WO2023008879A1 - Combined use of aucuba japonica extract and superoxide dismutase and/or bacillus strain spore in composition for improving oral health - Google Patents

Combined use of aucuba japonica extract and superoxide dismutase and/or bacillus strain spore in composition for improving oral health Download PDF

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WO2023008879A1
WO2023008879A1 PCT/KR2022/010973 KR2022010973W WO2023008879A1 WO 2023008879 A1 WO2023008879 A1 WO 2023008879A1 KR 2022010973 W KR2022010973 W KR 2022010973W WO 2023008879 A1 WO2023008879 A1 WO 2023008879A1
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composition
sod
oral
strain
spores
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PCT/KR2022/010973
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French (fr)
Korean (ko)
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반재구
김의중
박영찬
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주식회사 제노포커스
주식회사 바이옴로직
주식회사 벡스퍼트
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Publication of WO2023008879A1 publication Critical patent/WO2023008879A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9783—Angiosperms [Magnoliophyta]
    • A61K8/9789—Magnoliopsida [dicotyledons]
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/195—Proteins from microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A61K38/446—Superoxide dismutase (1.15)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A61K8/66—Enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q11/00—Preparations for care of the teeth, of the oral cavity or of dentures; Dentifrices, e.g. toothpastes; Mouth rinses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/312—Foods, ingredients or supplements having a functional effect on health having an effect on dental health
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
    • A61K2800/59—Mixtures
    • A61K2800/591—Mixtures of compounds not provided for by any of the codes A61K2800/592 - A61K2800/596

Definitions

  • the present invention relates to a composition
  • a composition comprising an extract of Aucuba japonica and superoxide dismutase and/or spores of a Bacillus sp. strain and its use for promoting oral health, maintaining oral hygiene, or preventing or treating oral diseases. .
  • Oral diseases most often begin with poor oral hygiene, leading to tartar, tooth decay, gingivitis, etc., and can be accompanied by severe bad breath.
  • the main causes of bad breath also called halitosis, are periodontal diseases such as gingivitis and periodontitis, odor-causing bacteria on the tongue, certain foods and alcoholic beverages, and smoking. If the food in the mouth is not properly removed, a soft deposit on the tooth surface called plaque is formed, and the plaque becomes tartar over time.
  • Tartar is a mineral in saliva and gingival crevicular fluid within plaque that is calcified and firmly adhered to the tooth surface. Tartar cannot be removed by brushing but can be removed only by scaling.
  • Periodontitis not only causes excruciating pain, but also requires special attention as bacteria can infect the heart or kidneys.
  • antibacterial agents include antibiotics.
  • antibiotics can cause the appearance of resistant bacteria in the oral cavity along with systemic side effects on our body, long-term use is difficult, so there is a disadvantage in that they must be used as a short-term treatment.
  • antibacterial agents used in mouth fresheners include Sangquinarine, Listerine, and Chlorhexidine. It has the disadvantage of being expensive, and although Listerine has alcohol as its main component and has some bacteriostatic action, it only shows a temporary effect in the oral cavity and may cause harm to tissues when used for a long time.
  • the object of the present invention is to solve the problems of the prior art described above.
  • Another object of the present invention is to improve oral health or manage oral hygiene by using a pine extract, superoxide dismutase and/or Bacillus sp. strain spores together.
  • Another object of the present invention is to prevent or treat oral diseases by using a pine extract, superoxide dismutase and/or spores of a Bacillus sp. strain together.
  • an oral composition comprising at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores.
  • an oral product comprising the composition is provided.
  • compositions for preventing or treating oral diseases comprising the composition.
  • a veterinary composition comprising the composition is provided.
  • a food composition comprising the composition is provided.
  • a feed composition comprising the composition is provided.
  • pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores are administered to the subject at least one selected from the group consisting of, or the subject.
  • SOD superoxide dismutase
  • Bacillus sp. strain spores are administered to the subject at least one selected from the group consisting of, or the subject.
  • composition according to the present invention can be usefully used for preventing or treating oral diseases such as bad breath, calculus, plaque, dental caries, gingivitis or periodontitis without causing side effects, as well as reducing pain caused by such oral diseases. can be usefully used for
  • Figure 1 shows the results of measuring the degree of edema and redness of the gums when a beagle dog with gingivitis according to Experimental Example 1.2 was treated with a cedar extract.
  • Figure 2 shows the results of measuring the degree of alveolar bone destruction when the cedar extract was administered to rats induced with periodontitis according to Experimental Example 1.3.
  • Figure 3 shows the criteria for converting the calculus index into a score.
  • Figure 4 shows the criteria for converting the gingivitis index into a score.
  • Figure 6 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 2.4. * indicates P ⁇ 0.05 versus week 0.
  • FIG. 7 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 2.4.
  • FIG. 10 is a graph showing the results of the halitosis index observed after administration of a test substance according to Experimental Example 3.4.
  • 11 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 3.4. * indicates P ⁇ 0.05 versus week 0, ** indicates P ⁇ 0.01 versus week 0.
  • Figure 12 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 3.4. * indicates P ⁇ 0.05 versus week 0.
  • FIG. 13 is a graph showing the results of the gingivitis index observed after administration of the test substance according to Experimental Example 3.4. ** indicates P ⁇ 0.01 versus week 0.
  • 16 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 4.4. * indicates P ⁇ 0.05 versus week 0.
  • 17 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 4.4. * indicates P ⁇ 0.05 versus week 0, ** indicates P ⁇ 0.01 versus week 0.
  • Figure 19 shows the overall procedure for preparing a recombinant production strain expressing SodA2 (BSBA310).
  • rrnB T1T2 represents a transcription terminator
  • rep(pBR322) represents the replicon from pBR322 that works in E. coli
  • rep(pUB110) is a replicon from pUB110 that works in B. subtilis
  • KanR stands for kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase)
  • the BJ27 promoter represents a strong promoter for B. subtilis.
  • subject is used interchangeably with “patient”, and is a mammal in need of oral hygiene or prevention or treatment of oral disease, such as a primate (eg, human, monkey, chimpanzee, etc.), companion It may be an animal (eg dog, cat, etc.), a domestic animal (eg cow, pig, horse, sheep, goat, etc.) or a laboratory animal (eg rat, mouse, guinea pig, etc.).
  • primate eg, human, monkey, chimpanzee, etc.
  • companion It may be an animal (eg dog, cat, etc.), a domestic animal (eg cow, pig, horse, sheep, goat, etc.) or a laboratory animal (eg rat, mouse, guinea pig, etc.).
  • treatment generally means obtaining a desired pharmacological and/or physiological effect.
  • This effect has a therapeutic effect in terms of partially or completely curing diseases and/or other unwanted or undesirable conditions (such as halitosis, tartar, plaque, dental caries, gingivitis, periodontitis, pain, etc.).
  • Desirable therapeutic effects include preventing occurrence or recurrence of the disease, amelioration of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or amelioration.
  • Prognosis includes, but is not limited to.
  • treatment may refer to medical intervention for a pre-existing disease or disorder.
  • prevention means obtaining a desired prophylactic pharmacological and/or physiological effect in terms of partially or completely preventing a disease or symptom thereof.
  • administration means providing an active ingredient to achieve a prophylactic or therapeutic purpose to a subject.
  • the present invention partially applies superoxide dismutase (SOD) or Bacillus sp. strain spores in combination with cedar extract to the oral cavity of a subject, or superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract. It is based on the finding that the spores, when applied to the oral cavity of a subject, are effective in promoting oral hygiene or preventing or treating oral diseases. Accordingly, according to one aspect of the present invention, there is provided an oral composition comprising (i) a cedar extract and (ii) superoxide dismutase (SOD), Bacillus sp. strain spores, or SOD and Bacillus sp.
  • SOD superoxide dismutase
  • SOD superoxide dismutase
  • Bacillus sp. strain spores or SOD and Bacillus sp.
  • the composition may include either superoxide dismutase or spores of a Bacillus sp. strain in combination with a pine extract.
  • the composition may include superoxide dismutase and spores of a Bacillus sp. strain in combination with a pine extract.
  • Aucuba japonica is a kind of evergreen plant of the dicotyledonous umbel dogwood family, and is mainly distributed in the southern part of Korea, especially in the Ulleungdo region. In other words, it is also referred to as Doyeop Coral, Cheongakpan, or Blue Tree.
  • extract refers to a liquid component obtained by immersing a desired material in various solvents and then extracting at room temperature, low temperature or elevated temperature for a certain period of time, and a liquid component obtained by removing the solvent from the liquid component. It means the result of solid content, etc.
  • it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof.
  • the pine tree extract may be an extract extracted from the whole of the aerial part of a pine tree, a part thereof, or a material derived therefrom.
  • the pine extract is preferably a hot water extract, but is not limited thereto.
  • the part of the tree may be a stem, leaf, flower, petal or seed of the tree.
  • the pine extract is preferably extracted from leaves, stems or mixtures thereof of pine trees, but is not limited thereto.
  • Whole, part of, or material derived therefrom used for extraction may be ground or shredded or suitably dried.
  • the extraction solvent is not particularly limited, and for example, water or an organic solvent may be used, and the organic solvent includes methanol, ethanol, propanol, isopropanol, alcohol having 1 to 4 carbon atoms including butanol, acetone, ether, and benzene. , Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination, but are not limited thereto.
  • any one of methods such as hot water extraction, cold brew extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used, preferably hot water extraction.
  • one or more of the above extraction methods may be used in combination.
  • the desired extract may be additionally subjected to a conventional fractionation process or may be purified using a conventional purification method.
  • any known method may be used.
  • the pine extract may be a hot water extract of pine tree, and 3 to 20 times the weight of the medicinal material is added, preferably 5 to 15 times the water, and the temperature is 80 to 120 °C, preferably 85 to 110 °C, more Preferably, it may be extracted at a temperature of 90 to 105 ° C. for 1 to 24 hours, preferably for 2 to 12 hours, and more preferably for 3 to 8 hours, but the hot water extraction method is not limited thereto.
  • the extract may be subjected to additional processes such as filtration, concentration, and drying as necessary after the extraction.
  • additional processes such as filtration, concentration, and drying as necessary after the extraction.
  • the method and conditions of the additional process are not particularly limited, and may be performed by a method known in the art or under commonly performed conditions.
  • the final concentration of the pine tree extract may be 100 to 50,000 mg/kg, preferably 5,000 to 25,000 mg/kg, but is not limited thereto.
  • Superoxide dismutase is an enzyme that alternately catalyzes the dismutation of superoxide (O 2 ⁇ - ) radicals into ordinary molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ).
  • SOD plays an important role in reducing oxidative stress by scavenging reactive oxygen species.
  • SODs are widely distributed in prokaryotic and eukaryotic cells and are classified into four classes according to the different types of metal centers (copper/zinc, nickel, manganese and iron).
  • Manganese-containing SOD [Mn-SOD] is widely present in the chloroplasts, mitochondria, and cytoplasm of many bacterial or eukaryotic cells.
  • SOD may be used interchangeably with (poly)peptide having superoxide dismutase activity.
  • SOD may include a polypeptide having superoxide dismutase activity or a fragment thereof, or a fusion product containing the same.
  • the SOD can be manganese bound (Mn-SOD).
  • the SOD may be deamidated Mn-SOD.
  • the SOD may comprise or consist of the amino acid sequence represented by SEQ ID NO: 2 (SodA).
  • SOD may be one in which amino acid residues at positions 74 and 137 of SEQ ID NO: 2 are substituted with Asp.
  • the SOD may include or consist of the amino acid sequence represented by SEQ ID NO: 4 (SodA2).
  • the SOD or polypeptide having SOD activity of the present invention also includes an amino acid sequence showing substantial identity to the above amino acid sequence.
  • the substantial identity is 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more when the aligned sequences are analyzed using an algorithm commonly used in the art. Means an amino acid sequence that exhibits identity.
  • the SOD is a modified or engineered polypeptide having SOD enzymatic activity that does not affect or affect various aspects (in vivo, in vitro or ex vivo stability, uniformity, and/or morphological alteration). may include one or more mutations, such as deletions, insertions or substitutions of one or more amino acids.
  • the polypeptide may be added with a heterologous material (eg, a tag known in the art, including a HIS tag, an HA tag, a myc tag, a GFC and/or an Fc domain of an antibody) for purification, detection, in vivo delivery, or increased stability. can be included with a heterologous material (eg, a tag known in the art, including a HIS tag, an HA tag, a myc tag, a GFC and/or an Fc domain of an antibody) for purification, detection, in vivo delivery, or increased stability. can be included with a heterologous material (eg, a tag known in the art, including
  • the SODs of the present invention may be derived from a variety of sources including natural, mutant or recombinant microorganisms.
  • SOD may be derived from bacteria.
  • the SOD may be derived from a bacterium that is generally regarded as safe (GRAS) for use in drugs or foods, such as a Bacillus sp. strain or variant thereof.
  • GRAS regarded as safe
  • SOD may be obtained from a Bacillus amyloliquesfaciens strain (eg, GF423 strain or GF424 strain) or a culture supernatant thereof.
  • the GF423 strain and the GF424 strain were deposited with the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017 and March 13, 2017, respectively (accession numbers KCTC 13222 BP and accession numbers KCTC 13227 BP, respectively).
  • the Bacillus amyloliquefaciens GF424 strain is a strain obtained by mutating the Bacillus amyloliquefaciens GF423 strain by UV irradiation in order to improve sod gene expression.
  • the SOD enzyme (SodA) derived from the Bacillus amyloliquefaciens GF423 or GF424 strain is Mn-SOD and has the amino acid sequence of SEQ ID NO: 2 (its nucleotide sequence is shown in SEQ ID NO: 1). Additionally, the SOD may be a recombinant polypeptide. For example, based on SEQ ID NO: 2, amino acid residues at positions 74 and 137 may be deamidated SOD in which Asp is substituted (SodA2), which has the amino acid sequence of SEQ ID NO: 4 (its nucleotide sequence is SEQ ID NO: marked 3). The sequences of SEQ ID NOs: 1 to 4 are as shown in Table 1.
  • SOD may be derived from other recombinant strains (eg, Bacillus Subtilis species strains) containing the SOD expression gene of the Bacillus amyloliquefaciens strain.
  • the recombinant strain may be prepared by recombinant technology using a conventional protein production strain known in the art.
  • the Bacillus subtilis strain (the parent strain of the recombinant strain) may be KCTC 3135, and the KCTC 3135 strain may be distributed from the Korea Research Institute of Bioscience and Biotechnology (KCTC).
  • KCTC Korea Research Institute of Bioscience and Biotechnology
  • one or more of the genes shown in Table 2 may be removed from the recombinant strain to facilitate post-processing.
  • the recombinant strain may be prepared through the process shown in FIG. 19 .
  • the recombinant strain may include the expression vector shown in FIG. 20 .
  • sodA and sodA2 represent genes encoding SOD.
  • SOD derived from the strain is an enzyme secreted outside the cell, when manufacturing SOD using the strain, it is possible to mass-produce SOD that is safe for individuals without going through an expensive purification process (eg, column purification). Because of this, efficient production is possible.
  • the SOD of the present invention can be obtained by culturing the natural, mutant or recombinant microorganism in various culture media.
  • SOD included in the oral composition may be isolated from the culture supernatant of Bacillus amyloliquefaciens strain GF423 or strain GF424.
  • a culture solution may be obtained by culturing the Bacillus amyloliquefaciens strain in various types of media.
  • the strain is grown at about 25° C. to about 42° C. for about 1 day to about 4 days using a complex medium (pH 6.0 to 7.0).
  • LB medium Luria-Bertani (LB) medium, International Streptomyces Project (ISP) medium, nutrient agar (NA) medium, brain heart infusion agar (BHI) medium, SDA (sabouraud dextrose agar) medium, PDA (potato dextrose agar) medium, NB (nutrient broth) medium, and the like.
  • ISP International Streptomyces Project
  • NA nutrient agar
  • BHI brain heart infusion agar
  • SDA sabouraud dextrose agar
  • PDA potato dextrose agar
  • NB nutrient broth
  • SODs can also be sourced from other natural, mutant or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda-enzymes.org on the World Wide Web).
  • the SODs of the present invention may be isolated or purified from cultures of natural, variant or recombinant strains. wherein the isolated or purified SOD or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it was derived.
  • the purified product may be a pure product separated from a strain culture through ultrafiltration, ammonium sulfate treatment, column purification, concentration, or the like, or a culture concentrate obtained through ultrafiltration, concentration, or the like.
  • substantially free of cellular material includes preparations of the protein in which the protein is isolated from cellular components of the cell from which the protein is isolated or recombinantly produced.
  • the phrase “substantially free of cellular material” means less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 10% by dry weight of unwanted proteins. Preferably it includes preparations of less than about 5% protein.
  • the SOD may be preferably purified by the following purification method, but is not limited thereto.
  • the culture supernatant is collected by centrifuging the culture solution obtained by previously culturing the Bacillus amyloliquefaciens strain.
  • the supernatant fraction is pretreated by solid phase extraction, then isolated and purified by chromatography.
  • SOD can be purified using a variety of methods of chromatography. Preferably, hydrophobic interaction chromatography is used.
  • the SOD may be included in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof.
  • strain lysate means obtained by mechanically or chemically disrupting the strain by culturing it, and may include all of the additional processes such as extraction, dilution, concentration, and purification therefrom.
  • strain culture may refer to a culture medium itself or a supernatant thereof obtained by culturing a strain.
  • strain culture concentrate refers to a culture concentrate obtained by ultrafiltration, ammonium sulfate treatment, column purification, concentration, or the like from a strain culture, or obtained through ultrafiltration, concentration, or the like.
  • strain culture extract refers to an extract obtained from the culture medium or a concentrate thereof, and may include an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof, or a fraction thereof.
  • the dried form may include a freeze-dried form.
  • the SOD may include cellular material from the cell or tissue source from which it was derived, such as extracellular vesicles.
  • the cellular material containing SOD can be separated from the culture solution by filtration, concentration, etc. by culturing various sources including natural, mutant or recombinant hosts using conventional techniques known in the art as described above. can
  • the spores of the Bacillus species strain may be obtained by culturing the Bacillus species strain in a suitable medium and isolating the spores produced after inducing sporulation.
  • spores of Bacillus sp. strains may be sourced from GRAS bacteria generally considered safe for use in drugs or foods.
  • Bacillus species strain spores are known to be resistant to proteases and low pH (Cutting SM. Bacillus probiotics. Food Microbiol. 2011;28:214-220.
  • spores of strains of Bacillus species are GRAS probiotics approved in several countries.
  • the spores of the Bacillus species strain may be derived from a Bacillus amyloliquefaciens strain (eg, GF423 or GF424 strain). Sporulation can be induced using conventional techniques known in the art.
  • spores of a strain obtained by inducing sporulation in a medium such as DSM, NB, SYP, LB2, etc. during main culture after pre-cultivation of the strain various sources including natural, mutant, or recombinant hosts are cultivated to produce feeder cells After removal, it can be separated through filtration, concentration, or centrifugation.
  • pine extract and the SOD enzyme and / or Bacillus sp. strain spores may be included in the composition in a form such that they can be administered sequentially, in reverse order or simultaneously with each other.
  • the oral composition may include pine extract, SOD, and Bacillus sp. strain spores, or may be included in a form in which they may be used in combination.
  • prevention of bad breath, calculus, dental plaque, dental caries or periodontal disease (eg, gingivitis or periodontitis) when a mixture of pine tree extract, SOD, and Bacillus species strain spores is applied to the oral cavity. , it was confirmed that it was effective for improving or treating oral health, oral hygiene management, or preventing or treating oral diseases. In addition, it was confirmed that the mixture was effective in reducing pain due to oral diseases.
  • the pine extract, SOD and spores of the Bacillus sp. strain may be mixed in an appropriate ratio to exert the effect.
  • the pine tree extract has an amount corresponding to a dose of 0.1 to 10,000 mg/kg, preferably 1 to 1,000 mg/kg, more preferably 1 to 100 mg/kg, and still more preferably 5 to 50 mg/kg per day.
  • SOD and Bacillus sp. strain spores are mixed in a ratio such that the SOD titer is 5,000 to 20,000 U per g dry weight and the spores are 0.1 x 10 ⁇ 10 to 1 x 10 ⁇ 11 cfu per g dry weight.
  • the final composition may be, for example, a mixture of 0.5% to 5% of Japanese oak extract and 0.5% to 12% of SOD and/or spores of a Bacillus species strain.
  • the final composition may preferably be a mixture of 2% of cedar extract and 2.5% of SOD and/or spores of a Bacillus species strain.
  • the composition can be used for oral health promotion or oral hygiene management.
  • the composition can be used for preventing or treating oral diseases.
  • the composition can be used to prevent, ameliorate, or treat one or more diseases selected from halitosis, tartar, plaque, dental caries, periodontal disease, and stomatitis (eg, chronic stomatitis).
  • diseases selected from halitosis, tartar, plaque, dental caries, periodontal disease, and stomatitis (eg, chronic stomatitis).
  • the composition can be used to reduce oral pain.
  • the composition may have various oral forms known in the art, including a liquid form, a solid form, a gel form, a powder form, a paste form, or a form impregnated or applied to a carrier.
  • an oral product containing the composition for oral cavity includes at least one selected from the group consisting of (i) pine extract and (ii) SOD and Bacillus sp. strain spores as described above.
  • the product may be one or more selected from the group consisting of toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral gel, oral ointment, oral patch, and mouthwash, but is not limited thereto.
  • the oral product of the present invention in addition to containing the pine extract and SOD and/or Bacillus sp. strain spores as active ingredients, may contain additional ingredients known in the art required for its formulation.
  • the oral product when the oral product is toothpaste, it may further include an abrasive, a moisturizing agent, a foaming agent, a sweetener, a whitening agent, or a flavoring agent.
  • the oral product when the oral product is an oral gel, it may further include a thickener such as a medium chain fatty acid for viscosity or adhesiveness of the gel.
  • a mouthwash it may further include a carrier such as non-toxic alcohol.
  • a pharmaceutical composition or a veterinary composition comprising a composition comprising at least one selected from the group consisting of pine extract as described above, superoxide dismutase (SOD) and spores of Bacillus sp. is provided.
  • the pharmaceutical composition of the present invention may be used interchangeably with the term veterinary composition when applied to animals other than humans.
  • the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
  • SOD superoxide dismutase
  • Bacillus sp. strain spores in combination with cedar extract.
  • the composition can be used for preventing or treating oral diseases.
  • the spherical disease may be one or more diseases selected from halitosis, calculus, dental plaque, dental caries, periodontal disease, and chronic stomatitis, but is not limited thereto.
  • the composition may be for preventing or treating oral pain.
  • the oral pain may be due to various oral diseases or undesirable conditions.
  • oral pain may be due to plaque, plaque, dental caries, periodontal disease or chronic stomatitis.
  • the pharmaceutical or veterinary composition of the present invention may further include one or more selected from the group consisting of pharmaceutically acceptable carriers, excipients and diluents.
  • the pharmaceutically acceptable carrier, excipient and/or diluent may be one commonly used in the art.
  • Carriers, excipients or diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Mineral oils such as cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and silicon dioxide, etc. It may include, but is not limited thereto.
  • additives such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • Additives for the formulation may be selected from those commonly used in the pharmaceutical field.
  • the pharmaceutical or veterinary composition of the present invention may be formulated in a preferred form depending on the method of use, and particularly known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated by adopting a method. Specific examples of such formulations include tablets, pills, powders, granules, syrups, solutions, capsules, suspensions, emulsions, injection solutions, plasters, lotions, liniments, limonade, aerosols, and extracts. There are extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, and patches.
  • compositions of the present invention can be prepared using a suitable method known in the art or using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA). can be formulated accordingly.
  • the pharmaceutical or veterinary composition of the present invention may be administered orally or parenterally depending on the desired method, and in the case of parenteral administration, nasal spray, external skin application or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection Injection or intrathoracic injection is preferred.
  • the pharmaceutical or veterinary composition of the present invention may be preferably applied through intraoral injection methods such as oral spray, oral application, and intraoral injection.
  • solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the complex composition, for example, starch, calcium carbonate, sucrose, lactose, It may be prepared by mixing gelatin or the like.
  • lubricants such as magnesium stearate and talc may also be used.
  • Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents.
  • SOD may be coated with a coating such as shellac, ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof.
  • a coating such as shellac, ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof.
  • the coating agent is not limited thereto.
  • Forms for parenteral administration include toothpastes, mouthwashes, topical formulations (eg, gels, creams, ointments, dressing solutions, sprays, and other coating agents).
  • topical formulations eg, gels, creams, ointments, dressing solutions, sprays, and other coating agents.
  • the active ingredient of the present invention may be impregnated with a carrier such as gauze made of natural or synthetic fibers.
  • the gel, cream or ointment it may be suitable for direct application on or around affected areas including teeth and gums or carious areas.
  • the spray it can be prepared by a conventional spray preparation method, and can be filled and packaged in a compression container or other spray container, and sprayed and applied to the oral disease area from time to time to prevent or treat oral diseases.
  • the dressing solution it can be prepared by a conventional dressing solution manufacturing method, and oral diseases can be prevented or treated by dressing an oral disease site or dressing another bacterial infection site.
  • the pharmaceutical or veterinary composition of the present invention is administered in a pharmaceutically or veterinarily effective amount.
  • pharmaceutically effective amount or “veterinarily effective amount” means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical or veterinary treatment, and the effective dosage level is the level of the patient or animal. Weight, gender, age, health condition, severity, activity of drug, sensitivity to drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical field. can
  • the pharmaceutical or veterinary composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously.
  • the pharmaceutical composition may be administered single or multiple times as needed. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and this amount can be easily determined by a person skilled in the art.
  • composition comprising at least one selected from the group consisting of (i) cedar extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above
  • SOD superoxide dismutase
  • the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
  • SOD superoxide dismutase
  • Bacillus sp. strain spores in combination with cedar extract.
  • Such food compositions include medical or nutraceutical food compositions.
  • medical food or “nutraceutical food” refers to food made from raw materials or ingredients that have the potential to have beneficial functions in the human body, which maintain or improve health by maintaining normal functions or activating the physiological functions of the human body. Refers to food, as defined by, but not limited to, the Ministry of Food and Drug Safety, and does not exclude any conventional health food in that sense.
  • the food includes, but is not limited to, various foods, food additives, beverages (eg, functional drinks, natural fruit juice and vegetable drinks), gum, tea, vitamin complexes, health functional foods, and other functional foods. .
  • beverages eg, functional drinks, natural fruit juice and vegetable drinks
  • gum e.g., tea
  • vitamin complexes e.g., vitamin complexes, health functional foods, and other functional foods.
  • the food may be prepared by a conventional method known in the art.
  • the medical food, nutraceutical food, or health functional food further includes at least one of a carrier, diluent, excipient, and additive in addition to the SOD for the purpose of improving oral hygiene or preventing or improving oral disease, tablets, It may be formulated into one selected from the group consisting of pills, powders, granules, powders, capsules and liquid formulations.
  • Specific examples of the carrier, excipient, diluent, and additive are well known in the art, and a person skilled in the art can prepare them by combining appropriate components according to the formulation.
  • the content of the extract of the present invention, superoxide dismutase and/or Bacillus species strain spores as active ingredients in the above-described formulation can be appropriately adjusted depending on the type and purpose of use, patient condition, type and severity of symptoms, etc. And, based on the weight of the solid content, it may be 0.001 to 99.9% by weight, preferably 0.01 to 50% by weight, but is not limited thereto.
  • the dosage of the food of the present invention may vary depending on the patient's age, weight, sex, dosage form, state of health and degree of disease, and divided administration once a day to several times at regular time intervals according to the judgment of a doctor or pharmacist You may.
  • the daily dosage may be 10 to 1,000 mg/kg based on the active ingredient content.
  • the above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences. If the daily dose of the health functional food of the present invention is less than the above dose, a significant effect may not be obtained, and if it exceeds more than that, it is uneconomical and may cause undesirable side effects because it is out of the range of the usual dose. there is.
  • a composition comprising at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above
  • a feed composition is provided.
  • the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
  • the feed composition of the present invention may be prepared in any formulation commonly used in the art.
  • the feed composition of the present invention may include auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes, and other probiotic-type microorganism preparations; grains such as ground or crushed wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; dry ingredients consisting of sugar and dairy products, such as various powdered milk and whey powder; main components such as lipids, for example, animal fats and vegetable fats optionally liquefied by heating; Additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventives may be further included.
  • the feed composition of the present invention may be in the form of a powder or liquid formulation, and may include a feed additive excipient (calcium carbonate, horse powder, zeolite, jade powder or rice bran, etc.).
  • a feed additive excipient calcium carbonate, horse powder, zeolite, jade powder or rice bran, etc.
  • administering to a subject at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above
  • a method for improving oral hygiene comprising a.
  • prevention of oral diseases comprising the step of administering to the subject at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above or method of treatment is provided.
  • the method may include administering SOD and Bacillus sp. strain spores to the subject in combination with a cedar extract.
  • the oral disease may include one or more selected from the group consisting of oral pain, halitosis, plaque, calculus, dental caries, periodontal disease, and chronic stomatitis.
  • the pine extract, SOD and Bacillus sp. strain spores when administered in combination, may be administered simultaneously as a single composition or as separate compositions containing each or any two components.
  • the pine extract, SOD and Bacillus sp. strain spores may be administered sequentially or in reverse order as separate compositions containing either or any two components.
  • the pine extract, SOD and Bacillus sp. strain spores may be administered by the same route or by different routes depending on the dosage form. The composition, dosage form, route of administration, etc. are as described above for the composition.
  • the leaves and stems of the cedar tree were collected from the forest in Jisepo-ri, Irun-myeon, Geoje-si, Gyeongsangnam-do in August 2020, and the evidence specimen is stored in the sample storage room of Chonbuk National University.
  • 900 L of distilled water was added to 100 kg of pine tree, shaken at 100 ° C. for 3 hours, concentrated under reduced pressure, and then dried to obtain a hot water extract powder of pine tree.
  • Bacillus amyloliqueurfaciens strains used in this example are the Bacillus amyloliqueurpaciens GF423 strain and the Bacillus amyloliqueurpaciens GF424 strain, and these strains were published by the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017 and 2017, respectively. It was deposited with accession number KCTC 13222 BP and accession number KCTC 13227BP as of March 13, 2018.
  • the Bacillus amyloliquefaciens GF424 strain is a strain obtained by inducing mutation by irradiating UV to the Bacillus amyloliquefaciens GF423 strain in order to improve the expression of the sodA gene.
  • Example 2.1 Bacillus amyloliquefaciens Cultivation of GF423 or GF424 strains
  • LB agar medium Lia-Bertani (LB) agar; tryptophan 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L
  • MnSO 4 manganese sulfate
  • the cell culture medium obtained in Example 2.1 was centrifuged at 4° C. and 3,578 x g for 20 minutes to obtain a supernatant, and then concentrated 10 times using ultrafiltration (UF, MWCO 10,000). The concentrated solution was filtered through a sterilization filter and lyophilized. The activity of SOD was analyzed using a SOD assay kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits superoxide radicals by 50%. The activity of the dried SOD enzyme was 30 ⁇ 6 U/mg.
  • the medium used in this example was SYP or DSM.
  • SYP medium contains 1.5% soy tone, 0.5% yeast extract, 0.5% K 2 HPO 4 , 0.1% MnSO 4 , 0.1% MgSO 4 , 10 mM FeSO 4 , 0.04% (NH 4 ) 2 SO 4 , 0.04% % (NH 4 ) 2 PO 4 , 0.1% CaCl 2 and 2% glucose.
  • the DSM medium contains Bacto nutrient broth 8 g/L, KCl 1 g/L, MgSO 4 0.25 g/L, Ca(NO 3 ) 2 0.16415 g/L, MnCl 2 0.9521 mg/L and FeSO 4 0.152 mg/L. .
  • MnSO 4 , MgSO 4 , FeSO 4 , (NH 4 ) 2 SO 4 , (NH 4 ) 2 PO 4 and CaCl 2 were dissolved in ddH 2 O and added before use.
  • a single colony of Bacillus amyloliquefaciens strain GF423 or GF424 was inoculated into 1 mL of LB in a 14 mL tube and incubated at 37° C. and 200 rpm for 12 hours.
  • 1 mL of the culture medium was transferred to 50 mL of LB medium in a 500 mL flask, and cultured at 37° C. and 200 rpm for 12 hours.
  • 20 mL of culture medium was transferred to 1 L of SYP or DSM in a 2.5 L baffled flask.
  • the inoculated culture was cultured at 37° C. and 200 rpm for 24 to 120 hours.
  • lysozyme (0.5 g/L) was added to the culture broth and incubated for 1 hour at 37° C. and 200 rpm to remove remaining feeder cells. Crude spores were harvested by centrifugation at 6000 rpm for 10 minutes. The harvested crude spores were purified by washing twice with water, then with 0.02% SDS, again with water twice, and then suspended in a PBS solution. The spore suspension was stored at -20 °C. The number of spores was determined by plating the diluted spore solution on LB agar plates and then counting colonies.
  • a test using beagle dogs or rats was performed to determine whether the pine tree extract exhibited oral health promotion and oral hygiene management effects.
  • Test materials were prepared as follows. An oral ointment containing 2% of the pine extract obtained in Example 1 (test group) and an oral ointment not containing the pine extract (control group, vehicle only) were prepared with the compositions shown in Table 3 below.
  • Test group contains 2% cedar extract
  • Control cedar extract 200 mg - Mucoadhesive (Sodium polyacrylate, PAA-Na) 300mg 300mg
  • Flavor enhancer Meltol 100 mg 100 mg Preservative (Methyl paraben) 18mg 18mg Preservative (Poly paraben) 2 mg 2 mg
  • the test substance of Experimental Example 1.1 was applied and administered to the gums (external gingival region of both maxillary molars) twice daily for 4 weeks.
  • the dosage was administered at 1.0 g/day by 0.5 g per time.
  • the content of the active ingredient contained in 1.0 g/day was 20 mg/day.
  • an oral examination was performed and the gingivitis index was evaluated according to the criteria shown in Table 4 below.
  • the gingivitis index deteriorated over time in the beagle dog administered with the control (vehicle) not containing the pine extract, whereas the oral ointment containing the pine extract was administered.
  • the gingivitis index was reduced by about 15%.
  • Anesthesia was induced by intraperitoneal injection of a mixture of zoletin and ketamine at a ratio of 1:2 (1.5 ml/kg) to the rats after the acclimatization process was completed.
  • periodontitis was induced by ligating the cervical region of the lower left second molar with a silk suture (0.5 mm).
  • a mandibular right second molar that was not ligated with a suture was set as a control group.
  • the day of inducing periodontitis was set as day 0, and the rats were sacrificed 2 weeks after the day of inducing periodontitis.
  • pine extract test substance in Experimental Example 1.1
  • DW vehicle
  • the rats were sacrificed and the mandibles were removed. Thereafter, all soft tissues were removed by treatment with 1M NaOH for about 1 hour, and the degree of alveolar bone loss was quantified by measuring the length from the cemento-enamel junction to the crest of the alveolar bone.
  • pine extract was useful for improving oral diseases such as gingivitis and destruction of the alveolar bone.
  • a test using beagle dogs was performed to determine whether a mixture of superoxide dismutase (SOD) and Bacillus amyloliquefaciens strain spores exhibits oral health promotion and oral hygiene management effects.
  • SOD superoxide dismutase
  • Bacillus amyloliquefaciens strain spores exhibits oral health promotion and oral hygiene management effects.
  • Superoxide dismutase (SOD) and Bacillus amyloliqueurfaciens strain spores were purified from Bacillus amyloliqueurfaciens GF424 in Example 2 and superoxide dismutase (SOD) prepared in Example 3. It was prepared by mixing the spores of the amiloliquefiaciens GF423 strain (2.5% mixture, SOD 10,000 U/g).
  • the mixture is adjusted to a desired viscosity by dissolving a medium-chain fatty acid such as lauric acid for maintaining the viscosity / adhesion of oral gel at room temperature, mixing tasty ingredients such as betaine, oligosaccharide, and egg yolk, and finally It was obtained by homogeneously mixing SOD and 2.5% of spores (SOD titer: 12,510 U/g, spores: 1.2 x 10 ⁇ 10 CFU/g). This mixture (SOD + spores) was used in a tube in the form of oral gel, which is a gel type (20 g/tube, 250 U/g gel).
  • a breeding room equipped with environmental conditions of temperature 23 ⁇ 3 °C, relative humidity 30 ⁇ 10 %, ventilation frequency 10 to 15 times / hour, lighting 12 hours, illumination intensity 150 to 200 Lux was used, and during the period of purification and testing, stainless steel was used. They were raised individually in cages, one per cage. Feed and drinking water were provided autonomously.
  • Test groups G1 (“G1_Vehicle”) and G2 (“G2_BASOD”) were constructed as follows.
  • test substance of Experimental Example 2.1 was applied to the gums (external gingival region of both maxillary molars) and administered every day for 4 weeks.
  • the dose was 0.25 g (SOD 62.5 U)/head twice a day.
  • Example 2 In order to confirm that the purified superoxide dismutase (SOD) in Example 2 exhibits oral health promotion and oral hygiene management effects, a test using a cat was performed.
  • SOD superoxide dismutase
  • Test materials used were prepared in the same manner as in Experimental Example 2.1, except for the spores of the Bacillus amyloliquefaciens GF423 strain.
  • test group G1 the breed is mixed, KSH (Korean short hair), Exotic, or Siamese, gender is neutered male (NM) or neutered female (SF), and age is 2 to 10 was three
  • test group G2 the breed was T.A. (Turkish Angora) or KSH (Korean short hair), gender was neutered male (NM) or neutered female (SF), and the age was 1 to 11 years.
  • test substance of Experimental Example 3.1 was applied and administered to the gingival region of the right maxillary molar every day for 4 weeks.
  • the dosage was 0.3 g (SOD 75 U)/head twice a day.
  • Feed intake of the test cats was evaluated at week 0 (before the start of administration), week 2, and week 4 (see table below).
  • the halitosis index did not change until the end of the test, but in the SOD group, it was confirmed that the halitosis index continued to decrease (FIG. 10).
  • the plaque index significantly increased during the test period, whereas in the SOD group, the plaque index decreased with statistical significance from the 2nd week to the end of the test (FIG. 11).
  • a test using cats was performed to determine whether a mixture of pine extract, superoxide dismutase (SOD), and spores of Bacillus amyloliquefaciens strain showed oral health promotion and oral hygiene management effects.
  • SOD superoxide dismutase
  • Test materials were prepared as follows. An oral gel containing a mixture of pine extract powder obtained in Example 1, SOD prepared in Example 2, and spores prepared in Example 3 was prepared with the composition shown in the table below.
  • the mixture dissolves pine extract powder in purified water at 60 ° C. for 1 hour, dissolves carboxymethylcellulose and sodium polyacrylate for maintaining the viscosity / adhesion of oral gel at room temperature, and contains vitamins, oligosaccharides, and egg yolk powder. After mixing such tasty ingredients, it was prepared by adding superoxide dismutase (SOD) and Bacillus amyloliquefaciens strain spore mixture at room temperature. Finally, the oral gel containing the above 2% pine tree extract and 2.5% SOD and spore mixture (SOD titer: 250 U/g, spores: 2 x 10 ⁇ 8 CFU/g) was put into a tube and used.
  • SOD superoxide dismutase
  • spore mixture 2 x 10 ⁇ 8 CFU/g
  • Test groups G1 (“G1_Vehicle”) and G2 (“G2_Vehicle+BASOD”) were constructed as follows.
  • test group G1 the breed is mixed, KSH (Korean short hair), Exotic, or Siamese, gender is neutered male (NM) or neutered female (SF), and age is 2 to 10
  • test substance of Experimental Example 4.1 was applied and administered to the gingival region of the right maxillary molar every day for 4 weeks.
  • the dose was administered twice a day at 0.3 g/head once.
  • Feed intake of the test cats was evaluated at week 0 (before the start of administration), week 2, and week 4 (see table below).
  • test group G1 activity was significantly improved from the 2nd week, and in test group G2, activity gradually increased, but there was no statistical significance (data not shown).
  • test group G1 there was little change in palpation pain until the end of the test, but in test group G2, the palpation pain index significantly decreased with statistical significance from 2 weeks after administration (FIG. 14).
  • test group G1 the halitosis index did not change until the end of the test, but in test group G2, the halitosis index decreased by about 50% compared to week 0 at the 2nd week of administration, and the bad breath was statistically significantly improved until the 4th week (FIG. 15) .
  • test group G1 the plaque index significantly increased during the test period, whereas in test group G2, the plaque index gradually decreased until the end of the test, although there was no statistical significance (FIG. 16).
  • test group G1 there was no significant change in the gingivitis index, but in test group G2, the gingivitis index decreased statistically significantly from week 2 to week 4 (FIG. 18).
  • a mixture of pine extract and SOD and spores as test substances was applied to the cat's gums twice daily at a dose of 0.3 g/head once. It was confirmed that the index, plaque index, calculus index, and gingivitis index all decreased. In particular, symptoms of palpable pain, halitosis, calculus, and gingivitis were improved statistically significantly compared to 0 weeks. In addition, it was confirmed that the test substance was not harmful because no special adverse reaction was observed in the entire test process.
  • the mixture of test substances, pine extract and SOD, and spores is not only useful for improving oral diseases such as bad breath, plaque, tartar and gingivitis, but also reduces pain caused by such oral diseases. It was also found to be useful.

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Abstract

The present invention relates to an oral composition comprising an Aucuba japonica extract and superoxide dismutase (SOD) and/or Bacillus strain spores and a use thereof for improving oral health or managing oral hygiene. This composition can be advantageously used for preventing or treating oral diseases, such as oral pain, halitosis, calculus, plaque, gingivitis, and periodontitis, without causing side effects in the oral cavity.

Description

식나무 추출물과 수퍼옥시드 디스뮤타제 및/또는 바실러스 균주 포자를 포함하는 조성물의 구강 건강 개선을 위한 병용 사용Concomitant use of a composition comprising pine extract and superoxide dismutase and/or spores of Bacillus strains to improve oral health
본 발명은 식나무(Aucuba japonica) 추출물과 수퍼옥시드 디스뮤타제 및/또는 바실러스 종 균주 포자를 포함하는 조성물 및 이의 구강 건강 증진, 구강 위생 관리, 또는 구강 질환의 예방 또는 치료를 위한 용도에 관한 것이다.The present invention relates to a composition comprising an extract of Aucuba japonica and superoxide dismutase and/or spores of a Bacillus sp. strain and its use for promoting oral health, maintaining oral hygiene, or preventing or treating oral diseases. .
구강 질환은 대부분 구강 위생 불량으로 시작되어, 치석, 충치, 치은염 등으로 이어지게 되며 심한 구취를 동반할 수 있다. 구취라 불리는 입 냄새의 주요 원인은 치은염 및 치주염과 같은 치주 질환, 냄새를 유발하는 혀의 박테리아, 특정 식품과 알코올성 음료, 흡연 등이 있다. 입안의 음식물 제거가 잘 이루어지지 않으면 치태라고 불리는 치아표면의 연성 부착물이 생기고 치태는 시간이 지나게 되면 치석이 된다. 치석은 치태 내에서 타액과 치은열구액 (gingival crevicular fluid)의 무기질이 석회화되어 치아표면에 단단하게 달라붙어 있는 것으로, 치석은 칫솔질로는 제거되지 않고, 치석제거술 (scaling)로만 제거할 수 있다. 치석을 제거하지 않으면, 치석으로 인해 잇몸에 치은염이라는 염증이 생기기 쉬워진다. 치은염의 상태가 나빠지게 되면 잇몸은 물론 인대나 치조골에까지 염증이 생기게 된다. 이 염증이 바로 치주염인데, 이로 인해 입 냄새가 유발되기도 한다. 치주염은 극심한 고통을 줄 뿐만 아니라 심장이나 신장에까지 세균이 감염될 수 있어 각별한 주의를 요한다.Oral diseases most often begin with poor oral hygiene, leading to tartar, tooth decay, gingivitis, etc., and can be accompanied by severe bad breath. The main causes of bad breath, also called halitosis, are periodontal diseases such as gingivitis and periodontitis, odor-causing bacteria on the tongue, certain foods and alcoholic beverages, and smoking. If the food in the mouth is not properly removed, a soft deposit on the tooth surface called plaque is formed, and the plaque becomes tartar over time. Tartar is a mineral in saliva and gingival crevicular fluid within plaque that is calcified and firmly adhered to the tooth surface. Tartar cannot be removed by brushing but can be removed only by scaling. If tartar is not removed, tartar makes the gums more prone to inflammation called gingivitis. When gingivitis gets worse, the gums as well as the ligaments and alveolar bone become inflamed. This inflammation is called periodontitis, which can also cause bad breath. Periodontitis not only causes excruciating pain, but also requires special attention as bacteria can infect the heart or kidneys.
치태 내에 서식하면서 각종 구강 질환을 유발하는 구강 병원균의 증식을 억제하는 물질로는 항생제를 포함한 항균제가 있다. 그러나, 항생제는 우리 몸에 대한 전신적인 부작용과 함께 구강 내 내성균의 출현을 유발할 수 있기 때문에 장기적인 사용이 곤란하여 단기간 동안 치료제로 이용해야 하는 단점이 있다. 또한, 구강 청정제에 사용되고 있는 항균제로는 생구이나린(Sangquinarine), 리스테린(Listerine), 클로르헥시딘(Chlorhexidine) 등이 있는데, 이 중에서 생구이나린은 구강 내에서 세균에 대한 효과가 불분명할 뿐만 아니라 가격도 고가라는 단점이 있고, 리스테린은 알코올이 주성분으로 약간의 정균 작용이 있으나 실제 구강 내에서는 일시적인 효과를 나타낼 뿐 장기간 사용 시 조직에 대해 위해 작용을 나타낼 수 있는 단점이 있다.Substances that inhibit the growth of oral pathogens that live in plaque and cause various oral diseases include antibacterial agents including antibiotics. However, since antibiotics can cause the appearance of resistant bacteria in the oral cavity along with systemic side effects on our body, long-term use is difficult, so there is a disadvantage in that they must be used as a short-term treatment. In addition, antibacterial agents used in mouth fresheners include Sangquinarine, Listerine, and Chlorhexidine. It has the disadvantage of being expensive, and although Listerine has alcohol as its main component and has some bacteriostatic action, it only shows a temporary effect in the oral cavity and may cause harm to tissues when used for a long time.
치석을 제거하는 효과적인 방법은 치석제거술이다. 그러나, 스케일링은 잇몸이 붓거나, 차거나 뜨거운 음식에 민감해질 수 있으며, 부적절한 스케일링은 잇몸 손상을 유발할 수 있다.An effective way to remove tartar is tartar removal. However, scaling can cause gums to swell and become sensitive to cold or hot food, and improper scaling can cause gum damage.
따라서, 치아 또는 잇몸에서 불리한 부작용을 일으킴이 없이 구강 위생 관리 및 구강 건강 증진에 사용될 수 있을 뿐만 아니라 치아 주위 조직의 염증으로 인한 치조골 파괴를 억제함으로써 구강 질환을 예방하거나 치료할 수 있는 물질 및 방법이 필요하다.Therefore, there is a need for materials and methods that can be used for oral hygiene management and oral health promotion without causing adverse side effects in teeth or gums, as well as prevent or treat oral diseases by inhibiting alveolar bone destruction due to inflammation of tissues around teeth. do.
본 발명은 상술한 종래 기술의 문제점을 해결하는 것을 그 목적으로 한다. The object of the present invention is to solve the problems of the prior art described above.
본 발명은 식나무 추출물과 수퍼옥시드 디스뮤타제 및/또는 바실러스 종 균주 포자를 함께 이용하여 구강 건강을 증진하거나 구강 위생을 관리하는 것을 다른 목적으로 한다. Another object of the present invention is to improve oral health or manage oral hygiene by using a pine extract, superoxide dismutase and/or Bacillus sp. strain spores together.
본 발명은 식나무 추출물과 수퍼옥시드 디스뮤타제 및/또는 바실러스 종 균주 포자를 함께 이용하여 구강 질환을 예방 또는 치료하는 것을 또 다른 목적으로 한다.Another object of the present invention is to prevent or treat oral diseases by using a pine extract, superoxide dismutase and/or spores of a Bacillus sp. strain together.
본 발명의 목적은 이상에서 언급한 목적으로 제한되지 않는다. 본 발명의 목적은 이하의 설명으로 보다 분명해질 것이며, 특허청구범위에 기재된 수단 및 그 조합으로 실현될 것이다.The object of the present invention is not limited to the object mentioned above. The objects of the present invention will become more apparent from the description that follows, and will be realized by means and combinations thereof set forth in the claims.
상기 목적을 달성하기 위한 본 발명의 대표적인 구성은 다음과 같다.Representative configurations of the present invention for achieving the above object are as follows.
본 발명의 일 태양에 따르면, (i) 식나무 추출물 및 (ii) 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자로 이루어진 군에서 선택되는 하나 이상을 포함하는 구강용 조성물이 제공된다.According to one aspect of the present invention, there is provided an oral composition comprising at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores.
본 발명의 다른 태양에 따르면, 상기 조성물을 포함하는 구강용 제품이 제공된다. According to another aspect of the present invention, an oral product comprising the composition is provided.
본 발명의 또 다른 태양에 따르면, 상기 조성물을 포함하는 구강 질환의 예방 또는 치료를 위한 약학 조성물이 제공된다. According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating oral diseases comprising the composition.
본 발명의 또 다른 태양에 따르면, 상기 조성물을 포함하는 수의학적 조성물이 제공된다. According to another aspect of the present invention, a veterinary composition comprising the composition is provided.
본 발명의 또 다른 태양에 따르면, 상기 조성물을 포함하는 식품 조성물이 제공된다. According to another aspect of the present invention, a food composition comprising the composition is provided.
본 발명의 또 다른 태양에 따르면, 상기 조성물을 포함하는 사료 조성물이 제공된다. According to another aspect of the present invention, a feed composition comprising the composition is provided.
본 발명의 다른 태양에 따르면, (i) 식나무 추출물 및 (ii) 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자로 이루어진 군에서 선택되는 하나 이상을 개체에 투여하는 단계 또는 상기 조성물을 개체에 투여하는 단계를 포함하는, 구강 위생을 증진하거나 구강 질환을 예방 또는 치료하는 방법이 제공된다.According to another aspect of the present invention, (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores are administered to the subject at least one selected from the group consisting of, or the subject There is provided a method for improving oral hygiene or preventing or treating oral disease, comprising the step of administering to.
본 발명에 따른 조성물은 부작용을 일으킴이 없이 구취, 치석, 치태, 치아 우식증, 치은염 또는 치주염과 같은 구강 질환을 예방하거나 치료하는 데에 유용하게 사용될 수 있을 뿐만 아니라 그러한 구강 질환으로 인한 통증을 감소시키는 데에 유용하게 사용될 수 있다.The composition according to the present invention can be usefully used for preventing or treating oral diseases such as bad breath, calculus, plaque, dental caries, gingivitis or periodontitis without causing side effects, as well as reducing pain caused by such oral diseases. can be usefully used for
도 1은 실험예 1.2에 따른 치은염을 가진 비글견(beagle dog)에 식나무 추출물을 처리했을 때 잇몸의 부종 및 발적 정도를 측정한 결과를 나타낸다.Figure 1 shows the results of measuring the degree of edema and redness of the gums when a beagle dog with gingivitis according to Experimental Example 1.2 was treated with a cedar extract.
도 2는 실험예 1.3에 따른 치주염을 유발한 랫트에 식나무 추출물을 투여했을 때 치조골의 파괴 정도를 측정한 결과를 나타낸다.Figure 2 shows the results of measuring the degree of alveolar bone destruction when the cedar extract was administered to rats induced with periodontitis according to Experimental Example 1.3.
도 3은 치석 지수를 점수로 환산하기 위한 기준을 나타낸다.Figure 3 shows the criteria for converting the calculus index into a score.
도 4는 치은염 지수를 점수로 환산하기 위한 기준을 나타낸다.Figure 4 shows the criteria for converting the gingivitis index into a score.
도 5는 실험예 2.4에 따른 시험 물질을 투여한 후에 관찰된 구취 지수에 관한 결과를 나타낸 그래프이다.5 is a graph showing the results of the halitosis index observed after administration of the test substance according to Experimental Example 2.4.
도 6은 실험예 2.4에 따른 시험 물질을 투여한 후에 관찰된 치석 지수에 관한 결과를 나타낸 그래프이다. *는 0주차 대비 P < 0.05를 나타낸다.Figure 6 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 2.4. * indicates P < 0.05 versus week 0.
도 7은 실험예 2.4에 따른 시험 물질을 투여한 후에 관찰된 치태 지수에 관한 결과를 나타낸 그래프이다.7 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 2.4.
도 8은 실험예 2.4에 따른 시험 물질을 투여한 후에 관찰된 치은염 지수에 관한 결과를 나타낸 그래프이다. **는 0주차 대비 P < 0.01을 나타낸다.8 is a graph showing the results of the gingivitis index observed after administration of the test substance according to Experimental Example 2.4. ** indicates P < 0.01 versus week 0.
도 9는 실험예 3.4에 따른 시험 물질의 투여 후에 관찰된 촉진성 통증 지수에 관한 결과를 나타낸 그래프이다. *는 0주차 대비 P < 0.05를 나타낸다.9 is a graph showing the results of the palpable pain index observed after administration of the test substance according to Experimental Example 3.4. * indicates P < 0.05 versus week 0.
도 10은 실험예 3.4에 따른 시험 물질의 투여 후에 관찰된 구취 지수에 관한 결과를 나타낸 그래프이다.10 is a graph showing the results of the halitosis index observed after administration of a test substance according to Experimental Example 3.4.
도 11은 실험예 3.4에 따른 시험 물질의 투여 후에 관찰된 치태 지수에 관한 결과를 나타낸 그래프이다. *는 0주차 대비 P < 0.05를 나타내고, **는 0주차 대비 P < 0.01을 나타낸다.11 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 3.4. * indicates P < 0.05 versus week 0, ** indicates P < 0.01 versus week 0.
도 12는 실험예 3.4에 따른 시험 물질의 투여 후에 관찰된 치석 지수에 관한 결과를 나타낸 그래프이다. *는 0주차 대비 P < 0.05를 나타낸다.Figure 12 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 3.4. * indicates P < 0.05 versus week 0.
도 13은 실험예 3.4에 따른 시험 물질의 투여 후에 관찰된 치은염 지수에 관한 결과를 나타낸 그래프이다. **는 0주차 대비 P < 0.01을 나타낸다.13 is a graph showing the results of the gingivitis index observed after administration of the test substance according to Experimental Example 3.4. ** indicates P < 0.01 versus week 0.
도 14는 실험예 4.4에 따른 시험 물질의 투여 후에 관찰된 촉진성 통증 지수에 관한 결과를 나타낸 그래프이다. **는 0주차 대비 P < 0.01을 나타낸다.14 is a graph showing the results of the palpable pain index observed after administration of the test substance according to Experimental Example 4.4. ** indicates P < 0.01 versus week 0.
도 15는 실험예 4.4에 따른 시험 물질의 투여 후에 관찰된 구취 지수에 관한 결과를 나타낸 그래프이다. **는 0주차 대비 P < 0.01을 나타낸다.15 is a graph showing the results of the halitosis index observed after administration of a test substance according to Experimental Example 4.4. ** indicates P < 0.01 versus week 0.
도 16은 실험예 4.4에 따른 시험 물질의 투여 후에 관찰된 치태 지수에 관한 결과를 나타낸 그래프이다. *는 0주차 대비 P < 0.05를 나타낸다.16 is a graph showing the results of plaque index observed after administration of a test substance according to Experimental Example 4.4. * indicates P < 0.05 versus week 0.
도 17은 실험예 4.4에 따른 시험 물질의 투여 후에 관찰된 치석 지수에 관한 결과를 나타낸 그래프이다. *는 0주차 대비 P < 0.05를 나타내고, **는 0주차 대비 P < 0.01을 나타낸다.17 is a graph showing the results of the calculus index observed after administration of the test substance according to Experimental Example 4.4. * indicates P < 0.05 versus week 0, ** indicates P < 0.01 versus week 0.
도 18은 실험예 4.4에 따른 시험 물질의 투여 후에 관찰된 치은염 지수에 관한 결과를 나타낸 그래프이다. *는 0주차 대비 P < 0.05를 나타내고, **는 0주차 대비 P < 0.01을 나타낸다.18 is a graph showing the results of the gingivitis index observed after administration of the test substance according to Experimental Example 4.4. * indicates P < 0.05 versus week 0, ** indicates P < 0.01 versus week 0.
도 19는 SodA2를 발현하는 재조합 생산 균주(BSBA310)을 제조하기 위한 전체 절차를 나타낸다.Figure 19 shows the overall procedure for preparing a recombinant production strain expressing SodA2 (BSBA310).
도 20은 sodA2 유전자의 과발현을 위한 발현 벡터를 나타낸다. 여기서, rrnB T1T2는 전사 종결인자를 나타내고; rep(pBR322)는 E. coli에서 작동하는 pBR322로부터의 레플리콘을 나타내고; rep(pUB110)는 B. subtilis에서 작동하는 pUB110로부터의 레플리콘이고; KanR은 카나마이신 내성 유전자(아미노글리코시드 O-뉴클레오티딜트랜스퍼라제)를 나타내고; BJ27 promoter는 B. subtilis에 대한 강력한 프로모터를 나타낸다.20 shows an expression vector for overexpression of the sodA2 gene. Here, rrnB T1T2 represents a transcription terminator; rep(pBR322) represents the replicon from pBR322 that works in E. coli; rep(pUB110) is a replicon from pUB110 that works in B. subtilis; KanR stands for kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); The BJ27 promoter represents a strong promoter for B. subtilis.
후술하는 본 발명에 대한 상세한 설명은, 본 발명이 실시될 수 있는 특정 구현예에 관하여 특정 도면을 참조하여 기술될 것이지만, 본 발명은 이에 한정되지 않고, 적절하게 설명된다면, 그 청구항들이 주장하는 것과 균등한 모든 범위와 더불어 첨부된 청구항에 의해서만 한정된다. 본 발명의 다양한 구현예/실시예는 서로 다르지만 상호 배타적일 필요는 없음이 이해되어야 한다. 예를 들어, 본 명세서에 기재되어 있는 특정 형상, 구조 및 특성은 본 발명의 기술적 사상과 범위를 벗어나지 않으면서 일 구현예/실시예에서 다른 구현예/실시예로 변경되거나 구현예/실시예들이 조합되어 구현될 수 있다. 본 명세서에 사용된 기술 및 학술 용어들은, 달리 정의되지 않는 한, 본 발명이 속하는 분야에서 일반적으로 사용되는 것과 같은 의미를 갖는다. 본 명세서를 해석할 목적으로 하기 정의들이 적용될 것이고, 단수로 사용된 용어는 적절한 경우에는 복수형을 포함할 것이며 그 반대도 마찬가지이다.The detailed description of the invention that follows will be described with reference to specific drawings in relation to specific embodiments in which the invention may be practiced, but the invention is not limited thereto and, if properly described, what the claims claim and All scopes of equivalence are limited only by the appended claims. It should be understood that the various implementations/embodiments of the present invention are different from each other but are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be changed from one implementation/embodiment to another or implementation/embodiment may be changed without departing from the spirit and scope of the present invention. can be implemented in combination. Technical and scientific terms used herein, unless defined otherwise, have the same meaning as commonly used in the field to which this invention belongs. For purposes of interpreting this specification, the following definitions will apply and terms used in the singular will include the plural as appropriate and vice versa.
정의Justice
본 명세서에서 사용되는 "개체"는 "환자"와 상호교환적으로 사용되고, 구강 위생 또는 구강 질환의 예방 또는 치료를 필요로 하는 포유동물, 예를 들어 영장류(예컨대 인간, 원숭이, 침팬지 등), 반려 동물(예컨대 개, 고양이 등), 가축 동물(예컨대 소, 돼지, 말, 양, 염소 등) 또는 실험실 동물(예컨대 랫트, 마우스, 기니피그 등)일 수 있다. As used herein, "subject" is used interchangeably with "patient", and is a mammal in need of oral hygiene or prevention or treatment of oral disease, such as a primate (eg, human, monkey, chimpanzee, etc.), companion It may be an animal (eg dog, cat, etc.), a domestic animal (eg cow, pig, horse, sheep, goat, etc.) or a laboratory animal (eg rat, mouse, guinea pig, etc.).
용어 "치료"는 일반적으로 목적하는 약리학적 효과 및/또는 생리학적 효과를 수득하는 것을 의미한다. 이러한 효과는 질환 및/또는 다른 원하지 않거나 바람직하지 않은 상태(예컨대 구취, 치석, 치태, 치아 우식증, 치은염, 치주염, 통증 등)를 부분적으로 또는 완전히 치유하는 점에서 치료적 효과를 가진다. 바람직한 치료적 효과는 질환의 발생 또는 재발 방지, 증상의 호전, 질환의 임의의 직접 또는 간접적인 병리학적 결과의 축소, 전이의 방지, 질환 진행 속도의 감소, 질환 상태의 호전 또는 완화 및 차도 또는 개선된 예후를 포함하지만 이에 제한되지 않는다. 바람직하게는 "치료"는 이미 나타난 질환 또는 장애의 의료적 개입을 의미할 수 있다. The term “treatment” generally means obtaining a desired pharmacological and/or physiological effect. This effect has a therapeutic effect in terms of partially or completely curing diseases and/or other unwanted or undesirable conditions (such as halitosis, tartar, plaque, dental caries, gingivitis, periodontitis, pain, etc.). Desirable therapeutic effects include preventing occurrence or recurrence of the disease, amelioration of symptoms, diminishment of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of the disease state, and remission or amelioration. Prognosis includes, but is not limited to. Preferably, “treatment” may refer to medical intervention for a pre-existing disease or disorder.
용어 "예방"은 질병 또는 이의 증상을 부분적으로 또는 완전히 예방한다는 관점에서 목적하는 예방적인 약리학적 효과 및/또는 생리학적 효과를 수득함을 의미한다. The term “prevention” means obtaining a desired prophylactic pharmacological and/or physiological effect in terms of partially or completely preventing a disease or symptom thereof.
용어 "투여"는 개체에 예방적 또는 치료적 목적을 달성하기 위한 유효성분을 제공하는 것을 의미한다.The term "administration" means providing an active ingredient to achieve a prophylactic or therapeutic purpose to a subject.
구강 조성물oral composition
본 발명은 부분적으로 식나무 추출물과 조합하여 수퍼옥시드 디스뮤타제(SOD) 또는 바실러스 종 균주 포자를 개체의 구강에 적용하거나, 식나무 추출물과 조합하여 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자를 개체의 구강에 적용하는 경우에 구강 위생의 증진 또는 구강 질환의 예방 또는 치료에 효과적이라는 발견에 기초한다. 따라서, 본 발명의 일 태양에 따르면, (i) 식나무 추출물 및 (ii) 수퍼옥시드 디스뮤타제(SOD), 바실러스 종 균주 포자, 또는 SOD 및 바실러스 종 균주 포자를 포함하는 구강용 조성물이 제공된다. 상기 조성물은 식나무 추출물과 조합하여 수퍼옥시드 디스뮤타제 또는 바실러스 종 균주 포자 중 어느 하나를 포함할 수 있다. 바람직하게는, 상기 조성물은 식나무 추출물과 조합하여 수퍼옥시드 디스뮤타제 및 바실러스 종 균주 포자를 포함할 수 있다.The present invention partially applies superoxide dismutase (SOD) or Bacillus sp. strain spores in combination with cedar extract to the oral cavity of a subject, or superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract. It is based on the finding that the spores, when applied to the oral cavity of a subject, are effective in promoting oral hygiene or preventing or treating oral diseases. Accordingly, according to one aspect of the present invention, there is provided an oral composition comprising (i) a cedar extract and (ii) superoxide dismutase (SOD), Bacillus sp. strain spores, or SOD and Bacillus sp. strain spores. . The composition may include either superoxide dismutase or spores of a Bacillus sp. strain in combination with a pine extract. Preferably, the composition may include superoxide dismutase and spores of a Bacillus sp. strain in combination with a pine extract.
식나무(Aucuba japonica)는 쌍떡잎식물 산형화목 층층나무과의 상록과목의 일종으로서, 한국 남부, 특히 울릉도 지역에 주로 분포하고 있다. 다른 말로 도엽산호(桃葉珊瑚), 천각판(天脚板) 혹은 청목(靑木)이라고도 지칭된다. Aucuba japonica is a kind of evergreen plant of the dicotyledonous umbel dogwood family, and is mainly distributed in the southern part of Korea, especially in the Ulleungdo region. In other words, it is also referred to as Doyeop Coral, Cheongakpan, or Blue Tree.
본 명세서에 사용되는 바와 같은 용어 "추출물"은 목적하는 물질을 다양한 용매에 침지한 다음, 상온, 저온 또는 가온 상태에서 일정시간 동안 추출하여 수득한 액상성분, 상기 액상성분으로부터 용매를 제거하여 수득한 고형분 등의 결과물을 의미한다. 뿐만 아니라, 상기 결과물에 더하여, 상기 결과물의 희석액, 이들의 농축액, 이들의 조정제물, 정제물 등을 모두 포함하는 것으로 포괄적으로 해석될 수 있다.As used herein, the term "extract" refers to a liquid component obtained by immersing a desired material in various solvents and then extracting at room temperature, low temperature or elevated temperature for a certain period of time, and a liquid component obtained by removing the solvent from the liquid component. It means the result of solid content, etc. In addition, in addition to the above results, it can be comprehensively interpreted as including all dilutions of the results, concentrates thereof, adjusted products, and purified products thereof.
일부 구현예에서, 상기 식나무 추출물은 식나무의 지상부의 전체, 그 일부분, 또는 이들로부터 유래된 재료로부터 추출된 추출물일 수 있다. 상기 식나무 추출물은 열수 추출물인 것이 바람직하지만, 이로 한정되지는 않는다. In some embodiments, the pine tree extract may be an extract extracted from the whole of the aerial part of a pine tree, a part thereof, or a material derived therefrom. The pine extract is preferably a hot water extract, but is not limited thereto.
상기 식나무의 일부분은 식나무의 줄기, 잎, 꽃, 꽃잎 또는 종실일 수 있다. 상기 식나무 추출물은 식나무의 잎, 줄기 또는 이의 혼합물로부터 추출된 것이 바람직하지만, 이로 한정되지는 않는다.The part of the tree may be a stem, leaf, flower, petal or seed of the tree. The pine extract is preferably extracted from leaves, stems or mixtures thereof of pine trees, but is not limited thereto.
추출에 사용된 상기 식나무의 전체, 그 일부분, 또는 이들로부터 유래된 재료는 분쇄 또는 세절되거나 적당하게 건조된 것일 수 있다. Whole, part of, or material derived therefrom used for extraction may be ground or shredded or suitably dried.
추출 용매는 특별히 한정되지 않으며, 예를 들면, 물 또는 유기용매가 사용될 수 있고, 유기용매로는 메탄올, 에탄올, 프로판올, 이소프로판올, 부탄올 등을 포함하는 탄소수 1 내지 4의 알코올, 아세톤, 에테르, 벤젠, 클로로포름, 에틸 아세테이트, 메틸렌 클로라이드, 헥산 및 시클로헥산 등의 각종 용매가 단독으로 또는 혼합하여 사용될 수 있지만, 이로 한정되지는 않는다.The extraction solvent is not particularly limited, and for example, water or an organic solvent may be used, and the organic solvent includes methanol, ethanol, propanol, isopropanol, alcohol having 1 to 4 carbon atoms including butanol, acetone, ether, and benzene. , Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or in combination, but are not limited thereto.
추출 방법으로는 열수추출법, 냉침추출법, 환류냉각추출법, 용매추출법, 수증기증류법, 초음파추출법, 용출법, 압착법 등의 방법 중 어느 하나를 선택하여 사용할 수 있고, 바람직하게는 열수추출법일 수 있다. 또한, 상기 추출 방법 중 하나 이상을 조합하여 사용할 수 있다. 목적하는 추출물은 추가로 통상의 분획 공정을 수행할 수도 있으며, 통상의 정제 방법을 이용하여 정제될 수도 있다. 본 발명에 따른 추출물의 제조방법에는 제한이 없으며, 공지되어 있는 어떠한 방법도 이용될 수 있다.As an extraction method, any one of methods such as hot water extraction, cold brew extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression may be selected and used, preferably hot water extraction. In addition, one or more of the above extraction methods may be used in combination. The desired extract may be additionally subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the preparation method of the extract according to the present invention, and any known method may be used.
일부 구현예에서, 식나무 추출물은 식나무의 열수 추출물일 수 있으며, 약재 중량의 3 내지 20배, 바람직하게는 5 내지 15배의 물을 가하여, 80 내지 120℃, 바람직하게는 85 내지 110℃, 보다 바람직하게는 90 내지 105℃의 온도로, 1 내지 24시간, 바람직하게는 2 내지 12 시간, 보다 바람직하게는 3 내지 8 시간 추출된 것일 수 있으나, 열수 추출방법은 이로 한정되지는 않는다.In some embodiments, the pine extract may be a hot water extract of pine tree, and 3 to 20 times the weight of the medicinal material is added, preferably 5 to 15 times the water, and the temperature is 80 to 120 ℃, preferably 85 to 110 ℃, more Preferably, it may be extracted at a temperature of 90 to 105 ° C. for 1 to 24 hours, preferably for 2 to 12 hours, and more preferably for 3 to 8 hours, but the hot water extraction method is not limited thereto.
다른 구현예에서, 추출물은 상기 추출 이후에 필요에 따라 여과, 농축, 건조 등의 추가 공정을 행한 것일 수 있다. 상기 추가 공정의 방법과 조건은 특별히 한정되지 않으며, 본 기술분야에 공지된 방법이나 통상적으로 행해지는 조건으로 수행될 수 있을 것이다.In another embodiment, the extract may be subjected to additional processes such as filtration, concentration, and drying as necessary after the extraction. The method and conditions of the additional process are not particularly limited, and may be performed by a method known in the art or under commonly performed conditions.
상기 식나무 추출물의 최종 농도는 100 내지 50,000 mg/kg일 수 있고, 바람직하게는 5,000 내지 25,000 mg/kg일 수 있지만, 이로 한정되지는 않는다.The final concentration of the pine tree extract may be 100 to 50,000 mg/kg, preferably 5,000 to 25,000 mg/kg, but is not limited thereto.
수퍼옥시드 디스뮤타제(Superoxide dismutase; SOD)는 수퍼옥시드(O2 ㆍ-) 라디칼의 일반 분자 산소(O2)와 과산화수소(H2O2)로의 디스뮤테이션(dismutation)을 번갈아 촉매하는 효소로서, SOD는 활성 산소 종을 제거하여 산화 스트레스를 줄이는 데 중요한 역할을 한다. SOD는 원핵 세포와 진핵 세포에 널리 분포하고 있으며, 다양한 유형의 금속 중심(구리/아연, 니켈, 망간 및 철)에 따라 4가지 부류로 분류된다. 망간 함유 SOD[Mn-SOD]가 많은 세균 또는 진핵 세포의 엽록체, 미토콘드리아, 및 세포질에 광범위하게 존재한다. 본 발명에서, 용어 "SOD"는 수퍼옥시드 디스뮤타아제 활성을 갖는 (폴리)펩티드와 상호 교환적으로 사용될 수 있다. 또한, SOD는 수퍼옥시드 디스뮤타제 활성을 갖는 폴리펩티드 또는 이의 단편, 또는 이를 포함하는 융합체를 포함할 수 있다.Superoxide dismutase (SOD) is an enzyme that alternately catalyzes the dismutation of superoxide (O 2 ㆍ - ) radicals into ordinary molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ). As an enzyme, SOD plays an important role in reducing oxidative stress by scavenging reactive oxygen species. SODs are widely distributed in prokaryotic and eukaryotic cells and are classified into four classes according to the different types of metal centers (copper/zinc, nickel, manganese and iron). Manganese-containing SOD [Mn-SOD] is widely present in the chloroplasts, mitochondria, and cytoplasm of many bacterial or eukaryotic cells. In the present invention, the term "SOD" may be used interchangeably with (poly)peptide having superoxide dismutase activity. In addition, SOD may include a polypeptide having superoxide dismutase activity or a fragment thereof, or a fusion product containing the same.
일부 구현예에서, SOD는 망간에 결합하는 것(Mn-SOD)일 수 있다. 바람직하게는, SOD는 탈아미드화된 Mn-SOD일 수 있다.In some embodiments, the SOD can be manganese bound (Mn-SOD). Preferably, the SOD may be deamidated Mn-SOD.
일부 구현예에서, SOD는 서열번호 2로 표시되는 아미노산 서열을 포함하거나 이로 이루어진 것일 수 있다(SodA). 바람직하게는, SOD는 서열번호 2를 기준으로 74번 및 137번 아미노산 잔기가 Asp로 치환된 것일 수 있다. 보다 바람직하게는, SOD는 서열번호 4로 표시되는 아미노산 서열을 포함하거나 이로 이루어질 수 있다(SodA2).In some embodiments, the SOD may comprise or consist of the amino acid sequence represented by SEQ ID NO: 2 (SodA). Preferably, SOD may be one in which amino acid residues at positions 74 and 137 of SEQ ID NO: 2 are substituted with Asp. More preferably, the SOD may include or consist of the amino acid sequence represented by SEQ ID NO: 4 (SodA2).
본 발명의 SOD 또는 SOD 활성을 갖는 폴리펩티드는 상기 아미노산 서열에 대하여 실질적인 동일성을 나타내는 아미노산 서열도 포함되는 것으로 해석된다. 상기 실질적인 동일성은 당업계에 통상적으로 이용되는 알고리즘을 이용하여 정렬된 서열을 분석하는 경우, 80% 이상, 바람직하게는 90% 이상, 보다 바람직하게는 95% 이상, 가장 바람직하게는 98% 이상의 서열 동일성을 나타내는 아미노산 서열을 의미한다. It is construed that the SOD or polypeptide having SOD activity of the present invention also includes an amino acid sequence showing substantial identity to the above amino acid sequence. The substantial identity is 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more when the aligned sequences are analyzed using an algorithm commonly used in the art. Means an amino acid sequence that exhibits identity.
일부 구현예에서, SOD는 SOD 효소 활성을 갖는 개질된(modified 또는 engineered) 폴리펩티드로서 다양한 측면(생체내, 시험관내 또는 생체외 안정성, 균일성, 및/또는 형태적 변경)에 영향을 미치거나 미치지 않는 하나 이상의 변이, 예컨대, 하나 이상의 아미노산의 결실, 삽입 또는 치환을 포함할 수 있다. 또한, 상기 폴리펩티드는 정제, 검출, 생체내 전달 또는 안정성 증가를 위한 이종성 물질(예컨대, HIS tag, HA tag, myc tag를 포함한 당업계에 공지된 tag, GFC 및/또는 항체의 Fc 도메인)을 추가로 포함할 수 있다.In some embodiments, the SOD is a modified or engineered polypeptide having SOD enzymatic activity that does not affect or affect various aspects (in vivo, in vitro or ex vivo stability, uniformity, and/or morphological alteration). may include one or more mutations, such as deletions, insertions or substitutions of one or more amino acids. In addition, the polypeptide may be added with a heterologous material (eg, a tag known in the art, including a HIS tag, an HA tag, a myc tag, a GFC and/or an Fc domain of an antibody) for purification, detection, in vivo delivery, or increased stability. can be included with
일부 구현예에서, 본 발명의 SOD는 천연, 변이체 또는 재조합 미생물을 비롯한 다양한 공급원으로부터 유래된 것일 수 있다. 예를 들어, SOD는 박테리아로부터 유래될 수 있다. 바람직하게는, SOD는 약물 또는 식품에 사용하기에 일반적으로 안전한 것으로 간주되는(generally regarded as safe, GRAS) 박테리아, 예를 들어 바실러스 종(Bacillus sp.) 균주 또는 그의 변이체로부터 유래될 수 있다. 보다 바람직하게는 SOD는 바실러스 아밀로리퀴파시엔스(Bacillus amyloliquesfaciens) 균주(예컨대, GF423 균주 또는 GF424 균주) 또는 이들의 배양 상등액으로부터 얻어질 수 있다. 상기 GF423 균주 및 GF424 균주는 한국생명공학연구원에 각각 2017년 3월 6일자 및 2017년 3월 13일자로 기탁된 것이다(각각 수탁번호 KCTC 13222 BP 및 수탁번호 KCTC 13227 BP). 상기 바실러스 아밀로리쿼파시엔스 GF424 균주는 sod 유전자의 발현을 개선하기 위해 바실러스 아밀로리쿼파시엔스 GF423 균주를 UV 조사에 의해 돌연변이시킴으로써 얻은 균주이다. 바실러스 아밀로리퀴파시엔스 GF423 또는 GF424 균주에서 유래된 SOD 효소(SodA)는 Mn-SOD이며, 서열번호 2의 아미노산 서열을 갖는다(그의 뉴클레오티드 서열은 서열번호 1로 표시됨). 또한, SOD는 재조합 폴리펩티드일 수 있다. 예를 들어, 서열번호 2를 기준으로 74번 및 137번 아미노산 잔기가 Asp로 치환된 탈아미드화된 SOD일 수 있고(SodA2), 이는 서열번호 4의 아미노산 서열을 갖는다(그의 뉴클레오티드 서열은 서열번호 3으로 표시됨). 서열번호 1 내지 4의 서열은 표 1에 나타낸 바와 같다.In some embodiments, the SODs of the present invention may be derived from a variety of sources including natural, mutant or recombinant microorganisms. For example, SOD may be derived from bacteria. Preferably, the SOD may be derived from a bacterium that is generally regarded as safe (GRAS) for use in drugs or foods, such as a Bacillus sp. strain or variant thereof. More preferably, SOD may be obtained from a Bacillus amyloliquesfaciens strain (eg, GF423 strain or GF424 strain) or a culture supernatant thereof. The GF423 strain and the GF424 strain were deposited with the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017 and March 13, 2017, respectively (accession numbers KCTC 13222 BP and accession numbers KCTC 13227 BP, respectively). The Bacillus amyloliquefaciens GF424 strain is a strain obtained by mutating the Bacillus amyloliquefaciens GF423 strain by UV irradiation in order to improve sod gene expression. The SOD enzyme (SodA) derived from the Bacillus amyloliquefaciens GF423 or GF424 strain is Mn-SOD and has the amino acid sequence of SEQ ID NO: 2 (its nucleotide sequence is shown in SEQ ID NO: 1). Additionally, the SOD may be a recombinant polypeptide. For example, based on SEQ ID NO: 2, amino acid residues at positions 74 and 137 may be deamidated SOD in which Asp is substituted (SodA2), which has the amino acid sequence of SEQ ID NO: 4 (its nucleotide sequence is SEQ ID NO: marked 3). The sequences of SEQ ID NOs: 1 to 4 are as shown in Table 1.
구분division 서열order 서열번호sequence number
SodA의 뉴클레오티드 서열Nucleotide sequence of SodA ATGGCTTACAAACTTCCAGAATTGCCTTACGCTTATGATGCTTTAGAACCTCATATCGATAAGGAAACGATGACGATTCACCATACGAAGCACCATAACACATACGTGACAAACCTCAACAAAGCGATCGAAGGATCTGCGCTTGCAGAGAAATCTGTAGATGAGCTTGTTGCTGATTTGAACGCAGTGCCGGAGGACATCCGCACGGCAGTCCGCAACAATGGCGGCGGACATGCAAACCACTCTTTATTCTGGACTCTTTTATCTCCGAACGGCGGAGGCGAACCGACTGGTGAGCTTGCTGAAGAGATCAAAAGCACGTTCGGAAGCTTCGATCAATTTAAAGAAAAATTCGCCGCAGCAGCTGCAGGCCGTTTCGGTTCAGGCTGGGCTTGGCTCGTTGTAAACAACGGCAAACTTGAAATTACAAGCACGCCAAACCAAGATTCACCGCTTTCAGAAGGTAAAACACCTGTTCTCGGTCTTGATGTTTGGGAGCATGCGTACTACCTGAACTACCAAAACCGCCGTCCTGATTACATTTCAGCTTTCTGGAATGTTGTGAACTGGGATGAAGTTGCCCGTCTTTACAGCGAAGCAAAATAAATGGCTTACAAACTTCCAGAATTGCCTTACGCTTATGATGCTTTAGAACCTCATATCGATAAGGAAACGATGACGATTCACCATACGAAGCACCATAACACATACGTGACAAACCTCAACAAAGCGATCGAAGGATCTGCGCTTGCAGAGAAATCTGTAGATGAGCTTGTTGCTGATTTGAACGCAGTGCCGGAGGACATCCGCACGGCAGTCCGCAACAATGGCGGCGGACATGCAAACCACTCTTTATTCTGGACTCTTTTATCTCCGAACGGCGGAGGCGAACCGACTGGTGAGCTTGCTGAAGAGATCAAAAGCACGTTCGGAAGCTTCGATCAATTTAAAGAAAAATTCGCCGCAGCAGCTGCAGGCCGTTTCGGTTCAGGCTGGGCTTGGCTCGTTGTAAACAACGGCAAACTTGAAATTACAAGCACGCCAAACCAAGATTCACCGCTTTCAGAAGGTAAAACACCTGTTCTCGGTCTTGATGTTTGGGAGCATGCGTACTACCTGAACTACCAAAACCGCCGTCCTGATTACATTTCAGCTTTCTGGAATGTTGTGAACTGGGATGAAGTTGCCCGTCTTTACAGCGAAGCAAAATAA 서열번호 1SEQ ID NO: 1
SodA의 아미노산 서열Amino acid sequence of SodA MAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNNGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNNGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAKMAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNNGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNNGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAK 서열번호 2SEQ ID NO: 2
SodA2의 뉴클레오티드 서열Nucleotide sequence of SodA2 ATGGCTTACAAACTTCCAGAATTGCCTTACGCTTATGATGCTTTAGAACCTCATATCGATAAGGAAACGATGACGATTCACCATACGAAGCACCATAACACATACGTGACAAACCTCAACAAAGCGATCGAAGGATCTGCGCTTGCAGAGAAATCTGTAGATGAGCTTGTTGCTGATTTGAACGCAGTGCCGGAGGACATCCGCACGGCAGTCCGCAACGATGGCGGCGGACATGCAAACCACTCTTTATTCTGGACTCTTTTATCTCCGAACGGCGGAGGCGAACCGACTGGTGAGCTTGCTGAAGAGATCAAAAGCACGTTCGGAAGCTTCGATCAATTTAAAGAAAAATTCGCCGCAGCAGCTGCAGGCCGTTTCGGTTCAGGCTGGGCTTGGCTCGTTGTAAACGACGGCAAACTTGAAATTACAAGCACGCCAAACCAAGATTCACCGCTTTCAGAAGGTAAAACACCTGTTCTCGGTCTTGATGTTTGGGAGCATGCGTACTACCTGAACTACCAAAACCGCCGTCCTGATTACATTTCAGCTTTCTGGAATGTTGTGAACTGGGATGAAGTTGCCCGTCTTTACAGCGAAGCAAAATAAATGGCTTACAAACTTCCAGAATTGCCTTACGCTTATGATGCTTTAGAACCTCATATCGATAAGGAAACGATGACGATTCACCATACGAAGCACCATAACACATACGTGACAAACCTCAACAAAGCGATCGAAGGATCTGCGCTTGCAGAGAAATCTGTAGATGAGCTTGTTGCTGATTTGAACGCAGTGCCGGAGGACATCCGCACGGCAGTCCGCAACGATGGCGGCGGACATGCAAACCACTCTTTATTCTGGACTCTTTTATCTCCGAACGGCGGAGGCGAACCGACTGGTGAGCTTGCTGAAGAGATCAAAAGCACGTTCGGAAGCTTCGATCAATTTAAAGAAAAATTCGCCGCAGCAGCTGCAGGCCGTTTCGGTTCAGGCTGGGCTTGGCTCGTTGTAAACGACGGCAAACTTGAAATTACAAGCACGCCAAACCAAGATTCACCGCTTTCAGAAGGTAAAACACCTGTTCTCGGTCTTGATGTTTGGGAGCATGCGTACTACCTGAACTACCAAAACCGCCGTCCTGATTACATTTCAGCTTTCTGGAATGTTGTGAACTGGGATGAAGTTGCCCGTCTTTACAGCGAAGCAAAATAA 서열번호 3SEQ ID NO: 3
SodA2의 아미노산 서열Amino acid sequence of SodA2 MAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNDGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNDGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAKMAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNDGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNDGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAK 서열번호 4SEQ ID NO: 4
또한, SOD는 상기 바실러스 아밀로리퀴파시엔스 균주의 SOD 발현 유전자를 포함하는 다른 재조합 균주(예컨대, 바실러스 서브틸리스(Bacillus Subtilis) 종 균주)로부터 유래될 수 있다. 상기 재조합 균주는 당업계에 알려진 통상적인 단백질 생산 균주를 이용하여 재조합 기술에 의해 제조될 수 있다. 예컨대, 상기 (재조합 균주의 모균주인) 바실러스 서브틸리스 균주는 KCTC 3135일 수 있으며, 상기 KCTC 3135 균주는 한국생명공학연구원 생물자원센터(KCTC)로부터 분양받을 수 있다. 또한, 상기 재조합 균주는 후공정을 유리하기 위해 하기 표 2에 나타낸 유전자 중 하나 이상이 제거될 수 있다. In addition, SOD may be derived from other recombinant strains (eg, Bacillus Subtilis species strains) containing the SOD expression gene of the Bacillus amyloliquefaciens strain. The recombinant strain may be prepared by recombinant technology using a conventional protein production strain known in the art. For example, the Bacillus subtilis strain (the parent strain of the recombinant strain) may be KCTC 3135, and the KCTC 3135 strain may be distributed from the Korea Research Institute of Bioscience and Biotechnology (KCTC). In addition, one or more of the genes shown in Table 2 may be removed from the recombinant strain to facilitate post-processing.
유전자 명칭gene name 단백질 명칭protein name 기능function
AprEAprE 세린 알칼리 프로테아제(서브틸리신 E)Serine alkaline protease (subtilisin E) 세포외 프로테아제extracellular protease
NprENprE 세포외 중성 메탈로프로테아제extracellular neutral metalloprotease 세포외 프로테아제extracellular protease
BprBpr 바실로펩티다제(bacillopeptidase) Fbacillopeptidase F 세포외 프로테아제extracellular protease
EprEpr 세포외 세린 프로테아제extracellular serine protease 세포외 프로테아제extracellular protease
NprBNprB 세포외 중성 프로테아제 Bextracellular neutral protease B 세포외 프로테아제extracellular protease
VprVpr 세포외 세린 프로테아제extracellular serine protease 세포외 프로테아제extracellular protease
MprMpr 세포외 메탈로프로테아제extracellular metalloprotease 세포외 프로테아제extracellular protease
IspAIspAs 세포내 세린 프로테아제intracellular serine protease 세포내 프로테아제intracellular protease
SrfACSrfAC 서팩틴 신타제surfactin synthase 서팩틴(surfactin) 합성Surfactin synthesis
spoIIACspoIIAC RNA 폴리머라제 포자형성-특이적 시그마 인자(시그마-F)RNA polymerase sporulation-specific sigma factor (sigma-F) 포자형성sporulation
EpsEEpsE 추정 글리코실트랜스퍼라제putative glycosyltransferase 세포외 다당류extracellular polysaccharide
XpfXpf RNA 폴리머라제 시그마 인자RNA polymerase sigma factor PBSX 프로파지 유전자의 전사Transcription of the PBSX prophage gene
예컨대, 상기 재조합 균주는 도 19에 도시된 과정을 거쳐 제조될 수 있다. 또한, 상기 재조합 균주는 도 20에 도시된 발현 벡터를 포함할 수 있다. 상기 도면에서 sodA 및 sodA2는 SOD를 코딩하는 유전자를 나타낸다. For example, the recombinant strain may be prepared through the process shown in FIG. 19 . In addition, the recombinant strain may include the expression vector shown in FIG. 20 . In the figure, sodA and sodA2 represent genes encoding SOD.
상기 균주 유래의 SOD는 세포 외부로 분비되는 효소이므로, 상기 균주를 이용하여 SOD를 제조하는 경우, 고가의 정제 과정(예를 들어 컬럼 정제)을 거치지 않고 개체에 안전성이 확보된 SOD를 대량으로 생산할 수 있기 때문에 효율적인 생산이 가능하다.Since SOD derived from the strain is an enzyme secreted outside the cell, when manufacturing SOD using the strain, it is possible to mass-produce SOD that is safe for individuals without going through an expensive purification process (eg, column purification). Because of this, efficient production is possible.
일부 구현예에서, 본 발명의 SOD는 상기 천연, 변이체 또는 재조합 미생물을 다양한 배양 배지에서 배양하여 수득될 수 있다. 예를 들어 구강 조성물에 포함되는 SOD는 바실러스 아밀로리퀴파시엔스 GF423 균주 또는 GF424 균주의 배양 상층액으로부터 분리될 수 있다. 구체적으로, 먼저 바실러스 아밀로리퀴파시엔스 균주를 다양한 유형의 배지에서 배양하여 배양액을 얻을 수 있다. 예를 들어, 복합 배지(pH 6.0 내지 7.0)를 사용하여 상기 균주를 약 25℃ 내지 약 42℃에서 약 1 일 내지 약 4 일 동안 성장시킨다. 상기 바실러스 아밀로리퀴파시엔스 균주를 배양하기 위한 다른 적합한 배지로는 LB(Luria-Bertani) 배지, ISP(International Streptomyces Project) 배지, NA(nutrient agar) 배지, BHI(brain heart infusion agar) 배지, SDA(sabouraud dextrose agar) 배지, PDA(potato dextrose agar) 배지, NB(nutrient broth) 배지 등이 포함된다. 바람직한 구현예에서, LB 배지, ISP 배지, BHI 배지, SDA 배지, 또는 NB 배지가 사용될 수 있다. 또한, SOD는 PubMed 또는 BRENDA(월드 와이드 웹의 brenda-enzymes.org)와 같은 데이터베이스에 제공된 정보를 사용하여 다른 천연, 변이체 또는 재조합 숙주로부터 공급될 수 있다.In some embodiments, the SOD of the present invention can be obtained by culturing the natural, mutant or recombinant microorganism in various culture media. For example, SOD included in the oral composition may be isolated from the culture supernatant of Bacillus amyloliquefaciens strain GF423 or strain GF424. Specifically, first, a culture solution may be obtained by culturing the Bacillus amyloliquefaciens strain in various types of media. For example, the strain is grown at about 25° C. to about 42° C. for about 1 day to about 4 days using a complex medium (pH 6.0 to 7.0). Other suitable media for culturing the Bacillus amyloliquefaciens strain include Luria-Bertani (LB) medium, International Streptomyces Project (ISP) medium, nutrient agar (NA) medium, brain heart infusion agar (BHI) medium, SDA (sabouraud dextrose agar) medium, PDA (potato dextrose agar) medium, NB (nutrient broth) medium, and the like. In a preferred embodiment, LB medium, ISP medium, BHI medium, SDA medium, or NB medium may be used. SODs can also be sourced from other natural, mutant or recombinant hosts using information provided in databases such as PubMed or BRENDA (brenda-enzymes.org on the World Wide Web).
일부 구현예에서, 본 발명의 SOD는 천연, 변이체 또는 재조합 균주의 배양물로부터 단리되거나 정제된 것일 수 있다. 이때 단리되거나 정제된 SOD 또는 그의 생물학적 활성 부분에는 그것이 유래된 세포 또는 조직 공급원으로부터의 세포 물질 또는 기타 오염 단백질을 실질적으로 포함하지 않는다. 예를 들어, 정제물은 균주 배양물로부터 한외 여과(Ultrafiltration), 황산암모늄 처리, 컬럼 정제, 농축 등을 통해 순수 분리된 것이거나, 한외 여과, 농축 등을 통해 얻어진 배양 농축액일 수 있다. "세포 물질이 실질적으로 없는"이라는 문구는, 단백질이 단리되거나 재조합적으로 생산되는 세포의 세포 성분으로부터 그러한 단백질이 분리된 단백질의 제조물을 포함한다. 일부 구현예에서, "세포 물질이 실질적으로 없는"이라는 문구는 원하지 않는 단백질이 건조 중량을 기준으로 약 30% 미만, 바람직하게는 약 20% 미만, 더욱 바람직하게는 약 10% 미만, 그리고 가장 바람직하게는 약 5% 미만인 단백질의 제조물을 포함한다.In some embodiments, the SODs of the present invention may be isolated or purified from cultures of natural, variant or recombinant strains. wherein the isolated or purified SOD or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which it was derived. For example, the purified product may be a pure product separated from a strain culture through ultrafiltration, ammonium sulfate treatment, column purification, concentration, or the like, or a culture concentrate obtained through ultrafiltration, concentration, or the like. The phrase "substantially free of cellular material" includes preparations of the protein in which the protein is isolated from cellular components of the cell from which the protein is isolated or recombinantly produced. In some embodiments, the phrase “substantially free of cellular material” means less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 10% by dry weight of unwanted proteins. Preferably it includes preparations of less than about 5% protein.
상기 SOD는 다음과 같은 정제 방법으로 정제하는 것이 바람직할 수 있지만, 이로 한정되지는 않는다. 예컨대, 앞서 바실러스 아밀로리퀴파시엔스 균주를 배양하여 얻은 배양액을 원심분리하여 배양 상등액을 수집한다. 상등액 분획을 고체상 추출로 전처리하고, 이어서 크로마토그래피로 단리하고 정제한다. 다양한 방식의 크로마토그래피를 사용하여 SOD를 정제할 수 있다. 바람직하게는, 소수성 상호작용 크로마토그래피가 사용된다.The SOD may be preferably purified by the following purification method, but is not limited thereto. For example, the culture supernatant is collected by centrifuging the culture solution obtained by previously culturing the Bacillus amyloliquefaciens strain. The supernatant fraction is pretreated by solid phase extraction, then isolated and purified by chromatography. SOD can be purified using a variety of methods of chromatography. Preferably, hydrophobic interaction chromatography is used.
일부 구현예에서, 상기 SOD는 균주 용해물(lysate), 균주 배양물, 균주 배양 농축액, 균주 배양 추출물, 또는 그의 건조 형태로 포함될 수 있다. 이때, "균주 용해물"은 균주를 배양하여 기계적 또는 화학적으로 파쇄하여 얻은 것을 의미하며 이로부터 추출, 희석, 농축, 정제 등의 추가 공정을 거친 것을 모두 포함할 수 있다. "균주 배양물"은 균주를 배양하여 얻은 배양액 그 자체 또는 그의 상층액을 의미할 수 있다. "균주 배양 농축액"은 균주 배양물로부터 한외 여과, 황산암모늄 처리, 컬럼 정제, 농축 등을 통해 순수 분리된 것이거나, 한외 여과, 농축 등을 통해 얻어진 배양 농축액을 지칭한다. "균주 배양 추출물"은 상기 배양액 또는 그의 농축액으로부터 추출한 것을 의미하며, 추출액, 추출액의 희석액 또는 농축액, 추출액을 건조하여 얻어지는 건조물, 또는 이의 조정제물 또는 정제물, 이를 분획한 분획물을 포함할 수 있다. 상기 건조 형태는 동결 건조 형태를 포함할 수 있다.In some embodiments, the SOD may be included in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof. At this time, the "strain lysate" means obtained by mechanically or chemically disrupting the strain by culturing it, and may include all of the additional processes such as extraction, dilution, concentration, and purification therefrom. "Strain culture" may refer to a culture medium itself or a supernatant thereof obtained by culturing a strain. "Strain culture concentrate" refers to a culture concentrate obtained by ultrafiltration, ammonium sulfate treatment, column purification, concentration, or the like from a strain culture, or obtained through ultrafiltration, concentration, or the like. "Strain culture extract" refers to an extract obtained from the culture medium or a concentrate thereof, and may include an extract, a dilution or concentrate of the extract, a dried product obtained by drying the extract, or a crude or purified product thereof, or a fraction thereof. The dried form may include a freeze-dried form.
일부 구현예에서, 상기 SOD는 그것이 유래된 세포 또는 조직 공급원으로부터의 세포 물질, 예컨대 세포외 소포체(Extracellular vesicle)를 포함할 수 있다. 이 경우에 SOD를 포함하는 세포 물질은 상기 기술한 바와 같이 당업계에 알려진 통상의 기술을 사용하여 천연, 변이체 또는 재조합 숙주를 비롯한 다양한 공급원을 배양하여 그 배양액으로부터 여과, 농축 등의 방법으로 분리될 수 있다. In some embodiments, the SOD may include cellular material from the cell or tissue source from which it was derived, such as extracellular vesicles. In this case, the cellular material containing SOD can be separated from the culture solution by filtration, concentration, etc. by culturing various sources including natural, mutant or recombinant hosts using conventional techniques known in the art as described above. can
한편, 상기 바실러스 종 균주 포자는 바실러스 종 균주를 적합한 배지에서 배양하고 포자 형성을 유도한 후에 생성된 포자를 분리하여 얻어질 수 있다. 일 구현예에서, 바실러스 종 균주 포자는 약물 또는 식품에 사용하기에 일반적으로 안전한 것으로 간주되는 GRAS 세균으로부터 공급될 수 있다. 바실러스 종 균주 포자는 프로테아제 및 낮은 pH에 내성이 있는 것으로 알려져 있다(Cutting SM. Bacillus probiotics. Food Microbiol. 2011;28:214-220. doi: 10.1016/j.fm.2010.03.007; 및 Wang Y, et al., In vitro assessment of probiotic properties of Bacillus isolated from naturally fermented congee from inner Mongolia of China. World J. Microb. Biot. 2010;26:1369-1377. doi: 10.1007/s11274-010-0309-7). 또한, 바실러스 종 균주 포자는 여러 국가에서 승인된 GRAS 프로바이오틱스(probiotics)이다. 구체적으로, 상기 바실러스 종 균주 포자는 바실러스 아밀로리퀴파시엔스 균주(예컨대, GF423 또는 GF424 균주)로부터 유래될 수 있다. 포자 형성은 당업계에 알려진 통상의 기술을 사용하여 유도될 수 있다. 예컨대, 균주를 전배양후 본배양시에 DSM, NB, SYP, LB2 등의 배지에서 포자 형성을 유도하여 얻어지는 균주의 포자로서, 천연, 변이체, 또는 재조합 숙주를 비롯한 다양한 공급원을 배양하여, 영양세포 제거 후, 여과, 농축, 혹은 원심분리를 통하여 분리할 수 있다.On the other hand, the spores of the Bacillus species strain may be obtained by culturing the Bacillus species strain in a suitable medium and isolating the spores produced after inducing sporulation. In one embodiment, spores of Bacillus sp. strains may be sourced from GRAS bacteria generally considered safe for use in drugs or foods. Bacillus species strain spores are known to be resistant to proteases and low pH (Cutting SM. Bacillus probiotics. Food Microbiol. 2011;28:214-220. doi: 10.1016/j.fm.2010.03.007; and Wang Y, et al., In vitro assessment of probiotic properties of Bacillus isolated from naturally fermented congee from inner Mongolia of China. World J. Microb. Biot. 2010;26:1369-1377. doi: 10.1007/s11274-010-0309-7) . In addition, spores of strains of Bacillus species are GRAS probiotics approved in several countries. Specifically, the spores of the Bacillus species strain may be derived from a Bacillus amyloliquefaciens strain (eg, GF423 or GF424 strain). Sporulation can be induced using conventional techniques known in the art. For example, as spores of a strain obtained by inducing sporulation in a medium such as DSM, NB, SYP, LB2, etc. during main culture after pre-cultivation of the strain, various sources including natural, mutant, or recombinant hosts are cultivated to produce feeder cells After removal, it can be separated through filtration, concentration, or centrifugation.
또한, 상기 식나무 추출물과 상기 SOD 효소 및/또는 바실러스 종 균주 포자는 서로 순차적으로, 역순으로 또는 동시에 투여될 수 있도록 하는 형태로 상기 조성물에 포함될 수 있다.In addition, the pine extract and the SOD enzyme and / or Bacillus sp. strain spores may be included in the composition in a form such that they can be administered sequentially, in reverse order or simultaneously with each other.
일부 구현예에서, 상기 구강용 조성물은 식나무 추출물과 SOD 및 바실러스 종 균주 포자를 포함하거나 이들이 병용될 수 있는 형태로 포함할 수 있다. 본 발명의 일 실시예에 따르면, 식나무 추출물과 SOD 및 바실러스 종 균주 포자가 조합된 혼합물을 구강에 적용하는 경우에 구취, 치석, 치태, 치아 우식증 또는 치주 질환(예컨대, 치은염 또는 치주염) 등의 예방, 개선 또는 치료에 우수한 효과를 나타내어 구강 건강 증진, 구강 위생 관리, 또는 구강 질환의 예방 또는 치료에 유효함을 확인하였다. 또한, 상기 혼합물은 구강 질환으로 인한 통증을 감소시키는 데에도 유효함을 확인하였다.In some embodiments, the oral composition may include pine extract, SOD, and Bacillus sp. strain spores, or may be included in a form in which they may be used in combination. According to one embodiment of the present invention, prevention of bad breath, calculus, dental plaque, dental caries or periodontal disease (eg, gingivitis or periodontitis) when a mixture of pine tree extract, SOD, and Bacillus species strain spores is applied to the oral cavity. , it was confirmed that it was effective for improving or treating oral health, oral hygiene management, or preventing or treating oral diseases. In addition, it was confirmed that the mixture was effective in reducing pain due to oral diseases.
일부 구현예에서, 상기 식나무 추출물과 SOD 및 바실러스 종 균주 포자는 상기 효과를 발휘하기에 적당한 비율로 혼합될 수 있다. 예컨대, 식나무 추출물은 일당 0.1 내지 10,000 mg/kg, 바람직하게는 1 내지 1,000 mg/kg, 더욱 바람직하게는 1 내지 100 mg/kg, 더욱더 바람직하게는 5 내지 50 mg/kg의 용량에 상응하는 함량으로 포함될 수 있고, SOD와 바실러스 종 균주 포자는 SOD 역가가 건조중량 g당 5,000 내지 20,000 U가 되고 포자가 건조중량 g당 0.1 x 10^10 내지 1 x 10^11 cfu가 되도록 하는 비율로 혼합될 수 있다. 또한, 최종 조성물은, 예를 들어 식나무 추출물 0.5% 내지 5%와 SOD 및/또는 바실러스 종 균주 포자 0.5% 내지 12%가 혼합된 것일 수 있다. 최종 조성물은 바람직하게는 식나무 추출물 2%와 SOD 및/또는 바실러스 종 균주 포자 2.5%가 혼합된 것일 수 있다. In some embodiments, the pine extract, SOD and spores of the Bacillus sp. strain may be mixed in an appropriate ratio to exert the effect. For example, the pine tree extract has an amount corresponding to a dose of 0.1 to 10,000 mg/kg, preferably 1 to 1,000 mg/kg, more preferably 1 to 100 mg/kg, and still more preferably 5 to 50 mg/kg per day. SOD and Bacillus sp. strain spores are mixed in a ratio such that the SOD titer is 5,000 to 20,000 U per g dry weight and the spores are 0.1 x 10^10 to 1 x 10^11 cfu per g dry weight. can In addition, the final composition may be, for example, a mixture of 0.5% to 5% of Japanese oak extract and 0.5% to 12% of SOD and/or spores of a Bacillus species strain. The final composition may preferably be a mixture of 2% of cedar extract and 2.5% of SOD and/or spores of a Bacillus species strain.
일부 구현예에서, 상기 조성물은 구강 건강 증진 또는 구강 위생 관리를 위해 사용될 수 있다. In some embodiments, the composition can be used for oral health promotion or oral hygiene management.
일부 구현예에서, 상기 조성물은 구강 질환의 예방 또는 치료를 위해 사용될 수 있다. In some embodiments, the composition can be used for preventing or treating oral diseases.
일부 구현예에서, 상기 조성물은 구취, 치석, 치태, 치아 우식증, 치주 질환 및 구내염(예컨대, 만성 구내염)으로부터 선택된 하나 이상의 질환을 예방, 개선, 또는 치료하기 위해 사용될 수 있다. In some embodiments, the composition can be used to prevent, ameliorate, or treat one or more diseases selected from halitosis, tartar, plaque, dental caries, periodontal disease, and stomatitis (eg, chronic stomatitis).
일부 구현예에서, 상기 조성물은 구강 통증을 감소시키기 위해 사용될 수 있다. In some embodiments, the composition can be used to reduce oral pain.
다른 구현예에서, 상기 조성물은 액체 형태, 고체 형태, 겔 형태, 분말 형태, 페이스트 형태, 또는 담체에 함침 또는 도포된 형태 등을 포함하여 당업계에 알려진 구강에 적용되는 다양한 형태를 가질 수 있다.In another embodiment, the composition may have various oral forms known in the art, including a liquid form, a solid form, a gel form, a powder form, a paste form, or a form impregnated or applied to a carrier.
구강용 제품oral products
본 발명의 다른 태양에 따르면, 상기 구강용 조성물을 포함하는 구강용 제품이 제공된다. 달리 말하면, 상기 구강용 제품은 상기 기술한 바와 같은 (i) 식나무 추출물과 (ii) SOD 및 바실러스 종 균주 포자로 이루어진 군에서 선택되는 하나 이상을 포함한다.According to another aspect of the present invention, an oral product containing the composition for oral cavity is provided. In other words, the oral product includes at least one selected from the group consisting of (i) pine extract and (ii) SOD and Bacillus sp. strain spores as described above.
상기 제품은 치약, 구강 세정제, 구강 청정제, 껌, 캔디류, 구강 스프레이, 구강 겔, 구강 연고제, 구강 패치, 및 구강 양치액로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 제한되지 않는다. The product may be one or more selected from the group consisting of toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral gel, oral ointment, oral patch, and mouthwash, but is not limited thereto.
본 발명의 구강용 제품은, 유효성분으로 식나무 추출물과 SOD 및/또는 바실러스 종 균주 포자를 포함하는 것 외에, 그 제형화에 필요한 당업계에 공지된 추가 성분을 포함할 수 있다. 예컨대, 구강용 제품이 치약인 경우에, 연마제, 보습제, 발포제, 감미료, 미백제 또는 향미제 등을 추가로 포함할 수 있다. 또한, 구강용 제품이 구강 겔인 경우에, 겔의 점성 또는 점착을 위한 중쇄 지방산 등의 증점제 등을 추가로 포함할 수 있다. 또한, 구강용 제품이 구강 세정제인 경우에, 비독성 알코올과 같은 담체를 추가로 포함할 수 있다. The oral product of the present invention, in addition to containing the pine extract and SOD and/or Bacillus sp. strain spores as active ingredients, may contain additional ingredients known in the art required for its formulation. For example, when the oral product is toothpaste, it may further include an abrasive, a moisturizing agent, a foaming agent, a sweetener, a whitening agent, or a flavoring agent. In addition, when the oral product is an oral gel, it may further include a thickener such as a medium chain fatty acid for viscosity or adhesiveness of the gel. In addition, when the oral product is a mouthwash, it may further include a carrier such as non-toxic alcohol.
약학 또는 수의학적 조성물pharmaceutical or veterinary composition
본 발명의 다른 태양에 따르면, 상기 기술한 바와 같은 식나무 추출물과 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자로 이루어진 군에서 선택된 하나 이상을 포함하는 조성물을 포함하는 약학 조성물 또는 수의학적 조성물이 제공된다. 본 발명의 약학 조성물은 인간을 제외한 동물에 적용되는 경우에 수의학적 조성물이라는 용어와 상호 교환적으로 사용될 수 있다. According to another aspect of the present invention, a pharmaceutical composition or a veterinary composition comprising a composition comprising at least one selected from the group consisting of pine extract as described above, superoxide dismutase (SOD) and spores of Bacillus sp. is provided. The pharmaceutical composition of the present invention may be used interchangeably with the term veterinary composition when applied to animals other than humans.
일부 구현예에서, 상기 조성물은 식나무 추출물과 조합하여 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자를 포함할 수 있다. In some embodiments, the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
일부 구현예에서, 상기 조성물은 구강 질환의 예방 또는 치료를 위해 사용될 수 있다. 구체적으로, 상기 구상 질환은 구취, 치석, 치태, 치아 우식증, 치주 질환, 및 만성 구내염으로부터 선택된 하나 이상의 질환일 수 있으나 이에 제한되지 않는다 In some embodiments, the composition can be used for preventing or treating oral diseases. Specifically, the spherical disease may be one or more diseases selected from halitosis, calculus, dental plaque, dental caries, periodontal disease, and chronic stomatitis, but is not limited thereto.
일부 구현예에서, 상기 조성물은 구강 통증의 예방 또는 치료를 위한 것일 수 있다. 상기 구강 통증은 다양한 구강내 질환 또는 바람직하지 않은 상태로 인한 것일 수 있다. 예컨대, 구강 통증은 치석, 치태, 치아 우식증, 치주 질환 또는 만성 구내염으로 인한 것일 수 있다.In some embodiments, the composition may be for preventing or treating oral pain. The oral pain may be due to various oral diseases or undesirable conditions. For example, oral pain may be due to plaque, plaque, dental caries, periodontal disease or chronic stomatitis.
본 발명의 약학 또는 수의학적 조성물은 약학적으로 허용되는 담체, 부형제 및 희석제로 이루어진 군에서 선택되는 하나 이상을 추가로 포함할 수 있다. 상기 약학적으로 허용되는 담체, 부형제 및/또는 희석제는 당업계에 통상적으로 사용되는 것일 수 있다. 담체, 부형제 또는 희석제로는, 예를 들어, 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 하이드록시 프로필 메틸 셀룰로오즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 이산화규소 등의 광물유 등을 들 수 있지만, 이에 제한되지 않는다.The pharmaceutical or veterinary composition of the present invention may further include one or more selected from the group consisting of pharmaceutically acceptable carriers, excipients and diluents. The pharmaceutically acceptable carrier, excipient and/or diluent may be one commonly used in the art. Carriers, excipients or diluents include, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Mineral oils such as cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and silicon dioxide, etc. It may include, but is not limited thereto.
제제화할 경우에는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 첨가제를 사용하여 조제될 수 있다. 상기 제제화를 위한 첨가제는 약제 분야에서 통상 사용하는 것 중에서 적합한 것을 선택할 수 있다.When formulated, it may be prepared using additives such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Additives for the formulation may be selected from those commonly used in the pharmaceutical field.
또한, 본 발명의 약학 또는 수의학적 조성물은 사용 방법에 따라 바람직한 형태로 제형화될 수 있으며, 특히 포유 동물에 투여된 후 활성 성분의 신속, 지속 또는 지연 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화될 수 있다. 그러한 제형의 구체적인 예로는 정제, 환제, 산제, 과립제, 시럽제, 액제, 캡슐제, 현탁제, 유제, 주사용액, 경고제(Plasters), 로션제, 리니멘트제, 리모나데제, 에어로졸제, 엑스제(Extracts), 엘릭실제, 연고제, 유동엑스제, 침제, 크림제, 연질 또는 경질 젤라틴 캡슐, 패치제 등이 있다.In addition, the pharmaceutical or veterinary composition of the present invention may be formulated in a preferred form depending on the method of use, and particularly known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated by adopting a method. Specific examples of such formulations include tablets, pills, powders, granules, syrups, solutions, capsules, suspensions, emulsions, injection solutions, plasters, lotions, liniments, limonade, aerosols, and extracts. There are extracts, elixirs, ointments, fluid extracts, infusions, creams, soft or hard gelatin capsules, and patches.
더 나아가 본 발명의 약학 또는 수의학적 조성물은 당해 기술 분야의 공지된 적절한 방법을 사용하여 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최신판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 바람직하게 제형화될 수 있다.Furthermore, the pharmaceutical or veterinary composition of the present invention can be prepared using a suitable method known in the art or using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA). can be formulated accordingly.
본 발명의 약학 또는 수의학적 조성물은 목적하는 방법에 따라 경구 투여 또는 비경구 투여될 수 있으며, 비경구 투여시 비강분무, 피부 외용 또는 복강내주사, 직장내주사, 피하주사, 정맥주사, 근육 내 주사 또는 흉부 내 주사 주입방식을 선택하는 것이 바람직하다. 또한, 본 발명의 약학 또는 수의학적 조성물은 구강 분무, 구강 도포, 구강내 주사 등 구강내 주입방식을 통해 적용되는 것이 바람직할 수 있다.The pharmaceutical or veterinary composition of the present invention may be administered orally or parenterally depending on the desired method, and in the case of parenteral administration, nasal spray, external skin application or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection Injection or intrathoracic injection is preferred. In addition, the pharmaceutical or veterinary composition of the present invention may be preferably applied through intraoral injection methods such as oral spray, oral application, and intraoral injection.
이때, 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 복합 조성물에 적어도 하나 이상의 부형제, 예를 들면, 전분, 탄산칼슘, 수크로스, 락토오스, 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제도 사용될 수 있다. 경구투여를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 포함되며, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면, 습윤제, 감미제, 방향제, 보존제 등이 사용될 수 있다. 경구 투여의 경우, SOD는 쉘락(shellac), 에틸 셀룰로오스, 히드록시프로필 메틸셀룰로스, 히드록시프로필 메틸셀루로스 프탈레이트, 제인(zein), 유드라짓(Eudragit) 및 이들의 조합과 같은 코팅제로 코팅될 수 있지만, 코팅제는 이로 한정되지 않는다.At this time, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the complex composition, for example, starch, calcium carbonate, sucrose, lactose, It may be prepared by mixing gelatin or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents. can be used For oral administration, SOD may be coated with a coating such as shellac, ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof. However, the coating agent is not limited thereto.
비경구 투여를 위한 형태로는 치약, 구강세정제, 국소 투여제(예를 들어, 겔, 크림, 연고, 드레싱 용액, 분무제, 기타 도포제) 등을 들 수 있다. 상기 국소 투여제의 제형의 일 예로는, 본 발명의 유효 성분을 천연 섬유 또는 합성 섬유로 만든 거즈 등의 담체에 함침시킨 것일 수 있다. 상기 겔, 크림 또는 연고의 경우, 치아 및 잇몸을 포함한 환부 또는 충치 부위나 그 주위에 직접 도포하는데 적합할 수 있다. 상기 분무제의 경우, 통상적인 분무제 제조 방법으로 제조될 수 있고, 압축용기나 기타 분무용기에 충전포장하여 구강 질환 부위에 수시로 분무 도포하여 구강 질환을 예방하거나 치료할 수 있다. 상기 드레싱 용액의 경우, 통상적인 드레싱 용액의 제조 방법으로 제조될 수 있고, 구강 질환 부위에 드레싱하거나 기타 세균 감염 부위에 드레싱하여 구강 질환을 예방하거나 치료할 수 있다.Forms for parenteral administration include toothpastes, mouthwashes, topical formulations (eg, gels, creams, ointments, dressing solutions, sprays, and other coating agents). As an example of the formulation of the topical administration agent, the active ingredient of the present invention may be impregnated with a carrier such as gauze made of natural or synthetic fibers. In the case of the gel, cream or ointment, it may be suitable for direct application on or around affected areas including teeth and gums or carious areas. In the case of the spray, it can be prepared by a conventional spray preparation method, and can be filled and packaged in a compression container or other spray container, and sprayed and applied to the oral disease area from time to time to prevent or treat oral diseases. In the case of the dressing solution, it can be prepared by a conventional dressing solution manufacturing method, and oral diseases can be prevented or treated by dressing an oral disease site or dressing another bacterial infection site.
본 발명의 약학 또는 수의학적 조성물은 약학적으로 또는 수의학적으로 유효한 양으로 투여된다. 용어 "약학적으로 유효한 양" 또는 "수의학적으로 유효한 양"은 의학적 또는 수의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자 또는 동물의 체중, 성별, 연령, 건강상태, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료 기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다.The pharmaceutical or veterinary composition of the present invention is administered in a pharmaceutically or veterinarily effective amount. The term "pharmaceutically effective amount" or "veterinarily effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical or veterinary treatment, and the effective dosage level is the level of the patient or animal. Weight, gender, age, health condition, severity, activity of drug, sensitivity to drug, time of administration, route of administration and rate of excretion, duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical field. can
본 발명의 약학 또는 수의학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 순차적 또는 동시에 투여될 수 있다. 또한, 상기 약학 조성물은 필요에 따라 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이러한 양은 통상의 기술자에 의해 용이하게 결정될 수 있다.The pharmaceutical or veterinary composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously. In addition, the pharmaceutical composition may be administered single or multiple times as needed. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, and this amount can be easily determined by a person skilled in the art.
식품 또는 사료 조성물food or feed composition
본 발명의 다른 태양에 따르면, 상기 기술한 바와 같은 (i) 식나무 추출물과 (ii) 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자로 이루어진 군에서 선택된 하나 이상을 포함하는 조성물을 포함하는 식품 조성물이 제공된다. According to another aspect of the present invention, a composition comprising at least one selected from the group consisting of (i) cedar extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above A food composition is provided.
일부 구현예에서, 상기 조성물은 식나무 추출물과 조합하여 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자를 포함할 수 있다. In some embodiments, the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
이러한 식품 조성물은 의료용 또는 뉴트라슈티컬(nutraceutical) 식품 조성물을 포함한다. 용어 "의료용 식품" 또는 "뉴트라슈티컬 식품"은 인체에 유익한 기능을 할 가능성이 있는 원료 또는 성분으로 제조된 식품으로서, 정상적인 기능을 유지하거나 인체의 생리적 기능을 활성화함으로써 건강을 유지시키거나 개선시키는 식품을 지칭하고, 식품의약품안전처에 의해 규정되지만 이로 한정되지 않으며, 임의의 통상적인 건강 식품을 그 의미에서 배제하지 않는다.Such food compositions include medical or nutraceutical food compositions. The term "medical food" or "nutraceutical food" refers to food made from raw materials or ingredients that have the potential to have beneficial functions in the human body, which maintain or improve health by maintaining normal functions or activating the physiological functions of the human body. Refers to food, as defined by, but not limited to, the Ministry of Food and Drug Safety, and does not exclude any conventional health food in that sense.
또한, 상기 식품에는 각종 식품류, 식품 첨가제, 음료(예를 들어, 기능성 음료, 천연 과일 주스 및 야채 음료), 껌, 차, 비타민 복합제, 건강기능식품, 기타 기능성 식품 등이 포함되지만 이로 한정되지 않는다.In addition, the food includes, but is not limited to, various foods, food additives, beverages (eg, functional drinks, natural fruit juice and vegetable drinks), gum, tea, vitamin complexes, health functional foods, and other functional foods. .
상기 식품은 당업계에 알려진 통상의 방법으로 제조될 수 있다. 예컨대, 상기 의료용 식품, 뉴트라슈티컬 식품 또는 건강기능식품은 구강 위생을 증진하거나 구강 질환을 예방하거나 개선시킬 목적으로 상기 SOD에 더하여 담체, 희석제, 부형제 및 첨가제 중 하나 이상을 추가로 포함하여 정제, 환제, 산제, 과립제, 분말제, 캡슐제 및 액제 제형으로 이루어진 군에서 선택된 하나로 제형화될 수 있다. 상기 담체, 부형제, 희석제 및 첨가제의 구체적인 예는 당업계에 잘 알려져 있으며 통상의 기술자는 그 제형에 따라 적절한 성분을 조합하여 제조할 수 있다.The food may be prepared by a conventional method known in the art. For example, the medical food, nutraceutical food, or health functional food further includes at least one of a carrier, diluent, excipient, and additive in addition to the SOD for the purpose of improving oral hygiene or preventing or improving oral disease, tablets, It may be formulated into one selected from the group consisting of pills, powders, granules, powders, capsules and liquid formulations. Specific examples of the carrier, excipient, diluent, and additive are well known in the art, and a person skilled in the art can prepare them by combining appropriate components according to the formulation.
상기 상술한 제형 내 유효성분으로서의 본 발명에 따른 식나무 추출물과 수퍼옥시드 디스뮤타제 및/또는 바실러스 종 균주 포자의 함량은 사용 형태 및 목적, 환자 상태, 증상의 종류 및 경중 등에 의하여 적절하게 조절할 수 있으며, 고형분 중량 기준으로 0.001 내지 99.9 중량%, 바람직하게는 0.01 내지 50 중량%일 수 있으나, 이에 한정되지 않는다The content of the extract of the present invention, superoxide dismutase and/or Bacillus species strain spores as active ingredients in the above-described formulation can be appropriately adjusted depending on the type and purpose of use, patient condition, type and severity of symptoms, etc. And, based on the weight of the solid content, it may be 0.001 to 99.9% by weight, preferably 0.01 to 50% by weight, but is not limited thereto.
본 발명의 식품의 투여 용량은 환자의 나이, 몸무게, 성별, 투여형태, 건강상태 및 질환 정도에 따라 달라질 수 있으며, 의사 또는 약사의 판단에 따라 일정 시간간격으로 1일 1회 내지 수회로 분할 투여할 수도 있다. 예컨대, 유효성분 함량을 기준으로 1일 투여량이 10 내지 1,000 ㎎/kg일 수 있다. 상기한 투여량은 평균적인 경우를 예시한 것으로서 개인적인 차이에 따라 그 투여량이 높거나 낮을 수 있다. 본 발명의 건강기능식품의 1일 투여량이 상기 투여 용량 미만이면 유의성 있는 효과를 얻을 수 없을 수 있으며, 그 이상을 초과하는 경우 비경제적일 뿐만 아니라 상용량의 범위를 벗어나므로 바람직하지 않은 부작용이 나타날 수 있다.The dosage of the food of the present invention may vary depending on the patient's age, weight, sex, dosage form, state of health and degree of disease, and divided administration once a day to several times at regular time intervals according to the judgment of a doctor or pharmacist You may. For example, the daily dosage may be 10 to 1,000 mg/kg based on the active ingredient content. The above dosage is an example of an average case, and the dosage may be higher or lower depending on individual differences. If the daily dose of the health functional food of the present invention is less than the above dose, a significant effect may not be obtained, and if it exceeds more than that, it is uneconomical and may cause undesirable side effects because it is out of the range of the usual dose. there is.
본 발명의 또 다른 태양에 따르면, 상기 기술한 바와 같은 (i) 식나무 추출물과 (ii) 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자로 이루어진 군에서 선택된 하나 이상을 포함하는 조성물을 포함하는 사료 조성물이 제공된다. 일부 구현예에서, 상기 조성물은 식나무 추출물과 조합하여 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자를 포함할 수 있다. According to another aspect of the present invention, a composition comprising at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above A feed composition is provided. In some embodiments, the composition may include superoxide dismutase (SOD) and Bacillus sp. strain spores in combination with cedar extract.
본 발명의 사료 조성물은 당업계에서 통상적으로 이용되는 어떠한 제형으로도 제조될 수 있다. 예를 들어, 본 발명의 사료 조성물은 아미노산, 무기염류, 비타민, 항생물질, 항균물질, 항산화, 항곰팡이 효소, 다른 생균 형태의 미생물 제제 등과 같은 보조제 성분; 곡물, 예를 들면 분쇄 또는 파쇄된 밀, 귀리, 보리, 옥수수 및 쌀; 식물성 단백질 사료, 예를 들면 평지, 콩 및 해바라기를 주성분으로 하는 것; 동물성 단백질 사료, 예를 들면 혈분, 육분, 골분 및 생선분; 당분 및 유제품, 예를 들면 각종 분유 및 유장 분말로 이루어지는 건조성분; 지질, 예를 들면 가열에 의해 임의로 액화시킨 동물성 지방 및 식물성 지방 등과 같은 주성분; 영양보충제, 소화 및 흡수향상제, 성장촉진제, 질병 예방제와 같은 첨가제를 추가로 포함할 수 있다.The feed composition of the present invention may be prepared in any formulation commonly used in the art. For example, the feed composition of the present invention may include auxiliary components such as amino acids, inorganic salts, vitamins, antibiotics, antimicrobial substances, antioxidants, antifungal enzymes, and other probiotic-type microorganism preparations; grains such as ground or crushed wheat, oats, barley, corn and rice; vegetable protein feeds such as those based on rape, soybean and sunflower; animal protein feed such as blood meal, meat meal, bone meal and fish meal; dry ingredients consisting of sugar and dairy products, such as various powdered milk and whey powder; main components such as lipids, for example, animal fats and vegetable fats optionally liquefied by heating; Additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease preventives may be further included.
본 발명의 사료 조성물은 분말 또는 액체 상태의 제제 형태일 수 있으며, 사료첨가용 부형제(탄산칼슘, 말분, 제올라이트, 옥분 또는 미강 등)를 포함할 수 있다. The feed composition of the present invention may be in the form of a powder or liquid formulation, and may include a feed additive excipient (calcium carbonate, horse powder, zeolite, jade powder or rice bran, etc.).
구강 위생의 증진 또는 구강 질환의 예방 또는 치료를 위한 방법Method for improving oral hygiene or preventing or treating oral disease
본 발명의 또 다른 태양에 따르면, 상기 기술한 바와 같은 (i) 식나무 추출물과 (ii) 수퍼옥시드 디스뮤타제(SOD)및 바실러스 종 균주 포자로 이루어진 군에서 선택된 하나 이상을 개체에 투여하는 단계를 포함하는 구강 위생의 증진을 위한 방법이 제공된다. 또한, 상기 기술한 바와 같은 (i) 식나무 추출물과 (ii) 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자로 이루어진 군에서 선택된 하나 이상을 개체에 투여하는 단계를 포함하는 구강 질환을 예방하거나 치료하는 방법이 제공된다. According to another aspect of the present invention, administering to a subject at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above There is provided a method for improving oral hygiene comprising a. In addition, prevention of oral diseases comprising the step of administering to the subject at least one selected from the group consisting of (i) pine extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above or method of treatment is provided.
일부 구현예에서, 상기 방법은 식나무 추출물과 조합하여 SOD 및 바실러스 종 균주 포자를 개체에 투여하는 단계를 포함할 수 있다.In some embodiments, the method may include administering SOD and Bacillus sp. strain spores to the subject in combination with a cedar extract.
일부 구현예에서, 상기 구강 질환은 구강 통증, 구취, 치태, 치석, 치아 우식증, 치주 질환 및 만성 구내염으로 이루어진 군에서 선택되는 하나 이상을 포함할 수 있다. In some embodiments, the oral disease may include one or more selected from the group consisting of oral pain, halitosis, plaque, calculus, dental caries, periodontal disease, and chronic stomatitis.
일 구현예에서, 상기 식나무 추출물, SOD 및 바실러스 종 균주 포자는, 조합 투여되는 경우에, 단일 조성물 또는 각각 또는 어느 두 성분을 함유한 별개 조성물로서 동시에 투여될 수 있다. 또한, 상기 식나무 추출물, SOD 및 바실러스 종 균주 포자는 각각 또는 어느 두 성분을 함유한 별개 조성물로서 순차적으로 또는 역순으로 투여될 수 있다. 동시 투여 또는 별개 투여의 경우에, 식나무 추출물, SOD 및 바실러스 종 균주 포자는 그 투약 형태에 따라 동일한 경로 또는 상이한 경로로 투여될 수 있다. 상기 조성물, 투약 형태, 투여 경로 등에 관해서는 조성물에 대해 상기 기술한 바와 같다.In one embodiment, the pine extract, SOD and Bacillus sp. strain spores, when administered in combination, may be administered simultaneously as a single composition or as separate compositions containing each or any two components. In addition, the pine extract, SOD and Bacillus sp. strain spores may be administered sequentially or in reverse order as separate compositions containing either or any two components. In the case of simultaneous administration or separate administration, the pine extract, SOD and Bacillus sp. strain spores may be administered by the same route or by different routes depending on the dosage form. The composition, dosage form, route of administration, etc. are as described above for the composition.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐이고 본 발명의 범위가 하기 실시예로 한정되는 것은 아니다. Hereinafter, examples will be described in detail to aid understanding of the present invention. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples.
실시예Example
실시예 1. 식나무 추출물의 제조Example 1. Preparation of cedar extract
식나무 잎과 줄기는 2020년 8월에 경남 거제시 일운면 지세포리 임야에서 채집하였으며, 증거표본은 전북대학교 시료보관실에 보관 중이다. 식나무 100 kg에 900 L의 증류수를 가한 다음 100℃에서 3시간 진탕하여 감압 농축한 후 건조하여 식나무 열수 추출물 분말을 얻었다.The leaves and stems of the cedar tree were collected from the forest in Jisepo-ri, Irun-myeon, Geoje-si, Gyeongsangnam-do in August 2020, and the evidence specimen is stored in the sample storage room of Chonbuk National University. 900 L of distilled water was added to 100 kg of pine tree, shaken at 100 ° C. for 3 hours, concentrated under reduced pressure, and then dried to obtain a hot water extract powder of pine tree.
실시예 2. Example 2. 바실러스 아밀로리쿼파시엔스Bacillus amyloliquefaciens 균주로부터 수퍼옥시드 디스뮤타아제의 분리 및 정제 Isolation and purification of superoxide dismutase from strains
본 실시예에 사용된 바실러스 아밀로리쿼파시엔스 균주는 바실러스 아밀로리쿼파시엔스 GF423 균주 및 바실러스 아밀로리쿼파시엔스 GF424 균주이며, 이들 균주는 한국생명공학연구원에 각각 2017년 3월 6일자 및 2017년 3월 13일자로 수탁번호 KCTC 13222 BP 및 수탁번호 KCTC 13227BP로 기탁된 것이다.The Bacillus amyloliqueurfaciens strains used in this example are the Bacillus amyloliqueurpaciens GF423 strain and the Bacillus amyloliqueurpaciens GF424 strain, and these strains were published by the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017 and 2017, respectively. It was deposited with accession number KCTC 13222 BP and accession number KCTC 13227BP as of March 13, 2018.
상기 바실러스 아밀로리쿼파시엔스 GF424 균주는 sodA 유전자의 발현을 개선하기 위해 바실러스 아밀로리쿼파시엔스 GF423 균주에 UV를 조사하여 돌연변이를 유발시켜 얻은 균주이다.The Bacillus amyloliquefaciens GF424 strain is a strain obtained by inducing mutation by irradiating UV to the Bacillus amyloliquefaciens GF423 strain in order to improve the expression of the sodA gene.
실시예 2.1. Example 2.1. 바실러스 아밀로리쿼파시엔스Bacillus amyloliquefaciens GF423 또는 GF424 균주의 배양 Cultivation of GF423 or GF424 strains
바실러스 아밀로리쿼파시엔스 GF423 또는 GF424 균주의 배양을 위해, LB 한천 배지(LB(Luria-Bertani) 한천; 트립토판 10 g/L, 효모 추출물 5 g/L, NaCl 10 g/L, 한천 15 g/L)에서 형성된 단일 콜로니를 LB 배지 30 ml에 접종하고, 37℃에서 12시간 동안 배양하였다. 이 종 배양액(seed culture)을 다시 1 mM 황산망간(MnSO4)이 포함된 LB 배지 3 L에 접종하고, 37℃에서 20시간 동안 배양하였다.For the cultivation of Bacillus amyloliquefaciens GF423 or GF424 strain, LB agar medium (Luria-Bertani (LB) agar; tryptophan 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L) A single colony formed in L) was inoculated into 30 ml of LB medium and cultured at 37° C. for 12 hours. The seed culture was again inoculated into 3 L of LB medium containing 1 mM manganese sulfate (MnSO 4 ) and cultured at 37° C. for 20 hours.
실시예 2.2. 수퍼옥시드 디스뮤타아제(SOD)의 분리 및 정제Example 2.2. Isolation and purification of superoxide dismutase (SOD)
실시예 2.1에서 얻은 세포 배양액을 4℃, 3,578 x g에서 20분간 원심분리하여 상등액을 취한 후, 초미세여과(Ultrafiltration, 이하 UF, MWCO 10,000)를 이용하여 10배 농축하였다. 농축액을 제균 필터로 여과한 후, 동결건조하였다. SOD의 활성은 SOD 분석 키트(Cayman Chemical, Michigan, USA)를 이용하여 분석하였다. SOD 활성 1 단위(unit)는 수퍼옥시드 라디칼을 50% 억제하는 효소의 양으로 정의된다. 건조된 SOD 효소의 활성은 30 ± 6 U/mg이었다. The cell culture medium obtained in Example 2.1 was centrifuged at 4° C. and 3,578 x g for 20 minutes to obtain a supernatant, and then concentrated 10 times using ultrafiltration (UF, MWCO 10,000). The concentrated solution was filtered through a sterilization filter and lyophilized. The activity of SOD was analyzed using a SOD assay kit (Cayman Chemical, Michigan, USA). One unit of SOD activity is defined as the amount of enzyme that inhibits superoxide radicals by 50%. The activity of the dried SOD enzyme was 30 ± 6 U/mg.
실시예 3. Example 3. 바실러스 아밀로리쿼파시엔스Bacillus amyloliquefaciens 균주 포자의 준비 Preparation of strain spores
실시예 3.1. 배지 조성Example 3.1. badge composition
본 실시예에 사용된 배지는 SYP 또는 DSM였다. SYP 배지는 1.5% 소이톤(soy tone), 0.5% 효모 추출물, 0.5 % K2HPO4, 0.1% MnSO4, 0.1% MgSO4, 10 mM FeSO4, 0.04% (NH4)2SO4, 0.04% (NH4)2PO4, 0.1% CaCl2 및 2% 포도당을 함유한다. DSM 배지는 박토 영양 브로쓰 8 g/L, KCl 1 g/L, MgSO4 0.25 g/L, Ca(NO3)2 0.16415 g/L, MnCl2 0.9521 mg/L 및 FeSO4 0.152 mg을 함유한다. MnSO4, MgSO4, FeSO4, (NH4)2SO4, (NH4)2PO4 및 CaCl2는 사용 전에 ddH2O에 용해시켜 첨가하였다.The medium used in this example was SYP or DSM. SYP medium contains 1.5% soy tone, 0.5% yeast extract, 0.5% K 2 HPO 4 , 0.1% MnSO 4 , 0.1% MgSO 4 , 10 mM FeSO 4 , 0.04% (NH 4 ) 2 SO 4 , 0.04% % (NH 4 ) 2 PO 4 , 0.1% CaCl 2 and 2% glucose. The DSM medium contains Bacto nutrient broth 8 g/L, KCl 1 g/L, MgSO 4 0.25 g/L, Ca(NO 3 ) 2 0.16415 g/L, MnCl 2 0.9521 mg/L and FeSO 4 0.152 mg/L. . MnSO 4 , MgSO 4 , FeSO 4 , (NH 4 ) 2 SO 4 , (NH 4 ) 2 PO 4 and CaCl 2 were dissolved in ddH 2 O and added before use.
실시예 3.2. 포자형성 유도Example 3.2. Induction of sporulation
바실러스 아밀로리쿼파시엔스 균주 GF423 또는 GF424의 단일 콜로니를 14 mL 튜브에 들어있는 LB 1 mL에 접종하고, 37℃ 및 200 rpm에서 12 시간 동안 배양하였다. 배양액 1 mL를 500 mL 플라스크 내의 LB 배지 50 mL로 옮기고, 37℃ 및 200 rpm에서 12 시간 동안 배양하였다. 이후, 20 mL의 배양 배지를 2.5 L 배플 플라스크 내의 SYP 또는 DSM 1 L로 옮겼다. 접종된 배양액을 37℃ 및 200 rpm에서 24 시간 내지 120 시간 동안 배양하였다.A single colony of Bacillus amyloliquefaciens strain GF423 or GF424 was inoculated into 1 mL of LB in a 14 mL tube and incubated at 37° C. and 200 rpm for 12 hours. 1 mL of the culture medium was transferred to 50 mL of LB medium in a 500 mL flask, and cultured at 37° C. and 200 rpm for 12 hours. Then, 20 mL of culture medium was transferred to 1 L of SYP or DSM in a 2.5 L baffled flask. The inoculated culture was cultured at 37° C. and 200 rpm for 24 to 120 hours.
실시예 3.3. 포자 세척Example 3.3. spore wash
배양 후, 라이소자임(0.5 g/L)을 배양 브로쓰에 첨가하고, 37℃ 및 200 rpm에서 1 시간 동안 배양하여 남아있는 영양 세포를 제거하였다. 6000 rpm에서 10 분 동안 원심분리하여 미정제 포자를 수거하였다. 수거된 미정제 포자를 물로 2회 세척하고, 0.02% SDS로 세척하고, 다시 물로 2회 세척하고, 이어서 PBS 용액에 현탁시킴으로써 정제하였다. 포자 현탁액을 -20℃에서 보관하였다. 희석된 포자 용액을 LB 한천 플레이트에 도말한 후 콜로니를 계수함으로써 포자 수를 결정하였다.After incubation, lysozyme (0.5 g/L) was added to the culture broth and incubated for 1 hour at 37° C. and 200 rpm to remove remaining feeder cells. Crude spores were harvested by centrifugation at 6000 rpm for 10 minutes. The harvested crude spores were purified by washing twice with water, then with 0.02% SDS, again with water twice, and then suspended in a PBS solution. The spore suspension was stored at -20 °C. The number of spores was determined by plating the diluted spore solution on LB agar plates and then counting colonies.
실험예 1. 식나무 추출물을 이용한 동물 시험Experimental Example 1. Animal test using pine extract
식나무 추출물이 구강 건강 증진 및 구강 위생 관리 효과를 나타내는지 확인하기 위해 비글견 또는 랫트를 이용한 시험을 수행하였다.A test using beagle dogs or rats was performed to determine whether the pine tree extract exhibited oral health promotion and oral hygiene management effects.
실험예 1.1. 시험 물질Experimental Example 1.1. test substance
시험 물질은 다음과 같이 준비하였다. 실시예 1에서 얻은 식나무 추출물이 2% 함유되어 있는 구강연고(시험군)와 식나무 추출물이 포함되어 있지 않은 구강연고(대조군, vehicle only)를 하기 표 3과 같은 조성으로 제조하였다.Test materials were prepared as follows. An oral ointment containing 2% of the pine extract obtained in Example 1 (test group) and an oral ointment not containing the pine extract (control group, vehicle only) were prepared with the compositions shown in Table 3 below.
성분ingredient 함량 (10 ml 기준)Content (based on 10 ml)
시험군(2% 식나무 추출물 함유)Test group (contains 2% cedar extract) 대조군(vehicle)Control (vehicle)
식나무 추출물cedar extract 200 mg200 mg --
점막부착제(Sodium polyacrylate, PAA-Na)Mucoadhesive (Sodium polyacrylate, PAA-Na) 300 mg300mg 300 mg300mg
향미증진제(Mannitol)Flavor enhancer (Mannitol) 100 mg100 mg 100 mg100 mg
보존제(Methyl paraben)Preservative (Methyl paraben) 18 mg18mg 18 mg18mg
보존제(Poly paraben)Preservative (Poly paraben) 2 mg2 mg 2 mg2 mg
실험예 1.2. 식나무 추출물을 이용한 치은염 지수 평가 시험Experimental Example 1.2. Gingivitis index evaluation test using pine tree extract
실험예 1.2.1. 시험 동물Experimental Example 1.2.1. test animal
㈜오리엔트 바이오로부터 3년령 이상이고 구강 임상 증상(구취, 치태, 치은염, 치주염 등)이 있는, 9 내지 13 kg의 수컷 비글견(non-naive)을 구입하고, 2주 이상의 휴약기를 가졌다.A non-naive male beagle dog weighing 9 to 13 kg, aged 3 years or older and having oral clinical symptoms (halitosis, plaque, gingivitis, periodontitis, etc.), was purchased from Orient Bio Co., Ltd. and had a drug holiday of 2 weeks or more.
시험 동물로 비글견을 선택한 이유는 비글견이 치은염 등 구강 관련 효능 평가 시험에 널리 사용되고 있으며, 풍부한 기초 시험 자료가 축적되어 있어 시험 결과의 해석 및 평가가 용이하기 때문이었다.The reason why the beagle dog was selected as the test animal was that the beagle dog is widely used in oral-related efficacy evaluation tests such as gingivitis, and it is easy to interpret and evaluate the test results due to the accumulation of abundant basic test data.
실험예 1.2.2. 시험 물질의 투여 및 평가 결과Experimental Example 1.2.2. Administration of test substance and evaluation result
실험예 1.1의 시험 물질을 4주간 매일 2회 잇몸(양쪽 상악 대구치 외측 치은 부위)에 도포하여 투여하였다. 투여량은 회당 0.5 g씩 1.0 g/day로 투여하였다. 1.0 g/day에 함유된 식나무 유효성분의 함량은 20 mg/day였다. 4주간의 식나무 추출물 투여 후 구강검사를 실시하여 하기 표 4에 제시된 기준에 따라 치은염 지수(Gingivitis index)를 평가하였다.The test substance of Experimental Example 1.1 was applied and administered to the gums (external gingival region of both maxillary molars) twice daily for 4 weeks. The dosage was administered at 1.0 g/day by 0.5 g per time. The content of the active ingredient contained in 1.0 g/day was 20 mg/day. After administration of the pine extract for 4 weeks, an oral examination was performed and the gingivitis index was evaluated according to the criteria shown in Table 4 below.
점수score 평가방법 Assessment Methods
00 질환 없음 no disease
1One 경미한 치은염
- 잇몸 : 약간의 발적
- 치아 : 약간의 치태 minor gingivitis
- Gums: Slight redness
- Teeth: Some plaque
22 초기 치주염
- 잇몸 : 발적과 부종
- 치아 : 잇몸아래 치태, 약간의 치석 early periodontitis
- Gums: redness and swelling
- Teeth: Plaque under the gums, some tartar
33 중등도 치주염
- 잇몸 : 발적, 부종, 출혈이 있을 수 있고, 단열 또는 증생
- 치아 : 중등도에서 심한 양의 치석, 잇몸하 치석, 치아가 흔들리거나 소실moderate periodontitis
- Gums: May have redness, swelling, bleeding, adiabatic or hyperplasia
- Teeth: Moderate to severe amounts of calculus, subgingival tartar, loosening or loss of teeth
44 심한 치주염
- 잇몸 : 심한 발적, 염증, 출혈, 치아 주변 pocket형성, 농이 있을 수 있음
- 치아 : 많은 양의 잇몸하 치석, 치아가 흔들리거나 소실severe periodontitis
- Gums: Severe redness, inflammation, bleeding, pocket formation around teeth, pus may be present
- Teeth: A large amount of subgingival calculus, teeth are loose or lost
그 결과, 도 1로부터 알 수 있는 바와 같이, 식나무 추출물이 포함되어 있지 않은 대조군(vehicle)을 투여한 비글견에서는 치은염 지수가 시간 경과에 따라 악화된 반면, 식나무 추출물이 포함된 구강연고를 투여한 비글견에서는 잇몸 부종 및 발적이 감소하여 결과적으로 치은염 지수가 약 15% 감소한 것을 확인할 수 있었다.As a result, as can be seen from FIG. 1, the gingivitis index deteriorated over time in the beagle dog administered with the control (vehicle) not containing the pine extract, whereas the oral ointment containing the pine extract was administered. In beagle dogs, it was confirmed that swelling and redness of the gums were reduced, and as a result, the gingivitis index was reduced by about 15%.
실험예 1.3. 식나무 추출물의 치조골 파괴 억제 효과 확인Experimental Example 1.3. Confirmation of alveolar bone destruction inhibitory effect of cedar extract
실험예 1.3.1. 시험 동물Experimental Example 1.3.1. test animal
6주령된 수컷 SD 랫트를 구입하였다(구입처: 다물사이언스, 대전, 대한민국). 입수한 랫트를 1주일간 순화시키고 물과 음식은 자유롭게 섭취시켰다.Six-week-old male SD rats were purchased (Purchased from Damul Science, Daejeon, Republic of Korea). Obtained rats were acclimatized for one week, and water and food were freely consumed.
순화과정이 끝난 랫트에 졸레틴과 케타민을 1:2 비율(1.5 ml/kg)로 섞어 복강내 주사하여 마취를 유도하였다. 마취 후 하악 좌측 제2 대구치 치경부를 silk 봉합사(0.5 mm)로 결찰시켜 치주염을 유발하였다. 봉합사로 결찰되지 않은 하악 우측 제2 대구치를 대조군으로 설정하였다.Anesthesia was induced by intraperitoneal injection of a mixture of zoletin and ketamine at a ratio of 1:2 (1.5 ml/kg) to the rats after the acclimatization process was completed. After anesthesia, periodontitis was induced by ligating the cervical region of the lower left second molar with a silk suture (0.5 mm). A mandibular right second molar that was not ligated with a suture was set as a control group.
실험예 1.3.2. 시험 물질의 투여 및 평가 결과Experimental Example 1.3.2. Administration of test substance and evaluation result
치주염을 유발시킨 날을 0일로 하였으며, 치주염을 유발한 날부터 2주 후에 랫트를 희생시켰다. 시험기간 동안 식나무 추출물(실험예 1.1.의 시험 물질)을 50, 100, 250 mg/kg의 농도로 1일 1회씩 경구 투여하였으며, 대조군에는 vehicle(DW)만 경구 투여하였다. 치주염을 유발한 날로부터 랫트를 희생시킨 날까지 봉합사가 유지되어 있는지 확인하였다.The day of inducing periodontitis was set as day 0, and the rats were sacrificed 2 weeks after the day of inducing periodontitis. During the test period, pine extract (test substance in Experimental Example 1.1) was orally administered once a day at concentrations of 50, 100, and 250 mg/kg, and only vehicle (DW) was orally administered to the control group. It was confirmed whether the suture was maintained from the day periodontitis was induced to the day the rats were sacrificed.
2주간의 식나무 추출물 투여 후 랫트를 희생시킨 후 하악을 적출하였다. 이후 1M NaOH에 1시간 정도 처리하여 연조직(soft tissue)을 모두 제거한 후 백악법랑경계(cemento-enamel junction)에서 치조골의 치조능(crest)까지 길이를 측정하여 치조골 소실 정도를 정량화 하였다. After administering the pine extract for 2 weeks, the rats were sacrificed and the mandibles were removed. Thereafter, all soft tissues were removed by treatment with 1M NaOH for about 1 hour, and the degree of alveolar bone loss was quantified by measuring the length from the cemento-enamel junction to the crest of the alveolar bone.
그 결과, 도 2로부터 알 수 있는 바와 같이, 치주염이 발생한 랫트에서는 치아의 뿌리가 외부로 노출될 정도로 치조골의 파괴가 심하게 발생했으나, 식나무 추출물을 투여한 랫트에서는 농도 의존적으로 치조골의 파괴가 억제되는 것을 확인할 수 있었다.As a result, as can be seen from FIG. 2, in rats with periodontitis, destruction of alveolar bone occurred so severely that the root of the tooth was exposed to the outside, but in rats administered with cedar extract, destruction of alveolar bone was suppressed in a concentration-dependent manner could confirm that
실험예 1.4. 고찰Experimental Example 1.4. Review
위와 같은 결과를 토대로 종합적으로 판단하였을 때, 식나무 추출물은 치은염 및 치골조 파괴와 같은 구강 질환을 개선하는 데에 유용함을 확인할 수 있었다.When comprehensively judged based on the above results, it was confirmed that pine extract was useful for improving oral diseases such as gingivitis and destruction of the alveolar bone.
실험예 2. 수퍼옥시드 디스뮤타아제(SOD) 및 Experimental Example 2. Superoxide dismutase (SOD) and 바실러스 아밀로리쿼파시엔스Bacillus amyloliquefaciens 균주 포자를 이용한 동물 시험 Animal testing using strain spores
수퍼옥시드 디스뮤타아제(SOD) 및 바실러스 아밀로리쿼파시엔스 균주 포자의 혼합물이 구강 건강 증진 및 구강 위생 관리 효과를 나타내는지 확인하기 위해 비글견을 이용한 시험을 수행하였다.A test using beagle dogs was performed to determine whether a mixture of superoxide dismutase (SOD) and Bacillus amyloliquefaciens strain spores exhibits oral health promotion and oral hygiene management effects.
실험예 2.1. 시험 물질Experimental Example 2.1. test substance
수퍼옥시드 디스뮤타아제(SOD) 및 바실러스 아밀로리쿼파시엔스 균주 포자는 실시예 2에서 바실러스 아밀로리쿼파시엔스 GF424로부터 정제된 수퍼옥시드 디스뮤타아제(SOD)와 실시예 3에서 제조된 바실러스 아밀로리쿼파시엔스 GF423 균주 포자를 혼합하여 제조하였다(2.5% 혼합물, SOD 10,000 U/g).Superoxide dismutase (SOD) and Bacillus amyloliqueurfaciens strain spores were purified from Bacillus amyloliqueurfaciens GF424 in Example 2 and superoxide dismutase (SOD) prepared in Example 3. It was prepared by mixing the spores of the amiloliquefiaciens GF423 strain (2.5% mixture, SOD 10,000 U/g).
구체적으로, 상기 혼합물은, 상온에서 구강 겔의 점성/점착 유지를 위한 라우르산과 같은 중쇄지방산 등을 용해하여 원하는 점도로 맞추고, 비테인, 올리고당, 난황 등 기호성 성분을 혼합한 후, 최종적으로 상기 SOD와 포자 2.5%(SOD 역가: 12,510 U/g, 포자: 1.2 x 10^10 CFU/g)를 균질 혼합함으로써 얻었다. 이러한 혼합물(SOD + 포자)을 겔 타입인 구강 겔 형태로 튜브에 담아 사용하였다(20 g/튜브, 250 U/g 겔).Specifically, the mixture is adjusted to a desired viscosity by dissolving a medium-chain fatty acid such as lauric acid for maintaining the viscosity / adhesion of oral gel at room temperature, mixing tasty ingredients such as betaine, oligosaccharide, and egg yolk, and finally It was obtained by homogeneously mixing SOD and 2.5% of spores (SOD titer: 12,510 U/g, spores: 1.2 x 10^10 CFU/g). This mixture (SOD + spores) was used in a tube in the form of oral gel, which is a gel type (20 g/tube, 250 U/g gel).
실험예 2.2. 시험 동물 및 사육 환경Experimental Example 2.2. Test animals and breeding environment
㈜오리엔트 바이오로부터 3년령 이상이고 구강 임상 증상이 없는, 9 내지 13 kg의 수컷 비글견(non-naive) 16마리를 구입하고, 1개월 이상의 휴약기를 가졌다.Sixteen non-naive male beagle dogs weighing 9 to 13 kg, aged 3 years or older and without oral clinical symptoms, were purchased from Orient Bio Co., Ltd., and had a drug holiday of at least 1 month.
온도 23±3℃, 상대 습도 30±10 %, 환기 횟수 10 내지 15 회/시간, 조명 12 시간, 조도 150 내지 200 Lux의 환경 조건을 갖춘 사육실을 이용하였고, 순화 및 시험 기간 동안 스테인레스로 제작된 케이지에 케이지당 1마리씩 개별 사육하였다. 사료와 음용수는 자율 급이하였다.A breeding room equipped with environmental conditions of temperature 23 ± 3 ℃, relative humidity 30 ± 10 %, ventilation frequency 10 to 15 times / hour, lighting 12 hours, illumination intensity 150 to 200 Lux was used, and during the period of purification and testing, stainless steel was used. They were raised individually in cages, one per cage. Feed and drinking water were provided autonomously.
시험 동물로 비글견을 선택한 이유는 비글견이 치은염 등 구강 관련 효능 평가 시험에 널리 사용되고 있으며, 풍부한 기초 시험 자료가 축적되어 있어 시험 결과의 해석 및 평가가 용이하기 때문이었다.The reason why the beagle dog was selected as the test animal was that the beagle dog is widely used in oral-related efficacy evaluation tests such as gingivitis, and it is easy to interpret and evaluate the test results due to the accumulation of abundant basic test data.
실험예 2.3. 시험군 구성Experimental Example 2.3. Test group composition
하기와 같이 시험군 G1("G1_Vehicle") 및 G2("G2_BASOD")를 구성하였다.Test groups G1 (“G1_Vehicle”) and G2 (“G2_BASOD”) were constructed as follows.
Figure PCTKR2022010973-appb-img-000001
Figure PCTKR2022010973-appb-img-000001
실험예 2.4. 시험 물질의 투여Experimental Example 2.4. Administration of test substance
실험예 2.1의 시험 물질을 4주간 매일 잇몸(양쪽 상악 대구치 외측 치은 부위)에 도포하여 투여하였다. 투여량은 1회 0.25 g(SOD 62.5 U)/head로 1일 2회 투여하였다.The test substance of Experimental Example 2.1 was applied to the gums (external gingival region of both maxillary molars) and administered every day for 4 weeks. The dose was 0.25 g (SOD 62.5 U)/head twice a day.
실험예 2.5. 시험 평가 항목Experimental Example 2.5. test items
(1) 구취 지수(Halitosis index)(1) Halitosis index
0주차(투여 시작 전), 2주차, 및 4주차에 구취를 관능검사에 의해 평가하였다(아래의 표 참조).At week 0 (before start of administration), week 2, and week 4, halitosis was evaluated by a sensory test (see table below).
Figure PCTKR2022010973-appb-img-000002
Figure PCTKR2022010973-appb-img-000002
(2) 치석 지수(Calculus index)(2) Calculus index
0주차(투여 시작 전), 2주차, 및 4주차에 치석 지수를 치면이 노출되게 입을 벌린 후, 치면의 사진을 촬영하고 육안으로 관찰하여 치석 지수를 산출하였다(아래의 표 및 도 3 참조).After opening the mouth so that the tooth surface was exposed for the calculus index at week 0 (before the start of administration), week 2, and week 4, a photograph of the tooth surface was taken and observed with the naked eye to calculate the calculus index (see table below and Figure 3) .
Figure PCTKR2022010973-appb-img-000003
Figure PCTKR2022010973-appb-img-000003
(3) 치태 지수(Plaque index)(3) Plaque index
0주차(투여 시작 전), 2주차, 및 4주차에 치태 착색제를 이용하여 치태를 염색한 후 치면을 사진 촬영하고, 치태 착색제가 치아 표면을 덮고 있는 부위를 전체 치아 표면의 % 비율로 환산하여 치태 지수를 산출하였다(아래의 표 참조).At week 0 (before the start of administration), week 2, and week 4, after staining the plaque with plaque staining agent, the tooth surface was photographed, and the area covered by the plaque staining agent on the tooth surface was converted into a % ratio of the total tooth surface. The plaque index was calculated (see table below).
Figure PCTKR2022010973-appb-img-000004
Figure PCTKR2022010973-appb-img-000004
(4) 치은염 지수(Gingival index)(4) Gingival index
0주차(투여 시작 전), 2주차, 및 4주차에 치면 및 치아 주위 잇몸이 노출되게 입을 벌린 후 사진을 촬영하고, 육안으로 관찰하여 치은염 지수를 산출하였다(아래의 표 및 도 4 참조).At week 0 (before the start of administration), week 2, and week 4, after opening the mouth to expose the tooth surface and gums around the teeth, photographs were taken, and the gingivitis index was calculated by observing with the naked eye (see the table below and FIG. 4).
Figure PCTKR2022010973-appb-img-000005
Figure PCTKR2022010973-appb-img-000005
(5) 기타 이상 반응(5) Other adverse reactions
시험 물질의 투여 기간 동안 기타 이상 반응이 관찰될 경우, 해당 증상을 기록하였다.If other adverse reactions were observed during the administration period of the test substance, the symptoms were recorded.
실험예 2.6. 통계학적 방법Experimental Example 2.6. statistical method
시험군 내에서 시험 물질 처리 전후의 비교를 위해 paired sample t-test를 사용하여 통계학적 유의성을 검정하였고, 통계 처리 방법으로는 상용으로 널리 사용되는 통계 패키지인 Graphpad prism 5.01 프로그램을 이용하였다.Statistical significance was tested using a paired sample t-test for comparison before and after treatment of the test substance within the test group, and the Graphpad prism 5.01 program, a statistical package widely used commercially, was used as a statistical processing method.
실험예 2.7. 시험 결과 및 고찰Experimental Example 2.7. Test results and considerations
(1) 구취 지수(1) Bad breath index
혼합물을 투여한 모든 동물의 경우, 투여 후 4주차까지 통계적으로 유의미한 관찰은 할 수 없었지만, 구취 지수가 감소하는 경향이 나타남을 확인할 수 있었다(표 5 및 도 5).In the case of all animals administered with the mixture, although statistically significant observations could not be made until the 4th week after administration, it was confirmed that the halitosis index tended to decrease (Table 5 and FIG. 5).
Figure PCTKR2022010973-appb-img-000006
Figure PCTKR2022010973-appb-img-000006
(2) 치석 지수(2) Calculus index
SOD와 포자의 혼합물을 투여한 모든 동물의 경우, 투여 후 4주차까지 치석이 점차 제거되는 경향이 나타났으며, 특히 4주차에는 0주차에 비해 통계적으로 치석 지수가 통계적으로 유의하게 감소하였다(p<0.05)(표 6 및 도 6). 표 6 및 도 6에서, 통계적 유의성은 paired sample t-test로 검증되었다.In the case of all animals administered with the mixture of SOD and spores, there was a tendency for tartar to be gradually removed until the 4th week after administration. <0.05) (Table 6 and Figure 6). In Table 6 and FIG. 6, statistical significance was verified by paired sample t-test.
Figure PCTKR2022010973-appb-img-000007
Figure PCTKR2022010973-appb-img-000007
(3) 치태 지수(3) plaque index
SOD와 포자의 혼합물을 투여한 모든 동물의 경우, 투여 후 4주차까지 통계적으로 유의한 것으로 관찰되지는 않았지만, 치태 지수가 감소하는 경향을 확인할 수 있었다(표 7 및 도 7).In the case of all animals administered with the mixture of SOD and spores, although statistical significance was not observed until 4 weeks after administration, a tendency for plaque index to decrease was confirmed (Table 7 and FIG. 7).
Figure PCTKR2022010973-appb-img-000008
Figure PCTKR2022010973-appb-img-000008
(4) 치은염 지수(4) Gingivitis index
SOD와 포자의 혼합물을 투여한 모든 동물의 경우, 투여 후 4주차까지 잇몸의 출혈이 없어지고, 부종 및 발적이 개선되는 효능이 관찰되었으며, 특히 4주차에는 0주차에 비해 치은염 지수가 통계적으로 유의하게 감소하였다(p<0.01)(표 8 및 도 8). 표 8 및 도 8에서, 통계적 유의성은 paired sample t-test로 검증되었다.In the case of all animals administered with the mixture of SOD and spores, the effect of eliminating gum bleeding and improving swelling and redness was observed up to the 4th week after administration, and in particular, the gingivitis index was statistically significant at the 4th week compared to the 0th week. (p<0.01) (Table 8 and Figure 8). In Table 8 and FIG. 8, statistical significance was verified by paired sample t-test.
Figure PCTKR2022010973-appb-img-000009
Figure PCTKR2022010973-appb-img-000009
(5) 고찰(5) Consideration
시험 물질인 SOD와 포자의 혼합물을 1회 0.25 g(SOD 62.5 U)/head의 용량으로 매일 2회 비글견의 잇몸에 도포한 결과, 투여 후 2주차부터 4주차까지 0주차에 비해 구취 지수, 치석 지수, 치태 지수, 및 치은염 지수 모두가 감소하는 경향을 확인할 수 있었다. 특히 치석 및 치은염은 0주차에 비해 통계적으로 유의하게 증상이 개선되었다. 또한, 시험 전과정에서 특별한 이상 반응은 나타나지 않아 시험 물질은 유해성이 없음을 확인하였다.As a result of applying the mixture of test substance SOD and spores to the gums of beagle dogs twice daily at a dose of 0.25 g (SOD 62.5 U)/head once, the halitosis index, It was confirmed that the calculus index, plaque index, and gingivitis index all decreased. In particular, symptoms of calculus and gingivitis improved statistically significantly compared to 0 weeks. In addition, it was confirmed that the test substance was not harmful because no special adverse reaction was observed in the entire test process.
위와 같은 결과를 토대로 종합적으로 판단하였을 때, 시험 물질인 SOD와 포자의 혼합물은 구취, 치석, 치태, 치은염과 같은 구강 질환을 개선하는 데에 유용함을 확인할 수 있었다.When comprehensively judged based on the above results, it was confirmed that the mixture of test substance SOD and spores is useful in improving oral diseases such as bad breath, tartar, plaque, and gingivitis.
실험예 3. 수퍼옥시드 디스뮤타아제(SOD)를 이용한 동물 시험Experimental Example 3. Animal test using superoxide dismutase (SOD)
실시예 2에서 정제된 수퍼옥시드 디스뮤타아제(SOD)가 구강 건강 증진 및 구강 위생 관리 효과를 나타내는지 확인하기 위해 반려묘를 이용한 시험을 수행하였다.In order to confirm that the purified superoxide dismutase (SOD) in Example 2 exhibits oral health promotion and oral hygiene management effects, a test using a cat was performed.
실험예 3.1. 시험 물질Experimental Example 3.1. test substance
사용한 시험 물질은 바실러스 아밀로리쿼파시엔스 GF423 균주 포자를 제외하고 실험예 2.1에서와 동일하게 제조하였다.Test materials used were prepared in the same manner as in Experimental Example 2.1, except for the spores of the Bacillus amyloliquefaciens GF423 strain.
실험예 3.2. 시험 동물Experimental Example 3.2. test animal
성별이나 품종과 무관하게 생후 6개월령 이상이고 체중 1 kg 이상 10 kg 이내인 반려묘 중에서 구취 및 통증으로 인한 기력 저하, 저작 곤란, 식욕 저하, 침 흘림, 잇몸 발적(미란 및 증식성 궤양의) 등의 구강 증상을 보이는 환묘를 피험묘로서 무작위로 선별하였다.Regardless of gender or breed, among cats aged 6 months or older and weighing 1 kg or more and 10 kg or less, lowered energy due to bad breath and pain, difficulty in chewing, decreased appetite, drooling, redness of the gums (erosion and proliferative ulcer), etc. Cats with oral symptoms were randomly selected as test cats.
실험예 3.3. 시험군 구성Experimental Example 3.3. Test group composition
각각의 피험묘 보호자로부터 자의에 의해 임상 시험에 참여한다는 동의를 받은 후, 피험묘들에 대해 무작위배정을 실시하였다.After obtaining consent to participate in the clinical trial voluntarily from each subject cat guardian, random assignment was performed for the subject cats.
Figure PCTKR2022010973-appb-img-000010
Figure PCTKR2022010973-appb-img-000010
시험군 G1의 경우, 품종은 믹스종(mixed), KSH(Korean short hair), Exotic, 또는 Siamese이고, 성별은 중성화된 수컷(NM) 또는 중성화된 암컷(SF)이고, 연령은 2세 내지 10세였다.In the case of test group G1, the breed is mixed, KSH (Korean short hair), Exotic, or Siamese, gender is neutered male (NM) or neutered female (SF), and age is 2 to 10 was three
시험군 G2의 경우, 품종은 T.A.(Turkish Angora) 또는 KSH(Korean short hair)이고, 성별은 중성화된 수컷(NM) 또는 중성화된 암컷(SF)이고, 연령은 1세 내지 11세였다.In the case of test group G2, the breed was T.A. (Turkish Angora) or KSH (Korean short hair), gender was neutered male (NM) or neutered female (SF), and the age was 1 to 11 years.
실험예 3.4. 시험 물질의 투여Experimental Example 3.4. Administration of test substance
실험예 3.1의 시험 물질을 4주간 매일 우측 상악 대구치 치은 부위에 도포하여 투여하였다. 투여량은 1회 0.3 g(SOD 75 U)/head로 1일 2회 투여하였다.The test substance of Experimental Example 3.1 was applied and administered to the gingival region of the right maxillary molar every day for 4 weeks. The dosage was 0.3 g (SOD 75 U)/head twice a day.
실험예 3.5. 시험 평가 항목Experimental Example 3.5. test items
임상 시험의 시작 전에 피험묘의 혈액 검사(ALT(알라닌 아미노전이효소) 등) 및 기본 신체 검사(체중 등)를 진행하였다.Before the start of the clinical trial, blood tests (ALT (alanine aminotransferase), etc.) and basic physical examinations (weight, etc.) of the subject cats were conducted.
(1) 사료 섭취(1) Feed intake
0주차(투여 시작 전), 2주차, 및 4주차에 피험묘의 사료 섭취량을 평가하였다(아래의 표 참조).Feed intake of the test cats was evaluated at week 0 (before the start of administration), week 2, and week 4 (see table below).
Figure PCTKR2022010973-appb-img-000011
Figure PCTKR2022010973-appb-img-000011
(2) 활동성(기분)(2) Activity (mood)
0주차(투여 시작 전), 2주차, 및 4주차에 피험묘의 활동성(기분)을 관찰하여 평가하였다(아래의 표 참조).At week 0 (before the start of administration), week 2, and week 4, the activity (mood) of the test cats was observed and evaluated (see the table below).
Figure PCTKR2022010973-appb-img-000012
Figure PCTKR2022010973-appb-img-000012
(3) 촉진성 통증 지수(3) palpable pain index
0주차(투여 시작 전), 2주차, 및 4주차에 피험묘의 질환부위를 손으로 만졌을 때, 나타나는 반응을 관찰하여 촉진성 통증을 평가하였다(아래의 표 참조).At week 0 (before the start of administration), week 2, and week 4, when the affected area of the test cat was touched with a hand, the palpation pain was evaluated by observing the reaction (see the table below).
Figure PCTKR2022010973-appb-img-000013
Figure PCTKR2022010973-appb-img-000013
(4) 구취 지수(4) Bad breath index
0주차(투여 시작 전), 2주차, 및 4주차에 구취를 관능검사에 의해 평가하였다(아래의 표 참조).At week 0 (before start of administration), week 2, and week 4, halitosis was evaluated by a sensory test (see table below).
Figure PCTKR2022010973-appb-img-000014
Figure PCTKR2022010973-appb-img-000014
(5) 치태 지수(5) plaque index
0주차(투여 시작 전), 2주차, 및 4주차에 치태 착색제를 이용하여 우측 대구치를 염색한 후, 치태 착색제가 치아 표면을 덮고 있는 부위를 전체 치아 표면의 % 비율로 환산하여 치태 지수를 산출하고(아래의 표 참조) 사진을 촬영하였다.After staining the right molar tooth with plaque staining agent at week 0 (before start of administration), week 2, and week 4, the area where plaque staining agent covers the tooth surface is converted into a percentage of the total tooth surface to calculate the plaque index (see table below) and photographs were taken.
Figure PCTKR2022010973-appb-img-000015
Figure PCTKR2022010973-appb-img-000015
(6) 치석 지수(6) Calculus index
0주차(투여 시작 전), 2주차, 및 4주차에 치석을 우측 상악 대구치에서 육안으로 관찰하여 치석 지수를 산출하고(아래의 표 및 도 3 참조) 사진을 촬영하였다.At week 0 (before start of administration), week 2, and week 4, calculus was observed with the naked eye in the right maxillary molar to calculate the calculus index (see the table below and FIG. 3), and photographs were taken.
Figure PCTKR2022010973-appb-img-000016
Figure PCTKR2022010973-appb-img-000016
(7) 치은염 지수(7) Gingivitis index
0주차(투여 시작 전), 2주차, 및 4주차에 우측 상악 대구치의 치은 부위를 육안으로 관찰하고, 아래 표의 기준으로 점수를 측정한 후 합계 점수를 계산하여 치은염 지수를 산출하고 사진을 촬영하였다.At week 0 (before start of administration), week 2, and week 4, the gingival area of the right maxillary molar was visually observed, the scores were measured based on the table below, the total score was calculated to calculate the gingivitis index, and photographs were taken. .
Figure PCTKR2022010973-appb-img-000017
Figure PCTKR2022010973-appb-img-000017
(8) 기타 이상 반응(8) Other adverse reactions
유효성 확인을 위한 시험 물질의 투여 기간 동안 나타나는 모든 이상 반응(식욕상실, 구토, 설사, 혈변, 토혈, 무기력, 위장관 장애 등)을 보호자 또는 시험 관리자가 관찰하여 기록하였다.All adverse reactions (loss of appetite, vomiting, diarrhea, bloody stool, hematemesis, lethargy, gastrointestinal disorder, etc.) occurring during the administration period of the test substance for validation were observed and recorded by the guardian or test manager.
실험예 3.6. 통계학적 방법Experimental Example 3.6. statistical method
시험군 내에서 시험 물질 처리 전후의 비교를 위해 paired sample t-test를 사용하여 통계학적 유의성을 검정하였고, 통계 처리 방법으로는 상용으로 널리 사용되는 통계 패키지인 Graphpad prism 5.01 프로그램을 이용하였다.Statistical significance was tested using a paired sample t-test for comparison before and after treatment of the test substance within the test group, and the Graphpad prism 5.01 program, a statistical package widely used commercially, was used as a statistical processing method.
실험예 3.7. 시험 결과 및 고찰Experimental Example 3.7. Test results and considerations
(1) 사료 섭취량 및 체중(1) Feed intake and body weight
사료 섭취량 및 체중은 시험 기간 동안 모든 군에서 유의한 차이가 관찰되지 않았다(데이터는 제시되지 않음).No significant differences were observed in feed intake and body weight in any group during the test period (data not shown).
(2) 활동성(2) activity
vehicle 대조군에서 활동성이 2주차부터 유의하게 개선되었고, SOD 군에서는 활동성이 점차적으로 증가하는 경향을 보였으나 통계적 유의성은 없었다(데이터는 제시되지 않음).In the vehicle control group, activity was significantly improved from the 2nd week, and in the SOD group, activity gradually increased, but there was no statistical significance (data not shown).
(3) 촉진성 통증 지수(3) palpable pain index
vehicle 대조군에서는 촉진성 통증이 시험 종료시까지 거의 변화가 없었으나, SOD 군에서는 촉진성 통증이 2주차부터 감소하여 4주차에 통계적으로 유의하게 개선되었다(도 9).In the vehicle control group, there was little change in palpation pain until the end of the test, but in the SOD group, palpation pain decreased from the 2nd week and was statistically significantly improved at the 4th week (FIG. 9).
(4) 구취 지수(4) Bad breath index
vehicle 대조군에서는 구취 지수가 시험 종료시까지 변화가 없었으나, SOD 군에서는 구취 지수가 지속적으로 감소하는 경향을 확인할 수 있었다(도 10).In the vehicle control group, the halitosis index did not change until the end of the test, but in the SOD group, it was confirmed that the halitosis index continued to decrease (FIG. 10).
(5) 치태 지수(5) plaque index
vehicle 대조군에서는 치태 지수가 시험 기간 동안 확연히 증가한 반면, SOD 군에서는 치태 지수가 2주차부터 시험 종료시까지 통계적 유의성을 보이며 감소하였다(도 11).In the vehicle control group, the plaque index significantly increased during the test period, whereas in the SOD group, the plaque index decreased with statistical significance from the 2nd week to the end of the test (FIG. 11).
(6) 치석 지수(6) Calculus index
vehicle 대조군에서는 치석 지수가 점차 증가한 반면, SOD 군에서는 치석 지수가 4주차에 통계적유의성을 보이며 감소하였다(도 12).While the calculus index gradually increased in the vehicle control group, the calculus index decreased with statistical significance at 4 weeks in the SOD group (FIG. 12).
(7) 치은염 지수(7) Gingivitis index
vehicle 대조군에서는 치은염 지수에 별다른 변화가 없었지만, SOD 군에서는 2주차부터 치은염 지수가 감소하여 4주차에 통계적으로 유의한 감소가 확인되었다(도 13).There was no significant change in the gingivitis index in the vehicle control group, but in the SOD group, the gingivitis index decreased from the 2nd week, and a statistically significant decrease was confirmed at the 4th week (FIG. 13).
(8) 고찰(8) Consideration
시험 물질인 SOD를 1회 0.3 g(SOD 75 U)/head의 용량으로 매일 2회 반려묘의 잇몸에 도포한 결과, 투여 후 2주차부터 4주차까지 0주차에 비해 구취 지수, 촉진성 통증 지수, 치태 지수, 치석 지수, 및 치은염 지수 모두가 감소하는 경향을 확인할 수 있었다. 특히, 촉진성 통증, 치태, 치석, 및 치은염은 0주차에 비해 통계적으로 유의하게 증상이 개선되었다. 또한, 시험 전과정에서 특별한 이상 반응은 나타나지 않아 시험 물질은 유해성이 없음을 확인하였다.As a result of applying SOD, a test substance, to the cat's gums twice daily at a dose of 0.3 g (SOD 75 U)/head, halitosis index, palpable pain index, palpation pain index, It was confirmed that the plaque index, calculus index, and gingivitis index all decreased. In particular, symptoms of palpable pain, plaque, calculus, and gingivitis were improved statistically significantly compared to the 0th week. In addition, it was confirmed that the test substance was not harmful because no special adverse reaction was observed during the entire test process.
위와 같은 결과를 토대로 판단하였을 때, 시험 물질인 SOD 단독 투여의 경우에도 구취, 치태, 치석 및 치은염과 같은 구강 질환을 개선하는 데에 유용할 뿐만 아니라 그러한 구강 질환으로 인한 통증을 감소시키는 데에도 유용함을 확인할 수 있었다.Judging from the above results, even in the case of the test substance SOD alone, it is not only useful for improving oral diseases such as bad breath, plaque, tartar and gingivitis, but also useful for reducing pain caused by such oral diseases was able to confirm
실험예 4. 식나무 추출물 및 수퍼옥시드 디스뮤타아제(SOD)와 Experimental Example 4. Cinnamon extract and superoxide dismutase (SOD) and 바실러스 아밀로리쿼파시엔스Bacillus amyloliquefaciens 균주 포자의 혼합물을 이용한 동물 시험 Animal testing using a mixture of strain spores
식나무 추출물 및 수퍼옥시드 디스뮤타아제(SOD)와 바실러스 아밀로리쿼파시엔스 균주 포자의 혼합물이 구강 건강 증진 및 구강 위생 관리 효과를 나타내는지 확인하기 위해 반려묘를 이용한 시험을 수행하였다.A test using cats was performed to determine whether a mixture of pine extract, superoxide dismutase (SOD), and spores of Bacillus amyloliquefaciens strain showed oral health promotion and oral hygiene management effects.
실험예 4.1. 시험 물질Experimental Example 4.1. test substance
시험 물질은 다음과 같이 준비하였다. 실시예 1에서 얻은 식나무 추출물 분말 및 실시예 2에서 제조된 SOD와 실시예 3에서 제조된 포자의 혼합물을 포함하는 구강 겔을 하기의 표와 같은 조성으로 제조하였다. Test materials were prepared as follows. An oral gel containing a mixture of pine extract powder obtained in Example 1, SOD prepared in Example 2, and spores prepared in Example 3 was prepared with the composition shown in the table below.
Figure PCTKR2022010973-appb-img-000018
Figure PCTKR2022010973-appb-img-000018
구체적으로, 상기 혼합물은, 60℃에서 식나무추출물 분말을 1시간 동안 정제수에 용해하고, 상온에서 구강 겔의 점성/점착 유지를 위한 카르복시메틸셀룰로오스 및 폴리아크릴산나트륨을 용해하고, 비테인, 올리고당, 난황분 등 기호성 성분을 혼합한 후, 상온에서 수퍼옥시드 디스뮤타아제(SOD) 및 바실러스 아밀로리쿼파시엔스 균주 포자 혼합물을 첨가하여 제조하였다. 최종적으로, 상기의 2% 식나무추출물 및 2.5% SOD와 포자 혼합물 (SOD 역가: 250 U/g, 포자: 2 x 10^8 CFU/g)을 함유한 구강 겔을 튜브에 담아 사용하였다.Specifically, the mixture dissolves pine extract powder in purified water at 60 ° C. for 1 hour, dissolves carboxymethylcellulose and sodium polyacrylate for maintaining the viscosity / adhesion of oral gel at room temperature, and contains vitamins, oligosaccharides, and egg yolk powder. After mixing such tasty ingredients, it was prepared by adding superoxide dismutase (SOD) and Bacillus amyloliquefaciens strain spore mixture at room temperature. Finally, the oral gel containing the above 2% pine tree extract and 2.5% SOD and spore mixture (SOD titer: 250 U/g, spores: 2 x 10^8 CFU/g) was put into a tube and used.
실험예 4.2. 시험 동물Experimental Example 4.2. test animal
성별이나 품종과 무관하게 생후 6개월령 이상이고 체중 1 kg 이상 10 kg 이내인 반려묘 중에서 구취 및 통증으로 인한 기력 저하, 저작 곤란, 식욕 저하, 침 흘림, 잇몸 발적(미란 및 증식성 궤양의) 등의 구강 증상을 보이는 환묘를 피험묘로서 무작위로 선별하였다.Regardless of gender or breed, among cats aged 6 months or older and weighing 1 kg or more and 10 kg or less, lowered energy due to bad breath and pain, difficulty in chewing, decreased appetite, drooling, redness of the gums (erosion and proliferative ulcer), etc. Cats with oral symptoms were randomly selected as test cats.
실험예 4.3. 시험군 구성Experimental Example 4.3. Test group composition
각각의 피험묘 보호자로부터 자의에 의해 임상 시험에 참여한다는 동의를 받은 후, 피험묘들에 대해 무작위배정을 실시하였다. 하기와 같이 시험군 G1("G1_Vehicle") 및 G2("G2_식나무 추출물+BASOD")를 구성하였다.After obtaining consent to participate in the clinical trial voluntarily from each subject cat guardian, random assignment was performed for the subject cats. Test groups G1 (“G1_Vehicle”) and G2 (“G2_Vehicle+BASOD”) were constructed as follows.
Figure PCTKR2022010973-appb-img-000019
Figure PCTKR2022010973-appb-img-000019
시험군 G1의 경우, 품종은 믹스종(mixed), KSH(Korean short hair), Exotic, 또는 Siamese이고, 성별은 중성화된 수컷(NM) 또는 중성화된 암컷(SF)이고, 연령은 2세 내지 10세였다.시험군 G2의 경우, 품종은 믹스종, KSH(Korean short hair), Exotic, 또는 Soti.F이고, 성별은 중성화된 수컷(NM) 또는 중성화된 암컷(SF)이고, 연령은 1세 내지 4세였다.In the case of test group G1, the breed is mixed, KSH (Korean short hair), Exotic, or Siamese, gender is neutered male (NM) or neutered female (SF), and age is 2 to 10 In the case of test group G2, the breed was mix, KSH (Korean short hair), Exotic, or Soti.F, gender was neutered male (NM) or neutered female (SF), and age was 1 year. was 4 years old.
실험예 4.4. 시험 물질의 투여Experimental Example 4.4. Administration of test substance
실험예 4.1의 시험 물질을 4주간 매일 우측 상악 대구치 치은 부위에 도포하여 투여하였다. 투여량은 1회 0.3 g/head로 1일 2회 투여하였다.The test substance of Experimental Example 4.1 was applied and administered to the gingival region of the right maxillary molar every day for 4 weeks. The dose was administered twice a day at 0.3 g/head once.
실험예 4.5. 시험 평가 항목Experimental Example 4.5. test items
임상 시험의 시작 전에 피험묘의 혈액 검사(ALT(알라닌 아미노전이효소) 등) 및 기본 신체 검사(체중 등)를 진행하였다.Before the start of the clinical trial, blood tests (ALT (alanine aminotransferase), etc.) and basic physical examinations (weight, etc.) of the subject cats were conducted.
(1) 사료 섭취(1) Feed intake
0주차(투여 시작 전), 2주차, 및 4주차에 피험묘의 사료 섭취량을 평가하였다(아래의 표 참조).Feed intake of the test cats was evaluated at week 0 (before the start of administration), week 2, and week 4 (see table below).
Figure PCTKR2022010973-appb-img-000020
Figure PCTKR2022010973-appb-img-000020
(2) 활동성(기분)(2) Activity (mood)
0주차(투여 시작 전), 2주차, 및 4주차에 피험묘의 활동성(기분)을 관찰하여 평가하였다(아래의 표 참조).At week 0 (before the start of administration), week 2, and week 4, the activity (mood) of the test cats was observed and evaluated (see the table below).
Figure PCTKR2022010973-appb-img-000021
Figure PCTKR2022010973-appb-img-000021
(3) 촉진성 통증 지수(3) palpable pain index
0주차(투여 시작 전), 2주차, 및 4주차에 피험묘의 질환부위를 손으로 만졌을 때 나타나는 반응을 관찰하여 촉진성 통증을 평가하였다(아래의 표 참조).At week 0 (before the start of administration), week 2, and week 4, palpation pain was evaluated by observing the reaction when the diseased area of the test cat was touched with a hand (see the table below).
Figure PCTKR2022010973-appb-img-000022
Figure PCTKR2022010973-appb-img-000022
(4) 구취 지수(4) Bad breath index
0주차(투여 시작 전), 2주차, 및 4주차에 구취를 관능검사에 의해 평가하였다(아래의 표 참조).At week 0 (before start of administration), week 2, and week 4, halitosis was evaluated by a sensory test (see table below).
Figure PCTKR2022010973-appb-img-000023
Figure PCTKR2022010973-appb-img-000023
(5) 치태 지수(5) plaque index
0주차(투여 시작 전), 2주차, 및 4주차에 치태 착색제를 이용하여 우측 대구치를 염색한 후, 치태 착색제가 치아 표면을 덮고 있는 부위를 전체 치아 표면의 % 비율로 환산하여 치태 지수를 산출하고(아래의 표 참조) 사진을 촬영하였다.After staining the right molar tooth with plaque staining agent at week 0 (before start of administration), week 2, and week 4, the area where plaque staining agent covers the tooth surface is converted into a percentage of the total tooth surface to calculate the plaque index (see table below) and photographs were taken.
Figure PCTKR2022010973-appb-img-000024
Figure PCTKR2022010973-appb-img-000024
(6) 치석 지수(6) Calculus index
0주차(투여 시작 전), 2주차, 및 4주차에 치석을 우측 상악 대구치에서 육안으로 관찰하여 치석 지수를 산출하고(아래의 표 및 도 3 참조) 사진을 촬영하였다.At week 0 (before start of administration), week 2, and week 4, calculus was observed with the naked eye in the right maxillary molar to calculate the calculus index (see the table below and FIG. 3), and photographs were taken.
Figure PCTKR2022010973-appb-img-000025
Figure PCTKR2022010973-appb-img-000025
(7) 치은염 지수(7) Gingivitis index
0주차(투여 시작 전), 2주차, 및 4주차에 우측 상악 대구치의 치은 부위를 육안으로 관찰하고, 아래 표의 기준으로 점수를 측정한 후 합계 점수를 계산하여 치은염 지수를 산출하고 사진을 촬영하였다.At week 0 (before start of administration), week 2, and week 4, the gingival area of the right maxillary molar was visually observed, the scores were measured based on the table below, the total score was calculated to calculate the gingivitis index, and photographs were taken. .
Figure PCTKR2022010973-appb-img-000026
Figure PCTKR2022010973-appb-img-000026
(8) 기타 이상 반응(8) Other adverse reactions
유효성 확인을 위한 시험 물질의 투여 기간 동안 나타나는 모든 이상 반응(식욕상실, 구토, 설사, 혈변, 토혈, 무기력, 위장관 장애 등)을 보호자 또는 시험 관리자가 관찰하여 기록하였다.All adverse reactions (loss of appetite, vomiting, diarrhea, bloody stool, hematemesis, lethargy, gastrointestinal disorder, etc.) occurring during the administration period of the test substance for validation were observed and recorded by the guardian or test manager.
실험예 4.6. 통계학적 방법Experimental Example 4.6. statistical method
시험군 내에서 시험 물질 처리 전후의 비교를 위해 paired sample t-test를 사용하여 통계학적 유의성을 검정하였고, 통계 처리 방법으로는 상용으로 널리 사용되는 통계 패키지인 Graphpad prism 5.01 프로그램을 이용하였다.Statistical significance was tested using a paired sample t-test for comparison before and after treatment of the test substance within the test group, and the Graphpad prism 5.01 program, a statistical package widely used commercially, was used as a statistical processing method.
실험예 4.7. 시험 결과 및 고찰Experimental Example 4.7. Test results and considerations
(1) 사료 섭취량 및 체중(1) Feed intake and body weight
사료 섭취량 및 체중은 시험 기간 동안 모든 군에서 유의한 차이가 관찰되지 않았다(데이터는 제시되지 않음).No significant differences were observed in feed intake and body weight in any group during the test period (data not shown).
(2) 활동성(2) activity
시험군 G1에서 활동성이 2주차부터 유의하게 개선되었고, 시험군 G2에서는 활동성이 점차적으로 증가하는 경향을 보였으나 통계적 유의성은 없었다(데이터는 제시되지 않음).In test group G1, activity was significantly improved from the 2nd week, and in test group G2, activity gradually increased, but there was no statistical significance (data not shown).
(3) 촉진성 통증 지수(3) palpable pain index
시험군 G1에서는 촉진성 통증이 시험 종료시까지 거의 변화가 없었으나, 시험군 G2에서는 투여 2주 후부터 촉진성 통증 지수가 통계적 유의성을 보이며 현저하게 감소하였다(도 14).In test group G1, there was little change in palpation pain until the end of the test, but in test group G2, the palpation pain index significantly decreased with statistical significance from 2 weeks after administration (FIG. 14).
(4) 구취 지수(4) Bad breath index
시험군 G1에서는 구취 지수가 시험 종료시까지 변화가 없었으나, 시험군 G2에서는 투여 2주차에 0주차에 비해 약 50% 구취 지수가 감소하였고 4주차까지 통계적으로 유의하게 구취가 개선되었다(도 15).In test group G1, the halitosis index did not change until the end of the test, but in test group G2, the halitosis index decreased by about 50% compared to week 0 at the 2nd week of administration, and the bad breath was statistically significantly improved until the 4th week (FIG. 15) .
(5) 치태 지수(5) plaque index
시험군 G1에서는 치태 지수가 시험 기간 동안 확연히 증가한 반면, 시험군 G2에서는 치태 지수가 시험 종료시까지 통계적 유의성은 없었지만 점차적으로 감소하는 경향을 보였다(도 16).In test group G1, the plaque index significantly increased during the test period, whereas in test group G2, the plaque index gradually decreased until the end of the test, although there was no statistical significance (FIG. 16).
(6) 치석 지수(6) Calculus index
시험군 G1에서는 치석 지수가 점차 증가한 반면, 시험군 G2에서는 치석 지수가 2주차와 4주차에 걸쳐 통계적 유의성을 보이며 현저하게 개선되었다(도 17).In test group G1, the calculus index gradually increased, whereas in test group G2, the calculus index was significantly improved with statistical significance over the 2nd and 4th weeks (FIG. 17).
(7) 치은염 지수(7) Gingivitis index
시험군 G1에서는 치은염 지수에 별다른 변화가 없었지만, 시험군 G2에서는 치은염 지수가 2주차부터 4주차까지 통계적으로 유의하게 감소하였다(도 18).In test group G1, there was no significant change in the gingivitis index, but in test group G2, the gingivitis index decreased statistically significantly from week 2 to week 4 (FIG. 18).
(8) 고찰(8) Consideration
시험 물질인 식나무 추출물 및 SOD와 포자의 혼합물을 1회 0.3 g/head의 용량으로 매일 2회 반려묘의 잇몸에 도포한 결과, 투여 후 2주차부터 4주차까지 0주차에 비해 촉진성 통증 지수, 구취 지수, 치태 지수, 치석 지수, 및 치은염 지수 모두가 감소하는 경향을 확인할 수 있었다. 특히, 촉진성 통증, 구취, 치석, 및 치은염은 0주차에 비해 통계적으로 유의하게 증상이 개선되었다. 또한, 시험 전과정에서 특별한 이상 반응은 나타나지 않아 시험 물질은 유해성이 없음을 확인하였다.A mixture of pine extract and SOD and spores as test substances was applied to the cat's gums twice daily at a dose of 0.3 g/head once. It was confirmed that the index, plaque index, calculus index, and gingivitis index all decreased. In particular, symptoms of palpable pain, halitosis, calculus, and gingivitis were improved statistically significantly compared to 0 weeks. In addition, it was confirmed that the test substance was not harmful because no special adverse reaction was observed in the entire test process.
위와 같은 결과를 토대로 판단하였을 때, 시험 물질인 식나무 추출물 및 SOD와 포자의 혼합물은 구취, 치태, 치석 및 치은염과 같은 구강 질환을 개선하는 데에 유용할 뿐만 아니라 그러한 구강 질환으로 인한 통증을 감소시키는 데에도 유용함을 확인할 수 있었다.Judging on the basis of the above results, the mixture of test substances, pine extract and SOD, and spores is not only useful for improving oral diseases such as bad breath, plaque, tartar and gingivitis, but also reduces pain caused by such oral diseases. It was also found to be useful.

Claims (23)

  1. (i) 식나무(Aucuba japonica) 추출물, 및 (i) an extract of Aucuba japonica , and
    (ii) 수퍼옥시드 디스뮤타제(SOD) 효소 및 바실러스 종 균주 포자로 이루어진 군에서 선택된 하나 이상을 유효 성분으로 포함하는(ii) containing as an active ingredient at least one selected from the group consisting of superoxide dismutase (SOD) enzyme and spores of Bacillus sp.
    구강용 조성물.Compositions for oral use.
  2. 제1항에 있어서, According to claim 1,
    상기 조성물은 (i) 식나무 추출물, 및 (ii) SOD 및 바실러스 종 균주 포자를 포함하는The composition comprises (i) cedar extract, and (ii) SOD and Bacillus species strain spores.
    조성물.composition.
  3. 제1항에 있어서, According to claim 1,
    상기 식나무 추출물은 열수 추출물인The pine extract is a hot water extract
    조성물.composition.
  4. 제1항에 있어서, According to claim 1,
    상기 식나무 추출물은 식나무의 잎, 줄기 또는 이의 혼합물로부터 추출된 것인The pine extract is extracted from the leaves, stems or mixtures thereof of pine trees.
    조성물.composition.
  5. 제1항에 있어서, According to claim 1,
    상기 SOD는 바실러스 종 균주로부터 유래된 것인 The SOD is derived from a Bacillus species strain
    조성물.composition.
  6. 제5항에 있어서, According to claim 5,
    상기 바실러스 종 균주는 바실러스 아밀로리퀴파시엔스 GF423 균주(KCTC 13222 BP) 또는 GF424 균주(KCTC 13227 BP)를 포함하는The Bacillus species strain includes Bacillus amyloliquefaciens GF423 strain (KCTC 13222 BP) or GF424 strain (KCTC 13227 BP)
    조성물.composition.
  7. 제5항에 있어서, According to claim 5,
    상기 SOD는 Mn-SOD 또는 탈아미드화된 Mn-SOD를 포함하는The SOD includes Mn-SOD or deamidated Mn-SOD
    조성물.composition.
  8. 제1항에 있어서, According to claim 1,
    상기 SOD는 서열번호 2 또는 서열번호 4의 아미노산 서열을 포함하는The SOD comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4
    조성물.composition.
  9. 제1항에 있어서, According to claim 1,
    상기 바실러스 종 균주 포자는 바실러스 아밀로리퀴파시엔스 GF423 균주(KCTC 13222 BP)의 포자 또는 GF424 균주(KCTC 13227 BP)의 포자를 포함하는The spores of the Bacillus species strain include spores of the Bacillus amyloliquefaciens GF423 strain (KCTC 13222 BP) or spores of the GF424 strain (KCTC 13227 BP)
    조성물.composition.
  10. 제1항에 있어서, According to claim 1,
    상기 조성물은 구강 건강 증진 또는 구강 위생 관리를 위해 사용되는The composition is used for oral health promotion or oral hygiene management
    조성물.composition.
  11. 제1항에 있어서,According to claim 1,
    상기 조성물은 구강 통증, 구취, 치석, 치태, 치아 우식증 또는 치주 질환의 예방 또는 개선을 위해 사용되는The composition is used for preventing or improving oral pain, bad breath, calculus, plaque, dental caries or periodontal disease
    조성물.composition.
  12. 제1항에 있어서, According to claim 1,
    상기 조성물은 겔 또는 페이스트 형태인The composition is in the form of a gel or paste
    조성물.composition.
  13. 제1항 내지 제12항 중 어느 한 항의 조성물을 포함하는Comprising the composition of any one of claims 1 to 12
    구강용 제품.oral products.
  14. 제13항에 있어서, According to claim 13,
    상기 제품은 치약, 구강 세정제, 구강 청정제, 껌, 캔디류, 구강 스프레이, 구강 겔, 구강 연고제, 구강 패치 및 구강 양치액으로부터 선택된 어느 하나인 The product is any one selected from toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral gel, oral ointment, oral patch, and mouthwash.
    제품.product.
  15. 제1항 내지 제12항 중 어느 한 항의 조성물을 포함하는Comprising the composition of any one of claims 1 to 12
    구강 질환의 예방 또는 치료를 위한 약학 조성물.A pharmaceutical composition for preventing or treating oral diseases.
  16. 제1항 내지 제12항 중 어느 한 항의 조성물을 포함하는Comprising the composition of any one of claims 1 to 12
    수의학적 조성물.Veterinary Compositions.
  17. 제1항 내지 제12항 중 어느 한 항의 조성물을 포함하는Comprising the composition of any one of claims 1 to 12
    식품 조성물.food composition.
  18. 제1항 내지 제12항 중 어느 한 항의 조성물을 포함하는Comprising the composition of any one of claims 1 to 12
    사료 조성물.feed composition.
  19. (i) 식나무 추출물, 및 (i) cedar extract, and
    (ii) 수퍼옥시드 디스뮤타제(SOD) 효소 및 바실러스 종 균주 포자로 이루어진 군에서 선택된 하나 이상을 개체에 투여하는 단계를 포함하는(ii) administering to the subject at least one selected from the group consisting of a superoxide dismutase (SOD) enzyme and spores of a Bacillus species strain;
    구강 질환을 예방하거나 치료하는 방법.How to prevent or treat oral disease.
  20. (i) 식나무 추출물, 및 (i) cedar extract, and
    (ii) 수퍼옥시드 디스뮤타제(SOD) 및 바실러스 종 균주 포자로 이루어진 군에서 선택된 하나 이상을 개체에 투여하는 단계를 포함하는(ii) administering to the subject at least one selected from the group consisting of superoxide dismutase (SOD) and spores of Bacillus sp.
    구강 위생을 증진하는 방법.How to Promote Oral Hygiene.
  21. 제19항 또는 제20항에 있어서, The method of claim 19 or 20,
    (i) 식나무 추출물과 (ii) SOD 및 바실러스 종 균주 포자를 개체에 투여하는 단계를 포함하는 (i) pine extract and (ii) SOD and Bacillus sp. strain spores are administered to the subject.
    방법.method.
  22. 제21항에 있어서, According to claim 21,
    상기 식나무 추출물, SOD 및 바실러스 종 균주 포자는 하나의 조성물로 투여되거나 또는 각각 또는 어느 두 성분을 함유하는 별개의 조성물로 투여되는 The pine extract, SOD and Bacillus sp. strain spores are administered as one composition or as separate compositions containing each or any two components.
    방법.method.
  23. 제21항에 있어서,According to claim 21,
    (i)의 성분 및 (ii)의 성분은 동시 투여되거나, 순차적으로 또는 역순으로 병용 투여되는Component (i) and component (ii) are administered simultaneously, sequentially or in reverse order.
    방법.method.
PCT/KR2022/010973 2021-07-27 2022-07-26 Combined use of aucuba japonica extract and superoxide dismutase and/or bacillus strain spore in composition for improving oral health WO2023008879A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4842846A (en) * 1986-11-22 1989-06-27 Minoru Nakano Superoxide dismutase composition for periodontal use
KR20110017533A (en) * 2009-08-14 2011-02-22 신미정 Composition of lactic acid
KR20160111646A (en) * 2015-03-17 2016-09-27 김상민 Method for producing of new material for Improving GOUT, Reducing URIC Acid
WO2020050460A1 (en) * 2018-09-04 2020-03-12 주식회사 제노포커스 Pharmaceutical composition for preventing or treating inflammatory bowel diseases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4842846A (en) * 1986-11-22 1989-06-27 Minoru Nakano Superoxide dismutase composition for periodontal use
KR20110017533A (en) * 2009-08-14 2011-02-22 신미정 Composition of lactic acid
KR20160111646A (en) * 2015-03-17 2016-09-27 김상민 Method for producing of new material for Improving GOUT, Reducing URIC Acid
WO2020050460A1 (en) * 2018-09-04 2020-03-12 주식회사 제노포커스 Pharmaceutical composition for preventing or treating inflammatory bowel diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SHIM KYUNG MI, SEOK HWA CHOI, MOON-JIN JEONG, SEONG SOO KANG: "Effects of Aucuba japonica Extract on Oral Wound Healing", IN VIVO, vol. 21, 1 January 2007 (2007-01-01), pages 1037 - 1042, XP093029618 *

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