KR101972070B1 - Composition comprising bee venom isolated from Vespa mandarinia worker for prevention or treating avian influenza - Google Patents

Abstract

The present invention relates to a composition containing purified bee venom collected from a long-life wasp work bee, wherein the composition containing the purified bee venom has an inhibitory effect of xanthine oxide, and a urate-induced MMP-9 metallopeptidase-9). It is confirmed that this effect is superior to the bee venom of beeswax of western species. Therefore, the tablet bee venom collected from the longevity wasp bee can be usefully used as a pharmaceutical composition or health functional food that can prevent, improve or treat gout.

Description

[0001] The present invention relates to a composition for preventing or treating gout containing bee venom isolated from a worker bee of a longevity wasp. (Composition for bee venom isolated from Vespa mandarinia worker for prevention or treating avian influenza)

The present invention relates to a composition for the prevention or treatment of gout containing bee venom isolated from a beetle of longevity wasp.

Most of the bee venom used in Korea is isolated from the bees of Apis mellifera , and has been mainly used as a needle therapy in oriental medicine. 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. In recent years, bee venom components have been confirmed to be different from each other, and their physiological activity studies are also continuing. However, the research on bee venom collected from the worker bees of loneliness was not progressing.

Recently, dietary changes such as meat intake and excessive drinking are increasing gout. Gout is an arthritis caused by monosodium urate crystals (MSU) produced by hyperuricemia. The remaining 10-15% of hyperuricemia except for reduced renal excretion is caused by excessive uric acid production Most of which are caused by genetic defects in the purine metabolism, problems with ATP metabolism, and diseases in which the rate of cell turnover increases.

The inventors of the present invention have been able to complete the present invention by studying various physiological activities of tablet bee venom collected from these various bees, confirming that bee venom of beeswax of longevity wasp is excellent in preventing or treating gout.

Korean Patent Laid-Open No. 10-1999-0039050 (Title: Acne Prevention and Therapeutic Agent Containing Horseradish Extract, Applicant: LG Chemical Co., Ltd., Disclosure Date: June 05, 1999) Korean Patent Laid-Open No. 10-2012-0100450 (Title of the Invention: Method of Manufacturing Functional Cosmetic Composition Using Longevity Wasp, Applicant: Jung Wook Chul, Publication Date: September 12, 2012)

The effects of apjugusun and bee poison treatment on ventilation induced rats, Thesis of Kyunghee University (MS), 1999.

It is an object of the present invention to provide a composition for the prevention or treatment of gout containing bee venom separated from a beeswax of longevity wasp.

The present invention relates to a composition for preventing or treating gout containing bee venom.

The bee venom can be a tablets rod isolated from a worker's beak of a lenticular wasp ( Vespa mandarinia ).

The bee venom may contain mandaractoxin as an active ingredient.

The bee venom is characterized by having an inhibitory effect of xanthine oxide.

The bee venom can inhibit the expression of MMP-9 (Matrix metallopeptidase-9) induced by uric acid.

The present invention also relates to a health functional food for preventing or ameliorating gout containing bee venom.

The health functional food is characterized in that it is selected from various drinks, meat, sausage, bread, candy, snack, noodle, ice cream, dairy product, soup, ionic drink, beverage, alcoholic beverage, gum, tea and vitamin complex.

Hereinafter, the present invention will be described in detail.

The bee venom of the present invention is collected from the beeswax of the longevity wasp collected after August, which is the summer adult time. More specifically, the bee venom of the present invention is a taboo bee poisoning, and the bee venom can be captured by capturing a wasp collecting in a wasp house or a bee barrel and separating the viscose or centrifuging the wasp by crushing. In particular, the tablet bee venom obtained by removing saccharides and volatile substances, which are foreign substances other than bee venom, in detail, the purified bee venom may be obtained by removing saccharides and volatile substances other than bee venom from bee venom, .

The present invention also provides a pharmaceutical composition for preventing or treating gout comprising an active ingredient of bee venom isolated from a gallbladder of the long-lived wasp. 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 composition of the present invention 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 a conventional method. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, 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 and the like, which may contain 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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex and weight of the subject to be treated, the specific disease or condition to be treated, the severity of the disease or pathological 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. Since the composition of the present invention has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

In addition, the present invention provides a health functional food for preventing or improving gout comprising bee venom and a food acceptable food-aid additive separated from the bees of said long wasp. At this time, the bee venom isolated from the bees of the longevity wasp may be added in an amount of 0.001 to 100% by weight to the health functional food of the present invention. The health functional food of the present invention includes forms such as tablets, capsules, pills, liquids and the like. Examples of foods to which the wasps poison of the present invention can be added include various kinds of drinks such as various kinds of drinks, meat, sausage, , Snacks, noodles, ice cream, dairy products, soups, ionic drinks, beverages, alcoholic beverages, gums, tea and vitamin complexes.

The present invention relates to a composition containing purified bee venom collected from a long-life wasp work bee, wherein the composition containing the purified bee venom has an inhibitory effect of xanthine oxide, and a urate-induced MMP-9 metallopeptidase-9). It is confirmed that this effect is superior to the bee venom of beeswax of western species. Therefore, the tablet bee venom collected from the longevity wasp bee can be usefully used as a pharmaceutical composition or health functional food that can prevent, improve or treat gout.

As for the bee venom of the longevity wasp, the effect of treating acne is disclosed in Korean Patent Laid-Open Publication No. 10-1999-0039050, and Korean Patent Laid-Open Publication No. 10-2012-0100450 discloses a cosmetic composition having an extract of wasp However, to date, there has been no evidence of the effect of bee venom inhibition on the ventilation of loneliness wasp.

1 is a photograph showing a tablet bee venom collected from a worker bees of a loneliness wasp.
Fig. 2 is a photograph showing a tablet bee venom collected from the beeswax of a Western species of bees.
Figure 3 shows the UPLC results for tablet bee venom collected from the worker bees of the longevity wasp (red marked peak-mandarotoxin).
4 is an ELISA showing the expression amount of MMP-9 according to the tablet bee venom treatment of the wasp.
FIG. 5 is a result of silver staining performed by electrophoresis of bee venom and mandarotoxin of longevity wasp.

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 &

Bee venom was collected from beeswax collected from beeswax collected from beeswax in the beeswax of longevity wasp, wyoming bee, oriental bee and western bees. Purification of bee venom by simple purification of sugar beet and volatile substances and freeze - . Longevity wasps were collected from the worker bees of the longevity wasps collected in August, the summer adult period, and the other beeons were collected from the worker bees 15 days old and older. In the case of western species, bee venom was found to be the most abundant in bees older than 15 days, and this phenomenon also occurred in other bees. FIG. 1 shows a process of collecting bee venom of a longevity wasp, and FIG. 2 shows a bee venom collecting process of a beeswax of a wyoming bee.

Table 1 shows the content of tablet bee venom per one bee collected in this manner, and it is confirmed that the content of tablet bee venom is different according to the bees.

Type of bee Western bees Oriental bell bees worker Westerner Longevity wasp Amount of tablets bee venom (mg / grains) 0.29 0.2 0.18 4.2

< Example  2. Tablets of beijing  Checking the active ingredients and quality>

Example 2-1. Determine the content of mandarotoxin

The quality of the collected bee venom was confirmed by confirming the content of mandarotoxin (MDTX) in bee venom by performing UPLC (Ultra Performance Liquid chromatography). 40 mg of purified bee venom (test bee venom) collected from longevity wasp was collected in 10 ml of distilled water and filtered through a PTFE 0.2 μm filter. The reference mandarotoxin (Reference.MD., 82545-77-5 Mandaratoxin) Ml and filtered using the same filter as the test bee venom. Detection limits were calculated according to ICH (International Conference on Harmonization) guidelines using Sepadex C-50 columns with good separation of enzyme peaks. The UPLC conditions were performed based on the following Table 2, and the results are shown in FIG.

The content of mandarin toxin in bee venom of longevity wasp was 11.3 ± 1.9%, which indicates that high quality bee venom was collected.

device Water I class column Sepadex C-50 Column temperature 50 ℃ Flow rate 0.5 ml / min Volume 1.0 ml Detection wavelength 280 nm This phase ammonium acetate buffer (0.05 M, pH 5.5)

In addition, SDS-PAGE analysis confirmed bee venom and mandarotoxin in the lobster wasp, SDS-polyacrylamide gels were prepared according to the method of Laemmli (1970) with 15% separation gel and 5% staking gel. Bee venom and mandarotoxin samples were diluted to 1 μg / ml with PBS and mixed with 4 × loading buffer. 10 ㎕ of sample was loaded with 7 ㎕ of size marker. Next, protein bands were identified by silver staining after separation at 100 V 400 mA for 1 hour and 30 minutes using Mini protein electrophoresis assay unit (Bio-Rad Laboratories, USA). The results are shown in Fig. 5, wherein CBV in Fig. 5 represents honeybee bee venom and MDTX represents mandarotoxin.

Example 2-2. Comparing the components of bee venom and bees

Table 3 compares the components of the bee venom of the Western and Oriental bees by reference to the description in the literature (Table 3) (Venom of the hymenoptera, by Tom Piek, Academic press, 1986; Journal of the Hymenoptera venom review focusing on Apis mellifera. (1997), and the degenerative changes of the vascular endothelial growth factor (VEGF) in vivo and in vivo.

ingredient Seoyangjong bee beeper Longevity Wasp Phospholipase A2 ○ × Hyaluronidase ○ × Acid phosphatase ○ × Melittin ○ × Dipeptidyleptidase IV ○ × 가타성 처리ase inhibitator ○ × CUB-serine protease ○ × Carboxylesterase ○ × Carboxypeptidase ○ × Carbohydrate-rich protein ○ × Phospholipase A1B × ○ Antigen 5 × ○ Histamine ○ ○ Dopamine ○ × Norepinephrine ○ × Vespakinin × ○ Mastoparan-M × ○ Ves-CP-M × ○

As shown in Table 3, the distribution of the active ingredients contained in the tablet bee venom collected from the western bees and the longevity wasps were found to be completely different from each other, and the pharmacological / physiological effects of the beeswax were expected to be different.

&Lt; Example 3: Activity confirmation of xanthine oxidase >

The inhibitory activity of xanthine oxidase, which is a gout inducer, was confirmed by treatment of purified bee venom with xanthine oxidase. For this purpose, 0.1 ml of Xanthine Oxidase (0.25 unit / ml) and 0.1 ml of various beeswax bees in various concentrations were added to 1 ml of the substrate solution in which 2 mM xanthine was dissolved in 0.1 M potassium phosphate buffer (pH 7.5). At this time, 0.1 ml of distilled water was added to the negative control, and allopurinol (0.5 μg / ml), which is a typical gout therapeutic agent, was used for the positive control. The reaction was then continued for 5 minutes at 37 ° C and 1 ml of 20% (v / v) trichloroacetic acid was added to terminate the reaction. After centrifugation at 3,500 rpm for 15 minutes, the absorbance was measured at 292 nm and the amount of uric acid produced in the reaction solution was determined according to the following equation (1).

(1): inhibition rate (%) = (1 - absorbance of purified bee venom / control absorbance) x 100

The results are shown in Table 4 below.

Condition voice
Control positivity
Control Bee venom treatment of longevity wasp Bee-bee treatment of western bees 0.1 mu g / ml 1 mu g / ml 10 mu g / ml 1 mu g / ml Uric acid (占 퐂 / ml) 23.7 6.5 15.6 10.2 7.1 16.3 % Inhibition - 71.5 34.1 56.9 70.4 33.2

Referring to Table 4, it was confirmed that the concentration of uric acid was decreased in a concentration-dependent manner to the bee venom of beeswax of long-lived wasp by the inhibition of xanthine oxidase activity.

Example 4 Confirmation of Matrix Metallopeptidase-9 Expression Inhibitory Activity Induced by Urea Salt

RAW 264.7 cells were purchased from Korean Cell Line Bank. The cells were subcultured in RPMI-1640 medium containing 10% FBS, and then grown in RPMI-1640 medium supplemented with 1% FBS. The expression level of MMP-9 was analyzed by ELISA (abcam, ab100732) after treatment with 2 mg / ml of endotoxin-free MSU (monosodium urate) Respectively.

Referring to FIG. 4, the amount of MMP-9 expression is remarkably reduced depending on the concentration of the bee venom of the long-life wasp, and thus, it is considered that the bee venom is effective for treatment of gout.

&Lt; Formulation Example 1 >

Formulation Example 1-1. Manufacture of tablets

200 g of purified bee venom collected from the worker bees of the longevity wasp of the present invention was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

Formulation Example 1-2. Injection preparation

1 g of tablets bee venom, 0.6 g of sodium chloride and 0.1 g of ascorbic acid, which were collected from the worker bees of the longevity wasp of the present invention, 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.

<Formulation Example 2: Food Preparation>

Formulation Example 2-1. Manufacture of cooking seasonings

Tablet bean sprouts collected from the worker bees of the longevity wasp of the present invention were added to the cooking sauce in an amount of 1% by weight to prepare sauces for health promotion.

Formulation Example 2-2. Manufacture of flour food products

The bee venom obtained from the beeswax of the present invention was added to the wheat flour in an amount of 0.1% by weight, and bread, cake, cookies, crackers and noodles were prepared using the mixture to prepare a food for health promotion.

Preparation Example 2-3. Manufacture of soups and gravies

The health enhancing soup and the juice were prepared by adding 0.1% by weight of purified beacon collected from the worker bees of the longevity wasp of the present invention to soup and juice.

Formulation Example 2-4. Manufacture of dairy products

Tablet bean poison taken from a worker bee of the longevity wasp of the present invention was added to milk in an amount of 0.1 wt%, and various dairy products such as butter and ice cream were prepared using the milk.

Formulation Example 2-5. Vegetable juice manufacturing

Vegetable juice for health promotion was prepared by adding 0.5 g of tablets bee venom collected from a worker bees of the longevity wasp of the present invention to 1,000 ml of tomato juice or carrot juice.

Formulation Example 2-6. Manufacture of fruit juice

Health enhancing fruit juice was prepared by adding 0.1 g of purified bee venom collected from a worker bee of the longevity wasp of the present invention to 1,000 ml of apple juice or grape juice.

Claims (11)

( Vespa mandarinia ) which is effective in inhibiting xanthine oxide or inhibiting the expression of uric acid-induced MMP-9 (Matrix metallopeptidase-9) Or a pharmaceutically acceptable salt thereof. delete The method according to claim 1,
The bee venom is a composition for preventing or treating gout, which comprises mandaroxin as an active ingredient. delete delete ( Vespa mandarinia ) which is effective in inhibiting xanthine oxide or inhibiting the expression of uric acid-induced MMP-9 (Matrix metallopeptidase-9) Wherein the functional food for preventing or improving gout is provided. delete The method according to claim 6,
The bee venom is a health functional food for preventing or improving gout, which comprises mandaroxin as an active ingredient. delete delete The method according to claim 6,
The health functional food is selected from a variety of drinks, meats, sausages, breads, candies, snacks, noodles, ice cream, dairy products, soups, ionic drinks, beverages, alcoholic beverages, gums, tea and vitamin complexes. Or health functional food for improvement.

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