KR20210050308A - Compositon comprising Tetragonia tetragonoides as an active ingredient for improving and treating Alzheimers disease - Google Patents

Abstract

The present invention relates to a composition comprising Tetragonia tetragonioides as an active ingredient for alleviating and treating Alzheimer's disease. Accordingly, the composition prevents accumulation of β-amyloid in the brain tissue, increases the expression of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) genes, and reduces the gene expression of Tau forming a nerve fiber mass (NFT), which is known as the cause of Alzheimer's disease, and in particular, can prevent, alleviate or treat β-amyloid accumulation type and Tau protein accumulation type Alzheimer's disease.

Description

Composition comprising Tetragonia tetragonoides as an active ingredient for improving and treating Alzheimers disease

The present invention relates to a composition for improving or treating Alzheimer's dementia comprising Bunhaengcho as an active ingredient, and more particularly, to a food composition for preventing or improving Alzheimer's dementia or a pharmaceutical composition for preventing or treating Alzheimer's dementia.

In Korea, the prevalence of dementia is also rapidly increasing due to the aging of the population. Accordingly, awareness of the risk of dementia is also increasing. In particular, since symptoms of dementia progress slowly, many people miss the treatment period and develop severe dementia symptoms in a short time, although there is a possibility of sufficient improvement if an early visit to a hospital and an accurate diagnosis is obtained. This causes the quality of life to rapidly deteriorate, as well as family members. Even the fields have had a great influence and are emerging as a social problem.

About 70% of dementia patients are known as Alzheimer's dementia, and proteins involved in Alzheimer's have been discovered through numerous studies, but the cause of the disease is not yet clearly known.

J Korean Neuropsychiatr Assoc 2018;57(1):30-42 Looking at the contents contributed to “The Current Status and Future of Alzheimer's Dementia Drug Treatment”, cholinergic enzyme inhibitors, memantine and other drugs for improving cognitive symptoms of Alzheimer's dementia. Antioxidants are used, and cholinergic enzyme inhibitors act as a mechanism to increase the amount of acetylcholine available in synapses by inhibiting cholinergic enzymes. Glutamate, a major excitatory amino acid neurotransmitter of neurons in the brain cortex and hippocampus, activates NMDA receptors, is involved in learning and memory functions, and is known to be effective in severe Alzheimer's rather than mild. However, studies have been conducted to see if the antioxidant effects of monovalent amine oxidase inhibitors selegiline or vitamin E (tocopherol) on antioxidants help prevent deterioration in Alzheimer's dementia patients. It was difficult to draw a consistent conclusion.

On the other hand, although the clinical trial evidence is weak, practically used drugs include estrogen, anti-inflammatory drugs, and ginkgo biloba extract. Among them, anti-inflammatory drugs are known and studied that amyloid induces inflammatory reactions, activates microglia, and promotes cytokine secretion, and epidemiological studies showing that taking nonsteroidal anti-inflammatory drugs lowers the risk of Alzheimer's dementia. there was.

However, a placebo-controlled trial of diclofenac/misoprostol in Alzheimer’s disease. As disclosed in Neurology 1999;53:197-201, etc., as a result of clinical trials, various anti-inflammatory drugs, including aspirin, do not prevent cognitive decline in Alzheimer's dementia, drug side effects are more common, and the risk of cardiovascular side effects in the case of Cox-2 inhibitors. Even so, it can be confirmed that anti-inflammatory drugs are not suitable for the prevention or treatment of Alzheimer's dementia.

Experimental drugs currently undergoing clinical trials include drugs based on the amyloid hypothesis and drugs based on the tau hypothesis.

The most basic pathophysiology in Alzheimer's dementia is the accumulation of amyloid in the brain and the resulting loss of synapses in neurons. Research is being conducted to prevent the accumulation of amyloid. On the other hand, tau lesions, the neurofibrillary tangle. ) Is more consistent with the clinical manifestations of dementia than amyloid neural plaques, and in recent years, the development of tau-based therapeutics is drawing attention.

Accordingly, the present invention has completed the present invention by discovering a substance that is particularly effective for β-amyloid accumulation type and tau protein accumulation type Alzheimer's by preventing β-amyloid accumulation and tau protein accumulation.

Korean Patent Registration No. 10-1638258 Korean Patent Publication No. 2018-0042885

An object of the present invention is to prevent the accumulation of β-amyloid in brain tissue, to increase the expression of brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF) genes, and to increase the expression of Alzheimer's dementia. By reducing the gene expression of Tau, which forms neurofibrillary tangles (NFTs) known as the cause, β-amyloid-accumulating Alzheimer's or Tau protein-accumulating Alzheimer's dementia can be prevented, improved or treated. It is intended to provide a food composition and a pharmaceutical composition containing as an active ingredient the extract of Bunhaengcho, which is harmless to the human body and can be prepared as various foods, beverages, and medicines.

According to one aspect of the present invention,

There is provided a food composition for preventing or improving Alzheimer's dementia comprising a beonhaengcho extract as an active ingredient.

The Bunhaengcho extract may be extracted with water, alcohol having 1-4 carbon atoms, or a mixed solvent thereof.

The Alzheimer's dementia may be β-amyloid-accumulating Alzheimer's or Tau protein-accumulating Alzheimer's.

The food composition for preventing or improving Alzheimer's dementia may increase the expression of one or more genes selected from Brain-derived neurotrophic factor (BDNF) and Ciliary neurotrophic factor (CNTF).

The food composition for preventing or improving Alzheimer's dementia may reduce the expression of the Tau gene.

The food composition for preventing or improving Alzheimer's dementia may be formulated in one or more selected from granules, powders, tablets, coated tablets, capsules, liquids, syrups, juices, suspensions, emulsions and drops, and may be added to food. .

According to another aspect of the present invention,

There is provided a pharmaceutical composition for the prevention or treatment of Alzheimer's dementia comprising the extract of Bunhaengcho as an active ingredient.

The Bunhaengcho extract may be extracted with water, alcohol having 1-4 carbon atoms, or a mixed solvent thereof.

The Alzheimer's dementia may be β-amyloid-accumulating Alzheimer's or Tau protein-accumulating Alzheimer's.

The pharmaceutical composition for preventing or treating Alzheimer's dementia may increase the expression of one or more genes selected from Brain-derived neurotrophic factor (BDNF) and Ciliary neurotrophic factor (CNTF).

The pharmaceutical composition for preventing or treating Alzheimer's dementia may reduce the expression of the Tau gene.

The pharmaceutical composition for preventing or treating Alzheimer's dementia may be formulated as one or more selected from granules, powders, tablets, coated tablets, capsules, solutions, syrups, juices, suspensions, emulsions and drops.

Food and pharmaceutical compositions containing the extract of Bunhaengcho of the present invention as an active ingredient prevent β-amyloid accumulation in brain tissue, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF, Ciliary neurotrophic). factor), and by reducing the expression of Tau, which forms neurofibrillary tangles (NFTs), which are known to cause Alzheimer's dementia, β-amyloid-accumulating Alzheimer's or Tau protein-accumulating Alzheimer's dementia is prevented. It can be improved or treated, and it is harmless to the human body by using beonhaengcho, a medicinal herb, and can be manufactured into various foods and medicines.

1 shows the results of cell viability analysis according to Experimental Example 1.
2 shows the degree of gene expression of BDNF according to Experimental Example 2.
3 shows the level of gene expression of CNTF according to Experimental Example 2.
4 shows the gene expression level of Tau according to Experimental Example 2.
5 shows the results of a passive avoidance experiment according to Experimental Example 3.
6 shows the results of the Y-shaped maze experiment according to Experimental Example 4.
7 shows the results of the underwater maze experiment according to Experimental Example 5.
8 shows the brain beta-amyloid accumulation analysis results according to Experimental Example 6.

Hereinafter, the present invention will be described in detail.

Typically, dementia refers to Alzheimer's dementia, vascular dementia, Lewis's dementia, frontotemporal dementia, Parkinson's disease dementia, Huntington's disease dementia, dementia due to normal pressure cerebral hydrocephalus, dementia due to head trauma, etc. The object is to improve or treat, and among Alzheimer's dementia, the object is to improve or treat β-amyloid-accumulating Alzheimer's and/or Tau protein-accumulating Alzheimer's.

The composition for the treatment or improvement of Alzheimer's dementia of the present invention comprises a beonhaengcho extract as an active ingredient.

The extraction solvent for extracting the extract is water, a lower alcohol having 1 to 4 carbon atoms, ethylene glycol, ethyl ether, or a mixed solvent thereof. The lower alcohol may include 20 to 80% of methanol, ethanol, butanol or propanol.

'Tetragonia tetragonoides' in the present invention is a perennial grass of the pomegranate family, a central dicotyledonous plant, and is grown and cultivated on the beach. It is 40 to 60 cm in height. There is no hair, but it has a wart-like protrusion, and branches are split from the bottom and stand at an angle or extend to the side. Leaves are alternate, egg-shaped, triangular, 4-6cm long and 3-44.5cm wide. The tip is dull and thick, and the petiole is about 2cm long, and leaves and stems can be used as medicinal materials. Fresh grass is sometimes used for harvesting from summer to autumn and drying in the sun, and it is known to be effective in antipyretic, detoxification, and small species. It is also used to treat gastroenteritis, gastric ulcer, gastric cancer, uterine cancer, and swelling caused by the invasion of purulent bacteria through the pores or oil holes of the skin, and it is known that it can cure chronic gastrointestinal diseases and intestinal diseases by boiling tea for a long time.

The term'extract' used in referring to beonhaengcho in the present specification includes not only crude extract obtained by treatment with an extraction solvent, but also a processed product of beunhaengcho extract. For example, the extract of Bunhaengcho may be prepared in a powder state by additional processes such as distillation under reduced pressure and freeze drying or spray drying.

In addition, the extract of beonhaengcho of the present invention has a meaning including a processed product of beunhaengcho, for example, powdered beonhaengcho, formulated so that the beonhaengcho can be administered to an animal in a broad sense. Although the experiment was carried out in the present invention as a beonhaengcho, it will be predictable to those skilled in the art that the desired effect can be achieved even in the same form as the beonhaengcho processed product.

On the other hand, in the present specification, the term'including as an active ingredient' means to include an amount sufficient to achieve the efficacy or activity of the extract of Bunhaengcho. As an example, the beonhaengcho extract is used in a concentration of 10 to 1500 ㎍ / ㎖, preferably 100 to 1000 ㎍ / ㎖. Bunhaengcho extract is a beunhaengcho extract and does not have side effects on the human body even if it is administered in an excessive amount, and thus the upper limit of the quantity of the beunhaengcho extract included in the composition of the present invention can be selected and carried out by a person skilled in the art within an appropriate range.

The present invention provides a food composition for preventing or improving Alzheimer's dementia, comprising the extract of Bunhaengcho as an active ingredient.

The extract of the present invention is effective in preventing or ameliorating β-amyloid-accumulating Alzheimer's or Tau protein-accumulating Alzheimer's.

The food composition according to the present invention may be formulated in the same manner as the following pharmaceutical composition to be used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, alcoholic beverages, confectionery, diet bars, dairy products, meat, chocolate, pizza, ramen, other noodles, gums, ice creams, vitamin complexes, health supplements. Etc.

The food composition of the present invention may include not only the extract of Bunhaengcho as an active ingredient, but also ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts, etc. may be further included in addition to the extract of Bunhaengcho of the present invention.

The present invention provides a health functional food comprising a food composition for preventing or improving Alzheimer's dementia comprising the extract of Bunhaengcho as an active ingredient. Health functional foods are foods prepared by adding Bunhaengcho extract to food ingredients such as beverages, teas, spices, chewing gums, confectionery, etc., or in encapsulation, powdering, suspension, etc., and ingesting them has specific health effects. However, unlike general drugs, it has the advantage of not having side effects that may occur when taking drugs for a long time by using food as a raw material. The health functional food of the present invention obtained in this way is very useful because it can be consumed on a daily basis. The added amount of the extract of Bunhaengcho in such health functional foods cannot be uniformly regulated depending on the type of health functional food to be targeted, but can be added within the range that does not damage the original taste of the food. To 50% by weight, preferably 0.1 to 20% by weight. In addition, in the case of health functional foods in the form of pills, granules, tablets or capsules, it is usually added in the range of 0.1 to 100% by weight, preferably 0.5 to 80% by weight. In one embodiment, the health functional food of the present invention may be in the form of a pill, tablet, capsule or beverage.

In addition, the present invention provides a pharmaceutical composition for the prevention or improvement of Alzheimer's dementia comprising the extract of Bunhaengcho as an active ingredient.

The extract of the present invention is particularly effective in preventing or treating β-amyloid-accumulating Alzheimer's or Tau protein-accumulating Alzheimer's.

The pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvants include excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants. Agents or flavoring agents may be used.

For administration, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients, and may be preferably formulated into a pharmaceutical composition.

The formulation form of the pharmaceutical composition may be granules, powders, tablets, coated tablets, capsules, suppositories, solutions, syrups, juices, suspensions, emulsions, drops, or injectable solutions. For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, natural sugars such as starch, gelatin, glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, trackcacanth or sodium oleate, sodium stearate, magnesium stearate, sodium Benzoate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, and the like.

Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterilized and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injection formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, etc., preferably oral administration.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, mode of administration, age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate and response sensitivity of the patient, and is usually As such, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10 g/kg.

The pharmaceutical composition of the present invention may be prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient, or may be prepared by incorporating it into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet or capsule, and may additionally include a dispersant or a stabilizer.

In addition, the present invention provides a use of the extract of Bunhaengcho for the manufacture of a medicine or food for improving or treating Alzheimer's dementia. As described above, the Bunhaengcho extract may be used for treatment or improvement of Alzheimer's dementia.

In addition, the present invention provides a method for improving, preventing or treating Alzheimer's dementia, comprising administering an effective amount of Bunhaengcho extract to a mammal.

The term "mammal" as used herein refers to a mammal that is an object of treatment, observation or experiment, and preferably refers to a human.

The term "effective amount" as used herein refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human thought by a researcher, veterinarian, doctor or other clinician, which Or an amount that induces relief of symptoms of the disorder. The effective amount and frequency of administration for the active ingredient of the present invention may be changed according to the desired effect. Therefore, the optimal dosage to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of active ingredients and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health of the patient. It can be adjusted according to various factors including condition, sex and diet, time of administration, route of administration and rate of secretion of the composition, duration of treatment, and drugs used concurrently. In the prevention, treatment or improvement method of the present invention, in the case of an adult, it is preferable to administer the extract at a dose of 0.001 g/kg to 10 g/kg when administered once to several times a day.

In the treatment method of the present invention, the composition containing the extract of Bunhaengcho as an active ingredient can be administered in a conventional manner through oral, rectal, intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, topical, or intradermal routes. I can.

Hereinafter, a preferred embodiment is presented to aid in the understanding of the present invention, but it is obvious to those skilled in the art that various changes and modifications are possible within the scope of the present invention and the scope of the technical idea, but the following examples are only illustrative of the present invention, It is natural that such modifications and modifications fall within the scope of the appended claims.

[ Example : First Extract preparation]

Manufacturing example One: First Alcohol extract manufacturing

2000 g of beonhaengcho was extracted with reflux cooling twice for 3 hours with 5 times the volume of 70% ethanol, and the filtered solution was concentrated using a vacuum concentrator and then freeze-dried to prepare an alcoholic extract of beonhaengcho.

Manufacturing example 2: First Hydrothermal Extract manufacturing

2000 g of beonhaengcho was hot-water extracted with 5 times the volume of water at 100°C, the filtered solution was concentrated using a vacuum concentrator, and then freeze-dried to prepare a hot-water extract of beonhaengcho.

[ Experimental example : In vitro experiment]

PC12 Adh cell Cultivation of

PC12 Adh cell (KCLB 21721, Korean Cell Line Bank, Seoul, Korea) is a cell line derived from pheochromocytoma arising in the adrenal medulla of rats and has neuronal characteristics. PC12 Adh cell is 75cm ² The flask was inoculated with High media (FBS + Horse serum + 4.5g/ml glucose DMEM) and cultured in a 37°C, 5% CO ₂ incubator. The media was replaced once every 2-3 days until the cell was in sufficient quantity for the experiment. After confirming that the amount of cell is about 70% ^{, transfer the cell from a 75cm 2} flask to a 10cm ² plate, and then add NGF (neuron growth) to high media (Horse serum 500µl, Penicillin 500µl in 50ml 4.5g/ml glucose DMEM). factor) 2.5µl was treated to observe that the length of the neuron became about 1 to 2 times the length of the cell.

Experimental example 1: Cell viability analysis

Scrap the ^{cells from a 10cm 2} plate and centrifuge (2000rpm, 5min at RT) so that 4 x 104 cells are in 200µl, and uniformly dispense into a 96-well plate. Low media (Horse serum 500µl, Penicillin 500µl in 1.0) g/ml glucose DMEM) at concentrations of 10 μM, 50 μM, 10 μg/ml, and 50 μg/ml, respectively, were treated with the alcohol extract and hot water extract of Bunhaengcho, respectively, and β-amyloid 25-35 was treated after 1 hour. After incubation for 3 days, 100µl of MTT solution diluted in PBS at a concentration of 1mg/ml of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide, SIGMA, USA) was dispensed. After 3 hours, it was left in an incubator at 37°C and 5% CO _2. After 3 hours, the supernatant was discarded, and 100 μl of DMSO (Dimethyl Sulfoxide, SIGMA, USA) was dispensed, and allowed to stand at room temperature for 10 minutes, followed by ELISA (VersaMax ^tm). , Molecular Devices) was measured at 490nm to confirm the cell viability.

Bunhaengcho extract was dissolved in cell water, and DMSO was used when it was not dissolved in cell water. The solvent in which the extract of Bunhaengcho was dissolved was recorded and the corresponding solvent was used as a control. When the molecular weight (M.W) was known, the concentration was treated with μM, and when the molecular weight was unknown, the concentration was treated with a concentration of ㎍/㎖.

The cell viability analysis results according to Experimental Example 1 are shown in FIG. 1. According to this, it was confirmed that the neuron bundle of PC12 Adh cells increased by 1.5 to 2 times by using NGF, and β-amyloid at concentrations of 2.5 μM, 5 μM, 10 μM, 25 μM, and 50 μM by dissolving β-amyloid 25-35 in cell water. After preparing 25-35, the same amount of cell water was used as a control. After treatment with Low NGF media, 24 hours later, MTT assay was conducted to investigate the survival rate of PC12 Adh cells. The treatment concentration of β-amyloid 25-35 was treated with 10 μM, which lowered the survival rate to 50% or less compared to the case where β-amyloid 25-35 was not treated.

The survival rate of cells was expressed as Control %. The survival rate of the 10 and 50 μg/ml Bunhaengcho extract treatment groups was significantly higher (P<0.05) than the control group, but the cell survival rate was significantly increased in the 50 μg/ml treatment group.

Experimental example 2: gene expression analysis

PC12 Adh cells were dispensed with 4 X 10 ⁴ cells/well in a 6 well plate and treated with High glucose DMEM supplemented with 10% Horse serum and 5% FBS for 2 hours. After confirming that the PC12 Adh cells were attached to the bottom of the plate, they were washed with PBS and replaced with 4.5mg/dl glucose DMEM treated with NGF, and the cells were grown by about 70% in _{a 37℃, 5% CO 2 incubator.} After washing with PBS, Bunhaengcho extract was diluted in NGF-treated 1.0mg/dl glucose DMEM at a concentration of 10µg/ml and 50µg/ml, and dispensed into each well. After 1 hour, β-amyloid 25-35 was dispensed into each well to a concentration of 10 μM.

(One) Total RNA Separation of

After 24 hours of treatment with the extract of Bunhaengcho of Example 1, the media residue was washed with PBS, and then ^{1 ml of Trizol®} Reagent (Ambion, USA) was dispensed, and the cells were scraped off and then autoclaved microcentrifuge. Transferred to the tube. After allowing to stand at room temperature for 10 minutes, 200 µl of Chloroform (SIGMA, USA) was added, followed by vortexing, followed by centrifugation at 4° C. at 1000 rpm for 15 minutes. 400 µl of the supernatant was vortexed in a new microcentrifuge tube with 500 µl of isopropyl alcohol (SIGMA, USA). After allowing it to stand at room temperature for 10 minutes, it was centrifuged for 10 minutes at 1000 rpm at 4°C. After centrifugation, 1 ml of 75% Pure ethanol (SIGMA, USA) diluted with RNAse free DEPC-treated water (DEPC-water, Ambion, USA) was added to the pellet, followed by centrifugation and supernatant removal twice. And washed. After removing the supernatant of the washed pellet, it was air-dried for about 5 minutes. The pellet was melted in a 55°C incubator using 40µl of DEPC-water. The obtained total RNA was diluted 50 times and measured at 260nm and 280nm using a spectrophotometer to confirm the purity and quantification of RNA to proceed with complementary DNA synthesis.

(2) Complementary DNA ( cDNA ) synthesis

cDNA synthesis was ^{performed using SuperScript TM} III Forst-strand (Invitrogen, USA). Total RNA was diluted to 1 μg/ml using DEPC-water. 1 µl of diluted RNA, 20 µl of 50 Oligo(dt), 1 µl of 10mM dNTP mix, and 7 µl of DEPC-water were mixed to make a total volume of 10 µl. After spin-down, incubation was performed at 65°C for 5 minutes, and then left at 4°C for 5 minutes. 10X RT buffer 2µl, 25mM MgCl ₂ , 0.1M DTT 2µl, RNAse Out 1µl, Super Scrit Ⅲ 1µl so that the total volume becomes 20µl, spin-down and MyCycler ^TM thermal cycler (Bio-Rad, USA) at 50°C for 50 minutes and 85°C for 5 minutes. cDNA was stored frozen at -20°C until qRT-PCR was performed.

(3) qRT - PCR practice

The prepared cDNA was diluted 15 times with DEPC-water and used. Diluted cDNA, iQ ^TM SYBER ^® Green Supermix (Bio-rad, USA), forward primer and reverse primer diluted to 10 pM, and DEPC-water were mixed so that the reaction volume was 20 μl. Using a Mini opicon ^TM Detector (Bio-rad, USA), qRT-PCR was performed by repeating 40 cycles of 40 cycles of maintaining 20 seconds at 94°C, 30 seconds at 65°C, and 20 seconds at 72°C.

The expression of each gene was compared using the calculation formula of the threshold cycle (Ct). The gene expression levels of β-actin, BDNF, Tau, and CNTF were confirmed, and the used Primer square is shown in Table 1 below.

β-actin Forward 5’-AGCGTGGCTACAGCTTCACC-3’ Reverse 5’-AAGTCTAGGGCAACATAGCAACAGC-3’ BDNF Forward 5’-ATGCCGAACTACCCAATCGT-3’ Reverse 5’-GCCAATTCTCTTTTTGCTATCCA-3’ CNTF Forward 5’-ATGGCTTTCGCAGAGCAATCACC-3’ Reverse 5’-GTCGTGTACTCTCGGTAATACC-3’ TAU Forward 5’-CGGCTAACGTGGCAAGCT-3’ Reverse 5’-AAGACAGACCATGGAGCAGAAATC-3’

(4) Experiment result

BDNF is a brain-derived neurotrophic factor, and when there are external stimuli such as education and learning in humans and animals, the activity of BDNF appears, and it has been shown that BDNF plays an important role in learning and memory. Although BDNF may also increase during exercise-induced stimulation, there are studies that show that BDNF is activated only in spontaneous exercise, and that in involuntary and compulsory cases, BDNF activity does not appear. The degree of expression of the BDNF gene is shown in FIG. 2, and according to this, when treated with 10 µg/ml and 50 µg/ml of Bunhaengcho extract, compared to β-amyloid-treated Control, Bunhaengcho extract-treated group was significant at p<0.05. Was highly expressed, and the Bunhaengcho extract did not increase the expression as much as normal-control without β-amyloid treatment.

CNTF is a ciliary neurotrophic factor and, like BDNF, plays a role in growing cranial nerves. The level of CNTF gene expression is shown in FIG. 3, and when treated with 10 µg/ml of Bunhaengcho extract, it was statistically significantly higher than that of the control treated with β-amyloid, which was significantly higher than the normal-control without treatment with β-amyloid. There was no difference.

In the case of Tau, NFT (Neurofibrillary tangle) is formed, which is one of the causes of Alzheimer's dementia. The degree of gene expression of Tau is shown in FIG. 4, and at concentrations of 10 μg/ml and 50 μg/ml, the Bunhaengcho extract-treated group showed statistically significantly lower expression compared to Control. This is normal without β-amyloid treatment. There was no significant difference from -control.

[ Experimental example : In vivo experiment (cognitive ability test)]

Experimental animal breeding

Experimental animals were Spargue-Dawley species, and 7-week-old male rats were purchased from Daehan Biolink and provided solid feed (DBL, Rod feed, Korea) to adapt to the breeding environment. Adaptation and growth were observed for 1 week during the adaptation period, and if there was a difference in growth during the adaptation period, they were excluded from the experiment.

(1) Application of experiment

After a 1-week adaptation period, the supply of rodfeed was restricted for 16 hours to make it into an empty stomach, and then divided into 7 groups of 10 animals per group by randomized complete block design according to weight and blood sugar.

(2) Management of experimental animals

The animal breeding room was maintained at 65±5% relative humidity, and the temperature was maintained at 25±5%, and an environment in which the photoperiod and dark cycle were controlled at 12 hour intervals was provided. A high-fat diet of 43 En% was provided to all of the experimental animals, and according to the experimental group, the corresponding ingredients were uniformly mixed so that 7.5 g of Bunhaengcho extract per 1 kg of the high-fat diet included, and there was no restriction on the intake amount. Dementia was induced 4 weeks after eating the experimental diet, and a water maze test and a Y maze test were conducted in the 6th week of the experiment for cognitive ability, and the passive avoidance experiment was conducted in the 7th week. test) was carried out.

(3) Induction of Alzheimer's dementia in experimental animals

For β-amyloid, Amyloid β-protein Fragment 25-35 (Sigma-Aldrich Corporation, A4559-1MG, USA) was used and diluted to 0.05mg/300µl with sterilized distilled water. The β-amyloid solution diluted using an osmotic pump (Alzet, Micro-osmotic pump 1003D, USA) was administered at a constant rate of 0.5 µl per hour for about 2 weeks by performing ICV (Intracerebroventricular) as a dementia control group (AD-con). and. The general control group (Normal-con) was observed by administering Saline without administering β-amyloid solution to the Osmotic pump.

(4) Diet of experimental animals

In the diet of the experimental animals, the energy ratio of carbohydrate, protein, and fat was 39.6%, 16.8%, and 43.6%, respectively, and a high-fat diet was provided with Mineral Mixture, Vitamin Mixture, DL-Methionine, Choline Chloride, and Cholesterol. In addition, the 70% ethanol extract of Bunhaengcho was extracted in 4L of 4kg of medicinal herbs and extracted with a yield of 20%, and the extract was provided to the experimental animals at 0.3g/kg BW/day.

Experimental example 3: passive avoidance experiment ( Passive avoidance test )

At the 6th week of the experiment, a passive avoidance reaction experiment was conducted. Using a passive avoidance test machine, a machine divided into room A and room B was used.After putting the experimental animals in room A, giving them an acclimatization time for 60 seconds, and then making light and noise generated in room A for 5 minutes to give them to the experiment animals. I made it stressful. While light and noise were generated, the passage leading to room B was opened, and when the experimental animals moved to room B to avoid stress, the passage was closed, and a current of 0.5 mA was allowed to flow through the feet for 3 seconds. Two hours after the first experiment was carried out, the same experiment was repeated and remembered, and after 24 hours, whether the experimental animal moved to the B room and the time it took to move were measured.

This passive avoidance experiment is an experiment to confirm the short-term memory ability of the test subject, and is an experiment in room A in a machine divided into room A, which causes stress with noise and light, and room B, which has no noise and light but has electrical stimulation. In order to observe the memory of the electrical stimulation of room B, the time it took for the subject to move to room B was observed, and cognitive ability was tested within 24 hours. The results are shown in FIG. 5. Compared to the dementia-inducing control group, the group administered the extract of Bunhaengcho showed a significant (P<0.05) long time, but lower than that of the normal control group.

Experimental example 4: Y-shaped maze experiment (Y- maze test )

In order to measure the cognitive ability of the experimental animals, a Y-shaped maze experiment was conducted. With a Y-shaped cage, three passages were divided into A, B, and C, respectively, and sawdust was applied, and the experimental animals were placed in passage A and the movement route was recorded for 8 minutes. If three different passages were entered in sequence, 1 point was given, and if the same passage was repeated, it was calculated as 0. After the experiment was over, sawdust in all passages was replaced so that there was no smell from other individuals.

This Y-shaped maze experiment was conducted in one time, and the short-term spatial memory ability to observe whether or not to remember the path they passed while passing through the labyrinth was confirmed. The result is shown in FIG. 6 by giving a score of 1 when finding a new path that is not.

According to this, the movement score in the Y-shaped maze was significantly higher (P<0.05) in the group intake of Bunhaengcho extract (AD-Beonhaengcho) compared to the dementia induction control group (AD-Con), and it is almost similar to that of the normal control group (Normal-con) I did.

Experimental example 5: underwater maze experiment ( Water maze )

The experiment was conducted at the 6th week of the experiment, and this was conducted at the 2nd week after the induction of dementia was made. Black food coloring was released in the pool so that the location of the island of the experimental animal could not be confirmed. After dividing the pool into 4 zones, the experimental animals were collected by each zone, and whether or not the island was searched for 2 minutes was observed. In the case of finding the island, it was decided that the experiment in the zone was terminated if it stayed for 10 seconds. For 3 consecutive days, each observation was made once in each of the four sections, and 48 hours after the last observation, the island was removed and acquired in the first section to observe whether or not to remember the island, and the degree of recovery of cognitive ability was observed.

This underwater maze experiment was conducted to find out the spatial memory ability of the subject's cognitive ability. It is an experiment that checks whether the subject remembers the existence and location of the island. The shorter the time it took to find the island, the more frequent the island was found, and the difference in cognitive ability by calculating how much time it stayed at the place where the island was. And the results are shown in FIG. 7. Here, (a) of FIG. 7 is a comparison of the time taken to find the island for the first time, and (b) is a comparison of the time spent on the island.

According to (a) of FIG. 7, the time taken to find the island for the first time was significantly (P<0.05) shorter in the normal control group and the Bunhaengcho extract intake group compared to the dementia-inducing control group. In addition, according to (b) of FIG. 7, the staying time on the island was shorter than that of the dementia-inducing control group (AD-Con) compared to the group ingesting the beonhaengcho extract (AD-beonhaengcho), and the normal control group (Normal-con) There was no statistically significant difference.

Experimental example 6: Brain β-amyloid accumulation assay

After all experiments were completed, brains of experimental animals treated with 4% paraformaldehyde were removed, fixed in 10% formaldehyde for 1 day, and replaced with 10% sugar water. At 1 day intervals, the brains were checked to see if they were sinking in sugar water, and in turn, they were replaced with 20% and 30% sugar water. Finally, after storing in 30% sugar water solution, the brain was frozen using dry ice and then frozen sectioned to a thickness of 40 μm. Before staining, the cut brain was divided into each part, put in a Storing solution (Glycerol, Butylene glycol) and stored at -20℃. In the case of staining, a section with a well-preserved shape of the hippocampus was separated, washed with 0.01M PBS, blocked with 10% normal serum (A-solution), and then diluted 200 times with β-amyloid antibody in DW for 18 hours. After reacting, washing with 0.01M PBS was carried out, and then a 50-fold dilution of the secondary antibody was reacted for 1 hour to perform fluorescence staining. The stained brain tissue was observed under a microscope, and the image is shown in FIG. 8. Like this Accumulation of β-amyloid plaques in the hippocampus was confirmed through fluorescence staining.

According to this, in the case of the dementia-inducing control group (AD-Con), the largest accumulation of β-amyloid plaques was shown, and the group in which the beonhaengcho extract intake group (AD-beonhaengcho) induces dementia together with the normal control group (Normal-con) that did not induce dementia. It can be observed that there was little accumulation of β-amyloid plaques compared to the control group.

In the above, embodiments of the present invention have been described, but those of ordinary skill in the relevant technical field add, change, delete or add components within the scope not departing from the spirit of the present invention described in the claims. Various modifications and changes can be made to the present invention by means of the like, and it will be said that this is also included within the scope of the present invention.

<110> Korea Institute of Oriental Medicine <120> Compositon comprising Tetragonia tetragonoides as an active ingredient for improving and treating Alzheimers disease <130> HPC-8709 <160> 8 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> beta actin Forward <400> 1 agcgtggcta cagcttcacc 20 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> beta actin Reverse <400> 2 aagtctaggg caacatagca acagc 25 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> BDNF Forward <400> 3 atgccgaact acccaatcgt 20 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> BDNF Reverse <400> 4 gccaattctc tttttgctat cca 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> CNTF Forward <400> 5 atggctttcg cagagcaatc acc 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CNTF Reverse <400> 6 gtcgtgtact ctcggtaata cc 22 <210> 7 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TAU Forward <400> 7 cggctaacgt ggcaagct 18 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> TAU Reverse <400> 8 aagacagacc atggagcaga aatc 24

Claims (12)

A food composition for preventing or improving Alzheimer's dementia, comprising the extract of Bunhaengcho as an active ingredient. The method of claim 1,
The Bunhaengcho extract is a food composition for preventing or improving Alzheimer's dementia, characterized in that it is extracted with water, alcohol having 1-4 carbon atoms, or a mixed solvent thereof. The method of claim 1,
The Alzheimer's dementia is β-amyloid-accumulating Alzheimer's, or Tau protein-accumulating Alzheimer's. A food composition for preventing or improving Alzheimer's dementia. The method of claim 1,
The food composition for preventing or improving Alzheimer's dementia is a food composition for preventing or improving Alzheimer's dementia, characterized in that it increases the expression of one or more genes selected from Brain-derived neurotrophic factor (BDNF) and Ciliary neurotrophic factor (CNTF). . The method of claim 1,
The food composition for preventing or improving Alzheimer's dementia food composition for preventing or improving Alzheimer's dementia, characterized in that reducing the expression of the Tau gene. The method of claim 1,
The food composition for preventing or improving Alzheimer's dementia is formulated in one or more selected from granules, powders, tablets, coated tablets, capsules, liquids, syrups, juices, suspensions, emulsions and drops, and added to food. Food composition for preventing or improving Alzheimer's dementia. A pharmaceutical composition for the prevention or treatment of Alzheimer's dementia comprising the extract of Bunhaengcho as an active ingredient. The method of claim 7,
The Bunhaengcho extract is a pharmaceutical composition for preventing or treating Alzheimer's dementia, characterized in that it is extracted with water, alcohol having 1-4 carbon atoms, or a mixed solvent thereof. The method of claim 7,
The Alzheimer's dementia is β-amyloid-accumulating Alzheimer's, or Tau protein-accumulating Alzheimer's. A pharmaceutical composition for the prevention or treatment of Alzheimer's dementia. The method of claim 7,
The pharmaceutical composition for preventing or treating Alzheimer's dementia is a pharmaceutical composition for preventing or treating Alzheimer's dementia, characterized in that it increases the expression of one or more genes selected from Brain-derived neurotrophic factor (BDNF) and Ciliary neurotrophic factor (CNTF). . The method of claim 7,
The pharmaceutical composition for preventing or treating Alzheimer's dementia is a pharmaceutical composition for preventing or treating Alzheimer's dementia, characterized in that it reduces the expression of the Tau gene. The method of claim 7,
The pharmaceutical composition for preventing or treating Alzheimer's dementia is Alzheimer's, characterized in that it is formulated as one or more selected from granules, powders, tablets, coated tablets, capsules, solutions, syrups, juices, suspensions, emulsions and drops. A pharmaceutical composition for preventing or treating dementia.

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