KR20190127410A - Composition for preventing and/or treating a neurodegenerative disease comprising extract or fraction of Aster koraiensis Nakai - Google Patents

Abstract

The present invention relates to: a pharmaceutical composition for preventing or treating Parkinson′s disease including Aster koraiensis extracts or fractions as active ingredients; a method for preventing or treating Parkinson′s disease using the pharmaceutical composition; and a food composition and a feed composition for preventing or alleviating Parkinson′s disease. The Aster koraiensis extracts or fractions by the present invention can be effectively used for preventing or treating Parkinson′s disease by having excellent effects of: specifically promoting the amount of protein involved in autophagy to prevent the death of dopamine neurons which is the cause of Parkinson′s disease; specifically increasing the expression of LC3 protein which is a membrane protein of autophagosome; increasing the expression of p-AMPK and p-ULK1 proteins which induce autophagy; and significantly reducing the expression of proteins of mTOR which inhibits autophagy. Moreover, the composition of the present invention is derived from natural products and thus, can be safely used without side effects in the prevention, treatment and alleviation of Parkinson′s disease.

Description

Composition for preventing and / or treating a neurodegenerative disease comprising extract or fraction of Aster koraiensis Nakai}

The present invention relates to a composition for the prevention or treatment of Parkinson's disease, which comprises the bee herb extract or fractions thereof as an active ingredient.

As the aging phenomena, which are increasing in proportion to the elderly population, have recently emerged as a social problem, there is a growing interest in neurodegenerative disorders. Degenerative brain diseases are disorders in which motor, memory, and cognitive disorders are caused by neurodegenerative diseases caused by various causes. Among them, Parkinson's disease is the most common neurodegenerative brain disease after Alzheimer's disease. . Parkinson's disease was first described by James Parkinson, a British physician in 1817, about 60 years later, the term Parkinson's disease was used by J.Charout, and by Kinner wilson in 1912, Parkinson's disease It was found that the disease is related to the extrapyramidal system and not the pyramidal system (Habibi et al., 2011).

Research into the cause of Parkinson's disease has been undertaken for decades, but the mechanism is not clear. As a result, Parkinson's disease is classified as a progressive neurological disorder intractable disease without effective and effective long-term treatment (Pan et al., 2008, Habibi et al., 2011). However, it is estimated that Parkinson's disease is caused by various pathological factors occurring in Parkinson's disease patients. As a factor, the abnormal killing of dopamine-releasing neurons among the neurotransmitters distributed in the melanoma of the midbrain is disrupted or destroyed by the substantia nigra par compacta (Camins et al., 2008, Smith). , 2008). Abnormal death of dopamine neurons occurs through a series of processes. First, mitochondria damaged by various factors, such as the environment, heredity, and aging, cause functional degradation of the proteolytic system, which causes abnormal proteins and organelles. It is caused by accumulation and not decomposition. These causes raise the importance of proteolytic systems in the treatment and prevention of Parkinson's disease.

To date, the highest prescription for Parkinson's disease patients is L-dopa, which is a treatment that increases the level of dopamine in the blood by direct injection of Levodopa, a precursor of dopamine neurons. Direct infusion of Levodopa results in substantial symptomatic relief in Parkinson's patients, but increased doses are essential for long-term use and are known to cause a variety of unforeseen side effects as the dose increases (Rock and Peterson, 2006). , McGeer and McGeer, 2008). Therefore, it is urgent to develop more fundamental treatments and drugs that can suppress the protection and death of nerve cells that facilitate dopamine secretion, rather than the direct administration of L-dopa, which increases dopamine blood levels. As one of these attempts, research into the development of therapeutic agents targeting autophagy, one of the proteolytic systems, has recently attracted attention.

Autophagy, or autophagy, is derived from the Greek word for "self," meaning "self," and "eating," meaning to eat. It not only maintains the metabolic balance of the cell but also affects the fate of the cell. It is known as a mechanism of self-sacrifice that plays an important role (Eisenberg-Lerner et al., 2009). This autophagy acts as a precursor to autophagosomes and an independent bilayer of phagophore, which gradually extends to completely surround unnecessary proteins in the cell, forming a morphogenetic autophagosome. It has a characteristic structure. In addition, autophagosomes move along microtubules to fuse with lysosomes in cells and form autolysosomes, which are reported to degrade proteins by proteolytic enzymes secreted inside lysosomes. It became.

On the other hand, Aster koraiensis Nakai is a compositae plant, also called Gymnaster koraiensis (Nakai) Kitamura, and is a native plant of Korea. Beetle odor is a perennial herb distributed mainly in Jeju Island and sub-Gyeonggi Province, and is known to grow well in valleys and wetlands with high humidity. Known so far the efficacy of these bee and herb extract and single compound (compound) of diabetic retinopathy induced mouse retinal vascular damage, protective effect of the detoxification enzymes known to have cancer prevention effect, bee anesthetic extract of Protein glycation and aldose reductase inhibitory activity against a single component and anti-apoptosis activity against diabetic mouse models, including inhibition of the development of diabetic nephropathy, colon cancer suppression, liver protection, and oxidative stress Effect. However, there have been no studies on autophagy, including Parkinson's disease, through bee anesthesia.

Accordingly, while the present inventors are conducting research on the development of a prophylactic and therapeutic agent for Parkinson's disease through induction of autophagy, the extract of Aster koraiensis Nakai plants induces autophagy and at the same time prevents and prevents Parkinson's disease. The present invention was completed by confirming that it is very effective for treatment.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of Parkinson's disease comprising a bee odor extract or fractions thereof as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating Parkinson's disease, comprising administering the pharmaceutical composition to an individual except a human.

Still another object of the present invention is to provide a food composition for preventing or improving Parkinson's disease, which comprises a bee anesthetist extract or a fraction thereof as an active ingredient.

Still another object of the present invention is to provide a feed composition for preventing or improving Parkinson's disease, which comprises a bee anesthetist extract or a fraction thereof as an active ingredient.

Still another object of the present invention is to provide a composition for inhibiting brain cell death by in vitro autophagy inducing effect, which comprises a bee stinger extract or a fraction thereof as an active ingredient.

Each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. In addition, the scope of the present invention is not to be limited by the specific description described below.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for the prevention or treatment of Parkinson's disease, including Aster koraiensis Nakai extract or a fraction thereof as an active ingredient.

As used herein, the term "beetle odor" is scientific name Aster koraiensis Nakai, but in the present invention can be used to extract the above-ground parts of the plant, more specifically, the leaves and stems of the plant can be used, preventing or treating Parkinson's disease As long as it has an effect, it is not specifically limited to this.

The hummingbird odor can be purchased commercially, or may be used collected or cultivated in nature.

As used herein, the term "extract" refers to an extract, such as an extract obtained by extracting a bee odor, a diluent or concentrate of the extract, a dried product obtained by drying the extract, a modifier or purified product of the extract, or a mixture thereof. It includes extracts of all formulations that can be formed using extracts.

The method of extracting the hummingbird odor is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method may include hot water extraction, ultrasonic extraction, filtration, reflux extraction, etc., these may be carried out alone or in combination of two or more methods.

In the present invention, the type of extraction solvent used to extract the bee odor is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent may include water, alcohols of C1 to C4, mixed solvents thereof, and the like, which may be used alone or in combination of one or more thereof. Specifically, preferably 95% ethanol may be used, but is not limited thereto.

As used herein, the term "fraction" refers to the result obtained by performing fractionation to separate a specific component or a specific group of components from a mixture comprising several various components.

The fractionation method of obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed through an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (size, charge, hydrophobicity). Or chromatographed fractions), and combinations thereof, which are prepared for separation according to affinity. Specifically, a method of obtaining a fraction from the extract by treating a predetermined solvent with an extract obtained by extracting the bee sting odor of the present invention.

The kind of the fractionation solvent used to obtain the fraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvents include polar solvents such as water and alcohols having 1 to 4 carbon atoms; Nonpolar solvents such as hexane, ethyl acetate, chloroform and dichloromethane; Or a mixed solvent thereof. These may be used alone or in combination of one or more thereof, but is not limited thereto. Specifically, hexane, ethyl acetate, butanol or water may be used alone or in combination of one or more thereof, but is not limited thereto. It doesn't happen.

In addition, the extract or fraction may be prepared and used in the form of a dry powder after extraction, but is not limited thereto.

As used herein, the term "Parkinson's disease" means a degenerative brain disease of the nervous system caused by the loss of dopamine neurons. Stability, stiffness, slowness of movement (slowness of motion) and postural instability are characteristic, and clinical symptoms usually begin after age 60.

As used herein, the term "prevention" means any action that inhibits or delays the progression of Parkinson's disease by the administration of a pharmaceutical composition of the present invention comprising a cane odor extract or a fraction thereof.

As used herein, the term "treatment" means any action in which Parkinson's disease is ameliorated or beneficially altered by administration of the pharmaceutical composition.

The "pharmaceutical composition" of the present invention may further comprise a pharmaceutically acceptable carrier, excipient or diluent commonly used in the manufacture of a pharmaceutical composition, which carrier is a non-naturally occuring carrier. It may include.

The pharmaceutical composition may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. .

"Pharmaceutically acceptable" means to exhibit properties that are not toxic to cells or humans exposed to the composition.

Specifically, the type of the carrier is not particularly limited, and any carrier can be used as long as it is a conventionally used and pharmaceutically acceptable carrier in the art. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol and the like. These may be used alone or in combination of two or more thereof. In addition, if necessary, other conventional additives such as antioxidants, buffers and / or bacteriostatic agents may be added and used, and diluents, dispersants, surfactants, binders, lubricants, and the like may be additionally added to provide a solution such as an aqueous solution, a suspension, an emulsion, or the like. It may be formulated into a use formulation, pills, capsules, granules or tablets.

The mode of administration of the pharmaceutical composition for preventing or treating Parkinson's disease according to the present invention is not particularly limited and may be in accordance with a method commonly used in the art. As a non-limiting example of the mode of administration, the composition may be administered by oral or parenteral administration. In addition, the composition for preventing or treating Parkinson's disease of the present invention can be prepared in various formulations according to the desired mode of administration.

Another aspect of the invention provides a method of preventing or treating Parkinson's disease comprising administering the pharmaceutical composition to a subject.

At this time, the definition of Parkinson's disease, prevention and treatment is as described above.

As used herein, the term "administration" means the introduction of certain substances into an individual in a suitable manner.

As used herein, the term "individual" means all animals, such as rats, mice, and livestock, including humans who may or may develop Parkinson's disease. As a specific example, it may be a mammal including a human.

More specifically, the "individual" may include a companion animal. The companion animal refers to an animal that lives together with a human, and includes, but is not limited to, a mammal such as a dog, a cat, a hamster, and a guinea pig, a bird such as a parrot, a canary, and the like.

Specifically, the method for preventing or treating Parkinson's disease of the present invention may include administering to the individual a pharmaceutically effective amount of a medicinal composition comprising a bee herb extract or a fraction thereof.

The "pharmaceutically effective amount" means an amount sufficient to treat the disease at a reasonable benefit / risk ratio applicable to the medical treatment and not causing side effects, and the effective dose level refers to the sex, age, weight, Well-known in the field and other medical fields, including health status, type of disease, severity, drug activity, drug sensitivity, method of administration, time of administration, route of administration, and rate of release, duration of treatment, combination or drug used simultaneously Depending on the factor, it can be readily determined by one skilled in the art.

The pharmaceutical composition of the present invention may be administered at 0.0001 to 500 mg / kg body weight per day, more specifically at 0.01 to 500 mg / kg body weight, based on solids. Dosing may include administering the recommended dose once a day or in several divided doses.

The pharmaceutical compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be single or multiple doses. Taking all of these factors into consideration it is important to administer an amount that will achieve the maximum effect in a minimum amount without causing side effects, which can be readily determined by one skilled in the art.

In the method for preventing or treating Parkinson's disease of the present invention, the route of administration and the mode of administration of the composition are not particularly limited, and any route of administration can be reached as long as the composition comprising the composition can be reached at the desired site. Depending on the mode of administration. Specifically, the composition may be administered through various routes, oral or parenteral, and non-limiting examples of the route of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, nasal What is administered through intralateral or inhalation etc. are mentioned.

Yet another aspect of the present invention provides a food composition for preventing or improving Parkinson's disease, comprising a bee odorous extract or a fraction thereof as an active ingredient.

At this time, the definition of bee odor, extract, fraction, Parkinson's disease and prevention is as described above.

As used herein, the term " improvement " means any action that at least reduces the parameters associated with the condition to be treated with the administration of the compositions of the present invention, such as the extent of symptoms.

The term "food" of the present invention, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products, including ice cream, various soups, drinks, tea, drinks, alcoholic beverages , Vitamin complexes, nutraceuticals and health foods, and includes all foods in the usual sense.

Since the food composition of the present invention can be consumed on a daily basis, a high Parkinson's disease improvement effect can be expected, and thus can be very useful for health promotion purposes.

The functional food (functional food) is the same term as a food for special health use (Food for special health use, FoSHU), in addition to the nutritional supply, the processed food, so that the bioregulatory function appears efficiently, high medical effect Means food. Here, the term 'function (sex)' refers to obtaining a useful effect for health purposes such as nutrient control or physiological action on the structure and function of the human body. The food of the present invention may be prepared by a method commonly used in the art, and the preparation may be performed by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the food may be prepared without limitation as long as the formulation is recognized as a food. Food composition of the present invention can be prepared in a variety of dosage forms, unlike the general medicine has the advantage that there is no side effect that can occur when taking a long-term use of the drug as a natural product, and because the portability is excellent, The food of the invention can be taken as an adjuvant for enhancing the Parkinson's disease improving effect.

The health food refers to foods having active health maintenance or promotion effect as compared to general foods, and the health supplement food refers to foods for health supplementation purposes. In some cases, the terms nutraceutical, health food, dietary supplement are used interchangeably.

Specifically, the health functional food is a food prepared by adding the extract of the present invention to food materials such as beverages, teas, spices, gums, confections, or the like, encapsulated, powdered, suspensions, etc. Means to bring effect, but unlike the general medicine has the advantage that there is no side effect that can occur when taking long-term use of the drug as a raw material.

The food composition may further include a physiologically acceptable carrier, and the type of carrier is not particularly limited and may be any carrier that is commonly used in the art.

In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve the smell, taste, time and the like. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu) and chromium (Cr); And amino acids such as lysine, tryptophan, cysteine, valine and the like.

In addition, the food composition is a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetic acid, etc.), fungicides (bleaching powder and highly bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisol (BHA), butylhydride) Oxytoluene (BHT), etc.), colorants (such as tar pigments), colorants (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (such as MSG glutamate), sweeteners (ducin, cyclate, saccharin Foods such as sodium, etc.), fragrances (vanillin, lactones, etc.), swelling agents (alum, potassium D-tartrate, etc.), reinforcing agents, emulsifiers, thickeners (foils), coatings, gum herbicides, foam inhibitors, solvents, modifiers, etc. It may include food additives. The additive may be selected according to the type of food and used in an appropriate amount.

Another aspect of the present invention provides a feed composition for the prevention or improvement of Parkinson's disease comprising a bee anesthetized extract or a fraction thereof as an active ingredient.

The bee odor, extract, fraction, prevention and improvement are as described above.

The composition comprising the honey bee odor extract or fractions thereof of the present invention can be used for the prevention or treatment of the development of Parkinson's disease in individuals other than humans, such as livestock or companion animals, can be utilized as a functional feed additive, or a feed composition.

The content of the bee-flavored extract or fractions thereof in the feed composition according to the present invention can be appropriately adjusted according to the type and age of the livestock, the type of application, the desired effect, and the like, for example, 1 to 99% by weight, preferably 10 to 90% by weight. %, More preferably 20 to 80% by weight, but is not limited thereto.

Feed composition of the present invention for administration, in addition to the bee anesthetic extract or fractions thereof, organic acids such as citric acid, fumaric acid, adipic acid, lactic acid; Phosphates such as potassium phosphate, sodium phosphate and polymerized phosphate; One or more of natural antioxidants such as polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan and tannic acid can be mixed and used. In addition, diluents, dispersants, surfactants, binders or lubricants may be additionally added to formulate injectable formulations, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.

In addition, the feed composition of the present invention is a supplementary ingredient such as amino acids, inorganic salts, vitamins, antioxidants, antifungal agents, antibacterial agents and vegetable protein feed such as milled or crushed wheat, barley, corn, blood meal, meat meal, fish meal as auxiliary ingredients In addition to the main components such as animal protein feed, animal fat and vegetable fat, such as can be used with nutritional supplements, growth promoters, digestive absorption accelerators, disease prevention agents.

When the feed composition of the present invention is used as a feed additive, the feed composition may be added as it is or used together with other ingredients, and may be suitably used according to a conventional method. Dosage forms of the feed composition may be prepared in an immediate release or sustained release formulation in combination with a nontoxic pharmaceutically acceptable carrier. Such edible carriers may be corn starch, lactose, sucrose, propylene glycol. In the case of a solid carrier, it may be a dosage form such as a tablet, a powder, a torokee agent, or the like, and in the case of a liquid carrier, it may be a dosage form of a syrup, a liquid suspension, an emulsion, or a solution. In addition, the dosage may contain preservatives, lubricants, solution promoters, stabilizers, and may also contain other inflammatory disease ameliorating agents and substances useful for virus prevention.

The feed composition of the present invention is applicable to a number of animal diets, ie feeds, including mammals, poultry, fish and shellfish. Commercially important mammals such as pigs, cows and goats, zoo animals such as elephants and camels, and livestock such as dogs and cats. Commercially important poultry includes chickens, ducks, geese and the like, and may include commercially raised fish and crustaceans such as trout and shrimp.

The feed composition according to the present invention may be mixed in a livestock feed in an amount of about 10 to 500 g, preferably 10 to 100 g per kg, on a dry weight basis, and then thoroughly mixed and then fed into a mesh or further processing. Palletization, expansion, extrusion through the process is preferred.

In a specific example of the present invention, as a result of treatment of the extract of bee anesthetized with SH-SY5Y cell line which is a dopaminergic cell, it was confirmed that the expression of LC3 protein, which is a membrane protein of autophagosome, was increased (see Example 1-2). It was also confirmed that the phosphorylated AMPK protein expression was increased (see Example 1-3).

In another specific embodiment of the present invention, it was confirmed by Western blotting through the cell experiment that the extract of the hummingbird odor increased phosphorylated ULK1 protein expression (see Example 1-4).

In another embodiment of the present invention, it was confirmed by Western blotting through the cell experiment that the extract of the hummingbird anesthetize reduces mTOR protein expression (see Example 1-5).

In another embodiment of the present invention, it was confirmed by Western blotting to suppress the autophagic flux through the autophagy inhibitor Bafilomycin A1 in order to confirm the effect of induction of autophagy action of the bee odorant extract (see Example 1-6).

In another embodiment of the present invention, as a result of confirming the expression level of the LC3 protein by the fraction of the antler odor, it was confirmed that the expression of the LC3 protein is increased by the BuOH fraction (see Examples 2-1 and 2).

In another embodiment of the present invention, it was confirmed by Western blotting through the cell experiment that the hummingbird BuOH fraction increased the expression of mTOR protein (see Example 2-3).

In another embodiment of the present invention, as a result of administering the extract of the hummingbird to the Parkinson's disease-induced animal model through MPTP, the activity that can effectively inhibit brain cell death through induction of autophagy by Western blotting It confirmed (refer Example 3).

As such, the composition of the present invention increases the protein expression level of phosphorylated ULK1 and inhibits mTOR protein expression by increasing the protein expression level of phosphorylated AMPK in dopaminergic cell lines, and autophagy in an animal model induced by Parkinson's disease through MPTP. Through the induction of the brain cell death can be effectively suppressed, it was confirmed that it has an excellent effect on the prevention, treatment and improvement of Parkinson's disease.

In other words, the bee anesthetized extract of the present invention has an excellent effect of inducing autophagy and promoting the degradation of alpha-synuclein protein, which is known as the ultimate cause of Parkinson's disease, thereby preventing or treating Parkinson's disease. It was confirmed that the composition can be effectively used for improvement.

Beetle odor extract of the present invention increases the expression of LC3 protein, a membrane protein of autophagosome, induces autophagy through the increase of phosphorylated AMPK and UK1 which induces autophagy, and the reduction of mTOR that inhibits autophagy. Therefore, the composition including the same can be effectively used for the prevention or treatment of Parkinson's disease through induction of autophagy action.

1 shows the results of Western blotting confirming the effect of increasing the expression level of LC3, an autophagosome membrane protein, according to the concentration of the bee odor extract in SH-SY5Y cell line (Control: control). ** indicates P <0.01 for control and *** indicates P <0.001.
Figure 2 is a result confirmed by Western blotting to increase the expression of the phosphorylated AMPK protein according to the concentration of the bee sting odor extract (Control: control). *** indicates P <0.001 for the control.
Figure 3 is a result confirmed by Western blotting to increase the expression of the phosphorylated ULK1 protein expression according to the concentration of the honey bee odor extract (Control: control). *** indicates P <0.001 for control
Figure 4 is a result confirmed by Western blotting to reduce the expression of mTOR protein expression according to the concentration of the bee sting odor extract (Control: control). * Indicates P <0.05 for control and *** indicates P <0.001.
5 is a result of confirming the change in the expression level of the LC3 protein according to the bee anesthesia extract treatment when Bafilomycin A1, an inhibitor of autophagy is administered (Control: control). *** indicates P <0.001 for the control group and ### indicates P <0.001 for the group treated with the bee-flavored extract at a concentration of 50 μg / ml.
FIG. 6 shows the results of Western blotting confirming the effect of increasing the expression level of LC3, an autophagy membrane protein, according to the bee-flavored fraction in SH-SY5Y cell line (Control: control).
Figure 7 is a result confirmed by Western blotting the reduction effect of the expression of mTOR protein according to the concentration of the bee sting odor fraction (Control: control). ** indicates P <0.01 for control and *** indicates P <0.001.
8 is a Western blotting effect of increasing the expression of Tyrosine hydroxylase (TH) protein according to bee hives extracts and fractions in substantia nigra of C57BL / 6 mice induced Parkinson's disease by MPTP administration. The result was confirmed (Control: control; MPTP: MPTP-treated group; Ropinirole: ropinirole + MPTP-treated group). *** indicates P <0.001 compared to the control group, ## indicates P <0.01 for the MPTP treatment group and ### indicates P <0.001 for the MPTP treatment group.
FIG. 9 shows the results of Western blotting of α-synuclein protein expression according to bee anesthetized extract and fraction administration in substantia nigra of C57BL / 6 mice induced Parkinson's disease by MPTP administration. (Control: control; MPTP: MPTP-only group; rofinirol: rofiniro + MPTP-treated group). *** indicates P <0.001 compared to the control group, ## indicates P <0.01 for the MPTP treatment group and ### indicates P <0.001 for the MPTP treatment group.
FIG. 10 shows the results of Western blotting confirming the effect of increasing the expression level of LC3 protein according to bee hives extracts and fractions in substantia nigra of C57BL / 6 mice induced Parkinson's disease by MPTP administration. Control: control; MPTP: MPTP alone group; ropinirol: rofiniro + MPTP treated group). # Indicates P <0.05 for MPTP alone treatment group.
FIG. 11 shows the results of Western blotting confirming the effect of increasing the amount of phosphorylated AMPK protein expression according to bee hives extracts and fractions in substantia nigra of C57BL / 6 mice induced Parkinson's disease by MPTP . (Control: control; MPTP: MPTP-only group; rofinirol: rofiniro + MPTP-treated group). # Indicates P <0.05 for MPTP alone treatment group.
Figure 12 Western blotting effect of the increase in the phosphorylated ULK1 (s555) protein expression according to bee hijab extract and fraction administration in substantia nigra of C57BL / 6 mice induced Parkinson's disease by MPTP administration This was confirmed by (Control: control group; MPTP: MPTP treatment group; ropinillol: ropinillol + MPTP treatment group). * Indicates P <0.05 compared to the control, ## indicates P <0.01 for the MPTP treatment group and ### indicates P <0.001 for the MPTP treatment group.
FIG. 13 shows the results of Western blotting confirming the reduction effect of mTOR protein expression according to bee hives extract and fraction administration in substantia nigra of C57BL / 6 mice induced Parkinson's disease by MPTP administration. Control: control; MPTP: MPTP alone group; ropinirol: rofiniro + MPTP treated group). # Indicates P <0.05 for the MPTP treatment group and ## indicates P <0.01 for the MPTP treatment group.
14 shows the results of Western blotting confirming the effect of beclin-1 protein expression according to bee anesthetized extract and fraction administration in substantia nigra of C57BL / 6 mice induced Parkinson's disease by MPTP administration. (Control: control; MPTP: MPTP-only group; rofinirol: rofiniro + MPTP-treated group). ## represents P <0.01 for the MPTP-only group and ### represents P <0.001 for the MPTP-only group.

Hereinafter, the present invention will be described in more detail with reference to Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes only and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example  1: Of Beetle Anesthetic Extract Self-feeding  Action-inducing effect

Example  1-1: Preparation of Beetle Anesthetic Extract

Beetle odor was cultivated in Bupyeong-myeon, Jeongseon-gu, Gangwon-do. After washing, completely dried, 15 kg of dry weight was obtained. kg was obtained.

Example  1-2: The effect of increasing the expression level of LC3 protein by the bee odor extract

In order to measure the effect of increasing the expression level of the LC3 protein of the honey bee extract obtained in Example 1, the dopaminergic cell line SH-SY5Y cell line was 10% heat-inactivated fetal bovine serum of Gibco (Grand Island, NY, USA) Dulbecco's modified eagle medium nutrient mixture F-12 (Ham) 1 × containing 1% penicillin / streptomycin (hereinafter referred to as P / S, Gibco); Cultured in DMEM / F12 (Gibco) medium was used. This cell line was purchased from ATCC: The Global Bioresource Center (CRL-2266 ™) (Manassas, Virgina 20108, USA).

Specifically, the bee-flavored extract obtained in Example 1 was added to the cell line (SH-SY5Y) in the amount shown in FIG. 1, then incubated at 5% CO ₂ , 37 ° C. for 24 hours, and collected for 6 minutes at 13,000 rpm. Centrifugation was washed twice with Dulbecco's phosphate buffered saline (DPBS). After lysis solution (1X lysis buffer, 1X Protease inhibitor cocktail, 1x Phenylmethyl sulfonyl fluoride) was added to the obtained cells, the cells were reacted at 4 ° C for 30 minutes, and then completely lysed while shaking at the same 4 ° C for 30 minutes. Centrifugation was performed at 13,000 rpm for 20 minutes to obtain the supernatant from which the protein was released. The supernatant was quantified by Bradford method using Protein assay dye Reagent concentrate (Bio-Rad).

The same amount of cell lysate was mixed with SDS loading buffer and heated at 99 ° C. for 5 minutes, then separated from 15% SDS-polyacrylamide gel (Bio-Rad), and the polyvinylidene fluoride membrane for about 45 minutes by semi dry transfer method. (PVDF) (Millipore). The transferred PVDF membrane was blocked by reacting with blocking buffer (5% skim milk, 3% bovine serum albumin) for 1 hour to 1 hour 30 minutes. After diluting each antibody 1,000-fold in blocking buffer, the PVDF membrane was reacted overnight at 4 ° C. Primary antibodies used in this experiment were LC3, GAPDH (Cell signaling technology), GAPDH was used as a loading control. The next day, washed with TBST buffer (Trizma base, sodium chloride, Tween 20) for 10 minutes / 1 time, 3 times, and then diluted anti-rabbit lgG secondary antibody (Cell signaling technology) in a ratio of 1: 2,000 in blocking buffer. ) Was reacted at room temperature for 2 hours, and washed under the same conditions. Afterwards, the membrane was fluorescently colored using an ECL solution (Thermo Fisher Scientific, Bio-Rad, Ab FRONTIRE) to confirm the expression of the protein using the LAS 4000 film (Fujifilm).

Then, the results of confirming the rate of transition from the LC3I protein to the LC3II protein are described in FIG. 1. At this time, the case was not added bee anesthesia extract as a control. As a result, as can be seen in Figure 1 , compared with the control group was confirmed that the expression level of the LC3 protein was increased by the addition of the honey-flavored extract, especially when the extract was treated at a concentration of 50 μg / ml, LC3 I The ratio of protein to LC3II protein was found to be 2.05. This resulted in increased expression of LC3 protein, which is one of autophagy membrane proteins, and increased transition from LC3 I to LC3 II protein, which is a marker of autophagy activity, As a result, it can be seen that it exhibits an autophagy induction effect.

Example  1-3: Phosphorylated by Honey Bee Extract AMPK  Increased protein expression

In order to measure the effect of increasing the expression level of phosphorylated AMPK protein of the bee-flavored extract obtained in Example 1, it was added to the cultured SH-SY5Y cell line in the amount of the bee-flavored extract described in Figure 2, at 5% CO ₂ , 37 ℃ Incubated for 24 hours, and experimented in the same manner as in Example 2. Primary antibodies used in this experiment are AMPK, p-AMPK, GAPDH (Cell signaling technology), GAPDH was used as a loading control.

Since the results showing the relative density of the phosphorylated AMPK (p-AMPK) protein in the total AMPK (t-AMPK) protein is shown in Figure 2, the case was not treated with the bee sting extract as a control. As a result, as can be seen in Figure 2 , compared with the control group it was confirmed that the expression level of the phosphorylated AMPK protein increased by the bee sting anesthetic extract. Through this, it can be confirmed that when the bee odor extract was treated to the dopaminergic cell line, the expression level of the phosphorylated AMPK protein that induces autophagy was increased, resulting in the effect of autophagy induction.

Example  1-4: Phosphorylated by Honey Bee Extract ULK1  Increased protein expression

In order to confirm the effect of increasing the expression level of phosphorylated ULK1 protein of the bee-flavored extract, it was added to the cultured SH-SY5Y cell line with the amount of the bee-flavored extract described in FIG. 3, followed by culturing for 24 hours at 5% CO ₂ , 37 ° C. The experiment was carried out in the same manner as in Example 2. The primary antibodies used in this experiment include ULK1, p-ULK1 (s555), GAPDH (Cell signaling technology), GAPDH was used as a loading control.

Then, the results showing the relative density of the phosphorylated ULK1 protein (p-ULK1) in the total ULK1 protein (t-ULK1) is described in Figure 3, the case was not treated with the bee hijab extract as a control. As a result, as can be seen in Figure 3 , compared with the control group was confirmed that the expression level of the phosphorylated ULK1 protein phosphorylated by the bee sting odor extract increased. Through this, it can be confirmed that when the bee odor extract was treated to the dopaminergic cell line, the expression level of the phosphorylated ULK1 protein that induces autophagy was increased, resulting in an autophagy induction effect.

Example  1-5: by honey bee anesthetic extract mTOR  Increased Protein Expression

In order to confirm the inhibitory effect of the mTOR protein expression of the bee odor extract, added to the cultured SH-SY5Y cell line with the addition of the bee odor ethanol extract shown in Figure 4, and then incubated for 24 hours at 5% CO ₂ , 37 ℃ always Experiment was carried out in the same manner as 2. Primary antibodies used in this experiment are mTOR, GAPDH (Cell signaling technology), GAPDH was used as a loading control.

Since the results showing the relative density of mTOR is described in Figure 4, the case was not treated with the bee herb extract was used as a control. As a result, as can be seen in Figure 4 , it was confirmed that the expression of mTOR protein is reduced in a concentration-dependent manner by the bee stinger extract compared to the control. Through this, it can be confirmed that when the bee sting odor extract was treated to the dopaminergic cell line, the expression level of mTOR protein that inhibits autophagy was reduced, resulting in an autophagy induction effect.

Example  1-6: Self-feeding  Inhibitor Bafilomycin  Of the Beetle Anesthetic Extract Through A Self-feeding  Action-inducing effect

In order to confirm whether the effect of inducing autophagy action of the bee-flavored extract was a non-specific result of false positive or true autophagy induction effect, the cultured SH-SY5Y cell line was obtained in Example 1 Beetle odor extract was treated for 18 hours at 50 μg / ml concentration as described in Figure 5 and then treated with 100nM bafilomycin A1 (hereinafter, baf A1) and incubated for 6 hours. Thereafter, LC3 protein expression levels were measured by Western blotting in the same manner as in Example 2.

Baf A1 is an autophagy inhibitor that inhibits the fusion of autophagosomes and lysosomes, thereby inhibiting the maturation of autophagic vacuoles. Specifically, autophagy flux occurs only at the last stage of the autophagy route, but autophagy induction occurs. However, when baf A1 is treated, LC3 protein expression is increased as autophagy flux is suppressed.

As a result, as can be seen in Figure 5 , it was confirmed that the autophagy flux was inhibited by the significantly increased expression of the LC3 protein of the Baf A1 treated group compared to the control. In addition, it was confirmed that the expression of LC3 protein (relative density of LC3 II relative to LC3 I) was significantly increased when the bee anesthetist extract and baf A1 were treated together. Through this, it can be seen that the effect of inducing the autophagy action of the bee anesthesia extract through the control of the autophagy flux.

Example  2: hummingbird Fraction Self-feeding  Action-inducing effect

Example  2-1: hummingbird Fraction  Produce

1 kg of the bee-flavored ethanol extract 1.7 kg obtained by 95% ethanol extraction was suspended with H ₂ O, and the mixture was left to shake with the same amount of hexane ( n -Hexane) to obtain a hexane fraction (149 g). The same amount of ethyl acetate was added to the aqueous layer. (EtOAc) was added to the mixture, and the mixture was left to shake to obtain an ethyl acetate fraction (175 g). An equal amount of butanol ( n -BuOH) was added to the aqueous layer and the mixture was left to shake to obtain a butanol fraction (190 g). H ₂ O) fractions were obtained.

Example  2-2: hummingbird In fractions  Increasing LC3 Protein Expression

In order to measure the effect of increasing the expression level of LC3 protein of the bee-flavored fraction obtained in Example 7, the bee-flavored fraction was added to the cell line (SH-SY5Y cell) at 5, 10, 25, 50, and 100 μg / ml depending on the fraction toxicity. After the addition, 5% CO ₂ , and incubated for 24 hours at 37 ℃, it was confirmed through the Western blotting experiment of the same method as Example 2.

The results of confirming the expression of the LC3 protein are shown in FIG. 6, and the case where no bee-flavored fraction was added was used as a control. As a result, as can be seen in Figure 6 , it was confirmed that the expression of the LC3 II protein significantly increased by the bee odor fraction. Through this, it was confirmed that when the hummingbird fraction was treated to the dopaminergic cell line, the transition from LC3 I, which is an indicator of autophagy activity, to LC3 II protein was increased, resulting in an autophagy induction effect.

Example  2-3: hummingbird In fractions  by mTOR  Increased Protein Expression

In order to determine the effect of reducing mTOR protein expression of the hummingbird BuOH fraction obtained in Example 7 at all, the hummingbird fraction was added to the SH-SY5Y cell line as described in FIG. 7, followed by 24% at 5% CO ₂ , 37 ° C. The culture was carried out in the same manner as in Example 2. Primary antibodies used in this experiment are mTOR, GAPDH (Cell signaling technology), GAPDH was used as a loading control.

The results showing the relative density of the mTOR is shown in FIG. 7, and the case where no bee sting fraction was added was used as a control. As a result, as can be seen in Figure 7 , it was confirmed that the expression of mTOR protein significantly reduced in the concentration-dependent treatment of the bee odor fraction. Through this, it can be confirmed that when the bee odor fraction was treated in the dopaminergic cell line, the expression level of mTOR protein that inhibited autophagy was reduced, resulting in an autophagy induction effect.

Example  3: MPTP  In Parkinson's Disease Animal Model by Administration, Self-feeding  Effect on brain cell death

C57BL / 6 male mice (8 weeks old) were used and feed and water were freely ingested. Mice were divided into seven groups, six in each group. The control group and the MPTP-treated group were orally administered with saline once 3 days, the bee odor extract (200 mg / kg) group obtained in Example 1-1 and the bee odor fraction obtained in Example 2-1 (25, 50, 100 mg / kg) group was dissolved orally in each saline extract and fractions and administered once orally for 3 days. In the positive control group, rofinirol was dissolved in physiological saline and administered once orally for 3 days. From 4th to 8th day of drug administration, MPTP (30 mg / kg) dissolved in physiological saline was administered orally to all groups except the control group 1 hour after drug administration, and to all groups except the control group on the 9th to 10th day of drug administration. The same amount of drug was administered orally once.

Seven days after the end of drug administration, mice in each group were killed and brain tissues were separated into substantia nigra and striatum. The isolated brain tissue was homogenized with PRO-PREPTM Lysis Buffer (iNtRON, Gyeonggi, Korea) supplemented with Phosphatase Inhibitor Cocktail Set I (Sigma-Aldrich, MO, USA). The homogenate was allowed to stand at 4 ° C. for 30 minutes and then the supernatant was collected by centrifugation at 13,000 rpm, 4 ° C. for 30 minutes, and the obtained supernatant was quantified by Bradford method using Protein assay dye Reagent concentrate (Bio-Rad). It was. Thereafter, the mixture was mixed with a predetermined amount of SDS loading buffer and heated at 99 ° C. for 5 minutes, and protein expression was measured by Western blotting in the same manner as in Example 1-2. Primary antibodies used in this experiment include TH, Beclin-1, p-mTOR, Mtor, p-ULK1 (s555), ULK1, p-AMPK, AMPK, LC3, α-synuclein, Cell signaling technology (GAPDH). GAPDH was used as loading control.

The results of confirming the expression of the proteins are described in FIGS . 8 to 14 , and the case of the addition of the bee-flavored extract, fractions and MPTP was used as a control, and the case of administration of ropinillol and MPTP as a positive control. Ropinirol is a substance used in the treatment of Parkinson's disease medication and is a dopamine agonist. MPTP is a substance used in animal models of Parkinson's disease and is a dopamine antagonist.

As a result, as shown in FIG . 8 , the TH expression of the MPTP-treated group was decreased compared to the control group, while the group administered the bee-flavored extract and fractions controlled the dopamine precursor (L-DOPA) in a concentration-dependent manner. It can be seen that the expression of TH protein, which is an enzyme, increased significantly.

As a result, as can be seen in Figure 9 , the aggregation of α-synuclein in the MPTP-treated group significantly increased compared to the control group, while in the group administered the hummingbird extract and fraction is known as the cause of Parkinson's disease protein It can be seen that aggregation of the α-synuclein protein present is reduced to or below the control level.

In addition, as can be seen in Figure 10 , compared with the control group it was confirmed that the concentration of the expression of LC3 protein, autophagosome membrane protein autologous phagocytic membrane protein of the group administered the extract and fraction.

In addition, as shown in FIG . 11 , in the MPTP-treated group, the expression of phosphorylated AMPK protein that induces autophagy was decreased in comparison with the control group, whereas in the group to which the bee-flavored extract and fractions were administered, the concentration-dependently increased. Could confirm.

As a result, as can be seen in FIG . 12 , in the MPTP-treated group, the expression of phosphorylated ULK1 protein that induces autophagy was decreased compared to the control group, whereas in the group to which the bee-flavored extract and fractions were administered, concentration-dependently Significantly increased.

As a result, as can be seen in Figure 13 , in the MPTP-treated group, the expression of phosphorylated mTOR protein that inhibits autophagy was increased compared to the control group, while in the group to which the bee-flavored extract and fractions were administered, the concentration was increased. It was confirmed that the decrease significantly.

As a result, as can be seen in Figure 14, in the case of the MPTP single treatment group. While the expression of Beclin-1 protein, a representative protein that induces autophagy, was decreased compared to the control group, it was confirmed that concentrations were significantly increased in the group administered bee anesthetized extract and fractions.

In the above results, the treatment of bee-flavored extracts and fractions in an animal model inducing Parkinson's disease with MPTP toxicants promotes the amount of TH, an enzyme that modulates the precursor of dopamine (L-DOPA) (FIG. 8), and in Parkinson's disease. Inhibits the aggregation of α-synuclein proteins in abnormally aggregated form (FIG. 9), increases the expression of autophagy membrane protein LC3 and p-AMPK, p-ULK1, Beclin-1 proteins that induce autophagy; It was confirmed that the expression of p-mTOR protein that inhibits autophagy is reduced (Figs. 10-14). Through this, bee-flavored extracts and fractions were administered in a concentration-dependent manner to treat and improve Parkinson's disease through induction of autophagy. It can be seen that the activity.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (9)

A pharmaceutical composition for the prevention or treatment of Parkinson's disease comprising bee odor extract or fractions thereof as an active ingredient. The pharmaceutical composition of claim 1, wherein the bee odor is extracted from leaves or stems of the bee odor. The pharmaceutical composition of claim 1, wherein the beetail odor extract is prepared by extraction with a solvent selected from the group consisting of water, alcohols having 1 to 4 carbon atoms, and mixed solvents thereof. The pharmaceutical composition of claim 1, wherein the prevention or treatment of Parkinson's disease is by inhibition of brain cell death through autophagy induction. A method for preventing or treating Parkinson's disease, comprising administering a pharmaceutical composition according to any one of claims 1 to 4 to a subject other than a human. Beetle odor extract or fractions thereof as an active ingredient, a food composition for preventing or improving Parkinson's disease. The food composition of claim 6, wherein the prevention or treatment of Parkinson's disease is by inhibition of brain cell death through autophagy induction. Beetle odor extract or fraction comprising as an active ingredient, Parkinson's disease prevention or improvement of feed composition. A composition for inhibiting brain cell death by in vitro autophagy induction, comprising bee anesthetized extract or fraction as an active ingredient.

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