KR101972074B1 - Composition comprising bee venom isolated from Vespa mandarinia worker for prevention or treating Alzheimer's disease - Google Patents

Abstract

The present invention relates to a composition containing purified bee venom collected from a longevity wasp work bee, wherein the bee poisoned composition is excellent in an aggregation inhibition effect of amyloid beta and an acetylcholine estrogen inhibitory effect, Can be used as a pharmaceutical composition, a food composition, etc. which can prevent or treat Alzheimer's disease.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating Alzheimer's disease, which comprises bee venom isolated from a worker bee of a loneliness wasp,

The present invention relates to a composition for preventing or treating Alzheimer's disease 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.

In Korea, the prevalence of dementia is also rapidly increasing due to the rapid aging of the population. Although the awareness of dementia risk is high, the perception of dementia symptom is lower than the world average. In particular, dementia is a symptom that gradually develops, so if you are diagnosed early and have a good diagnosis, there is a possibility of improvement. However, many people miss the treatment period and develop into severe dementia symptom in a short time. The early diagnosis of dementia is a very important factor as it has a very large impact and is developing into a social problem. About 70% of patients with Alzheimer's disease are dementia patients, and a number of studies have found proteins involved in Alzheimer's disease, but the cause of the disease is not yet clear. However, neurofibrillary tangles formed by amyloid plaques and hyperphosphorylated tau protein filaments in nerve cells, which were formed by accumulation of beta-amyloid peptides between nerve cells, Have been reported. Beta amyloid is composed of 36-43 amino acids of peptide. Accumulation of beta amyloid peptide is an early etiology that precedes development of brain damage and nerve fiber masses. Methods of inhibiting or eliminating amyloid formation are being studied as a treatment for Alzheimer's disease.

The present inventors have studied various physiological activities of tablet bee venom collected from these various bees, and confirmed that the bee venom of beeswax in the longevity wasp is excellent in the prevention or treatment of Alzheimer's disease, thereby completing the present invention.

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) Korean Registered Patent No. 10-1219551 (Title of the invention: Composition for preventing and treating degenerative neurological diseases containing bee venom as an active ingredient Applicant: Korea, registered on January 02, 2013) Korean Patent Laid-Open No. 10-2009-0096019 (Title of the Invention: Composition for preventing or treating Alzheimer's disease containing bee venom as an active ingredient Applicant: Dongguk University Industry-Academic Cooperation Agency Publication Date: September 10, 2009)

It is an object of the present invention to provide a composition for preventing or treating Alzheimer's disease containing bee venom isolated from a beetle of longevity wasp.

The present invention relates to a composition for preventing or treating Alzheimer's disease 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 in that it has an anti-aggregation effect of amyloid beta.

The bee venom has an acetylcholinesterase inhibitory activity.

The present invention also relates to a health functional food for preventing or improving Alzheimer's disease 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 Alzheimer's disease comprising bee venom isolated from the capsules 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.

The present invention also provides a health functional food for preventing or ameliorating Alzheimer's disease, comprising bee venom and a food-acceptable food-aid additive separated from the bees of the long-life 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 the foods to which bee venom of the longevity wasp of the present invention can be added include various drinks, meat, sausages, Breads, candies, 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 longevity wasp work bee, wherein the bee poisoned composition is excellent in an aggregation inhibition effect of amyloid beta and an acetylcholine estrogen inhibitory effect, Can be used as a pharmaceutical composition, a food composition, etc. which can prevent or treat Alzheimer's disease.

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, the inhibitory effect of bee venom of Alzheimer's disease in the lobster wasp has not been confirmed to date. On the other hand, Korean Patent No. 10-1219551 and Korean Patent Laid-Open No. 10-2009-0096019 disclose the therapeutic effect of bee venom derived from beeswax beetle, which is mainly composed of melitin, on Alzheimer's disease, Derived bee venom is more superior to the bee venom derived from the bees wax of the western species of bee than the bee venom of the amyloid beta and the inhibitory effect of acetylcholinesterase.

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 a graph showing the inhibition effect of amyloid beta on the coagulation inhibition of tablet bee venom collected from a worker bees of longevity wasps.
5 is a graph showing the acetylcholinesterase inhibitory activity of tablet bee venom collected from the bees of the loneliness wasp.
FIG. 6 is a graph showing the effect of inhibiting NO production in neurons of purified bee venom collected from the bees of long-lived wasp.
FIG. 7 is a graph showing the inhibitory effect of TNF-.alpha. On neurons in purified bee venom collected from a beeswax of longevity wasps.
FIG. 8 is a graph showing the effect of inhibiting IL-1β generation in neurons of purified bee venom collected from the bees of long-lived wasp.
9 is a graph showing the effect of inhibiting IL-6 production in neurons of purified bee venom collected from a worker bees of longevity wasps.
FIG. 10 shows results of silver staining performed by electrophoresis of bee venom and mandarotoxin of longevity wasps.

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. 10, wherein CBV in FIG. 10 represents wasp honeybee 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: Coagulation inhibitory activity of amyloid beta >

 The coagulation - mitigating effect of amyloid beta, a causative agent of neurodegenerative diseases in tablets bee venom, was investigated by ThioflavinT assay. Amyloid beta peptide (Aβ1-42) was used at a concentration of 5 μM and stored at -80 ° C before use. Tablet bee venom collected from longevity wasp was diluted in distilled water by test concentration. To determine the degree of inhibition of A.beta.1-42 aggregation, 5 .mu.l of bee venom solution was added to 92.5 .mu.l of 0.01 M sodium phosphate buffer solution, 2.5 .mu.l of 5 .mu.M Aβ1-42 was added, the mixture was reacted at 37.degree. C. for 26 hours, T was added thereto and the result was measured by a fluorescence spectrometer.

The results are shown in FIG. 4. Referring to FIG. 4, it is confirmed that the tablet bee venom collected from the longevity wasp is excellent in the aggregation inhibition effect of amyloid beta.

At the same time, the same experiment was conducted using bee venom of beeswax of western species, and the bee venom of bees on western bees showed about 50% of aggregation of amyloid beta at a concentration of 0.1 / / ㎖, %, Indicating that the bee venom of the longevity wasp has a better inhibitory effect.

&Lt; Example 4. Confirmation of acetylcholinesterase inhibitory activity >

It was confirmed that the tablet bee venom collected from the wasp was inhibited the activity of acetylcholinesterase inducing the destruction of the neurotransmitter acetylcholine. The acetylcholinesterase inhibitory activity was measured by the modified Ellman method. The concentration of acetylcholinesterase was diluted to 0.03 unit and stored at -80 ° C before use. Purified bee venom collected from longevity wasp beetles was treated with 1,000 μM acetylcholine iodide dissolved in 0.1 M sodium phosphate buffer (pH 8.0), and the coloring reagent used was 39.6 mg of 5,5-dithio-bis (2-nitrobenzoic acid) and 15 mg of sodium bicarbonate were dissolved in 10 ml of 0.1 M sodium phosphate buffer (pH 7.0). 200 μl of DTNB solution and 100 μl of enzyme were added to 2 ml of sodium phosphate buffer (pH 8.0), followed by reaction at 37 ° C for 10 minutes. 200 μl of bee venom was added thereto for 3 minutes, and the absorbance was measured at 412 nm.

The results are shown in FIG. 5. Referring to FIG. 5, it is confirmed that the tablet bee venom collected from the longevity wasp is excellent in the inhibitory effect of acetylcholinesterase.

<Example 5: Confirmation of nerve cell inflammation inhibitory effect>

Mouse neurons (BV-2) cells were subcultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS and 1% penicillin / streptomycin at 37 ° C in a 5% CO ₂ incubator Lt; / RTI &gt; When the cultured cells reached 90% of the floor area, they were subcultured and cells of logarithmic growth period were used for the experiment. BV-2 cells were plated at a density of 5 × 10 ⁵ cells / ml in a 96-well plate, cultured for one day, adhered, replaced with fresh medium, and treated with various concentrations of bee venom. Cell viability was measured by MTT (methylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) reagent after incubation at 37 ° C in a 5% CO ₂ incubator for 48 hours. The absorbance was measured at 540 nm using an ELISA reader The cytotoxicity was evaluated. BV-2 cells were plated on a 24-well plate at a density of 5 × 10 ⁵ cells / ml, and the cells were cultured for 1 day. The cells were then replaced with fresh medium. LPS (100 ng / Range bee venom. After culturing in a 5% CO ₂ incubator at 37 ° C, NO, IL-1β, IL-6 and TNF-α released in the culture medium of each well were measured. IL-1β, IL-6 and TNF-α production were measured by ELISA kit (Pierce Biotechnology, USA). The BV-2 cell line was treated with LPS and bee venom for 20 hours, and then 50 μl of the culture was taken to react the primary antibody and the secondary antibody in order and 3,3 ', 5,5'-tetramethyl benzidine The IL-1β, IL-6 and TNF-α concentrations of the samples were calculated from the graphs using IL-1β, IL-6 and TNF-α standard solutions after reading the absorbance with an ELISA reader. NO measurement The BV-2 cell line was treated with LPS and bee venom for 20 hours, and 50 μl of the culture was placed in a 96-well plate. The same amount of the grease reagent was added, and the mixture was incubated at room temperature for 10 minutes. Were measured. Sodium nitrite was used as a NO ₂ standard and a calibration curve was prepared.

The results are shown in FIG. 6 to FIG. 9, wherein the substances produced in the inflammatory reaction such as NO (FIG. 6), TNF-α (FIG. 7), IL-1β It is confirmed that it is remarkably reduced by processing the tablet bee venom collected from the bee venom. Therefore, it is considered that the tablet bee can be used as a composition capable of inhibiting inflammation in neuronal cells and treating Alzheimer's disease.

&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)

A composition for preventing or treating Alzheimer's disease, which comprises purified bee venom isolated from a gallbladder of a long-lived wase ( Vespa mandarinia ) worker having an anti-aggregation inhibitory activity or acetylcholinesterase inhibitory activity of amyloid beta. delete The method according to claim 1,
The bee venom is a composition for preventing or treating Alzheimer's disease, which comprises mandaroxin as an active ingredient. delete delete A health functional food for preventing or ameliorating Alzheimer's disease, characterized by containing purified bee venom isolated from a gallbladder of a long-lived wase ( Vespa mandarinia ) worker having an agglutination inhibitory effect or an acetylcholinesterase inhibitory activity of amyloid beta. delete The method according to claim 6,
The bee venom is a health functional food for preventing or ameliorating Alzheimer's disease, 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 vitamins. Health functional foods for prevention or improvement.

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