KR20110066400A - Pharmaceutical composition comprising gintonin for prevention and treatment of neurodegenerative disease - Google Patents

Abstract

PURPOSE: A composition containing gintonin for preventing and treating neurodegenerative diseases is provided to enhance soluble amyloid precursor protein(sAPPalpha) release and to suppress memory impairment. CONSTITUTION: A composition for preventing and treating neurodegenerative diseases contains 0.0001-50 weight% of gintonin as an active ingredient. The ginseng is roots, leaves, stems, fruits, or flowers of ginseng. A pharmaceutical composition is manufactured in the form of powder, granule, tablet, capsule, suspension, emulsion, syrup, suppository, or sterilized injection.

Description

Pharmaceutical composition comprising gintonin for prevention and treatment of neurodegenerative disease}

The present invention relates to a composition for the prevention and treatment of neurodegenerative diseases of the neurodegenerative system comprising a glycoprotein isolated from ginseng gintonin (gintonin) as an active ingredient.

Alzheimer's disease (AD) was first reported by Alois Alzheimer in 1907 and is one of the leading degenerative dementia diseases in the elderly (Plassman, et al. Neuroepidemiology , 29, 125-132, 2007; Alzheimer, Allg Z Psychiat . 64, 146-148, 1907).

Dementia is characterized by impaired memory and other cognitive intellects essential to social life, such as damage to the central nervous system, which weakens the perception of language, time, and place (Van Der Flier, et al, Neurology , 59, 874-879, 2002). Histologically, neurofibrillary tangles, senile plaques, and cerebrovascular amyloid deposits appear in the cerebral neocortex and limbic system of Alzheimer's patients, which are markers for confirming Alzheimer's patients ( Braak, et al, Neurosci Lett . 65, 351-354, 1986).

Among the components related to Alzheimer's, beta-amyloid β-protein (Aβ) is the most common feature in patients with dementia, and beta-amyloid has neurotoxicity that kills nerve cells, which leads to memory loss. Most commonly found in senile plaques (Glenner and Wong, Biochem. Biophys. Res. Commun. 120, 885-890, 1984; Yankner, Neuron , 16, 921-932, 1996).

Formation of neurotoxic beta-amyloid protein is made from amyloid precursor protein (APP), a transmembrane protein through amyloidogenic proteolytic cleavage. APP is first cleaved by β-secretase and then cleaved by γ-sectretase to produce the neurotoxic Aβ-protein (Vassar, et al Science. 286) . , 735-741, 1999).

As another pathway, APP produces a soluble amyloid proprotein (sAPPα) instead of the Aβ protein via a non-amyloidogenic pathway. This pathway is made by the action of α-secretase first followed by the action of γ-secretase. The sAPPα protein produced by the non-amyloid pathway has been reported to have cell proliferation, anti-apoptosis and neuroprotective effects as opposed to Aβ protein (Fahrenholz, Curr Alzheimer Res. 4, 412-417, 2007;. Yuan and Yankner , Nature 407, 802-809, 2000).

As a result, drugs that promote the production of sAPPα protein via the non-amyloid pathway are considered candidates for protecting the nervous system from dementia drugs or Aβ proteins.

Recent studies have shown that receptors coupled with GTP-binding protein (G protein), in particular Gαq / 11 → phospholipase C → inositol 1,4,5-trisphosphate (IP _3). ) → Ca ²⁺ pathway → activity of receptor leading to protein kinase C (PKC) activity increases intracellular free calcium (Ca ²⁺ ) concentration ([Ca ²⁺ ] _i ), followed by non- It has been found to promote the release of sAPPα through amyloid pathway activity (Petryniak, et al, Biochem J. 320, 957-963, 1996; Rosner, et al., Prog Neurobiol. 56, 541-569, 1998; Kim, et al, J Neurochem. 97, 245-254, 2006).

Moreover, increased sAPPα release through the non-amyloid pathway following receptor activity linked to Gαq / 11-protein is closely associated with decreased Aβ production, which is associated with the process of sAPPα production. Aβ production is inversely correlated (Hung, et al., J. Biol. Chem. 268, 22959-22962, 1993; Gabuzda, et al, J. Neurochem. 61, 2326-2329, 1993; Busciglio, et al, Proc. natl Acad. Sci. USA 90, 2092-2096, 1993).

Meanwhile, ginseng has been widely used as a tonic for a long time, and ginseng saponins (ginsenosides), known as active ingredients of ginseng, are well known for protecting the nervous system from various neurotoxins including Aβ protein (Kim et al. , J Neurosci Res 53: 426432, 1998; Kim, et al Neuropharmacology 48: 743756, 2005; Lin et al, J Neural Transm Suppl. 72, 105-112, 2007; Wang, et al, J Ethnopharmacol. 103, 103- 108. 2006; Ji et al, J Ethnopharmacol. 107, 48-52, 2006; Song et al, Yao Xue Xue Bao. 43, 29-34, 2008; Wei et al, Neurosci Lett. 443 (3): 145- 149, 2008).

Until now, the main physiological and pharmacologically active ingredient of ginseng was known to be ginseng saponin. However, the present inventors have demonstrated in recent studies that the characteristics of the conventional ginseng are due to a new glycolipoprotein composed of proteins, carbohydrates and fats that are not ginsenosides, and the glycoproteins are isolated and purified from ginseng. It was named gintonin (see Korean Patent Application No. 2009-0110662).

Accordingly, the inventors of the present invention, while studying the physiological activity of the gintonin isolated from the ginseng, the gintonin is a Gαq / 11 → phospholipase C → IP ₃ → Ca ²⁺ pathway activity Increasing the intracellular free calcium concentration, not only activates the non-amyloid pathway of APP, but also prevents the reduction of Aβ formation and prevents neurotoxicity by Aβ in vitro and in vivo , showing that it exhibits neuroprotective activity The invention was completed.

After all, the main object of the present invention is to provide a composition for the prevention or treatment of degenerative cerebral nervous system disease, using the gintonin isolated from ginseng as an active ingredient.

In order to achieve the above object, the present invention provides gintonin isolated from ginseng.

In the present invention, the gintonin has a molecular weight of about 67 kDa, the ginseng is characterized in that at least one selected from the group consisting of red ginseng, ginseng, white ginseng, cultivated ginseng, camphor ginseng and wild ginseng, preferably 4 to 6 Red ginseng manufactured with Panax ginseng CA Meyer is recommended, but is not limited thereto.

In addition, the ginseng may be the root (ginseng) of the ginseng, as well as the leaves (leaves), stem (stem), fruit and / or flowers of the ginseng.

Gintonin is not a pathway for the production of beta-amyloid (Aβ), but a pathway that causes the production of soluble amyloid precursor protein (sAPPα), which is known to have neurological protection, neurotrophic efficacy and anti-apoptosis. It activates the amyloidogenic pathway (non-amyloidogenic pathway) to increase the release of sAPPα, and is effective in preventing nervous system toxicity by Aβ.

Accordingly, the present invention provides novel uses for the prevention and / or treatment of degenerative cerebral nervous system disease.

Specifically, the present invention provides a pharmaceutical composition for the prevention and treatment of degenerative neurological diseases, including gintonin as an active ingredient.

In the present invention, the gintonin is a glycolipid protein isolated from ginseng, the degenerative neurological disease is stroke, stroke, memory loss, memory impairment, dementia, forgetfulness, Parkinson's disease, Alzheimer's disease caused by neuronal cell death , Pick bottles, or Creutzfeld-Jakob bottles, and the like.

The pharmaceutical composition for the prevention and treatment of degenerative cerebral nervous system diseases of the present invention comprises 0.0001 to 50% by weight, preferably 0.001 to 50% by weight of gintonin, based on the total weight of the composition.

In addition, the pharmaceutical compositions according to the invention may further comprise suitable carriers, excipients or diluents commonly used in the manufacture of pharmaceutical compositions.

Carriers, excipients or diluents usable in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil.

In addition, the pharmaceutical compositions according to 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 sterile injections, respectively, according to conventional methods. Can be used.

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 preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may be mixed with at least one excipient, for example, starch calcium carbonate, sucrose or lactose, gelatin, or the like. To prepare. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Oral liquid preparations include suspensions, solvents, 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. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In the present invention, the pharmaceutical composition may be administered by varying the dose depending on the progress of the disease, the age, sex, physical condition, administration period, administration method, weight of the patient, diet, discharge rate, and the like of the disease. However, for the desired effect, the pharmaceutical composition of the present invention is preferably administered parenterally or orally in the range of 0.0001 to 100 mg / kg, more preferably in the range of 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

In addition, the pharmaceutical dosage forms of gintonin of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active substances, as well as in a suitable collection.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention also provides a health functional food for the prevention and improvement of cerebral nervous system disease comprising the gintonin as an active ingredient.

The dietary supplement of the present invention may further include food supplements which are food acceptable in addition to gintonin, and may be appropriately used according to a conventional method, and the mixed amount of the active ingredient may be used for its purpose, for example, prevention. It may be appropriately determined according to health, or therapeutic treatment.

The effective amount of gintonin contained in the dietary supplement may be used in accordance with the effective amount of the pharmaceutical composition, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or for health control. It is evident that the component can be used in an amount above the range because there is no problem in terms of stability.

In addition, there are no particular restrictions on the type of health functional food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various Soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and health functional foods.

It may also be added to food or beverages for the purpose of preventing the effects of cerebral nervous system disease. At this time, the amount of the gintonin in the food or beverage may be added in an amount of 0.01 to 30% by weight, preferably 0.3 to 15% by weight of the total food weight, the health beverage composition is 0.01 to 5 g, preferably based on 100ml Can be added at a ratio of 0.03 to 1 g.

The health functional food of the present invention can be manufactured and processed into health functional foods in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing and improving cerebral nervous system diseases.

The functional beverage composition of the present invention is not particularly limited to other ingredients other than containing the gintonin as an essential ingredient in the indicated ratio, and may further include various flavors or natural carbohydrates, such as ordinary drinks. At this time, the natural carbohydrate is, for example, monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; And conventional sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (tauumatin, stevia extract, for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the gintonin of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the gintonin of the present invention may contain natural fruit juice and pulp for preparing fruit juice beverages and vegetable beverages, and these ingredients may be used independently or in combination. In addition, the ratio of the additive is not so important, but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of gintonin of the present invention.

Gintonin isolated from ginseng of the present invention activates the non-amyloidogenic pathway to increase the release of sAPPα and protects the neurotoxicity caused by Aβ, thereby damaging neurons as well as cytotoxicity and It can effectively suppress cell death. In addition, there is an effect of suppressing short- and long-term memory and working memory damage due to neurotoxicity.

Therefore, the composition containing the gintonin of the present invention as an active ingredient can be usefully used as a medicine or health functional food for the prevention and treatment of degenerative neurological diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Preparation of Gintonin

20 kg of 6 years old red ginseng ( Panax ginseng CA meyer) purchased from Korea Ginseng Corporation (Daejeon, Korea) was finely pulverized, and 30 ℓ of 80% methanol was added and reflux-cooled at 80 ° C for 8 hours, followed by vacuum filtration. Was recovered. This process was repeated three times to collect the supernatant and concentrated under reduced pressure to give 6.2 kg of methanol extract.

The methanol extract was solvent fractionated with a mixed solvent of water and n-butanol (1: 1) to obtain 908 g of water and n-butanol fractions, and the n-butanol fractions were concentrated to give chloroform: methanol: water (CHCl ₃ : Elution with a mixed solvent of MeOH: H ₂ O = 13: 7: 2) performed silica gel column chromatography to obtain eight small fractions.

The small fraction C7 was further purified by silica gel column chromatography eluting with ethyl acetate: ethanol: water (EtOAc: EtOH: H ₂ O = 1: 3: 0.5) mixed solvent to obtain two fractions. Crude earth after dialysis using excess distilled water and Spectra / Por dialysis membrane (molecular wight cut off 6,000 ~ 8,000) (Spectrum Lavoratories, Inc., CA, USA) for 8 hours at ℃ and removing ginsenosides and other small molecule materials 18 g of crude gintini was obtained.

Preparation Example 1 Cell Culture

Human neuroblastoma SH-SY5Y cells (ATCC CRL-2266) purchased from Korea Cell Line Bank were 10% (v / v) fetal bovine serum and 100 units / ml penicillin and 100 ㎍ / ml. Incubated in streptomycin-added Dulbecco's modified Eagle's medium (DMEM; Gibco-BRL Technologies, Calif., USA) medium with a temperature of 37 ° C., 95% air and 5% CO ₂ .

Preparation Example 2 Preparation of Experimental Animal

18 ~ 20 g of ICR-based, 4 week old male mice were purchased from Koatech Co., Ltd. (Korea), and 10 animals per cage were used to freely feed water and feed, maintaining a temperature of 23 ± 1 ℃ and a humidity of 55 ± 5%. The contrast cycle was adapted to the experimental environment adjusted to 12 hours, and then the experiment was started.

Experimental Example 1

1-1. sAPPα emission measurement

sAPPα release was measured according to Kim et al. (Kim, JH, Choi, S., Jung, JE, Roh, EJ, and Kim, HJ (2006) Capacitative Ca ²⁺ entry is involved in regulating soluble amyloid precursor protein (sAPPalpha) release mediated by muscarinic acetylcholine receptor activation in neuroblastoma SH-SY5Y cells.J Neurochem. 97, 245-254).

Specifically, cells (2.5 × 10 ⁶ / well) were aliquoted into 6-well plates and incubated for two days. Prior to gintonin treatment, cells were washed twice with serum-free medium and then at timed intervals, inhibitors Dec-PVRK-CMK (furin inhibitor), TAPI-2 (TNF-α processing inhibitor) (above, Biomol, Plymouth Meeting). , PA, USA), Brefeldin, BAPTA-AM (above, purchased from Sigma-Aldrich (St Louis, MO, USA)), wortmannin and GF109203X (from Calbiochem (Sunnyvale, CA, USA)) Treated.

After pretreatment, cells were treated with gintonin at intervals. Protease inhibitor cocktail was added to the culture medium of each well and centrifuged for 2 minutes at 1000g, 4 ° C to remove tissue debris and then desalted with PD-10 (GE healthcare). (desalting). Desalted samples were freeze dried and re-dissolved in sodium dodecyl sulfate sample buffer. Cells were washed with cold phosphate-buffered saline (PBS) and pooled to collect RIPA-modified buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EGTA, 2 mM sodium orthovanadate, protease inhibitor cocktail, total protein amount in the cell lysate was quantified by Bradford assay, Biorad. Cell lysates were subjected to 10% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes, which were anti-pre-A4 App monoclonal antibodies (clone 22C11, 1: 1000). Immunoblots were performed using (Millipore, Billerica, MA, USA) or anti- (amyloid β) monoclonal antibodies to sAPPα (clone 6E10, 1: 1000) (Convace, Buckinghamshire, UK), and bands Is a chemiluminescence method (GE health) care) Data collection and processing were performed with a luminescent image analyzer (LAS-3000) and multi gauge software (Fujifilm, Tokyo, Japan).

1-2. Measurement of SH-SY5Y Intracellular ADAM

Cells treated with gintonin as described above were washed with cold PBS, collected and lysed with RIPA modified buffer to obtain whole cell lysate. To obtain the membrane fraction, the cells treated with gintonin were washed with cold PBS, pooled with buffer containing 20 mM Tris-HCl (pH 7.5), 2 mM EDTA, 5 mM dithiothreitol, 0.32 M sucrose, and a protease inhibitor cocktail. Dissolved. Cell lysates were centrifuged at 12000 g , 4 ° C. for 30 minutes, pellets were resuspended in the same lysis buffer containing 1.0% Triton X-100, incubated on ice for 45 minutes, and again 12000 g. g , centrifuged at 4 ° C for 30 minutes. Supernatants were collected and used for Western blot analysis using anti-ADAM10 polyclonal antibody (Milipore).

1-3. Measurement of Intracellular Calcium Concentration

Intracellular free calcium concentration was measured by dual excitation spectrofluorometric anlysis of cell suspension loaded with Fura-2 AM (Sigma-Aldrich, St Louis, MO, USA).

Cells were HEPES buffer (120 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 1.5 mM CaCl ₂ , 10 mM glucose, 25 mM HEPES, pH 7.4) or Ca ²⁺ free HEPES buffer (120 mM NaCl, 5 mM KCl, 1 mM MgCl ₂ , 10 mM glucose, 0.2 mM EGTA, 25 mM HEPES, pH 7.4). In addition, changes in intracellular calcium concentration were also tested in cell suspensions treated with Fura-2 AM and BAPTA-AM and calculated according to the method of Grynkiewicz et al. (Grynkiewicz, G., Poenie, M., and Tsien, RY ( 1985) A new generation of Ca ²⁺ indicators with greatly improved fluorescence properties.J Biol Chem. 260, 3440-3450).

1-4. SH-SY5Y transfection of wt-APP or swe-APP genes

Dispense 2 × 10 ³ cells per well into a 96 well plate, and after 48 hours, culture medium was added to N2 fertilized medium (0.5 μg / ml insulin, 100 μg / ml apo-transferrin, 20 nM progesterone, 1 mM carnitine, 30 nM selenium). , DMEM supplemented with 100 units / ml penicillin and 100 μg / ml streptomycin (Bottenstein, JE, and Sato, GH (1979) Growth of a rat neuroblastoma cell line in serum-free supplemented medium.Proc Natl Acad Sci US A. 76, 514-517).

Cells were transiently transfected with 1 μg of plasmid DNA and 3 μl of Fugene 6 (Roche Diagnostics, Mannheim, Germany) together in 1 mL of N2 fertilization medium according to the manufacturer's instructions. Six hours after the introduction of the gene into cells, different concentrations of gintonin were added to the cells.

After 48 hours, cell viability was assayed by XTT assay.

1-5. Aβ _1-42  Peptide Preparation and Aβ _1-42  The cytoprotective effect of gintonin against toxicity

Aβ _1-42 peptide was dissolved in sterile distilled water and aliquoted and stored at -20 ° C (stock solution). The stock solution is phosphate buffered saline (PBS, pH 7.4) diluted to 100 μM, and aggregated Aβ _1-42 peptide (aggregated Aβ _1-42 peptide) 4 ilgan pretreatment (pre-aging) at 37 ℃ to obtain a It was.

SH-SY5Y cells were dispensed 2 × 10 ³ per well in 96-well plate, and after 48 hours the culture medium was incubated with gintonin by exchange with N2 fertilization medium. After 1 hour of culture, the aggregated Aβ _1-42 peptide (pre-aggregated Aβ _1-42 ), which had been pre-aggregated to the cells, was added, and further cultured for 48 hours, the cell activity was assayed by XTT assay.

1-6. Cell viability analysis using XTT

To assess cell activity, an XTT assay was performed. Sodium 3 '-[1- (phenylamino-carbonyl) -3,4-tetrazolium-bis (4-methoxy-6-nitro) benzene in the presence of phenazine methosulfate (PMS) Sulfonate (sodium 3 '-[1- (phenylamino-carbonyl) -3, 4-tetrazolium] -bis (4-methoxy-6-nitro) benzene sulfonate, XTT) is soluble in mitochondrial dehydrogenase in living cells. It can be broken down into soluble orange formazan crystals.

The cell culture medium of each well in a 96 well plate was exchanged with 200 μl of serum free medium except phenol red and XTT reaction solution (containing 1 mg / ml XTT and 0.0306 PMS) was added.

After incubation for 4 hours, orange formazan absorbance, which correlates with cell activity, was measured at 450 nm.

1-7. Aβ _1-42  Release

3 × 10 ⁵ cells per well were dispensed into 24-well plates and after 48 hours the culture medium was exchanged with N2 fertilization medium. Cells were transiently transfected with 2 ug of wild-type or swe-APP plasmid DNA and 6 Fu of Fugene 6 (Roche Diagnostics, Mannheim, Germany) in 1 ml of N2 fertilization medium according to the manufacturer's instructions. . Six hours after transduction, different concentrations of gintonin were added to the cells.

After 48 hours of culture, the culture medium was collected and treated with a protease inhibitor cocktail and stored at -70 ° C until use.

For analysis, samples were lyophilized and resuspended in sample buffer. Aβ _1-42 release was assayed by sandwich enzyme-linked immunosorbent assay (ELISA) using the β amyloid 1-42 immunoassay kit (Invitrogen, Camarillo, Calif., USA) according to the manufacturer's instructions. The result obtained by the total protein amount of each sample was corrected.

1-8. Aβ _25-35 Intracerebroventricular injection (ICV)

Aβ _25-35 (Sigma-Aldrich, MO, USA) was dissolved in sterile distilled water and stored at -20 ° C until use. To induce aggregation, lysed Aβ _25-35 was incubated at 37 ° C. for 4 days. Cohesive _{Aβ 25-35 (aggregated Aβ 25-35; 6} mmol) was intraventricular injection according to a conventional method (Maurice, T., Lockhart, BP , and Privat, A. (1996) Amnesia induced in mice by centrally administered beta amyloid peptides involves cholinergic dysfunction.Brain Res. 706, 181-193), a disposable 30-gauge 4.0 mm needle (Dong-Hwa C & N, Korea) and Hamilton microsyringe were used.

5 μl injection was performed within 3 seconds by inserting a needle at an equal distance between the eyes and ears perpendicular to the cranial plane and 1 mm to the right of the midpoint equidistant from each eye. The control group was injected with sterile double-distilled water.

9. Drug treatment

First, gintonin was dissolved in distilled water and administered to the mice by intraventricular injection at a dose of 0.125, 0.25 and 0.5 μg / 5 μl once daily for 8 days, but Aβ _25-35 intraventricular injection was the first Once every 60 minutes after injection.

The anti-amnesic effect of gintonin on Aβ _25-35 -induced loss of working memory was tested by Y-maze 7 days after Aβ _25-35 administration. In addition, the neuroprotective effect of gintonin on long-term memory was tested by passive avoidance test after 8 days (training) and 9 days (test) after administration of Aβ _25-35 .

10. Spontaneous alternation behavior Y-maze experiment

The Y-maze consists of three arms of the same angle, each consisting of a 30 cm long, 5 cm wide and 12 cm high wall, with the labyrinth floor and walls being dark gray polyvinyl plastic. Was made.

Mice were initially placed in one passage, and the entry order and number of passages were recorded by hand for each mouse for 8 minutes.

One hour before the test, mice received gintonin by intraventricular injection, and the control group received distilled water by the amount of gintonin. The labyrinth's passages were cleaned with 10% ethanol each time they were examined to remove odors and residues.

The alteration score (%) of each mouse was converted into a percentage of the actual number of alteration relative to the maximum possible number of crossings (defined as "full passage entry-2"). The number of arm entries is used as an indicator of locomotor activity.

11.Step-through passive avoidance test

The passive evacuation device consists of a clear chamber and a dark chamber separated by a guillotine door, and the bottom of each room (12 × 10 × 12 cm) has a 2 mm stainless steal (stainless steal). rods) are spaced 0.5 cm apart. The apparatus was also equipped with a 50 W lamp 1 meter upward.

The mice were trained and final tested 24 hours later. For the training test, the mice were initially placed in a transparent chamber, each time the mouse went to a dark place, the door was closed and through a stainless steel rod. An electric foot shock (0.5 kPa, 3 sec) was applied to the bottom.

One hour prior to the test, mice were administered intraventricularly with gintonin at 0.125, 0.25 and 0.5 μg / μl, and the control group received distilled water instead of gintonin. After 24 hours of the training test, the mouse was placed in an illuminated part for the final test, and the time taken for the mouse to enter the dark after opening the door was defined as the latency for training and the final test. At this time, the maximum time limit of the incubation period until entering the dark place was 300 seconds, and the passive avoidance reaction time was determined to be 300 seconds unless the entry into the dark place over 300 seconds.

12. Statistical Analysis

All of the above data were statistically compared between control and experimental groups using Student's t-test. Statistical significance was considered at p <0.05. Animal experimental data were analyzed using Newman-Keuls test followed by one-way analysis of variance (ANOVA).

Experimental Example 2. Consideration of Results

2-1. Confirmation of Gintonine's Increased sAPPα Release (1)

When SH-SY5Y cells, which are human neuroblastoma cells, were treated with gintonin at different concentrations for 1 hour, it was confirmed that sAPPα release was increased depending on the administration concentration (see FIG. 1A). In this case, the EC 50 was 21.7 ± 0.8 μg / ml, and the concentration corresponds to about 320 nM when the native molecular weight of gintonin was 67 kDa.

In addition, 100 μg / ml (1.5 μM) of gintonin was expressed in SH-SY5Y cells using 6E10 antibody with sAPPα released in the medium as 1 mM carbachol (muscarin receptor antagonist) as a positive control. ) Was detected by immunoblot after treatment according to the indicated time, and sAPPα release was found to increase maximally within 2 hours after gintonin treatment (see FIG. 1B).

On the other hand, to determine whether the increase in sAPPα release by gintonin affects the cellular total full length APP expression, it was tested using 22C11 antibody (Millipore, Billerica, MA, USA) . As a result, gintonin did not appear to affect the cell total length APP expression (see Figures 2A and B).

The results indicate that the effect of increasing sAPPα release by gintonin is not due to an increase in cell total length APP expression, demonstrating the possibility that gintonin will stimulate the non-amyloid pathway.

However, administration of different gintonin concentrations and treatment times did not show any cytotoxicity (not shown).

Increased sAPPα release via the non-amyloid pathway in SH-SY5Y cells is known to reduce Aβ formation (Hung, et al., J. Biol. Chem. 268, 22959-22962, 1993; Gabuzda, et al, J Neurochem. 61, 2326-2329, 1993; Busciglio, et al, Proc. Natl Acad. Sci. USA 90, 2092-2096, 1993).

In addition, the present invention confirmed the effect of _{gintonin on} the release of Aβ _1-42 protein. The level released in the medium was quantified by the sandwich ELISA method, and as shown in FIG. 3, in the SH-SY5Y cells transfected with SH-SY5Y cells, wild-type-APP and swe-APP for 48 hours. It has been shown to inhibit Aβ _1-42 formation depending on the gintonin dose concentration. This shows the possibility that the promotion of the non-amyloid pathway by gintonin may be associated with the reduction of the amyloid pathway for Aβ production.

2-2. Confirmation of Gintonine's Increased Release of sAPPα (2)

To determine whether ginsenosides known to be active ingredients of ginseng release sAPPα, the untreated (UT) control, 10 μM ginsenoside Rb ₁ , 10 μM ginsenoside Rg ₁ and 100 μg / ml gintonin SY5Y cells were treated. As a result, gintonin increased the release of sAPPα, whereas no other ginsenoside treated group showed a significant effect (see FIG. 4).

2-3. Confirmation of Gintonin's Increased sAPPα Release (3)

Phosphatidylinositol 3-kinase (PI3K) is activated by GTP-binding proteins and tyrosine kinase and is associated with neurological protective signaling pathways (Maurice, et al. , Brain Res. 706, 181-193, 1996; Peng, et al, Eur J Neurosci. 28, 1255-1264, 2008; Marone, et al, Biochim Biophys Acta. 1784, 159-185, 2008; Shioda, et al , Neuroscience. 148, 221-229, 2007).

In the present invention, to determine whether the PI3K pathway is associated with sAPP release of gintonin, the effect of wortmannin, a PI3K inhibitor, on gintonin treatment was examined.

As a result, after pretreatment with wortmannin (wort, PI3K inhibitor, 500 nM) for 30 minutes in SH-SY5Y cells, the sAPPα released by gintonin was reduced by about 40% when treated with gintonin for 1 hour. Appeared (see FIG. 5A).

2-4. Confirmation of Gintonine's Increased sAPPα Release (4)

In addition, PKC activity through the Gαq / 11-phospholipase C-IP ₃ -Ca ²⁺ pathway is known to increase the release of sAPPα (Yang et al, Eur J Neurosci. 26, 381-391 , 2007), In the present invention, to confirm whether the increase in sAPPα release by gintonin is through PKC activity, gintonin with GF109203X (GF, PKC inhibitor, 10 μM) purchased from Calbiochem (Gibbstown, NJ, USA) The effect of increasing sAPPα release was examined. PMA (1 μM), a well-known PKC activator, was used as a positive control.

As a result, after GF109203X pretreatment with SH-SY5Y cells for 30 minutes, sAPPα released by treatment with 100 μg / ml of gintonin for 1 hour was reduced by about 50%. It was confirmed to increase (see FIG. 5B).

2-5. Confirmation of Gintonine's Increased sAPPα Release (5)

Intracellular [Ca ²⁺ ] _i elevation via the Gαq / 11-phospholipase C-IP ₃ -Ca ²⁺ pathway plays a major role in increasing sAPPα release (Rosner, et al, Prog Neurobiol. 56, 541-69, 1998; Kim, et al, J Neurochem. 97, 245-254, 2006).

In the present invention, in order to determine whether gintonin increases calcium of SH-SY5Y cells, SH-SY5Y cells treated with Fura 2-AM (4 μM) in HEPES buffer containing calcium and HEPES buffer containing no calcium Fura-2 AM (4 μM) treated SH-SY5Y cells were treated with 30 μg / ml (corresponding to about 500 nM) of gintonin, which resulted in intracellular [Ca ^{2 +} ] _i EC ₅₀ is 0.28 ± 0.02 μg / mL (4.2 nM) with extracellular calcium, and EC ₅₀ is 0.38 ± 0.02 μg / mL (5.7) without extracellular calcium. nM) (see FIG. 6D).

However, when Gintonin (30 μg / ml) was treated to SH-SY5Y cells treated with Fura 2-AM (4 μM, 40 minutes) in HEPES buffer containing cell membrane permeable BAPTA-AM (25 μM) and calcium There was confirmed that the effect of increasing the cytoplasm in [Ca ^{2 +]} _i caused by the dust non-destroyed (see Fig. 6C).

In addition, gintonin induced increased release of sAPPα even in the absence of extracellular calcium (see FIGS. 6B and D), but was found to significantly decrease when treated with BAPTA-AM (see FIG. 6C). These results suggest that synergism of [Ca ²⁺ ] _i in the cytoplasm by gintonin is associated with increased sAPPα release.

2-6. Confirmation of the Effect of Gintonin on sAPPα Release (6)

Metalloprotease, an enzyme that catalyzes the cleavage of the extracellular APP domain, includes ADAM10 and ADAM17 / TACE, also called α-secretase (Fahrenholz, Curr Alzheimer Res) 4, 412-417, 2007).

Pretreatment of SH-SY5Y cells with TAPI-2 (20 μM) for 30 minutes resulted in about 50% inhibition of sAPPα release by gintonin (see FIG. 7A).

On the other hand, the activation of ADAM10 and ADAM17 requires proteolytic processing and protein secretion via Golgi apparatus, and the treatment of chloromethyl ketone (CMK) and brefledin that interferes with this is caused by gintonin The release of sAPPα inhibited about 50% and 70%, respectively (see Figures 7B and C).

These results indicate that the release of sAPPα by gintonin is mediated by metalloprotease activation, proteolytic processes through Golgi and protein secretion.

2-7. Confirmation of Gintonine's Increased sAPPα Release (7)

In order to reconfirm the involvement of ADAMs in the increased release of sAPPα by gintonin, the ADAM concentration in cells was measured and ADAM10, ADAM17 in whole cell homogenates after 1 hour of gintonin treatment There was no change in the level of / TACE (see Figure 8A).

In addition, ADAM10 is known to be translocation from the cytosol to the membrane when activated by GPCR ligand or PKC activator treatment (Yang et al, Eur J Neurosci. 26, 381-391, 2007), In the present invention, it was confirmed whether the gintonin treatment induced the translocation of ADAM10.

As a result, it was found that the active form of ADAM10 was increased in the membrane fraction when treated with gintonin, compared to the group without gintonin treatment (see FIG. 8B).

These results indicate that gintonin induces the activity of ADAM10 and induced ADAM10 to translocate from the cytoplasm to the membrane.

2-8. Confirmation of Gintonin's Cytotoxic Inhibitory Effect

Gintonin treatment in SH-SY5Y cells was confirmed to have an effect of inhibiting cytotoxicity induced by Aβ _1-42 and swe-APP transfection.

As shown in Figure 9, SH-SY5Y cells were treated with 1 ~ 100 ㎍ / ㎖ of gintonin and then cultured for 48 hours did not affect the cell activity when assayed by XTT assay. However, cytotoxicity was induced by Aβ _1-42 (30 μM) treatment, which reduced cell viability by about 20% and confirmed that the cell viability was recovered by gintonin treatment (see FIG. 9A).

The mutated APP protein (swe-APP) gene found in Swedish patients with dementia is known to increase Aβ formation and cytotoxicity when introduced into cells and expressed (Kim et al, J Neurosci). Res. 85, 1528-1537, 2007; Sheng, et al, Free Radic Biol Med. 46, 1362-1375, 2009).

However, gintonin has been shown to reduce cell death of cells into which the swe-APP gene has been introduced according to the concentration of administration. (See Fig. 9B).

2-9. Identifying the Effect of Gintonin in Animal Experiments

Intraperitoneal administration of Aβ _25-35 to laboratory animals results in decreased memory due to toxicity (Maurice et al, Brain Res. 706, 181-193, 1996; Yamada et al, Behav Brain Res. 164, 139-146). , 2006; Stepanichev et al, Neurosci.Behav.Physiology, 36, 102-106, 2006).

In the present invention, as a result of confirming the effect of gintonin in spontaneous cross-acting Y-maze experiments and passive avoidance experiments using mice, the decrease in short-term and long-term memory and working memory due to Aβ _25-35 intraventricular administration according to the treatment with gintonin It has been shown to decrease (see FIG. 10). This suggests that the gintonin of the present invention has an effect of alleviating memory impairment caused by intraventricular administration of Aβ _25-35 .

Examples of the pharmaceutical composition containing the gintonin of the present invention are illustrated as follows, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation Example 1 Preparation of Tablet

Gintonin 100mg

Lactose 100mg

Starch 100mg

2 mg magnesium stearate

The tablets were prepared by mixing the above components and tableting according to a conventional method for producing tablets.

Formulation Example 2 Preparation of Powder

Gintonin 100mg

Lactose 100mg

Talc 10mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 3 Preparation of Capsule

Gintonin 100mg

3mg of crystalline cellulose

Lactose 14.8mg

0.2 mg magnesium stearate

According to a conventional method for preparing capsules, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Gintonin 20mg

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Gintonin 1000mg

10 g of isomerized sugar

Mannitol 5g

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Health Food

Gintonin 100mg

Vitamin mixture proper amount

70 μl of Vitamin A Acetate

Vitamin E 1.0mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15mg

Vitamin B ₆ 0.5mg

0.2 μg of vitamin B ₁₂

Vitamin C 10mg

10 μg biotin

Nicotinamide 1.7mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75mg

Zinc Oxide 0.82mg

Magnesium Carbonate 25.3mg

15 mg potassium monophosphate

Dicalcium Phosphate 55mg

Potassium Citrate 90mg

Calcium Carbonate 100mg

Magnesium chloride 24.8mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

Formulation Example 7 Preparation of Health Beverage

Gintonin 100 g

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

Zinc Oxide 3.5g

Nicotinic Acid 3.5g

1.0 g of vitamin A

0.13 g of vitamin B ₁

0.15 g of vitamin B ₂

After mixing the above components according to a conventional healthy beverage production method, and then 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 in the present invention Used to prepare healthy beverage compositions.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

As described above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a graph confirming sAPPα release (SH) of SH-SY5Y cells according to the gintonin treatment (A) gintonin concentration and (B) gintonin treatment time.

2 is a graph confirming cellular full length APP expression of SH-SY5Y cells according to gintonin treatment by (A) gintonin concentration and (B) gintonin treatment time.

Figure 3 is a graph confirming the reduction of Aβ _1-42 formation according to the gintonin treatment (UT: untreated media control group; wt-APP: wild-type-APP treatment group; swe-APP: swe-APP treatment group).

Figure 4 is a graph confirming the release of sAPPα of SH-SY5Y cells following the treatment of gintonin and ginsenosides (UT: untreated media control; Rb1: ginsenoside Rb1 treatment group; Rg1: ginsenoside Rg1 treatment group; Gintonin : Gintonin treatment group).

Figure 5 is a graph confirming that the increase in sAPPα release following gintonin treatment is reduced by (A) PI3K inhibitor (wortmannin) or (B) PKC inhibitor (GF109203X).

Figure 6 is a graph confirming the increase of [Ca ²⁺ ] _i according to the gintonin treatment, (A) is a case of treatment in a buffer containing calcium, (B) is a case of treatment in a buffer containing no calcium (C) is the case of treatment with a buffer containing cell membrane permeable BAPTA-AM and calcium. In addition, (D) is the case of treatment with a buffer containing / without calcium at each concentration of gintonin concentration, (E) is treated with gintonin treatment after treatment with a buffer or cell membrane-permeable BAPTA-AM containing / without calcium Increasing sAPPα release was confirmed.

Figure 7 shows that the increased sAPPα release following gintonin treatment is (A) metalloprotease inhibitor (TAPI-2), (B) proteolytic process inhibitor (docanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK)), and (C) is a graph confirming the decrease by the protein secretion inhibitor (brefeldin).

Figure 8 is a graph confirming the activation of ADMA10 in (A) whole cell homogeneous suspension or (B) cell membrane fractions following gintonin treatment.

Figure 9 is a graph confirming the cytotoxicity caused by (A) Aβ _1-42 treatment according to the gintonin treatment and (B) cytotoxic inhibitory effect induced by swe-APP infusion.

Figure 10 confirms the effects of the gintonin treatment in animal experiments, (A) is a schedule of animal experiments, (B) and (C) is the gintonin effect in the spontaneous cross-acting Y-maze experiment, (D ) Gintonin effect in passive avoidance experiment.

Claims (6)

A composition for the prevention and treatment of degenerative neurological diseases, including gintonin as an active ingredient. The method of claim 1, The gintonin is a glycolipid protein isolated from ginseng, the composition for the prevention and treatment of degenerative neurological diseases. 3. The method of claim 2, The ginseng is red ginseng, ginseng, white ginseng, cultivated ginseng, camphor ginseng, and wild ginseng composition for the prevention and treatment of neurodegenerative diseases, characterized in that at least one selected from the group consisting of. The method of claim 2 or 3, The ginseng is a root (leaves), leaves (leaves), stems (stem), fruit and flowers of the ginseng composition for the prevention and treatment of degenerative neurological disease, characterized in that at least one selected from the group consisting of. The method of claim 1, The neurodegenerative disorders include one or more diseases selected from stroke, stroke, memory loss, memory impairment, dementia, forgetfulness, Parkinson's disease, Alzheimer's disease, Pick's disease, and Creutzfeld-Kacob disease. Composition for the prevention and treatment of neurodegenerative diseases, characterized in that the. Gintonin and foods have the effect of activating the non-amyloidogenic pathway to increase the release of soluble amyloid proprotein (sAPPα) and preventing neurotoxicity by beta-amyloid (Aβ) Functional foods containing acceptable food supplement additives.

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