Composition comprising lycii radicis cortex extract for preventing, alleviating, or treating periodontal disease

Abstract

The present invention relates to a composition comprising a Lycii radicis cortex extract for preventing, alleviating, or treating periodontal disease. The composition of the present invention exhibits antioxidant, antibacterial, anti-inflammatory, and periodontal muscle protection activities against periodontal diseases, and has effects of preventing, alleviating, or treating periodontal diseases through periodontal bone loss inhibition and gum tissue protection.　

Description

Composition for preventing, improving or treating periodontal disease comprising extract of Ginkgo biloba

[0001] This application claims priority to Republic of Korea Application No. 10-2023-0122083, filed September 13, 2023, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a composition for preventing, improving or treating periodontal disease, comprising a natural extract of Ginkgo biloba as an active ingredient.

[0003] Periodontal disease (PD) is a chronic inflammatory disease characterized by destruction of the tooth-supporting connective tissue and cementum, bone resorption, leukocyte infiltration, and periodontal pocket formation. Periodontal disease, a common cause of tooth loss in adults, is characterized by localized bone resorption. The presence of bacterial plaque has been considered a contributing factor to the pathogenesis of associated periodontal disease and can induce a local inflammatory response. This inflammatory response leads to edema, leukocyte infiltration, and the release of inflammatory mediators, which in turn trigger periodontal pocket formation, connective tissue separation, and alveolar bone resorption, ultimately leading to tooth loss. Recently, nitric oxide activation and oxidative stress have been implicated in the pathogenesis of periodontitis, and many antioxidants have shown beneficial effects on periodontitis and associated alveolar bone loss.

[0004] Currently, periodontitis is treated by scaling to remove plaque and tartar, or by resecting the gums to reduce the depth of periodontal pockets between the teeth and gums. In severe cases, antibiotics may be prescribed, or antibiotic ointments may be injected into the periodontal pockets between the teeth and gums. However, due to the problems associated with antibiotic overuse and the side effects associated with chemical prescriptions, efforts are being made to develop various alternative therapies derived from natural products with relatively fewer side effects. In particular, natural ingredients contain various phenolic compounds, vitamins, carotenoids, and flavonoids, which are known to exhibit various pharmacological effects, including immunomodulatory and anti-inflammatory effects via antioxidant mechanisms. In this context, recent efforts are focused on finding natural treatments for periodontal disease with relatively low side effects and relatively potent anti-inflammatory effects. Currently, various natural products are known to modulate the body's immune system and exhibit antioxidant and anti-inflammatory effects. In particular, various natural extracts have been reported to have therapeutic or inhibitory effects on periodontal disease and inflammation.

[0005] Numerous references and citations are provided throughout this specification. The disclosures of these references are incorporated herein by reference in their entirety to further clarify the state of the art and the scope of the present invention.

[0006] The present inventors have conducted research efforts to develop a natural product that has no side effects and exhibits effects in preventing, improving, and treating periodontal diseases such as gingivitis and periodontitis, and as a result, have discovered that the extract of Golpi has excellent effects in preventing, improving, or treating periodontal diseases, thereby completing the present invention.

[0007] Accordingly, the purpose of the present invention is to provide the following implementation examples.

[0008] Embodiment example 1. A composition for preventing, improving, or treating periodontal disease comprising a rehmannia root extract as an active ingredient; use of a rehmannia root extract or a composition comprising a rehmannia root extract as an active ingredient for manufacturing a pharmaceutical, quasi-drug, food, health functional food, toothpaste, product, veterinary drug, or veterinary product for preventing, improving, or treating periodontal disease; or a method for preventing, improving, or treating periodontal disease comprising administering a rehmannia root extract or a composition comprising a rehmannia root extract as an active ingredient to a subject in need thereof.

[0009] Embodiment 2. A composition; application; or method according to Embodiment 1, characterized in that the extract is extracted using any one selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixtures thereof as an extraction solvent.

[0010] Embodiment 3. In any one of the preceding embodiments, the extract is a composition; application; or method characterized in that the extract is obtained by first extracting the raw material by grinding the raw material and then extracting it at 85±5°C for 8±1 hours using 30 to 50% of the raw material as an extraction solvent in an amount of 10 times the raw material, or by mixing the first extract with a second extract obtained by extracting it at 85±5°C for 4±1 hours using 30 to 50% of the raw material as an extraction solvent in an amount of 8 times the raw material, and drying the mixture.

[0011] Embodiment 4. A composition; application; or method according to any one of the preceding embodiments, wherein the periodontal disease is gingivitis or periodontitis.

[0012] Embodiment 5. A composition; application; or method according to any one of the preceding embodiments, wherein the prevention, improvement, or treatment of periodontal disease is achieved through a complex mechanism through antioxidant, antibacterial, anti-inflammatory, and periodontal root protection activities.

[0013] Embodiment 6. In any of the preceding embodiments, the composition comprising the extract of the root ...

[0014] Embodiment 7. In any one of the preceding embodiments, the composition comprising the extract of the root ...

[0015] Embodiment 8. A composition; application; or method according to any one of the preceding embodiments, characterized in that the composition is a food composition for preventing or improving periodontal disease.

[0016] Embodiment 9. A composition; application; or method according to any of the preceding embodiments, wherein the composition is a pharmaceutical composition.

[0017] Embodiment 10. A composition; application; or method according to any one of the preceding embodiments, characterized in that the composition is a health functional food composition for preventing or improving periodontal disease.

[0018] Embodiment 11. A composition; application; or method according to any one of the preceding embodiments, wherein the composition is a toothpaste composition.

[0019] Embodiment 12. A composition; application; or method according to any one of the preceding embodiments, characterized in that the composition is for improving gum health in an animal.

[0020] Embodiment 13. A pharmaceutical, quasi-drug, food, health functional food, toothpaste, product, veterinary drug, or veterinary product comprising a composition comprising a Ginkgo biloba extract or Ginkgo biloba extract as an active ingredient as described in any of the preceding embodiments.

[0021]

[0022] One aspect of the present invention is to provide a composition for preventing, improving or treating periodontal disease, comprising an extract of G. chinensis as an active ingredient.

[0023] Lycium chinense (Lycii Radicis Cortex, root bark of Lycium chinense Mill.) is the root bark of the wolfberry, a deciduous small tree in the Solanaceae family. The dried fruit of the wolfberry is called wolfberry, and the dried root bark is called jigolpi. Jigolpi is non-toxic and has a mild flavor, and is known to exert its effects by entering the lungs, liver, and nerves. In oriental medicine, Jigolpi is mainly used as a tonic and antipyretic to treat pulmonary tuberculosis and diabetes.

[0024] The composition of the present invention includes an extract of Ginkgo biloba as an effective ingredient. The term 'extract' used in this specification includes an extraction result obtained by juicing a raw material or treating a raw material with an extraction solvent, or a processed product formulated (e.g., powdered) thereof.

[0025] When the extract used in the composition of the present invention is obtained by treating the raw material with an extraction solvent, various extraction solvents can be used, for example, a polar solvent or a non-polar solvent can be used. Polar solvents include (i) water, (ii) alcohol (preferably, methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol, normal-butanol, 1-pentanol, 2-butoxyethanol or ethylene glycol), (iii) acetic acid, (iv) DMF (dimethyl-formamide) and (v) DMSO (dimethyl sulfoxide), and non-polar solvents include acetone, acetonitrile, ethyl acetate, methyl acetate, fluoroalkane, pentane, hexane, 2,2,4-trimethylpentane, decane, cyclohexane, cyclopentane, diisobutylene, 1-pentene, 1-chlorobutane, 1-chloropentane, o-xylene, diisopropyl ether, 2-chloropropane, toluene, 1-chloropropane, Chlorobenzene, benzene, diethyl ether, diethyl sulfide, chloroform, dichloromethane, 1,2-dichloroethane, aniline, diethylamine, ether, carbon tetrachloride, and THF can also be used.

[0026] Preferably, the extract used in the present invention is one obtained by extracting using any one selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixtures thereof as an extraction solvent.

[0027] In addition, the term 'extract' used in this specification has the meaning commonly used in the art as a crude extract as described above, but in a broad sense, it also includes a fraction obtained by further fractionating an extract. That is, it includes not only an extract obtained by juicing the raw material or using the above-mentioned extraction solvent, but also one obtained by additionally applying a purification process thereto. For example, a fraction obtained by passing the extract through an ultrafiltration membrane having a certain molecular weight cut-off value, separation by various chromatographies (designed for separation according to size, charge, hydrophobicity, or affinity), supplementation of a specific component, etc., fractions obtained through various additional purification methods, and products obtained through the addition of a specific component are also included in the extract of the present invention.

[0028] In addition, the extract of the present invention may be obtained by removing the solvent through an additional process, such as filtration, concentration, or drying, or by performing all of filtration, concentration, and drying. Filtration may be performed, for example, using filter paper or a vacuum filter, concentration may be performed using a vacuum concentrator, and drying may be performed using spray drying or freeze drying to obtain a powdered extract.

[0029] In a preferred embodiment, the extract of the present invention may be obtained by first extracting the raw material by grinding it and then extracting it at 85±5°C for 8±1 hours using 30 to 50% of the raw material as an extraction solvent in an amount of 10 times that of the raw material, or by mixing the first extract with a second extract obtained by extracting it at 85±5°C for 4±1 hours using 30 to 50% of the raw material as an extraction solvent in an amount of 8 times that of the raw material, and drying the mixture.

[0030] In the composition of the present invention, the active ingredient, the extract of the root ...

[0031] In one embodiment, the extract of the present invention or a composition comprising the extract of the present invention as an active ingredient may contain 4 to 20 mg / g of Kukoamine A and 8 to 40 mg / g of Kukoamine B.

[0032] In another embodiment, the extract of the present invention or a composition comprising the extract of the present invention as an active ingredient may contain 15.0 to 32 mg / g of Kukoamine B.

[0033] The above-mentioned effective ingredient may be included in an amount of 0.1 to 5% by volume based on the total volume of the composition of the present invention, and the prevention, improvement, or treatment of periodontal disease may be achieved through a complex mechanism through antioxidant, antibacterial, anti-inflammatory, and periodontal root protection activities. In a preferred embodiment, the periodontal disease is gingivitis or periodontitis.

[0034] In one embodiment, the composition of the present invention may be a food composition. The food composition of the present invention includes processed forms of all natural materials, such as foods, functional foods, nutritional supplements, health foods, and food additives. The above-mentioned types of food compositions can be prepared in various forms using conventional methods known in the art.

[0035] For example, as a health food, the extract of the present invention itself can be manufactured in the form of tea, juice, and drink and consumed, or can be granulated, encapsulated, or powdered and consumed. In addition, in addition to the extract of the present invention, white peony root, cornelian cherry, scutellaria baicalensis, reishi mushroom, tangerine peel, ginseng root, angelica root, gardenia fruit, astragalus membranaceus, malt, tangerine seed, vitamin C, fructooligosaccharide, stevioside, purified water, maltodextrin, etc. can be further included alone or in mixture within a range that does not inhibit the purpose of the present invention; however, other medicinal ingredients and / or additives that the food composition of the present invention can additionally include are not limited to the above examples.

[0036] For example, the food composition according to the present invention may include water-soluble vitamins such as thiamine (vitamin B1), riboflavin, ascorbic acid, niacin, and vitamin B6; fatty acids such as myristic acid, palmitic acid, stearic acid, oleic acid, and linoleic acid; weak acids such as glycolic acid and acetic acid; and amino acids such as eight essential amino acids, threonine, valine, methionine, isoleucine, leucine, phenylalanine, tryptophan, and lysine, as well as aspartic acid, serine, glutamic acid, proline, glycine, alanine, cysteine, tyrosine, histidine, and arginine.

[0037] In another embodiment, the composition of the present invention may be a pharmaceutical composition. The pharmaceutical composition may include the above-described active ingredient and may be formulated into a pharmaceutical unit dosage form by adding a pharmaceutically acceptable carrier, excipient, or diluent.

[0038] The above “pharmaceutically acceptable” refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not typically cause allergic reactions such as gastrointestinal upset, dizziness, or similar reactions.

[0039] Examples of the carrier, excipient or diluent may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition may further include a filler, an anticoagulant, a lubricant, a wetting agent, a fragrance, an emulsifier or a preservative.

[0040] The term "pharmaceutically effective amount" refers to an amount that produces a response greater than that of a negative control, and preferably an amount sufficient to produce an effect in the prevention and / or treatment of periodontal disease.

[0041] Additionally, the pharmaceutical composition of the present invention can be formulated using methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to a mammal. The formulation may be, for example, a formulation selected from the group consisting of gels, pastes, ointments, powders, emulsions, sprays, and aerosols, but is not limited thereto.

[0042] In addition, the pharmaceutical composition of the present invention can be administered in combination with a known compound having a preventive and / or therapeutic effect on periodontal disease.

[0043] In another embodiment, the composition of the present invention may be a toothpaste composition.

[0044] The toothpaste composition of the present invention may further include other additives commonly used in toothpaste compositions in the art, depending on the formulation and intended use, as long as they do not impede the purpose of the present invention. Such other additives may further include, for example, abrasives, flavoring agents, sweeteners, medicinal agents, pigments, pH adjusters, preservatives, and whitening agents.

[0045] In addition, the composition of the present invention can be a composition for improving gum health in animals as well as humans.

[0046] All ingredients described in the present invention preferably do not exceed the maximum usage levels stipulated in relevant laws and regulations of Korea, China, the United States, Europe, Japan, etc. (e.g., Regulations on Cosmetic Safety Standards (Korea), Cosmetic Safety Technical Standards (China), Food Code (Korea), Food Additives Code (Korea), Health Functional Food Code (Korea)). That is, preferably, the cosmetic, food, or personal care composition according to the present invention contains the ingredients according to the present invention in the content limits permitted by relevant laws and regulations of each country.

[0047]

[0048] The composition of the present invention exhibits antioxidant, antibacterial, anti-inflammatory and periodontal root protection activities against periodontal disease, and exhibits effects of preventing, improving or treating periodontal disease through inhibition of periodontal bone loss and protection of gum tissue.

[0049]

[0050] Figure 1 shows the results of analyzing the composition of cucurbita lucidum B in the extract of Ginkgo biloba using the HPLC method.

[0051] Figure 2 shows the results of analyzing the composition of cucurbita lucidum A in the extract of Ginkgo biloba using the HPLC method.

[0052] Figure 3 shows the results of comparing the NO production inhibition ability according to the weight ratio of cucurbitamine A and B in the extract of Ginkgo biloba.

[0053] Figure 4 shows the results of comparing the PGE2 production inhibition ability according to the weight ratio of cucurbita lucidum extract and cucurbita lucidum extract.

[0054] Figure 5 shows the results of comparing the changes in periodontal bone loss rate measured using a ligation EPD rat model.

[0055] Figure 6 shows the results of comparing the changes in the appearance and volume of periodontal bone observed using a ligation EPD rat model.

[0056] Figure 7 shows the results of comparing the numerical changes in infiltrating inflammatory cells in gum tissue measured using a ligation EPD rat model.

[0057] Figure 8 shows the results of comparing the oral anaerobic bacteria inhibition activity measured using a ligation EPD rat model.

[0058] Figure 9 shows the results of comparing the changes in MDA content in gingival tissue measured using a ligation EPD rat model.

[0059] Figure 10 shows the results of comparing MMP-8 inhibitory activity in gingival tissue measured using a ligation EPD rat model.

[0060]

[0061] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0062]

[0063] Example

[0064] I. Extraction of the root of the plant and confirmation of the optimal composition

[0065] 500 g of Lycii Radicis Cortex (Root bark of Lycium chinense Mill.) purchased from a herbal medicine store was prepared and ground, and then extracted at 85℃ for 8 hours using 10 times the amount of 30% alcohol compared to the original material as a solvent, and then filtered to obtain the first extract (recovery rate 15.74%). Using 7 times the amount of 30% alcohol compared to the first extract, the extraction was performed at 85℃ for 4 hours, and then filtered to obtain the second extract (recovery rate 6.49%). Afterwards, it was concentrated to 15-20 brix at 60-65℃, and then spray dried under the conditions of inlet 165℃, outlet 80℃ to obtain brown powders.

[0066] Among the various active substances contained in the extract of Ginkgo biloba, Kukoamine A and B are included. For the analysis of Kukoamine B contained in the extract, 2.1 mg of Kukoamine B standard was weighed and added to 50% methanol (containing 0.5% acetic acid) to make 20 mL to prepare a standard. Approximately 200 mL of Ginkgo biloba extract powder and approximately 1 g of concentrate were weighed and added to 50% methanol (containing 0.5% acetic acid) to make 20 mL (ultrasonic extraction for 30 min), filtered, and used as samples. The HPLC conditions for the analysis of Kukoamine B are shown in Table 1 below.

[0067] [Table 1]

[0068] [Correction pursuant to Rule 91, November 20, 2024]

[0069]

[0070] In addition, for the analysis of Kukoamine A contained in the extract, 1.4 mg of Kukoamine A standard was prepared by adding 40% methanol (containing 0.5% acetic acid) to make 25 mL, and approximately 199.7 mg of the extract powder of Rehmannia glutinosa was prepared by adding 40% methanol (containing 0.5% acetic acid) to make 25 mL (ultrasonic extraction for 30 min), filtering, and then used as a sample. The HPLC conditions for the analysis of Kukoamine A are shown in Table 2 below.

[0071] [Table 2]

[0072]

[0073]

[0074] In order to secure the optimal composition of the extract of Ginkgo biloba, a composition was prepared by combining cucurbita lucidum A (KA) and cucurbita lucidum B (KB) in weight ratios of 2:1, 1:1, 1:2, and 1:3, respectively, using column chromatography, and the oxidative stress inhibition activity through NO inhibition and the anti-inflammatory activity through PGE2 inhibition were evaluated as follows.

[0075] (1) Cell culture

[0076] Human keratinocyte cell line (HaCaT), human (neonatal) dermal fibroblast cell line (HDFn), and mouse macrophage-derived cell line (Raw264.7) were each supplied by ATCC (Rockville, MD, USA). HaCaT cells were cultured in Dulbecco's modified Eagle's medium (DMEM; HyClone Laboratories, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Lonza, Walkersville, MD, USA), 100 μg / mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 100 U / mL penicillin (Sigma-Aldrich). HDFn cells were cultured in fibroblast basal medium (FBM; ATCC, PCS-201-030) and the FBM low serum kit (ATCC, PCS-201-041). Raw264.7 cells were cultured in DMEM supplemented with 10% FBS, 50 units / mL penicillin, and 50 μg / mL streptomycin. All cells were cultured at 37°C in 5% CO2.

[0077] (2) Measurement of nitric oxide production

[0078] Raw264.7 cells were pretreated with 0.3 to 3 mg / mL of LRC or 1 μM of Kukoamine A, B and a 1:2 composition and DEXA (Dexamethasone) for 1 hour, and then treated with 1 μg / mL of LPS for 18 hours. An equal volume of Griess reagent was added to the recovered culture medium, and the absorbance was measured at 540 nm using an automated microplate reader to evaluate the amount of nitric oxide production, and the results are shown in Fig. 3. The experimental results showed that when Kukoamine A (KA) and Kukoamine B (KB) were included in a weight ratio of 1:2 (KA:KB), they exhibited a significantly superior effect on reducing NO production within the cells compared to when they were included in other weight ratios.

[0079] (3) Measurement of PGE2 content

[0080] The PGE2 content in the cell culture medium was measured using a prostaglandin E2 parameter analysis kit. Briefly, a 96-well plate coated with goat anti-mouse PGE2 antibody was reacted with cell culture medium and primary antibody, and then additionally reacted with horseradish peroxidase-conjugated PGE2. The 96 wells were washed, reacted with 3,3',5,5'-tetramethylbenzidine (TMB) to develop color, and the reaction was stopped with sulfuric acid solution. The absorbance of the reaction solution was measured at a wavelength of 450 nm using an automated microplate reader, and the results are shown in Figure 4. The experimental results showed that when cucurbit A (KA) and cucurbit B (KB) were included in a weight ratio of 1:2 (KA:KB), they exhibited significantly superior anti-inflammatory activity through PGE2 inhibition in cells compared to when they were included in other weight ratios.

[0081] Accordingly, an additional in vivo efficacy evaluation was performed using the 1:2 complex (a licorice extract containing KA and KB in a weight ratio of 1:2 (KA:KB)) (hereinafter referred to as LRC), which was confirmed to have the most remarkable effect in oxidative stress inhibition activity and anti-inflammatory activity.

[0082]

[0083] II. In vivo efficacy evaluation

[0084] 1. Laboratory animals

[0085] A total of 77 SPF / VAF Outbred Crl:CD [SD] male rats (OrientBio, Seungnam, Korea) were obtained and acclimatized for 9 days. After that, experimental animals with a constant body weight were selected and divided into 7 groups of 10 rats per group (normal medium control group: average 282.90±13.08 g, 262.00–302.00 g; EPD-induced group: average 282.87±12.97 g, 254.00–312.00 g). This animal experiment was conducted with prior approval from the Animal Experiment Ethics Committee of Daegu Haany University.

[0086]

[0087] Military detachments (7 groups total; 10 horses per group):

[0088] 1. Normal control group (medium control group without dental cervical ligation; oral administration of sterile distilled water)

[0089] 2. EPD (Experimental periodontitis diseases) control group (cervical ligation medium control group; oral administration of sterile distilled water)

[0090] 3. IND (Indomethacin) administered EPD control drug group (group administered 5 mg / kg of IND orally after cervical ligation)

[0091] 4. INP (Insadol Plus TM) EPD control drug group (63 mg / kg (0.60 tablet / kg) oral administration group as the total amount of INP active ingredient after cervical ligation)

[0092] 5. LRC high-dose EPD experimental group (200 mg / kg LRC oral administration group after cervical ligation)

[0093] 6. LRC intermediate dose EPD experimental group (group administered LRC 100 mg / kg orally after cervical ligation)

[0094] 7. LRC low-dose EPD experimental group (50 mg / kg LRC oral administration group after cervical ligation)

[0095]

[0096] 2. Experimental method

[0097] (1) Cause of periodontal disease

[0098] According to the previous methods of researchers et al. [Lee et al., 2014; Park et al., 2016b; Kim et al., 2022], the experimental animals were acclimated to the laboratory environment for 9 days, and then whole body anesthesia was performed with a mixture of 3% isoflurane (Hana Pharm. Co., Hwasung, Korea), 70% N2O, and 28.5% O2 using a rodent inhalation anesthesia machine (Surgivet, Waukesha, WI, USA) and a ventilator (Model 687, Harvard Apparatus, Cambridge, UK). Periodontitis and gingival disease were induced by ligation of the left maxillary incisor neck using a 3-0 nylon suture. Meanwhile, in the normal medium control group, only the tooth neck of the maxillary incisor was checked and ligation was not performed.

[0099] (2) Dose Frequency

[0100] The highest dose of LRC, 200 mg / kg, was established based on the efficacy exploration experiment of a single dose of LRC (200 mg / kg) using a previous dental cervical ligation EPD rat model by our researchers, and 100 mg / kg and 50 mg / kg were established as the intermediate and low doses, respectively, with a ratio of 2. In addition, the administration dose of IND was also set to 5 mg / kg based on the results of efficacy evaluation experiments in previous EPD models [Ku et al., 2011; Kim et al., 2012; Park et al., 2016b; Kim et al., 2022], and the administration dose of INP was selected as 63 mg / kg (0.60 tablet / kg) as the total amount of active ingredients based on the clinical application dose and the body surface area ratio of the rat (1 / 6). LRC was dissolved in sterile distilled water at concentrations of 40, 20, and 10 mg / ml, and administered orally once daily for 10 days, starting 24 hours after dental cervical ligation, at a dose of 5 ml / kg (200, 100, and 50 mg / kg, respectively), using a metal sonde attached to a 5 ml syringe. IND was also dissolved in sterile distilled water at a concentration of 1 mg / ml, and administered orally once daily for 10 days, starting 24 hours after dental cervical ligation, at a dose of 5 ml / kg (5 mg / kg). In addition, the commercially available INP tablet was ground using a regular mortar and pestle, then suspended in sterile distilled water at a concentration of 12.6 mg / ml (0.12 tablet / ml) as the total amount of active ingredient, and administered orally once daily for 10 days starting 24 hours after dental cervical ligation at a dose of 5 ml / kg (total amount of active ingredient: 63 mg / kg; 0.60 tablet / kg). In the normal medium and EPD control groups, only sterile distilled water as a medium was orally administered at the same dose and frequency instead of LRC, IND, or INP to induce the same corrective stress according to oral administration.

[0101] (3) Observation items

[0102] On the final day of sacrifice, periodontal bone loss was measured, and the total number of anaerobic bacteria in the oral cavity was analyzed. In addition, some gingival tissues were excised on the final day of sacrifice, and changes in the number of infiltrating inflammatory cells and the contents of MDA and MMP-8 in the gingival tissues were observed.

[0103]

[0104] 3. Experimental results

[0105] (1) Changes in periodontal bone loss rate

[0106] In the case of the EPD control group, a significant (p<0.01) increase in the exposed area of ​​the incisor root due to periodontal bone loss, i.e., an increase in the periodontal bone loss rate, was observed compared to the normal medium control group. However, a significant (p<0.01) decrease in the periodontal bone loss rate was observed in a dose-dependent manner in all three doses of LRC 200, 100, and 50 mg / kg compared to the EPD control group. In particular, LRC 200 mg / kg and LRC 50 mg / kg showed inhibitory activity against the increase in the periodontal bone loss rate associated with EPD due to tooth neck ligation comparable to IND 5 mg / kg and INP 50 mg / kg, respectively, as a total amount of the active ingredient (Figs. 5 and 6). The periodontal bone loss rate showed a change of 249.24% in the EPD control group compared to the normal medium control group, but in the groups administered 5 mg / kg of IND, 63 mg / kg of the total amount of INP active ingredient, and 200, 100, and 50 mg / kg of LRC, changes of -34.00, -22.36, -34.91, -29.90, and -23.15%, respectively, compared to the EPD control group.

[0107]

[0108] (2) Numerical changes in infiltrating inflammatory cells in gingival tissue

[0109] In the control group, a significant increase (p<0.01) in the number of inflammatory cells infiltrating the gingival tissue was observed compared to the normal medium control group, but a significant decrease (p<0.01) in the number of inflammatory cells infiltrating the gingival tissue was observed in a dose-dependent manner in all three doses of LRC 200, 100, and 50 mg / kg groups compared to the EPD control group. In particular, LRC 200 mg / kg and LRC 50 mg / kg showed inhibitory activity against inflammatory cell infiltration into the gingival tissue related to EPD by tooth neck ligation comparable to IND 5 mg / kg and INP 50 mg / kg, respectively, as a total amount of effective ingredients (Fig. 7). The number of infiltrating inflammatory cells in the gingival tissue showed a change of 1168.60% in the EPD control group compared to the normal medium control group, but in the groups administered 5 mg / kg of IND, 63 mg / kg of the total amount of INP active ingredient, and 200, 100, and 50 mg / kg of LRC, changes of -65.38, -28.33, -67.42, -47.83, and -29.39%, respectively, compared to the EPD control group.

[0110]

[0111] (3) Changes in the total number of anaerobic bacteria in the oral cavity

[0112] In the EPD control group, a significant increase (p<0.01) in the total number of anaerobic bacteria in the oral buccal tissue was observed compared to the normal medium control group, but a significant decrease (p<0.01) in the number of anaerobic bacteria in the oral buccal tissue compared to the EPD control group was observed in a dose-dependent manner in all three doses of LRC 200, 100, and 50 mg / kg groups, and in particular, LRC 50 mg / kg showed an inhibitory activity against the increase in the total number of anaerobic bacteria in the oral buccal tissue related to EPD caused by dental neck ligation comparable to 63 mg / kg as the total amount of INP active ingredient, but in the IND 5 mg / kg group, no significant change in the total number of anaerobic bacteria in the oral buccal tissue was observed compared to the EPD control group (Fig. 8). The total number of oral anaerobic bacteria in the EPD control group showed a change of 819.15% compared to the normal medium control group, but in the groups administered 5 mg / kg of IND, 63 mg / kg of the total amount of INP active ingredient, and 200, 100, and 50 mg / kg of LRC, the changes were -3.01, -27.31, -44.68, -38.19, and -28.94% compared to the EPD control group, respectively.

[0113]

[0114] (4) Changes in MDA content in gingival tissue

[0115] In the case of the EPD control group, a significant increase (p<0.01) in lipid peroxidation and MDA content in the gingival tissue was observed compared to the normal medium control group, but a significant decrease (p<0.01 or p<0.05) in MDA content was observed in a dose-dependent manner in all three doses of LRC 200, 100, and 50 mg / kg groups compared to the EPD control group. In particular, LRC 200 mg / kg and LRC 50 mg / kg showed inhibitory activity against the increase in MDA content in the gingival tissue related to EPD caused by tooth neck ligation comparable to IND 5 mg / kg and INP 50 mg / kg, respectively, as a total amount of effective ingredients (Fig. 9). The MDA content in the gingival tissue showed a change of 433.28% in the EPD control group compared to the normal medium control group, but in the groups administered IND 5 mg / kg, INP 63 mg / kg as the total amount of effective ingredient, and LRC 200, 100, and 50 mg / kg, the changes were -53.46, -29.73, -55.02, -42.31, and -30.53%, respectively, compared to the EPD control group.

[0116]

[0117] (5) Changes in MMP-8 content in gingival tissue

[0118] In the case of the EPD control group, a significant increase (p<0.01) in the MMP-8 content in the gingival tissue was observed compared to the normal medium control group, but a significant decrease in the MMP-8 content compared to the EPD control group was observed in a dose-dependent manner in all three doses of LRC 200, 100, and 50 mg / kg, respectively. In particular, LRC 200 mg / kg and LRC 50 mg / kg showed inhibitory activity against the increase in the MMP-8 content in the gingival tissue related to EPD caused by tooth neck ligation comparable to IND 5 mg / kg and INP 50 mg / kg, respectively, as a total amount of the active ingredient (Fig. 10). The MMP-8 content in the gingival tissue showed a change of 94.44% in the EPD control group compared to the normal medium control group, but in the groups administered 5 mg / kg of IND, 63 mg / kg of the total amount of INP active ingredient, and 200, 100, and 50 mg / kg of LRC, changes of -36.28, -23.03, -37.71, -29.56, and -23.24%, respectively, compared to the EPD control group.

[0119]

[0120] As described above, the efficacy of the extract of Golpi rhizome was comparatively evaluated using the EPD rat model of dental cervical ligation for periodontitis and periodontal bone loss, and it was confirmed that the extract of Golpi rhizome has the efficacy as a natural effective ingredient for improving periodontitis and periodontal bone damage.

Claims

1. A composition for preventing, improving or treating periodontal disease, comprising an extract of Golpi rhizome as an active ingredient.

2. A composition according to claim 1, characterized in that the extract is extracted using any one selected from the group consisting of water, lower alcohols having 1 to 4 carbon atoms, and mixtures thereof as an extraction solvent.

3. In the first paragraph, the extract is a composition characterized in that the extract is first extracted at 85±5℃ for 8±1 hours using 30 to 50% of the original amount as an extraction solvent after crushing the original material, or the first extract is mixed with a second extract obtained by extracting at 85±5℃ for 4±1 hours using 30 to 50% of the original amount as an extraction solvent and drying the first extract.

4. A composition according to claim 1, characterized in that the periodontal disease is gingivitis or periodontitis.

5. A composition according to claim 1, characterized in that the prevention, improvement or treatment of periodontal disease is achieved through a complex mechanism through antioxidant, antibacterial, anti-inflammatory and periodontal root protection activities.

6. A composition according to claim 1, wherein the extract of the root of the plant contains 4 to 20 mg / g of Kukoamine A and 8 to 40 mg / g of Kukoamine B, or contains 15 to 32 mg / g of Kukoamine B.

7. A composition according to claim 1, wherein the extract of the root of the plant contains Kukoamine A (KA) and Kukoamine B (KA) in a weight ratio of 1:2±0.2 (KA:KB).

8. A composition according to any one of claims 1 to 7, characterized in that the composition is a food composition for preventing or improving periodontal disease.

9. A composition according to any one of claims 1 to 7, characterized in that the composition is a pharmaceutical composition.

10. A composition according to any one of claims 1 to 7, characterized in that the composition is a health functional food composition for preventing or improving periodontal disease.

11. A composition according to any one of claims 1 to 7, characterized in that the composition is a toothpaste composition.

12. A composition according to any one of claims 1 to 7, characterized in that the composition is for improving the gum health of an animal.

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