KR102632704B1 - Combined use for improving oral health of aucuba japonica extracts and compositions comprising superoxide dismutase and/or bacillus strain spores - Google Patents

Abstract

The present invention relates to an oral composition comprising an extract of the chinensis tree and superoxide dismutase (SOD) enzyme and/or spores of a Bacillus species strain, and its use for improving oral health or managing oral hygiene. These compositions can be usefully used to prevent or treat oral diseases such as oral pain, bad breath, tartar, plaque, gingivitis, and periodontitis without causing side effects in the oral cavity.

Description

Combination use for improving oral health of a composition comprising AUCUBA JAPONICA EXTRACTS AND COMPOSITIONS COMPRISING SUPEROXIDE DISMUTASE AND/OR BACILLUS STRAIN SPORES}

The present invention relates to a composition comprising Aucuba japonica extract and superoxide dismutase and/or Bacillus species strain spores and its use for promoting oral health, managing oral hygiene, or preventing or treating oral diseases. .

Most oral diseases begin with poor oral hygiene, leading to tartar, cavities, gingivitis, etc., and can be accompanied by severe bad breath. The main causes of bad breath, also called bad breath, include periodontal disease such as gingivitis and periodontitis, odor-causing bacteria on the tongue, certain foods and alcoholic beverages, and smoking. If food in the mouth is not properly removed, soft deposits called plaque form on the tooth surface, and over time, plaque turns into tartar. Tartar is made up of minerals from saliva and gingival crevicular fluid that are calcified and firmly adhered to the tooth surface. Tartar cannot be removed by toothbrushing and can only be removed by scaling. If tartar is not removed, the tartar can easily cause an inflammation called gingivitis in the gums. As gingivitis worsens, inflammation occurs not only in the gums but also in the ligaments and alveolar bone. This inflammation is periodontitis, which also causes bad breath. Periodontitis not only causes extreme pain, but also requires special attention because bacteria can infect the heart or kidneys.

Substances that inhibit the growth of oral pathogens that live in plaque and cause various oral diseases include antibacterial agents, including antibiotics. However, antibiotics have the disadvantage of having to be used as a treatment for a short period of time as it is difficult to use them long-term because they can cause systemic side effects to our body and the emergence of resistant bacteria in the oral cavity. In addition, antibacterial agents used in mouthwashes include Sangquinarine, Listerine, and Chlorhexidine. Among these, Sangquinarine is not only effective against bacteria in the oral cavity but is also expensive. It has the disadvantage of being expensive, and although Listerine has a slight bacteriostatic effect as its main ingredient is alcohol, it only has a temporary effect in the oral cavity and may have a harmful effect on tissues when used for a long period of time.

An effective way to remove tartar is tartar removal. However, scaling can cause gum swelling, sensitivity to cold or hot foods, and improper scaling can cause gum damage.

Therefore, there is a need for substances and methods that can not only be used for oral hygiene management and oral health promotion without causing adverse side effects on teeth or gums, but can also prevent or treat oral diseases by suppressing alveolar bone destruction caused by inflammation of tissues around teeth. do.

The purpose of the present invention is to solve the problems of the prior art described above.

Another purpose of the present invention is to promote oral health or manage oral hygiene by using a combination of Chinese Cuisine extract, superoxide dismutase, and/or Bacillus species strain spores.

Another object of the present invention is to prevent or treat oral diseases by using a combination of Chinese chinensis extract, superoxide dismutase, and/or Bacillus species strain spores.

The object of the present invention is not limited to the objects mentioned above. The object of the present invention will become clearer from the following description and may be realized by means and combinations thereof as set forth in the claims.

A representative configuration of the present invention to achieve the above object is as follows.

According to one aspect of the present invention, an oral composition is provided comprising (i) an extract of chinensis chinensis and (ii) one or more selected from the group consisting of superoxide dismutase (SOD) and Bacillus species strain spores.

According to another aspect of the present invention, an oral product comprising the composition is provided.

According to another aspect of the present invention, a pharmaceutical composition for preventing or treating oral diseases comprising the composition is provided.

According to another aspect of the present invention, a veterinary composition comprising the composition is provided.

According to another aspect of the present invention, a food composition comprising the composition is provided.

According to another aspect of the present invention, a feed composition comprising the above composition is provided.

According to another aspect of the present invention, administering to an individual at least one selected from the group consisting of (i) a Chinese oak extract and (ii) superoxide dismutase (SOD) and Bacillus species strain spores, or administering the composition to an individual. A method for promoting oral hygiene or preventing or treating oral disease is provided, including the step of administering to.

The composition according to the present invention can be useful in preventing or treating oral diseases such as bad breath, tartar, plaque, dental caries, gingivitis, or periodontitis without causing side effects, as well as reducing pain caused by such oral diseases. It can be useful for

Figure 1 shows the results of measuring the degree of swelling and redness of the gums when a beagle dog with gingivitis according to Experimental Example 1.2 was treated with a Chinese oak extract.
Figure 2 shows the results of measuring the degree of destruction of alveolar bone when the alveolar tree extract was administered to rats suffering from periodontitis according to Experimental Example 1.3.
Figure 3 shows the standards for converting the tartar index into a score.
Figure 4 shows the criteria for converting the gingivitis index into a score.
Figure 5 is a graph showing the results of the bad breath index observed after administering the test substance according to Experimental Example 2.4.
Figure 6 is a graph showing the results of the tartar index observed after administering the test substance according to Experimental Example 2.4. * indicates P < 0.05 compared to week 0.
Figure 7 is a graph showing the results of the plaque index observed after administering the test substance according to Experimental Example 2.4.
Figure 8 is a graph showing the results of the gingivitis index observed after administering the test substance according to Experimental Example 2.4. ** indicates P < 0.01 compared to week 0.
Figure 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 compared to week 0.
Figure 10 is a graph showing the results of the bad breath index observed after administration of the test substance according to Experimental Example 3.4.
Figure 11 is a graph showing the results of the plaque index observed after administration of the test substance according to Experimental Example 3.4. * indicates P < 0.05 compared to week 0, and ** indicates P < 0.01 compared to week 0.
Figure 12 is a graph showing the results of the tartar index observed after administration of the test substance according to Experimental Example 3.4. * indicates P < 0.05 compared to week 0.
Figure 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 compared to week 0.
Figure 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 compared to week 0.
Figure 15 is a graph showing the results of the bad breath index observed after administration of the test substance according to Experimental Example 4.4. ** indicates P < 0.01 compared to week 0.
Figure 16 is a graph showing the results of the plaque index observed after administration of the test substance according to Experimental Example 4.4. * indicates P < 0.05 compared to week 0.
Figure 17 is a graph showing the results of the tartar index observed after administration of the test substance according to Experimental Example 4.4. * indicates P < 0.05 compared to week 0, and ** indicates P < 0.01 compared to week 0.
Figure 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 compared to week 0, and ** indicates P < 0.01 compared to week 0.
Figure 19 shows the overall procedure for producing a recombinant production strain (BSBA310) expressing SodA2.
Figure 20 shows an expression vector for overexpression of the sodA2 gene. Here, rrnB T1T2 represents a transcription terminator; rep(pBR322) represents a replicon from pBR322 that operates in E. coli; rep(pUB110) is a replicon from pUB110 that operates in B. subtilis; KanR stands for kanamycin resistance gene (aminoglycoside O-nucleotidyltransferase); The BJ27 promoter represents a strong promoter for B. subtilis.

The detailed description of the invention set forth below will be described with reference to specific drawings with respect to specific embodiments in which the invention may be practiced, but the invention is not limited thereto and, if properly described, is as claimed in the claims. It is limited only by the appended claims, along with all equivalents. It should be understood that the various implementations/embodiments of the present invention are different from one another but are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be changed from one embodiment/embodiment to another embodiment/embodiment, or the embodiments/embodiments may be changed without departing from the technical spirit and scope of the present invention. It can be implemented in combination. Technical and academic terms used in this specification have the same meaning as generally used in the field to which the present invention pertains, unless otherwise defined. For purposes of interpreting this specification, the following definitions will apply and terms used in the singular will include the plural where appropriate and vice versa.

Justice

As used herein, “individual” is used interchangeably with “patient,” and refers to mammals, such as primates (e.g., humans, monkeys, chimpanzees, etc.), companion animals in need of oral hygiene or prevention or treatment of oral disease. It may be an animal (eg, dog, cat, etc.), domestic animal (eg, cow, pig, horse, sheep, goat, etc.), or laboratory animal (eg, rat, mouse, guinea pig, etc.).

The term “treatment” generally refers to obtaining a desired pharmacological and/or physiological effect. This effect has a therapeutic effect in that it partially or completely cures the disease and/or other unwanted or undesirable conditions (e.g., bad breath, tartar, plaque, dental caries, gingivitis, periodontitis, pain, etc.). Desirable therapeutic effects include preventing the occurrence or recurrence of the disease, improving symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, improving or alleviating the disease state, and remission or improvement. Including, but not limited to, prognosis. Preferably, “treatment” may mean medical intervention for an already present disease or disorder.

The term “prophylaxis” means obtaining the desired prophylactic pharmacological and/or physiological effect with a view to partially or completely preventing the disease or its symptoms.

The term “administration” means providing an active ingredient to achieve a preventive or therapeutic purpose to an individual.

oral composition

The present invention provides, in part, the application of superoxide dismutase (SOD) or Bacillus sp. strain spores in combination with a cucumber extract to the oral cavity of an individual, or the application of superoxide dismutase (SOD) and Bacillus sp. strain in combination with a cucumber extract. It is based on the discovery that applying spores to an individual's oral cavity is effective in improving oral hygiene or preventing or treating oral disease. Accordingly, according to one aspect of the present invention, there is provided an oral composition comprising (i) an extract of the chinensis tree and (ii) superoxide dismutase (SOD), Bacillus sp. strain spores, or SOD and Bacillus sp. strain spores. . The composition may include either superoxide dismutase or Bacillus species strain spores in combination with a carnivore extract. Preferably, the composition may comprise superoxide dismutase and Bacillus sp. strain spores in combination with a carnivora extract.

Aucuba japonica is a type of evergreen tree of the dicotyledonous umbel plant Dogwood family, and is mainly distributed in southern Korea, especially the Ulleungdo region. In other words, it is also called maple coral, celestial plate, or blue tree.

As used herein, the term "extract" refers to a liquid component obtained by immersing the desired substance in various solvents and then extracting it at room temperature, low temperature, or at a heated state for a certain period of time, and a liquid component obtained by removing the solvent from the liquid component. It refers to the result of solid content, etc. In addition, in addition to the above results, it can be comprehensively interpreted to include all dilutions of the results, concentrates thereof, crude preparations, purifications, etc. of the above results.

In some embodiments, the chinensis extract may be an extract extracted from the entire aerial part of the chinensis tree, a portion thereof, or materials derived therefrom. It is preferable that the chinensis extract is a hot water extract, but it is not limited thereto.

The part of the tree may be the stem, leaf, flower, petal or seed of the tree. The extract of the Chinese oak tree is preferably extracted from the leaves, stems, or mixtures thereof, but is not limited thereto.

The whole of the oak trees, parts thereof, or materials derived therefrom used for extraction may be ground, shredded, or suitably dried.

The extraction solvent is not particularly limited, and for example, water or an organic solvent can be used. Organic solvents include alcohols with 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, and benzene. , chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane may be used alone or in combination, but are not limited thereto.

As an extraction method, any one of the following methods can be selected and used, such as hot water extraction, cold needle extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression, and preferably hot water extraction. Additionally, one or more of the above extraction methods may be used in combination. The desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation to the method for producing the extract according to the present invention, and any known method can be used.

In some embodiments, the camphor tree extract may be a hot water extract of the camphor tree, and 3 to 20 times, preferably 5 to 15 times, the weight of the medicinal material is added to the water, and the mixture is incubated at 80 to 120° C., preferably 85 to 110° C., or more. Preferably, the extract 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 to this.

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 using methods known in the art or under commonly used conditions.

The final concentration of the chinensis extract may be 100 to 50,000 mg/kg, preferably 5,000 to 25,000 mg/kg, but is not limited thereto.

Superoxide dismutase (SOD) catalyzes the dismutation of superoxide (O ₂ ^ㆍ- ) radicals alternately into general 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 based on 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” can be used interchangeably with (poly)peptide having superoxide dismutase activity. Additionally, the SOD may include a polypeptide having superoxide dismutase activity, a fragment thereof, or a fusion containing the same.

In some embodiments, the SOD can be one that binds manganese (Mn-SOD). Preferably, the SOD may be deamidated Mn-SOD.

In some embodiments, the SOD may comprise or consist of the amino acid sequence represented by SEQ ID NO: 2 (SodA). Preferably, the SOD may be one in which amino acid residues 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).

The SOD or polypeptide having SOD activity of the present invention is interpreted to include amino acid sequences showing substantial identity to the above amino acid sequences. The substantial identity is 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more of the sequence when analyzing the aligned sequences using an algorithm commonly used in the art. It refers to an amino acid sequence showing identity.

In some embodiments, the SOD is a modified or engineered polypeptide with SOD enzymatic activity that may or may not affect various aspects (in vivo, in vitro, or in vitro stability, uniformity, and/or conformational changes). may include one or more mutations, such as deletion, insertion, or substitution of one or more amino acids. In addition, the polypeptide may be added with a heterologous material (e.g., a tag known in the art including HIS tag, HA tag, myc tag, GFC and/or Fc domain of an antibody) for purification, detection, in vivo delivery or increased stability. It can be included as .

In some embodiments, the SOD of the invention may be derived from a variety of sources, including natural, mutant, or recombinant microorganisms. For example, SOD can 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 food, such as a strain of Bacillus sp. or a variant thereof. More preferably, SOD can be obtained from Bacillus amyloliquesfaciens strains (eg, GF423 strains or GF424 strains) or their culture supernatants. The GF423 strain and 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 KCTC 13227 BP, respectively). The Bacillus amyloliquefaciens GF424 strain is a strain obtained by mutating the Bacillus amyloliquefaciens GF423 strain by UV irradiation to improve the expression of the sod gene. 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 as SEQ ID NO: 1). Additionally, SOD may be a recombinant polypeptide. For example, based on SEQ ID NO: 2, it may be a deamidated SOD in which amino acid residues 74 and 137 are replaced with Asp (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.

division order sequence number Nucleotide sequence of SodA ATGGCTTACAAACTTCCAGAATTGCCTTACGCTTATGATGCTTTAGAACCTCATATCGATAAGGAAACGATGACGATTCACCATACGAAGCACCATAACACATACGTGACAAACCTCAACAAGGCGATCGAAGGATCTGCGCTTGCAGAGAAATCTGTAGATGAGCTTGTTGCTGATTTGAACGCAGTGCCGGAGGACATCCGCACGGCAGTCCGCAACAATGGCGGCGGACATGCAAACCACTCTTTATTCTGGACTCTTTTATCTC CGAACGGCGGAGGCGAACCGACTGGTGAGCTTGCTGAAGAGATCAAAAGCACGTTCGGAAGCTTCGATCAATTTAAAGAAAAATTCGCCGCAGCAGCTGCAGGCCGTTTCGGTTCAGGCTGGGCTTGGCTCGTTGTAAACAACGGCAAACTTGAAATTACAAGCACGCCAAACCAAGATTCACCGCTTTCAGAAGGTAAAACACCTGTTCTCGGTCTTGATGTTTGGGAGCATGCGTACTACCTGAACTACCAAAACCGCCGTC CTGATTACATTTCAGCTTTCTGGAATGTTGTGAACTGGGATGAAGTTGCCCGTCTTTACAGCGAAGCAAAAATAA SEQ ID NO: 1 Amino acid sequence of SodA MAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNNGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNNGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAK SEQ ID NO: 2 Nucleotide sequence of SodA2 ATGGCTTACAAACTTCCAGAATTGCCTTACGCTTATGATGCTTTAGAACCTCATATCGATAAGGAAACGATGACGATTCACCATACGAAGCACCATAACACATACGTGACAAACCTCAACAAGCGATCGAAGGATCTGCGCTTGCAGAGAAATCTGTAGATGAGCTTGTTGCTGATTTGAACGCAGTGCCGGAGGACATCCGCACGGCAGTCCGCAACGATGGCGGCGGACATGCAAACCACTCTTTATTCTGGACTCTTTTATC TCCGAACGGCGGAGGCGAACCGACTGGTGAGCTTGCTGAAGAGATCAAAAGCACGTTCGGAAGCTTCGATCAATTTAAAGAAAAATTCGCCGCAGCAGCTGCAGGCCGTTTCGGTTCAGGCTGGGCTTGGCTCGTTGTAAACGACGGCAAACTTGAAATTACAAGCACGCCAAACCAAGATTCACCGCTTTCAGAAGGTAAAACACCTGTTCTCGGTCTTGATGTTTGGGAGCATGCGTACTACCTGAACTACCAAAACCGCCG TCCTGATTACATTTCAGCTTTCTGGAATGTTGTGAACTGGGATGAAGTTGCCCGTCTTTACAGCGAAGCAAAAATAA SEQ ID NO: 3 Amino acid sequence of SodA2 MAYKLPELPYAYDALEPHIDKETMTIHHTKHHNTYVTNLNKAIEGSALAEKSVDELVADLNAVPEDIRTAVRNDGGGHANHSLFWTLLSPNGGGEPTGELAEEIKSTFGSFDQFKEKFAAAAAGRFGSGWAWLVVNDGKLEITSTPNQDSPLSEGKTPVLGLDVWEHAYYLNYQNRRPDYISAFWNVVNWDEVARLYSEAK SEQ ID NO: 4

Additionally, SOD may be derived from another recombinant strain (eg, Bacillus Subtilis species strain) containing the SOD expression gene of the Bacillus amyloliquefaciens strain. The recombinant strain can be produced by recombinant technology using conventional protein producing strains 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 purchased from the Korea Research Institute of Bioscience and Biotechnology Biological Resources Center (KCTC). In addition, the recombinant strain may have one or more of the genes shown in Table 2 removed to facilitate subsequent processes.

gene name protein name function AprE Serine alkaline protease (subtilisin E) extracellular protease NprE Extracellular neutral metal protease extracellular protease Bpr bacillopeptidase F extracellular protease Epr extracellular serine protease extracellular protease NprB Extracellular neutral protease B extracellular protease Vpr extracellular serine protease extracellular protease Mpr Extracellular metalloprotease extracellular protease IspA Intracellular serine protease intracellular protease SrfAC Surfactin synthase Surfactin synthesis spoIIAC RNA polymerase sporulation-specific sigma factor (Sigma-F) spore formation EpsE Putative glycosyltransferase extracellular polysaccharide Xpf RNA polymerase sigma factor Transcription of PBSX prophage genes

For example, the recombinant strain can be produced through the process shown in Figure 19. Additionally, the recombinant strain may include the expression vector shown in FIG. 20. In the figure, sodA and sodA2 represent genes encoding SOD.

Since SOD derived from the above strain is an enzyme secreted to the outside of the cell, when producing SOD using the above strain, it is possible to mass produce SOD that is safe for individuals without going through an expensive purification process (for example, column purification). This makes efficient production possible.

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 GF423 strain or GF424 strain. Specifically, a culture medium can be obtained by first cultivating Bacillus amyloliquefaciens strains in various types of media. For example, the strain is grown at about 25°C to about 42°C for about 1 to about 4 days using complex medium (pH 6.0 to 7.0). Other suitable media for culturing the Bacillus amyloliquefaciens strain include LB (Luria-Bertani) medium, ISP (International Streptomyces Project) medium, NA (nutrient agar) medium, BHI (brain heart infusion agar) medium, and SDA. (sabouraud dextrose agar) medium, PDA (potato dextrose agar) medium, NB (nutrient broth) medium, etc. In a preferred embodiment, LB medium, ISP medium, BHI medium, SDA medium, or NB medium can be used. Additionally, SODs can 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).

In some embodiments, the SOD of the present invention may be isolated or purified from a culture of a natural, mutant, or recombinant strain. 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 is derived. For example, the purified product may be purified from a strain culture through ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or may be a culture concentrate obtained through ultrafiltration, concentration, etc. The phrase “substantially free of cellular material” includes preparations of a protein in which such protein has been separated from cellular components of the cell from which the protein was isolated or recombinantly produced. In some embodiments, the phrase “substantially free of cellular material” refers to 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. It includes preparations containing less than about 5% protein.

The SOD may be preferably purified by the following purification method, but is not limited thereto. For example, the culture medium obtained by previously culturing the Bacillus amyloliquefaciens strain is centrifuged to collect the culture supernatant. The supernatant fraction is pretreated by solid phase extraction and then isolated and purified by chromatography. SOD can be purified using various methods of chromatography. Preferably, hydrophobic interaction chromatography is used.

In some embodiments, the SOD may be included in the form of a strain lysate, strain culture, strain culture concentrate, strain culture extract, or dried form thereof. At this time, “strain lysate” refers to a product obtained by cultivating a strain and crushing it mechanically or chemically, and may include anything that has undergone additional processes such as extraction, dilution, concentration, and purification. “Strain culture” may refer to the culture itself or its supernatant obtained by cultivating a strain. “Strain culture concentrate” refers to a culture concentrate purified from a strain culture through ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or obtained through ultrafiltration, concentration, etc. “Strain culture extract” refers to an extract from the culture medium or its concentrate, and may include an extract, a diluted or concentrated liquid 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.

In some embodiments, the SOD may comprise cellular material from the cell or tissue source from which it was derived, such as extracellular vesicles. In this case, cellular material containing SOD can be cultured from various sources, including natural, mutant, or recombinant hosts, using conventional techniques known in the art as described above, and isolated from the culture medium by methods such as filtration and concentration. You can.

Meanwhile, the Bacillus species strain spores can be obtained by culturing the Bacillus species strain in a suitable medium, inducing spore formation, and then isolating the resulting spores. In one embodiment, Bacillus species strain spores may be sourced from GRAS bacteria that are generally considered safe for use in drugs or food. 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) . Additionally, Bacillus species strain spores 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). Spore formation can be induced using conventional techniques known in the art. For example, spores of a strain obtained by inducing spore formation in media such as DSM, NB, SYP, and LB2 during main culture after pre-culturing the strain, and culturing various sources including natural, mutant, or recombinant hosts to form vegetative cells. After removal, it can be separated through filtration, concentration, or centrifugation.

In addition, the cypress extract and the SOD enzyme and/or Bacillus species strain spores may be included in the composition in a form that allows them to be administered sequentially, in reverse order, or simultaneously with each other.

In some embodiments, the oral composition may include an extract of the chinensis tree, SOD, and Bacillus species strain spores, or may be used in combination. According to one embodiment of the present invention, when a mixture of algae extract, SOD, and Bacillus species spores is applied to the oral cavity, bad breath, tartar, plaque, dental caries, or periodontal disease (e.g., gingivitis or periodontitis) are prevented. , it was confirmed to be effective in improving or treating oral health, managing oral hygiene, or preventing or treating oral diseases. In addition, the mixture was confirmed to be effective in reducing pain caused by oral diseases.

In some embodiments, the cypress extract, SOD, and Bacillus species strain spores may be mixed in an appropriate ratio to exert the above effects. For example, the extract of the chinensis tree corresponds to a dose of 0.1 to 10,000 mg/kg per day, preferably 1 to 1,000 mg/kg, more preferably 1 to 100 mg/kg, and even more preferably 5 to 50 mg/kg. It may be included, and SOD and Bacillus species strain spores may be 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. You can. Additionally, the final composition may be, for example, a mixture of 0.5% to 5% of carob tree extract and 0.5% to 12% of SOD and/or Bacillus species strain spores. The final composition may preferably be a mixture of 2% of carob tree extract and 2.5% of SOD and/or Bacillus species strain spores.

In some embodiments, the compositions can be used to promote oral health or maintain oral hygiene.

In some embodiments, the composition can be used for preventing or treating oral diseases.

In some embodiments, the composition can be used to prevent, improve, or treat one or more diseases selected from bad breath, calculus, plaque, dental caries, periodontal disease, and stomatitis (e.g., chronic stomatitis).

In some embodiments, the composition can be used to reduce oral pain.

In other embodiments, the composition may have various forms applied to the oral cavity known in the art, including liquid form, solid form, gel form, powder form, paste form, or form impregnated or applied to a carrier.

oral products

According to another aspect of the present invention, an oral product containing the oral composition is provided. In other words, the oral product comprises one or more selected from the group consisting of (i) a Chinese oak extract and (ii) SOD and Bacillus species 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 oral rinse, but is not limited thereto.

The oral product of the present invention may contain additional ingredients known in the art necessary for its formulation, in addition to containing the cypress extract and SOD and/or Bacillus species strain spores as active ingredients. For example, when the oral product is toothpaste, it may additionally contain abrasives, moisturizers, foaming agents, sweeteners, whitening agents, or flavoring agents. Additionally, when the oral product is an oral gel, it may additionally contain a thickener such as medium chain fatty acid for the viscosity or adhesion of the gel. Additionally, when the oral product is an oral rinse, it may additionally contain a carrier such as non-toxic alcohol.

Pharmaceutical or veterinary compositions

According to another aspect of the present invention, a pharmaceutical composition or a veterinary composition comprising a composition comprising an extract of the chinensis tree as described above and one or more selected from the group consisting of superoxide dismutase (SOD) and Bacillus species strain spores. This 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.

In some embodiments, the composition may include superoxide dismutase (SOD) and Bacillus species strain spores in combination with a carnivora extract.

In some embodiments, the composition can be used for preventing or treating oral diseases. Specifically, the globular disease may be one or more diseases selected from bad breath, tartar, plaque, dental caries, periodontal disease, and chronic stomatitis, but is not limited thereto.

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 calculus, 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 those 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. Cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, mineral oils such as methylhydroxybenzoate, propylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and silicon dioxide, etc. Examples include, but are not limited to.

When formulated, it can 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.

In addition, the pharmaceutical or veterinary composition of the present invention may be formulated in a desired form depending on the method of use, and may be formulated in a form known in the art to provide rapid, sustained or delayed release of the active ingredient, especially after administration to a mammal. It can be formulated by adopting a method. Specific examples of such dosage forms 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, needles, creams, soft or hard gelatin capsules, patches, etc.

Furthermore, the pharmaceutical or veterinary composition of the present invention is preferably prepared using appropriate methods known in the art or methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA. It can be formulated as follows.

The pharmaceutical or veterinary composition of the present invention can be administered orally or parenterally depending on the desired method. When administered parenterally, it is administered by nasal spray, external application to the skin, or intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, or intramuscular injection. It is advisable to select injection or intrathoracic injection. In addition, the pharmaceutical or veterinary composition of the present invention may be preferably applied through an intraoral injection method such as oral spray, oral application, or intraoral injection.

At this time, solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient in a complex composition, such as starch, calcium carbonate, sucrose, lactose, It can be prepared by mixing gelatin, etc. Additionally, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are included. can be used For oral administration, SOD may be coated with coatings such as shellac, ethyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, zein, Eudragit, and combinations thereof. However, the coating agent is not limited to this.

Forms for parenteral administration include toothpaste, mouthwash, topical administration (eg, gel, cream, ointment, dressing solution, spray, and other coating agents). An example of the formulation of the topical administration agent may be one in which the active ingredient of the present invention is impregnated into a carrier such as gauze made of natural fiber or synthetic fiber. The gel, cream or ointment may be suitable for direct application to or around affected or decayed areas, including teeth and gums. In the case of the above spray, it can be manufactured by a typical spray manufacturing method, and can be filled and packaged in a compression container or other spray container and sprayed and applied to oral disease areas at any time to prevent or treat oral disease. In the case of the dressing solution, it can be prepared by a conventional dressing solution manufacturing method, and can be used to prevent or treat oral disease 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. The term "pharmaceutically effective amount" or "veterinary 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 dose level is defined as that of the patient or animal. Weight, gender, age, health status, severity, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors well known in the medical field. You 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. Additionally, the pharmaceutical composition may be administered singly or multiple times as needed. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this amount can be easily determined by a person skilled in the art.

Food or feed composition

According to another aspect of the invention, comprising a composition comprising at least one selected from the group consisting of (i) a Chinese oak extract and (ii) superoxide dismutase (SOD) and Bacillus sp. strain spores as described above. A food composition is provided.

In some embodiments, the composition may include superoxide dismutase (SOD) and Bacillus species strain spores in combination with a carnivora extract.

Such food compositions include medical or nutraceutical food compositions. The term “medical food” or “nutraceutical food” refers to food manufactured from raw materials or ingredients that have the potential to perform beneficial functions in the human body, and that maintain or improve health by maintaining normal function or activating physiological functions of the human body. It refers to food, and is regulated by the Ministry of Food and Drug Safety, but is not limited thereto, and does not exclude any common health food from its meaning.

In addition, the above foods include, but are not limited to, various foods, food additives, beverages (e.g., functional drinks, natural fruit juices and vegetable drinks), gum, tea, vitamin complexes, health functional foods, and other functional foods. .

The food can be manufactured by conventional methods known in the art. For example, the medical food, nutraceutical food, or health functional food may further include one or more of carriers, diluents, excipients, and additives in addition to the SOD for the purpose of promoting oral hygiene or preventing or improving oral disease. It may be formulated as one selected from the group consisting of pills, powders, granules, powders, capsules, and liquid formulations. Specific examples of the carriers, excipients, diluents and additives are well known in the art, and those skilled in the art can prepare them by combining appropriate ingredients according to the formulation.

In the above-mentioned formulation, the content of the Chinese maple extract and superoxide dismutase and/or Bacillus species strain spores according to the present invention as active ingredients can be appropriately adjusted depending on the form and purpose of use, patient condition, type and severity of symptoms, etc. It may be 0.001 to 99.9% by weight, preferably 0.01 to 50% by weight, based on the weight of solid content, but is not limited thereto.

The administered dose of the food of the present invention may vary depending on the patient's age, weight, gender, dosage form, health condition, and degree of disease, and is administered in divided doses from once to several times a day 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 dosage of the health functional food of the present invention is less than the above dosage, significant effects may not be obtained, and if it exceeds the dosage, it is not only uneconomical but also outside the range of the usual dosage, so undesirable side effects may occur. there is.

According to another aspect of the present invention, it comprises a composition comprising at least one selected from the group consisting of (i) an extract of the chinensis tree and (ii) superoxide dismutase (SOD) and spores of a Bacillus species strain, as described above. A feed composition is provided. In some embodiments, the composition may include superoxide dismutase (SOD) and Bacillus species strain spores in combination with a carnivora extract.

The feed composition of the present invention can be prepared in any formulation commonly used in the art. For example, the feed composition of the present invention includes auxiliary ingredients such as amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, and other microbial agents in the form of live bacteria; Grains such as ground or broken wheat, oats, barley, corn and rice; Vegetable protein feeds, such as those based on rapeseed, soybean and sunflower; Animal protein feeds 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 ingredients such as lipids, such as animal fats and vegetable fats optionally liquefied by heating; It may additionally contain additives such as nutritional supplements, digestion and absorption enhancers, growth promoters, and disease prevention agents.

The feed composition of the present invention may be in the form of a powder or liquid preparation, and may contain excipients for adding feed (calcium carbonate, horse meal, zeolite, jade meal, or rice bran, etc.).

Methods for promoting oral hygiene or preventing or treating oral disease

According to another aspect of the present invention, administering to an individual at least one selected from the group consisting of (i) a Chinese oak extract and (ii) superoxide dismutase (SOD) and Bacillus species strain spores as described above. A method for promoting oral hygiene comprising a is provided. In addition, preventing oral diseases comprising the step of administering to an individual at least one selected from the group consisting of (i) Chinese fern extract and (ii) superoxide dismutase (SOD) and Bacillus species strain spores as described above. or treatment methods are provided.

In some embodiments, the method may include administering to the individual SOD and Bacillus species strain spores in combination with a Culinium sinensis extract.

In some embodiments, the oral disease may include one or more selected from the group consisting of oral pain, bad breath, plaque, tartar, dental caries, periodontal disease, and chronic stomatitis.

In one embodiment, the chinensis 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 both components. Additionally, the alveolar extract, SOD, and Bacillus sp. strain spores may be administered sequentially or in reverse order, respectively, or as separate compositions containing either of the two components. In the case of simultaneous or separate administration, the cypress extract, SOD, and Bacillus species strain spores may be administered by the same route or different routes depending on the dosage form. The composition, dosage form, route of administration, etc. are as described above for the composition.

Hereinafter, the present invention will be described in detail through examples to aid understanding. However, the following examples only illustrate the content of the present invention and the scope of the present invention is not limited to the following examples.

Example

Example 1. Preparation of Peach Tree Extract

Arborvitae leaves and stems were collected from Jisepo-ri Forest, Irun-myeon, Geoje-si, Gyeongsangnam-do in August 2020, and evidence samples are stored in the sample storage room of Chonbuk National University. 900 L of distilled water was added to 100 kg of Chinese oak, shaken at 100°C for 3 hours, concentrated under reduced pressure, and then dried to obtain a powdered Chinese oak hot water extract.

Example 2. Bacillus amyloliquefaciens Isolation and purification of superoxide dismutase from strains

The Bacillus amyloliquefaciens strains used in this example are Bacillus amyloliquefaciens GF423 strain and Bacillus amyloliquefaciens GF424 strain, and these strains were registered with the Korea Research Institute of Bioscience and Biotechnology on March 6, 2017 and 2017, respectively. It was deposited on March 13, 2018 with accession number KCTC 13222 BP and accession number KCTC 13227BP.

The Bacillus amyloliquefaciens GF424 strain is a strain obtained by causing mutations in the Bacillus amyloliquefaciens GF423 strain by irradiating UV to improve the expression of the sodA gene.

Example 2.1. Bacillus amyloliquefaciens Cultivation of GF423 or GF424 strains

For culturing of Bacillus amyloliquefaciens GF423 or GF424 strains, LB agar medium (LB (Luria-Bertani) agar; tryptophan 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, agar 15 g/L) L) The single colony formed was inoculated into 30 ml of LB medium and cultured at 37°C for 12 hours. This 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.

Example 2.2. Isolation and purification of superoxide dismutase (SOD)

The cell culture obtained in Example 2.1 was centrifuged at 4°C and 3,578 The concentrate was filtered through a sterilization filter and then freeze-dried. 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 dried SOD enzyme was 30 ± 6 U/mg.

Example 3. Bacillus amyloliquefaciens Preparation of strain spores

Example 3.1. Badge composition

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% Contains % (NH ₄ ) ₂ PO ₄ , 0.1% CaCl ₂ and 2% glucose. DSM medium contains 8 g/L bactonutrient broth, 1 g/L KCl, 0.25 g/L MgSO ₄ , 0.16415 g/L Ca(NO ₃ ) ₂ , 0.9521 mg/L MnCl ₂ and 0.152 mg FeSO ₄ . 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.

Example 3.2. Induction of spore formation

A single colony of Bacillus amyloliquefaciens strain GF423 or GF424 was inoculated into 1 mL of LB contained in a 14 mL tube and cultured at 37°C and 200 rpm for 12 hours. 1 mL of culture 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.

Example 3.3. Spore washing

After incubation, lysozyme (0.5 g/L) was added to the culture broth and incubated at 37°C and 200 rpm for 1 hour to remove remaining vegetative cells. Crude spores were collected by centrifugation at 6000 rpm for 10 minutes. The collected crude spores were washed twice with water, washed with 0.02% SDS, washed again with water twice, and then purified by suspending in a PBS solution. The spore suspension was stored at -20°C. The number of spores was determined by spreading the diluted spore solution on an LB agar plate and counting the colonies.

Experimental Example 1. Animal test using extract of Culinium chinensis

A test using beagle dogs or rats was conducted to determine whether the extract of the Chinese maple tree had an effect on improving oral health and managing oral hygiene.

Experimental Example 1.1. test substance

The test material was prepared as follows. An oral ointment (test group) containing 2% of the chinensis tree extract obtained in Example 1 and an oral ointment (control group, vehicle only) containing no china tree extract (control group, vehicle only) were prepared with the compositions shown in Table 3 below.

ingredient Content (based on 10 ml) Test group (containing 2% algae extract) Control group (vehicle) Acacia tree extract 200mg - Mucoadhesive (Sodium polyacrylate, PAA-Na) 300mg 300mg Flavor enhancer (Mannitol) 100 mg 100 mg Preservative (Methyl paraben) 18mg 18mg Preservative (Poly paraben) 2mg 2mg

Experimental Example 1.2. Gingivitis index evaluation test using Cuca sinensis extract

Experimental Example 1.2.1. test animals

Male beagle dogs (non-naive), weighing 9 to 13 kg, over 3 years of age and with oral clinical symptoms (bad breath, plaque, gingivitis, periodontitis, etc.) were purchased from Orient Bio Co., Ltd. and had a medication holiday period of at least 2 weeks.

The reason why beagle dogs were chosen as test animals was because they are widely used in oral-related efficacy evaluation tests such as gingivitis, and because they have accumulated a wealth of basic test data, it is easy to interpret and evaluate test results.

Experimental Example 1.2.2. Administration and evaluation results of test substances

The test substance of Experimental Example 1.1 was administered by applying it to the gums (outside gingiva of both maxillary molars) twice a day for 4 weeks. The dosage was 0.5 g per time, 1.0 g/day. The content of the active ingredient contained in 1.0 g/day was 20 mg/day. After administration of the Chinese maple extract for 4 weeks, an oral examination was performed to evaluate the gingivitis index according to the standards shown in Table 4 below.

score Assessment Methods 0 no disease One Mild gingivitis - Gums: slight redness
- Teeth: A little bit of plaque 2 Early periodontitis - gums: redness and swelling
- Teeth: Plaque under the gums, some tartar 3 Moderate periodontitis - Gums: May be red, swollen, bleeding, insulated or thickened.
- Teeth: Moderate to severe amount of tartar, subgingival tartar, loose or missing teeth 4 Severe periodontitis - Gums: Severe redness, inflammation, bleeding, formation of pockets around teeth, and pus may occur.
- Teeth: Large amount of subgingival tartar, teeth are loose or missing

As a result, as can be seen from Figure 1, the gingivitis index worsened over time in beagle dogs administered a control vehicle (vehicle) that did not contain the chinensis extract, whereas the gingivitis index worsened over time in the beagle dogs administered the oral ointment containing the chinensis tree extract. In beagle dogs, gum swelling and redness were reduced, resulting in a 15% decrease in the gingivitis index.

Experimental Example 1.3. Confirmation of the effect of alveolar bone destruction inhibition of alveolar tree extract

Experimental Example 1.3.1. test animals

Six-week-old male SD rats were purchased (purchased from Damul Science, Daejeon, Korea). The obtained rats were acclimatized for one week and allowed to freely consume water and food.

After the acclimation process was completed, anesthesia was induced by intraperitoneal injection of zoletine and ketamine mixed in a 1:2 ratio (1.5 ml/kg). After anesthesia, the cervical region of the mandibular left second molar was ligated with silk suture (0.5 mm) to induce periodontitis. The mandibular right second molar that was not ligated with suture was set as the control group.

Experimental Example 1.3.2. Administration and evaluation results of test substances

The day periodontitis was induced was designated as day 0, and the rats were sacrificed 2 weeks after the periodontitis was induced. During the test period, algae extract (test material in Experimental Example 1.1) was orally administered once a day at concentrations of 50, 100, and 250 mg/kg, and only the vehicle (DW) was orally administered to the control group. It was checked whether the sutures were maintained from the day periodontitis was induced until the day the rat was sacrificed.

After 2 weeks of administration of the chinensis extract, the rats were sacrificed and their lower jaws were removed. Afterwards, all soft tissues were removed by treatment with 1M NaOH for about 1 hour, and then the length from the cemento-enamel junction to the crest of the alveolar bone was measured to quantify the degree of alveolar bone loss.

As a result, as can be seen from Figure 2, in rats with periodontitis, the destruction of the alveolar bone occurred so severely that the roots of the teeth were exposed to the outside, but in rats administered the alveolar extract, the destruction of the alveolar bone was suppressed in a concentration-dependent manner. could be confirmed.

Experimental Example 1.4. Review

When comprehensively judged based on the above results, it was confirmed that fern extract is useful in improving oral diseases such as gingivitis and pubic bone destruction.

Experimental Example 2. Superoxide dismutase (SOD) and Bacillus amyloliquefaciens Animal testing using strain spores

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.

Experimental Example 2.1. test substance

Superoxide dismutase (SOD) and Bacillus amyloliquefaciens strain spores were prepared from superoxide dismutase (SOD) purified from Bacillus amyloliquefaciens GF424 in Example 2 and Bacillus prepared in Example 3. It was prepared by mixing spores of Amyloliquefaciens GF423 strain (2.5% mixture, SOD 10,000 U/g).

Specifically, the mixture is adjusted to the desired viscosity by dissolving medium-chain fatty acids such as lauric acid to maintain the viscosity/adhesion of the oral gel at room temperature, mixing palatable ingredients such as betaine, oligosaccharides, and egg yolk, and finally, SOD and 2.5% spores (SOD titer: 12,510 U/g, spores: 1.2 x 10^10 CFU/g) were obtained by homogeneously mixing. This mixture (SOD + spores) was used in a gel-type oral gel form in a tube (20 g/tube, 250 U/g gel).

Experimental Example 2.2. Test animals and breeding environment

Sixteen male beagle dogs (non-naive), over 3 years old and without oral clinical symptoms, weighing 9 to 13 kg, were purchased from Orient Bio Co., Ltd., and were given a drug-free period of at least 1 month.

A breeding room with environmental conditions of 23 ± 3°C, relative humidity 30 ± 10%, ventilation frequency of 10 to 15 times/hour, lighting for 12 hours, and illuminance of 150 to 200 Lux was used. During the acclimatization and testing period, the cages made of stainless steel were used. They were raised individually in cages, one per cage. Feed and drinking water were provided autonomously.

The reason why beagle dogs were chosen as test animals was because they are widely used in oral-related efficacy evaluation tests such as gingivitis, and because they have accumulated a wealth of basic test data, it is easy to interpret and evaluate test results.

Experimental Example 2.3. Test group composition

Test groups G1 (“G1_Vehicle”) and G2 (“G2_BASOD”) were formed as follows.

Experimental Example 2.4. Administration of test substance

The test substance of Experimental Example 2.1 was administered by applying it to the gums (outside gingiva of both maxillary molars) every day for 4 weeks. The dosage was 0.25 g (SOD 62.5 U)/head twice a day.

Experimental Example 2.5. Test evaluation items

(1) Halitosis index

Bad breath was evaluated by sensory tests at week 0 (before starting administration), week 2, and week 4 (see table below).

(2) Calculus index

At week 0 (before starting administration), week 2, and week 4, the tartar index was calculated by opening the mouth to expose the tooth surface, taking pictures of the tooth surface, and observing it with the naked eye (see table below and Figure 3). .

(3) Plaque index

At week 0 (before starting administration), week 2, and week 4, plaque was stained using a plaque stainer, the tooth surface was photographed, and the area covered by the plaque stainer was converted to a percentage of the total tooth surface. The plaque index was calculated (see table below).

(4) Gingival index

At week 0 (before starting administration), week 2, and week 4, photos were taken after opening the mouth to expose the tooth surfaces and gums around the teeth, and the gingivitis index was calculated by visual observation (see table below and Figure 4).

(5) Other adverse reactions

If other adverse reactions were observed during the administration period of the test substance, the symptoms were recorded.

Experimental Example 2.6. statistical methods

To compare before and after treatment of the test substance within the test group, statistical significance was tested using paired sample t-test, and the Graphpad prism 5.01 program, a widely used commercial statistical package, was used as a statistical processing method.

Experimental Example 2.7. Test results and considerations

(1) Bad breath index

For all animals administered the mixture, no statistically significant observation could be made until the 4th week after administration, but it was confirmed that the bad breath index tended to decrease (Table 5 and Figure 5).

(2) Tartar index

For all animals administered the mixture of SOD and spores, there was a tendency for tartar to be gradually removed until the 4th week after administration, and in particular, at week 4, there was a statistically significant decrease in the tartar index compared to week 0 (p <0.05) (Table 6 and Figure 6). In Table 6 and Figure 6, statistical significance was verified by paired sample t-test.

(3) Plaque index

For all animals administered the mixture of SOD and spores, a tendency for the plaque index to decrease was confirmed, although it was not observed to be statistically significant until the 4th week after administration (Table 7 and Figure 7).

(4) Gingivitis index

For all animals administered the mixture of SOD and spores, the efficacy of eliminating gum bleeding and improving swelling and redness was observed by the 4th week after administration. In particular, the gingivitis index was statistically significant at the 4th week compared to the 0th week. decreased significantly (p<0.01) (Table 8 and Figure 8). In Table 8 and Figure 8, statistical significance was verified by paired sample t-test.

(5) Consideration

As a result of applying the test substance, a mixture of SOD and spores, to the gums of a beagle dog twice daily at a dose of 0.25 g (SOD 62.5 U)/head, the bad breath index was higher from the 2nd to the 4th week after administration compared to the 0th week. A tendency for the tartar index, plaque index, and gingivitis index to all decrease was confirmed. In particular, symptoms of tartar and gingivitis were statistically significantly improved compared to week 0. In addition, no special adverse reactions occurred during the entire test process, confirming that the test substance was not harmful.

When comprehensively judged based on the above results, it was confirmed that the test substance, a mixture of SOD and spores, is useful in improving oral diseases such as bad breath, tartar, plaque, and gingivitis.

Experimental Example 3. Animal test using superoxide dismutase (SOD)

A test using companion cats was performed to determine whether the superoxide dismutase (SOD) purified in Example 2 exhibited oral health promotion and oral hygiene management effects.

Experimental Example 3.1. test substance

The test material used was prepared in the same manner as in Experimental Example 2.1, except for the spores of the Bacillus amyloliquefaciens GF423 strain.

Experimental Example 3.2. test animals

Regardless of gender or breed, among cats over 6 months old and weighing between 1 kg and 10 kg, there are cases of bad breath, decreased energy due to pain, difficulty chewing, decreased appetite, drooling, red gums (erosions and proliferative ulcers), etc. Cats showing oral symptoms were randomly selected as test cats.

Experimental Example 3.3. Test group composition

After obtaining consent to voluntarily participate in the clinical trial from each cat's guardian, random allocation was performed on the test cats.

For test group G1, the breed was mixed, KSH (Korean short hair), Exotic, or Siamese, the sex was neutered male (NM) or neutered female (SF), and the age was 2 to 10 years. It was three years old.

For test group G2, the breed was T.A. (Turkish Angora) or KSH (Korean short hair), the sex was neutered male (NM) or neutered female (SF), and the age was 1 to 11 years old.

Experimental Example 3.4. Administration of test substance

The test substance of Experimental Example 3.1 was administered by applying it to the gingival area of the right maxillary molar every day for 4 weeks. The dosage was 0.3 g (SOD 75 U)/head twice a day.

Experimental Example 3.5. Test evaluation items

Before the start of the clinical trial, blood tests (alanine aminotransferase (ALT), etc.) and basic physical examinations (body weight, etc.) of the test cats were conducted.

(1) Feed intake

The feed intake of the test cats was evaluated at week 0 (before starting administration), week 2, and week 4 (see table below).

(2) Activity (mood)

The activity (mood) of the test cat was observed and evaluated at week 0 (before starting administration), week 2, and week 4 (see table below).

(3) Palpable pain index

Palpable pain was evaluated by observing the reaction when touching the diseased area of the test cat with the hand at week 0 (before starting administration), week 2, and week 4 (see table below).

(4) Bad breath index

Bad breath was evaluated by sensory tests at week 0 (before starting administration), week 2, and week 4 (see table below).

(5) Plaque index

After staining the right molar using a plaque stain at week 0 (before starting administration), week 2, and week 4, the plaque index was calculated by converting the area covered by the plaque stain on the tooth surface into a percentage of the total tooth surface. and took a picture (see table below).

(6) Tartar index

At week 0 (before starting administration), week 2, and week 4, tartar was visually observed on the right maxillary molar, the tartar index was calculated (see table below and Figure 3), and photographs were taken.

(7) Gingivitis index

At week 0 (before starting administration), week 2, and week 4, the gingival area of the right maxillary molar was observed with the naked eye, the score was measured based on the table below, the total score was calculated, the gingivitis index was calculated, and photographs were taken. .

(8) Other adverse reactions

All adverse reactions (loss of appetite, vomiting, diarrhea, hematochezia, hematemesis, lethargy, gastrointestinal disorders, etc.) that occurred during the administration of the test substance for validation were observed and recorded by the guardian or test administrator.

Experimental Example 3.6. statistical methods

To compare before and after treatment of the test substance within the test group, statistical significance was tested using paired sample t-test, and the Graphpad prism 5.01 program, a widely used commercial statistical package, was used as a statistical processing method.

Experimental Example 3.7. Test results and considerations

(1) Feed intake and body weight

No significant differences in feed intake and body weight were observed in any group during the study period (data not shown).

(2) Activity

In the vehicle control group, activity significantly improved from the second week, and in the SOD group, activity tended to gradually increase, but there was no statistical significance (data not shown).

(3) Palpable pain index

In the vehicle control group, there was little change in palpable pain until the end of the test, but in the SOD group, palpable pain decreased from the 2nd week and improved statistically significantly at the 4th week (FIG. 9).

(4) Bad breath index

In the vehicle control group, the bad breath index did not change until the end of the test, but in the SOD group, the bad breath index continued to decrease (FIG. 10).

(5) Plaque index

In the vehicle control group, the plaque index clearly increased during the test period, while in the SOD group, the plaque index decreased with statistical significance from the second week to the end of the test (FIG. 11).

(6) Tartar index

In the vehicle control group, the tartar index gradually increased, while in the SOD group, the tartar index decreased with statistical significance at 4 weeks (FIG. 12).

(7) Gingivitis index

In the vehicle control group, there was no significant change in the gingivitis index, 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) Considerations

As a result of applying the test substance SOD to the cat's gums twice daily at a dose of 0.3 g (SOD 75 U)/head, bad breath index, palpable pain index, A tendency for the plaque index, tartar index, and gingivitis index to all decrease was confirmed. In particular, symptoms of palpable pain, plaque, calculus, and gingivitis were statistically significantly improved compared to week 0. In addition, no special adverse reactions occurred during the entire test process, confirming that the test substance was not harmful.

Judging based on the above results, even in the case of SOD, the test substance administered alone, it is not only useful in improving oral diseases such as bad breath, plaque, tartar, and gingivitis, but is also useful in reducing pain caused by such oral diseases. was able to confirm.

Experimental Example 4. Chrysanthemum extract and superoxide dismutase (SOD) Bacillus amyloliquefaciens Animal testing using mixtures of strain spores

A test using companion cats was conducted to determine whether a mixture of algae extract, superoxide dismutase (SOD), and Bacillus amyloliquefaciens strain spores exhibits oral health promotion and oral hygiene management effects.

Experimental Example 4.1. test substance

The test material was prepared as follows. An oral gel containing a mixture of the chinensis extract powder obtained in Example 1, the SOD prepared in Example 2, and the spore prepared in Example 3 was prepared with the composition shown in the table below.

Specifically, the mixture was prepared by dissolving algae extract powder in purified water at 60°C for 1 hour, dissolving carboxymethylcellulose and sodium polyacrylate to maintain the viscosity/adhesion of the oral gel at room temperature, and containing vitamine, oligosaccharides, and egg yolk powder. After mixing the palatable 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% Chinese acacia extract and 2.5% SOD and spore mixture (SOD titer: 250 U/g, spores: 2 x 10^8 CFU/g) was used in a tube.

Experimental Example 4.2. test animals

Regardless of gender or breed, among cats over 6 months old and weighing between 1 kg and 10 kg, symptoms such as bad breath and pain, decreased energy, difficulty chewing, decreased appetite, drooling, and red gums (erosions and proliferative ulcers) Cats showing oral symptoms were randomly selected as test cats.

Experimental Example 4.3. Test group composition

After obtaining consent to voluntarily participate in the clinical trial from each cat's guardian, random allocation was performed on the test cats. Test groups G1 (“G1_Vehicle”) and G2 (“G2_Coronavirus extract + BASOD”) were formed as follows.

For test group G1, the breed was mixed, KSH (Korean short hair), Exotic, or Siamese, the sex was neutered male (NM) or neutered female (SF), and the age was 2 to 10 years. It was three years old.

For test group G2, the breed is mixed breed, KSH (Korean short hair), Exotic, or Soti.F, the sex is neutered male (NM) or neutered female (SF), and the age is 1 to 4 years old. It was.

Experimental Example 4.4. Administration of test substance

The test substance of Experimental Example 4.1 was administered by applying it to the gingival area of the right maxillary molar every day for 4 weeks. The dosage was 0.3 g/head twice a day.

Experimental Example 4.5. Test evaluation items

Before the start of the clinical trial, blood tests (alanine aminotransferase (ALT), etc.) and basic physical examinations (body weight, etc.) of the test cats were conducted.

(1) Feed intake

The feed intake of the test cats was evaluated at week 0 (before starting administration), week 2, and week 4 (see table below).

(2) Activity (mood)

The activity (mood) of the test cat was observed and evaluated at week 0 (before starting administration), week 2, and week 4 (see table below).

(3) Palpable pain index

Palpable pain was evaluated by observing the response when touching the diseased area of the test cat with the hand at week 0 (before starting administration), week 2, and week 4 (see table below).

(4) Bad breath index

Bad breath was evaluated by sensory tests at week 0 (before starting administration), week 2, and week 4 (see table below).

(5) Plaque index

After staining the right molar using a plaque stain at week 0 (before starting administration), week 2, and week 4, the plaque index was calculated by converting the area covered by the plaque stain on the tooth surface into a percentage of the total tooth surface. and took a picture (see table below).

(6) Tartar index

At week 0 (before starting administration), week 2, and week 4, tartar was visually observed on the right maxillary molar, the tartar index was calculated (see table below and Figure 3), and photographs were taken.

(7) Gingivitis index

At week 0 (before starting administration), week 2, and week 4, the gingival area of the right maxillary molar was observed with the naked eye, the score was measured based on the table below, the total score was calculated, the gingivitis index was calculated, and photographs were taken. .

(8) Other adverse reactions

All adverse reactions (loss of appetite, vomiting, diarrhea, hematochezia, hematemesis, lethargy, gastrointestinal disorders, etc.) that occurred during the administration of the test substance for validation were observed and recorded by the guardian or test administrator.

Experimental Example 4.6. statistical methods

To compare before and after treatment of the test substance within the test group, statistical significance was tested using paired sample t-test, and the Graphpad prism 5.01 program, a widely used commercial statistical package, was used as a statistical processing method.

Experimental Example 4.7. Test results and considerations

(1) Feed intake and body weight

No significant differences in feed intake and body weight were observed in any group during the study period (data not shown).

(2) Activity

In test group G1, activity significantly improved from the second week, and in test group G2, activity tended to gradually increase, but there was no statistical significance (data not shown).

(3) Palpable pain index

In test group G1, there was little change in palpable pain until the end of the test, but in test group G2, the palpable pain index decreased significantly with statistical significance two weeks after administration (FIG. 14).

(4) Bad breath index

In test group G1, the bad breath index did not change until the end of the test, but in test group G2, the bad breath index decreased by about 50% in the 2nd week of administration compared to week 0, and bad breath was statistically significantly improved by the 4th week (Figure 15) .

(5) Plaque index

In test group G1, the plaque index clearly increased during the test period, while in test group G2, the plaque index tended to gradually decrease, although there was no statistical significance until the end of the test (FIG. 16).

(6) Tartar index

In test group G1, the tartar index gradually increased, while in test group G2, the tartar index significantly improved with statistical significance over the 2nd and 4th weeks (Figure 17).

(7) Gingivitis index

There was no significant change in the gingivitis index in test group G1, but in test group G2, the gingivitis index decreased statistically significantly from week 2 to week 4 (Figure 18).

(8) Considerations

As a result of applying the test substance, a mixture of algae extract and SOD and spores, to the cat's gums at a dose of 0.3 g/head twice daily, palpable pain index and bad breath were observed from the 2nd to the 4th week after administration compared to the 0th week. It was confirmed that the index, plaque index, tartar index, and gingivitis index all showed a decreasing trend. In particular, symptoms of palpable pain, bad breath, tartar, and gingivitis were statistically significantly improved compared to week 0. In addition, no special adverse reactions occurred during the entire test process, confirming that the test substance was not harmful.

Judging based on the above results, the test substances, a mixture of fir extract and SOD with spores, are not only useful in improving oral diseases such as bad breath, plaque, tartar, and gingivitis, but also reduce pain caused by such oral diseases. It was also found to be useful.

Claims (23)

(i) Aucuba japonica extract, and
(ii) containing as an active ingredient at least one selected from the group consisting of superoxide dismutase (SOD) enzyme and Bacillus species strain spores
An oral composition for preventing or improving one or more diseases selected from oral pain, bad breath, tartar, plaque, and periodontal disease,
The SOD is derived from a Bacillus species strain.
Oral composition. According to paragraph 1,
The composition comprises (i) a carnivore extract, and (ii) SOD and Bacillus species strain spores.
Composition. According to paragraph 1,
The Chinese oak extract is a hot water extract.
Composition. According to paragraph 1,
The Chinese oak extract is extracted from the leaves, stems, or mixtures thereof.
Composition. delete According to paragraph 1,
The Bacillus species strain includes Bacillus amyloliquefaciens GF423 strain (KCTC 13222 BP) or GF424 strain (KCTC 13227 BP).
Composition. According to paragraph 1,
The SOD includes Mn-SOD or deamidated Mn-SOD.
Composition. According to paragraph 1,
The SOD contains the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
Composition. According to paragraph 1,
The Bacillus species strain spores include spores of the Bacillus amyloliquefaciens GF423 strain (KCTC 13222 BP) or spores of the GF424 strain (KCTC 13227 BP).
Composition. According to paragraph 1,
The composition is used for oral health promotion or oral hygiene management.
Composition. delete According to paragraph 1,
The composition is in gel or paste form.
Composition. Comprising the composition of any one of claims 1 to 4, 6 to 10, and 12.
Oral products. According to clause 13,
The product is any one selected from toothpaste, mouthwash, mouthwash, gum, candy, oral spray, oral gel, oral ointment, oral patch, and oral rinse.
product. Comprising the composition of any one of claims 1 to 4, 6 to 10, and 12.
A pharmaceutical composition for the prevention or treatment of one or more oral diseases selected from oral pain, bad breath, tartar, plaque, and periodontal disease. Comprising the composition of any one of claims 1 to 4, 6 to 10, and 12.
A veterinary composition for the prevention or treatment of one or more oral diseases selected from oral pain, bad breath, tartar, plaque, and periodontal disease. Comprising the composition of any one of claims 1 to 4, 6 to 10, and 12.
Food composition. Comprising the composition of any one of claims 1 to 4, 6 to 10, and 12.
Feed composition. (i) carob tree extract, and
(ii) administering to a non-human subject at least one selected from the group consisting of superoxide dismutase (SOD) enzyme and Bacillus species strain spores.
A method for preventing or treating one or more oral diseases selected from oral pain, bad breath, tartar, plaque, and periodontal disease, comprising:
The SOD is derived from a Bacillus species strain.
method. delete According to clause 19,
Comprising the step of administering (i) a carnivorous tree extract and (ii) SOD and Bacillus species strain spores to an individual.
method. According to clause 21,
The cypress extract, SOD, and Bacillus species strain spores are administered as one composition or as separate compositions containing each or both components.
method. According to clause 21,
The components of (i) and (ii) are administered simultaneously or in combination sequentially or in reverse order.
method.