KR102048471B1 - Oral Composition Comprising Nano-capsule Comprising Natural antimicrobials - Google Patents

Abstract

The present invention provides an oral cleaning composition having an antimicrobial activity against oral microorganisms, including nanocapsules containing natural antibacterial extracts as an active ingredient, a pharmaceutical composition for preventing or treating periodontal disease, and preventing or improving periodontal disease. Health functional foods for use.
The nanocapsules containing natural antibacterial extracts and the composition for oral cavity containing the same according to the present invention greatly increase the antimicrobial activity by complexly capturing the natural extracts exhibiting synergistic action, and by controlling the coating properties of the capsules to improve oral mucosa adhesion. Not only is it possible to increase, by confirming the synergistic effect of the natural extracts and nanocapsules, there is an effect that the antimicrobial activity provides a composition for the oral cavity that is long lasting in the oral cavity. Therefore, the composition for oral cavity of the present invention can be applied as a safe antibacterial product for dental health as a composition for oral cleaning, various food groups or pharmaceutical compositions.

Description

Oral composition Comprising Nano-capsule Comprising Natural antimicrobials}

The present invention relates to a composition for oral cavity comprising a nano capsule containing a natural antimicrobial substance as an active ingredient, and more particularly, to an oral microorganism comprising a nano capsule having a natural antimicrobial extract collected therein as an active ingredient. Oral cleaning composition having an antimicrobial activity, a pharmaceutical composition for preventing or treating periodontal disease, and a health functional food for preventing or improving periodontal disease.

Caries and periodontitis are diseases caused by bacterial infections and tooth plaques. Streptococcus bacteria such as non-immunogenic Tansu caries (Streptococcus mutans) and Streptococcus small debris Taunus (Streptococcus sobrinus) is known to be the major causative agent of dental caries. This family of caries is known as dental plaque, and it is a general view of dentistry that it accumulates on the surface of teeth and causes tooth decay and periodontal disease.

For this reason, chemical antibacterial agents such as fluorine, chlorhexidine, xylitol and penicillin are known to play an important role in reducing the growth of bacteria in teeth. However, excessive use of these chemicals can lead to disturbance of the oral microbial community, and can cause side effects such as microbial sensitivity and tooth discoloration, diarrhea, vomiting.

Although fluoride is an essential factor in preventing tooth decay, excessive use of fluoride can increase the risk of developing cramps. In addition, chlorhexidine is an antimicrobial agent that has been commonly used to suppress oral diseases, but it has been found that it can cause side effects such as deterioration of taste function or tooth discoloration. Xylitol may also inhibit in vitro growth by inhibiting the degradation of S. mutans and some other oral bacteria, but bacteria exposed to xylitol for extended periods of time lose sensitivity and are no longer inhibited by xylitol. .

Therefore, there is a need for additional research on natural antimicrobial agents that are harmless to humans and have fewer side effects.

Natural antimicrobial extracts have weak antimicrobial activity, narrow antimicrobial spectrum, and low safety compared to chemical antimicrobial agents. They also have low solubility, unique taste, smell, and irritation. Remains, and only a few products have been developed and commercialized as natural antibacterial agents.

In this regard, encapsulation technology has the potential to improve the antimicrobial activity and stability of natural extracts and their application to food ingredients. Encapsulation techniques have been used to protect unstable compounds from environmental factors including light and oxygen, moisture and temperature during food processing and storage. For example, Japanese Patent Application Laid-Open No. 10-2004-0064286 discloses a capsule that can be used for oral hygiene products, and includes a capsule including a core and a film-forming polymer as an outer shell, and in Korean Patent Publication No. 10-1420483 As a soft capsule applicable to food, it describes a low-action soft capsule which softens in oral cavity and has good chewability and solubility, and gives a soft taste to gelatinous film.

As a result of studying natural antibacterial extract and encapsulation technology for oral health, the inventors of the present invention have found that a combination of a specific natural antibacterial extract and capsule coating material can provide a nanocapsule showing excellent antimicrobial activity. The invention was completed.

An object of the present invention for solving the above problems is an oral cleaning composition having an antimicrobial activity against oral microorganisms comprising nanocapsules containing natural extracts as antimicrobial substances as an active ingredient, prevention or treatment of periodontal disease To provide a pharmaceutical composition, and a food composition for the prevention or improvement of periodontal disease.

In order to achieve the above object, the present invention is an antibacterial against oral microorganisms having an antimicrobial material collected therein, including chitosan, and a nano capsule consisting of arabic gum or carrageenan as an active ingredient. It provides a composition for oral cleaning having activity.

In the present invention, the antimicrobial material is grapefruit seed extract, clove oil (clove oil), cinnamon oil (cinnamon oil), thyme oil (thyme oil), black pepper oil (black pepper oil) and ginger oil (ginger oil) It may be characterized in that at least one selected from the group consisting of, preferably, grapefruit seed extract, and may be characterized in that clove oil or cinnamon oil.

In the present invention, the nanocapsule may be characterized by consisting of chitosan and carrageenan.

In the present invention, the oral cleaning composition may be any one or more formulations selected from the group consisting of toothpaste, mouthwash, mouthwash, mouthwash, and mouthwash.

In the present invention, the oral microorganisms are Streptococcus mutans ( Streptococcus mutans ), Streptococcus sanguinis , Streptococcus sobrinus , Streptococcus ratti ( Streptococcus ratti ) (Streptococcus criceti), host Gino not tepto Caucus Seuss (Streptococcus anginosus), Streptococcus altitude you (Streptococcus gordonii), liquid Tino Bacillus solution Tino My semtem Komi Tansu (Actinobacillus actinomycetemcomitans), Fu earthy Te Leeum New Clegg Atum (Fusobacterium nucleatum) , Prevotella Intermedia intermedia ) and Popphylomonas gingivalis ) may be one or more oral microorganisms selected from the group consisting of.

In another aspect, the present invention is a pharmaceutical composition for preventing or treating periodontal disease, wherein the antimicrobial substance is collected therein and comprises a nanocapsule consisting of chitosan and arabic gum or carrageenan as an active ingredient. To provide.

In the present invention, the periodontal disease may be characterized in that any one or more diseases selected from the group consisting of dental caries, stomatitis, gingivitis, periodontitis, alveolar bone fracture, alveolar osteoporosis, alveolar softening, alveolar bone reduction, alveolar bone remodeling disorder and bad breath have.

The present invention is also a health functional food for preventing or improving periodontal disease, wherein the antimicrobial substance is collected therein and comprises a nanocapsule consisting of chitosan, and arabic gum or carrageenan as an active ingredient. To provide.

In the present invention, the health functional food may be characterized in the form of beverages, confectionery, bakery, chewing gum, tea, vitamin complexes or health supplements.

The nanocapsules containing natural antibacterial extracts according to the present invention and the composition for oral cavity containing the same have an antimicrobial activity greatly increased by complexly capturing a natural extract exhibiting synergistic action, and the coating property of the capsule is controlled to improve the oral mucosa adhesion. Not only is it possible to increase, by confirming the synergistic effect of the natural extracts and nanocapsules, there is an effect that the antimicrobial activity provides a composition for the oral cavity that is long lasting in the oral cavity.

Therefore, the composition for oral cavity of the present invention can be applied as a safe antibacterial product for dental health as a composition for oral cleaning, various food groups or pharmaceutical compositions.

Figure 1 shows the mucoadhesive properties of natural antimicrobial material and nanoparticles comprising the same.
2 shows a transmission electron microscope (TEM) image of chitosan / carrageenan nanocapsules in which grapefruit seed extract and cinnamon oil are collected.
Figure 3 (a) shows the grapefruit seed extract and clove oil, respectively, combinations of S. mutans , and the time-kill curve of the nanocapsules.
Figure 3 (b) shows the grapefruit seed extract and clove oil, respectively, combinations of S. sobrinus , and the time-kill curve of the nanocapsules.
Figure 4 (a) shows the grapefruit seed extract and cinnamon oil, combinations thereof, and time-kill curves of nanocapsules for S. mutans , respectively.
Figure 4 (b) shows the grapefruit seed extract and cinnamon oil, respectively, combinations of S. sobrinus , and the time-kill curve of the nanocapsules.
Figure 5 (a) shows the change in the total saliva of saliva with time after oral cleaning.
Figure 5 (b) shows the change of S.mutans with time after oral lavage.
Figure 6 (a) shows the change in total saliva with saliva after drinking yogurt intake.
Figure 6 (b) shows the change of S. mutans with time after drinking yoghurt intake.

EMBODIMENT OF THE INVENTION Hereinafter, the specific aspect of this invention is demonstrated in detail. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention relates to a composition for oral cavity comprising a nanocapsules containing the natural extract as an antimicrobial substance as an active ingredient.

In the present invention, the natural extract having the antimicrobial activity is Grape Seed Extracts (Grape Seed Extracts; GSE), clove oil (clove oil), cinnamon oil (cinnamon oil), thyme oil (thyme oil), black pepper oil (black pepper oil) and ginger oil (ginger oil) may be one or more selected from the group consisting of.

The natural extracts are known to have antimicrobial activity against oral bacteria, but it is difficult to survive in the oral cavity for a long time due to their low antimicrobial activity and low stability, and the strong taste and fragrance make them practically difficult to apply. . The inventors of the present invention have found that by forming the natural extracts in the form of nanocapsules, they can survive for a long time in the oral cavity, inhibit strong taste and aroma, and further improve antimicrobial activity and persistence.

The natural extract is preferably used grapefruit seed extract, it is more preferable to use a combination of grapefruit seed extract and other natural extract as an antimicrobial material collected in the core of the nanocapsules.

In one embodiment of the present invention, grapefruit seed extract and clove oil or grapefruit seed extract and cinnamon oil may exert a synergistic effect (synergy) in antibacterial activity, in particular, the combination of grapefruit seed extract and cinnamon oil nanoencapsulated It was confirmed that synergistic effects on antimicrobial activity and persistence can be greatly exerted.

Langveld et al. (2013)], the mechanisms by which pharmacological synergies can be generated are:

(1) multiple target effects in which the compound targets different sites of the bacterium;

(2) physicochemical effects such as improved solubility and bioavailability; or

(3) Target the specific resistance mechanism of the fungus.

Grapefruit seed extract is known to cause cell death by destroying the cell membrane of the bacteria. In addition, thymol and eugenol, which are the main compounds of clove oil, are known to have an antimicrobial activity by breaking down the outer membrane of bacteria and binding to cytosolic proteins, and cinnamaldehyde, a component of cinnamon oil. The carbonyl group of) is known to have an antimicrobial activity through binding to a protein and interfering with enzyme activity.

Therefore, in the present invention, the combination of grapefruit seed extract and clove oil or cinnamon oil exhibiting the synergistic effect of the antimicrobial activity, grapefruit seed extract increases the penetration of the bacterial cell membrane, and eugenol or cinnamic aldehyde is combined with the protein into the cell It means the possibility of a mechanism that can be carried more easily.

In the present invention, the mixing ratio of the grapefruit seed extract and clove oil or cinnamon oil is a weight ratio of 1: 1 to 1: 2, preferably a weight ratio of 1: 2 to 1:10, most preferably with the grapefruit seed extract The weight ratio of quantitative oil is about 1: 4, and the weight ratio of grapefruit seed extract and cinnamon oil is about 1: 8.

In the present invention, as a coating material for forming the nanocapsules by collecting the natural antibacterial extract therein, a combination of chitosan, arabic gum or carrageenan may be used.

Chitosan is widely distributed in the cell walls of crustaceans, insects, mushrooms and filamentous fungi, and is a non-toxic, pollution-free, biodegradable natural material produced by deacetylating chitin, which is the second most produced after cellulose in biological resources.

Chitosan's anti-cancer, immune-boosting, cholesterol-lowering, and antibacterial effects have been reported, and are being used as food materials, cosmetic materials, and medical materials. The mechanism of antimicrobial activity is not clear, but chitosan forms a dense, positively charged linear polyelectrolyte structure, which affects the permeability of the material by attaching amine groups of cationic chitosan to negatively charged microorganisms. It has been reported to exhibit antimicrobial activity and to bind to DNA of microorganisms directly and inhibit the formation of mRNA and protein.

It is a hardened mucus obtained from the Arabian gum tree, an evergreen broad-leaved arborescent of Arabian black legumes. It is used in chemicals, cooking, and confectionery by using the viscosity produced when dissolved in water.

Carrageenan is a food additive mainly used to increase the adhesiveness and viscosity of foods, to improve the emulsion stability and to improve the physical properties and feel of foods. Used as a thickener, gelling agent, stabilizer and the like. White to pale brown powder or particles with no odor or slightly peculiar odor. It is obtained by extracting seaweeds of the genus Red, Chondrus, Eucheuma, Gigartina, Hypnea or Iridaea, called Irish moss, with hot water or a hot alkaline aqueous solution, followed by purification.

In the present invention, paying attention to the mucoadhesive properties of chitosan, gum arabic, and carrageenan, when coating the antimicrobial material with these materials to produce nanocapsules, it is possible to prepare an oral composition having excellent antibacterial activity and long-term survival in the oral cavity. Found.

In one embodiment of the present invention, it was confirmed that the combination of chitosan and gum arabic (CS / AR) or the combination of chitosan and carrageenan (CS / CR) can form nanoparticles having excellent mucoadhesive properties. It was confirmed that the combination of carrageenan has more excellent mucoadhesion property.

In the present invention, the nanoparticles may be prepared by ion bonding of chitosan having a cation and gum arabic or carrageenan having an anion, and the nanocapsule containing a natural antimicrobial extract may first mix chitosan and a natural antimicrobial substance, and the mixture It can be prepared by adding gum arabic or carrageenan.

Chitosan has a (+) charge by an amine group in the molecular structure, and gum arabic and carrageenan have a (-) charge by a carboxyl group and a sulfate group, respectively. Nanoparticles are produced by bonding together by electrostatic attraction by the opposite charge. The size of the nanoparticles is influenced by the molecular weight and concentration of the coating material. In general, when the molecular weight is high and the concentration is high, the size of the nanoparticles increases because the amount of particles to be bonded increases. In the present invention, when the chitosan is 0.10 mg / mL or less, preferably 0.05 mg / mL or less, and gum arabic and carrageenan are 1.0 mg / mL or less, preferably 0.50 mg / mL or less, Easy to make

In the present invention, chitosan and gum arabic or carrageenan are preferably included in a weight ratio of 1: 1 to 1:20, more preferably 1: 1.5 to 1:10. Most preferably, chitosan and arabian gum are contained in a weight ratio of about 1: 5, and chitosan and carrageenan have a weight ratio of about 1: 2.

In addition, the natural antimicrobial material is preferably included 10 to 90 parts by weight based on the total weight of the coating material, in the case of grapefruit seed extract 10 to 50 parts by weight, preferably about 30 parts by weight, clove oil and thyme About 63 parts by weight of oil, about 78 parts by weight of cinnamon oil, black pepper oil and ginger oil are preferred.

Nanoparticles prepared by chitosan, and gum arabic or carrageenan in the present invention may have a particle size of 90 to 200nm, nanoparticles made of chitosan and arabic gum may have a particle size of 90 to 150nm, Nanoparticles made by chitosan and carrageenan may have a particle size of 150-200 nm.

In the present invention, when the coating material is made of nanocapsules in which the natural antimicrobial extract is collected, the particle size may represent an average particle size of 150 to 900 nm, and preferably may have an average particle size of 300 to 400 nm. .

Much of the current research on encapsulation deals with micrometer sized capsules to protect the active compound. However, microcapsules generally do not affect antimicrobial activity, while nanometer sized delivery systems can increase antimicrobial activity due to subcellular size. In general, nanoparticles with small particle size have a high surface area to volume ratio and are advantageous in contact with pathogenic bacteria, which may result in increased dispersibility in aqueous solution and thus exhibit better antimicrobial activity. Since it can be increased antimicrobial activity.

The composition for oral cavity according to the present invention may be used as an oral cleaning composition having an antimicrobial activity against oral microorganisms by containing the nanocapsules trapped therein as an active ingredient.

Oral cleaning composition of the present invention exhibits an antimicrobial effect against oral microorganisms, the oral microorganisms include cariogenic bacterial strains (periodontopathogenic bacterial strains), preferably Streptococcus mutans ( Streptococcus mutans , Streptococcus sanguinis , Streptococcus sobrinus , Streptococcus ratti , Streptococcus ratti , Streptococcus criceti , Streptococcus anus ), Streptococcus gordonii , Actinobacillus actinomycetemcomitans , Fusobacterium nucleatum , Prevotella intermedia intermedia ), Porphylomonas gingivalis , and the like.

Oral cleaning composition of the present invention can be used in the formulation of toothpaste for oral hygiene, oral cleaner, oral cleaner, oral spray, oral solution, but is not limited thereto.

The toothpaste may include one or more components such as abrasives, caking agents, moisturizers, foaming agents, sweeteners, flavoring agents and the like. Paste and liquid oral compositions in dentifrices include sorbitol, glycerin, ethylene glycol, propylene glycol, polyether glycol, polypropylene glycol, and the like as a moisturizing agent in the preparation of about 10 to 70% of the total composition. It can be used in combination.

The present invention also provides a pharmaceutical composition for the prevention or treatment of diseases in the oral cavity.

The disease in the oral cavity means a disease that may occur due to the bacteria or cell cultures present in the oral cavity, for example, dental caries, stomatitis, gingivitis, periodontitis, alveolar bone breakage, alveolar osteoporosis, alveolar osteomalacia, alveolar osteopathy, alveolar bone Remodeling disorders, bad breath, and the like.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.

In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and formulations may be preferably formulated according to each component using the methods disclosed in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be oral or parenteral administration, and parenteral administration includes intravenous infusion, subcutaneous infusion, intramuscular infusion, intraperitoneal infusion, transdermal administration and the like.

The composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level means the type of disease, the severity, and the activity of the drug. , Drug sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts. The composition according to the present invention may be administered as a separate therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and weight of the patient, and generally 0.001 to 150 mg, preferably 0.01 to 100 mg per 1 kg of body weight is administered daily or every other day or 1 to 1 day. It can be divided into three doses. However, the dosage may be increased or decreased depending on the route of administration, sex, weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or it may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers for the prevention or treatment of periodontal disease.

The present invention also relates to a food composition for preventing or improving periodontal disease.

In the present invention, "health functional food" refers to a food having a bioregulatory function, such as prevention and improvement of disease, biodefence, immunity, recovery of disease, inhibition of aging, and should be harmless to the human body when taken for a long time. Nanocapsules containing natural antibacterial extract according to the present invention may be added to health functional food for the purpose of preventing or improving periodontal disease.

When the composition of the present invention is used as a food additive, the composition may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the compositions of the invention are added in an amount of up to 15% by weight, preferably up to 10% by weight relative to the raw materials. However, in the case of long-term intake for the purpose of health and hygiene or for health control, it may be below the above range, and if there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The health functional food of the present invention is not particularly limited as long as it can include the nanocapsules, for example, beverages, bakery, bakery, gum, tea, vitamin complexes, health supplements, more specifically, dairy products , Confectionery, condiments, beverages and drinks, snacks, candies, jellies, ice cream and frozen desserts, breakfast cereals, nutrition bars, snack bars chocolate products, processed foods, grain products and pastas, soups, sauces and dressings, confectionery products , Oils and fats, dairy drinks and milk drinks, tea, soy milk and soy dairy-like products, frozen foods, cooked foods and alternative foods, meat products, cheese, yogurt, bread and buns, It may be any one selected from the group consisting of cakes, cookies and crackers.

In one embodiment of the present invention as a result of applying the nano-capsules containing the natural antimicrobial material of the present invention as a core (core) to drinking yogurt, it was confirmed that the growth of oral bacteria can be suppressed.

In addition, the health functional food may be prepared in the form of capsules, tablets, powders, liquid suspensions, pills and granules.

In addition, the health functional food may further include ingredients that are commonly added at the time of food production as an active ingredient.

The health functional food of the present invention may contain various flavors or natural carbohydrates as additional ingredients. The natural carbohydrates described above are sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose and polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol. As the sweetener, natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin, aspartame, and the like can be used. The ratio of the natural carbohydrate can be selected from 0.01 to 0.04 parts by weight, specifically about 0.02 to 0.03 parts by weight per 100 parts by weight of the health functional food of the present invention.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, And a carbonation agent used for the carbonated beverage. These components can be used independently or in combination. The proportion of such additives is not critical but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health food of the present invention.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only to illustrate the invention, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as limited by these examples.

Production Example  1: Preparation of Nanoparticles

Chitosan / Arabian gum (CS / AR) and chitosan / carrageenan (CS / CR) nanoparticles were prepared by ionic bonding of chitosan as a cation and Arabian gum or carrageenan as anion.

Chitosan (Kittolife Co., water-soluble, 24 cps, 95% deacetylated) was dissolved in distilled water at a concentration of 0.0625 mg / mL, and Arabian gum (ESS Ingredients Co.) and carrageenan (ESS Ingredients Co.) were dissolved in distilled water, respectively. Solutions at concentrations of 0.50, 1.25 and 2.50 mg / mL were prepared. After magnetic stirring at 1,000 rpm using WiseStir MS-MP8 (Wisd Laboratory Instruments, Germany) for 10 minutes, 1 mL of Arabic gum or carrageenan solution was dropped into 4 mL of chitosan solution to prepare nanoparticles corresponding to each concentration.

Example  1: Investigation of physical properties of nanoparticles

Particle size and polydispersity index (PDI) of CS / AR and CS / CR nanoparticles prepared in Preparation Example 1 were measured using Zetasizer Nano ZS (Malvern Instruments Ltd., UK). Measurement was performed in multiple narrow mode at 25 ± 1 ℃, the results are shown in Table 1 below.

All analyzes were repeated three more times as independent samples. All data are expressed as mean ± standard deviation (SD) of values of 3 replicates. Data were analyzed using one-way analysis of variance (ANOVA) and the significant difference was p <0.05. Significant differences between means were determined by Duncan's multi-range analysis. Statistical analysis was performed using Statistical Package for Social Science (SPSS, Version 21.0, SPSS Inc., USA).

As shown in Table 1, the particle sizes of the CS / AR and CS / CR nanoparticles ranged from 95 to 148 nm and 151 to 190 nm, respectively. As the AR and CR concentrations increased from 0.10 to 0.50 mg / mL, the particle size of the nanoparticles increased significantly in both CS / AR and CS / CR nanoparticles.

PDI is defined as the particle size distribution of nanoparticles. The closer the PDI value is to 0.0, the more uniform the distribution, while a value above 0.3 indicates a nonuniform distribution. Since the PDI of the nanoparticles prepared in the present preparation ranges from 0.1 to 0.4, it can be seen that uniform nanoparticles are formed by ion bonding. As the AR and CR concentrations increased, the PDI of CS / AR nanoparticles decreased from 0.2 to 0.1, but the CS / CR nanoparticles increased significantly from 0.2 to 0.4.

Example 2 investigation of mucoadhesion of nanoparticles

To analyze mucoadhesion of nanoparticles to mucin, Malaekeh-Nikouei et al. (2008)] was modified and applied.

0.05 mg / mL chitosan; Arabic gum and carrageenan at 0.10, 0.25 and 0.50 mg / mL; And 0.8 mL of a dispersion was prepared for nanoparticles prepared from a combination of chitosan and gum arabic or carrageenan. 0.8 mL of a mucin (Sigma-Aldrich Co., USA) solution at a concentration of 0.5 mg / mL was added to the dispersion, vortexed, and incubated in a constant temperature bath at 37 ° C. for 1 hour. After centrifugation at 10,000 rpm for 4 minutes, the supernatant was taken to measure the concentration of free mucin. Determination of mucin concentration in the supernatant was carried out by Bradford protein assay.

The mucin concentration was calculated using a standard curve, and the amount of mucin attached to the nanoparticles was calculated by the difference between the total amount of mucin added and the total amount of free mucin in the supernatant, and the results are shown in FIG. 1.

In FIG. 1, the amount of carrageenan and attached mucin increased significantly as the concentration of carrageenan increased from 0.10 to 0.50 mg / mL, while the increase in arabic gum concentration was slightly increased but was not significant.

It was also confirmed that the mucin adhesion of carrageenan at all concentrations was higher than that of Arabian gum. It is believed that the thiol bonds of carrageenan show bioadhesion because they form huge mucin oligomers through disulfide bonds.

Although the amount of mucin attached to the nanoparticles after the nanoparticles was significantly decreased in both CS / AR and CS / CR, the antimicrobial activity was increased by the advantages such as the reduction of particle size, the improvement of solubility and the protection of the core material. You can expect

Preparation Example 2 Preparation of Antimicrobial-Nano Capsules

As antibacterial substance, Clove oil (Newdia Co.), Time oil (Newdia Co.), Cinnamon oil (Absolute Aromas Co. Ltd., UK), Black pepper oil (Absolute Aromas Co. Ltd., UK), Ginger oil (Absolute Aromas Co. Ltd., UK) and grapefruit seed extract (GSE, ESS Ingredients Co.) were prepared respectively.

Chitosan was dissolved in distilled water and prepared at 0.0833 mg / mL, and the combination of the six antibacterial substances, GSE and clove oil, and a combination of GSE and cinnamon oil were added to 6 mL of chitosan to make a total of 8 mL. GSE and clove oil were mixed at a weight ratio of 1: 4, and GSE and cinnamon oil were mixed at a weight ratio of 1: 8. While stirring magnetically for 10 minutes, 2 mL of carrageenan solution (0.5 mg / mL) was dropped into the chitosan and antimicrobial mixture to prepare nanocapsule particles containing the antimicrobial substance.

Example 3: Physical Characterization of Antimicrobial-Nano Capsules

The particle size and polydispersity index (PDI) of the prepared antimicrobial-nano capsules were investigated and shown in Table 2 below.

In Table 2, the presence of natural extracts inside the nanoparticles was found to increase the particle size from 172nm to 884nm.

The particle size of the GSE-nano capsule was the smallest and the particle size of the black pepper oil-nano capsule was the largest.

It can be seen that the CS / CR nanoparticles in which the natural extracts except the time oil and the black pepper oil are collected have a uniform particle distribution.

TEM was used to investigate detailed appearance and morphological information of CS / CR nanocapsules containing GSE and cinnamon oil.

Transmission electron microscopy (TEM, JEM 2100F, JEOL, Japan) was used for morphological analysis of the antimicrobial-nanoparticles. Samples for TEM analysis were prepared by placing nanoparticle droplets on a 200mesh copper coated grid and then naturally drying at 37 ° C. And stained with 2% phosphotungstic acid (Sigma-Aldrich Co.) solution for 30 minutes before measuring under a microscope. TEM images are shown in FIG. 2.

From FIG. 2, nanocapsule particles were observed to have a spherical, regular appearance with a diameter of 300-400 nm.

Example  4: minimum inhibitory concentration ( MIC ) analysis

Minimum inhibitory concentrations (MIC) measurements were performed to evaluate the antimicrobial activity of the antimicrobial and antimicrobial-nanocapsules. MIC is defined as the lowest concentration of antimicrobial material that inhibited visible growth after culture.

Streptococcus mutans (Korea Microorganism Conservation Center, KCCM 40105) and Streptococcus sobrinus (KCCM 11898) were used as strains for antimicrobial activity evaluation. These two Gram-positive bacteria are known to be a major cause of dental caries.

Strains were stored at −80 ° C. with Brain Heart Infusion Broth (BHIB, Becton Dickinson and Company, USA) containing 87% glycerol. After incubation at 37 ° C. for 24 hours with BHIB, the required concentration was adjusted to 10 ⁶ CFU / mL.

The minimum inhibitory concentration (MIC) of the antimicrobial material is described in Fu et al. (2007)] was determined using a 96-well microtiter plate. 100 μL of antibacterial material dissolved in pure ethanol was dispensed into stock solution in the first row of each plate. The remaining wells were dispensed with 100 μL of sterile BHIB. The stock solution of the first row was diluted 2-fold serially along each column with BHIB. Finally, 100 μL of 10 ⁶ CFU / mL bacteria was dispensed into all wells.

At least two rows were used as a control. The positive control group was BHIB inoculated without the antimicrobial material. As a negative control, only antibacterial material and BHIB were used. The highest pure ethanol dose was used as a control to prove that the antimicrobial effect was antimicrobial rather than ethanol. Plates were incubated at 37 ° C. for 24 hours.

The MIC values for S. mutans and S. sobrinus of each of the measured antimicrobial substances are shown in Table 3 below.

In Table 3, the average activity of the antimicrobial substances was GSE> clove oil> time oil> cinnamon oil = black pepper oil = ginger oil.

GSE showed the highest antimicrobial activity in both S. mutans and S. sobrinus . The MIC of GSE for S. mutans was 0.13 mg / mL and the MIC for S. sobrinus was 0.03 mg / mL. Clove oil showed the second highest activity, MIC for S. mutans was 1.00 mg / mL and 0.13 mg / mL for S. sobrinus .

GSE nanoparticles showed 2 times lower MIC values for S. mutans than free GSE. This means that grapefruit seed extracts have an improved transport mechanism to the cell membrane of target microorganisms when used in nanoencapsulation.

Example  5: Synergy Analysis of Antibacterial Substances

In order to numerically define the synergistic effects of antimicrobial substances, the FIC Index (Fractional Inhibitory Concentration Index) for natural antimicrobial substances containing GSE showed the highest antimicrobial activity in MIC test.

To check the FIC, a checkerboard method was performed using a 96-well microtiter plate. Microplate analysis was arranged as follows. GSE was diluted 2-fold along the x-axis and other extracts were diluted 2-fold along the y-axis. The final volume of each well was 100 μL consisting of 50 μL of each antimicrobial diluent.

Subsequently, 100 μL of BHIB containing 10 ⁶ CFU / mL of bacteria was dispensed into each well. Plates were incubated at 37 ° C. for 24 hours.

FIC was calculated using the following equation.

The experimental results were interpreted as synergistic effect (S, FIC <0.5), additive effect (A, 0.5≤FIC≤1.0), void effect (I, 1.0 <FIC≤4.0), and antagonism (AN, FIC <4.0), It is shown in Table 4 below.

In Table 4, none showed synergy (FIC <0.5) for S. mutans and S. sobrinus , but showed an additive effect (0.5 ≦ FIC ≦ 1.0) in most combinations. Ginger oil and GSE combinations were the only effective against the two oral bacteria, and none of the evaluated combinations showed antagonistic action.

Clove oil and cinnamon oil showed the lowest FIC values in combination with GSE.

Example  6: Encapsulation-Synergy Effects Over Time

Time-kill experiments were conducted to examine the antimicrobial effects of encapsulation with GSE and clove oil and GSE and cinnamon oil over time.

Time-kill analysis is described in Koo et al. (2002). Initial inoculation concentrations of S. mutans KCCM 40105 and S. sobrinus KCCM 11898 were 10 ⁶ CFU / mL. Final concentrations of GSE, clove oil and cinnamon oil were set at 1/16 to 1/4 x MIC. The flask containing BHIB containing bacteria and antibacterial materials was incubated at 37 ° C.

Samples were taken every 0, 3, 6, 12 and 24 hours to analyze survival rate over time. Samples were serially diluted 10-fold serially to Brain Heart Infusion agar (BHIA, Becton, Dickinson and company) and incubated at 37 ° C for 48 hours to count CFU / mL. Time-kill curves were obtained using log CFU / mL for 24 hours.

The synergistic effect of the time-kill method is defined as a reduction in colony counts of more than 2 log ₁₀ in a 24-hour culture of combined antimicrobials compared to a single antimicrobial. The null and / or additive effects are defined as an increase or decrease of colony numbers less than 2 log ₁₀ in a 24-hour incubation of combined antimicrobials compared to a single antimicrobial. Antagonism is defined as an increase in colony numbers of at least 2 log ₁₀ at 24 hours compared to antimicrobial in combination with a single antimicrobial group.

To evaluate the synergistic effects of the combination of GSE and clove oil (GSE + Cl) and the combination of GSE and cinnamon oil (GSE + Cn), the use of antimicrobial agents alone and combinations of antimicrobial agents against S. mutans and S. sobrinus , and antimicrobial agents The antimicrobial activity of the nanoparticles in which the combination of was collected was tested.

3 shows a significant decrease in log CFu / mL when the S. mutans (a) and S. sobrinus (b) bacteria were exposed to the GSE + clove oil (Cl) combination. After 24 hours, GSE + Cl combination showed significantly lower colony than GSE or Cl alone. This result means that GSE + Cl combination shows synergistic effect.

GSE + Cl nanocapsules showed slightly antibacterial activity than GSE + Cl combination.

Figure 4 shows the antimicrobial activity of the combination of GSE + cinnamon oil (Cn) against S. mutans (a) and S. sobrinus (b).

After 24 hours, the GSE + Cn combination decreased more than 2 log CFU / mL compared to GSE or Cn alone for all bacteria used in the experiment, indicating that the GSE + Cn combination had a synergistic effect.

There is also a significant difference in log CFU / mL between GSE + Cn free and GSE + Cn nanoparticles for both bacteria at 24 hours. This result indicates that the encapsulated antimicrobial material can maintain antimicrobial activity for a long time.

Example  7: Oral Wash Test

Oral lavage test was performed to evaluate the antimicrobial activity of nanoparticles containing antimicrobial agents against saliva bacteria.

We recruited 18 healthy adults (mean age 25.9 years) between 23 and 29 years of age. The experiment was conducted under the approval of the Hanyang University Institutional Audit Committee, and all subjects agreed to provide the information. All volunteers were non-smokers and currently had at least 28 natural teeth without dental caries, periodontal disease or other oral pathologies. None of the subjects took antibiotics.

The following two solutions were prepared by mixing 1.25 mL of GSE and cinnamon oil or encapsulated GSE and cinnamon oil in a total of 10 mL in 8.75 mL of sterile distilled water. 2.5% ethanol diluted with distilled water was prepared. Used as control solution:

(1) a combination of 1/16 x MIC GSE and 1/16 x MIC cinnamon oil, and

(2) a combination of CS / CR nanoencapsulated 1/16 x MIC GSE and 1/16 x MIC cinnamon oil

Subjects were divided into three random groups. Subjects brush their teeth immediately after lunch and collect saliva before the experiment 2 hours later. Thereafter, the subject rinsed his mouth for 1 minute using 10 mL of the mouthwash solution or the control solution. After the experiment, the subject's saliva was taken every 30, 60 and 90 minutes.

Saliva samples collected from each subject were vortexed for 2 minutes. Samples were serially diluted 10-fold in PBS. Appropriate diluted samples are plated on 3M petrifilm (3M, USA) for total bacterial counts and S. mutans Mitis Salivarius Sucrose Bacitracin Agar (MSBA, MBcell, USA) was plated for bacterial counting. Plates were incubated at 37 ° C. for 48 hours.

Changes in total saliva and S. mutans after oral lavage in three groups are shown in FIGS. 5 (a) and (b), respectively. The total bacterial counts in the control group were nearly constant in all casts, while the GSE + CN free and GSE + Cn nanoparticle groups decreased slightly and then increased again. Up to 60 minutes, no significant difference was observed between the experimental groups. At 90 minutes, the total cell count of the GSE + Cn nanoparticle group was significantly lower than the control group.

Changes in the number of S. mutans in the control group showed a similar trend to the total bacterial counts during the test period. However, GSE + Cn free and GSE + Cn nanoparticles significantly reduced salivary S. mutans growth at all three sampling times compared to the control.

The log CFU / mL values of the GSE + Cn free group decreased significantly up to 60 minutes but gradually increased over time, while the GSE + Cn nanoparticle group remained decreasing.

These results indicate that nanoparticles with GSE and cinnamon oil can maintain antimicrobial activity in the oral cavity for a long time compared to the simple combination without nanoencapsulation.

Example  8: Antimicrobial Activity of Drinking Yogurt

The antimicrobial activity of drinking yogurt containing the antimicrobial-nano capsule of the present invention was evaluated.

The experiment was conducted under the same conditions as in Example 7, but two yogurts were prepared and used in the experiment:

(1) a drinking yoghurt with a 1/16 x MIC GSE plus a 1/16 x MIC cinnamon oil combination, and

(2) Drinking yogurt with a combination of 1/16 x MIC GSE and 1/16 x MIC cinnamon oil encapsulated with CS / CR nano.

In addition, untreated drinking yoghurt was used as a control.

To study the antimicrobial activity of yogurt, subjects consumed 10 mL of yogurt with or without antimicrobial material. Saliva was collected every 30, 60 and 90 minutes after the experiment after oral lavage or yogurt ingestion.

6 shows the inhibitory effect of GSE + Cn free and GSE + Cn nanoparticles on saliva total bacteria and S. mutans in drinking yogurt.

In FIG. 6 (a), the total bacterial count after ingestion of the control-treated untreated yoghurt rapidly increased to 60 minutes and then decreased to baseline. Oral bacteria are believed to be multiplied by other ingredients of yogurt such as sugar.

GSE + Cn free group showed a total number of bacteria significantly decreased up to 30 minutes and then gradually increased until 90 minutes. On the other hand, in the GSE + Cn nanoparticle group, the total cell count gradually decreased until 60 minutes, and then maintained for the last 90 minutes. A significant decrease in total saliva bacteria after 90 minutes was observed only in the GSE + Cn nanoparticle group.

In FIG. 6 (b), the S. mutans number changes of the control and GSE + Cn free groups tend to be similar to the total bacterial counts during all test periods. However, GSE + Cn nanoparticles rapidly reduced the growth of S. mutans by 60 minutes and maintained antimicrobial activity by 90 minutes.

The GSE + Cn nanoparticle group significantly inhibited the growth of saliva S. mutans compared to the other two groups. These results show that nano-encapsulation of antimicrobial substances effectively inhibits the growth of oral bacteria in food applications.

As described above in detail the specific part of the content of the present invention, for those skilled in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

An antibacterial substance is collected therein, and (i) contains chitosan and arabic gum, or ii) chitosan and carrageenan as an active ingredient.
The antimicrobial material iii) grapefruit seed extract and clove oil, or ii) grapefruit seed extract and cinnamon oil,
Oral cleaning composition having an antimicrobial activity against oral microorganisms.
delete delete The method of claim 1,
The nano-capsule, characterized in that consisting of chitosan and carrageenan, oral cleaning composition having an antimicrobial activity against oral microorganisms.
The method of claim 1,
The oral cleansing composition is a toothpaste, oral cleansing agent, oral cleaning agent, oral spray or oral dentifrice formulation, characterized in that, oral cleaning composition having an antimicrobial activity against oral microorganisms.
The method of claim 1,
The oral microorganisms include Streptococcus mutans , Streptococcus sanguinis , Streptococcus sobrinus , Streptococcus ratti , Streptococcus creti , Streptococcus cristi Streptococcus anginosus , Streptococcus gordonii , Actinobacillus Actinobacillus 　 actinomycetemcomitans ), Fusobacterium 　 nucleatum ), Prevotella intermedia ( Prevotella intermedia ) and Popphylomonas gingivalis ( Porphylomonas gingivalis ), characterized in that at least one microorganism selected from the group consisting of, oral cleaning composition having an antimicrobial activity against oral microorganisms.
Antibacterial substances are collected therein, and (i) prevention or treatment of periodontal disease comprising chitosan and arabic gum, or ii) nanocapsules consisting of chitosan and carrageenan as active ingredients. As a pharmaceutical composition for
The antimicrobial material iii) grapefruit seed extract and clove oil, or ii) grapefruit seed extract and cinnamon oil,
Pharmaceutical composition for the prevention or treatment of periodontal disease.
delete delete The method of claim 7, wherein
The periodontal disease is characterized in that any one or more diseases selected from the group consisting of dental caries, gingivitis, periodontitis, alveolar bone fracture, alveolar osteoporosis, alveolar osteomalacia, alveolar bone reduction, alveolar bone remodeling disorder and bad breath, prevention or treatment of periodontal disease Pharmaceutical composition for.
Antibacterial substances are collected therein, and (i) prevention or improvement of periodontal disease comprising chitosan and arabic gum, or ii) nanocapsules consisting of chitosan and carrageenan as active ingredients. As a dietary supplement for
The antimicrobial material iii) grapefruit seed extract and clove oil, or ii) grapefruit seed extract and cinnamon oil,
Health functional food for the prevention or improvement of periodontal disease.
delete delete The method of claim 11,
The periodontal disease is characterized in that any one or more diseases selected from the group consisting of dental caries, gingivitis, periodontitis, alveolar bone fracture, alveolar osteoporosis, alveolar osteomalacia, alveolar bone reduction, alveolar bone remodeling disorder and bad breath, prevention or improvement of periodontal disease Dietary supplements.
The method of claim 11,
The health functional food is characterized in that the form of beverages, confectionery, bakery, gum, tea, vitamin complex or health supplement foods, health functional foods for the prevention or improvement of periodontal disease.

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