KR20190087797A - COMPOSITION COMPRISING Medinilla assamica (C.B. Clarke) C. Chen EXTRACT AS AN EFFECTIVE INGREDIENT FOR PREVENTING OR TREATING NEURODEGENERATIVE DISEASE - Google Patents

Abstract

The present invention relates to a composition for preventing, treating or alleviating neurodegenerative diseases containing a Medinilla assamica extract as an active component. The Medinilla assamica extract has an excellent effect of preventing, treating or alleviating neurodegenerative diseases, thereby being able to be usefully used as a medicine or health functional food.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating degenerative neurological diseases comprising Medinilla asamyca extract as an active ingredient,

The present invention relates to a composition for preventing or treating degenerative neurological diseases comprising Medinilla asamyca extract as an active ingredient.

The control imbalance of the neuroimmune system is known to be a pathogenesis of various neurodegenerative diseases. Microglial cells are present in the central nervous system and play an immune regulatory role. They are activated by neurotoxin or external infection. Activated microglial cells produce various kinds of neuroinflammatory factors and neurotoxins. Microgliosis means that microglial cells, which are activated and activated, help or increase the damage of nerve cells by pathologically releasing toxins and stimulation, which causes damage to other neighboring neurons. Lipopolysaccharide (LPS), a neurotoxin, is a common study model for neuronal damage caused by general inflammation induction. The BV-2 microglial cells synthesize factors such as various inflammation-related factors, nitrite, iNOS, COX-2, TNF-α, IL1β and IL-6 by the endotoxicity of LPS. These materials have been reported to be involved in pathological mechanisms of degenerative neurodegenerative diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease and Huntington's disease. Therefore, it is thought that the regulation of such neuroinflammation plays an important role in the prevention of degenerative neurodegenerative diseases.

The present invention provides a pharmaceutical composition for preventing or treating degenerative neurological diseases comprising Medinilla asamyca extract as an active ingredient.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

The present invention provides a pharmaceutical composition for preventing or treating degenerative neurological diseases comprising Medinilla asamyca extract as an active ingredient.

The Medililla asamyca extract may be obtained by extracting Medililla asamycar with water, C1 to C4 lower alcohols or a mixture thereof.

The medililla asamyca extract may inhibit inflammation induced in microglial cells activated by lipopolysaccharide (LPS).

The inhibition of inflammation may include inhibiting the production of nitrogen monoxide (NO); inhibition of iNOS and COX-2 gene or protein expression; Inhibition of the expression of inflammatory cytokine genes; Or by inhibiting the expression of ERK1 / 2, JNK, p38 and IkB-alpha protein.

The concentration of Medinilla asamyca extract may be 10 μg / ml to 100 μg / ml.

The degenerative neurological disease is selected from the group consisting of stroke, paralysis, memory loss, memory impairment, dementia, forgetfulness, Parkinson's disease, Alzheimer's disease, Pick's disease, Creutzfeld-Kacob disease, Huntington's disease, It may be selected.

In one embodiment of the present invention, there is provided a health functional food for preventing or ameliorating a neurodegenerative disease comprising Medinilla asamyca extract as an active ingredient.

The present invention relates to a composition for preventing, treating or ameliorating degenerative neurological diseases comprising Medinilla asamyca extract as an active ingredient, wherein the Medinilla asamyca extract is a composition for preventing, treating, or ameliorating neurodegenerative diseases in microglial cells activated by lipopolysaccharide (LPS) By inhibiting the induced inflammation, it is excellent in the prevention, treatment or amelioration effect of degenerative neurological diseases and can be useful as a medicine or a health functional food.

FIG. 1 is a graph showing the inhibitory effect on the production of nitrogen monoxide (NO) and the cell survival rate in BV-2 microglia stimulated with LPS in the treatment of methanol extract of Asaminica methanol.
FIG. 2 is a graph showing inhibitory effects of iNOS and COX-2 gene and protein on LPS-stimulated BV-2 microglia upon treatment with medinilla asamycat methanol extract.
FIG. 3 is a graph showing the inhibitory effect of the inflammatory cytokine gene on the LPS-stimulated BV-2 microglial cells in the treatment of Medinilla asamycatum methanol extract. FIG.
FIG. 4 is a graph showing the inhibitory effect of ERK1 / 2, JNK, p38 and IκB-alpha protein on LPS-stimulated BV-2 microglia cells upon treatment with medinilla asamycat methanol extract.

The inventors of the present invention found that treatment of Medinilla asamyca extract with BV-2 microglia cells activated by lipopolysaccharide (LPS) can inhibit neuroinflammation in a concentration-dependent manner with high cell viability Thereby completing the present invention.

Hereinafter, the present invention will be described in detail.

Pharmaceutical compositions for preventing or treating degenerative neurological diseases

The present invention provides a pharmaceutical composition for preventing or treating degenerative neurological diseases comprising Medinilla asamyca extract as an active ingredient.

The pharmaceutical composition for preventing or treating degenerative neurological diseases according to the present invention comprises Medinilla asamyca extract as an active ingredient.

The Medinilla assamica ( CB . Clarke) C. Chen is a shrub belonging to the genus Melastomataceae and Medinilla, and is mainly grown in the Madagascar region, Africa, and tropical regions of the South Asian belt. On the other hand, the neuroinflammation-related efficacy of Medilla asaminica has not been reported.

The medinilla asamyca extract may be obtained by extracting Medinilla asamycar with water, C1 to C4 lower alcohols or a mixture thereof (particularly, methanol), and the extract of Medinilla asamyca extract may contain stem, leaf, Flowers, fruits and roots, but is not limited thereto.

Specifically, the extraction is preferably performed by adding water, C1 to C4 lower alcohols or a mixture thereof to the medinilla asamycar after 2 to 20 times the volume of the Medinilla asaminica, To 10 times the amount of water to be extracted, but is not limited thereto. The extraction temperature is preferably 30 ° C to 100 ° C, more preferably 70 ° C to 100 ° C, but is not limited thereto. The extraction time is preferably 1 hour to 20 hours, more preferably 10 hours to 15 hours, but is not limited thereto. The extraction method may be a cold extraction, an ultrasonic extraction, or a reflux cooling extraction method, but is not limited thereto. The number of times of extraction is preferably 1 to 5 times, more preferably 2 to 3 times, but is not limited thereto. In addition, the Medililla asamyca extract may be diluted, concentrated, diluted or concentrated, and then purified and used.

The medililla asamyca extract inhibits inflammation induced in microglial cells activated by lipopolysaccharide (LPS). Specifically, the suppression of inflammation inhibits formation of nitrogen monoxide (NO). inhibition of iNOS and COX-2 gene or protein expression; Inhibition of the expression of inflammatory cytokine genes; Or by inhibiting the expression of ERK1 / 2, JNK, p38 and IkB-alpha proteins.

The concentration of the medicinal asamyceta extract is preferably 10 μg / ml to 100 μg / ml, more preferably 20 μg / ml to 100 μg / ml, more preferably 50 μg / ml to 100 μg / But is not limited thereto. At this time, when the concentration of Medilla asamyceta extract is too low, the inflammation induced by lipopolysaccharide (LPS) -activated microglia can not be effectively inhibited, so that prevention, treatment or improvement of degenerative neuropathy And when the concentration of the medicinal asamyca extract is too high, toxicity including cytotoxicity may be a concern.

The degenerative neurological disease is an inflammation mediated by microglial cells activated by lipopolysaccharide (LPS), and is used as an inflammatory mediator of stroke, paralysis, memory loss, memory loss, dementia, forgetfulness, Parkinson's disease, Alzheimer's disease, ) Bottle, Creutzfeld-Kacob disease, Huntington's disease and Lou Gehrig's disease, but is not limited thereto.

The pharmaceutical compositions for the prevention or treatment of degenerative neurological diseases according to the present invention can be administered orally or parenterally in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories, And may contain suitable carriers, excipients or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

The carrier or the excipient or diluent includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

In the case of formulation, a diluent or excipient such as a commonly used filler, a weight agent, a binder, a wetting agent, a disintegrant or a surfactant may be used.

The solid preparation for oral administration can be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like in the Medililla asamyca extract. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included .

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As a base for suppositories, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol gelatin and the like can be used.

The preferred dosage of the pharmaceutical composition for the prevention or treatment of degenerative neurological diseases according to the present invention varies depending on the condition of the patient, the body weight, the degree of disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. However, for a desired effect, the dose may be 0.0001 to 2,000 mg / kg, preferably 0.001 to 2,000 mg / kg per day. The administration may be carried out once a day or divided into several doses. However, the scope of the present invention is not limited by the dosage.

The pharmaceutical composition for preventing or treating degenerative neurological diseases according to the present invention can be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

Health functional foods for the prevention or improvement of degenerative neurological diseases

The present invention also provides a health functional food for preventing or ameliorating a neurodegenerative disease comprising Medinilla asamyca extract as an active ingredient.

The health functional food for preventing or ameliorating a neurodegenerative disease according to the present invention comprises the Medinilla asamyca extract as an active ingredient, and the Medinilla asamyca extract has been described above, so that duplicate description will be omitted.

The degenerative neurological disease is an inflammation mediated by microglial cells activated by lipopolysaccharide (LPS), and is used as an inflammatory mediator of stroke, paralysis, memory loss, memory loss, dementia, forgetfulness, Parkinson's disease, Alzheimer's disease, ) Bottle, Creutzfeld-Kacob disease, Huntington's disease and Lou Gehrig's disease, but is not limited thereto.

In the health functional food for preventing or improving degenerative neurological diseases according to the present invention, when the Medicina asamyca extract is used as an additive for a health functional food, it can be added as it is or can be used together with other food or food ingredients, It can be used appropriately according to the method. The amount of the active ingredient to be mixed may be suitably determined according to each use purpose such as prevention, health, or treatment.

The health functional food may be in the form of powders, granules, pills, tablets, capsules, as well as in the form of ordinary foods or beverages.

There is no particular limitation on the type of the food, and examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, , Various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include foods in a conventional sense.

Generally, the Medinilla asamyca extract may be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the raw material when the food or beverage is produced. However, in the case of long-term consumption intended for health or hygiene purposes or for the purpose of controlling health, the amount may be less than the above range. Further, since the present invention uses natural products, there is no problem in terms of safety. Can also be used.

The beverage in the health functional food according to the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the beverage according to the present invention.

In addition to the above, the health functional food for preventing or ameliorating a neurodegenerative disease according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, , Stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages. In addition, the health functional food for preventing or ameliorating the degenerative neurological disease of the present invention may contain natural fruit juice, fruit juice drink, and pulp for the production of vegetable drinks. These components may be used independently or in combination. The ratio of such additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight based on 100 parts by weight of the health functional food of the present invention.

Accordingly, the present invention relates to a composition for preventing, treating or ameliorating a neurodegenerative degenerative disease comprising Medinilla asamyca extract as an active ingredient, wherein the Medinilla asamyca extract is a microglia activated by lipopolysaccharide (LPS) By inhibiting the inflammation induced by cells, it is excellent in the prevention, treatment or amelioration effect of degenerative neurological diseases and can be usefully used as medicines or health functional foods.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Example]

Preparation of natural materials

Medinilla asamica extract ( Medinilla assamica ( CB . Clarke) C. Chen ), which has been harvested, dried and extracted with the help of a local manpower associated with the International Biological Material Center (IBMRC) Were purchased from Korea Plant Extract Bank (KPEB).

Cell culture

BV-2 microglial cells were obtained from Kyungpook National University, and DMEM medium supplemented with 5% FBS inactivated and 100 units / ml 1% penicillin / streptomycin (Gibco, BRL) and 5% ₂ incubator. The number of cells in each experiment was 2.5 × 10 ⁵ cells / ㎖. The treatment with medinilla asamycatum methanol extract was diluted in the medium and pretreated for 1 hour at concentration (10, 50 and 100 μg / ml), and 200 ng / ml of LPS was used.

The cytotoxicity of MedNila asaminica methanol extract was confirmed by MTT assays. After MTT assays were performed, 20 μl of 5 mg / ml MTT was added to the cells in the microplate and reacted for 1 hour in an incubator. The formazan crystals were dissolved in 400 μl of DMSO, and 200 μl of the supernatant was transferred to a 96-well plate, and the absorbance was measured with a microplate reader at a wavelength of 540 nm.

NO Assay

A 24-well assay is a method for quantitatively measuring the production of nitric oxide (NO). After seeding the cells in microplates, LPS and Medinilla asamycatum methanol extracts were added at different concentrations and reacted in a 5% CO ₂ incubator. After 24 hours, the supernatant was reacted with a Griess reagent [1% sulfanilamide, 0.1% N- (1-naphthyl) -ethylenediamine dihydrochloride, 2.5% H3PO4] Respectively.

Identification of total RNA and RT-PCR

All RNAs in BV-2 microglial cell line were isolated using RNAiso Plus (TaKaRa, Japan) according to the manufacturer's instructions. 1 ml of RNAiso Plus and 200 μl of chloroform were added to the BV-2 microglial cell line and mixed well. The mixture was allowed to react at room temperature for 5 minutes and then centrifuged (4 ° C, 16,000 rpm, 15 minutes). To the new tube, 500 μl of the supernatant and the same amount of isopropanol were added, followed by reaction at room temperature for 10 minutes, followed by centrifugation (4 ° C., 16,000 rpm, 15 minutes). The supernatant was removed and washed twice with 75% EtOH. The pellet was well dried and dissolved in DEPC-treated water. RT (reverse transcription) PCR was performed so that the concentration of the sample was 1 μg / μl and the cDNA was synthesized according to the cDNA synthesis method of ReverTra Ace (TOYOBO, JAPAN). Each PCR reaction was carried out using 2 μl of cDNA (0.5 μg / μl). The reaction composition was AccuPower® RocketPlex RT-PCR PreMix (BIONEER), 2 μl of template, 2 μl of PreMix, 1 μl of primer- -R 1 μl and 16 μl of distilled water were reacted in a total of 22 μl under the reaction conditions of 94 ° C for 5 minutes, 94 ° C for 30 seconds to 54 ° C for 1 minute, 72 ° C for 1 minute, 25 times, 72 ° C for 5 minutes and 4 ° C . The primers used were listed in Table 1 below.

target  gene The sequence (5 '- > 3') Size (bp) iNOS Forward primer
Reverse primer 5'-GAGGTACTCAGCGTGCTCCA-3 '
5'-AGGGAGGAAAGGGAGAGAGG-3 ' 444 COX-2 Forward primer
Reverse primer 5'-TGAGTGGTAGCCAGCAAAGC-3 '
5'-CTGCAGTCCAGGTTCAATGG-3 ' 319 IL-1? Forward primer
Reverse primer 5'-CAAGGAGAACCAAGCAACGA-3 '
5'-TTGGCCGAGGACTAAGGAGT-3 ' 428 IL-6 Forward primer
Reverse primer 5'-GGAGGCTTAATTACACATGTT-3 '
5'-TGATTTCAAGATGAATTGGAT-3 ' 435 TNF-a Forward primer
Reverse primer 5'-AGGGAGAGTGGTCAGGTTGC-3 '
5'-CAGCCTGGTCACCAAATCAG-3 ' 392 GAPDH Forward primer
Reverse primer 5'-CCAGTAGACTCCACTCACG-3 '
5'-CCTTCCACAATGCCAAAGTT-3 ' 160

Western blot analysis

Identification of proteins in BV-2 microglial cells was performed using 2X sample buffer [Glycerol, 10% SDS, b-Mercaptoethanol, Bromophenol blue, 0.5M Tris-Hcl]. The same amount of protein was electrophoresed on 10% SDS-PAGE, and then transferred to 0.2 μm nitrocellulose (NC: GE Healthcare Life Sciences) membrane. Anti-β-actin, anti-COX-2 (1: 2000; Santa Cruz) and anti-iNOS (1: 2000; Cell Signaling) were used as primary antibodies and O / N (overnight). Antibodies with Horseradish peroxidase (HRP) as a secondary antibody were reacted for 1 hour at room temperature, and the results were confirmed using ECL kit (pierce, CA) and LAS-3000 (Fuji Co, Japan).

Statistical analysis

To confirm the reproducibility of the test results, the mean value is expressed as ± standard deviation, and each experiment was performed three times. Statistical analysis of the experimental results was performed using Graphpad Prism V5.01. One-way ANOVA was performed using Dunnett's method using software.

Experimental Example  One: Medinilla Asamika  In the treatment of methanol extracts, By LPS  Stimulated BV -2 Measurement of inhibitory effect of nitric oxide (NO) on microglial cells

NO treatment was performed to measure inhibition of nitric oxide (NO) production in BV-2 microglia cells stimulated with LPS during the treatment with medinilla asamycat methanol extract, and the results are shown in Fig.

Specifically, BV-2 microglia cells were placed on a 24-well plate at 1.25 × 10 ⁵ cells / well, and the Medinilla asamycatum methanol extract was pretreated for 1 hour by concentration. Cells were then stimulated with LPS (100 ng / ml). After 24 hours, 100 μl of the cell supernatant was dispensed into 96 wells and the amount of released nitric oxide was measured by Griess assay. The MTT reagent was added to the remaining supernatant, and the survived cells were counted as a percentage, and the experimental results were subjected to three experiments.

FIG. 1 is a graph showing the inhibitory effect on the production of nitrogen monoxide (NO) and the cell survival rate in BV-2 microglia stimulated with LPS in the treatment of methanol extract of Asaminica methanol.

As shown in Fig. 1, the cell survival rate of BV-2 microglia stimulated with LPS was high in treatment of medinilla asamycat methanol extract (10, 50 and 100 / / ml) NO) production inhibitory effect.

Experimental Example  2: Medinilla Asamika  In the treatment of methanol extracts, By LPS  Stimulated BV -2 in microglia iNOS  And COX-2 gene or protein

Inhibition of the expression of iNOS and COX-2 gene in BV-2 microglia stimulated with LPS was measured during the treatment with medinilla asamycat methanol extract. The results are shown in FIGS. 2 (A) and (C). Specifically, BV-2 microglial cells were cultured on a 6-well plate at 5 × 10 ⁵ cells / well. After 6 hours, the supernatant was removed and RNA was identified from the cells. The iNOS and COX- PCR.

FIGS. 2 (A) and 2 (C) are graphs showing the inhibitory effect of iNOS and COX-2 gene on LPS-stimulated BV-2 microglia upon treatment with Medinilla asamycat methanol extract.

As shown in FIGS. 2 (A) and 2 (C), the expression levels of iNOS and COX-2 gene in BV-2 microglia stimulated with LPS were increased compared to the control group, but the treatment with Medinilla asamycat methanol extract 10, 50 and 100 占 퐂 / ml), and the amount of iNOS and COX-2 gene expression was decreased in a concentration-dependent manner. At this time, gapdh was used as a control group to analyze the expression amount of the gene.

Inhibition of iNOS and COX-2 protein expression was also measured in BV-2 microglia stimulated with LPS at the time of treatment with Medinilla asamycat methanol extract. The results are shown in FIGS. 2 (B) and 2 (D) . Specifically, BV-2 microglial cells were cultured on a 6-well plate at 5 × 10 ⁵ cells / well. Twenty hours later, the proteins were identified in the cells and the cells were treated with monoclonal antibodies iNOS and COX-2 using western blot Respectively.

FIGS. 2 (B) and 2 (D) are graphs showing the inhibitory effect of iNOS and COX-2 protein on LPS-stimulated BV-2 microglia when treated with medinilla asamycat methanol extract.

As shown in FIGS. 2 (B) and 2 (D), the expression levels of iNOS and COX-2 gene in BV-2 microglia stimulated with LPS were increased compared with the control group, but the treatment with Medinilla asaminica methanol extract 10, 50 and 100 占 퐂 / ml), and the amount of expression of iNOS and COX-2 protein was found to decrease in a concentration-dependent manner. At this time, β-actin was used as a control to analyze the amount of protein expression.

Experimental Example  3: Medinilla Asamika  In the treatment of methanol extracts, By LPS  Stimulated BV -2 Inhibitory Effect of Inflammatory Cytokine Gene on Microglial Cells

Inhibition of the expression of inflammatory cytokines (TNF-α, IL-1β and IL-6) in BV-2 microglia stimulated with LPS was measured during the treatment with medinilla asamycat methanol extract, Respectively.

Specifically, BV-2 microglia cells were plated at 5 × 10 ⁵ cells / well on a 6-well plate, and Medinilla asamycatum methanol extract was pretreated for 1 hour by concentration. Cells were then stimulated with LPS (200 ng / ml). Six hours later, RNA was isolated from the cells and analyzed for TNF-α, IL-1β and IL-6 genes using RT-PCR.

FIG. 3 is a graph showing the inhibitory effect of the inflammatory cytokine gene on the LPS-stimulated BV-2 microglial cells in the treatment of Medinilla asamycatum methanol extract. FIG.

As shown in FIG. 3, the expression levels of TNF-α, IL-1β and IL-6 genes in BV-2 microglia stimulated with LPS were increased compared to the control group, , 50 and 100 占 퐂 / ml), and the amounts of IL-1? And IL-6 gene expression were found to decrease in a concentration-dependent manner. In particular, the expression level of the TNF-α gene was found to be good at 10 μg / ml, the smallest concentration. At this time, gapdh was used as a control group to analyze the expression amount of the gene.

Experimental Example  4: Medinilla Asamika  In the treatment of methanol extracts, By LPS  Stimulated BV -2 in microglia ERK  1/2, JNK, p38 and IκB-α protein

Inhibition of ERK1 / 2, JNK, p38 and IκB-α protein expression in BV-2 microglia stimulated with LPS was measured during the treatment with methanol extract of Asaminaka methanol. The results are shown in FIG. At this time, ERK1 / 2, JNK and p38 proteins are constituents of MARKs (mitogen-activated protein kinase), and inhibition of this protein expression means inhibition of neuroinflammation through MARKs pathway. Inhibition of IκB-α protein expression also inhibits NF-κB activation, which produces iNOS and COX-2 and inflammatory cytokines.

Specifically, BV-2 microglial cells were plated on a 6-well plate at 5 × 10 ⁵ cells / well, and cultured for 24 hours to pre-treat Medinilla asamycana methanol extract for 1 hour at each concentration. Cells were then stimulated with LPS (200 ng / ml). One hour later, proteins were identified in the cells and analyzed using western blot using monoclonal antibodies p-ERK 1/2, p-JNK, p-p38 and p-IκB-α.

FIG. 4 is a graph showing the inhibitory effect of ERK1 / 2, JNK, p38 and IκB-alpha protein on LPS-stimulated BV-2 microglia cells upon treatment with medinilla asamycat methanol extract.

As shown in FIG. 4, the phosphorylation of ERK1 / 2, JNK, p38 and IκB-α proteins in BV-2 microglia stimulated with LPS was increased compared to the control group, but the treatment with the medinilla asamycat methanol extract 10, 50 and 100 μg / ml), ERK1 / 2, JNK, p38 and IκB-α proteins were found to decrease, indicating that these are signaling mechanisms that suppress neuroinflammation, .

Hereinafter, formulation examples of the medinilla asamyca extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation Example 1: Preparation of powder

Medinilla asamyca extract 20 mg

Lactose hydrate 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight container to prepare powders.

Formulation Example 2: Preparation of tablets

Medinilla asamyca extract 10 mg

Corn starch 100 mg

100mg of lactose hydrate

Magnesium stearate 2 mg

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

Formulation Example 3: Preparation of capsules

Medinilla asamyca extract 10 mg

Microcrystalline cellulose 3 mg

Lactose hydrate 14.8 mg

Magnesium stearate 0.2 mg

After mixing the above components, the capsules were filled in gelatin capsules according to the usual preparation method of capsules.

Formulation Example 4: Preparation of injection

Medinilla asamyca extract 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Sodium dihydrogenphosphate 26 mg

After the above components were mixed, they were prepared with the above ingredient contents per ampoule (2 mL) according to the usual injection preparation method.

Formulation Example 5: Preparation of a liquid preparation

Medinilla asamyca extract 10 mg

10 g per isomer

5 g mannitol

Purified water quantity

Lemon incense quantity

The components are dissolved in purified water according to the usual preparation method, and the lemon flavor is added in an appropriate amount. Then, purified water is added to adjust the total volume to 100 mL, sterilized and filled in a brown bottle to prepare a liquid preparation.

Formulation Example 6: Preparation of Health Functional Foods

Medinilla asamyca extract 10 mg

Vitamin mixture quantity

Vitamin A acetate  70 [mu] g

Vitamin E  1.0 mg

0.13 mg of vitamin B ₁

0.15 mg of vitamin B ₂

0.5 mg of vitamin B ₆

Vitamin B ₁₂ 0.2 g

Vitamin C  10 mg

Biotin  10 [mu] g

Nicotinic acid amide  1.7 mg

Folic acid  50 [mu] g

Calcium pantothenate  0.5 mg

Mineral mixture  Suitable amount

Ferrous sulfate  1.75 mg

Zinc oxide  0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate  15 mg

Dicalcium phosphate  55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

Magnesium chloride  24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a component suitable for a health functional food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above components may be mixed And then granules are prepared and used in the manufacture of health functional foods according to conventional methods.

Formulation Example 7: Preparation of health drinks

Medinilla asamyca extract 10 mg

Vitamin C  15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B ₁ 0.25 g

Vitamin B ₂ 0.3 g

Purified water quantitation

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, It is used in the production of the health beverage composition of the invention.

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.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

Claims (7)

A pharmaceutical composition for preventing or treating degenerative neurological diseases, comprising Medinilla asamyca extract as an active ingredient.
The method according to claim 1,
Wherein the medililla asamyca extract is obtained by extracting medililla asamycar with water, C1 to C4 lower alcohol or a mixture thereof.
The method according to claim 1,
Wherein the medililla asamyca extract inhibits inflammation induced in microglial cells activated by lipopolysaccharide (LPS). ≪ RTI ID = 0.0 > 11. < / RTI >
5. The method of claim 4,
The inhibition of inflammation
Inhibiting the generation of nitrogen monoxide (NO);
inhibition of iNOS and COX-2 gene or protein expression;
Inhibition of the expression of inflammatory cytokine genes; or
ERK < / RTI > 1/2, JNK, p38 and < RTI ID = 0.0 > IkB-a < / RTI > proteins.
The method according to claim 1,
Wherein the concentration of Medinilla asamyca extract is 10 占 퐂 / ml to 100 占 퐂 / ml.
The method according to claim 1,
The degenerative neurological disease is selected from the group consisting of stroke, paralysis, memory loss, memory impairment, dementia, forgetfulness, Parkinson's disease, Alzheimer's disease, Pick's disease, Creutzfeld-Kacob disease, Huntington's disease, Lt; RTI ID = 0.0 > (I) < / RTI > for the prevention or treatment of neurodegenerative diseases.
A health functional food for preventing or improving a degenerative neurological disease comprising Medinilla asamyca extract as an active ingredient.

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