WO2018080158A1 - Composition for preventing, improving or treating cognitive impairment, containing elaeagnus glabra extract as active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating cognitive impairment, the composition containing a Elaeagnus glabra extract as an active ingredient, and more specifically, the Elaeagnus glabra extract, according to the present invention, exhibits antioxidant activity, amyloid-beta aggregation inhibition activity, a nerve cell protection effect and a brain cell inflammation inhibition effect, and thus may be usefully employed as a composition for preventing, improving or treating cognitive impairment.

Description

Composition for the prevention, improvement or treatment of cognitive impairment containing narrow leaf barley berry extract as an active ingredient

The present invention is narrow leaf barley ( Elaeagnus) glabra ) relates to a composition for the prevention, improvement or treatment of cognitive impairment containing the extract as an active ingredient.

Cognition refers to all processes in which humans use their brains to think, speak, remember, judge, and execute. Therefore, 'cognitive function' includes attention, perception, memory, language ability, executive ability, etc., 'cognitive impairment' is the most prominent phenomenon of the aging of the brain, characterized by a decrease in learning, memory, and judgment It is a physiological phenomenon that is erased. In particular, these cognitive impairments may appear in various ways. Among them, dementia is markedly impaired in interpersonal relations, occupational functions, and daily life functions due to the decline in cognitive functions such as memory, language skills, visual perception and space-time composition ability, and executive functions. It is diagnosed as a complex disease that results. Geriatric dementia is mainly caused by Alzheimer's dementia. Alzheimer's dementia is commonly referred to as Alzheimer's disease (AD), and it can be said to die slowly without any treatment efforts. The disease was known in 1906 by Alois Alzheimer, a German neuropathologist. In 1960, the elderly population of 72 million people was 720,000 in 1960, accounting for only 2.9%, but it is expected to become a full-fledged aging society, and after 2026, the population aged 65 or older will increase to 20% of the total population. . The number of patients with senile dementia increases sharply in proportion to this. Experts predict that 10% of people over 65 years of age will suffer from senile dementia, and 40% to 50% of people over 85 years of age.

Cognitive dysfunction, such as the loss of memory and learning ability caused by Alzheimer's disease (AD), a type of senile dementia, is caused by the aggregation and deposition of two insoluble proteins in the hippocampus and cortex. Protein aggregation is the accumulation of neurofibrillary tangles consisting of senile plaque composed of amyloid beta (Aβ) and hyperphosphorylated tau protein in neurons. Despite many efforts, the cause and invention of Alzheimer's disease are still not fully understood. As the aging population is accelerating, the demand for the development of more effective formulations (including health functional food ingredients) to improve cognitive or memory decline is more than ever.

On the other hand, the narrow-leaved barley grows on the coast of the island, and its leaves are alternate, thick, glossy, and narrow. Both ends are narrow, 4 ~ 8cm long, petiole is short. The outer surface of the brown virgin (star hair: star-shaped split hair) disappears and dense on the back. At the edges there are wavy teeth. Flowers bloom in white in October-December, with 2-3 leaves hanging on the axilla. Calyx tube is long bell-shaped, end is divided into 4 branches, and stamen is 4. Fruits are elliptical nucleus, 10-18mm long, silvery brown. Our lady is dense on the outside and ripens red in April-May. Breeding is by seed or folding. It is weak to cold, grows well in sunny places, and is strong in salt. Fruit is sweet and eats raw or dipped fruit liquor. It is distributed in Korea (Jeju).

As an example of a narrow-leaf barley biloba extract related art, but disclosed in Chinese Patent Laid-Open No. 103652170 relates to a tea and a method for preparing the same, including the elaeagnus pungens, which is the same as that of the narrow-leaf barley biloba, There is no disclosure about the composition for the prevention, improvement or treatment of cognitive impairment containing leaf barley extract as an active ingredient.

The present invention is derived by the above requirements, relates to a composition for the prevention, improvement or treatment of cognitive impairment containing narrow leaf barley extract as an active ingredient, inhibiting amyloid beta aggregation of the narrow leaf barley extract, The present invention was completed by confirming the antioxidant activity, neuronal cell protection and the inhibitory effect of brain cells.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of cognitive impairment containing narrow leaf barley berry extract as an active ingredient.

In addition, the present invention provides a health functional food composition for the prevention or improvement of cognitive impairment containing a narrow leaf barley berry extract as an active ingredient.

The present invention relates to a composition for the prevention, improvement or treatment of cognitive impairment containing a narrow barley barberry extract as an active ingredient, the narrow barley barberry extract inhibits amyloid beta aggregation, antioxidant activity, neuronal cell protection and brain cells Inflammatory effect and cognitive function because it has excellent effect in manual evasion experiment and Morris underwater maze experiment using Alzheimer's dementia animal model, and also in manual avoidance experiment and Y-maze experiment using short term memory disorder animal model. It can be used to prevent, ameliorate or treat a disorder.

1 is to confirm the amyloid beta aggregation inhibitory effect of the narrow barley barberry extract of the present invention, (A) is a narrow leaf barley biloba branch extract, (B) to confirm the amyloid beta aggregation inhibitory effect of the narrow barley barberry leaf extract 100 μM of Morin compound was used as a positive control.

Figure 2 confirms the antioxidant activity of the narrow leaf barley biloba extract of the present invention, (A) is a result of confirming the ABTS radical scavenging activity of the narrow leaf barley biloba branch extract, (B) DPPH of the narrow leaf barley biloba branch extract The result of confirming the radical scavenging activity, (C) is the result of confirming the ABTS radical scavenging activity of the narrow leaf barley berry leaf extract, (D) is the result of confirming the DPPH radical scavenging activity of the narrow leaf barley berry leaf extract. Vitamin C (vit. C) was used as a positive control.

Figure 3 confirms the neuronal protective effect (CCK) of the narrow leaf barley bran branch extract of the present invention, (A) to determine the cytotoxicity to the neuronal hippocampal cell line, treated with narrow leaf barley bran branch extract Survival rate is the result, (B) is a result of the treatment of H ₂ O ₂ to the nerve cells induced nerve cell damage, the treatment of narrow barley biloba branch extract to confirm the cytoprotective effect, (C) cell membrane damage The results of measuring LDH outflow and mitigating the effects of LDH outflow due to the treatment of Narrow-leaf Barley bran branch extract. In comparison to the normal group, H ₂ O ₂ is statistically significant difference in cell survival rate or LDH release due to neuronal damage following treatment, p <0.001. **, *** is compared with the H ₂ O ₂ treatment group, by the treatment of the narrow barley bark branch extract showed that there is a statistically significant difference in the cell survival rate or LDH release of neurons, ** is p < 0.01 and *** means p <0.001.

Figure 4 as a result of confirming the inhibitory effect on the brain cell inflammation according to the treatment of the narrow barley bark branch extract of the present invention, (A) is the result of confirming the toxicity of microglia cells, (B) is inflammation of BV-2 cells After induction, the result of NO assay according to the treatment of Narrow barley bran branch extract, (C) induces inflammation in BV-2 cells, PGE ₂ assay according to the treatment of Narrow barley bran branch extract The result is. ### means that the production of nitric oxide (NO) or PGE ₂ was significantly increased by damage to neurons following LPS treatment, which means p <0.001. ** and *** indicate that NO (nitric oxide) or PGE ₂ production was statistically significantly decreased by treatment of N. barberry extract compared to LPS treatment group, ** is p <0.01 , *** means p <0.001.

5 is a result of confirming the effect of the narrow leaf barley bran branch extract according to the present invention in a passive avoidance experiment using the Alzheimer's dementia animal model. # Is a statistically significant difference in the manual avoidance time of the control group treated with amyloid beta compared to the normal group (group not treated with amyloid beta), which means that p <0.05, *, ** means amyloid beta and Negative leaf barley branch extract administration (50, 200mg / kg) group or positive control group (morin administration group) significantly improved passive avoidance time compared to the control group, * is p <0.05, ** is p <0.01.

6 is a result of confirming the effect of the narrow leaf barley bran extract according to the present invention in the Morris water maze test using the Alzheimer's dementia animal model. # Indicates statistically significant difference in the time of staying on the platform of the control group treated with amyloid beta compared to the normal group (group not treated with amyloid beta), which means p <0.05, and *, ** indicates amyloid beta And the time that the NF extract (50, 200 mg / kg) or the positive control group (morine administration group) stayed on the platform significantly statistically compared to the control group, * is p <0.05, * * Means p <0.01.

Figure 7 is a result of confirming the effect of the narrow leaf barley bran branch extract according to the present invention in a passive avoidance experiment using a short-term memory disorder animal model. # Is a statistically significant difference in the passive avoidance time of the scopolamine-treated control group compared to the normal group (the group not treated with scopolamine), where p <0.05, and ** is the scopolamine And the control group (50 mg / kg) or narrow control barley bran branch extract (Tacrine administration group) statistically significantly increased passive avoidance time compared to the control group, ** means p <0.01.

8 is a Y-maze experiment using a short-term memory impairment animal model, it is a result confirming the effect of the narrow leaf barley biloba branch extract according to the present invention. ### is statistically significant difference in behavioral change of the scopolamine-treated control group compared to the normal group (the group not treated with scopolamine), which means p <0.001, **, * ** shows that behavioral change in the scopolamine and narrow-leaf barley bran extract (50 mg / kg) or positive control group (tacrine administration group) was significantly improved compared to the control group, ** is p Means <0.01, *** means p <0.001.

The present invention relates to a pharmaceutical composition for the prevention or treatment of cognitive impairment containing narrow leaf barley berry extract as an active ingredient.

The narrow leaf barley berry extract may be prepared by a method comprising the following steps, but is not limited thereto:

1) extracting by adding an extracting solvent to the narrow leaf barley plant;

2) filtering the extract of step 1); And

3) concentration of the filtered extract of step 2) under reduced pressure and drying to prepare an extract.

The extraction solvent in step 1) is preferably water, a lower alcohol of C ₁ ~ C ₄ or a mixture thereof, more preferably an ethanol extract, even more preferably 70% (v / v) ethanol ultrasonic extract It is not limited to this.

In the above production method, the narrow leaf barley may be extracted using any conventional method known in the art, such as filtration, hot water extraction, dipping extraction, reflux cooling extraction, and ultrasonic extraction. The extraction solvent is preferably extracted by adding 1 to 20 times the volume of dried narrow leaf barley biloba, more preferably 3 to 10 times. Extraction temperature is preferably 20 to 50 ℃ but is not limited thereto. In addition, the extraction time is preferably 0.5 to 10 hours, more preferably 0.5 to 5 hours, most preferably 1 hour is not limited thereto. In the above method, the reduced pressure concentration in step 3) is preferably a vacuum reduced pressure concentrator or a vacuum rotary evaporator, but is not limited thereto. In addition, the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, but is not limited thereto.

The narrow-leaf barley berry extract may be an extract of an outpost, a flower, a leaf, a branch, a root or a seed, but is preferably a leaf or branch extract of a narrow-leaf barley.

The cognitive impairment is preferably any one selected from dementia, Alzheimer's, ischemic stroke, traumatic brain injury, forgetfulness, Parkinson's disease, pick disease, Creutzfeldt-Jakob disease, and mild cognitive impairment Do not.

In the present invention, mild cognitive impairment is defined as a pre-dementia clinical stage that does not cause a state in which memory and cognitive function is significantly lower than age and education level or disruption in life.

The composition of the present invention may include a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient, and may be various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include capsules, powders, granules, tablets, pills, and the like, which may comprise at least one excipient such as starch, calcium carbonate, sucrose or lactose (at least one compound). lactose) and gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, emulsions, syrups, aerosols and the like, and may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to commonly used simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, 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, glycero gelatin and the like can be used. For parenteral administration, it is desirable to select either external skin or intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections, most preferably for external skin use.

The pharmaceutical composition according to the invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, the effective amount of the level of the disease, the severity, the drug activity of the patient , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of the drug, and other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The dosage of the composition of the present invention varies depending on the body weight, age, sex, health status, diet, time of administration, administration method, excretion rate and severity of the disease, the daily dosage of the narrow leaf barley extract 0.01-2,000 mg / kg, preferably 30-500 mg / kg, more preferably 50-300 mg / kg, based on the amount, can be administered 1-6 times per day. The compositions of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy and biological response modifiers.

In addition, the present invention relates to a health functional food composition for the prevention or improvement of cognitive impairment containing a narrow leaf barley berry extract as an active ingredient. The composition is characterized by having antioxidant activity, the composition is preferably prepared in any one of the formulations selected from powders, granules, pills, tablets, capsules, candy, syrups and beverages, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the narrow barley barley extract may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). Generally, in the preparation of food or beverages, the composition of the present invention is added in an amount of up to 15 parts by weight, preferably up to 10 parts by weight based on the raw materials. However, in the case of long-term intake for health and hygiene or health control purposes, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. There is no particular limitation on the kind of food. Examples of foods to which the extract or fractions thereof may be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, drinks, tea , Drinks, alcoholic beverages and vitamin complexes, and includes all the health functional foods in the conventional sense.

When the composition of the present invention is used as a health beverage, various flavors, natural carbohydrates, and the like may be contained as additional components, as in general beverages. The above-mentioned natural carbohydrates are sugars such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as textine and cyclotenstrin, xylitol, sorbitol and erythritol. As the sweetening agent, natural sweetening agents such as tautin and stevia extract, synthetic sweetening agents such as saccharin and aspartame, and the like can be used. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 g of the composition of the present invention. In addition to the above, the composition of the present invention includes various nutrients, vitamins, electrolytes, flavors, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols. And carbonation agents used in carbonated beverages. In addition, the composition of the present invention may contain a pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components can be used independently or in combination. The proportion of such additives is not critical, but the composition of the present invention is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.

1. Preparation method of extract

Narrow leaf barley leaves and branches were used from the Medicinal Plant Material Bank of Gachon University. 5 liters of 70% (v / v) ethanol was added to 500.0 g of each dried sample of the barley bark leaves and branches and ultrasonically extracted three times for 1 hour using an ultrasonic extractor. The ultrasonic extract was filtered using Advantec No. 2 (110 mm) filter paper to remove insoluble matters, and then concentrated under reduced pressure at 40 ° C. with a condenser equipped with a cooling condenser. To completely remove the solvent of the concentrated extract under reduced pressure, 500 ml of purified water was added and suspended, and then concentrated by using a lyophilizer.

2. Determination of the Effect of Amyloid Beta Aggregation Inhibition

Amyloid beta aggregation inhibitory activity was measured by fluorescence assay. Amyloid beta (Aβ _1-42 ) peptide was stored at minus 80 ℃, the final concentration used in the measurement was used to 100㎛ / ㎖. Thioflavin T, used as a fluorescent substance, was dissolved in assay buffer (50 mM Tris / 150 mM NaCl (pH 7.2), 20 mM HEPES / 150 mM NaCl (pH 7.2), 10 mM phosphate / 150 mM NaCl (pH 8.0)). It was prepared and used at a concentration of 2 mM. Samples were dissolved in assay buffer and used at a final concentration of 100 μg / ml. In order to measure the degree of Aβ _1-42 aggregation, 10 μl of thioflavin T and 85 μl of Aβ _1-42 were added to a 96-well plate and mixed with 5 μl of a sample prepared at a constant concentration. Measured with a spectrometer. The wavelength value of the fluorescence spectrometer used at this time was 440 nm / 484 nm (absorption / emission), and it measured in total for 2 hours at 37 degreeC 20 minutes. Morin compounds were used as positive controls for activity comparison.

3. Antioxidant Activity Assay

(A) Determination of ABTS free radical scavenging ability

Antioxidant efficacy measurement using ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) radical was carried out by modifying the ABTS + cation decolorization assay method to 96 well plates. 7 mM ABTS and 2.45 mM potassium persulfate were mixed at the final concentration and left for 24 hours in the dark at room temperature to form ABTS +. And diluted with PBS to have an absorbance value of 0.7 at 743 nm. After mixing ABTS + solution and the sample in a 96 well plate and reacting at room temperature for 30 minutes, the absorbance was measured at 743 nm using an Epoch Microplate Spectophotometer. The radical scavenging activity of each sample was expressed as a percentage of the radical scavenging ability with respect to the control group using PBS as a control group. Vitamin C (vit. C) was used as a positive control for activity comparison.

(B) Measurement of DPPH free radical scavenging ability

Antioxidant potency measurements using DPPH (1-1-diphenyl-2-picrylhydrazyl) radicals were performed using 96 well plates. 0.15 mM DPPH solution and the sample were mixed in a 96 well plate and reacted at room temperature for 30 minutes, and then the absorbance was measured at 517 nm. The antioxidant activity of each treatment extract was expressed as a percentage of radical scavenging ability with respect to the control group using the solvent DMSO as a control. Vitamin C (vit. C) was used as a positive control for activity comparison.

4. Measurement of neuronal protective effect

(A) Cytotoxicity measurements on neuronal hippocampal cell lines

In order to determine the cytotoxicity of the sample, Cell Counting Kit-8 (CCK-8) was used according to the manufacturer's instructions. Samples were treated by concentration in HT22 cell lines (5 × 10 ³ cells / well) dispensed in 96-well plates and incubated for 24 hours. After incubating for 4 hours with 10 μl of CCK-8 solution, absorbance was measured at 450 nm and relative cell viability (% of control) was calculated by comparison with the control group.

(B) Measurement of neuronal cell protective efficacy

In order to induce neuronal damage, HT22 cells were treated with 250 μM hydrogen peroxide (H ₂ O ₂ ) for 6 hours. H ₂ O ₂ via CCK Assay The cell protection efficacy was determined by comparing the change in cell viability of the treatment group alone and the sample and H ₂ O ₂ simultaneous treatment group. Carvedilol was used as a positive control.

(C) LDH assay

Cell viability was measured by measuring LDH efflux due to cell membrane damage. In order to induce nerve cell damage, HT22 cells were treated with 250 μM of hydrogen peroxide (H ₂ O ₂ ) for 6 hours. After the culture supernatant was collected, the mixture was mixed with the LDH substrate solution in the same amount and reacted at room temperature for 30 minutes. Then, 1N HCl was added to stop the reaction, and the absorbance was measured at 490 nm (experimental LDH release).

After removing the supernatant, lysis solution was added to the remaining cells and reacted at 37 ° C. for 45 minutes, followed by centrifugation at 250 g for 4 minutes. The supernatant was collected and subjected to LDH reaction as described above to measure absorbance at 490 nm (Maximum LDH release). Cell viability was calculated by the following equation (1).

Formula (1)

% Cell survival rate = {[experimental LDH release (OD ₄₉₀ )] / [Maximum LDH release (OD ₄₉₀ )]} × 100

5. Inhibition of brain cell inflammation

(A) Determination of microglial toxicity

Samples were treated by concentration in BV-2 cell lines (1 × 10 ⁴ cells / well) dispensed in 96-well plates and incubated for 24 hours. After incubating for 4 hours with 10 μl of CCK-8 solution, absorbance was measured at 450 nm and relative cell viability (% of control) was calculated by comparison with the control group.

(B) NO (nitric oxide) assay

After pretreatment with BV-2 cells for 2 hours, LPS (lipopolysaccharide, 1 µg / ml) was treated and cultured for 24 hours to induce an inflammatory response, and then the supernatant was recovered. After 5-10 minutes of reaction with the same amount of the supernatant and Griess reagent (sulfanilamide: N- 1-napthylethylenediamine dihydrochloride = 1: 1), the absorbance at 540 nm was measured to calculate the amount of nitric oxide (NO) based on the standard curve. . Ibuprofen (ibuprofen) was used as a positive control.

(C) PGE ₂ (Prostaglandin E ₂ ) Assay

After pretreatment of the sample to BV-2 cells for 2 hours, in order to induce an inflammatory response, LPS (lipopolysaccharide, 1㎍ / ㎖) was treated and incubated for 24 hours, and then the supernatant was recovered. Experiments were performed using an enzyme-linked immunosorbent assay (ELISA) kit to measure the amount of PGE ₂ in cell culture. The supernatant was added to a 96-well plate coated with goat anti-mouse IgG, 50 μl each, and 50 μl of the primary antibody solution and 50 μl of PGE ₂ conjugate were added thereto, and reacted at 4 ° C. for 18 hours. After washing 5 times with a washing buffer and 200μL reaction of the substrate solution was reacted for 90 minutes, the absorbance was measured at 412nm.

6. Efficacy Evaluation on Animal Models of Alzheimer's Dementia

(A) Animal model

 12-week-old male mouse (C57BL / 6N, coretech) of 23-28 g has temperature 23 ± 3 degrees Celsius, relative humidity 55 ± 15%, ventilation times 10-20 times / hr, lighting time 12 hours (8 am lighting-afternoon 8 o'clock) and roughness of 150-300 Lux were kept in the animal room. All experimental procedures were conducted in accordance with the NIH Guidelines for the Management and Use of Laboratory Animals. Animal handling was conducted in accordance with the National Animal Welfare Law of Korea.

(B) Administration of amyloid beta and test substance

Mice were randomly divided into 5 groups after 1 week of accrual (n = 8). Induction of Alzheimer's dementia was measured after storage of amyloid beta, dissolved in sterile 0.1 M phosphate-buffered saline (pH 7.4) and prepared at a concentration of 1 μg / ml for at least 3 weeks before dosing to induce aggregation. And administered to the third ventricle of the brain (intracerebroventricular injection, icv). Narrow barley bark branch extract was used in PBS was dissolved orally administered for 20 days from the day after the amyloid beta administration. Morin, an amyloid aggregation inhibitor, was used as a positive control.

(C) passive avoidance test

Passive avoidance experiments were performed at the same time for three consecutive days at 24 hour intervals. On the 8th day of the administration of the test substance, the animals were kept in the shaded area for 2 minutes, and then put back in the illuminated area and immediately taken out to the shaded area for adaptive training. Twenty-four hours after the 9th day of the test substance administration, two training sessions were performed every two minutes. In the first training, animals were placed in the chamber of a passive avoidance test instrument for 60 seconds to adjust the instrument. At this time, the guillotine door was opened without lighting so that the animals could freely enter and exit. After 60 seconds of acclimatization, they were re-lit and allowed to freely enter and exit for 120 seconds, then moved to the shaded area, closing the guillotine door and receiving a 0.20 mA scrambled shock for 2 seconds. At this time, animals that did not move were excluded from the experiment. 24 hours later, on the 10th day of the administration of the test substance, the time to move to the shaded area by measuring the time at the same time to open the guillotine door (guillotine door).

(D) Morris water maze test

Morris water maze experiments were conducted for 7 days from day 15. The platform was placed in the pool of one of the tank's four designated release points, allowing it to be found for 60 seconds. After finding the platform, he was allowed to rest on it for about 10 seconds. If the platform was not found within 60 seconds, the animal was placed on the platform and then rested for about 30 seconds. After all animals had completed the trial one time, the next trial started again, and two trials were performed per day. However, the release point was randomly selected so as not to overlap. In this manner, the procedure was repeated for 7 days and the time to find the platform was measured (training: 2 days, behavioral test: 4 days, probe trial: 1 day). On the last 7 days, the platform was removed 1 hour after administration and a 60-second probe trial was conducted to determine the time spent in each quadrant, the time spent in the platform and the cross number.

7. Efficacy measurement for short term memory disorder animal model

(A) Animal model

7-week-old male mouse (ICR, central laboratory animal) of 23-28 g has temperature 23 ± 3 ℃, relative humidity 55 ± 15%, ventilation times 10-20 times / hr, lighting time 12 hours (8 am lighting-8 pm Litter) and was maintained in the animal room maintained at roughness 150 ~ 300 Lux. All experimental procedures were conducted in accordance with the NIH Guidelines for the Management and Use of Laboratory Animals. Animal handling was conducted in accordance with the National Animal Welfare Law of Korea.

(B) Administration of Narrow Leaf Barley Extract

Mice were randomly divided into 5 groups after 1 week of accrual (n = 8). Narrow leaf barley extract was used in PBS, and orally administered for 2 weeks. Tacrine was used as a positive control for the narrow barley berry extract. Scopolamine, a memory loss substance, was intraperitoneally administered 30 minutes before the behavioral experiment.

(C) passive avoidance test

Passive avoidance experiments were performed at the same time for three consecutive days at 24 hour intervals. On the 10th day of administration of the test substance, the animals were allowed to stay in the shaded area for 2 minutes, and then placed in the illuminated area again and immediately taken out to the shaded area for adaptive training. Twenty-four hours after the 11th day of test substance administration, two training sessions were performed every two minutes. In the first training, animals were placed in a passive avoidance test instrument chamber for 60 seconds to adjust the instrument. At this time, the guillotine door was opened without lighting so that the animals could freely enter and exit. After 60 seconds of acclimatization, they were re-lit and allowed to freely enter and exit for 120 seconds, then moved to the shaded area, closing the guillotine door and receiving a 0.20 mA scrambled shock for 2 seconds. At this time, animals that did not move were excluded from the experiment. On the 12th day of the test substance administration, which was again 24 hours later, it was placed in an illuminated area and the guillotine door was opened, and the time was measured to move to the shaded area.

As a result, as shown in FIG. 7, the manual avoidance time of the control group treated with scoflavin (1 mg / kg) was statistically significant compared to the manual avoidance time of the normal group not treated with scoflavin (1 mg / kg). Although it was decreased, both the experimental group and the positive control group treated with Narrow barley bark branch extract of the present invention was confirmed that the passive avoidance time is restored to the level of the normal group.

(D) Y-maze test

Y-maze experiments were conducted on day 14 of test substance administration. Y-maze is made of black acrylic in the shape of a four-sided maze that is made of four, each of the three A, B, C, then carefully placed the mouse on one branch and let it move freely for 8 minutes The branches containing the mice were recorded in order. It continued when the tail had been completely entered, even when it had entered the branch that had once entered. One point was awarded for three different branches. Change behavior was calculated by the following equation.

Spontaneous alternation (%) = [Actual Changes / Total Entry-2] × 100

8. Statistical Analysis

Statistical analysis on the results obtained in the embodiment of the present invention is SPSS Statistics 22 or GraphPad Prism Ver. 6 was used and tested for significance with the control. The test group was tested by one-way ANOVA and tested by Duncan or Dunnett t-test . In addition, the normal group and the control group were tested for significance using Student's t-test . P <0.05 was determined to be statistically significant.

Example  One. Narrowleaf barley  Confirmation of the Amyloid-beta Aggregation Inhibitory Effect of Extracts

6.25, 12.5, 25, 50 and 100 ㎍ / ㎖ narrow leaf barley bark and leaf extract was treated to determine the degree of aggregation inhibition of amyloid beta. As a result, as shown in Figure 1, narrow barley bark branches and leaf extracts inhibited amyloid beta aggregation in a concentration-dependent manner, and inhibited amyloid beta aggregation when treated with narrow barley bark branches extract of 50 µg / ml or more. It was confirmed that is more than 60%.

Example  2. Narrowleaf barley  Confirmation of Antioxidant Activity of Extracts

In Example 2, ABTS radical scavenging activity and DPPH radical scavenging activity were confirmed in order to confirm the antioxidant activity of the narrow barley barberry and leaf extract. As shown in FIG. At 25 µg / ml or more, 100% ABTS radical scavenging activity and DPPH radical scavenging activity were observed, and when the concentration of the narrow leaf barberry leaf extract was 100 µg / ml, it showed almost 100% ABTS radical scavenging activity.

Example  3. Narrowleaf barley  Confirmation of neuroprotective effect of extract

In Example 3, it was confirmed that the narrow barley bark branch extract has little cytotoxicity against normal neurons (FIG. 3 (A)), and due to H ₂ O ₂ damage to neurons, cell survival rate is reduced. , By treating the narrow leaf barley bran extract to damage the neurons caused by H ₂ O ₂ , it was confirmed that there is an effect of suppressing the decrease in cell viability (FIG. 3 (B)), by H ₂ O ₂ Neuronal cell membranes are damaged and LDH release is increased, but by treating the narrow leaf barley bran extract, the effect of mitigating the increase in LDH release was confirmed (Fig. 3 (C)).

Example  4. Confirmation of inhibitory effect on brain cell inflammation

In Example 4 confirmed the inhibitory effect on brain cell inflammation, and as a result, as shown in Figure 4, the narrow barley bark branch extract of the present invention is almost non-toxic to the brain cells, the cell survival rate is maintained at 100% level In addition, by inducing inflammation in brain cells by treating LPS, the amount of nitric oxide (NO) is increased, whereas the amount of nitric oxide (NO) is significantly reduced when the barley bark branch extract of the present invention is treated. . In addition, as a result of measuring the amount of PGE _{2 in the} cell culture, the amount of PGE ₂ was increased when inflammation was induced, whereas the treatment group of the Narrow bark branch extract was found to decrease PGE ₂ in a concentration-dependent manner.

Example  5. Efficacy Assessment on Animal Models of Alzheimer's Dementia

(1) passive avoidance test

As a result of the manual evasion experiment on the animal model of Alzheimer's dementia, as shown in FIG. 5, the manual evasion time of the control group treated with amyloid beta was significantly decreased compared to the manual avoidance time of the normal group not treated with amyloid beta. , The experimental group and the positive control group treated with the narrow leaf barley bran branch extract of the present invention was confirmed that the passive avoidance time is restored to the level of the normal group.

(2) Morris underwater maze experiment ( Morris water maze test )

Morris water maze experiments on Day 19 and Day 20 of the Alzheimer's dementia animal model showed a statistically significant increase in retention time on the platform of the amyloid beta-treated control compared to the normal (non-amyloid beta-treated) group. , The administration of amyloid beta and narrow barley bark branch extract (50, 200mg / kg) group or positive control group (morin administration group) significantly reduced the time to stay on the platform compared to the control group (Fig. 6).

Example  6. Measuring efficacy on animal models of short-term memory impairment

(1) passive avoidance test

As a result of the passive avoidance experiment for the short-term memory impairment animal model, as shown in FIG. 7, the treatment with scoflavin (1mg / kg) compared to the passive avoidance time of the normal group not treated with scoflavin (1mg / kg) Although the passive avoidance time of the control group was statistically significantly reduced, both the experimental group and the positive control group treated with the narrow leaf barley bran extract of the present invention confirmed that the passive evacuation time was restored to the level of the normal group.

(2) Y-maze test

As a result of the passive avoidance experiment for the short-term memory impairment animal model, as shown in FIG. Although the altered behavior of the control group treated with scoflavin (1 mg / kg) was statistically significantly reduced compared to the spontaneous alternation (%) of the normal group not treated with scoflavin (1 mg / kg), the present invention It was confirmed that both the experimental group and the positive control group treated with the narrow leaf barley bran extract were able to recover their altered behavior.

Claims (10)

1. Narrow-leaf barley ( Elaeagnus) glabra ) A pharmaceutical composition for the prevention or treatment of cognitive impairment containing the extract as an active ingredient.
2. The pharmaceutical composition for preventing or treating cognitive impairment of claim 1, wherein the narrow leaf barley berry extract is a leaf or branch extract of the narrow leaf barley berry.
3. The pharmaceutical composition for preventing or treating cognitive impairment according to claim 1, wherein the solvent of the Narrow barley berry extract is water, C ₁ to C ₄ lower alcohols, or a mixture thereof.
4. The method of claim 1, wherein the cognitive impairment is any one selected from dementia, Alzheimer's disease, ischemic stroke, traumatic brain injury, forgetfulness, Parkinson's disease, pick disease, Crutzfeldt-Jakob disease and mild cognitive impairment. Pharmaceutical composition for the prevention or treatment of cognitive disorders, characterized in that.
5. The pharmaceutical composition for preventing or treating cognitive impairment according to claim 1, comprising a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.
6. According to claim 1, wherein the composition is a capsule, powder, granules, tablets, suspensions, emulsions, syrups, aerosol pharmaceutical composition for the prevention or treatment of cognitive impairment, characterized in that it is prepared in any one of the formulations.
7. Health functional food composition for the prevention or improvement of cognitive impairment containing narrow leaf barley berry extract as an active ingredient.
8. 8. The dietary supplement composition for preventing or improving cognitive dysfunction according to claim 7, wherein the solvent of the narrow barley bark extract is water, C ₁ ~ C ₄ lower alcohol, or a mixture thereof.
9. 8. The health functional food composition for preventing or improving cognitive impairment according to claim 7, wherein the composition has antioxidant activity.
10. According to claim 7, wherein the composition is a health functional food composition for the prevention or improvement of cognitive impairment, characterized in that the formulation is made of any one selected from powder, granules, pills, tablets, capsules, candy, syrups and beverages.

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