KR20110080383A - Composition of comprising bee-venoms for neuroprotective properties - Google Patents

Abstract

PURPOSE: A composition containing bee venom for preventing and treating neurodegenerative diseases is provides to suppress amyloid accumulation. CONSTITUTION: A composition for preventing and treating neurodegenerative diseases contains bee venom as an active ingredient. The neurodegenerative diseases are dementia, Alzheimer's disease, or stroke. A functional food for preventing and treating neurodegenerative diseases contains 0.0001-10%(w/v) of bee venom as an active ingredient. The composition is manufactured in the form of oral formulation or parenteral formulation.

Description

Composition of comprising bee-venoms for neuroprotective properties

The present invention relates to a composition for preventing and treating neurodegenerative diseases comprising bee venom as an active ingredient.

As the standard of living and the development of the medical industry made it possible to treat cancer and other intractable diseases, the lifespan of humans increased, but the aging of the population resulted in the increase of chronic degenerative brain diseases such as stroke and degenerative neuropathy. The quality of life is showing a relative decline. Degenerative neurological disease represented by dementia is a chronic progressive degenerative brain disease, and stroke is also the third cause of death in the industrial society following cancer and heart attack. The prognosis is ischemic and hemorrhagic stroke caused by infarction. And stroke are emerging as serious medical and social problems. Despite the rapid development of modern medical and life sciences, brain disease can be understood at the molecular level, and the incidence and prevalence have continued to increase, and no clear preventive and therapeutic agents have been developed. The mechanisms by which degenerative brain diseases are known so far vary. Among the drugs currently on the market, it is found that the function of acetylcholine, a neurotransmitter involved in cell memory, is significantly reduced in brain tissues of Alzheimer's neurodegenerative diseases. Therefore, drugs that reduce the in vivo destruction of acetylcholine or acetylcholine precursors, ie, acetylcholinesterase enzyme inhibitors, have been developed as a method of supplementing the function of insufficient acetylcholine that acts centrally. Amyloid beta, another causative agent, is a neurodegenerative disorder in which neurofibrillary tangles and neural plaques are found in various parts of brain tissues of Alzheimer's patients. Known as a toxic fragment of. Therefore, drugs that can alleviate such amyloid accumulation have been developed. Recently, advances in molecular biology have made it possible to identify epidermal markers of cells that play an important role in immune-inflammatory mechanisms, and as the identity and function of immune-inflammatory mediators have been identified, immune-inflammatory mechanisms have been shown to affect neurological damage. In addition to non-specific secondary reactions, it may be involved in the early stages of neurodegenerative disease progression, and is known to actively control pathogenesis and actively participate in the survival and death of neurons. Thus, substances that regulate the function of microglia cells, which play a central role in the immune-inflammatory response, and amyloid P, the immune-inflammatory mediators NO, IL-1β, IL-6 and TNF-α acute phase response proteins And factors regulating cyclooxygenase and the like have been reported. Thus, understanding how glioblastoma cell activation is regulated, and inhibiting inflammatory mediators secreted from activated glioblastoma cells, uses an effective therapeutic approach to prevent neuronal damage in neurodegenerative diseases caused by multiple causes. To develop drugs.

While these drugs show some effect, they cause liver toxicity during long-term administration or have frequent side effects and recurrences. Thus, there are no clear degenerative neurodegenerative prevention and cure products.

Bee venom of domestic bees ( Apis mellifera ) can be obtained by collecting only pure bee venom without damaging bees by using bee venom gathering device using electric shock method, and freeze drying after removing foreign substances to obtain purified bee venom. The bee venom obtained in this way is composed of various components, and these components play the role of anti-inflammatory, antibacterial, analgesic and immune enhancement.

The effects of bee venom on degenerative neurological diseases have not been studied.

Therefore, the present inventors have conducted research to solve the above problems, as a result, bee venom inhibits the accumulation of miloids and destroys acetylcholine (Ach), a neurotransmitter involved in cell memory in brain tissue. By inhibiting cholinesterase (acetylcholinesterase, AchE) to confirm that the present invention can be used as an active ingredient for preventing and treating neurodegenerative diseases was completed the present invention.

Accordingly, an object of the present invention is to provide a composition for preventing and treating neurodegenerative diseases containing bee venom as an active ingredient.

The present invention is characterized by a composition for preventing and treating neurodegenerative diseases containing bee venom as an active ingredient.

The present invention also includes functional foods for the prevention and improvement of degenerative neurological diseases containing bee venom as an active ingredient.

Due to the development of a preventive and therapeutic agent for degenerative neurological diseases containing bee venom according to the present invention, it is thought to contribute greatly to the treatment of patients suffering from dementia, Alzheimer's, Parkin's, stroke, etc. It will contribute to the income increase of beekeeping farmers.

Figure 1 shows the amyloid beta aggregation inhibitory effect of bee venom.
Figure 2 shows the effect of bee venom acetylcholinesterase formation.
Figure 3 shows the cytotoxicity of bee venom on activated neuroinflammatory cells (BV-2).
Figure 4a shows the effect of inhibiting NO production after activated neuroinflammatory cells (BV-2) bee venom treatment.
Figure 4b shows the inhibitory effect of IL-1β production after activated neuroinflammatory cells (BV-2) bee venom treatment.
Figure 4c shows the inhibitory effect of IL-6 production after activated neuroinflammatory cells (BV-2) bee venom treatment.
Figure 4d shows the inhibitory effect of TNF-α production after activated neuroinflammatory cells (BV-2) bee venom treatment.

Hereinafter, the present invention will be described in detail.

The present invention includes bee venom that inhibits the accumulation of amyloid and inhibits choline esterase (acetylcholinesterase (AchE)), which destroys acetylcholine (Ach), a neurotransmitter involved in cell memory in brain tissue. It relates to a composition for preventing and treating neurodegenerative diseases.

In the present invention, bee venom strongly inhibited amyloid beta aggregation, which causes neurodegenerative diseases, and strongly inhibited the formation of acetylcholinesterase, which induces the destruction of acetylcholine, a neurotransmitter. In addition, it effectively inhibited the production of inflammatory cytokines such as NO, IL-1, IL-6, and TNF-α, which induce cranial nerve damage due to inflammatory responses.

As a result, it is possible to develop medicines containing bee venom and functional foods containing the same in the prevention and treatment of neurodegenerative diseases.

In the composition for preventing and treating neurodegenerative diseases according to the present invention, the content of bee venom is preferably 0.0001 to 10% (w / v) based on tablet bee venom, and more preferably 0.01 to 1% (w / v). It is suitable to contain. If less than 0.0001% (w / v) is used to reduce amyloid accumulation and inhibition of acetylcholinesterase activity can be halved efficacy, and when used more than 10% (w / v) may cause cytotoxicity.

The composition containing bee venom as an active ingredient can be used for the prevention and treatment of neurodegenerative diseases. As the neurodegenerative disease, dementia, Alzheimer's, Parkin's, stroke, etc. are preferable.

The composition according to the present invention may include, in addition to the active ingredient, a pharmaceutically acceptable carrier, excipient or diluent.

The compositions of the present invention may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

The composition of the present invention may be administered orally or parenterally, and may be used as an external skin preparation or injection such as a dressing agent, a patch, a bandage, an ointment, and the like during parenteral administration.

The dosage of the composition of the present invention varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate and severity of the disease of the patient, the daily dosage is gelatin degrading product 0.1 to 100 mg / kg, preferably 30 to 80 mg / kg, more preferably 50 to 60 mg / kg, based on the amount, can be administered 1 to 6 times per day.

In addition, it is also possible to use in combination with existing degenerative neurological diseases prevention and treatment.

In addition, the present invention includes a functional food for preventing and improving degenerative neurological disease containing bee venom as an active ingredient.

Functional foods according to the present invention, for example, chewing gum, caramel products, candy, ice cream, confectionery, such as various food products, soft drinks, mineral water, alcoholic beverages, beverage products such as vitamins and minerals Can be.

The bee venom of the present invention may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or hygiene).

The functional food of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients. The above-mentioned natural carbohydrates are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is preferably selected in the range of 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight, per 100 parts by weight of the health food of the present invention.

In addition to the above, the functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the functional food of the present invention may contain fruit flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components can be used independently or in combination.

Hereinafter, the present invention will be described in detail based on the following examples, but the present invention is not limited thereto.

Example 1 Measurement of Active Ingredients of Bee Venom

Western bee ( Apis meliffera L.) was collected by bee venom using the bee venom collection device, and the collected dried bee venom was dissolved in distilled water, centrifuged and filtered, and then lyophilized to obtain pure bee venom.

It was confirmed using liquid chromatography (AKTA explorer, Pharmacia, USA). Sephadex TM75 and Source 15RPC ST comum were used, and the melittin, apamin, phospholipase A2 content in the bee venom component was determined by the following equation (1).

The results are shown in Table 1 below.

In the bee venom melittin, apamin, phospholipase A2 content was used in the purified bee venom in the range as shown in Table 1.

[Equation 1]

ingredient content Apamine 2.8 ^a (2.0-3.2) ^b Phospholipase A2 12.8 (10.8-14.5) Melittin 50.7 (48.5 ～ 67.2) a: mean
b: Average range of the highest and lowest

Example 2 Amyloid-beta Aggregation Inhibitory Activity

Amyloid beta aggregation, a substance that causes degenerative neuropathy of bee venom, was measured by fluorescence analysis (ThioflavinT assay). Amyloidbeta peptide (Aβ1-42) was used at a concentration of 5 uM and stored at −80 ° C. before use. Bee venom was diluted in distilled water by test concentration. In order to specify the degree of inhibition of Aβ1-42 aggregation, 5 μl of bee venom solution was added to 92.5 μl of 0.01 M sodium phosphate buffer solution, 2.5 μl of 5 uM Aβ1-42 was added, followed by reaction at 37 ° C. for 26 hours, followed by 5 uM thioflavin. 2,000 μl of T was added and measured by fluorescence spectrometer.

The results are shown in FIG.

Example 3: Acetylcholinesterase Inhibitory Activity

It was confirmed that bee venom inhibited the activity of acetylcholinesterase, which induces the destruction of acetylcholine, a neurotransmitter. Acetylcholinesterase inhibitory activity measurement was modified Ellman method, the concentration of acetylcholinesterase was diluted to 0.03 unit and stored at -80 ℃ before use. Bee venom used 1,000 uM of acetylcholine iodide dissolved in 0.1 M sodium phosphate buffer (pH 8.0), and the color development reagent was 39.6 mg of 5,5-dithio-bis (2-nitrobenzoic acid). And 15 mg of sodium bicarbonate were prepared by dissolving 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 for 10 minutes at 37 ° C., 200 μl of bee venom for 3 minutes, and then absorbance at 412 nm.

The results are shown in FIG.

Example 4 Neuronal Inflammation Inhibitory Effect

(1) Neuronal cytotoxicity measurement

Mouse microglia (BV-2) cells, which are neurons, were passaged in a 37 ° C., 5% CO ₂ incubator using Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS and 1% penicillin / streptomycin. Used for. When cultured cells were grown to about 90% of the floor area, subcultures were used and cells in logarithmic growth phase were used for the experiment. BV-2 cells were aliquoted into 5 × 10 ⁵ cells / ml in 96 well plates, cultured for one day, adhered to fresh medium, and treated with various concentrations of bee venom. After 48 hours of incubation in a 37 ° C., 5% CO ₂ incubator, cell viability was treated with MTT (methylthiazol-2-yl-2.5-diphenyl tetrazolium bromide) reagent and the absorbance at 540 nm was measured by ELISA reader. Was evaluated.

The results are shown in Fig.

(2) NO, IL-1β, IL-6 and TNF-α expression test

Dissolve 1 ml of BV-2 cell line into 5 × 10 ⁵ cells / ml in a 24 well plate, incubate for 1 day, attach and replace with fresh medium, and show LPS (100 ng / ml) and no cytotoxic concentration. A range of bee venom was treated. After culturing in a 37 ° C., 5% CO ₂ incubator, the free NO, IL-1β, IL-6 and TNF-α were measured as cultures in each well. IL-1β, IL-6, and TNF-α were produced using an ELISA kit (Pierce Biotechnology. USA). After incubation for 20 hours with LPS and bee venom treated with BV-2 cell line, 50 ul of the culture medium was taken to react the primary and secondary antibodies in order and 3,3 ', 5,5'-tetramethyl benzidine was added. After the color development, the absorbance was read with an ELISA reader, and the IL-1β, IL-6 and TNF-α concentrations of the samples were converted from the graph using IL-1β, IL-6 and TNF-α standard solutions. 50 μl of culture medium incubated for 20 hours with LPS and bee venom treated with BV-2 cell line for NO measurement was added to a 96-well plate, the same amount of grease reagent was added and reacted at room temperature for 10 minutes, followed by absorbance at 540 nm with an ELISA reader. Was measured. A calibration curve was prepared using sodium nitrite as the NO ₂ standard.

The results are shown in Figures 4a to 4d.

Example 5: Toxicity Test of Bee Venom

To determine the toxicity of bee venom, bee venom 0.5-5 mg / kg was administered to 24 mice intraperitoneally to confirm survival for 24 hours after behavior observation. As a result, it was found that 3 out of 6 mice treated with bee venom 2 to 3 mg / kg survived and the remaining 3 were sacrificed. On the other hand, all the mice administered with a dose less than 1 mg / kg survived, and showed no statistically significant difference compared to the mice that did not receive the drug in the behavioral observation. Considering the above results, a toxic dose of approximately half of bee venom in mice is estimated to be 2.5 mg / kg.

Formulation Example 1 Preparation of Dressing Agent

Mix 0.1 g of bee venom obtained in Example 1 and 1 g of alginate evenly, mix 5 ml of distilled water, mix well, mix well until clear on a hot plate at 60 ° C, transfer to a glass plate, and thin using a glass rod. Spread it out. At this time, the ratio of bee venom and alginate and water can be adjusted according to the wound or burn site and condition to be treated, and the size, thickness, voids, etc. of the dressing can be prepared in any form. In addition, conventional gel matrix materials such as hyaluronic acid and the like can be used in place of alginate. The wound or burn dressing prepared as described above is illustrated in FIG. 5.

Formulation Example 2: Preparation of Ointment

Using the bee venom as an active ingredient, an ointment was prepared in the following composition.

[Furtherance]

Bee venom 1.0%, beeswax 10% by weight, polysorbate-60 5.0% by weight, Fiji-60 cured castor oil 2.0%, sorbitassesquioleate 0.5% by weight, petrolatum 5.0%, liquid paraffin 10.0%.

Formulation Example 3 Preparation of Patch

Using the bee venom as an active ingredient, a patch was prepared in the following composition.

[Furtherance]

Bee venom 1.2 wt%, Hexylene glycol 20.0 wt%, Dietylamine 0.7 wt%, Polyacrylic acid 1.0 wt%, Sodium sulfite 0.1 wt%, Polyoxyethylene lauryl ether 1.0 wt%, Polyhydroxyethylene cetyl stearyl ether 1.0 Wt%, viscous paraffin oil 2.5 wt%, caprylic acid ester / capric acid ester 2.5 wt%, polyethylene glycol-400 3.0 wt%, deionized water 100 wt%.

Formulation Example 4 Preparation of Functional Food

< Production of Drinks>

Bee Venom 100 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare beverage compositions of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (3)

Degenerative neurological disease prevention and treatment composition comprising bee venom as an active ingredient.
The composition of claim 1, wherein the neurodegenerative disease is dementia, Alzheimer's disease, Parkin's disease or paralysis.
Functional food for preventing and improving degenerative neurological disease, comprising bee venom as an active ingredient.

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