KR20070054915A - Composition comprising flavone type flavonoid for treating and preventing cognitive dysfunction - Google Patents

Abstract

The present invention relates to a composition containing a flavonoid compound having a beta amyloid aggregation inhibitory effect, a beta amyloid toxicity inhibitory effect and a cognitive function recovery effect, the compound of the present invention is a flavonoid component present in nature, beta amyloid aggregation inhibition, beta amyloid It can be usefully used as a pharmaceutical composition and health functional food for preventing and treating cognitive impairment by showing toxic inhibitory activity.

Beta amyloid, flavonoids, memory repair effect, cognitive impairment

Description

Composition comprising flavone type flavonoid for treating and preventing cognitive dysfunction

1 is a diagram showing the structure of a flavonoid compound,

2 is a diagram showing beta amyloid coagulation inhibitory activity by fiscetin (fisetin),

3 is a diagram showing the beta amyloid coagulation inhibitory activity by flavone (flavone),

4 is a diagram showing beta amyloid coagulation inhibitory activity by camphorol (kaempferol),

5 is a diagram showing the beta amyloid coagulation inhibitory activity by morin,

6 is a diagram showing beta amyloid coagulation inhibitory activity by quercetin,

7 is a diagram showing beta amyloid coagulation inhibitory activity by rhamnetin,

8 is a diagram showing the beta amyloid toxicity inhibitory activity by the flavonoid compound,

Figure 9 is a measure of the self-toxicity of the flavonoid compounds.

The present invention relates to a composition for the prevention and treatment of cognitive disorders, including flavonoid compounds present in natural plants.

The central nervous system, composed of the brain and spinal cord, is the central center for life phenomena, which is responsible for all functions of the human body, from sensory and (involuntary) movements to thinking, memory, emotions, and language. It is an essential organ. Therefore, in all cases such as rapid death of nerve cells caused by stroke, trauma, death of slow neurons that cause central nervous system degenerative diseases such as senile dementia and Parkinson's disease, which are represented by Alzheimer's disease This results in irreversible dysfunction of the network and eventually a permanent loss of the human body's function. Geriatric dementia, represented by Alzheimer's disease, has a characteristic of increasing proportionately with the extension of the average human life expectancy and the modernization of medical welfare facilities. According to the statistics of the Korea Institute of Health and Social Affairs, since the elderly population in Korea entered the aged society in 2000, the population reached 3.97 million in 2003, accounting for 8.3% of the elderly population, and 14.4% in 2019. It is expected to enter. Among the elderly population aged 65 years or older, the elderly had at least one chronic disease, and the prevalence of dementia among the elderly aged 65 and older was estimated to be 8.2%. In Western society, Alzheimer's disease occurs in about 10% of the population aged 65 and over, and about 40-50% of the population aged 80 and older.In the United States, more than 5 million people have this disease. It is estimated in dollars. In Korea, more than 200,000 people have dementia. In the United States, it is estimated to reach 16 million people by 2030, up from the current double, and more than 350% by 2050. (Korea Health Industry Development Institute; USA, Alzheimer's Association www.alz.org)

Alzheimer's disease, which begins as a cognitive dysfunction, is a degenerative disease that destroys human nature and is a long-term degenerative disease. Therefore, passive methods of accommodating patients cannot afford socioeconomic burdens. Should. However, to date, no therapeutic agents have been developed to treat the underlying causes of Alzheimer's disease.Available as general therapeutic agents are Aricept, Pfizer's Aricept, Novartis's Exelon, and Janssen. Reminyl and Lundbeck's Ebixa (Memantine), the mechanism of antagonists of NMDA receptors recently approved by the US FDA. However, acetylcholine esterase inhibitors only improve the cognitive ability that has been impaired and do not cure the underlying cause of Alzheimer's disease. In addition, only some patients (approximately 40-50%) show temporary relief of symptoms, and the drug does not last long, and thus is not a fundamental treatment. In addition, the nature of the disease requires long-term administration, the drug is also accompanied by various side effects, including liver toxicity, vomiting, loss of appetite has also been revealed as a problem. Therefore, the development of a therapeutic agent that can prevent the progress of the disease is an urgent task. Many multinational pharmaceutical companies are investing heavily in research and development in this area, especially beta or gamma secretase inhibitors, which reduce the production of beta amyloid, which consists of about 40 amino acids that are believed to be the underlying cause of Alzheimer's disease. Development is the dominant. In Korea, basic research on Alzheimer's disease has been conducted to some extent, but there is almost no case in developing dementia treatment itself. Gamma secretase inhibitors are opaque, with significant toxicity not only in animal experimental models but also in recent clinical trials. Although the research and development period is relatively short, beta cyclase is promising as a target for the development of safer and more effective treatment for dementia as shown in the result of transgenic animal model. In addition, targeting a factor involved in the aggregation of beta amyloid is thought to be relatively safe. Recently, in the development of a new drug targeting beta amyloid, it has been confirmed that Ansonix, USA, has undergone a phase 2 clinical trial for a drug called Phenserine, which has a dual function, and is a cholineesterase. It has also been reported to have inhibitory effects (Greig et al., J. Med . Chem . 44 , pp.4062-4071, 2001; Medical News Today September 4, 2004 article, www.medicalnewstoday.com; Alzheimer's Association homepage, www.alzforum.org/drg/drc).

Another possibility is the development of a vaccine using beta amyloid. As a result of a series of studies and clinical trials conducted by Elan, it was reported that beta amyloid peptide could be used as a vaccine, but the encephalopathy reaction was stopped in a few patients in phase II. Currently, vaccine development using various beta amyloid structures is in progress. As a result of animal testing, beta amyloid vaccine has been shown to reduce the number of senile plaques formed in the brain and improve the cognition of model animals. It is also possible to mitigate Alzheimer's by enhancing the activity of brain cells and enhancing the activity of damaged brain neurons ( Gelinas et al ., Proc . Natl . Acad . Sci . USA , 101 , pp14657 ). 14662 )

Flavonoids are derived from the Greek word flavus, which is yellow, and is a natural substance widely found in plants such as fruit peels, leaves, stems, roots, seeds, and flowers. It is a polyphenol compound based on C6-C3-C6 as a backbone. It is known that about 4,000 species exist in plants and about 60 species exist in citrus fruits. The first clinical use of these flavonoid compounds was by Albert Szent-Gyorgyi, Hungary, in 1936. These substances were also called vitamin P because of their vascular permeability control and vitamin C co-activation. The average intake of the body is about 25-1000 mg / It is known as work. Flavonoids in oriental medicine have been used as anti-inflammatory and immunomodulators in the form of mixed extracts for a long time, but after further research, antibacterial, antifungal / antiviral, vascular control, hepatic, anti-inflammatory / antiallergic effects , Anti-cancer activity has been shown to be used as a diet in the treatment of various diseases (Russell L. et al., American chemical society . pp 83-107, 1980; Citrus science and technology , ed . Steven Nagy , Westport Conn ., Avi Co. ISBN 087055221X , 1977).

As these effects of flavonoids are known, application to various diseases has been attempted, but cognitive dysfunction, especially senile dementia, through the inhibition of the beta amyloid aggregation mechanism by the flavonoid-based compound was confirmed in the present invention for the first time.

In the present invention, a natural product library was screened for the purpose of separating substances having a therapeutic and prophylactic effect of cognitive dysfunction by an established screening method. Surprisingly, beta amyloid aggregation inhibitory activity was observed from flavonoid flavonoids, followed by beta amyloid aggregation inhibitory activity by concentration, and natural products inhibiting cytotoxicity by aggregated beta amyloid. The beta amyloid aggregation inhibitory activity and cytotoxicity inhibitory activity by each flavonoid were expressed as a value. Until now, many studies have been made on various physiological activities of flavonoid compounds, but the physiological activities of cognitive dysfunction have not been identified.

Therefore, the present inventors, in order to determine the effect of the flavonoid-based compound, through the beta amyloid aggregation inhibitory effect and memory learning recovery experiments, the compound of the present invention inhibits the beta amyloid toxicity and aggregation, and inhibits beta amyloid cell death The present invention was completed by confirming to promote the proliferation of nerve cells.

The present invention is to provide a composition for the prevention and treatment of cognitive dysfunction containing a flavonoid compound.

In order to achieve the above object, the present invention includes a flavonoid compound represented by the following general formula (I) having a beta amyloid aggregation inhibitory effect and a memory learning recovery effect as an active ingredient for the prevention or treatment of diseases related to cognitive dysfunction It provides a pharmaceutical composition for.

Wherein R ₁ to R ₇ are each independently H, OH or OCH ₃ .

Preferred compounds of the general formula (I) are R ₁ , R ₂ , R ₄ , R ₅ and R ₆ are H or OH, R ₃ is H, OH or OCH ₃ and R ₇ is H. Preferred compounds include fisetin, flavone, kaempferol, morin, quercetin and rhamnetin represented by the following structural formulas (1) to (6) (See Figure 1)

The compound provides a pharmaceutical composition comprising 0.1 to 50% by weight of the compound based on the total weight of the composition.

The cognitive dysfunction related diseases include Alzheimer's dementia, cerebrovascular dementia, pick name, Creutzfeldt-jakob disease, dementia due to head injury or Parkinson's disease, specifically Alzheimer's dementia, cerebrovascular dementia.

Hereinafter, the flavonoid compounds of the present invention are commercially available (e.g., Sigma) and also from plants containing the flavonoid compounds, fisetin, flavone, kaempferol, and moline ( morin), quercetin, and rhamnetin may be isolated and purified by methods of compound purification well known in the art.

Flavonoid compounds of the present invention represented by the general formula (I) may be prepared as pharmaceutically acceptable salts and solvates according to conventional methods in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluene sulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid ( gluconic acid), galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanic acid, hydroiodic acid, and the like.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Pharmaceutically acceptable salts of the above general formula (I) include salts of acidic or basic groups which may be present in compounds of general formula (I) unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

The present invention also provides a pharmaceutical composition for the prevention and treatment of diseases related to cognitive dysfunction, including the flavonoid compound obtained by the above method.

The composition of the present invention preferably contains 0.01 to 99.9% of the flavonoid compound, and more preferably 0.1 to 90%. However, the composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.

The composition comprising the flavonoid compound of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Compositions comprising the flavonoid compounds according to the present invention are in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods The carriers, excipients and diluents which may be used in the formulation, and may be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. 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 tablets, pills, powders, granules, capsules and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate and sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the flavonoid compounds of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. Administration may be administered once a day or may be divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

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

In another aspect, the present invention provides a health functional food containing a flavonoid compound represented by the general formula (I) as an active ingredient exhibiting the effect of preventing and improving cognitive impairment-related diseases.

The flavonoid compounds include fisetin, flavone, flavone, kaempferol, morin, quercetin, and rhamnetin.

The composition comprising the flavonoid compound of the present invention can be used in various ways, such as drugs, food and beverages effective for the prevention and improvement of cognitive impairment-related diseases. Foods to which the flavonoid compound of the present invention may be added include, for example, various foods, beverages, gums, teas, vitamin complexes, health supplements, and the like, and are in the form of powders, granules, tablets, capsules, or beverages. Can be used.

The flavonoid compound itself of the present invention is a drug that can be used with confidence even when taken for a long time for the purpose of prevention and improvement because there is little toxicity and side effects.

The flavonoid compound of the present invention may be added to food or beverage for the purpose of preventing and improving cognitive impairment. In this case, the amount of the compound in the food or beverage is generally added to the health food composition of the present invention to 0.01 to 15% by weight of the total food weight, the health beverage composition is 0.02 to 10 g, preferably based on 100 ml Can be added in a ratio of 0.3 to 1 g.

The health beverage composition of the present invention, in addition to containing the compound as an essential ingredient in the indicated proportions, has no particular limitation on the liquid component and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates are monosaccharides such as disaccharides such as glucose and fructose, such as maltose, sucrose and the like, and polysaccharides such as dextrin, cyclodextrin and the like. Phosphorous sugar and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. The compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples.

However, the following Examples and Experimental Examples are only illustrative of the present invention, and the content of the present invention is not limited to the following Examples and Experimental Examples.

Example  1. Preparation of Compounds

Fiscetin (fisetin (F4043)), flavone (flavone, F2003), camphorol (kaempferol, K0133), morin (M4008), quercetin (quercetin, Q0125) are Sigma Chemical Co., ST, Louis, MO , USA) was used. Rhamnetin was used separately in the laboratory.

Experimental Example  1. Beta-amyloid coagulation inhibition experiment

In this experiment, the flavonoid compound obtained in Example 1 was used as a sample to confirm the efficacy of inhibiting the aggregation of beta amyloid, which is a fundamental cause of Alzheimer's disease.

1-1. Preparation of the experiment

Synthesized beta amyloid 1-42 (BACHEM) completely dissolved in 250μM in DMSO and diluted 1/10 with PBS in a fluorescent black plate to induce aggregation. Here, the beta amyloid aggregation inhibitory ability of each flavonoid was compared, and 50 μg inhibitory effect was selected at 10 µg / ml, and the mixture was added as a sample and reacted at room temperature for 1 hour. After diluting ThT (Thioflavin T) in 50 mM glycine buffer, 150 μl per well was measured at an excitation wavelength of 450 nm / emission wavelength of 480 nm in a microplate reader (SAFIRE, TECAN).

1-2. Phystine ( fisetin Inhibition Effect of Beta Amyloid Coagulation

Inhibition of beta amyloid aggregation of the fiscetin in the flavonoid compound was measured at a concentration of 10 ㎍ / ㎖ confirmed a 72.7% inhibitory effect. It is seen more than 50% inhibition, the IC ₅₀ through test concentrations By determining the value, the aggregation inhibitory ability was confirmed at various concentrations, such as 0.05 µg / ml, 0.5 µg / ml, 5 µg / ml, and 50 µg / ml. It exhibited a degree of bending by inhibiting aggregation concentration of paroxetine in Figure 2 IC ₅₀ The value was found to be 12.5 µg / ml.

1-3. Flavones flavone Inhibition Effect of Beta Amyloid Coagulation

Inhibition of beta amyloid aggregation of flavones in the flavonoid compound was measured at a concentration of 10 ㎍ / ㎖ confirmed the 43.1% inhibitory effect. IC ₅₀ through concentration experiment By determining the value, the aggregation inhibitory ability was confirmed at various concentrations, such as 0.05 µg / ml, 0.5 µg / ml, 5 µg / ml, and 50 µg / ml. It exhibited a degree of aggregation inhibition by the concentration of isoflavones in Figure 3 IC ₅₀ The value was confirmed to be 20.0 µg / ml or more.

1-4. Camphorol ( kaempferol Inhibition Effect of Beta Amyloid Coagulation

The beta amyloid aggregation inhibitory activity of camphorol in the flavonoid compound was measured at a concentration of 10 µg / ml, and the inhibitory effect was 65.9%. It is seen more than 50% inhibition, the IC ₅₀ through test concentrations By obtaining the values, the aggregation inhibitory ability was confirmed at various concentrations such as 0.05 µg / ml, 0.5 µg / ml, 5 µg / ml, and 50 µg / ml. It exhibited a degree of aggregation inhibition by the concentration of Kam Ferrol in Figure 4 IC ₅₀ The value was found to be 7.0 µg / ml.

1-5. Maureen ( morin Inhibition Effect of Beta Amyloid Coagulation

Inhibition of beta amyloid aggregation of the morphine in the flavonoid compound was measured at a concentration of 10 ㎍ / ㎖ confirmed a 79.9% inhibitory effect. It is seen more than 50% inhibition, the IC ₅₀ through test concentrations By determining the value, the aggregation inhibitory ability was confirmed at various concentrations, such as 0.05 µg / ml, 0.5 µg / ml, 5 µg / ml, and 50 µg / ml. It exhibited a degree of suppression by Maureen aggregation concentration of IC ₅₀ in Figure 5 The value was found to be 7.4 µg / ml.

1-6. Quercetin ( quercetin Inhibition Effect of Beta Amyloid Coagulation

The beta amyloid aggregation inhibitory effect of quercetin in the flavonoid compound was measured at a concentration of 10 µg / ml to confirm the inhibitory effect of 92.6%. It is seen more than 50% inhibition, the IC ₅₀ through test concentrations By determining the value, the aggregation inhibitory ability was confirmed at various concentrations, such as 0.05 µg / ml, 0.5 µg / ml, 5 µg / ml, and 50 µg / ml. It exhibited a degree of aggregation inhibition by the concentration of kwosetin in Figure 6 IC ₅₀ The value was confirmed as 2.4 µg / ml.

1-7. Ramnetine ( rhamnetin Inhibition Effect of Beta Amyloid Coagulation

Inhibition of beta amyloid agglutination of ramnetine in the flavonoid compound was measured at a concentration of 10㎍ / ㎖ confirmed the 92.7% inhibitory effect. It is seen more than 50% inhibition, the IC ₅₀ through test concentrations By determining the value, the aggregation inhibitory ability was confirmed at various concentrations, such as 0.05 µg / ml, 0.5 µg / ml, 5 µg / ml, and 50 µg / ml. It exhibited a degree of aggregation inhibition by the concentration of the indigo plant netin in Figure 7 IC ₅₀ The value was found to be 2.3 µg / ml.

Experimental Example  2. Experimental Inhibition of Beta Amyloid Toxicity of Flavonoid Flavonoids

In this experiment, the flavonoid compounds obtained in Example 1 were used as samples to confirm the efficacy of inhibiting the toxicity of beta amyloid, which is a fundamental cause of Alzheimer's disease.

2-1. Experiment preparation

HT22, a mouse neuronal cell line, was cultured at 37 ° C using DMEM (Dulbecco's Modified Eagle's Medium, Gibco-BRL) medium supplemented with 10% FBS (Fetal Bovine Serum, Hyclone) and 1% penicillin / streptomycin (Sigma). Incubated in a 5% CO ₂ condition incubator (Forma). Before entering the experiment, HT22 cells are plated in 96-well plates at a density of 5 × 10 ³ cells / well and then incubated for 1 hour in serum-free DMEM medium before processing the samples. Incubate for 1 hour by adding samples for each concentration, incubated for 18 hours after incubating beta amyloid 25-35 (US peptide) at a concentration of 25μM, and then induced cell necrosis, 5mg / ml MTT 15 μl of (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide) solution was added per well, followed by 4 hours of incubation and solubilization buffer (10% SDS, 100% of 50% dimethylformamide, pH 4.7) was added and allowed to react overnight. After 18 hours, absorbance was measured at 570 nm / 630 nm using a microplate reader (Sunrise, TECAN) (Gillardon, F. et al., Brain Research , 706 (1) , pp. 169-172, 1996).

2-2. Experiment result

The aggregated beta amyloid was used to stimulate HT22 cells to induce necrosis of the cells, and when the flavonoid compounds were treated together, it was measured how much the cells could be saved. The experimental concentration was 10 µg / ml, respectively, and after 1 hour of pretreatment, beta amyloid was added to the cells to induce 18 hours of cytotoxicity. Cell death was measured by the method using MTT and the results are shown in FIG. 8. Among the flavonoids tested, flavonoids except flavones were found to inhibit beta amyloid toxicity.

Experimental Example  3. Self-Cytotoxicity Test of Flavonoid Flavonoids

3-1. Experiment preparation

In order to determine whether the sample itself is toxic, incubate HT22 cells in the same manner as in Experimental Example 1-1-2, add samples according to concentrations, and incubate for 18 hours. Measured with a reader.

3-2. Experiment result

Self-toxicity of flavonoid compounds was measured to detect side effects due to self-toxicity of samples. The experiment was carried out similarly to the procedure for confirming cytotoxicity caused by beta amyloid, except for beta amyloid. Each sample was treated at a concentration of 10 μg / ml, and its own toxicity was measured. It was confirmed that most of the flavonoid compounds do not have their own cytotoxicity. Flavonoid compounds are not only cytotoxic but are thought to assist cell proliferation.

As a result of these experiments, among the flavonoid-based compounds, whicetin, flavone, camphorol, morphine, quercetin, and ramnetine have two functions of inhibiting beta amyloid aggregation and neuronal cell necrosis caused by beta amyloid. In practice it can be used for the purpose of eliminating and treating Alzheimer's disease.

Examples of the pharmaceutical compositions containing the compounds of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation example  One. Powder  Produce

Fisetin 20 mg

Lactose 100 mg

Talc 10 mg

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

Formulation example  2. Preparation of Tablets

Flavone 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  3. Manufacture of capsule

10 mg of kaempferol

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2mg

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

Formulation example  4. Preparation of Injectables

10 mg morin

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

_{Na 2 HPO 4 · 12H 2 O} 26 ㎎

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

Formulation example  5. Liquid  Produce

Quercetin 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of healthy food

Rhamnetin 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

50 μg folic acid

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

Morin 100mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B ₁

0.3 g of vitamin B ₂

Water quantification

After mixing the above components in accordance with the conventional healthy beverage preparation method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As mentioned above, the flavonoid compound of the present invention has a beta amyloid coagulation inhibitory effect, beta amyloid toxicity inhibitory effect and cognitive function recovery effect, which is estimated to cause Alzheimer's disease, thereby preventing and treating cognitive impairment. And it can be usefully used as a dietary supplement.

Claims (7)

 A pharmaceutical composition for the prevention or treatment of diseases related to cognitive dysfunction comprising a flavonoid compound represented by the following general formula (I) as an active ingredient:

Wherein R ₁ to R ₇ are each independently H, OH, or OCH ₃ . The pharmaceutical composition of claim 1, wherein R ₁ , R ₂ , R ₄ , R ₅ and R ₆ are H or OH, R ₃ is H, OH or OCH ₃ and R ₇ is H. The pharmaceutical composition of claim 1, wherein the compound comprises fisetin, flavone, kaempferol, morin, quercetin or rhamnetin. The pharmaceutical composition of claim 1, wherein the compound comprises 0.1 to 50% by weight of the compound, based on the total weight of the composition. The method of claim 1, wherein the cognitive dysfunction disorder is Alzheimer's dementia, cerebrovascular dementia, pick name, Creutzfeldt-jakob disease, dementia due to head injury or Parkinson's disease. Pharmaceutical composition characterized in that. A health functional food containing the compound as an active ingredient of claim 1, which shows the effect of preventing and improving a disease related to cognitive dysfunction. 7. The dietary supplement of claim 6 which is a powder, granule, tablet, capsule or beverage.

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