver nanoparticles prepared using the tomato extract of the present invention on biofilm inhibition. To this end, the suspension of the Candida strain was adjusted to 0.5 McFarland in RPMI 1640 medium and inoculated into a 6-well plate containing a custom-made 20 mm x 20 mm polystyrene cover slip. Polystyrene coverslips were pre-incubated at 37°C for 2 hours to allow Candida strains to adhere, and after pre-cultivation, the coverslips were gently washed with PBS to remove non-adherent cells, and test wells of 64 μg/mL of the present invention Was filled with a medium containing silver nanoparticles, and then incubated at 37° C. for 48 hours to form a biofilm. At this time, the control well was used as a group treated with fresh RPMI 1640 medium that does not contain the silver nanoparticles of the present invention. After the biofilm was formed, the biofilm on the polystyrene cover slip was washed with PBS and fixed overnight using 2.5% glutaraldehyde using PBS buffer. Thereafter, the coverslips were dehydrated using ethanol (70, 80, 90, and 100%) having different ethanol contents and dried overnight in a dryer. The sample was then coated with platinum using an automatic magnetron sputter coater system and observed with a scanning electron microscope. A representative image of the effect of silver nanoparticles on the entire sample surface was confirmed.
그 결과, 상기 방법으로 관찰한 주사 전자현미경의 사진을 도 8에 나타내었는데, 도 8은 48 시간 동안 본 발명의 은 나노입자로 처리하기 전과 후의 칸디다 종에 대한 SEM 현미경 사진을 나타낸 것이다. 칸디다 종의 세포는 기질 또는 경계면에 비가역적으로 부착되어 미생물 군집에서 바이오필름이 형성되며 이는 칸디다 병원성의 주요 원인으로 작용한다. 이러한 생리학적 구조는 칸디다 종이 다양한 항균제들에 대한 내성을 가질 수 있도록 한다. 칸디다 종은 세포학적 형태로서 효모, 유사 균사(pseudo-hyphae) 및 균사(hyphae)의 3가지 종류를 가지고 있는 것으로 알려져 있다. C. glabrata는 이질적인 형태를 나타내지 않으며 오로지 효모의 형태를 형성하며, C. parasilopsis는 종종 거대 세포라고 불리는 효모 및 유사 균사 형태를 형성하고, 대조적으로, C. albicans는 효모, 유사 균사 및 균사 형태를 형성할 수 있다. 발아관 특징(germ tube feature)을 갖는 균사는 C. albicans의 진단에 임상적 중요성을 가지고 있다. As a result, a photograph of the scanning electron microscope observed by the above method is shown in FIG. 8, and FIG. 8 shows SEM micrographs of Candida species before and after treatment with the silver nanoparticles of the present invention for 48 hours. Cells of Candida species are irreversibly attached to the substrate or interface to form a biofilm in the microbial community, which acts as a major cause of Candida pathogenicity. This physiological structure makes Candida species resistant to various antimicrobial agents. Candida species are known to have three types of yeast, pseudo-hyphae, and hyphae as a cytological form. C. glabrata does not exhibit heterogeneous morphology and only forms yeast morphology, C. parasilopsis forms yeast and pseudohyphae morphologies, often called giant cells, whereas C. albicans forms yeast, pseudohyphae, and hyphae morphologies, often called giant cells. Can be formed. Hyphae with germ tube features have clinical importance in the diagnosis of C. albicans.
도 8에 나타난 주사 전자현미경 분석 결과, 대조군 C. albicans(8a)은 전형적이고 부드러운 형태를 보였으며 효모와 균사 형태가 바이오필름에서 발견되었다. 반면, 본 발명의 은 나노입자가 처리된C. albicans(8b)에서 균사는 관찰되지 않았고, rimose 막이 있는 단일 또는 버딩 효모만이 관찰되었다. 본 발명의 은 나노입자가 처리된 C. albicans를 고배율로 확인한 이미지(8c)를 살펴보니 은 나노입자가 근처 효모의 표면에 부착 및 응집되어 있는 것으로 나타났다(이미지에서 검은색 화살표). As a result of scanning electron microscopy analysis shown in FIG. 8, the control C. albicans (8a) showed a typical and smooth morphology, and yeast and hyphae morphology were found in the biofilm. On the other hand, the silver nanoparticles of the present invention treated C. albicans (8b), no hyphae was observed, and only single or budding yeasts with rimose membranes were observed. Looking at the image (8c) confirming the C. albicans treated with the silver nanoparticles of the present invention at high magnification, it was found that the silver nanoparticles adhered and aggregated on the surface of the nearby yeast (black arrow in the image).
C. parasilopsis에 대한 주사 전자현미경 분석 결과, 대조군 바이오필름 (8d)은 주로 규칙적인 형태를 가진 효모와 유사 균사로 구성되어 있었고, 본 발명의 은 나노입자를 처리한 경우(8e), 견고한 막을 가진 효모는 샘플 전체에 걸쳐 존재하고 있었으나 유사 균사는 관찰되지 않았다. 20,000 배 확대한 이미지 (8f)에는 본 발명의 은 나노입자가 주름진 비정상적인 효모 및 유사 균사의 표면에 위치하고 있는 것으로 나타났다(이미지에서 검은색 화살표).As a result of scanning electron microscopy analysis of C. parasilopsis, the control biofilm (8d) was mainly composed of yeast and similar hyphae with a regular shape, and when the silver nanoparticles of the present invention were treated (8e), it had a solid film. Yeast was present throughout the sample, but no similar hyphae was observed. In the image (8f) magnified 20,000 times, the silver nanoparticles of the present invention were found to be located on the surface of wrinkled abnormal yeast and similar hyphae (black arrows in the image).
C. glabrata 에 대한 주사 전자현미경 분석 결과, 대조군 C. glabrata의 바이오필름(8g)은 C. albicans 및 C. parasilopsis 보다 상당히 작은 구형 또는 타원형인 blastoconidia로만 구성되어 있는 것으로 나타났다. 본 발명의 은 나노입자를 처리한 C. glabrata (8h)의 경우에는 비정상적인 형태를 가지며, 일부 기공 및 파괴된 막이 효모에서 관찰되었다. 20,000 x 배율로 관찰한 도 8i는 C. albicans 및 C. parasilopsis에서도 관찰된 C. glabrata의 천공된 막 주위에 본 발명의 은 나노입자가(이미지의 검은색 화살표)가 붙어있는 것을 관찰할 수 있었다. As a result of scanning electron microscopy analysis of C. glabrata, it was found that the biofilm (8g) of the control C. glabrata consisted only of blastoconidia, which are spherical or elliptical, which is significantly smaller than that of C. albicans and C. parasilopsis. In the case of C. glabrata (8h) treated with silver nanoparticles of the present invention, it has an abnormal shape, and some pores and broken membranes were observed in yeast. 8i observed at 20,000 x magnification, it could be observed that silver nanoparticles of the present invention (black arrows in the image) adhered around the perforated membrane of C. glabrata observed in C. albicans and C. parasilopsis. .
이러한 결과를 통해 본 발명자들은 본 발명의 토마토 추출물을 이용한 은 나노입자가 유해균 또는 곰팡이에 대하여 막 전위를 방해하고 막 구조의 상호 작용에 영향을 줄 수 있는데, 특히 막에 큰 구멍을 형성하게 할 수 있고 추가의 기공 형성도 가능하게 하여 궁극적으로 유해균 또는 곰팡이의 세포사멸을 초래할 수 있음을 알 수 있었다. 또한 본 발명의 은 나노입자는 유해균 또는 곰팡이의 표면에 결합하여 정상적인 성장을 억제하고 세포막에 이상을 유발시키며 바이오필름의 형성을 억제하여 우수한 항균 활성을 갖는다는 것을 알 수 있었다.Through these results, the present inventors believe that the silver nanoparticles using the tomato extract of the present invention can interfere with the membrane potential and affect the interaction of the membrane structure with respect to harmful bacteria or fungi, in particular, it is possible to form a large hole in the membrane. In addition, it was found that the formation of additional pores could ultimately lead to apoptosis of harmful bacteria or fungi. In addition, it was found that the silver nanoparticles of the present invention bind to the surface of harmful bacteria or fungi to inhibit normal growth, cause abnormalities in cell membranes, and inhibit the formation of biofilms, thereby having excellent antibacterial activity.
이제까지 본 발명에 대하여 그 바람직한 실시 예들을 중심으로 살펴보았다. 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자는 본 발명이 본 발명의 본질적인 특성에서 벗어나지 않는 범위에서 변형된 형태로 구현될 수 있음을 이해할 수 있을 것이다. 그러므로 개시된 실시 예들은 한정적인 관점이 아니라 설명적인 관점에서 고려되어야 한다. 본 발명의 범위는 전술한 설명이 아니라 특허 청구범위에 나타나 있으며, 그와 동등한 범위 내에 있는 모든 차이점은 본 발명에 포함된 것으로 해석되어야 할 것이다.So far, the present invention has been looked at around its preferred embodiments. Those of ordinary skill in the art to which the present invention pertains will be able to understand that the present invention may be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from a descriptive point of view rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (13)

  1. 상온에서 토마토 추출물을 질산은 용액에 첨가하고 교반하는 단계를 포함하는, 토마토 추출물을 이용한 항균성 은 나노입자의 제조방법.A method for producing antimicrobial silver nanoparticles using tomato extract comprising the step of adding and stirring the tomato extract to the silver nitrate solution at room temperature.
  2. 제1항에 있어서,The method of claim 1,
    상기 토마토 추출물은,The tomato extract,
    토마토의 표면을 세척하고 껍질을 제거한 열매 부위를 균질화시켜 토마토 즙을 수득한 다음, 상기 토마토 즙을 원심 분리하여 상층액을 수득한 후, 여과시켜 얻은 토마토 추출물인 것을 특징으로 하는, 토마토 추출물을 이용한 항균성 은 나노입자의 제조방법.After washing the surface of the tomato and homogenizing the fruit portion from which the skin was removed to obtain tomato juice, the tomato juice was centrifuged to obtain a supernatant, and then filtered to obtain a tomato extract. Method for producing antimicrobial silver nanoparticles.
  3. 제1항에 있어서,The method of claim 1,
    상기 질산은 용액은 질산은의 농도가 3~7mM로 포함된 용액인 것을 특징으로 하는, 토마토 추출물을 이용한 항균성 은 나노입자의 제조방법.The silver nitrate solution is a method of producing antimicrobial silver nanoparticles using a tomato extract, characterized in that the silver nitrate is a solution containing 3 to 7 mM.
  4. 제1항에 있어서,The method of claim 1,
    상기 토마토는 에어룸 토마토(Heirloom Tomatoes), 비프스테이크 토마토(Beefsteak Tomatoes), 플럼 토마토(Plum Tomatoes), 노란 토마토(Yellow Tomatoes), 체리 토마토(Cherry Tomatoes), 그린 토마토(Green Tomatoes), 캄파리 토마토(Campari Tomatoes), 방울 토마토(Pear Tomatoes), 브랜드와인 토마토(Brandywine Tomatoes) 및 체로키퍼플 토마토(Cherokee Purple Tomatoes)로 이루어진 군 중에서 선택되는 것을 특징으로 하는, 토마토 추출물을 이용한 항균성 은 나노입자의 제조방법.The tomatoes are Heirloom Tomatoes, Beefsteak Tomatoes, Plum Tomatoes, Yellow Tomatoes, Cherry Tomatoes, Green Tomatoes, Campari Tomatoes (Campari Tomatoes), Cherry Tomatoes (Pear Tomatoes), Brandywine Tomatoes (Brandywine Tomatoes) and Cherokee Purple Tomatoes (Cherokee Purple Tomatoes), characterized in that selected from the group consisting of, a method for producing antimicrobial silver nanoparticles using tomato extract .
  5. 제1항 내지 제4항 중 어느 한 항의 방법으로 제조된 토마토 추출물을 이용한 항균성 은 나노입자.Antimicrobial silver nanoparticles using a tomato extract prepared by the method of any one of claims 1 to 4.
  6. 제5항에 있어서,The method of claim 5,
    상기 은 나노입자는 칸디다 균에 대한 항균활성을 갖는 것을 특징으로 하는, 토마토 추출물을 이용한 항균성 은 나노입자.The silver nanoparticles are antimicrobial silver nanoparticles using a tomato extract, characterized in that having antibacterial activity against Candida bacteria.
  7. 제5항에 있어서,The method of claim 5,
    상기 은 나노입자는 입자의 크기가 100 nm의 직경을 갖는 것을 특징으로 하는, 토마토 추출물을 이용한 항균성 은 나노입자.The silver nanoparticles are antimicrobial silver nanoparticles using tomato extract, characterized in that the particle size has a diameter of 100 nm.
  8. 제6항에 있어서,The method of claim 6,
    상기 항균활성은 칸디다 균에 대한 생장억제, 생착억제 또는 바이오필름 형성억제를 통해 항균활성을 갖는 것을 특징으로 하는, 토마토 추출물을 이용한 항균성 은 나노입자.The antimicrobial activity is antimicrobial silver nanoparticles using tomato extract, characterized in that it has antimicrobial activity through inhibition of growth, inhibition of engraftment, or inhibition of biofilm formation against Candida bacteria.
  9. 제8항에 있어서,The method of claim 8,
    상기 칸디다 균은 칸디다 알비칸스(C. albicans), 칸디다 트로피칼리스(Candidan tropicalis), 칸디다 글라브라타(Candida glabrata), 칸디다 크루제(C.krusei) 및 칸디다 파라피실로시스(C. parapsilosis)으로 이루어진 군 중에서 선택되는 것을 특징으로 하는, 토마토 추출물을 이용한 항균성 은 나노입자.The Candida fungus is composed of C. albicans, Candidan tropicalis, Candida glabrata, Candida glabrata, C. krusei, and C. parapsilosis. It characterized in that it is selected from the group, antimicrobial silver nanoparticles using a tomato extract.
  10. 제5항의 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다증 예방 및 치료용 약학 조성물.A pharmaceutical composition for preventing and treating candidiasis, comprising antimicrobial silver nanoparticles using the tomato extract of claim 5.
  11. 제10항에 있어서,The method of claim 10,
    상기 조성물은 칸디다 알비칸스(C. albicans), 칸디다 트로피칼리스(Candidan tropicalis), 칸디다 글라브라타(Candida glabrata), 칸디다 크루제(C.krusei) 및 칸디다 파라피실로시스(C. parapsilosis)으로 이루어진 군 중에서 선택되는 칸디다 균에 대한 항진균 활성을 갖는 것을 특징으로 하는, 칸디다증 예방 및 치료용 약학적 조성물.The composition is a group consisting of C. albicans, Candidan tropicalis, Candida glabrata, C. krusei, and C. parapsilosis. A pharmaceutical composition for preventing and treating candidiasis, characterized in that it has antifungal activity against Candida bacteria selected from.
  12. 제5항의 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다증 개선용 피부 외용제 조성물.A composition for external application for skin for improving candidiasis, including antimicrobial silver nanoparticles using the tomato extract of claim 5.
  13. 제5항의 토마토 추출물을 이용한 항균성 은 나노입자를 포함하는, 칸디다 균이 형성하는 바이오필름 생성억제용 조성물.A composition for inhibiting biofilm formation formed by Candida bacteria, comprising antimicrobial silver nanoparticles using the tomato extract of claim 5.
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