KR102319712B1 - Composition for improvement of memory or cognition ability, or for prevention, delay, treatment or improvement of dementia comprising extracts of Rosa multiflora Thunb. - Google Patents

Abstract

The present invention relates to a composition for improving memory, improving cognitive ability, or preventing, delaying, treating, or improving dementia, which contains an extract of chrysanthemum flower as an active ingredient.
The composition of the present invention can improve memory and cognitive ability through acetylcholinesterase inhibitory activity, beta-amyloid secretion inhibitory activity, and control activity and antioxidant activity of dementia-related proteins such as beta-secretase, and reduce dementia effectively prevent, delay, treat or ameliorate.

Description

Composition for improvement of memory or cognition ability, or for prevention, delay, treatment or improvement of dementia comprising extracts of Rosa multiflora Thunb.}

The present invention relates to a composition for improving memory, improving cognitive ability, or preventing, delaying, treating, or improving dementia, which contains an extract of chrysanthemum as an active ingredient.

Korea, which has already entered an aging society, accounted for 15% of the elderly population in 2019, and the number of dementia patients is also rapidly increasing. According to the data released by the Central Dementia Center in the '2018 Dementia Status in Korea', it is estimated that there are 70,5473 dementia patients among the elderly population over 65 in Korea, and the prevalence of dementia is 10%. It is estimated that the number will increase to about 1 million in 2024 and 2 million in 2039. This reality not only deteriorates the quality of life of individuals, but is also emerging as a great social and national problem due to the decrease in national competitiveness due to excessive medical expenses.

Currently, acetylcholinesterase (AChE) inhibitors, such as donepezil, galantamine, and rivastigmine, are used in clinical trials as drugs for improving dementia, but their treatment efficiency is low and side effects are severe. There is no effective treatment.

Dementia is caused by the irreversible destruction of cranial nerves due to atrophy of the brain, a decrease in nerve cells and the appearance of senile plaques, and is accompanied by various acquired cognitive dysfunction symptoms such as memory, speech, and behavioral disorders. syndrome is referred to as

Dementia is classified into Alzheimer's disease, vascular dementia and degenerative diseases caused by Parkinson's disease, metabolic diseases caused by hypothyroidism, and other dementias caused by brain tumors or infectious diseases.

In the brain tissue of Alzheimer's disease patients, pathological features such as senile plaques generated around nerve cells and neurofibrillary tangles generated inside cells can be observed.

Neurofibrillary tangles are formed by hyperphosphorylation of tau protein, and in older adults, beta-amyloid (Aβ) secreted out of the cell is aggregated around nerve cells.

Alzheimer's disease is classified into a familial type and a sporadic type, and more than 90% of all diseases are sporadic, mainly in the elderly over 65 years of age. In the case of the familial type, mutations in genes such as APP (amyloid precursor protein) and presenilin (PS) are known. The elderly half due to the accumulation of Aβ is a common pathology in both familial and sporadic types. Aβ is produced by decomposition of APP (Amyloid precursor protein), a beta-amyloid precursor protein, by two types of proteases. It is gamma-secretase.

Gamma-secretase, which is one of the targets for the development of Alzheimer's disease therapeutics, is produced by presenilin, nicastrin, APH-1, a protein product of an anterior pharynx defective gene, and presenilin enhancing gene. It exists in a complex structure composed of a protein product, such as PEN-2. However, since this enzyme reacts with many substances such as Notch1, APLP1, ErbB4, Jagged, and CD44 as a substrate, there is a problem in that an APP-specific inhibitor needs to be developed.

On the other hand, plants existing in nature are very diverse and often contain useful physiologically active ingredients, and these plant-derived ingredients are generally superior in stability to compounds created through artificial synthesis or transformation. Since it is a substance that exists in nature, it can be decomposed naturally, so there is little problem of accumulation in the body because it is not decomposed or discharged. Therefore, the present inventors have been searching for substances effective for dementia from plants to develop drugs that can effectively prevent, delay, treat or improve dementia without side effects, and through these results, Korean Patent Registration Nos. 10-1194091, 10- 1480899 has developed the same technology.

The present inventors tried to study various plants in order to develop a new and more effective substance for dementia, and among them, it was found that the flower of brier is a possibility. Based on this, through a more detailed and full-scale study, the flower extract can improve memory and cognitive ability through AChE inhibitory activity, Aβ secretion inhibitory activity, modulating activity of dementia-related protein and antioxidant activity, etc., and effectively prevent and delay dementia , it was confirmed that it can be treated or improved, and the present invention has been completed.

Republic of Korea Patent Registration No. 10-1194091 Republic of Korea Patent Registration No. 10-1480899

Accordingly, the main object of the present invention is to provide a pharmaceutical composition and a food composition containing a plant-derived material that can improve memory and cognitive ability, and can effectively prevent, delay, improve or treat dementia without side effects.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for improving memory, improving cognitive ability, or preventing, delaying, or treating dementia, containing the extract of chrysanthemum flower as an active ingredient.

In the pharmaceutical composition of the present invention, the extract is preferably extracted using 10 to 90% (v/v) of ethanol as a solvent.

According to another aspect of the present invention, the present invention provides a food composition for improving memory, improving cognitive ability, or preventing, delaying or improving dementia, which contains an extract of Violet flower as an active ingredient.

In the food composition of the present invention, the extract is preferably extracted using 10 to 90% (v/v) of ethanol as a solvent.

The composition of the present invention can improve memory and cognitive ability through AChE inhibitory activity, Aβ secretion inhibitory activity, regulatory activity and antioxidant activity of dementia-related proteins such as BACE, and can effectively prevent, delay, treat or improve dementia can

1 is a graph showing the results of an acetcholinesterase inhibitory activity experiment of an extract of chrysanthemum according to an embodiment of the present invention. con, negative control; 10 μM gal, 10 μM galantamine treatment group (positive control).
Figure 2 is a graph showing the results of the beta-amyloid secretion inhibitory activity of the extract of chrysanthemum according to an embodiment of the present invention. con, negative control; β-si 10 μM, beta-secretase inhibitor III 10 μM treatment group (positive control).
Figure 3 is a Western blot result and graph showing the expression control experiment results of the dementia-related protein of the extract according to an embodiment of the present invention. con, negative control; β-si 10 μM, beta-secretase inhibitor III 10 μM treatment group (positive control).
Figure 4 is a graph showing the results of the BACE inhibitory activity of the extract according to an embodiment of the present invention. con, negative control; β-si 10 μM, beta-secretase inhibitor III 10 μM treatment group (positive control).
Figure 4 is a graph showing the results of the antioxidant activity experiment of the extract according to an embodiment of the present invention. con, negative control.

The present invention provides a pharmaceutical composition for improving memory, improving cognitive ability, or preventing, delaying, or treating dementia, which contains an extract of chrysanthemum flower as an active ingredient.

The brier flower means the flower of the brier tree, and the brier tree is a deciduous shrub of the Rosa family Rosa with the scientific name of Rosa multiflora Thunb. In the present invention, the flower of the brier tree is used as an extraction raw material. Preferably, those harvested in October based on Jeollabuk-do, Korea are used. In the present invention, the brier can be used without limitation in the collected, cultivated, or commercially available ones, and preferably, after collecting, washing with water to remove foreign substances, etc., and then drying it before use. At this time, it is preferable to dry in the shade in a well-ventilated place. This is because, when drying under direct sunlight, useful components may be physically and chemically changed or volatilized by light. However, for steam distillation or supercritical extraction, it is preferable to use fresh ingredients without drying them immediately after washing.

In the present invention, the brier flower extract can be prepared by conventional extraction methods in the art, such as hot water extraction, ultrasonic extraction, filtration, and reflux extraction using brier flower as an extraction raw material, and is preferably extracted using a polar solvent or a non-polar solvent. do. In this case, the polar solvent may be selected from the group consisting of C1 to C4 lower alcohols and water, and the non-polar solvent may be selected from the group consisting of chloroform, petroleum ether, ether, butanol, hexane, toluene, and these.

For the preparation of the extract in the present invention, preferably 10 to 90% (v/v) of ethanol is used as a solvent. More preferably 20 to 80% (v/v) ethanol, more preferably 30 to 70% (v/v) ethanol, more preferably 40 to 60% (v/v) ethanol, more preferably Preferably 45 to 55% (v/v) of ethanol is used. Also preferably, the remainder other than ethanol in the ethanol solvent is water.

In addition, the amount of the solvent as described above is preferably 1 to 100 times, more preferably 5 to 50 times, more preferably 10 to 20 times, based on the weight of the extraction raw material. The extraction temperature is preferably 0 to 120°C, more preferably 50 to 100°C, more preferably 60 to 90°C, more preferably 70 to 90°C, and still more preferably 80 to 90°C. The extraction time is preferably 1 to 20 hours, more preferably 2 to 10 hours, more preferably 3 to 6 hours. According to these extraction conditions, it is possible to efficiently extract a component exhibiting the desired effect in the present invention from the raw material.

The extract of the present invention may be further subjected to a method such as concentration, reduced pressure drying, freeze-drying, etc. after extraction as described above.

The composition of the present invention may be administered orally or parenterally during clinical administration, and may be used in the form of general pharmaceutical formulations. The pharmaceutical composition of the present invention may contain 0.1 to 100% by weight of the extract as an active ingredient based on the total weight of the composition.

The pharmaceutical composition of the present invention may be formulated in a conventional pharmaceutical formulation by selecting one or more pharmaceutically acceptable conventional carriers and one or two or more additives to the extract as the main component. The pharmaceutical composition of the present invention can be used alone or in combination with other pharmaceutically active compounds as well as in combination with suitable substances.

The pharmaceutical composition of the present invention may be in various oral or parenteral formulations. In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose or lactose ( lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like may also be used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

In the pharmaceutical composition of the present invention, in addition to the main components, as auxiliary components, minerals-containing compounds such as vitamins B, C, E, beta-carotene, Ca, Mg, Zn, phospholipids such as lecithin, or compounds such as maltol and amino acids may be included, Among them, it is more preferable because it is possible to increase or reinforce the bioactive effect when two to three components are mixed and used in vitamins C, E, beta-carotene, maltol, and the like.

In addition to the above ingredients, as known additives, natural flavors such as plum, lemon flavor, pineapple flavor or herb flavor, natural fruit juice, natural pigments such as chlorophyllin, flavonoid, and fructose, a sweetening ingredient, It can be used by mixing honey, sugar alcohol, sugar, and acidifying agents such as citric acid and sodium citrate.

The subject to which the pharmaceutical composition of the present invention can be applied is a vertebrate, preferably a mammal, more preferably a rat, a mouse, a rabbit, a guinea pig, a hamster, a dog, and a cat, and most preferably an experimental animal. are primates, including humans, such as chimpanzees and gorillas.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and it is preferable to select an external skin or intraperitoneal, rectal, intravenous, muscle, subcutaneous, intrauterine dural or intracerebrovascular injection when parenteral administration is performed. Most preferably, it is good to use for oral administration.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease, but based on the dry weight of the extract 0.01 to 10 mg/kg may be administered once a day or divided into 2 to 6 times.

Dosage units may be, for example, 1, 2, 3 or 4 times the individual dose, or 1/2, 1/3 or 1/4 times the individual dose. The individual dosage is preferably such that the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3, or 1/4 times the daily dose.

The pharmaceutical composition of the present invention may further contain one or more active ingredients exhibiting the same or similar function.

The pharmaceutical composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

In addition, the present invention provides a food composition for improving memory, improving cognitive ability, or preventing, delaying, or improving dementia, which contains the extract of chrysanthemum as an active ingredient.

The food composition according to the present invention is, for example, various foods such as chewing gum, caramel products, candies, frozen desserts, and confectionery, beverage products such as soft drinks, mineral water and alcoholic beverages, and health functional foods including vitamins and minerals. can

The extract of the present invention may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or hygiene). In general, in the production of food or beverage, the extract of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term ingestion for health and hygiene purposes or for health control, the above amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

The food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients. 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. As the sweetener, natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used.

The proportion of the natural carbohydrate is preferably selected from 0.01 to 0.04 parts by weight, preferably from about 0.02 to 0.03 parts by weight, per 100 parts by weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , a carbonation agent used in carbonated beverages, and the like. In addition, the functional food of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the food composition of the present invention.

The compositions of the present invention are particularly capable of inhibiting beta-secretase. Beta-secretase is a beta-site APP cleaving enzyme (BACE) with aspartyl protease properties. In a previous study, BACE knockout mice developed normally. No specific abnormalities were observed in the brain. Therefore, being able to inhibit beta-secretase means that there is a high possibility of preventing, delaying, treating, or ameliorating dementia, particularly Alzheimer's, without being harmful to the living body.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention should not be construed as being limited by these examples.

[Example]

1. Manufacture of brier flower extract

The brier flower is a flower of the brier tree that grows wild in Namwon-si, Jeollabuk-do and harvested in October was used. 10 kg of dried ivy flower was put in an extractor, 10 times the weight of 50% (v/v) ethanol (a mixed solution of ethanol and water) was added, and extracted at 80 to 90° C. for 3 hours. Then, the resulting extract was filtered using a microfilter of 5 μm (pore size), and the residue was used as an extraction material and extracted once more under the same conditions (extracted twice in total). The extraction filtrates obtained in the two extractions were collected, and then concentrated under reduced pressure at 60° C. or less to obtain 2.24 kg of dry extract.

2. Evaluation of the physiological activity of the birch flower extract

2-1. Acetylcholinesterase inhibitory activity evaluation

This is an experiment to confirm that the activity is inhibited through acetylcholinesterase, which is the most important enzyme for nerve reaction and function.

The inhibitory activity on acetylcholinesterase was measured using an Acetylcholinesterase Activity Colorimetric assay kit (BioVision, Milpitas, CA).

In a 96-well plate, 30 and 10 μl of each of the sample solution (concentration of 50 or 100 μg/ml) and acetylcholinesterase enzyme solution obtained by dissolving the flower extract of 1 above in dimethyl sulfoxide (DMSO) were added, followed by 50 μl of the reaction mixture. Each was added and the light was blocked, and the reaction was carried out at room temperature. In this case, DMSO was added to the control group instead of the sample solution. Absorbance was measured at 570 nm using a Model 680 Microplate Reader (Bio-Rad, Hercules, CA).

When treated with the flower extract, it inhibited by 69.21% compared to the negative control group (con) at a concentration of 100 μg/ml, and showed AChE inhibitory activity at a similar level by 74.10% when compared with 10 μM of galantamine, a positive control. was (Fig. 1).

2-2. Aβ secretion inhibitory activity

APPswe (human APP with the Swedish mutation) cells were used.

Similar to the pathology of dementia patients, the APPswe cell line used in the experiment increased the secretion of Aβ42 more than twice that of normal cells, making it easy to detect Aβ secretion inhibitory activity.

Sandwich ELISA was performed to measure the amount of Aβ secreted from the APPswe cell line. 1×10 ⁶ cells were cultured in a 60 mm culture dish, exchanged for serum-free DMEM, and 16 hours later, the extract of the flower 1 above was added to the medium so as to have a concentration of 10 or 50 μg/ml. In the positive control group, beta-secretase inhibitor III (Calbiochem, Darmstadt, Germany) was added so that 10 μM instead of the ivy extract. The extract was used by diluting it dissolved in DMSO at a concentration of 100 mg/ml.

After culturing for 24 hours, each culture medium was recovered in the presence of PMSF (phenylmethanesulfonylfluoride) and used as a sample. Plates coated with affinity purified anti-human Aβ(38-42) rabbit IgG were used for Aβ42 analysis, and plates coated with affinity purified human Aβ(35-40)(1A10) mouse IgG MoAb were used for Aβ40 analysis. 100 μl of a sample was placed in each plate, reacted at 4° C. for 16 hours, washed 7 times, and then HRP (enzyme horseradish peroxidase)-conjugated Aβ (11-28) specific affinity purification anti-human Aβ (11-28) ) Mouse IgG MoAb was reacted at 4°C for 1 hour. After washing again 9 times, TMB (tetramethyl benzidine) substrate solution was added and reacted at room temperature for 30 minutes. Then _{, 100 μl of 1N H 2} SO ₄ as a stop solution was added, and the Model 680 Microplate Reader (Bio-Rad, California) was operated at 450 nm. was used to measure the absorbance.

When the birch flower extract was treated, the secretion of Aβ40 and 42 was inhibited at a concentration of 50 μg/ml, and the degree of inhibition was 67.94±9.03% and 57.30±14.05%, respectively, compared to the negative control group (con). In other words, the extract of chrysanthemum flower significantly decreased the secretion of Aβ in a concentration-dependent manner. A positive control, beta-secretase inhibitor III (β-si) 10 μM also exhibited an inhibitory activity of about 80% against Aβ of the two species ( FIG. 2 ).

2-3. Expression regulation of dementia-related proteins

Protein analysis was performed by Western blot method. 1×10 ⁶ APPswe cells were cultured in a 60 mm culture dish, and 24 hours later, the extract of 1 above was treated to a concentration of 10 or 50 μg/ml. In the positive control group, beta-secretase inhibitor III (Calbiochem, Darmstadt, Germany) was treated so as to become 10 μM, instead of the ivy extract. The extract was used by diluting it dissolved in DMSO at a concentration of 100 mg/ml.

After 24 hours, the culture medium and cell fraction were recovered in the presence of phenylmethanesulfonylfluoride (PMSF) and a cell lysis buffer (150 mM NaCl, 50 mM Tris) in which a protease inhibitor (Sigma, St. Louis, MO) was added to the cells washed with phosphate buffered saline (PBS). -HCl, pH 7.4, 0.5% NP-40, 0.5% sodium deoxycholate, 5mM EDTA) was added and pulverized and used as a sample. After 50 μg of protein was separated by 7% SDS-PAGE, antibodies of anti-human sAPPα monoclonal antibody 2B3 (IBL, Minnesota, USA) and rabbit anti-amyloid precursor protein polyclonal antibody CT (Stressgen, Victoria, Canada) were isolated. The protein patterns of APP and sAPP (soluble APP) were detected by immunoblot using the Protein bands were quantified with the Image J analysis program (provided by NIH).

The results of analyzing the protein patterns of APP and sAPPα in the cell fraction and cell culture are shown in FIG. 3 . Wildflower extract showed a tendency to significantly increase the amount of intracellular APP.

As a result of analyzing the pattern of sAPPα protein from the culture medium of the flower extract-treated cell line, it was significantly increased in a concentration-dependent manner by 1.05 and 1.19 times, respectively, compared to the negative control group (con) when treated with 10 and 50 μg/ml. The increased secretion of sAPPα means that the activity of alpha-secretase was increased, and at the same time, the activity of beta-secretase, which reacts competitively with the substrate, was decreased. When combined with the results in 2-4 below, it can be concluded from the above results that the iris extract inhibits BACE.

2-4. BACE inhibitory activity evaluation

In order to confirm the BACE (beta-secretase) inhibitory activity of the wild flower extract, a BACE-1 fluorescence resonance energy transfer (FRET) assay kit (PanVera Co, Madison, Wisconsin) was used.

In a 96-well plate, a sample solution (concentration of 50 or 100 μg/ml), BACE-1 substrate (Rh-EVNLDAEFK quencher, in 50 nmol/ℓ ammonium bicarbonate), and BACE in which the extract of the birch flower 1 was dissolved in DMSO (dimethyl sulfoxide) -1 enzyme [in 50 mM Tris (pH 7.5), 10% glycerol) (1.0 U/ml)] was added in an amount of 10 μl each, and reacted at room temperature after blocking light. After 1 hour of reaction, 10 μl of stop solution (2.5 mol/L sodium acetate) was added, and fluorescence was measured at emission 585 nm and excitation 545 nm using a multi-well spectrofluorometer (infinite F200, TECAN, Switzerland). did. In this case, as a positive control, 10 μM of beta-secretase inhibitor III (β-SI) was used. As a result of the experiment, it showed significant BACE inhibitory activity at 68.24% at 100 μg/ml of the flower extract.

2-5. Antioxidant activity evaluation

Dementia is a disease related to aging, and aging is closely related to oxidative stress. Therefore, it was attempted to confirm the antioxidant activity of the extract by evaluating the DPPH radical scavenging activity. After dilution with DMSO so that the final concentration of the extract and the positive control group in 1 above is 10, 50, 100㎍ / ㎖, 2.5㎍ and DPPH solution 100μM, respectively, 247.5㎍, respectively, put into 96 wells. After reacting for 20 minutes, absorbance was measured at a wavelength of 415 nm, and the degree of inhibition of oxidation activity was calculated.

As a result, as shown in FIG. 5 , the extract of chrysanthemum flower exhibited excellent antioxidant activity when compared with L-ascorbic acid used as a positive control.

Claims (4)

Improving memory, improving cognitive ability, or preventing, delaying, or dementia containing an extract extracted at 70 to 90° C. for 2 to 10 hours at 70 to 90° C. using ethanol of 40 to 60% (v/v) as a solvent A therapeutic pharmaceutical composition. delete Improving memory, improving cognitive ability, or preventing, delaying, or dementia containing an extract extracted at 70 to 90° C. for 2 to 10 hours at 70 to 90° C. using ethanol of 40 to 60% (v/v) as a solvent A food composition for improvement. delete

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