WO2025058370A1 - Composition comprising lycii radicis cortex extract for preventing, alleviating, or treating periodontal disease - Google Patents

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 containing extract of Golpi

This application claims priority to Republic of Korea Application No. 10-2023-0122083, filed September 13, 2023, the entire contents of which are incorporated herein by reference.

The present invention relates to a composition for preventing, improving or treating periodontal disease, comprising a natural extract, a licorice root extract, as an effective ingredient.

Periodontal disease is a chronic inflammatory disease characterized by destruction of the supporting connective tissue and cementum, bone resorption, leukocyte infiltration, and periodontal pocket formation. Periodontal disease, a related cause of tooth loss in adults, is characterized by localized bone resorption. The presence of bacterial plaque has been considered as a pathogenesis of associated periodontal disease, which can induce a local inflammatory response. This inflammatory response leads to edema, leukocyte infiltration, and release of inflammatory mediators, which induce periodontal pocket formation, connective tissue separation, and alveolar bone resorption, ultimately resulting in tooth loss. Recently, nitric oxide activation and oxidative stress have been shown to be involved in the pathogenesis of periodontitis, and many antioxidants have shown beneficial effects on periodontitis and associated alveolar bone loss.

Currently, periodontitis is treated by scaling to remove plaque and tartar, or by excising the gums to reduce the depth of the periodontal pockets between the teeth and gums. If periodontitis is severe, antibiotics are prescribed or antibiotic ointments are inserted into the periodontal pockets between the teeth and gums. However, due to problems caused by antibiotic abuse and side effects caused by chemical drug prescriptions, attempts are being made to develop various alternative therapies derived from natural products that are expected to have relatively low side effects. In particular, natural products contain various phenolic compounds, vitamins, carotenoids, and flavonoids, and are known to exhibit various pharmacological actions, including immune regulation and anti-inflammatory effects through antioxidant mechanisms. In this context, efforts are being made to find periodontal disease treatments with low side effects and relatively strong anti-inflammatory effects from natural products. Currently, various natural product preparations are known to regulate the body's immune system and exhibit antioxidant and anti-inflammatory effects, and in particular, various natural extracts have been reported to have therapeutic or inhibitory effects on periodontal disease and inflammation.

Throughout this specification, numerous references are made and cited. The disclosures of the cited references are incorporated herein by reference in their entirety so as to better explain the state of the art to which the present invention pertains and the content of the present invention.

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

Therefore, the purpose of the present invention is to provide the following implementation examples.

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

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.

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

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

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 via antioxidant, antibacterial, anti-inflammatory, and periodontal root protective activities.

Embodiment 6. A composition; application; or method according to any one of the preceding embodiments, wherein the extract of Ginkgo biloba or a composition comprising the extract of Ginkgo biloba as an active ingredient comprises 4 to 20 mg/g of Kukoamine A and 8 to 40 mg/g of Kukoamine B, or comprises 15 to 32 mg/g of Kukoamine B.

Embodiment 7. In any one of the preceding embodiments, the composition comprising the extract of Ginkgo biloba or the extract of Ginkgo biloba as an active ingredient comprises Kukoamine A (KA) and Kukoamine B (KA) in a weight ratio of 1:2±0.2 (KA:KB); application; or method.

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.

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

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.

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

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

Embodiment 13. A pharmaceutical, quasi-drug, food, health functional food, toothpaste, product, veterinary drug, or veterinary product comprising the extract of G. chinensis or a composition comprising the extract of G. chinensis as an active ingredient as described in any one of the preceding embodiments.

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 effective ingredient.

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 wolfberry. Lycii Radicis Cortex is non-toxic and has a sweet taste, and is known to be effective mainly by entering the lungs, liver, and nerves. In oriental medicine, Lycii Radicis is mainly used to treat pulmonary tuberculosis and diabetes as a tonic and antipyretic.

The composition of the present invention contains 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.

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 nonpolar 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 nonpolar 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.

Preferably, the extract used in the present invention 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.

In addition, the term 'extract' used in the present 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 a raw material or using the above-described 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, a fraction obtained through various additional purification methods such as separation by various chromatographies (designed for separation according to size, charge, hydrophobicity or affinity), supplementation of specific components, and a product obtained through the addition of specific components are also included in the extract of the present invention.

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. For example, filtration may be performed using a 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 powder extract.

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

In the composition of the present invention, the active ingredient, the Ginkgo biloba extract, or the composition including the Ginkgo biloba extract as an active ingredient may include Kukoamine A (KA) and Kukoamine B (KA), and when Kukoamine A (KA) and Kukoamine B (KA) are included in a weight ratio of 1:2±0.5 (KA:KB), preferably in a weight ratio of 1:2±0.2 (KA:KB), and more preferably in a weight ratio of 1:2±0.1 (KA:KB), it is characterized in that a more remarkable synergistic effect is exhibited compared to cases where they are included in other weight ratios.

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.

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.

The above 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.

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 feeds, and food additives. The food composition of the above type can be prepared in various forms according to conventional methods known in the art.

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 it 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, gardenia, astragalus membranaceus, malt, tangerine seed, vitamin C, fructooligosaccharide, stevioside, purified water, maltodextrin, and the like 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.

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.

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

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.

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 flavoring agent, an emulsifier or a preservative.

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.

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

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.

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

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, within a range that does not impede the purpose of the present invention. The other additives may further include, for example, abrasives, flavoring agents, sweeteners, medicinal agents, pigments, pH adjusters, preservatives, and whitening agents.

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

All of the ingredients described in the present invention preferably do not exceed the maximum usage levels specified in relevant laws and regulations of Korea, China, the United States, Europe, Japan, etc. (for example, 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.

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.

Figure 1 shows the results of analyzing the composition of cucurbita oleracea B in the extract of Ginkgo biloba using the HPLC method.

Figure 2 shows the results of analyzing the composition of cucurbita oleifera A in the extract of Ginkgo biloba using the HPLC method.

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

Figure 4 shows the results of comparing the PGE ₂ production inhibition ability according to the weight ratio of cucurbit A and B in the extract of Ginkgo biloba.

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

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

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

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

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

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

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain 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.

Example

I. Jigolpi Extraction and optimal composition confirmation

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

Among the various active substances, the extract of Ginkgo biloba contains Kukoamine A and B. For the analysis of Kukoamine B contained in the extract, 2.1 mg of the standard Kukoamine B was weighed and added to 50% methanol (containing 0.5% acetic acid) to make 20 mL to prepare a standard, and about 200 mL of the extract powder of Ginkgo biloba and about 1 g of the 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.

[Table 1]

[Correction under Rule 91 20.11.2024]

In addition, for the analysis of Kukoamine A contained in the extract, 1.4 mg of the standard Kukoamine A was weighed and diluted with 40% methanol (containing 0.5% acetic acid) to make a standard by diluting the volume to 25 mL, and approximately 199.7 mg of the extract powder of Ginkgo biloba was weighed and diluted with 40% methanol (containing 0.5% acetic acid) to make a standard by diluting the volume to 25 mL (ultrasonic extraction for 30 min), filtering, and using as a sample. The HPLC conditions for the analysis of Kukoamine A are shown in Table 2 below.

[Table 2]

In order to secure the optimal composition of the extract of Ginkgo biloba, column chromatography was used to prepare a composition in which cucurbita amine A (KA) and cucurbita amine B (KB) were combined in weight ratios of 2:1, 1:1, 1:2, and 1:3, respectively, and the oxidative stress inhibition activity through NO inhibition and the anti-inflammatory activity through PGE2 inhibition were evaluated as follows.

(1) Cell culture

Human-derived keratinocyte cell line (HaCaT), human (neonatal) dermal fibroblast cell line (HDFn), and mouse-derived 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% CO ₂ .

(2) Measurement of nitric oxide production

Raw264.7 cells were pretreated with 0.3 to 3 mg/mL of LRC or 1 μM Kukoamine A, B and a 1:2 composition and DEXA (Dexamethasone) for 1 hour, then treated with 1 μg/mL LPS for 18 hours. An equal volume of Griess reagent was added to the harvested 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. As a result of the experiment, it was confirmed that when Kukoamine A (KA) and Kukoamine B (KB) were included in a weight ratio of 1:2 (KA:KB), it exhibited a significantly superior effect on reducing NO production within the cells compared to when they were included in other weight ratios.

(3) Measurement of PGE ₂ content

The PGE ₂ 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 PGE ₂ antibody was reacted with the cell culture medium and the primary antibody, and then additionally reacted with horseradish peroxidase-conjugated PGE _2. After washing the 96 wells, the color was developed by reacting with 3,3',5,5'-tetramethylbenzidine (TMB), 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 Fig. 4. As a result of the experiment, it was confirmed that when cucurbit A (KA) and cucurbit B (KB) were included in a weight ratio of 1:2 (KA:KB), it exhibited significantly superior anti-inflammatory activity through PGE ₂ inhibition in cells compared to when they were included in other weight ratios.

Thus, the 1:2 complex (a lily of the valley 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, was used to conduct additional in vivo efficacy evaluation.

II. In Vivo ( in vivo ) Efficacy evaluation

1. Laboratory animals

A total of 77 SPF/VAF Outbred Crl:CD [SD] male rats (OrientBio, Seungnam, Korea) were obtained and, after 9 days of acclimatization, experimental animals with constant body weight were selected and divided into 7 groups with 10 animals per group (normal medium control group: mean 282.90±13.08 g, 262.00 ~ 302.00 g; EPD-induced group: mean 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.

Military detachments (7 total; 10 horses per group):

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

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

3. IND (Indomethacin) administered EPD control drug group (IND oral administration group of 5 mg/kg after dental cervical ligation)

4. INP (Insadol Plus ^TM ) administered EPD control drug group (Oral administration of 63 mg/kg (0.60 tablet/kg) of total INP effective ingredient after cervical ligation)

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

6. LRC intermediate dose EPD experimental group (after cervical ligation, LRC 100 mg/kg oral administration group)

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

2. Experimental method

(1) Cause of periodontal disease

According to the previous method of researchers et al. [Lee et al., 2014; Park et al., 2016b; Kim et al., 2022], the experimental animals were acclimatized to the laboratory environment for 9 days, and then whole body inhalation anesthesia was performed with a mixture of 3% isoflurane (Hana Pharm. Co., Hwasung, Korea), 70% N ₂ O, and 28.5% O ₂ using a rodent inhalation anesthesia device (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.

(2) Dose Frequency

The maximum administration dose of LRC, 200 mg/kg, was set based on the efficacy exploration experiment at 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 set as the intermediate and low doses with a dose ratio of 2, respectively. 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 effective 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 orally administered 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 orally administered 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, suspended in sterile distilled water at a concentration of 12.6 mg/ml (0.12 tablet/ml) as the total amount of active ingredients, and orally administered once daily for 10 days starting 24 hours after dental cervical ligation at a dose of 5 ml/kg (total amount of active ingredients: 63 mg/kg; 0.60 tablet/kg). In the normal medium and EPD control groups, sterile distilled water as a medium was orally administered at the same dose and frequency instead of LRC, IND, or INP in order to apply the same corrective stress according to oral administration.

(3) Observation items

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

3. Experimental Results

(1) Changes in periodontal bone loss rate

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 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 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 effective 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 effective ingredient, and 200, 100, and 50 mg/kg of LRC, changes of -34.00, -22.36, -34.91, -29.90, and -23.15% compared to the EPD control group, respectively.

(2) Numerical changes in infiltrating inflammatory cells in gingival tissue

In the control group, a significant (p<0.01) increase in the number of inflammatory cells infiltrating the gingival tissue was observed compared to the normal medium control group, but a significant (p<0.01) decrease in the number of inflammatory cells infiltrating the gingival tissue was observed compared to the EPD control group 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 comparable activity in inhibiting inflammatory cell infiltration into the gingival tissue associated with tooth neck ligation compared to IND 5 mg/kg and INP 50 mg/kg, respectively, as a total amount of effective ingredients (Fig. 7). The number of inflammatory cells infiltrating 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 the 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.

(3) Changes in total anaerobic bacterial counts in the oral cavity

In the EPD control group, a significant (p<0.01) increase in the total anaerobic bacterial count in the oral buccal tissue was observed compared to the normal medium control group, but a significant (p<0.01) decrease in the anaerobic bacterial count 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 anaerobic bacterial count in the oral buccal tissue related to EPD by dental neck ligation comparable to that of 63 mg/kg as the total amount of INP effective ingredient, but in the IND 5 mg/kg group, no significant change in the total anaerobic bacterial count in the oral buccal tissue was observed compared to the EPD control group (Fig. 8). The total anaerobic bacterial count in the oral cavity showed a change of 819.15% 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 the INP effective ingredient, and 200, 100, and 50 mg/kg of LRC, the changes were -3.01, -27.31, -44.68, -38.19, and -28.94%, respectively, compared to the EPD control group.

(4) Changes in MDA content in gingival tissue

In the case of the EPD control group, a significant (p<0.01) increase in lipid peroxidation and MDA content in the gingival tissue was observed compared to the normal medium control group, but a significant (p<0.01 or p<0.05) decrease 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 5 mg/kg of IND, 63 mg/kg of the total amount of the INP active ingredient, and 200, 100, and 50 mg/kg of LRC, changes of -53.46, -29.73, -55.02, -42.31, and -30.53% compared to the EPD control group, respectively.

(5) Changes in MMP-8 content in gingival tissue

In the case of the EPD control group, a significant (p<0.01) increase in the MMP-8 content in the gingival tissue was recognized compared to the normal medium control group, but a remarkable decrease in the MMP-8 content compared to the EPD control group was recognized in a dose-dependent manner in all three doses of LRC 200, 100, and 50 mg/kg groups, 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 by tooth neck ligation comparable to IND 5 mg/kg and INP 50 mg/kg, respectively, as a total amount of effective ingredients (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 the 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% compared to the EPD control group, respectively.

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

Claims (12)

A composition for preventing, improving or treating periodontal disease, comprising an extract of Golpi rhizome as an effective ingredient. 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. In the first paragraph, the extract is a composition characterized in that the extract is obtained by first extracting the raw material by grinding it and using 30 to 50% of ethanol as an extraction solvent in an amount 10 times that of the raw material at 85±5℃ for 8±1 hours, or by mixing the first extract with a second extract obtained by extracting the raw material by using 30 to 50% of ethanol as an extraction solvent in an amount 8 times that of the raw material at 85±5℃ for 4±1 hours, and drying the mixture. A composition according to claim 1, characterized in that the periodontal disease is gingivitis or periodontitis. 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. In claim 1, a composition 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. A composition in 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). 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. A composition according to any one of claims 1 to 7, characterized in that the composition is a pharmaceutical composition. 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. A composition according to any one of claims 1 to 7, characterized in that the composition is a toothpaste composition. A composition according to any one of claims 1 to 7, characterized in that the composition is for improving gum health in an animal.

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