Bone protective agent, RANKL expression inhibitor, and OPG expression enhancer

Abstract

To provide a bone protectant having a bone protection effect, an RANKL expression inhibitor, and an OPG expression enhancer.SOLUTION: The present invention relates to a bone protectant comprising extract from Equisetum arvense, an RANKL expression inhibitor, and an OPG expression enhancer.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a bone protectant, an RANKL expression inhibitor, and an OPG expression enhancer.

Background Art

[0002] Bone maintains bone density and bone strength by repeating bone metabolism, which involves the absorption of aged bone (bone resorption) by osteoclasts and the formation of new bone (bone formation) by osteoblasts. When the balance between bone resorption and bone formation is disrupted and the function of osteoclasts is enhanced, the bone mechanical properties such as bone density and bone structure decrease, and bone destructive lesions such as Paget's disease of bone, osteoporosis, alveolar bone resorption in periodontal disease, and bone destruction in rheumatoid arthritis are known to occur.

[0003] On the other hand, periodontal disease is a lesion that occurs in the periodontal tissues (gingiva, periodontal ligament, cementum, alveolar bone), and most of it is an inflammatory disease of the periodontal tissues derived from bacteria in dental plaque. Periodontal disease is roughly classified into gingivitis in which inflammation is limited to the gingiva and periodontitis in which inflammation spreads to the alveolar bone, cementum, and periodontal ligament. When the state where dental plaque adheres near the gingival sulcus at the junction between teeth and the gingiva persists due to poor oral hygiene, pockets are formed due to continuous infection with periodontal pathogenic bacteria (gingivitis). If gingivitis is left untreated, the pockets become deeper, inflammation spreads to deep periodontal tissues such as the periodontal ligament, and alveolar bone resorption begins, leading to periodontitis. If it progresses further, alveolar bone resorption progresses, the teeth become loose, and eventually the teeth may fall out. For alveolar bone resorption associated with periodontal disease, the removal of physical infection sources (dental plaque) by brushing has conventionally been the basic treatment. However, due to time limitations, brushing skills, dentition, human issues, etc., there are many cases where a sufficient brushing effect cannot be obtained. Therefore, bactericides for chemically sterilizing periodontal disease-causing bacteria and anti-inflammatory agents for suppressing inflammation of periodontal tissues have been used complementarily.

[0004] Furthermore, research is being conducted on inhibiting alveolar bone resorption, which is effective in preventing and improving periodontal disease. For example, Patent Document 1 describes the use of egg yolk-derived protein to inhibit alveolar bone resorption. Patent Document 2 also describes a technique for obtaining an oral composition that inhibits alveolar bone resorption by osteoclasts by suppressing osteoclast formation, thereby promoting alveolar bone formation and contributing to the prevention and treatment of alveolar bone destruction caused by periodontal disease.

[0005] Furthermore, RANKL (receptor activator of nuclear factor-kappa B ligand), which is mainly expressed by osteoblast cells, is known to induce differentiation into osteoclasts and promote bone resorption by binding to RANK (receptor activator of nuclear factor-kappa B) on osteoclast precursor cells. In addition, OPG (osteoprotegerin; osteoclast differentiation inhibitor), which is mainly expressed by osteoblast cells, is known to inhibit the function of RANKL by acting as a decoy receptor for RANKL. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2010-260794 [Patent Document 2] Japanese Patent Publication No. 2002-128686 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Osteoclasts are the only cells responsible for the absorption of calcified bone. In bone-destructive diseases such as periodontal disease, excessive bone resorption by osteoclasts disrupts the balance of bone metabolism. Therefore, treatment and prevention methods require effects that restore or maintain the degree of bone resorption by osteoclasts to a normal state. Maintaining a balance between OPG expression and RANKL expression is crucial for maintaining the balance of bone metabolism. However, conventional techniques have only focused on suppressing bone resorption through RANKL regulation, and have not considered how to properly maintain the above balance. Therefore, the object of the present invention is to provide a bone protective agent that can maintain the balance of bone metabolism and protect bone. The present invention also relates to a RANKL expression inhibitor that can suppress the expression of RANKL, and an OPG expression enhancer that can enhance the expression of OPG. [Means for solving the problem]

[0008] The inventors of this invention conducted extensive research to solve the above problems and, as a result, discovered that by including horsetail extract, the balance of bone metabolism can be maintained and a bone-protective effect can be obtained, thus completing the present invention.

[0009] In other words, the present invention has been able to solve the above problems by the means described below. [1] A bone protectant containing horsetail extract. [2] A bone protective agent as described in [1], for use on the alveolar bone. [3] A RANKL expression inhibitor containing horsetail extract. [4] An OPG expression enhancer containing horsetail extract. [Effects of the Invention]

[0010] The bone protective agent of the present invention can achieve a bone protective effect by containing horsetail extract. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the number of osteoclasts (osteoclast count / mm) formed along the alveolar bone surface obtained in Experimental Example 1. [Figure 2]Figure 2 shows an immunohistochemistry (IHC) image (Scale bars = 100 μm) obtained in Experimental Example 2. [Figure 3] (a) to (d) of Figure 3 show the expression levels of LPS-induced inflammatory cytokines and RANKL with respect to the expression level of GAPDH obtained in Experimental Example 3. [Figure 4] Figure 4 shows the expression level of OPG (OPG / GAPDH) with respect to the expression level of GAPDH obtained in Experimental Example 3. [Figure 5] Figure 5 shows the number of osteoclasts (number of osteoclasts / mm) formed along the alveolar bone surface obtained in Experimental Example 4.

Mode for Carrying Out the Invention

[0012] Hereinafter, the present invention will be described in more detail.

[0013] <Bone Protective Agent> The bone protective agent according to an embodiment of the present invention is characterized by containing an extract of Equisetum arvense.

[0014] The extract of Equisetum arvense (Equisetum arvense extract) is obtained by extracting the components contained in the whole herb of Equisetum arvense, a kind of crude drug, belonging to the genus Equisetum of the family Equisetaceae. Although the Equisetum arvense extract has been known to have an anti-inflammatory effect, the present inventors have first found that it has a bone protective effect through the suppression of osteoclast formation. Although the mechanism is not clear, the inventors speculate as follows. That is, it is presumed that the shepherd's purse extract acts on osteoblast-lineage cells, and can obtain a bone protection effect such as bone resorption inhibition mainly by enhancing the expression of osteoprotegerin (OPG), which is expressed by osteoblast-lineage cells. In addition, the shepherd's purse extract can obtain a bone protection effect by suppressing the expression of RANKL (receptor activator of nuclear factor kappa-B ligand), which is a factor that induces differentiation into osteoclasts and is mainly expressed by osteoblast-lineage cells. Osteoblast-lineage cells including osteoblasts that form bone express RANKL, which is a differentiation-inducing factor for osteoclasts, and bind to RANK expressed on osteoclast precursor cells, thereby transmitting a signal to RANK and inducing differentiation and activation into osteoclasts. On the other hand, OPG expressed by osteoblast-lineage cells binds to RANKL and inhibits signal transduction to RANK, thereby inhibiting the induction of differentiation into osteoclasts. That is, OPG plays a role in protecting the alveolar bone from excessive bone resorption by strongly suppressing the RANKL-RANK interaction, and maintaining the balance between bone resorption by osteoclasts and bone formation by osteoblast-lineage cells. In addition, the formation of osteoclasts is controlled by the ratio of RANKL to OPG (referred to as the RANKL / OPG ratio) produced by osteoblast-lineage cells.

[0015] The shepherd's purse extract (in terms of solids) can be contained in an amount of, for example, 0.00001 to 1.0% by mass, preferably 0.00005 to 0.1% by mass, more preferably 0.0001 to 0.05% by mass, based on the total amount of the bone protectant of the present invention. By setting the content of the shepherd's purse extract to 0.00001% by mass or more, an enhancement of the bone protection effect of the shepherd's purse extract is expected, and by setting it to 1.0% by mass or less, formulation becomes easy, and particularly a good feeling of use can be obtained in an oral composition.

[0016] As described later in the examples, commercially available shepherd's purse extract can be used, or extract obtained by extracting shepherd's purse by the methods described later can also be used.

[0017] In the bone protective agent according to the embodiment of the present invention, the method for extracting the horsetail extract is not particularly limited and can be any conventionally known method. For example, any part of the horsetail can be used as is, or after being cut, crushed, etc., and then the extract can be obtained by squeezing, solvent extraction, water distillation, steam distillation, etc. As for the solvent extraction method, any method known in the art can be used, for example, conventionally known extraction methods such as water (including hot water and boiling water) extraction, alcohol extraction, supercritical fluid extraction, microwave extraction, and pressing extraction can be used.

[0018] Examples of solvents used in solvent extraction include water; alcohols such as methanol, ethanol, isopropyl alcohol, ethylene glycol, propylene glycol, and 1,3-butylene glycol (whether anhydrous or hydrated); ketones such as acetone; ethers such as diethyl ether and dioxane; nitriles such as acetonitrile; esters such as ethyl acetate; hexane, xylene, benzene, and chloroform. Preferably, the solvent used in solvent extraction is water, alcohols, ketones, and hexane, and more preferably water, alcohols, and ketones. These solvents may be used individually or in combination of two or more.

[0019] The obtained extract may be used as is or after drying. Furthermore, the obtained extract may be purified and concentrated as needed. Examples of purification methods include filtration or adsorption, decolorization, and separation using ion exchange resins or activated carbon columns. Examples of concentration methods include conventional methods such as evaporators. Further drying treatments, such as freeze-drying, may be performed, or the extract may be powdered according to conventionally known methods. The extract thus obtained may also be dissolved in water, ethanol, or other solvents as needed before use.

[0020] Horsetail extract, which is the active ingredient of the bone protective agent according to the embodiment of the present invention, is readily absorbed by the mucous membrane and exhibits excellent bone protective effects through transmucosal absorption; therefore, it is preferable to use the bone protective agent as an oral composition. When the bone protective agent according to the embodiment of the present invention is an oral composition, it can be applied to the prevention and treatment of various bone diseases occurring in the oral and maxillofacial region, such as periodontal diseases including gingivitis and periodontitis, osteonecrosis of the jaw including anti-osteoresorption drug-associated osteonecrosis of the jaw (ARONJ), peri-implantitis, etc., and can also be applied to promote the fixation of dental implants.

[0021] Here, "periodontal disease" refers to an inflammatory disease in which inflammation is caused in the periodontal tissues by bacteria in dental plaque, and includes gingivitis and periodontitis.

[0022] Osteoblast-derived cells express RANKL and OPG, which regulate their differentiation and activation into osteoclasts. As mentioned above, OPG expressed by osteoblast-derived cells acts as a decoy receptor, binding to RANKL and inhibiting signal transduction to RANK. However, in periodontal disease, LPS derived from periodontal pathogens contained in dental plaque causes inflammation in the periodontal pocket epithelium, leading to excessive production of inflammatory cytokines. Furthermore, LPS reaches deep into the periodontal tissue via the highly permeable junctional epithelium, enhancing the expression of RANKL in osteoblast-derived cells while suppressing the expression of OPG. As a result, the expression level of RANKL in the periodontal tissue far exceeds that of OPG, leading to the formation and activation of excessive osteoclasts and pathological alveolar bone resorption.

[0023] Oral compositions can take the form of, for example, mouthwash, oral fresheners, gargles (mouthwashes), toothpastes such as liquid toothpaste and paste toothpaste, gels, ointments, foams, lozenges, chewing gum, etc. In particular, it is suitable for use as a mouthwash, where an appropriate amount is placed in the mouth for use, in order to ensure that the oral composition is thoroughly distributed throughout the oral cavity. To prepare a mouthwash, for example, water or ethanol can be used as a solvent and prepared by a conventional method. Furthermore, for example, liquid toothpaste can be used that contains abrasives such as calcium hydrogen phosphate, aluminum hydroxide, anhydrous silicic acid, and calcium carbonate as needed.

[0024] Furthermore, the oral composition may contain any other components as long as they do not impair the effects of the present invention. For example, fluorides such as sodium fluoride and sodium monofluorophosphate; anti-inflammatory agents such as azulene, azulene sulfonate, glycyrrhizic acid or its salts, β-glycyrrhetinic acid, dihydrocholesterol, epidihydrocholesterol, dl-α-tocopherol acetate, dl-α-tocopherol nicotinate, ε-aminocaproic acid, tranexamic acid, allantoin, and ascorbic acid; tartar preventative agents such as phosphates, polyphosphates, methoxyethylene maleic anhydride copolymer, zinc chloride, and zinc organic acid; astringents such as hinokitiol, allantoin chlorohydroxyaluminum, allantoin dihydroxyaluminum (aldioxa), allantoin chlorohydroxyaluminum (alcloxa), and sodium chloride; humectants such as glycerin, sorbitol, and polyethylene glycol; foaming agents such as sodium lauryl sulfate; pinene, peppermint oil, spearmint oil, Japanese peppermint oil, cinnamon oil, clove oil, eugenol, lemon oil, vanilla Fragrances such as phosphorus, cineole, and eucalyptus oil; plant extracts such as hawthorn, peony, witch hazel, birch, and sage; sweeteners such as saccharin, sodium saccharin, sucralose, xylitol, erythritol, sorbitol, maltitol, and stevia; colorants such as Blue No. 1, Yellow No. 5, Yellow No. 4, Yellow No. 203, Green No. 3, Green No. 201, and Red No. 102; preservatives such as parabens and sodium benzoate; pH adjusters such as monosodium phosphate, disodium phosphate, citric acid, and sodium citrate; nonionic surfactants such as POE hydrogenated castor oil, POE-POP block polymer, POE-POP alkyl ether, POE alkyl ether, POE alkylphenyl ether, POE fatty acid ester, POE higher alcohol ether, POE-POP fatty acid ester, POE sorbitan fatty acid ester, sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, and propylene glycol fatty acid ester;Examples include anionic surfactants such as sodium lauryl sulfate, sodium myristyl sulfate, POE alkyl ether sulfate, sodium lauroyl sarcosinate, sodium myristoyl sarcosinate, alkyl ether carboxylates, alkyl phosphates, POE alkyl ether phosphates, N-acyl taurine salts, POE alkyl ether phosphates, sulfonates, etc.; cationic surfactants such as alkyltrimethylammonium chloride, dialkyldimethylammonium chloride, POE alkylamine fatty acid amides, etc.; and amphoteric surfactants such as 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, coconut oil fatty acid amidopropyl betaine, and sodium lauryldiaminoethylglycinate.

[0025] The bone-protective effect of the bone-protective agent according to the embodiment of the present invention can be evaluated, for example, by measuring the number of tissues or cells collected from various organs, or by measuring the expression levels of RANKL and OPG, as described later in the examples. For example, the bone-protective effect of including horsetail extract can be evaluated by comparing the case in which a composition containing only glycolipids that induce inflammation of periodontal tissue and alveolar bone resorption is applied with the case in which a bone-protective agent according to the embodiment of the present invention, which contains glycolipids and horsetail extract, is applied. Examples of glycolipids include lipopolysaccharide (LPS). LPS is a component of the outer membrane surrounding peptidoglycan on the surface of Gram-negative bacteria, and is a glycolipid composed of lipids and polysaccharides. LPS induces inflammation of periodontal tissue and alveolar bone resorption via Toll-like receptors (TLRs). Furthermore, as mentioned above, osteoclast formation is mainly controlled by the RANKL / OPG ratio (the ratio of RANKL expression to OPG expression) produced by osteoblast cells. In periodontal disease, the RANKL / OPG ratio increases due to the influence of LPS, etc., leading to excessive osteoclast formation and accelerated bone resorption. More specific details will be provided in the examples.

[0026] The bone protective agent according to the embodiment of the present invention is preferably intended for use on the alveolar bone. The bone protective agent according to an embodiment of the present invention contains horsetail extract as an active ingredient and can be used to protect the alveolar bone, thereby preventing or improving periodontal disease.

[0027] The RANKL expression inhibitor according to an embodiment of the present invention contains an extract of horsetail. That is, it contains horsetail extract as an active ingredient and can suppress the expression of RANKL.

[0028] The OPG expression enhancer according to the embodiment of the present invention contains an extract of horsetail. That is, the OPG expression enhancer according to the embodiment of the present invention contains horsetail extract as an active ingredient and can enhance OPG expression.

[0029] In the bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to embodiments of the present invention, the content of the horsetail extract, which is the active ingredient, is, for example, 0.0001 to 1.0% by mass, preferably 0.0005 to 0.1% by mass, and more preferably 0.001 to 0.015% by mass, based on the total amount of the bone protective agent, RANKL expression inhibitor, or OPG expression enhancer.

[0030] The bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to the embodiments of the present invention can be used as a food composition, a pharmaceutical product, or a quasi-drug. The food composition may be provided in the form of, for example, health foods, foods for specified health uses, functional foods, nutritional functional foods, supplements, etc. In other words, according to the present invention, a bone-protective food composition, a bone-protective pharmaceutical, or a bone-protective quasi-drug can be provided.

[0031] The bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to the embodiment of the present invention may consist solely of the bone protective agent, or may further contain materials usable in food compositions, quasi-drugs, or pharmaceuticals. The materials usable in food compositions, quasi-drugs, or pharmaceuticals are not particularly limited, but examples include amino acids, proteins, carbohydrates, oils and fats, sweeteners, minerals, vitamins, flavorings, excipients, binders, lubricants, disintegrants, emulsifiers, surfactants, bases, solubilizers, suspending agents, and the like.

[0032] The shape of the bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to the embodiment of the present invention is not limited and may be in any form such as solid (powder, granules, etc.), liquid (solution, suspension, etc.), or paste, and may be in any dosage form such as powder, pill, granule, tablet, capsule, lozenge, spray, liquid, or suspension. A liquid form is preferred because it is easy to apply to mucous membranes due to its excellent absorption in mucous membranes.

[0033] The bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to the embodiment of the present invention may be applied directly to the affected area and absorbed transdermally, or they may be administered parenterally, such as orally or intravenously.

[0034] When the bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to the embodiment of the present invention are applied directly to the affected area, the amount used is preferably such that, for example, the horsetail extract (in terms of solid matter) is 5 pg / kg (body weight) or more per application, more preferably 50 pg / kg (body weight) or more, and even more preferably 500 pg / kg (body weight) or more. Furthermore, it is preferable that the horsetail extract is used at a rate of 5 pg / kg (body weight) or more per day, more preferably 50 pg / kg (body weight) or more, and even more preferably 500 pg / kg (body weight) or more. Furthermore, it is preferable that the horsetail extract is used at a rate of 2000 mg / kg (body weight) or less per application, more preferably 1500 mg / kg (body weight) or less, and even more preferably 1000 mg / kg (body weight) or less. Furthermore, it is preferable that the horsetail extract is used at a dose of 3000 mg / kg (body weight) or less per day, more preferably 2500 mg / kg (body weight) or less, and even more preferably 2000 mg / kg (body weight) or less. Within this range, sufficient blood concentrations can be achieved, and the bone-protective effect can be expressed more effectively.

[0035] When the bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to the embodiment of the present invention are administered orally, the dosage is preferably such that the horsetail extract (solid matter equivalent) is 5 pg / kg (body weight) or more per dose, more preferably 50 pg / kg (body weight) or more, and even more preferably 500 pg / kg (body weight) or more. Furthermore, the horsetail extract is preferably administered at a rate of 15 pg / kg (body weight) or more per day, more preferably 50 pg / kg (body weight) or more, and even more preferably 500 pg / kg (body weight) or more. Furthermore, the horsetail extract is preferably administered at a rate of 2000 mg / kg (body weight) or less per dose, more preferably 1500 mg / kg (body weight) or less, and even more preferably 1000 mg / kg (body weight) or less. Furthermore, it is preferable that the horsetail extract be administered at a dose of 3000 mg / kg (body weight) or less per day, more preferably 2500 mg / kg (body weight) or less, and even more preferably 2000 mg / kg (body weight) or less. Within this range, sufficient blood concentrations can be achieved, and the bone-protective effect can be expressed more effectively.

[0036] When the bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to the embodiments of the present invention are administered parenterally, the dosage is preferably such that, for example, horsetail extract (on a solid basis) is administered at a dose of 5 pg / kg (body weight) or more per dose, more preferably at a dose of 50 pg / kg (body weight) or more, and even more preferably at a dose of 500 pg / kg (body weight) or more. Furthermore, it is preferable that the horsetail extract be administered at a dose of 5 pg / kg (body weight) or more per day, more preferably at a dose of 50 pg / kg (body weight) or more, and even more preferably at a dose of 500 pg / kg (body weight) or more. Furthermore, it is preferable that the horsetail extract be administered at a dose of 4000 mg / kg (body weight) or less per dose, more preferably at a dose of 3000 mg / kg (body weight) or less, and even more preferably at a dose of 2000 mg / kg (body weight) or less. Furthermore, it is preferable that the horsetail extract be administered at a dose of 4000 mg / kg (body weight) or less per day, more preferably 3000 mg / kg (body weight) or less, and even more preferably 2000 mg / kg (body weight) or less. Within this range, sufficient blood concentrations can be achieved, and the bone-protective effect can be expressed more effectively.

[0037] The bone protectant, RANKL expression inhibitor, and OPG expression enhancer according to embodiments of the present invention can be used in humans or animals. When the bone protectant, RANKL expression inhibitor, and OPG expression enhancer are used in animals, they can be used as feed or feed additives. Examples of feed include companion animal feed such as dog food and cat food, livestock feed, poultry feed, and farmed fish and shellfish feed. "Feed" includes everything that animals ingest orally for nutritional purposes. More specifically, when classified in terms of nutrient content, it includes roughage, concentrated feed, inorganic feed, and specialty feed, and when classified in terms of official standards, it includes compound feed, mixed feed, and single-component feed. Furthermore, when classified in terms of feeding method, it includes feed that is fed directly, feed that is mixed with other feeds, or feed that is added to drinking water to supplement nutrients.

[0038] The bone protective agent, RANKL expression inhibitor, and OPG expression enhancer according to the embodiments of the present invention can improve bone metabolism in humans or non-human animals, and can therefore be used for the prevention and treatment of bone-related diseases such as fractures, osteoporosis, and osteomalacia, as well as periodontal disease, and as agents to promote the fixation of dental implants. [Examples]

[0039] The present invention will be further explained below with reference to the following experimental examples, but the present invention is not limited to these examples.

[0040] <Experimental Example 1: Evaluation of the effect of bone protective agents on inhibiting osteoclast formation on the alveolar bone surface> In this study, the inhibitory effect of bone protective agents on osteoclast formation on the alveolar bone surface was evaluated using a periodontal disease model rat.

[0041] [Preparation of specimens] First, an Ec-LPS solution (LPS solution) with an Ec-LPS concentration of 5 mg / mL was prepared using E. coli-derived lipopolysaccharides (Ec-LPS) (manufactured by SIGMA-Aldrich) and purified water. Furthermore, an Ec-LPS + horsetail extract solution (LPS / EA solution) was prepared using Ec-LPS (manufactured by SIGMA-Aldrich), horsetail extract (manufactured by Maruzen Pharmaceutical Co., Ltd., product name: Horsetail Extract BG), and purified water, with an Ec-LPS concentration of 5 mg / mL and a horsetail extract (EA) (solids equivalent) concentration of 15 μg / mL. Physiological saline was used as the control solution.

[0042] 〔method〕 Seven-week-old male Wistar rats were kept in cages for one week as a preliminary period, and then raised to eight weeks of age (n=7). Rats were anesthetized and fixed to a cork board with their mouths open. Each sample (control solution, LPS solution, and LPS / EA solution) was then dropped at a rate of 2 μL every 10 minutes for 1 hour into the palatal gingival sulcus of the maxillary molars. Three days later, periodontal tissue was collected, and tissue fragments including the maxillary molar region were fixed in paraformaldehyde periodate solution at 4°C for 5 days. Then, demineralization was performed at 4°C for 1 month using 1 mM PBS (Phosphate-buffered saline) (pH 7.4) containing 10% EDTA. Tissue fragments including the first or second molar were divided, embedded in paraffin, and then sections (4.5 μm thick) including the root apex were cut parallel to the long axis of the tooth using a sliding microtome (manufactured by Yamato Koki Kogyo Co., Ltd.) and mounted on glass slides.

[0043] Next, to investigate the localization of osteoclasts, immunohistochemical staining was performed as follows: After defatting and rehydration, sections were incubated in 0.3% hydrogen peroxide (methanol) at room temperature for 30 minutes to reduce endogenous peroxidase activity. Protein blocks were added and incubated at room temperature for 10 minutes, then cathepsin K antibody (1:500 dilution) was added and incubated overnight at 4°C. After two treatments with PBS for 5 minutes each, secondary antibody was added and incubated for 30 minutes. After two treatments with PBS for 5 minutes each, staining with DAB was performed, counterstaining with hematoxylin was done, followed by dehydration, clearing, and mounting of coverslips.

[0044] Anti-rat cathepsin K rabbit antibody (primary antibody): Abcam / 1:500 Anti-rabbit IgG antibody (secondary antibody): EnVision+System-HRP Labeled Polymer Anti-Rabbit, Dako Protein Block: Dako DAB:DAB Peroxidase (HRP) Substrate Kit,Dako

[0045] After capturing tissue images with an optical microscope, the number of osteoclasts formed along the alveolar bone margin on the periodontal ligament side, up to 1 mm from the alveolar bone crest, was counted. This is considered to be the area where the subgingival inflammation induced by LPS spread and the osteoclast formation-promoting effect is felt. The data for one individual was the average of the first and second molars, and measurements were taken for seven individuals, with the average value used as the osteoclast count per individual (osteoclast count / mm).

[0046] 〔result〕 Figure 1 shows the number of osteoclasts (osteoclast count / mm) on the alveolar bone surface of each sample that was treated. As can be seen from the results in Figure 1, the number of osteoclasts formed along the alveolar bone margin on the periodontal ligament side tended to increase in the LPS solution administration group compared to the control solution administration group, but tended to be suppressed in the LPS / EA solution administration group containing LPS and horsetail extract. In other words, the formation of osteoclasts induced by LPS was suppressed by the active ingredient, horsetail extract, demonstrating the inhibitory effect of the bone protective agent on osteoclast formation.

[0047] <Experimental Example 2: Evaluation of RANKL and OPG expression in periodontal tissue> In this study, the inhibitory effect of horsetail extract on osteoclast formation on the alveolar bone surface was investigated by evaluating the expression of RANKL and OPG in periodontal tissue using prepared samples (control solution, LPS solution, and LPS / EA solution) in the same manner as in Experimental Example 1, and verifying the mechanism of action.

[0048] 〔method〕 Immunohistochemical staining was performed on 8-week-old male Wistar rats using the same method as in Experimental Example 1, employing antibodies that specifically stain OPG (anti-osteoprotegrin, abcam (OPG antibody)) and antibodies that specifically stain RANKL (anti-sRANKL, abcam (sRANKL)) to visualize and evaluate the expression of OPG and RANKL in periodontal tissue.

[0049] • Anti-rat osteoprotegerin antibody (primary antibody): Abcam / 1:400 ·Anti-rat RANKL antibody (primary antibody): anti-sRANKL,abcam / 1:2000 • Rabbit IgG antibody (secondary antibody): EnVision+System-HRP Labeled Polymer Anti-Rabbit,Dako

[0050] 〔result〕 The immunohistochemical (IHC) staining images (Scale bars = 100 μm) obtained after applying the staining to each sample are shown in Figure 2. By observing Figure 2, the expression localization of RANKL and OPG was confirmed. Compared to the control solution administration group (a), the LPS solution administration group (b) showed a tendency for enhanced RANKL expression from the junctional epithelium to the upper layer of the periodontal ligament, while OPG expression on the periodontal ligament tended to be suppressed. On the other hand, in the LPS / EA solution administration group (c), RANKL expression was suppressed to the same extent as the control solution administration group (a), while OPG expression tended to recover.

[0051] RANKL expression showed a positive correlation with the increase or decrease in osteoclast number. Specifically, RANKL expression was enhanced in the LPS solution administration group (b) compared to the control solution administration group (a), but tended to be suppressed to a similar degree in the LPS / EA solution administration group (c) as in the control solution administration group (a). On the other hand, OPG expression showed a negative correlation with the increase or decrease in osteoclast number. Specifically, OPG expression was suppressed in the LPS solution administration group (b) compared to the control solution administration group (a), but tended to be enhanced in the LPS / EA solution administration group (c) to an extent even greater than in the control solution administration group (a).

[0052] The results above indicate that the "RANKL / OPG ratio," which controls osteoclast formation and was elevated by LPS solution administration, tended to normalize with treatment using a bone protective agent containing horsetail extract. Therefore, it was suggested that the bone protective agent may suppress osteoclast formation on the alveolar bone surface by normalizing the RANKL / OPG ratio disrupted by the LPS solution.

[0053] <Experimental Example 3: Evaluation of the effect of horsetail extract on increasing the RANKL / OPG ratio> In this study, we used mouse bone marrow-derived osteoblast-like cells (ST2 cells) to evaluate the effect of horsetail extract on the increase in the RANKL / OPG ratio induced by LPS.

[0054] [Preparation of specimens] The following culture media were prepared as samples. First, α-MEM medium (LPS medium) containing 1 μg / mL of Ec-LPS and 10 v / v% FBS was prepared. In addition, 10v / v% FBS-supplemented α-MEM medium (LPS / EA medium) containing 1 μg / mL Ec-LPS and 3 μg / mL horsetail extract was prepared. Furthermore, α-MEM medium supplemented with 10v / v% FBS was used as the control medium.

[0055] 〔method〕 Place 3 mL of culture medium (10v / v% FBS-added α-MEM medium) into each Φ6 cm plate, and divide into 8 × 10 5 ST2 cells were seeded in cells / plate and cultured at 37°C for 48 hours. After washing with PBS (Phosphate-buffered saline) (pH 7.4), 3 mL of each sample (control medium, LPS medium, LPS / EA medium) was added to the plate. Cells were collected after 2 hours, 12 hours, and 24 hours, and total RNA was extracted and purified from the cells using the RNeasy mini Kit (Qiagen) according to the protocol. The concentration of total RNA extracted from cells was measured using Nano Drop (manufactured by Thermo Scientific). From RNA extracted from cells, reverse transcriptase (ReverTra Ace TM cDNA was synthesized according to the protocol using a device manufactured by TOYOBO Corporation.

[0056] ST2 cells were collected after 2 hours for TNF-α, IL-6, and RANKL, and after 12 hours for IL-1β. OPG was collected after 24 hours.

[0057] (Real-time PCR) Applied Biosystems® StepOne TM Real-time PCR was performed using the SYBR Green Assay with a Real-time PCR System (Thermo Fisher Scientific). The FastStart Essential DNA Green Master (Roche) was used for the test, and the procedure was followed according to the protocol under the following conditions.

[0058] (Primer information) • Sequence ID 1: GAPDH Forward 5'-TGAACGGGAAGCTCACTGG-3' • Sequence ID 2: GAPDH Reverse 5'-TCCACCACCCTGTTGCTGTA-3' • Sequence ID 3: OPG Forward 5'-CAGAGACTAATAGATCAAAGGCA-3' • Sequence ID 4: OPG Reverse 5'-ATGAAGTCTCACCTGAGAAGAAC-3' • Sequence ID 5: RANKL Forward 5'-GCACACCTCACCATCAATGC-3' • Sequence ID 6: RANKL Reverse 5'-GTCTGTAGGTACGCTTCCCG-3' • Sequence ID 7: IL-6 Forward 5'-TTACACATGTTCTCTGGGAAATCGT-3' • Sequence ID 8: IL-6 Reverse 5'-TGGTAGCATCCATCATTTCTTTGT-3' • Sequence ID 9: IL-1β Forward 5'-AGAGAGCCTGTGTTTTCCTCCTTG-3' • SEQ ID NO: 10: IL-1β Reverse 5'-GCTTCAATGAAAGACCTCAGTGCAG-3' • Sequence ID 11: TNF-α Forward 5'-ATGAGCACAGAAAGCATGATC-3' • Sequence ID 12: TNF-α Reverse 5'-TACAGGCTTGTCACTCGAATT-3'

[0059] (Real-time PCR conditions) • 10 minutes at 95°C per cycle 45 cycles in total: 10 seconds at 95°C, 10 seconds at 57°C, and 45 seconds at 72°C. • 5 minutes at 72℃ constitutes one cycle.

[0060] 〔result〕 Figures 3(a) to (d) show the TNF-α expression level relative to the GAPDH expression level (TNF-α / GAPDH), the IL-6 expression level relative to the GAPDH expression level (IL-6 / GAPDH), the IL-1β expression level relative to the GAPDH expression level (IL-1β / GAPDH), and the RANKL expression level relative to the GAPDH expression level (RANKL / GAPDH) when each sample is applied.

[0061] Furthermore, Figure 4 shows the ratio of OPG expression levels to GAPDH expression levels (OPG / GAPDH) when each sample is applied.

[0062] Inflammatory cytokines and RANKL, whose expression was induced by LPS medium containing LPS, tended to be suppressed by LPS / EA medium containing horsetail extract (Figure 3(a)-(d)). On the other hand, OPG expression was suppressed by LPS medium, but tended to recover in LPS / EA medium in the presence of horsetail extract (Figure 4). These results suggest that bone protective agents containing horsetail extract may act on osteoblasts, which are the main source of RANKL and OPG, and suppress LPS-induced changes in RANKL and OPG expression, thereby maintaining a normal RANKL / OPG ratio.

[0063] <Experimental Example 4: Evaluation of the inhibitory effect of LPS on osteoclast formation> The inhibitory effect of LPS on osteoclast formation was compared and investigated using Houttuynia cordata extract, known as an anti-inflammatory component, using the same method as in Experimental Example 1.

[0064] [Preparation of specimens] Using the same method as in Experimental Example 1, a 5 mg / mL Ec-LPS solution (LPS solution) and an Ec-LPS + horsetail extract (EA) solution (LPS / EA solution) with an Ec-LPS concentration of 5 mg / mL and a horsetail extract (EA) (solids equivalent) concentration of 15 μg / mL were prepared. Furthermore, an Ec-LPS + Houttuynia cordata extract solution (LPS / Houttuynia cordata solution) was prepared using Ec-LPS (manufactured by SIGMA-Aldrich), Houttuynia cordata extract (manufactured by Ichimaru Falcos, product name: Falcorex Houttuynia cordata B), and purified water, with an Ec-LPS concentration of 5 mg / mL and a Houttuynia cordata extract (solid matter equivalent) concentration of 15 μg / mL. Physiological saline was used as the control solution.

[0065] 〔method〕 Using 8-week-old male Wistar rats, the inhibitory effect of Houttuynia cordata extract on osteoclast formation on the alveolar bone surface was evaluated using the same method as in Experimental Example 1 (n=2).

[0066] 〔result〕 Figure 5 shows the number of osteoclasts (osteoclast count / mm) on the alveolar bone surface after applying each sample. For the control solution group, LPS solution group, and LPS / EA solution group, n=2 of each group were extracted from the results of Experimental Example 1 in order of the earliest number of trials and are shown in the graph in Figure 5. As can be seen from the results in Figure 5, the inhibitory effect of LPS on osteoclast formation was investigated using a periodontal disease model rat. The results showed that when an LPS / EA solution, a bone protective agent containing horsetail extract, was dropped into the gingival sulcus of the periodontal disease model rats, the number of osteoclasts per unit area formed along the alveolar bone margin on the periodontal ligament side decreased compared to the control group. On the other hand, an increasing trend was observed in the LPS solution administration group and the LPS / Houttuynia cordata solution administration group compared to the control group. This suggests that bone protective agents containing horsetail extract have an inhibitory effect on osteoclast formation, potentially controlling alveolar bone resorption due to the progression of periodontitis and protecting the alveolar bone.

[0067] On the other hand, while the anti-inflammatory effects of Houttuynia cordata extract have been reported previously, these results suggest that Houttuynia cordata extract may not possess the osteoclast formation inhibitory effect seen in horsetail extract. This suggests that the osteoclast formation inhibitory effect may be unique to horsetail extract among plant extracts that have been shown to have anti-inflammatory effects.

[0068] Based on the results of the above experiments, it was found that bone protectants containing horsetail extract have a bone-protective effect.

Claims

1. An oral bone protectant containing horsetail extract, applied to the oral mucosa to suppress bone resorption caused by inflammation.

2. An oral bone protectant according to claim 1, for use on the alveolar bone or jawbone.

3. An agent containing horsetail extract that suppresses RANKL expression and enhances OPG expression.

4. An oral bone protectant containing horsetail extract, applied to the oral mucosa, for suppressing RANKL expression and enhancing OPG expression, thereby suppressing alveolar bone or jawbone resorption due to inflammation.

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