KR100991857B1 - Pharmaceutical composition for preventing or treating periodontal disease comprising extracts of Pharbitidis Semen - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the prevention or treatment of periodontal disease (periodontal disease) comprising Pharbitidis Semen extract as an active ingredient. The nut extract is useful as a preventive and therapeutic agent for periodontal disease such as periodontitis, alveolar bone breakdown, alveolar bone dysfunction, as well as inhibiting NO production and inducing the activity of alkaline phosphatase to promote osteoblast differentiation and mineralization. .

Dog, periodontal disease

Description

Pharmaceutical composition for preventing or treating periodontal disease comprising extracts of Pharbitidis Semen}

The present invention relates to a pharmaceutical composition for preventing or treating periodontal disease (periodontal disease) comprising Pharbitidis Semen extract as an active ingredient.

Teeth are structurally supported by the alveolar bone as the hardest hard tissue in the human body. The alveolar bone is firmly attached to the base bone of the jaw bone, and the 2-3 mm portion immediately adjacent to the root is called the periodontal bone, but the alveolar bone generally refers to all of the hard tissue including the periodontal bone. Alveolar bone gradually decreases with age, and the roots of teeth are exposed. In addition, when the teeth are lost, the alveolar bone is gradually lost. In the oral alveolar bone, the teeth are attached and supported by a layer containing an undifferentiated mesenchymal cell called periodontal ligament, which has an average thickness of 0.2 mm. It also transmits the sense of teeth to the alveolar bone through the periodontal ligament. In addition, the periodontal ligament is a layer in which undifferentiated mesenchymal cells exist, and is structured to adapt to pressure while continuing bone remodeling without losing the structure and function of the entire fiber. The undifferentiated mesenchymal cells in the periodontal ligament move to the adjacent tissues, which are directed to the alveolar bone and are responsible for the remodeling of the alveolar bone.

As the age increases, congenital and acquired deterioration of the alveolar bone causes periodontal disease. Representative periodontal disease is periodontal inflammation or alveolar bone osteodystrophy. The alveolar bone dysplasia may occur in the form of alveolar bone osteoporosis, alveolar bone osteomalacia, alveolar bone osteopenia and the like, resulting in tooth loss. In the treatment of periodontal disease, artificial alveolar bone graft or alveolar bone formation is the predominant predominant method. However, there is no drug that can effectively treat periodontal disease, and it relies on drugs to relieve individual symptoms such as anti-inflammatory drugs.

Alveolar bone is also balanced through continuous remodeling, which involves bone formation by osteoblasts and bone resorption by osteoclasts. This metabolism is controlled by physical effects, hormonal systems and local factors. Alveolar osteoporosis, such as alveolar osteoporosis, occurs when bone resorption exceeds bone formation and bone volume decreases below the limit by various factors. Osteoblasts make bone formation by depositing the bone's organic substance, osteoid, which is composed of collagen type 1, osteocalcin, osteonectin, and sialoprotein. Consists of. The formed osteoid is later subjected to mineral deposition, in which osteoblasts induce minerals to form hydroxyapatite, which is a calcium phosphate crystal, to be deposited on the osteoid. Alkaline phosphatase, type 1 collagen, osteocalcin and the like are used clinically as biochemical indicators of osteoblasts, among which alkaline phosphatase is involved in mineralization of the substrate.

The present inventors searched for various natural extracts in order to develop substances that can prevent and treat periodontal disease. As a result, extracts from cows not only inhibit NO production but also induce the activity of alkaline phosphatase to promote osteoblast differentiation and mineralization, thereby preventing periodontal disease such as periodontitis, alveolar bone breakage, alveolar bone dysplasia, etc. And therapeutic activity.

Therefore, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of periodontal disease, including the nut extract as an active ingredient.

According to one aspect of the invention, there is provided a pharmaceutical composition for the prevention or treatment of periodontal disease (periodontal disease) comprising Pharbitidis Semen extract as an active ingredient.

In the pharmaceutical composition of the present invention, the cow's milk extract can be obtained by extracting the cow's milk with an extraction solvent, preferably an aqueous solution of ethanol, selected from the group consisting of water, C ₁ -C ₄ alcohol, and C ₁ -C ₄ alcohol solution. have.

In addition, the nut extract is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ ~ C ₄ alcohol, and C _{1 ~} C ₄ alcohol aqueous solution; And (b) may be obtained by performing an extraction process comprising the step of extracting the extract obtained from step (a) with n-hexane.

In addition, the nut extract is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ ~ C ₄ alcohol, and C _{1 ~} C ₄ alcohol aqueous solution; (b ') adding water and n-hexane to the extract obtained from step (a) and separating the aqueous layer; And (c) adding ethyl acetate to the aqueous layer obtained from step (b '), and separating the ethyl acetate layer.

In addition, the nut extract is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ ~ C ₄ alcohol, and C _{1 ~} C ₄ alcohol aqueous solution; (b ') adding water and n-hexane to the extract obtained from step (a) and separating the aqueous layer; (c ') adding ethyl acetate to the aqueous layer obtained from step (b') and separating the aqueous layer; And (d) adding n-butanol to the aqueous layer obtained in step (c '), and separating the aqueous layer and the n-butanol layer.

The periodontal disease includes periodontal inflammation (periodontal inflammation); Alveolar bone breakage; Or alveolar bone osteodystrophy, such as alveolar bone osteoporosis, alveolar bone osteomalacia, or alveolar bone osteopenia.

The extract of the nutmeg contained as an active ingredient in the pharmaceutical composition according to the present invention not only inhibits NO production but also induces the activity of alkaline phosphatase to promote osteoblast differentiation and mineralization, periodontitis, alveolar bone breakage, alveolar bone formation disorder It is useful as an agent for the prevention and treatment of periodontal diseases.

The present invention provides a pharmaceutical composition for the prevention or treatment of periodontal disease (periodontal disease) comprising Pharbitidis Semen extract as an active ingredient.

Pharbitidis Semen (는 子) is a morning glory ( Pharbitis) nil Choisy) seed, also known as black axis. The morning glory is native to tropical Asia such as India and about 256 varieties are known, and black seeds are called black axes and white seeds are called white axes. Broiled cows have a slight smell, irritating taste, bitterness and coldness. Geunwoo is a lower-disease (瀉 下) and diuretic effect is strong, when the body swells down, chronic pyelonephritis, liver cirrhosis, etc. is used when the ascites. Effective for seawater and asthma and used for stubborn constipation and parasite removal. Pharmacological action, intestinal peristalsis, has been reported to have strong hypoactivity. The nut contains about 2% pharbitin, a type of polysaccharide, and 11% fatty acids such as olein, palmitine, and stearin. (Painting, Ingredients and Use of Herbs, p589, 1984) .

In the pharmaceutical composition of the present invention, the nut extract can be obtained by extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ ~ C ₄ alcohol, and C _{1 ~} C ₄ alcohol aqueous solution. Preferably, the extraction solvent may be an aqueous solution of ethanol, the concentration of ethanol is not limited significantly, about 70% aqueous ethanol can be used. Extraction using the ethanol aqueous solution or the like may be performed at room temperature for 5 days to 10 days, preferably about 7 days. It may also be carried out under shading and / or warming (eg warming) conditions as needed. The extraction process may be performed once or plural times, preferably about three times. The extract can be obtained in a conventional manner, for example, by concentrating under reduced pressure or drying under reduced pressure at 20 to 40 ° C., to obtain the extract of the cow in liquid or powder form.

The nut extract can be obtained by performing an additional extraction process to increase the content of the active ingredient. In other words, by extracting the extract obtained using water, alcohol, or an aqueous solution of alcohol with an organic solvent such as n-hexane, ethyl acetate, butanol or the like as described above, a cow extract having a high content of the active ingredient is obtained. Can be.

For example, the nut extract is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ ~ C ₄ alcohol, and C _{1 ~} C ₄ alcohol aqueous solution; And (b) may be obtained by performing an extraction process comprising the step of extracting the extract obtained from step (a) with n-hexane. Extracting with n-hexane [step (b)] may be performed by sequentially adding water and n-hexane to the extract obtained from step (a) or simultaneously, and separating the n-hexane layer. The process may be performed once or multiple times, preferably two or more times. The obtained n-hexane layer can be obtained in a liquid or powder form by a conventional method, for example, by concentrating under reduced pressure or drying under reduced pressure.

In addition, the nut extract is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ ~ C ₄ alcohol, and C _{1 ~} C ₄ alcohol aqueous solution; (b ') adding water and n-hexane to the extract obtained from step (a) and separating the aqueous layer; And (c) adding ethyl acetate to the aqueous layer obtained from step (b '), and separating the ethyl acetate layer.

In addition, the nut extract is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ ~ C ₄ alcohol, and C _{1 ~} C ₄ alcohol aqueous solution; (b ') adding water and n-hexane to the extract obtained from step (a) and separating the aqueous layer; (c ') adding ethyl acetate to the aqueous layer obtained from step (b') and separating the aqueous layer; And (d) adding n-butanol to the aqueous layer obtained in step (c '), and separating the aqueous layer and the n-butanol layer.

The extract using the aqueous alcohol solution, n-hexane extract, ethyl acetate extract, and n-butanol extract may be performed by a series of continuous extraction process, an example of the continuous extraction process is shown in FIG. same.

The pharmaceutical composition of the present invention containing a nut extract as an active ingredient as described above inhibits the inflammatory response and also induces the activation of alkaline phosphatase to promote differentiation and mineralization of osteoblasts, thereby preventing or preventing various periodontal diseases. It can be used for treatment. The periodontal disease preferably includes periodontal inflammation, alveolar bone breakage or alveolar bone osteodystrophy, wherein the alveolar bone dysplasia is alveolar bone osteoporosis, alveolar osteomalacia. bone osteomalacia), or alveolar bone osteopenia.

The pharmaceutical composition of the present invention contains the nut extract as an active ingredient, may include a pharmaceutically acceptable carrier, powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols according to conventional methods Oral dosage forms, external preparations, and sterile injectable solutions. The pharmaceutically acceptable carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline Cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. Also included are diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like. Oral solid preparations include tablets, pills, powders, granules, capsules and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, and the like, and may include lubricants such as magnesium stearate, talc, and the like. Oral liquid preparations include suspensions, solvents, emulsions, syrups, and the like, and may include water, diluents such as liquid paraffin, wetting agents, sweeteners, fragrances, preservatives, and the like. Parenteral preparations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized preparations. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, and vegetable oils such as olive oil and ethyl oleate. Injectable esters such as the like and the like.

The dosage of the nut extract contained in the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. For example, the nut extract may be administered at a dose of 0.0001 to 100 mg / kg, preferably 0.01 to 100 mg / kg, and the administration may be administered once or several times a day. In addition, the pharmaceutical composition of the present invention may include 0.001 to 50% by weight of the nut extract, relative to the total weight of the composition.

The pharmaceutical compositions of the present invention can be administered to mammals, such as humans, by various routes, for example by oral, intravenous, intramuscular, or subcutaneous injection.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Preparation of Peanut Butter Extract

The cow was washed with running water to remove impurities and then ground to obtain a powder. 4 L of 70% ethanol aqueous solution was added to 400 g of the obtained powder, and extracted with stirring at room temperature for 7 days. The process was repeated three times, and the extracts were combined and concentrated under reduced pressure at 40 ° C. or lower to obtain 14.9 g (yield 3.75%) of the semi-solid extract.

About 5 g of the extract was suspended in 100 mL of water, then 100 mL of n-hexane was added, stirred at room temperature for 10 minutes, and the n-hexane layer was separated. The process was repeated twice. The resulting n-hexane layers were combined and concentrated under reduced pressure to obtain 1.98 g of hexane extract. Ethyl acetate and n-butanol were extracted in the remaining solution in the same manner, and each ethyl acetate layer, n-butanol layer, and residual water fraction layer were concentrated under reduced pressure, 0.45 g of ethyl acetate extract, 1.93 g of n-butanol extract, And 0.82 g of residual water fraction were obtained, respectively.

The manufacturing process of each of the extracts is shown in the schematic diagram as shown in FIG.

Example  2. By extract NO  Production inhibitory activity assessment

The activity of each extract obtained in Example 1, ie, ethanol extract, n-hexane extract, ethyl acetate extract, n-butanol extract, and water fraction, on NO production was measured. That is, Sherman et al. [Sherman et al., Biochem. Biophys. Res. Commun. 191, p1301-1308, 1993], a mouse monocyte / macrophage cell line RAW 264.7 (hereinafter RAW 274.7 cells, ATCC # TIB-71) which induced activation with lipopolysaccharide (LPS). The effect of each extract on the production of nitrate ions (NO ₃ ⁻ ), a stable oxidation product of NO, was determined as follows.

RAW 264.7 cells incubated in DMEM (Dulbecco's Modified Eagle's medium, Cambrex, USA) containing 10% fetal bovine serum were seeded in 48 well plates at a concentration of 2.5 × 10 ⁵ cells / ml, followed by LPS. (10 ng / ml) was added to induce activation and incubated for 24 hours in a carbon dioxide incubator (5% carbon dioxide, 95% relative humidity, 37 ° C.). In 50 ul of each culture, grease reagent (37.5 mM sulphanilic acid), 12.5 mM N- (1-naphthyl) ethylenediamine dihydrochloride (N- (1-naphthyl) ethylenediamine dihydride), 6.5 mM hydrochloric acid] 50 ul was added and reacted at room temperature for 10 minutes, and then absorbance was measured at 550 nm using a spectrophotometer. The NO ₃ using the nitrate produced by each concentration of 0 ~ 50 uM ^- Fill the absorbance coefficient, the generated NO ₃ in the RAW 264.7 ^{cell-concentration} was calculated concentration. Each extract prepared in Example 1 was dissolved in DMSO, and then added to RAW 264.7 cells at the final concentration of 5 ug / ml simultaneously with LPS to measure NO ₃ ⁻ concentration after the reaction. The result is shown in FIG.

As can be seen in Figure 2, the nut extracts obtained in Example 1 all showed NO production inhibitory activity, especially ethyl acetate extract and butanol extract showed a strong NO production inhibitory activity. Higher concentrations of NO inhibit osteoblast proliferation and differentiation, while lower concentrations of NO stimulate osteoblast function [Raltson SH et al. Endocrinol. 135, p 330-336, 1994; Riancho JA et al. J. Bone Miner Res. 10, p439-446, 1995], it can be seen that the extract of the cows has a prophylactic and therapeutic activity against periodontitis by inhibiting the inflammatory response.

Example  3. Cytotoxicity Test

Each dog extract obtained in Example 1 was administered at a concentration of 5 ug / ml, cells were cultured in the same manner as in Example 2, and cell viability was measured using an MTT assay. That is, cells treated with or without each cow extract for 24 hours in a 48 well microplate were incubated with a final concentration of 0.5 mg / ml MTT for 4 hours, then dissolved in 200 ul DMSO solution and spectrophotometrically at 570 nm. Measured. The result is shown in FIG. 3.

From the results of Figure 3, the cell viability after the administration of each of the nut extract (cell viability) is similar to the control group, it can be confirmed that the cytotoxicity by the nut extract in the above concentration is not induced.

< Example  4> Evaluation of alkaline phosphatase activity

<Cell Culture>

Mouse-derived osteoblasts (MC3T3-E1, US Cell Line Bank ATCC # CRL-2593) were treated with DMEM medium supplemented with 10% fetal bovine serum (FBS), 100 IU / ml penicillin and 100 ug / ml streptomycin. And cultured in a cell incubator maintained at 5% CO ₂ , 37 ℃. In order to rule out the effect of the components in the serum, the cow's ethanol extract obtained in Example 1 was added to the cell culture after replacing the medium with the serum added with 0.1% of bovine serum albumin instead of fetal calf serum 24 hours before administration. To identify osteoblast calcification nodules, the cells were administered at a concentration of 5 ug / ml, exchanged for 3 days, and replaced with a medium containing the nut extract and incubated for 25 days. In addition, ascorbic acid and beta-glycerophosphate, which are known to promote the formation of calcified nodules in osteoblasts, were added to the medium at 50 ug / ml and 10 mM concentrations, respectively. Used as. In addition, in order to confirm the alkaline phosphatase activity in osteoblasts, ethanol extract was treated with 2, 10 and 50 ug / ml concentration, and the change in activity according to the change in concentration was measured.

<Measurement of alkaline phosphatase activity>

The mouse-derived osteoblasts treated with the ethanol extract of the cow obtained in Example 1 were fixed with a 10% formaldehyde (neutral formaldehyde) solution, then treated for 5 minutes in an Alizarin red-S staining solution and washed. The formation of red calcified nodules was visually observed by comparison with the control, and the results are shown in FIG. 4.

In addition, after removing the medium from the cell culture, PBS (phosphate buffered saline) was added and scraped with a cell scraper (cell scraper). This was decomposed by ultrasound for 30 seconds, centrifuged at 5,000 rpm for 3 minutes, and the supernatant was taken as a sample for measurement. In order to measure the activity of the alkaline phosphatase, the absorbance was measured at 500 nm using an alkaline phosphatase assay kit (Yongdong Pharm.), And the results are shown in FIG. 5.

As can be seen in Figure 4, after culturing for 25 days after administration of the extract of the dog, the formation of calcified nodules was observed by a phase contrast microscope, and alizarin red-S staining was performed to confirm whether the nodules observed on the microscope were calcified nodules. The calcified nodules were observed in the negative control group, the positive control group and the cow extract treatment group, and the degree of nodule was increased in the cow control extract group compared to the negative control group and the positive control group.

In addition, as can be seen in Figure 5, the activity of alkaline phosphatase, which is an indicator of osteoblast differentiation, increased in a concentration-dependent manner compared to the control. Therefore, the extract of the cows induces increased activity of alkaline phosphatase, induces differentiation of osteoblasts and promotes mineralization of bone, resulting in alveolar bone breakage, alveolar osteoporosis, alveolar osteomalacia, alveolar osteopenia It can be seen that it has a prophylactic and therapeutic activity against alveolar bone dysplasia.

1 is a schematic diagram showing an example of the extraction process of the cow nut extract.

Figure 2 shows the results of measuring the change in nitrite produced when the cow extract was co-treated with LPS in RAW 264.7 cells.

Figure 3 shows the results of measuring the cell viability when treated with the extract of RAW 264.7 cells.

Figure 4 is a photograph of the calcified crystals formed by the nut extract extracted through alizarin red-S staining.

Figure 5 shows the results of measuring the activity of the alkaline phosphatase, which is a osteoblast differentiation marker by treating the nut extract.

Claims (8)

A pharmaceutical composition for the prevention or treatment of periodontal disease (periodontal disease), comprising Pharbitidis Semen extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the extract of the cow is obtained by extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ -C ₄ alcohol, and C ₁ -C ₄ alcohol aqueous solution. The pharmaceutical composition according to claim 2, wherein the extractant is an aqueous ethanol solution. The method of claim 1, wherein the extract of the cow is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ -C ₄ alcohol, and C ₁ -C ₄ alcohol aqueous solution; And (b) extracting the extract obtained from step (a) with n-hexane. The method of claim 1, wherein the extract of the cow is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ -C ₄ alcohol, and C ₁ -C ₄ alcohol aqueous solution; (b ') adding water and n-hexane to the extract obtained from step (a) and separating the aqueous layer; And (c) adding ethyl acetate to the aqueous layer obtained from step (b '), and performing an extraction process comprising separating the ethyl acetate layer. The method of claim 1, wherein the extract of the cow is (a) extracting the cow with an extraction solvent selected from the group consisting of water, C ₁ -C ₄ alcohol, and C ₁ -C ₄ alcohol aqueous solution; (b ') adding water and n-hexane to the extract obtained from step (a) and separating the aqueous layer; (c ') adding ethyl acetate to the aqueous layer obtained from step (b') and separating the aqueous layer; And (d) adding n-butanol to the aqueous layer obtained in step (c ') and separating the aqueous layer and the n-butanol layer. The pharmaceutical composition according to any one of claims 1 to 6, wherein the periodontal disease is periodontal inflammation, alveolar bone breakage, or alveolar bone osteodystrophy. 8. The pharmaceutical composition of claim 7, wherein the alveolar bone dysplasia is alveolar bone osteoporosis, alveolar bone osteomalacia, or alveolar bone osteopenia.

Images