KR20180021569A - Composition containing a bee venom for prevention or treating periodontal diseases - Google Patents

Abstract

The present invention relates to a composition containing bee venom, which is isolated from venom sac of Apis cerana working bee, wherein the composition has excellent antibacterial effect against Prevotella intermedia, which is a causative organism of periodontal diseases, and can be easily used as a therapeutic agent for preventing and alleviating the periodontal diseases.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating periodontitis including bee venom as an active ingredient,

The present invention relates to a composition for the prevention or treatment of periodontitis including bee venom as an active ingredient, which is isolated from a bee venom of an Oriental bee ( Apis cerana L.).

There are four species of bees: Apis mellifera , Apis cerana , Apis dorsata , Apis florea , and so on. At present, there are western and oriental bees in Korea. Especially, oriental bees have been reared since the Goryeo Dynasty, and have been used as a needle therapy in the private sector and in the oriental medicine. However, the western varieties of bees were introduced into Korea in 1892 by German missionaries during the Emperor Gojong period of Joseon Dynasty.

Bee venom is contained in the bee venom of the bee, which is composed of about 40 substances and has been used for the treatment of diseases of the human body since BC. The bee venom shows differences in its components depending on the bees, and even in the same bee, there is a difference in composition depending on the age. Currently, studies on the action and mechanism of bee venom component, sensitivity and toxicity of bee venom, immunotherapy and treatment of diseases such as arthritis, herpes simplex, multiple sclerosis and tumor have been reported. Especially, It is known.

Many types of microorganisms live in human oral cavity, and oral microorganisms causing oral disease are known to exist in plaque which is a bacterial membrane formed between tooth and gum. The oral microorganisms produce toxins in the praes, and the toxins produced by the oral microorganisms enter the periodontal tissues to cause inflammation of the gingiva, destruction of the alveolar bone, and infection, resulting in periodontitis. When these bacteria form an acid, . As such, microorganisms distributed in the oral cavity cause postnatal and periodontal disease, and some of these microorganisms are opportunistic pathogens and are known to have the possibility of disease development. Several species are known as pathogenic microorganisms that cause periodontal disease in the oral cavity. Among them, Prevotella intermedia , Streptococcus mutans and Porphyromonas gingivalis have been known to cause periodontitis Is known to be the leading pathogen.

Periodontal disease is also a complex cause of symptoms and a variety of symptoms. One of the major causes of periodontitis is caused by microorganisms in the oral cavity. They primarily produce insoluble glucan by using saccharides, etc., and they attach to the surface of teeth like various cells to form tartar. These calculus causes tooth decay and periodontitis. Therefore, the most effective way to prevent periodontitis can be achieved by using a substance that inhibits the activity of these microorganisms and improves the blood flow of the inflamed gums. That is, in order to prevent periodontitis, an oral composition in which various medicines are blended for the purpose of preventive treatment is proposed.

On the other hand, antibiotics such as penicillin, erythromycin and tetracycline are effective for the inhibition of sterilization and growth among drugs used for the preventive treatment of periodontitis, but the adverse effects such as inducing the appearance of resistant bacteria are significant. In addition, antimicrobial agents such as chlorhexidine have an effect of inhibiting tooth decay, but they are known to be highly toxic. In recent years, in order to solve the above-mentioned problems, a composition for oral cavity excellent in pharmacological action and stability has been developed in addition to a conventional oral cleaning action.

Under these circumstances, the present inventors have studied various physiological activities possessed by tablet bee venom, and confirmed that bee venom of oriental bees has a large difference in content and composition from bees poisoned bees of western bees, ( Prevotella intermedia ), which is a major causative agent of periodontitis, is excellent in antibacterial effect.

Korean Registered Patent No. 10-1202302 (Name of invention: Composition for prevention and treatment of periodontal disease and tooth decay containing bee venom, Applicant: Dong Sung Pharmaceutical Co., Ltd., Korea, Registered: November 12, 2012) Korean Patent No. 10-1061257, entitled "Natural Antibiotics Using Bee Venom and Herbal Extracts as Active Ingredients and Method for Producing the Same, Applicant: Korea, Date of Registration: August 25, 2011)

It is an object of the present invention to provide a composition for the prevention or treatment of periodontitis containing bee venom as an active ingredient, which is isolated from a bee venom of an Oriental bee ( Apis cerana L.).

The present invention relates to a composition for the prevention or treatment of periodontitis including bee venom as an active ingredient, which is isolated from a bee venom of an Oriental bee ( Apis cerana L.).

The periodontitis is caused by Prevotella intermedia ) as causative bacteria.

In another aspect, the present invention provides a pharmaceutical preparation for oral use to which a fluorine compound is added to the composition.

The present invention also provides a pharmaceutical formulation for oral use in a formulation selected from the group consisting of toothpaste, mouthwash, mouthwash, and oral spray containing the composition.

The beeing concentration in the formulation is preferably 0.1 to 10 mg / ml.

The present invention also provides a health functional food for preventing or ameliorating periodontal disease, which comprises bee venom isolated from a bee venom of a bee bee of the Oriental species bee ( Apis cerana L.).

Hereinafter, the present invention will be described in detail.

The present invention relates to a composition for the prevention or treatment of periodontitis including bee venom as an active ingredient, which is isolated from a bee venom of an Oriental bee ( Apis cerana L.). The bee venom is a taboo bee venom, and the collecting process may be a method used in a conventional farmhouse. In particular, the tablet bee venom obtained by removing saccharides and volatile substances, which are foreign substances other than bee venom, is described in detail. In detail, the above-mentioned tablet bee poison is a foreign substance other than bee venom in bee venom, It may be obtained by removing saccharides and volatile substances. The periodontitis is characterized by causing Prevotella intermedia as a causative organism.

When the composition of the present invention is in the form of a liquid, the beesing concentration is preferably 0.1 to 10 mg / ml. When the bee venom concentration is less than 0.1 mg / ml, the effective ingredient in bee venom is changed and the pharmacological effect may be changed. If the bee venom concentration is more than 10 mg / ml, the effective ingredient in bee venom may not be stable. In addition, since bee venom of high concentration may be difficult to be dispersed outside the human or animal body, it is more preferable that the bee venom concentration is 10 mg / ml or less. On the other hand, it is preferable that the active concentration of the bee venom for inhibiting periodontitis is 0.1 to 5 占 퐂 / ml, and it is preferable to dilute the bee venom immediately before use.

The present invention also provides a pharmaceutical composition for the prevention or treatment of periodontal disease, which comprises bee venom isolated from the bee venom of the bees of the Oriental species bee ( Apis cerana L.). The bee venom may be added to the pharmaceutical composition of the present invention in an amount of 0.001 to 99.9% by weight.

The pharmaceutical compositions may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to conventional methods.

Such formulations may be manufactured in particular from the group consisting of toothpaste, mouthwash, mouthwash and mouthwash. In another aspect, the present invention provides a pharmaceutical preparation for oral use to which a fluorine compound capable of increasing the periodontitis inhibitory effect is added to the composition. The composition of the present invention containing bee venom can further enhance the periodontitis inhibitory effect of the fluorine compound.

Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations can be prepared by mixing the bee venom containing pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, sucrose Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition of the present invention will depend on the age, sex, body weight of the subject to be treated, the particular disease or condition to be treated, the severity of the disease or condition, the route of administration and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

The pharmaceutical composition of the present invention can be administered to mammals such as rats, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

The present invention also provides a health functional food for preventing or ameliorating periodontitis including bee venom and a food acceptable food-aid additive isolated from a bee venom of a bee-bee from an Oriental bee ( Apis cerana L.). The bee venom can be added to the health food in an amount of 0.001 to 99.9% by weight. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various kinds of drinks, meat, sausage, bread, Snacks, noodles, ice cream, dairy products, soups, ionic drinks, beverages, alcoholic beverages, gums, tea and vitamin complexes.

The present invention relates to a composition comprising bee venom isolated from a bee venom of an Oriental bee ( Apis cerana L.) worker bees as an active ingredient, wherein the composition has an antimicrobial effect on Prevotella intermedia which is a causative organism of periodontitis Can be easily used as a therapeutic agent for prevention and improvement of periodontitis. Korean Patent No. 10-1202302 discloses a composition for the prevention and treatment of periodontal disease and tooth decay containing bee venom. However, this is a technique related to bee venom of beeswax of western species, Was found to be significantly higher than bee venom of western species. Korean Patent No. 10-1061257 also discloses the antibacterial effect of bee venom but also discloses only the antibacterial effect of bee venom derived from bees waxy against diarrhea inducing bacteria.

Fig. 1 is a photograph showing the process of collecting bee venom in a work bee of oriental species.
FIG. 2 is a photograph showing the process of collecting bee venom in a worker bee of a Western species.
FIG. 3 is a liquid chromatogram showing the distribution of bee venom components collected from bees of oriental and honey bees (A-Oriental bee bee venom and B-Western bee bee venom).
Fig. 4 is a graph showing hyaluronidase inhibitory activity of bee venom collected from bees of oriental species.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art.

≪ Example 1: Preparation of tablet bee venom &

The bee venom was collected from the bees that were more than 15 days old by using the bee venom collecting device from the oriental bees ( Apis cerana L.) and the western bees ( Apis mellifera L.), and the bee venom was purified through the simple purification method to remove saccharides and volatile substances And then lyophilized to prepare a purified beacon product having pure water solubility. The bee venom was collected from bees that were over 15 days old because the bee venom was found to be the most in the bees of more than 15 days old in the case of the western bees. FIG. 1 shows the process of collecting bee venom of Oriental species, and FIG. 2 shows a process of collecting bee venom of bees of the Western species.

≪ Example 2: LC analysis for confirming the components of tablet bee venom >

When the bee venom contents collected in Example 1 were confirmed, it was confirmed that bee venom contents of Oriental and Western bees differed as shown in Table 1 below.

Type of bee Bee venom content (mg / h) Oriental bell bees worker 0.2 Western bees 0.3

The difference in the content of bee venom according to the kind of bees may be due to the difference in effective ingredients contained in the bee venom. Therefore, 40 mg of the purified bee venom collected in Example 1 was dissolved in 10 ml of the third distilled water, , And the components of bee venom were compared and confirmed by liquid chromatography (LC). The LC analyzer used was the AKTA Explorer 10 model and equipped with a superdex G200 column. The mobile phase was 0.1M Ammonium formate, pH 4.5. The phase detection limit was calculated according to the ICH guidelines (International Conference on Harmonization Guideline).

FIG. 3A shows the results of the analysis of the bee venom solution of oriental species, and FIG. 3B shows the results of the analysis of the bees poison solution of the bees of the Western species. 3A and 3B, the peaks of the bee venom in bee venom of oriental and honey bees are different from each other, and it is confirmed that there is a difference in the content of each component contained in the bee venom.

≪ Example 3: Confirmation of antibacterial effect of tablet bee venom &

Prevotella intermedia was purchased from the Korea Research Institute of Bioscience and Biotechnology to confirm the antibacterial effect of the tablets bee venom collected from the bees of Oriental bees with more than 15 days old. (MIC) and minimum inhibitory concentration (MBC) were measured after incubation in a trypticase soy agar medium.

The minimal inhibitory concentration assay was performed using a broth-dilution method. The aqueous solution of bee venom was diluted to a concentration of 0 to 20 μg / ml, and each strain (bacterium) was used at a concentration adjusted to O.D.600 = 0.2. 100 ml of TSB (trypticase soy broth), 50 ml of bee venom solution and 50 ml of the dilution solution of the bean culture used in the present invention, which was used in the present invention, were sequentially added to a 96-well plate (96-well plate, Falcon, USA) After the addition, incubation was carried out at 37 캜 for 24 hours while measuring the absorbance value using a UV-spectrophotometer every 4 hours. At this time, the minimum concentration at which no bacterial growth was detected on the growth curve of the bacteria was set as the minimum growth inhibitory concentration.

For the minimum inhibition concentration, 50 ml of the cultured bacterial sample at each time zone was taken on the TSA plate to which hemin menadione was added, and cultured for 48 hours at 37 ° C in the analysis of the minimum inhibitory concentration. colony counts were observed, the minimum concentration at which the bacteria were completely killed (99.9%) was set as the minimum inhibitory concentration. The results are shown in Table 2 below.

Condition MIC (占 퐂 / ml) for Preboter Intermedia MBC (占 퐂 / ml) for Prebotella intermedia Bees of oriental bell bee 4.3 ± 0.3 23.5 ± 1.9 Bee's bee 15.2 ± 0.6 85.4 ± 2.5

Referring to the results of Table 2 above, it is confirmed that the tablets of bee poison collected from the bees of oriental bees have excellent antimicrobial activity against Prebotella intermedia, which causes periodontitis.

The antibacterial duration (PAE) was also confirmed by treating the bee venom collected from the bees of Oriental species of bees with twice the concentration of MIC (8.6 占 퐂 / ml) to Prebotella intermedia. , It can be confirmed that the antimicrobial activity is maintained for about 4 hours even at a low concentration of 8.6 ㎍ / ml, and the antibacterial power retention time is remarkably longer than that of the Western species.

Condition PAE for Prevotella Intermedia Bees of oriental bell bee 3.9 ± 0.6 hours Bee's bee 1.2 ± 0.3 hours

<Example 4: Confirmation of hyaluronidase inhibitory activity against tablet bee venom>

The hyaluronidase inhibitory activity of the oriental bees obtained in Example 1 at a bee venom concentration of 0.01 to 100 占 퐂 / ml was measured using a hyaluronidase assay kit (amsbio), and the result was shown in Fig.

4, when the bee venom of bees of Oriental species is 0.1 ~ 5 / / ㎖, the hyaluronidase inhibitory activity is very high, and if the bee venom content is very low or high, the inhibition of hyaluronidase Activity is lowered. On the other hand, as shown in Table 4, when hyaluronidase inhibitory activity at 0.1 占 퐂 / ml was compared between bee venom of bees of Oriental and honey bees, it was also confirmed that hyaluronidase inhibitory activity of bee venom of oriental bees More excellent.

Condition Hyaluronidase inhibitory activity (%) at 0.1 / / ml Bees of oriental bell bee 61.4 Bee's bee 15.2

<Example 5: Confirmation of stability of tablet bee poisoning>

The bee venom component in solid ecology is highly stabilized, but when powder or solid tablet bee venom is dissolved in water or physiological saline, it needs to be stably maintained without changing the effective ingredient. Therefore, it was confirmed whether the concentration of melittin, which is a standard substance of bee venom in aqueous solution, changes with time.

The concentration of melittin in 50 ml (1 mg / ml) of the bee venom aqueous solution of Oriental bees stored at room temperature was measured. As a result, the concentration of melittin was measured by collecting samples in the 'upper layer / middle layer / lower layer' The concentration was found to be maintained evenly for 24 hours (no result graph attached). Therefore, it can be seen that 1 mg / ml of the aqueous solution of bean poison is stably stored at room temperature, and thus it can be confirmed that bee venom is stable because it is used as a pharmaceutical composition or food. In addition, 50 ml of purified bee venom was prepared at a concentration of 0.1 to 100 mg / ml, and the result of measurement of the content of melitin in 'upper layer / middle layer / lower layer' after 6 hours by the same method was confirmed. Bee venom is most unstable in its condition, and it is confirmed that bee venom of 0.1 ~ 10 mg / ml is stable, and it is most stable at 1 mg / ml. Therefore, when bee venom is present in an aqueous solution, it is preferable to prepare 0.1 to 10 mg / ml and appropriately dilute it before use.

&Lt; Preparation Example 1 >

100 mg of purified bee venom of Oriental species bees of Example 1, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes to prepare a liquid for a dog gag that can be diluted 10 times or more with water.

&Lt; Preparation Example 2 > Preparation of toothpaste &

A toothpaste for the prevention of periodontitis was prepared by incorporating the bee venom of oriental species of Example 1 into a dentifrice composition so as to contain 1 mg / ml of tablet bee venom.

Claims (10)

A composition for preventing or treating periodontal disease, characterized in that the bee venom isolated from the bee venom of an insect bee ( Apis cerana L.) is contained as an active ingredient. The method according to claim 1,
Wherein the bee venom is a tablet bee gum obtained by removing saccharides and volatile substances, which are foreign substances other than bee venom, to prevent or treat periodontal disease. The method according to claim 1,
A composition for preventing or treating periodontal disease, wherein the periodontitis is caused by Prevotella intermedia . A pharmaceutical preparation for oral use to which a fluorine compound is added to the composition according to any one of claims 1 to 3. A pharmaceutical formulation for oral use in the form of a toothpaste, a mouth rinse, an oral cleanser and an oral spray containing the composition according to any one of claims 1 to 3. 6. The method of claim 5,
Wherein the beeing concentration in the formulation is 0.1 to 10 mg / ml. A functional food for preventing or ameliorating periodontal disease, characterized by comprising bee venom isolated from a bee venom of a bee beetle ( Apis cerana L.). 8. The method of claim 7,
Wherein the bee venom is a tablet bee venom obtained by removing sugars and volatile substances, which are foreign substances other than bee venom, to prevent or ameliorate periodontal disease. 8. The method of claim 7,
Wherein the periodontitis is caused by Prevotella intermedia . The present invention relates to a health functional food for preventing or ameliorating periodontal disease. 8. The method of claim 7,
The health functional food is selected from the group consisting of various drinks, meats, sausages, bread, candy, snacks, noodles, ice cream, dairy products, soups, ionic drinks, beverages, alcoholic beverages, gums, tea and vitamins Health functional food for prevention or improvement of periodontitis.

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