KR20200052669A - Pharmaceutical composition including black berry extract for preventing or treating dementia - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating dementia containing a blackberry extract as an active component. The pharmaceutical composition for preventing or treating dementia of the present invention contains the blackberry extract as the active ingredient. The pharmaceutical composition containing the blackberry extract can be provided for stable and effective dementia prevention or treatment.

Description

Pharmaceutical composition for preventing or treating dementia containing blackberry extract as an active ingredient {PHARMACEUTICAL COMPOSITION INCLUDING BLACK BERRY EXTRACT FOR PREVENTING OR TREATING DEMENTIA}

The present invention relates to a pharmaceutical composition for preventing or treating dementia comprising blackberry extract as an active ingredient.

Raspberry (Rubus) is a berry fruit and is a shrub plant belonging to the family Rosaceae (Thompson 1997). Rubus, commonly known as raspberry (bramble, raspberry), has over 400 plants, and is largely classified into raspberry, blackberry, and dewberry. Raspberries native to Korea have been reported in a variety of ways, including bokbunja (R. coreanus) and yolk (R. parvifolius). Bokbunja is widely cultivated in sub-Central, especially Gochang, Jeongeup, and Jeju Island in Jeollabuk-do. It is estimated that this is a kind of black raspberry introduced from North America as a similar species of native Bokbunja strawberry. Raspberry fruits contain ingredients such as flavonoids, tannins, phenolic compounds, and β-cystosterols, which have high antioxidant and anti-cancer effects, and their demand is increasing as a food and beverage material.

Blackberry ( Rubus fruticosus ) is a subgenus of Eubatus, the fruit is black, about 1 to 2 cm in diameter, and much more complex than the raspberry in genetic background, growth characteristics, and number of species.

As a prior art patent using blackberry fruit, Korean Patent Publication No. 10-2017-0073778 describes a berry drink having an antioxidant function, including a blackberry juice solution and a juice of another fruit, and Korean Patent Application No. 10 -2006-0003218 describes that blackberry has a protective effect on liver function, and Korean Patent Publication No. 10-2012-0027040 discloses that blackberry extract has an anti-influenza effect. In addition, Korean Patent Application No. 10-2014-0167578 describes that the extract of blackberry fermentation relieves gum inflammation and suppresses gum disease bacteria.

Thus, the present inventors confirmed the effect of preventing or treating dementia as a new effect different from the existing one of the blackberry extract, and completed the present invention.

The present invention is to provide a pharmaceutical composition for preventing or treating dementia comprising a blackberry extract as an active ingredient.

The pharmaceutical composition for preventing or treating dementia of the present invention for achieving the above object includes a blackberry extract as an active ingredient.

The blackberry extract contained in the pharmaceutical composition defined in the present invention includes not only blackberry extract, but also a solvent fraction thereof, or a mixture thereof.

In one embodiment, the blackberry extract may be extracted with water, a lower alcohol having 1 to 6 carbon atoms, or a mixed solvent thereof.

In one embodiment, the blackberry extract may be a blackberry extract with 30% to 95% ethanol or a hot water extract.

In one embodiment, the blackberry extract may be a blackberry extract with 50% ethanol.

In one embodiment, the blackberry extract may be a fraction obtained by fractionating a blackberry extract with 30% to 95% ethanol into at least one of hexane, dichloromethane (methylene chloride), ethyl acetate, saturated butanol, and water.

In one embodiment, the blackberry extract exhibits the ability to inhibit beta amyloid aggregation or inflammation of brain microglia.

In one embodiment, the blackberry extract exhibits the ability to inhibit vascular dementia.

In one embodiment, the blackberry extract shows the ability to inhibit phosphorylation of Tau protein.

In one embodiment, the blackberry extract may be a blackberry leaf 50% alcoholic extract.

In the pharmaceutical composition for preventing or treating dementia comprising the blackberry extract described above as an active ingredient, the blackberry extract, which is an active ingredient, exhibits an inhibitory effect on beta amyloid aggregation or inflammation of brain microglia, and inhibits vascular dementia and Tau protein. It was confirmed that the phosphorylation inhibitory ability was exhibited. Pharmaceutical compositions comprising such blackberry extract may be provided for stable and effective dementia prevention or treatment. In particular, the new Blackberry variety of "Black Pearl" was the final selection of fully mutants with increased yield and sugar content among 80 gy gamma-irradiated plants (20,000 individuals) derived from the offspring of blackberry varieties (Black V3). Thereafter, as a new breed of blackberry obtained by propagating through in-flight culture and fixing the trait, the new breed of blackberry extract exhibits the ability to inhibit beta amyloid aggregation or to suppress inflammation of brain microglia, and has an excellent ability to inhibit vascular dementia and phosphorylation of Tau protein. Was confirmed.

1 is a view showing the results of a cell survival rate evaluation experiment for confirming the effect of inhibiting brain cell inflammation of the blackberry leaf 50% alcohol extract according to an embodiment of the present invention.
2 is a view showing the results of NO secretion suppression experiments to confirm the effect of inhibiting brain cell inflammation of the blackberry leaf 50% alcohol extract according to an embodiment of the present invention.
3 is a view showing the results of an inflammatory cytokine secretion inhibition experiment for confirming the effect of inhibiting brain cell inflammation of a blackberry leaf 50% alcohol extract according to an embodiment of the present invention.
4 is a view showing the results of the evaluation of the inhibition of hyperphosphorylation in the brain neurons of blackberry leaf 50% alcohol extract according to an embodiment of the present invention.
5 is a view showing the results of passive evasion evaluation of an animal model for inducing vascular dementia administered with 50% alcoholic extract of blackberry leaves according to an embodiment of the present invention.
FIG. 6 is a diagram showing the results of a Y-type maze test in an animal model of vascular dementia inducing administration of 50% alcoholic extract of blackberry leaves according to an embodiment of the present invention.
7 is a view showing the results of a maze test (acquisition experiment) in an vascular dementia-induced animal model to which 50% alcoholic extract of blackberry leaves was administered according to an embodiment of the present invention.
8 is a view showing changes in the number of cranial nerve cells in the brain (hippocampus) of an animal model inducing vascular dementia to which 50% alcoholic extract of blackberry leaves is administered according to an embodiment of the present invention.

Blackberry for obtaining a blackberry extract in the present invention may include those obtained from any one or two or more parts of leaves, stems, fruits, roots, husks, and seeds in Korea, preferably blackberry leaf extract Or a stem extract is used.

The blackberry variety used in the present invention is a “Black Pearl” new breed of blackberry, and among the individuals irradiated with ⁶⁰ Co gamma ray 80 Gy on the predators (20,000 individuals) derived from an offspring of the existing blackberry variety (Black V3). It is a new breed of blackberry obtained by genetically fixing traits by propagating through in-flight culture after final selection of mutants. The mineral content, sugar content, vitamin C content, total amino acid content, total anthocyanin content, total flavonoid content and total phenol content of the Black Pearl variety are as shown in FIG. 9. Referring to FIG. 9, it can be seen that the new pearl black varieties have a different content of each component compared to the V3 before the improvement, and it can be confirmed that the new varieties are different from the V3 before the improvement.

In addition, preferably, the leaf extract and the stem extract may be a leaf / stem harvested during a specific time period or a specific period during a period between spring and autumn, and a leaf / stem harvested during a period between spring and autumn. It may be used. Extraction of the active ingredient from the blackberry in the present invention can be performed by a commonly known solvent extraction method. In the present invention, it may preferably be a blackberry leaf alcoholic extract. In particular, the blackberry leaf or stem alcohol extract in the present invention is preferred in preventing or treating dementia than the blackberry fruit extract.

In order to obtain a blackberry extract, the blackberry is picked and cut, washed, dried and crushed to a desired site, for example a blackberry leaf and / or stem; 3 to 30 times, preferably 10 to 20 times the weight of the blackberry leaf or stem pulverized, water or lower alcohol (e.g. methanol, ethanol, butanol, etc.), or a mixture thereof to extract at 60 ° C to 75 ° C The extraction process is performed for at least 1 hour at a temperature. At this time, the extraction of alcohol using lower alcohol may be 30% alcohol to 95% alcohol. If it is less than 30% alcohol, it is preferable to perform at 30% to 95% alcohol because more practically no alcohol soluble active ingredients can be obtained, and when it is more than 95% alcohol, there are few water-soluble active ingredients, more preferably May be 30% to 70% alcohol, and most preferably 70% alcohol. At this time, the extract can be extracted until the concentration of the soluble solids exceeds 0 brix but reaches 30 brix. As an example, the extract may be extracted until the concentration of the soluble solids reaches 20 to 21 brix. At this time, as a mixed solvent, the mixing ratio of water and lower alcohol may be 1: 1 to 1:10. As the extraction method, hot water extraction, cold immersion extraction, reflux cooling extraction, ultrasonic extraction, steam distillation extraction, or the like may be used to continuously extract 1 to 5 times. After extraction, it is filtered at a temperature of 40 ° C to 60 ° C, the filtrate is concentrated under reduced pressure, and then dried by a freeze dryer to obtain a blackberry extract.

After suspending the thus obtained blackberry extract in water, the system is separated using an extraction solvent, and concentrated under reduced pressure to obtain a fraction for each extraction solvent of the blackberry extract. As an example of a solvent used to obtain a blackberry extract, purified water or an organic solvent having 1 to 6 carbon atoms can be used. Examples of the organic solvent, ethanol, methanol, propanol, butanol, glycerin, ethyl acetate, ethyl acetate, butylene glycol, propylene glycol (propylene glycol) glycol), dichloromethane, chloroform, ethyl ether, hexane, and the like. These may be used alone or in combination of two or more.

In one embodiment, in order to obtain a fraction of a blackberry extract, hexane, dichloromethane (methylene chloride), ethyl acetate, water-saturated butanol, and water can be used for system separation, through which the hexane layer of the blackberry extract, dichloromethane Fractions of layers, ethyl acetate layers, butanol layers and water layers can be obtained. That is, hexane treatment can be performed to obtain a hexane-soluble fraction and a water-soluble fraction, and again, the water-soluble fraction can be extracted with dichloromethane to obtain a water-soluble fraction and a dichloromethane-soluble fraction, ethyl in the water-soluble fraction Acetate can be added to obtain an ethyl acetate soluble fraction and a water soluble fraction, and the water soluble fraction can be extracted with butanol to obtain a butanol soluble fraction and a water soluble fraction.

In order to confirm the efficacy of the blackberry extract as an active ingredient of the pharmaceutical composition according to the present invention as a pharmaceutical composition for preventing or treating dementia, a result of evaluating beta amyloid aggregation inhibition and inflammatory inhibitory effect of BV-2 brain microglia, It was confirmed that the blackberry extract of the present invention has an effect of inhibiting beta amyloid aggregation. Through this, it is known as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Multiple sclerosis (MS), and amyotrophic lateral axons known as Lou Gehrig's disease. Amyotrophic lateral sclerosis (ALS) can be improved or treated. In addition, since it has no cytotoxicity and has little toxicity and side effects as a natural substance, it can be used safely for a certain period to prevent or treat dementia, and it has been confirmed that it inhibits the secretion of NO, an inflammatory marker, and also has cytokine suppression ability. Was confirmed. In addition, it was confirmed that the blackberry extract acts to suppress the hyperphosphorylation of Tau protein in neurons and can effectively improve vascular dementia.

Through the efficacy of such a blackberry extract, blackberry extract may be provided as a pharmaceutical composition for preventing or treating dementia.

The pharmaceutical composition according to the present invention, the blackberry extract as an active ingredient, may include a pharmaceutically acceptable excipient together.

The pharmaceutical composition according to the present invention, the blackberry extract as an active ingredient, may include one or more known active ingredients having a dementia prevention or treatment effect.

The composition of the present invention may be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. Pharmaceutically acceptable carriers can be used by mixing one or more of these components: saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and antioxidants, buffers if necessary. , Other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into injectable formulations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets.

The blackberry extract according to the present invention may be included as an active ingredient in the total pharmaceutical composition of 0.001 to 90% by weight, preferably 0.01 to 15% by weight, more preferably 0.01 to 10% by weight. The dosage amount of the pharmaceutical composition thus prepared may be appropriately adjusted according to the individual difference formulation such as age and degree of lesion, and the daily dosage for an adult may be used in an effective amount of 0.01 mg to 10,000 mg. There is, for example, the daily dosage may be from 0.01 mg to 1,000 mg, it is preferable to be divided into once to several times a day.

The pharmaceutical composition of the present invention may further contain a preservative, a stabilizer, a hydrating or emulsifying accelerator, a salt or buffer for controlling osmotic pressure, a disintegrant, a binder, a lubricant, a diluent, and other therapeutically useful substances. It can be formulated in various oral or parenteral dosage forms according to conventional methods.

Formulations for oral administration include, for example, tablets, pills, hard and soft capsules, liquids, suspensions, emulsifiers, syrups, granules, etc. These formulations, in addition to active ingredients, diluents such as lactose, dextrose, sucrose , Mannitol, sorbitol, cellulose and glycine, lubricants such as silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycol. Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, and optionally starch, agar, alginic acid or sodium salts thereof It may contain pharmaceutical additives such as disintegrants, absorbents, colorants, flavoring agents, and sweetening agents. Tablets can be prepared by conventional mixing, granulating or coating methods. In addition, a representative formulation for parenteral administration is an injectable formulation, preferably an isotonic aqueous solution or suspension.

However, it should be understood that the actual dosage of the active ingredient should be determined in light of a number of related factors such as the severity of the symptoms, the route of administration chosen, the age, sex, weight and health of the subject.

Dosages suitable as pharmaceutical compositions according to the present invention are usually readily available to the skilled physician to determine and prescribe effective doses for the skin. The daily dosage of the drug according to the present invention varies depending on various factors such as the degree of obesity progression, the onset time, age, health condition, complications, etc. of the subject to be administered, but is generally the above-mentioned weight ratio when based on adults. Compositions combined with 1 to 500 mg / kg, preferably 30 to 200 mg / kg can be administered in divided portions 1 to 2 times a day, and the dosage is to limit the scope of the present invention in any way. no.

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

Below, the reagents used for the preparation and experiment of the extract were purchased from Sigma Aldrich and used as they are.

In addition, the blackberry varieties used in the present invention were pre-sold new "Black Peal" blackberry seedlings developed by Bioplus Co., Ltd., and used materials cultivated and produced by Prime Farm Co., Ltd. The "Black Pearl" variety was mutated as reported in the Journal of Radiation Industry, 6 (3): 257 to 265 (2012), a predatory plant derived from an offspring of the existing Blackberry variety (Black V3) ( It is a new breed of blackberry obtained by genetically fixing traits by propagating through in-flight culture after final selection of a complete mutant among individuals who have irradiated gamma ray 80 Gy to 20,000 individuals). In the present invention, the leaves, berries and stems of the corresponding blackberry varieties were purchased from Prime Farm Co., Ltd. and used.

Blackberry  Preparation of extract

(One) Blackberry Hydrothermal  Preparation of extract

Blackberry leaves obtained as described above were added to 40 kg of distilled water in 2 kg of leaf powder obtained after crushing and crushing the blackberry leaves, filtered for 4 hours at 100 ° C., filtered with filter paper, and concentrated at 50 ° C. under reduced pressure to 2 L. Did. The concentrated extract was dried with a freeze dryer to obtain a blackberry leaf hot water extract.

For each of the blueberry fruits and stems, a substantially identical process to the step of obtaining the blackberry leaf hot water extract was performed, thereby obtaining the blackberry fruit and stem hot water extract, respectively.

(2) Blackberry  Preparation of alcoholic beverage extract

The blackberry leaves obtained as described above were added to 40 kg of 30%, 50%, 70%, and 95% ethanol into 2 kg of leaf powder obtained after crushing and crushing, and then filtered for 4 hours at 75 ° C. At 50 ° C., the extract was concentrated to 2 L under reduced pressure. The concentrated extract was dried with a freeze dryer to obtain a blackberry leaf extract (30% alcohol (30% ethanol): 0.68 kg, 50% alcohol: 0.61 kg, 70% alcohol: 0.56 kg, 95% alcohol: 0.27 kg).

In addition, for each of the blueberry fruits and stems, the blackberry leaves 30%, 50%, 70% alcohol and 95% alcohol extracts were subjected to substantially the same process as the process to obtain the blackberry fruits and stems, respectively. Eggplant spirit extract was obtained.

(3) Blackberry Fraction  Preparations

20 g of the blackberry leaf alcohol 50% extract obtained as described above was suspended in 200 mL of distilled water, systemically separated with hexane, dichloromethane (methylene chloride), ethyl acetate, water-saturated butanol and water, and concentrated under reduced pressure to extract Fractions of hexane layer (0.4 g), dichloromethane layer (1.5 g), ethyl acetate layer (2.0 g), butanol layer (10.0 g), and water layer (6.1 g) were obtained. All extracts were used by dissolving the fractions obtained for cell experiments in DMSO (dimethyl sulfoxide) at a concentration of 100 mg / mL.

Experimental Example  1: beta amyloid aggregation Restraint  evaluation

The beta amyloid aggregation inhibitory ability evaluation experiment was performed using the blackberry extract obtained according to the above example. Fluorescence analysis (ThioflavinT assay) was used to measure the degree of aggregation of beta amyloid (β-amyloid, Aβ). The beta amyloid (Aβ1-40) peptide was stored at minus 80 ° C, and the final concentration used in the measurement was used at a concentration of 5 μM. The blackberry extract obtained according to the present invention was dissolved in 99.5% DMSO immediately before the experiment and diluted with buffer solution for concentration, and 5 μM of thioflavin T used as a fluorescent material was used as a 0.01M sodium phosphate buffer solution. It was prepared by dissolving in.

In order to measure the degree of Aβ1-40 aggregation inhibition, 5 µl of various blackberry extracts (hot water, alcohol, and fractions by concentration) were added to 92.5 µl of 0.01 M sodium phosphate buffer solution, and 2.5 µl of 5 µM Aβ1-40 was added, and then 37 Incubation was performed at 26 ° C. for 26 hours, and 2,000 μl of 5 μM thioflavin T was added and measured by a fluorescence spectrometer. At this time, the wavelength value of the fluorescence spectrometer used was Ex = 446 nm (slit width = 5 nm), Em = 490 nm (slit width = 10 nm), and the same experiment was repeated at least three times or more to represent the average value. The results are shown in Table 1 below.

Sample classification Sample Concentration (μg / mL) β-amyloid
Inhibition of aggregation (%) Ab 0 0 Ab + 0.4% DMSO 0 0 Ab + ovanillin 100 40.2 Ab + Blackberry Leaf Hot Water 100 48.7 Ab + Blackberry Fruit Water 100 20.7 Ab + Blackberry Stem Hot Water 100 28.7 Ab + Blackberry leaf alcohol (50%) 100 68.3 Ab + Blackberry Fruit Alcohol (50%) 100 34.1 Ab + Blackberry Stem alcohol (50%) 100 42.4 Ab + Blackberry leaf alcohol (30%) 100 40.3 Ab + Blackberry leaf alcohol (50%) 100 67.6 Ab + Blackberry leaf alcohol (70%) 100 42.3 Ab + Blackberry Leaf Alcohol (95%) 100 50.7 Ab + Blackberry Fruit Alcohol (30%) 100 20.2 Ab + Blackberry Fruit Alcohol (50%) 100 40.8 Ab + Blackberry Fruit Alcohol (70%) 100 25.4 Ab + Blackberry Fruit Alcohol (95%) 100 31.7 Ab + Blackberry Stem alcohol (30%) 100 32.8 Ab + Blackberry Stem alcohol (50%) 100 48.7 Ab + Blackberry Stem alcohol (70%) 100 30.1 Ab + Blackberry Stem alcohol (95%) 100 35.7 Ab + blackberry leaf nucleic acid fraction 100 11.4 Ab + Blackberry Leaf Methyl Chloride Fraction 100 15.7 Ab + Blackberry Leaf Ethyl Acetate Fraction 100 58.4 Ab + Blackberry Leaf Butanol Fraction 100 47.6 Ab + Blackberry Leaf Water Fraction 100 42.5

Table 1 is a table showing the experimental results for confirming the extraction method of blackberry extract obtained according to an embodiment of the present invention (hot water, alcohol) and the inhibitory effect of beta amyloid aggregation by region.

In Table 1, "Ab" represents beta amyloid, "Ab + 0.4% DMSO" represents a group treated with DMSO on beta amyloid, and "Ab + ovanillin" represents a group treated with beta amyloid, a positive control ovanillin. "Ab + Blackberry Leaf Hydrothermal Water" refers to a group treated with an extract obtained by extracting hot water from a blackberry leaf to beta amyloid, and "Ab + Blackberry Fruit Water" refers to a group treated with an extract obtained by extracting hot water from a blackberry fruit to beta amyloid. "Ab + Blackberry Stem Hydrothermal Water" refers to a group of beta amyloid treated with an extract obtained by hot water extraction of a blackberry stem. Ab + Blackberry Leaf Alcohol (30%), (50%), (75%), (90%) treated extracts extracted with 30%, 50%, 75% and 90% of blackberry leaves in beta amyloid, respectively. One group, Ab + Blackberry Stem and Ab + Blackberry Fruit Sake represent groups treated with the extract of stem and fruit, but the numerical value in each sample represents the concentration of alcohol. In addition, the Ab + solvent fraction represents the groups treated with the fraction.

Referring to Table 1, it can be seen that the aggregation inhibitory effect of beta amyloid by region of the blackberry extract was highest in the blackberry leaf.

For extracts of blackberry leaves, berries, and stems by extraction method (hot water, 30%, 50%, 75%, and 90% alcohol), it was confirmed that the aggregation inhibition of beta amyloid was highest in the 50% alcohol extract treatment group. have.

When comparing these results with the same amount of ovanillin treatment, it was confirmed that the aggregation inhibitory effect of beta amyloid was obtained by the blackberry extract according to the present invention, and the most effective extraction method of the blackberry extract according to the present invention was alcohol (50 %), But compared to the fruit, it can be seen that the aggregation inhibitory effect of beta amyloid appears in the stem and leaves.

In addition, among the extract treatments by blackberry leaf 50% alcoholic extract and solvent fractions (nucleic acid, methyl chloride, ethyl acetate, butanol, water), the aggregation inhibition of beta amyloid was the highest in the blackberry leaf 50% alcoholic extract treatment, In the blackberry leaf ethyl acetate, butanol, and water fractions, compared to the positive control ovanillin, it can be seen that it is similar or highly observed, and it can be seen that the blackberry extract according to the present invention has a beta amyloid aggregation inhibitory effect.

Experimental Example  2: BV -2 Inflammation inhibitory effect of brain microglia-Cell survival rate

Cytotoxicity evaluation of the blackberry extract was performed by observing the cytotoxicity of BV-2 by treating the blackberry extract by concentration in brain microglial cells (BV-2 microglial cells).

Specifically, after culturing the microglial cells in DMEM cell culture medium (Dulbecco's Modified Eagle's medium) containing 5% FBS at 37 ° C and 5% CO ₂ conditions, spread them on a 96 well plate to be 2 × 10 ⁴ cells per well. , The cells were completely adhered while incubating for 12 hours, and the group treated with the blackberry leaf 50% alcohol extract at a concentration of 50, 100 and 200 μg / mL for 24 hours and the 50% alcohol extract of the blackberry leaf 50, 100 and At the same time as 200 μg / mL treatment, cell viability of the treatment group treated with LPS (Lipopolysaccharide) at a concentration of 1 μg / mL for 24 hours was evaluated. Evaluation of cell viability was performed according to the MTT method, and the survival rate was calculated as% survival compared to the untreated group. The results are shown in FIG. 1.

1 is a view showing the results of a cell survival rate evaluation experiment for confirming the effect of inhibiting brain cell inflammation of the blackberry leaf 50% alcohol extract according to an embodiment of the present invention.

Referring to FIG. 1, brain microglia through cell viability as a result of treatment by concentration (50, 100, 200 μg / mL) of blackberry leaf 50% alcoholic extract by treatment and non-treatment conditions of inflammation-inducing LPS (BV-2 microglial cell) can be confirmed that there is no cytotoxicity.

Experimental Example  3: BV -2 Inflammatory inhibitory effect of brain microglia -NO Restraint

After dispensing BV-2 cells into a 96 well plate at 2 ㅧ 10 ⁴ cells / well, cells are completely adhered while incubating for 12 hours in a 37 ° C, 5% CO ₂ incubator, and 50% of blackberry leaves extract 50, The group treated for 24 hours at a concentration of 100, 200 μg / mL and the 50% alcoholic extract of blackberry leaves were treated at 50, 100, 200 μg / mL, and at the same time, LPS, an inflammatory substance, at a concentration of 1 μg / mL for 24 hours. In the meantime, the culture supernatant of the treated group was separated. The content of NO was measured in the separated culture supernatant. The content of NO was treated with the same amount of Griess (Sigma) reagent in 100 μL of the separated culture supernatant, reacted for 10 minutes, and absorbance at 517 nm was measured using a microplate reader. The concentration of nitric oxide was calculated by comparison with a standard straight line obtained using sodium nitrite (NaNO ₂ , Sigma). The results are shown in FIG. 2.

2 is a view showing the results of NO secretion suppression experiments to confirm the effect of inhibiting brain cell inflammation of the blackberry leaf 50% alcohol extract according to an embodiment of the present invention.

Referring to Figure 2, when the treatment of the inflammation-inducing substance LPS, the secretion of NO was increased in the blackberry extract-free treatment, but the concentration of 50% of the blackberry leaf extract in the brain microglial cells (BV-2 microglial cells) (50, 100, 200 μg / mL), it can be seen that the concentration of the extract is decreased by the concentration of the extract, an inflammation marker that increases secretion by LPS. The treatment of the 50% alcoholic extract of blackberry leaves reduces the secretion capacity of NO by the treatment of LPS, and it can be confirmed that it is effective in the inflammatory reaction.

Experimental Example  4: BV -2 Inflammatory inhibitory effect of brain microglia-Cytokine Restraint

After dispensing BV-2 cells into a 96 well plate at 2 ㅧ 10 ⁴ cells / well, cells are completely adhered while incubating for 12 hours in a 37 ° C, 5% CO ₂ incubator, and 50% of blackberry leaves extract 50, The group treated for 24 hours at a concentration of 100, 200 μg / mL and the 50% alcoholic extract of blackberry leaves were treated at 50, 100, 200 μg / mL, and at the same time, LPS, an inflammatory substance, at a concentration of 1 μg / mL for 24 hours. In the meantime, the culture supernatant of the treated group was separated. The content of inflammatory cytokines TNF-α and IL-6 in the separated culture supernatant was measured. The cytokine content was measured using an ELISA kit (eBioscience Co. (Company name), San Diege, CA, USA), and the concentration of the cytokine was calculated from a standard curve calculated from a standard solution included in the kit. The results are shown in FIG. 3.

3 is a view showing the results of an inflammatory cytokine secretion inhibition experiment for confirming the effect of inhibiting brain cell inflammation of a blackberry leaf 50% alcohol extract according to an embodiment of the present invention.

Referring to FIG. 3, when the LPS, which is an inflammation-causing substance, was treated, the secretion of inflammatory cytokines (TNF-α and IL-6) was increased in the blackberry leaf 50% alcoholic extract, but the blackberry leaf 50% alcoholic extract was brain-treated. When the microglia cells (BV-2 microglial cells) are treated at different concentrations (50, 100, 200 μg / mL), TNF-α and IL-6, which are inflammation markers that are secreted by LPS, are decreased in a concentration-dependent manner. You can confirm that. It can be seen that the treatment of the blackberry leaf 50% alcoholic extract was effective in the inflammatory reaction, as it was observed to reduce the secretory ability of inflammatory cytokines by treatment of LPS.

Experimental Example  5: Tau Protein superphosphorylation  Inhibitory effect

After dispensing SH-SY5Y cells, which are brain neurons, into 24 well plates at 2 × 10 ⁵ cells / well, incubate for 24 hours in a 37 ° C, 5% CO ₂ incubator, attach the cells completely, and extract 50% of blackberry leaves Was pretreated at a concentration of 50, 100, 200 μg / mL for 4 hours, and okadaic acid was treated at a concentration of 10 nM for 16 hours. In this experiment, it was used to induce hyperphosphorylation of brain neurons, SHSY5Y.

After extracting the protein with RIPA lysis buffer (Thermo) and quantifying with BCA Protein Assays kit (Thermo), a sample was prepared for electrophoresis. The prepared samples were subjected to electrophoresis and Western blotting to react with p-Tau antibody and beta-actin antibody (abcam) to analyze the protein expression levels of p-Tau and beta-actin, and the effect of inhibiting the hyperphosphorylation of Tau protein was okada It was evaluated in comparison with the protein expression level of the dispersed sample. The results are shown in Fig. 4.

4 is a view showing the results of the evaluation of inhibition of hyperphosphorylation in brain neurons of a blackberry leaf 50% alcohol extract according to an embodiment of the present invention.

In FIG. 4, “OA” indicates okadaic acid, “CON” indicates okadaic acid-free treatment, “0”, “25”, “50”, and “100” represents okadaic acid and blackberry leaf alcoholic extract 50% 25, Groups treated together at concentrations of 50 and 100 μg / ml are shown.

Referring to FIG. 4, it can be seen that Tau protein hyperphosphorylation was induced in the “0” group (Okada acid mono-treatment group) compared to the “CON” group, and that hyperphosphorylation was inhibited in the blackberry leaf alcohol 50% extract treatment group. Therefore, it can be seen that the 50% alcoholic extract of blackberry leaves is effective in inhibiting phosphorylation of Tau protein.

Experimental Example  6: Efficacy in inhibiting vascular dementia

In the 8-week-old SD male rats, it was administered at 10 mg / kg / day for 5 days of blackberry leaf alcoholic 50% extract, and vascular dementia was induced on the 3rd day of administration. For the induction of vascular dementia, a model was used to limit the blood flow to the brain to less than 30% of the physiological blood flow by ligating both sides of the carotid artery for 10 minutes.

① Manual evasion experiment

Passive evasion experiments were conducted on the 10th to 11th days after induction of vascular dementia, and experiments using GEMINITM Avoidance as an experiment using the characteristics of a mouse preferring dark places instinctively. There are two acrylic boxes, and a door is installed between the boxes for the moving animals. A stainless steel rod is placed on the bottom of the box to apply an electric shock. Before starting the experiment, adapt to one box for 60 seconds, then shine the light through the light, make noise, and open the door, causing the mouse to move to the other box. On the 10th day of inducing vascular dementia, the experimental animal is placed in a box in the same location, and after 20 seconds, the light is lit again and makes a noise. When the experimental animal enters the opposite box, an electric shock (0.1 mA / 10 g body) is given after 2 seconds. After 24 hours, the same experiment was conducted and the time for the test animal to move to the opposite box was measured as step through latency. Step through latency was measured up to 180 seconds, after which the experiment was stopped if there was no movement. The results are shown in Fig. 5.

5 is a view showing the results of passive evasion evaluation of an animal model for inducing vascular dementia administered with 50% alcoholic extract of blackberry leaves according to an embodiment of the present invention.

In FIG. 5, “SHAM” indicates gastric surgery, “VD” indicates vascular dementia inducer, and “VD + Blackberry leaf alcohol 50% extract” refers to vascular dementia milk tool and blackberry leaf alcohol 50% extract 10 mg / kg body weight refers to the group administered orally once a day for 5 days.

Referring to FIG. 5, the electric shock was recognized in the gastric surgery tool, and the time to move to the opposite box was long, and it was confirmed that the electric shock was continuously recognized by the vascular dementia inducer and was continuously moved to the opposite box.

In the vascular dementia treatment tool administered with 50% extract of blackberry leaf, the electric shock was recognized, and the time to move to the opposite box was long. Therefore, it can be seen that administration of the 50% alcoholic extract of blackberry leaves improves cognitive ability according to the induction of vascular dementia.

② Y-maze experiment

The Y-maze experiment was conducted on the 9th and 10th days after the induction of vascular dementia, and the device of the Y-maze experiment consisted of three branches (each branch being designated as A, B, and C). Each arm passage has a length of 40 cm, a width of 12 cm, and a height of 30 cm, and the angle formed by the three branches with each adjacent branch is 120 degrees. All testing devices consisted of black polyvinyl plastic. The experimental animal was carefully placed on one branch of the maze, allowed to move freely for 8 minutes, and the branch containing the experimental animal was recorded. The record was limited to the case where the animal's tail was completely retracted, and the case of re-entering the branch was also recorded. The results are shown in Fig. 6.

FIG. 6 is a diagram showing the results of a Y-type maze test in an animal model of vascular dementia inducing administration of 50% alcoholic extract of blackberry leaves according to an embodiment of the present invention.

In FIG. 6, “SHAM” indicates a gastric surgical instrument, “VD” indicates a vascular dementia inducer, and “VD + Blackberry Leaf Alcohol 50% Extract” refers to a blackberry leaf alcohol 50% extract in a vascular dementia milk tool 10 mg / kg body weight refers to the group administered orally once a day for 5 days, Total arm entries represents the number of times the experimental animals entered into different (or the same) arms for a total of 8 minutes, and "spontaneous alternation" represents the unit time ( The number of movements (eg, A → B → C or B → C → A or C → A → B, etc.) based on a sequence of arms with unequal arms per 8 minutes). This number was converted to a ratio by the following equation: alternation rate (%) = number of alternations / (number of total arm entries-2) x 100. The results are shown in Fig. 6.

Referring to FIG. 6, all groups of experimental animals show the number of times they entered with different (or the same) arms, and spontaneous alternation value was measured high in gastric surgery, but significantly decreased in vascular dementia inducers and vascular dementia In the group administered with the blackberry leaf alcohol 50% extract, it can be seen that it shows an increasing trend. That is, it is judged that administration of the 50% alcoholic extract of the blackberry leaves of the present invention improves cognitive ability due to vascular dementia.

③ Acquisition phase experiment during Barnes maze inspection

During the Barnes maze test, the acquirer experiment was conducted on the 6th to 10th day after induction of vascular dementia, and the rat was placed on the opposite side of the platform of the Barnes maze (122 Cm wide, 90 Cm high, hole diameter 10 Cm, number of holes 18). . In a situation where a strong light is shined on the ceiling, and the disgust is stimulated, the only one of the holes is found and the time it takes to hide it is measured. Each animal was measured for 5 minutes (300 sec), averaged by measuring three times, and converted to an average value per group. The copper wire was tracked using the Ethovision XT (Noldus) video tracking system, and the tracking was terminated when it was successfully hidden in the platform. The results are shown in FIG. 7.

7 is a view showing the results of a maze test (acquisition experiment) in an vascular dementia-induced animal model to which 50% alcoholic extract of blackberry leaves was administered according to an embodiment of the present invention.

In FIG. 7, "SHAM" represents the gastric surgical instrument, "VD" represents the vascular dementia inducer, and "VD + Blackberry Leaf Alcohol 50% Extract" is 10 mg of blackberry leaf alcohol 50% extract in the vascular dementia milk tool. The group that was orally administered once a day for 5 days with / kg body weight was measured, and the time to find a way out of the maze was measured in all groups, and the experiment was repeated for a total of 5 days once a day. .

Referring to FIG. 7, the time from the gastric surgery tool to finding the exit through a five-day repetitive experiment was rapidly reduced, while in the vascular dementia treatment tool, the time from finding the exit through the repeated experiment was gradually reduced. In the group administered with 50% of blackberry leaf extract to the vascular dementia treatment tool, the time to find an exit was reduced similarly to the gastric surgery tool. That is, it can be seen that administration of 50% extract of blackberry leaf improves cognitive ability according to the induction of vascular dementia.

④ Organizational analysis

9 days after the end of the 5th administration, cardiac perfusion was fixed with 4% paraformaldehyde to extract the brain from the experimental animals, and the extracted brain was fixed with the same solution for 24 hours. The washed brain was dehydrated and paraffinized through an alcohol series. It was cut into 7 um using a microtome, and the part with the hippocampus was selected and made into a slide. Among these, 5 or more slides were randomly selected per H-E staining, and the number of live neurons in the hippocampus CA1 region was expressed as a value. The results are shown in FIG. 8.

8 is a view showing changes in the number of cranial nerve cells in the brain (hippocampus) of an animal model inducing vascular dementia to which 50% alcoholic extract of blackberry leaves is administered according to an embodiment of the present invention.

In FIG. 8, “SHAM” refers to a gastric surgical instrument, “VD” refers to a vascular dementia inducer, and “VD + Blackberry Leaf Alcohol 50% Extract” refers to a vascular dementia oil tool and extracts 50% of Blackberry Leaf Alcohol 10 mg. / kg body weight indicates the group orally administered once a day for 5 days.

Referring to FIG. 8, the number of live brain neurons in the gastric surgery zone was the highest, and in the case of vascular dementia induction, the number of brain neurons was markedly reduced, and the number of live brain neurons in the blackberry leaf 50% alcohol extract administration was It was found to be higher than that of vascular dementia. Therefore, it is thought that administration of 50% alcoholic extract of blackberry leaves shows an improvement effect of vascular dementia by blocking cell death of brain neurons caused by vascular dementia.

The following is an example of a formulation for the pharmaceutical composition of the present invention.

Formulation example  1: Preparation of pharmaceutical preparations

One. Wild  Produce

A powder was prepared by mixing 500 mg of blackberry leaf extract 500 mg, lactose 300 mg, corn starch 190 mg, and magnesium stearate 10 mg according to a conventional powder manufacturing method and filling in a gas-tight bag. The final weight of the contents was 1,000 mg.

2. Preparation of tablets

According to a conventional tablet manufacturing method, blackberry leaves 50% alcohol extract 250 mg, lactose 100 mg, microcrystalline cellulose 130 mg, sodium starch glycolate 15 mg and magnesium stearate 5 mg were mixed and granulated and tableted with a tablet press.

3. Preparation of Injection

According to the conventional injection preparation method, blackberry leaf 50% alcoholic extract 10 mg, mannitol 180 mg, sterile distilled water 2974 mg, Na ₂ HPO ₄ · 12H ₂ 0 26 mg were prepared and dispensed at 2 mL per ampoule.

Claims (10)

A pharmaceutical composition for preventing or treating dementia comprising blackberry extract as an active ingredient.
According to claim 1,
The blackberry extract is characterized in that the blackberry extract with water, a lower alcohol having 1 to 6 carbon atoms or a mixed solvent thereof,
Pharmaceutical composition for preventing or treating dementia.
According to claim 1,
The blackberry extract is characterized in that the blackberry extract with 30% to 95% ethanol or hot water extract,
Pharmaceutical composition for preventing or treating dementia.
According to claim 1,
The blackberry extract is characterized in that the blackberry extract by 50% ethanol,
Pharmaceutical composition for preventing or treating dementia.
According to claim 1,
The blackberry extract
Characterized in that the blackberry extract with 30% to 95% ethanol is a fraction fractionated with at least one of hexane, dichloromethane (methylene chloride), ethyl acetate, water-saturated butanol, and water,
Pharmaceutical composition for preventing or treating dementia.
According to claim 1,
The blackberry extract is characterized in that it exhibits the ability to inhibit beta amyloid aggregation,
Pharmaceutical composition for preventing or treating dementia.
According to claim 1,
The blackberry extract is characterized in that it exhibits the ability to inhibit inflammation of the brain microglial cells,
Pharmaceutical composition for preventing or treating dementia.
According to claim 1,
The blackberry extract is characterized in that it exhibits the ability to inhibit vascular dementia,
Pharmaceutical composition for preventing or treating dementia.
According to claim 1,
The blackberry extract is characterized in that it represents the ability to inhibit the phosphorylation of Tau protein,
Pharmaceutical composition for preventing or treating dementia.
According to claim 1,
The blackberry extract is characterized in that the blackberry leaf 50% alcohol extract,
Pharmaceutical composition for preventing or treating dementia.

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