KR101576352B1 - Pharmaceutical composition comprising extract of lycium root bark as an effective ingredient for the prevention or the treatment of the periodontitis diseases - Google Patents

Abstract

There is provided a pharmaceutical composition for the prevention or treatment of periodontal disease comprising an extract of Ganoderma lucidum as an active ingredient.

Description

FIELD OF THE INVENTION The present invention relates to an oral composition for preventing or treating periodontal disease,

The present invention relates to a pharmaceutical composition capable of preventing or treating periodontal disease, and more particularly, to a pharmaceutical composition for preventing or treating periodontal disease, which comprises an extract of Ganoderma lucidum as an active ingredient. Specifically, the present invention relates to a pharmaceutical composition for preventing or treating periodontal diseases such as gingivitis, periodontitis, and alveolar bone destruction.

Periodontal disease refers to any disease that occurs in periodontal mattes, and can be divided into gingivitis and periodontitis depending on the severity of the disease. It is a relatively light and fast-acting periodontal disease called gingivitis that is limited to gingiva or soft tissue, and it is called periodontitis when the inflammation progresses to the gums and the gums. Periodontal disease is a major cause of tooth loss after adulthood and is considered to be important as it enters the aging society.

There is a V-shaped gap between the gum (gums) and the teeth. Periodontal disease is caused by bacteria attacking the lower part of the gum line of this sulcus and damaging the periodontal ligament and adjacent tissues. As the inflammation progresses and more tissue is damaged, the groove develops into a periodontal pocket, and the more the periodontal disease becomes, the deeper the depth of the periodontal pocket becomes. Periodontal disease is a periodontal disease that causes inflammation and bone loss in the periodontal ligament.

The periodontal tissue consists of alveolar bone, gingiva, and periodontal ligament. The direct cause of periodontal disease is the plaque, which is formed continuously in the teeth. The plaque is sticky and colorless, and it becomes tartar when it is hardened without being removed. When plaque and tartar build up, the gums fall from the teeth, and as a result, a gap is formed and a periodontal pouch is formed between the tooth and the gum.

As the inflammation progresses, gums and teeth become more open and germs that cause periodontal disease multiply and periodontitis develops. Such inflammation reduces osteoporosis, increases the ability to absorb bone, destroys alveolar bone and periodontal ligament, and eventually shakes teeth.

That is, it is understood that periodontal disease is expressed through the following process.

Normal teeth → oral microbial proliferation → adhesion → dental plaque formation (plaque) → tartar formation → inflammation progression → destruction of alveolar bone and periodontal ligament → periodontal disease

Local factors related to periodontal disease are the accumulation of plaque in the periodontal pouch, which becomes a host of anaerobic gram negative bacteria present in the surrounding area. Bacteria in the gut create toxins and free oxygen that breaks the gums, destroying the tissue surrounding the teeth, or stimulating the immune system, causing inflammation along with periodontal tissue destruction by various actions of the stimulated immune system. This causes irritation of the gums, redness, swelling easily, and often blood.

The incidence of periodontal disease due to changes in diet and social stress is increasing. As the age increases, congenital and acquired alveolar bone weakens and periodontal disease develops. Alveolar bone is remodeled by osteoblast-induced osteoclast and osteoclast-induced bone resorption. This metabolic process is regulated by the physical effects and local factors. When the balance is broken and bone absorption exceeds the bone formation and the bone mass decreases, the alveolar bone formation disorder occurs.

Recently, research has been reported that there is a high correlation between periodontal disease and systemic diseases such as dementia, atherosclerosis, myocardial infarction, and brain dysfunction. Currently, non-surgical treatment (non-steroidal anti-inflammatory drug) and surgical treatment (dental plaque removal, gingival curettage, periodontal regeneration) are known to treat periodontal disease. However, the most effective treatment, surgical treatment, is the hassle of having to visit the dentist and there is a limit to how long after the disease has progressed. Because of the side effects of non-steroidal anti-inflammatory drugs, treatment of periodontal disease is not effective and general periodontal disease is chronic disease.

To overcome the limitations of these surgical and non-surgical treatments and to improve the prevention and treatment effects of periodontal disease, it is required to develop a preparation that combines safety with the destruction of alveolar bone tissue and the recovery of lost tissue.

The gill bark is the bark of the gugija root. Gugija is a deciduous shrub that is planted on the roadside or on the roadside in various parts of Korea. It grows about 1 ~ 4m long and grows more than 1m long. The ends of the branches are stretched, and the short branches are often deformed by the thorns. Leaves are ovate lanceolate, ovate, have no sawtooth on the edge, and long branch leaves are common horticulture, but short branches produce several leaves. Flowers are purple, bell shaped, 5 for upper part, 5 for stamen, 1 for pistil, and the fruit ripens red to long ovate, 1.5 ~ 2cm long. Flowering period is from October to October, and fertility period is from October to November.

The fasciocutaneous follicles are cylindrical, semicylindrical or sculpted, and have a length of 3 to 10 cm, a width of 5 to 15 mm, and a thickness of 1 to 3 mm. Outer surface is yellow to orange-yellow, roughly irregularly torn, and the bark is easily scaly. The inside surface is yellowish white to yellowish yellow, has a relatively flat and thin vertical pattern. The body is lightweight and vulnerable in quality, making it easy to break. The folded surface is gray to yellowish brown, not fibrous, and the vagina is light and rough. The cross section of this drug is microscopically seen on the outer layer. The anchovy layer consists of two to three rows of cork tissue layers, and the innermost one layer forms a perfect, well-coordinated cork band, occurring in the deep part of the body. Decaded cadavers and repair cells can be seen in the fallow layer. The calves occupy half of the thickness of the skin, and the repair has become one row of cells. Flow cells contain calcium oxalate and pre-break. It can be seen that the fibers and stones are scattered. The fibers are individual or bundled and the cell walls are cotton or slightly cotton.

Gyobolpyu is a taste of Korean medicine, sweet and cool, the quality is cold, there is no venom. It works in the lungs and kidneys. It has the effect of lowering the heat of the blood and lungs and releasing the heat of osteoporosis (hot bones), so that the body feels weak fever, internal fever to the bone, hot symptoms (blemish), nosebleeds, Cough, asthma, and other pathologies.

The fact that the extracts of gingival bark have an effect on gingivitis, periodontitis and the like is not known to date, and thus a therapeutic composition containing it as a main active ingredient is not known at present.

Korean Patent Publication No. 2001-0036152 " Method for producing plant extract powder and oral composition containing plant extract powder prepared by this method ", includes plant extracts of 38 kinds of licorice and the like. However, the above patent is based on antibacterial action only as a test example of green tea extract, and does not disclose any reason why the remaining 38 kinds are effective for periodontal disease, and the patent does not disclose specific examples of toothpaste, mouthwash, mouthwash And the like are all applied to the inside of oral cavity, whereas the present invention is to prevent and treat periodontal disease by oral administration, and it can be understood that the dosage form and route of administration are different.

In the meantime, Gyobolpi and Gugija refer to the roots and fruits of the same plants, respectively, in the clinical herbology (Shin Min-gyo and Clinical Herbology, Yeonglim, 2006.) and the Chinese Medicine Dictionary (Zhejiang New School, Chinese Medicine Dictionary, Shanghai Science and Technology Publishing Co., The Chinese pharmacopoeia (Chinese Pharmacopoeia, China Pharmacopoeia, 22010.) has two different pharmacological properties, which are different from each other and have different pharmacological properties. Although it is originated from one plant, it is different from the symptom of weakness and efficacy treatment, and Korean medicine and Chinese medicine classify it as different medicine.

Accordingly, the inventors of the present invention have shown that the extract of Gingkoffi prevents the destruction of the alveolar bone, effectively inhibits the proliferation of gingival cell lines, and can effectively regenerate periodontal tissue while performing various physiological activities of Gingkoffia extract. And thus the present invention has been completed.

Korean Patent Laid-Open Publication No. 2001-0036152

There is provided an oral administration composition for effectively preventing or treating periodontal disease.

The present invention provides a pharmaceutical composition for preventing or treating gingivitis, periodontitis, and alveolar bone destruction containing an extract of gingival bark as an active ingredient.

The extract according to the present invention is effective in preventing alveolar bone destruction, promoting bone formation, and inhibiting the growth of gingival germ cells. Also, it has no toxicity and is safe for human body, so it is harmless even for a long time.

It is possible to provide a pharmaceutical composition which is effective for the treatment and prevention of periodontal diseases including periodontal disease and gingivitis by reducing inflammation in the oral cavity and having few side effects.

FIG. 1 is a graph showing that the extract according to the present invention has no cytotoxicity on rat macrophage cell line (Raw 264.7 cell) after 24 hours.
FIG. 2 is a graph showing that the extract according to the present invention shows no cytotoxicity on rat macrophage cell line (Raw 264.7 cell) after 48 hours.
FIG. 3 is a graph showing the effect of the extract according to the present invention on cell proliferation after 24 hours in a gingival cell line (YD-38 cell).
4 is a graph showing the effect of the extract according to the present invention on cell proliferation after 48 hours in a gingival cell line (YD-38 cell).
FIG. 5 is a photograph showing the effect of the extract according to the present invention on the inhibition of osteoclast differentiation by concentration.
FIG. 6 is a graph showing the effect of the extract of the present invention on osteoclast differentiation inhibition by measuring the number of osteoclasts.
7 is a graph showing the activity of the extract of the present invention for inhibiting osteoclast differentiation.
FIG. 8 is a graph showing the distance between the root and the root ligament of the mesial and apical roots of the mesial and distal roots in a rat model in which the extract according to the present invention induces periodontal disease.
9 is a graph showing the degree of histopathological inflammation in a rat model in which the extract according to the present invention causes periodontal disease.
10 is a histological photograph showing the extent of inflammation infiltration in a rat model causing periodontal disease of the extract according to the present invention.

The corolla used in the present invention can be cultivated or commercially available corpuscles can be used without limitation. For example, the corpuscle is washed and dried, followed by pulverization with a dry herbal powder or a pulverizer to obtain pulverized herbal medicine, To 20 times, preferably from 5 times to 10 times the amount of the extracting solvent in a solvent such as water, a lower alcohol such as ethanol or methanol or a mixed solvent thereof at a temperature of about 50 to 150 DEG C for about 1 to 3 hours, , Reflux cooling extraction, ultrasonic extraction, and the like, extracted once to five times, filtered under reduced pressure, and lyophilized to obtain an extract of Ganoderma lucidum.

As the extraction solvent used in the present invention, water, a lower alcohol, or a mixed solvent thereof may be used. The lower alcohol may be an alcohol having 1 to 6 carbon atoms, for example, methanol, ethanol, propanol, isopropanol, butanol, pentanol, heptanol and the like.

A pharmaceutical composition for preventing or treating alveolar bone destruction or gingivitis including a pharmacologically acceptable carrier, diluent or excipient containing the obtained extract of corollas as an active ingredient can be prepared.

The composition of the present invention comprises 1 to 99% by weight, preferably 10 to 90% by weight of said extract, based on the total weight of the composition.

The present invention relates to a method of treating periodontal disease, which comprises administering to a patient in need thereof an effective amount of a periodontal disease, which is effectively selected from gingivitis, periodontitis, alveolar bone destruction, alveolar osteoporosis, alveolar osteoporosis, alveolar bone loss, alveolar bone formation, gingival bleeding, Addition or deletion of the constituent herbicides, suitably adjusting the mixing ratio thereof, and may be suitably used for the purpose of the present invention.

The composition comprising the herbal extract of the present invention may further comprise an appropriate carrier, excipient or diluent according to a conventional method. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols or the like, oral preparations, suppositories or sterilized injection solutions according to a conventional method .

More specifically, when formulating the composition, it can be prepared using a diluent or an excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, syrups and the like, and various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used diluents such as water and liquid paraffin .

The present invention further provides the use of a gingival extract for the prevention or treatment of periodontal disease selected from gingivitis, periodontal disease, alveolar bone destruction, alveolar bone disorder, alveolar bone disorder, alveolar bone disorder, alveolar bone disorder, gingival bleeding, do.

The present invention also relates to a pharmaceutical composition comprising the herbal extract of the present invention in a therapeutically effective amount to a mammal including a human body. The gingivitis, periodontitis, alveolar bone destruction, alveolar bone disorder, alveolar bone loss, A method for preventing and treating periodontal disease selected from alveolar bone formation disorder, gum bleeding, and bad breath due to gingival inflammation.

The dosage of the composition containing the herbal extract of the present invention may vary depending on the age, sex and body weight of the patient, but is generally in the range of 1 mg to 10 g / kg, preferably 10 mg to 1000 mg / Kg can be administered once or several times per day. The dosage can also be increased or decreased according to route of administration, degree of disease, sex, weight, age, health condition, diet, administration time, administration method, excretion rate and the like. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

Since the herbal medicine extract of the present invention has little toxicity and side effects, it can be safely used for long-term use for preventive purposes.

The present inventors confirmed selective and excellent effects of inhibiting the proliferation of gingival keratinocytes, inhibiting osteoclastogenesis and toxicity, in order to confirm the preventive or therapeutic effect of periodontal disease on the gingival fibroblast extract.

Example 1: Preparation of extract

Gingival fibroblasts were purchased from the medicines of Daejeon. After finely crushing the dried product, the purified water corresponding to 10 times the total weight of the pulverized product is added and refluxed at an extraction temperature of 100 ° C for about 2 hours, filtered and lyophilized to obtain a leaf extract. The yield of the extract was 5.3%.

Example 2: Preparation of extract

Gingival fibroblasts were purchased from the medicines of Daejeon. The dried product is finely ground and then added to 95% ethanol corresponding to 10 times the total weight of the pulverized product, refluxed for extraction at 80-85 ° C for about 2 hours, filtered, and lyophilized to obtain an extract of the crustacea. The yield of the extract was 3.30%.

Comparative Examples 1 to 20

The water extracts and 95% ethanol extracts were prepared by using the same procedures as in Examples 1 and 2, but using the same conditions as in the following table for licorice, gobon, angelica, windbreak, white gingiva, ginger, .

Herbal medicine Extraction solvent Extraction temperature and time Extraction method yield Comparative Example 1 licorice water 95-100 ° C, 2 hr Reflux extraction 21.46% Comparative Example 2 licorice 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 19.70% Comparative Example 3 Gobon water 95-100 ° C, 2 hr Reflux extraction 17.56% Comparative Example 4 Gobon 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 19.42% Comparative Example 5 Angelica water 95-100 ° C, 2 hr Reflux extraction 20.32% Comparative Example 6 Angelica 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 30.54% Comparative Example 7 Wind wind water 95-100 ° C, 2 hr Reflux extraction 27.39% Comparative Example 8 Wind wind 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 21.31% Comparative Example 9 blank water 95-100 ° C, 2 hr Reflux extraction 43.08% Comparative Example 10 blank 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 23.30% Comparative Example 11 ginger water 95-100 ° C, 2 hr Reflux extraction 2.89% Comparative Example 12 ginger 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 1.30% Comparative Example 13 Seisin water 95-100 ° C, 2 hr Reflux extraction 14.69% Comparative Example 14 Seisin 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 6.50% Comparative Example 15 riding water 95-100 ° C, 2 hr Reflux extraction 9.76% Comparative Example 16 riding 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 10.20% Comparative Example 17 Mold water 95-100 ° C, 2 hr Reflux extraction 6.90% Comparative Example 18 Mold 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 2.69% Comparative Example 19 Black water 95-100 ° C, 2 hr Reflux extraction 1.20% Comparative Example 20 Black 95% ethanol 80-85 < 0 > C, 2hr Reflux extraction 3.70%

Experimental Example 1 Toxicity experiment

The MTT assay, which is commonly used for cytotoxicity evaluation, was used to determine the stability of the extracts. The extracts of the bark bark extract prepared in Example 1 were dissolved in 10% DMSO solution and further dissolved in tertiary distilled water.

Mouse macrophage cell line Raw 264.7 macrophage cell (American Cell Line Bank ATCC # TIB-71) was used, and 1% antibiotic (Penicillin G / Streptomycin) and 10% FBS were added to DMEM medium for cell culture. These cell lines were cultured at 37 ° C in 5% CO 2 and were used for experiments while subculturing every 2-3 days.

To determine the toxicity of the extracts from Raw 264.7 cells, each experimental group and control group was cultured for 24 hours and 48 hours, followed by MTT reagent at 5 mg / ml for 4 hours. After incubation, the supernatant was removed and DMSO was added to dissolve the cells. The formazan produced by MTT reduction in the intracellular mitochondria was measured at 570 nm using an ELISA microplate reader (680, Bio-Rad, Tokyo, Japan) Respectively. The average absorbance value of each sample group was obtained and compared with the absorbance value of the control group, the cytotoxicity was examined. The results are shown in Figs. 1 and 2.

From the results of FIGS. 1 and 2, it was confirmed that the cell viability after administration of the extracts of GPC was similar to that of the control group, so that the cytotoxicity by the GPC extract was not induced. The extracts prepared in Example 2 and Comparative Examples 1 to 20 were evaluated for toxicity in the same manner as above, and the results are summarized in Table 2 below.

Herbal medicine Concentration (ug / ml) Crude extracting solvent In macrophages
Cytotoxicity (%) Gill pulpit Example 1 500 Hot water extract 158.13 1000 150.13 Example 2 500 95% ethanol extract 88.14 1000 91.35 licorice Comparative Example 1 500 Hot water extract 117.27 1000 110.34 Comparative Example 2 500 95% ethanol extract 3.41 1000 4.71 Gobon Comparative Example 3 500 Hot water extract 87.60 1000 88.54 Comparative Example 4 500 95% ethanol extract 64.77 1000 62.35 Angelica Comparative Example 5 500 Hot water extract 107.34 1000 106.98 Comparative Example 6 500 95% ethanol extract 2.71 1000 3.85 Wind wind Comparative Example 7 500 Hot water extract 67.89 1000 68.34 Comparative Example 8 500 95% ethanol extract 80.19 1000 66.58 blank Comparative Example 9 500 Hot water extract 95.24 1000 78.25 Comparative Example 10 500 95% ethanol extract 78.34 1000 81.34 ginger Comparative Example 11 500 Hot water extract 66.35 1000 65.36 Comparative Example 12 500 95% ethanol extract 6.89 1000 8.84 Seisin Comparative Example 13 500 Hot water extract 90.25 1000 88.65 Comparative Example 14 500 95% ethanol extract 18.65 1000 16.65 riding Comparative Example 15 500 Hot water extract 98.56 1000 90.25 Comparative Example 16 500 95% ethanol extract 11.39 1000 9.95 Mold Comparative Example 17 500 Hot water extract 5.29 1000 8.48 Comparative Example 18 500 95% ethanol extract 100.09 1000 113.17 · Black Pearl Comparative Example 19 500 Hot water extract 113.12 1000 99.65 Comparative Example 20 500 95% ethanol extract 80.03 1000 77.59

Experimental Example 2: Gingival cell proliferation experiment (in vitro)

In order to examine the effect of the extract according to the present invention on the proliferation and cell activity of gingival germ cells, the following experiment was conducted.

MTT assay was used to evaluate the proliferation of gingival cell proliferation. The extracts of the extracts of Example 1 were dissolved in 10% DMSO solution and further dissolved in tertiary distilled water.

Yi-38 cell (human oral squamous cell carcinoma cell line), a gingivitis cell, was purchased from Korea cell line bank (Seoul, Korea). Cell culture was performed in RPMI medium supplemented with 1% antibiotic Penicillin G / Streptomycin ) And 10% FBS. These cell lines were cultured at 37 ° C in 5% CO 2 and were used for experiments while subculturing every 2-3 days.

In order to confirm the inhibition of cell growth, the extracts were incubated for 24 hours and 48 hours in MTT reagent and 4 hours, respectively. After incubation, the supernatant was removed and DMSO was added to dissolve the cells. The formazan produced by MTT reduction in the intracellular mitochondria was measured at 570 nm using an ELISA microplate reader (680, Bio-Rad, Tokyo, Japan) Respectively. The average absorbance value of each sample group was obtained and compared with the absorbance value of the control group, the cytotoxicity was examined. The results are shown in FIGS. 3 and 4.

From the results shown in Figs. 3 and 4, it was confirmed that the production, growth, and cell activity of the gingival goblet cells were suppressed after administration of the extracts of the gingival bark. The extracts prepared in Example 2 and Comparative Examples 1 to 20 were tested for gingival cell proliferation in the same manner as above, and the results are summarized in Table 3 below.

Herbal medicine Concentration (ug / ml) Crude extracting solvent Growth inhibition in gingivitis cells (%) Gill pulpit Example 1 500 Hot water extract 79.52 1000 42.98 Example 2 500 95% ethanol extract 19.13 1000 22.38 licorice Comparative Example 1 500 Hot water extract 91.54 1000 87.36 Comparative Example 2 500 95% ethanol extract 28.45 1000 23.11 Gobon Comparative Example 3 500 Hot water extract 88.47 1000 83.54 Comparative Example 4 500 95% ethanol extract 18.16 1000 11.24 Angelica Comparative Example 5 500 Hot water extract 88.47 1000 89.35 Comparative Example 6 500 95% ethanol extract 24.36 1000 10.88 Wind wind Comparative Example 7 500 Hot water extract 48.54 1000 45.55 Comparative Example 8 500 95% ethanol extract 36.69 1000 41.34 blank Comparative Example 9 500 Hot water extract 71.04 1000 73.25 Comparative Example 10 500 95% ethanol extract 62.43 1000 65.35 ginger Comparative Example 11 500 Hot water extract 6.20 1000 11.35 Comparative Example 12 500 95% ethanol extract 3.38 1000 5.59 Seisin Comparative Example 13 500 Hot water extract 67.36 1000 55.54 Comparative Example 14 500 95% ethanol extract 11.34 1000 12.16 riding Comparative Example 15 500 Hot water extract 22.43 1000 33.58 Comparative Example 16 500 95% ethanol extract 36.69 1000 45.62 Mold Comparative Example 17 500 Hot water extract 84.84 1000 81.45 Comparative Example 18 500 95% ethanol extract 80.27 1000 81.77 · Black Pearl Comparative Example 19 500 Hot water extract 153.57 1000 127.25 Comparative Example 20 500 95% ethanol extract 47.43 1000 33.58

Experimental Example 3 Measurement of osteoclast production

In order to differentiate Raw 264.7 cells into osteoclasts, the cells were seeded in a 96-well plate at a density of 104 per well, and cultured for 24 hours using α-MEM containing 10% FBS as a culture medium. RANKL of 100 ng / ml was replaced with the culture solution containing the above-mentioned extract, which was continuously diluted to a constant concentration in Example 1, and cultured for 7 days while confirming the state of differentiation.

Adherent cells in the cultured cells were washed with PBS, fixed with citrate-acetate-formaldehyde, and subjected to TRAP staining to evaluate osteoclast production.

TRAP staining was carried out according to the manufacturer's instructions (TRAP staining kit, KAMIYA), using Naphthol AS-BI Phosphate as a substrate and Fast Garnet GBC solution as a staining agent Respectively. After staining, TRAP - positive polynuclear cells with three or more nuclei were counted using an optical microscope and used as an index of osteoclastogenesis.

As a result, in FIGS. 5 and 6, the number of TRAP (+) polynuclear cells in the group treated with the extract according to the present invention was significantly reduced as compared with that in the control group. Thus, it was confirmed that the extract according to the present invention inhibited osteoclast formation.

EXPERIMENTAL EXAMPLE 4 Measurement of Polyclonal Cell Activity as TRAP (+)

In order to differentiate Raw 264.7 cells into osteoclasts, the cells were seeded in a 96-well plate at a density of 104 per well, and cultured for 24 hours using α-MEM containing 10% FBS as a culture medium. 100ng / ml of RANKL and the extract of Example 1, which had been continuously diluted to a constant concentration, and cultured for 7 days while confirming the state of differentiation.

TRAP activity was measured by the TRAP staining kit, KAMIYA. After incubation for 3 hours in a supernatant of cultured cells, a buffer containing a Tartrate-containing buffer and a chromogenic substrate, it was measured at 504 nm using a spectrophotometer and expressed as a percentage of the control.

As a result, in the group treated with the extract according to the present invention, TRAP activity was inhibited in comparison with the control group in FIG. Thus, it was confirmed that the extract of the present invention inhibited the activity of osteoclasts.

Experimental Example 5 Animal Experiment

Periodontal disease is an inflammatory disease that leads to the destruction of connective tissue and bone tissue supporting the tooth. Early lesions are those in which the upper part of the gum is separated from the teeth, the inflammation spreads to the alveolar bone, and at the end, the gingiva falls and the alveolar bone is destroyed by exposing the root.

In this model, periodontal lesions were induced by ligating the perimeter of the periodontal ligament of the left mandibular molar with a suture. The extracts were administered to the rats in which periodontal disease was induced, and then the mandible with the teeth was observed by imaging and the cut surfaces were stained to observe the tissues.

The extracts of Example 1 were administered at a dose of 500 mg / kg / day for 7 days after the termination of the purification period of the rats in the rabbits. At the 8th day after administration, zoletil (1 mg / ml) kg was intraperitoneally administered. Sterile nylon (3-0) was used for oral administration of the rim gingiva and periodontal border for 21 days after ligation.

On the day of the autopsy, all animals were planed with CO2 gas, and the mandible of each animal was separated, and the gingival region was separated. After fixation with 10% neutral buffered formalin solution, Micro CT was performed and histopathological examination was performed using H & E staining. However, only the left mandible that caused periodontitis was measured in all animals.

 Imaging pathologic analysis of periodontal ligament was performed by measuring the distance between the mesiobuccal root (MBR) of the mandibular first molar and the periodontal ligament (PDL) of the distal bifurcation root (DBR) And compared the induced group with the experimental group based on the normal group. The results are shown in Fig.

 In FIG. 9-10, the pathologic and histopathological findings of the lesions showed significant differences compared to the control group and the normal control group. Particularly, there was a statistically significant difference in the results of the periodontitis-induced group, the normal group, and the administration group in the DBR.

Thus, it was confirmed that the extract of the present invention is effective for the prevention and treatment of periodontal disease in rats that induced periodontal disease.

Experimental Example 6 Toxicity experiment

The extract of Example 1 of the present invention was administered to Sprague-Dawley rats in single oral doses to conduct toxicity tests. The control group was divided into two groups: 1, 2, and 5 mg / kg, 2,500 and 5,000 mg / kg, respectively. General symptoms, weight and autopsy findings were observed and compared with the vehicle control group.

The results showed that no animals died during the test period, and no abnormalities in weight change associated with the administration of the test substance were observed as a result of observing the weight change. No abnormal findings were observed in the autopsy results.

As a result, when the extract of Example 1 of the present invention was administered to Sprague-Dawley rats in single oral doses, the approximate lethal dose (ALD) was judged to exceed 5,000 mg / kg in both sexes, Material.

Claims (6)

delete delete delete delete A pharmaceutical composition for oral administration for the prevention or treatment of gingivitis or periodontitis comprising water as an effective ingredient, extract of Ganoderma lucidum extracted with water, C1-C6 alcohols or a mixed solvent thereof. delete

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