KR20240040886A - Composition for improvement of memory or cognition ability, or for prevention, delay, treatment or improvement of dementia comprising extracts of Campbell early grape leave - Google Patents

Abstract

The pharmaceutical composition for improving memory, improving cognitive ability, or preventing, delaying, or treating dementia and the functional food composition for improving memory, improving cognitive ability, or preventing, delaying, or treating dementia of the present invention contain Campbell's Early grape leaf extract as an active ingredient. It contains.

Description

Composition for improving memory, improving cognitive ability, or preventing, delaying, treating or improving dementia containing Campbell's Early grape leaf extract as an active ingredient {Composition for improvement of memory or cognition ability, or for prevention, delay, treatment or improvement of dementia comprising extracts of Campbell early grape leaves}

The present invention relates to a composition for improving memory, improving cognitive ability, or preventing, delaying, treating or improving dementia, containing Campbell Early grape leaf extract as an active ingredient. Specifically, the present invention contains Campbell Early grape leaf extract as an active ingredient, thereby effectively preventing, delaying, treating or treating dementia through acetylcholinesterase inhibitory activity, beta-amyloid secretion inhibitory activity, and amyloid precursor protein-related protein expression regulatory activity. It relates to pharmaceutical compositions and functional food compositions that can improve memory and cognitive abilities effectively.

Aging is a global phenomenon, and it is more pronounced in the seven developed countries (G7: USA, Canada, UK, France, Germany, Italy, Japan). Korea is aging the fastest among OECD countries, and the proportion of the population aged 65 or older is expected to increase from 16.5% in 2021 to 43.9% in 2060.

Alzheimer's disease (AD) is the most common form of dementia. As the elderly population increases, more than 50 million patients are reported worldwide. It is one of the major diseases causing death in the elderly, and is also caused by cancer and cerebrovascular disease. Deaths due to disease are decreasing, but deaths due to Alzheimer's disease continue to increase, ranking as the 5th leading cause of death for the elderly for the first time in 2020.

According to data, the estimated number of dementia patients among the elderly population aged 65 or older in 2021 is 890,000, with a prevalence rate of 10.33%.

Dementia is caused by irreversible destruction of cranial nerves due to brain atrophy, decrease in nerve cells, and appearance of senile plaques, causing various symptoms of acquired cognitive dysfunction such as memory, speech, and behavioral disorders. Refers to a concomitant syndrome. Dementia is classified into other dementias caused by Alzheimer's disease, degenerative diseases caused by vascular dementia and Parkinson's disease, metabolic diseases caused by hypothyroidism, brain tumors, or infectious diseases.

Alzheimer's disease is a degenerative neurological disease of the brain that causes abnormalities in the central nervous system due to loss of nerve cells, leads to loss of intellectual abilities such as memory decline and language impairment, and ultimately leads to death.

Beta amyloid (β-amyloid, Aβ) is a major pathological marker in Alzheimer's. Amyloid Precursor Protein (APP) is activated by β-secretase (BACE, β-site APP cleaving enzyme) and γ-secretase (BACE). It is a peptide produced by decomposition by secretase (γ-secretase). γ-Secretase, one of the targets for developing a treatment for Alzheimer's disease, is known to be a complex composed of presenilin (PS), nicastrin (NCT), APH-1, and PEN-2. there is.

Beta-amyloid is known to be neurotoxic and cause dementia, and if you look at the brains of dementia patients, you can see beta-amyloid deposited along with the loss of neurons, which are nerve cells. In particular, neuronal loss and beta-amyloid deposition appear to be severe in the hippocampus, which is responsible for memory and cognitive abilities.

Additionally, dementia patients show a decrease in the neurotransmitter acetylcholine (ACh) by about 50% compared to normal people. This is believed to be due to an abnormal concentration of acetylcholinesterase (AChE), an enzyme that decomposes acetylcholine, and in fact, dementia patients have higher concentrations of acetylcholinesterase than normal people.

Therefore, inhibition of beta-amyloid production and aggregation and inhibition of acetylcholinesterase may be potential strategies for the treatment of Alzheimer's disease.

Currently, acetylcholinesterase inhibitors such as donepezil, galantamine, and rivastigmine are used clinically as drugs to improve dementia, but due to low treatment efficiency and severe side effects, there is no effective treatment. .

In addition, Aducanumab, a beta amyloid targeting antibody treatment that recently received conditional approval from the FDA, is an important result proving the beta amyloid theory. However, it is expected that it will take time to commercialize due to the high treatment cost.

Accordingly, the present inventor has been searching for substances effective against dementia from plants in order to develop drugs that can effectively prevent, delay, treat or improve dementia without side effects. Through these results, Korean Patent Nos. 10-1804212 and 10- The same technology as No. 1828379 has been developed.

The present inventor attempted to conduct research on various plants in order to develop new and more effective substances for dementia, and found that among these, edible Campbell's Early grape leaves had potential. Based on this, through a more detailed and full-scale study, it was discovered that the ethanol extract of Campbell Early grape leaves inhibits acetylcholinesterase activity in vitro and reduces the secretion of beta-amyloid. In addition, it was confirmed in animal behavioral experiments that memory and cognitive ability were improved, and it was also found to reduce the accumulation of beta-amyloid plaques in the brain. Through this, it was confirmed that Campbell Early grape leaf extract can prevent, delay, treat, or improve dementia without side effects and effectively, and the present invention was completed.

Republic of Korea Patent No. 10-1804212 Republic of Korea Patent No. 10-1828379

Therefore, the main object of the present invention is to provide a plant-derived substance that has physiological activities such as acetylcholinesterase inhibitory activity and beta-amyloid secretion inhibitory activity, can prevent, delay, improve or treat dementia, and can improve memory and cognitive ability. The aim is to provide pharmaceutical compositions and functional food compositions containing active ingredients.

In one aspect, the present invention provides a pharmaceutical composition for improving memory, improving cognitive ability, or preventing, delaying, or treating dementia, containing Campbell's Early grape leaf extract as an active ingredient.

In another aspect, the present invention provides a functional food composition for improving memory, improving cognitive ability, or preventing, delaying, or improving dementia, containing Campbell's Early grape leaf extract as an active ingredient.

The composition of the present invention can effectively prevent, delay, treat or improve dementia through acetylcholinesterase inhibitory activity, beta-amyloid secretion inhibitory activity, and amyloid precursor protein-related protein expression regulation activity, and also effectively improves memory and cognitive ability. can do. In addition, since the composition of the present invention uses plant raw materials, it can be free from restrictions due to safety issues such as side effects, and since it uses raw materials that are rarely used and discarded, it increases the usability of plant resources and improves the environment. It can also provide significant advantages in terms of protection.

Figure 1 shows the manufacturing process of Campbell Early grape leaf extract (VLL) according to an embodiment of the present invention.
Figure 2 is a graph showing the results of an experiment on the secretion inhibition activity of beta-amyloid (Aβ) 40 and 42 of Campbell Early grape leaf extract (VLL) according to an embodiment of the present invention. C, negative control; 1 to 50, VLL treatment group by concentration (㎍/㎖); β-si 10μM, positive control.
Figure 3 is a graph showing the results of an amyloid precursor protein (APP)-related protein expression activity test of Campbell Early grape leaf extract (VLL) according to an embodiment of the present invention. C, negative control; 1 to 50, VLL treatment group by concentration (㎍/㎖); β-si 10μM, positive control.
Figure 4 is a graph showing the results of an mRNA expression activity test of Campbell Early grape leaf extract (VLL) according to an embodiment of the present invention. C, negative control; 1 to 50, VLL treatment group by concentration (㎍/㎖); β-si 10μM, positive control.
Figure 5 is a graph showing the acetylcholinesterase (AChE) inhibitory activity of Campbell's Early grape leaf extract (VLL) according to an embodiment of the present invention. C, negative control; 10 ~ 100, VLL treatment group by concentration (㎍/㎖); Gal 10μM, positive control.
Figure 6 is a graph showing the results of an experiment analyzing the effect of Campbell Early grape leaf extract (VLL) on improving spatial learning ability according to an embodiment of the present invention. A, distance to target; B, number of passes through the target; Aβ-, normal group; Aβ+, negative control; Aβ+VLL, VLL administration group; Aβ+PG, positive control.

The composition of the present invention is characterized by containing Campbell Early grape leaf extract as an active ingredient.

Campbell early ( Vitis labrusca B., Campbell early) is a grape variety mainly cultivated in Korea and is a cross between European ( Vitis vinifera L.) and American ( Vitis labrusca L.) belonging to the Central Asian and Mediterranean groups. In the present invention, the leaves of these Campbell Early grapes are used as an extraction raw material. Grape leaves are mostly discarded and have very low utilization, but since the present invention utilizes these grape leaves, it can provide significant advantages in terms of increasing the utilization of plant resources and protecting the environment.

In the present invention, Campbell's Early grape leaves can be used without limitation whether they are cultivated, commercially available, or collected from the wild. Preferably, they are washed with water to remove foreign substances, etc. before extraction and then dried. However, in the case of steam distillation or supercritical extraction, it can be used immediately after washing without drying.

According to the present invention, the extract of Campbell's Early grape leaves can exhibit acetylcholinesterase inhibitory activity and beta-amyloid secretion inhibitory activity.

The present invention is significant in that it provides the potential of Campbell Early grape leaf extract as a candidate material for drugs and functional foods for improving memory, improving cognitive ability, or preventing, delaying, treating or improving dementia, especially Alzheimer's disease.

In the present invention, the term “extract” refers to the result, such as a liquid component obtained by immersing the extraction raw material in a solvent and extracting it for a certain period of time at room temperature or at a heated state, and a solid content obtained by removing the solvent from the liquid component. It can mean. In addition, in addition to the above results, it can be comprehensively interpreted to include dilutions of the above results, concentrates thereof, crude preparations, purifications, etc. of the above results.

The Campbell Early grape leaf extract of the present invention can be prepared according to an extraction method commonly used in this field using Campbell Early grape leaves as an extraction raw material. For example, extraction can be performed using a polar solvent or a non-polar solvent. 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 chloroform, petroleum ether, ether, butanol, and hexane. , toluene, and the group consisting of these. For the effect aimed at in the present invention, the Campbell Early grape leaf extract of the present invention is preferably extracted using water, ethanol, or a mixed solution thereof as a solvent, and more preferably, a mixed solution of water and ethanol is used as a solvent. and extract it. The mixed solution of water and ethanol is preferably 10 to 90% (v/v) ethanol aqueous solution, more preferably 30 to 70% (v/v) ethanol aqueous solution, more preferably 40 to 60% ( v/v) ethanol aqueous solution, more preferably 45 to 55% (v/v) ethanol aqueous solution. Also, more preferably, extraction is performed using a solution containing acetic acid as a solvent. Preferably, a solvent to which acetic acid is added at 0.1 to 10% (v/v) is used, more preferably a solvent to which acetic acid is added at 1 to 5% (v/v) is used, and more preferably 2 A solvent to which acetic acid is added at 4% (v/v) is used.

During extraction, for example, a solvent may be used in an amount of 1 to 20 times the weight of the dry matter of the Campbell Early grape leaf extract, and preferably 5 to 15 times the weight of the dried matter of the Campbell Early grape leaf extract. Preferably 7 to 13 times the weight of the solvent is used, more preferably 8 to 12 times the weight, and even more preferably 9 to 11 times the weight of the solvent.

The extraction temperature can be set to, for example, 0 to 120°C, and is preferably set to 50 to 100°C, more preferably 60 to 90°C, more preferably 70 to 90°C, and more preferably 80 to 90°C.

The extraction time can be set to, for example, 1 to 12 hours, preferably 1 to 10 hours, more preferably 2 to 7 hours, and more preferably 2 to 4 hours.

According to the above extraction conditions, an extract showing the desired effect of the present invention can be efficiently prepared from Campbell Early grape leaf extract.

In addition, the extract can be obtained by repeating the extraction, for example, after the first extraction, the extract is separated from the residue, recovered, a new solvent is added to the residue, and a second extraction is performed to obtain the extract again. When repeating extraction, the number of repetitions can be, for example, 2 to 5 times, preferably 2 to 4 times, more preferably 2 to 3 times, and even more preferably 2 times.

To prepare the extract in the form of dried product, a drying process can be performed after extraction. Preferably, a method of concentrating under reduced pressure and then freeze-drying is used.

When concentrating under reduced pressure, it is preferably carried out under conditions of 50 to 70°C and a vacuum degree of 100 to 200 torr, and when freeze-drying, it is preferably carried out under conditions of -50 to -100°C for 24 to 48 hours.

The composition of the present invention can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical preparation. The pharmaceutical composition of the present invention may contain, for example, 0.1 to 50% by weight of the extract as an active ingredient based on the total weight of the composition, but is not limited thereto.

The pharmaceutical composition of the present invention can be formulated into a conventional pharmaceutical formulation by selecting the active ingredient, Campbell's Early grape leaf extract, one or two or more pharmaceutically acceptable carriers and one or two or more additives. there is. 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 appropriate substances.

The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulated, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch, calcium carbonate, sucrose, or lactose ( It can be prepared by mixing lactose), gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate, talc, etc. may also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Preparations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerogelatin, etc. can be used.

In addition to the active ingredients, the pharmaceutical composition of the present invention may include mineral-containing compounds such as vitamins B, C, E, beta-carotene, Ca, Mg, and Zn, phospholipids such as lecithin, or compounds such as maltol and amino acids as auxiliary ingredients. Among these, it is more preferable to use a mixture of 2 to 3 ingredients such as vitamins C, E, beta-carotene, maltol, etc. because the bioactive effect can be increased or reinforced.

In addition to the above ingredients, known additives include natural flavors such as plum, lemon, pineapple, or herb flavors to enhance the sense of taste, natural fruit juices, natural colorings such as chlorophyllin and flavonoids, and fructose, a sweetener, It can be used by mixing honey, sugar alcohol, sugar, and acidulants 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, and more preferably a human.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered externally to the skin or by intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine, intrathecal, or intracerebrovascular injection. is administered orally.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease, but the daily dosage is Campbell Early Grape Leaf Based on the amount of extract, it is preferably 0.1 to 10 mg/kg, more preferably 3 to 9 mg/kg, and more preferably 5 to 6 mg/kg, and can be administered 1 to 6 times a day.

Dosage units can be, for example, 1, 2, 3 or 4 times the individual dosage, or 1/2, 1/3 or 1/4 times the amount. The individual dosage is preferably the amount of the active ingredient administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dosage.

The pharmaceutical composition of the present invention may further contain one or more active ingredients that exhibit the same or similar functions.

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

The food composition of the present invention may be, for example, various food products such as chewing gum, caramel products, candy, ice cream, and confectionery, beverage products such as soft drinks, mineral water, and alcoholic beverages, and health functional foods containing vitamins and minerals, etc. there is.

The Campbell Early grape leaf extract of the present invention can be added as is or used with other foods or food ingredients, and can be used appropriately according to conventional methods. The mixing amount of the active ingredients can be appropriately determined depending on the purpose of use. In general, when manufacturing food or beverages, the active ingredient 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 materials. However, in case of long-term intake, the amount may be below 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. 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 a sweetener, natural sweeteners such as thaumatin and stevia extract or synthetic sweeteners such as saccharin and aspartame can be used.

The ratio of natural carbohydrates is preferably selected in the range of 0.01 to 0.04 parts by weight, preferably about 0.02 to 0.03 parts by weight, per 100 parts by weight of the food composition of the present invention.

In addition, the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, and alcohol. , may contain carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The ratio 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.

Hereinafter, the present invention will be described in more detail through examples. Since these examples are merely for illustrating the present invention, the scope of the present invention is not to be construed as limited by these examples.

[Example]

Example 1. Preparation of Campbell Early Grape Leaf Extract

The raw material, Campbell's Early grape leaves, were dried, crushed, and used to produce the extract. Ten times the weight of the extraction solvent, i.e., 50% (v/v) ethanol solution with 3% (v/v) acetic acid, was added to the Campbell Early grape leaf dried sample, followed by reflux extraction twice for 3 hours at 85°C. The extract was obtained by filtration through a 1㎛ pore filter. More specifically, after the first extraction, the extract was obtained, an extraction solvent was added to the extraction residue from which the extract was removed, and a second extraction was performed under the same conditions (85°C, 3 hours). The extract obtained through two extractions was concentrated under reduced pressure and dried under vacuum at about 60°C or lower to obtain an extract (yield 13.6%). The final prepared extract was named 'VLL'.

The manufacturing process of the extract is shown in Figure 1.

Example 2. Evaluation of physiological activity of Campbell Early grape leaf extract

(1) Evaluation of beta-amyloid (Aβ) secretion inhibitory activity

APPswe (human APP with the Swedish mutation) cells were used. The APPswe cell line used in the experiment has a more than two-fold increase in Aβ42 secretion compared to normal cells, similar to the pathology of dementia patients, making it easy to search for beta-amyloid secretion inhibitory activity. Sandwich ELISA was performed to measure the amount of beta amyloid secreted from the APPswe cell line.

^Specifically , 1 Treated at a concentration of 10, 25 or 50㎍/㎖. The positive control group was treated with 10 μM of β-si (β-secretase inhibitor IV) (Calbiochem, Darmstadt, Germany). After culturing for 24 hours, the culture medium was recovered in the presence of PMSF (phenylmethanesulfonylfluoride) and used as a sample for sandwich ELISA.

As a result, as shown in Figure 2, VLL was found to inhibit the secretion of beta-amyloid in a concentration-dependent manner, and the IC50 of Aβ40 was calculated to be 46.04 μg/ml and the IC50 of Aβ42 was calculated to be 36.78 μg/ml.

(2) Evaluation of APP and related protein expression activity

Cell fractions or culture fluids were recovered from the APPswe cell line, and Western blot was performed to measure the levels of APP and related metabolic proteins, sAPPα and CTF.

Specifically, the APPswe cell line was cultured in a ⁶ -well plate at 1 , the positive control group was treated with 10 μM of β-si. After culturing for 16 hours, the cells were incubated in cell lysis buffer (150mM NaCl, 50mM Tris-HCl, pH 7.4, 0.5%) containing protease inhibitor (GenDEPOT, Katy, Texas, USA). sodium deoxycholate, 0.5% NP-40, 5mM EDTA) was added, ultrasonic pulverized, and used as a sample. After quantifying the protein using BCA (Bicinchonic acid) protein analysis reagent (Thermo Fisher Inc., San Jose, CA, USA), 50 μg of protein was subjected to 7%, 10% tris-glycine, or 16.5% tris-tricine SDS-PAGE. After separation, protein patterns such as APP, sAPPα, and APP carboxyl-terminal fragment (CTF) were measured through immunoblotting using Azure C-600 (Azure Biosystems, Dublin, CA, USA). Protein bands obtained from three repeated experiments were quantified using Image J software (National Institutes of Health, Bethesda, Maryland, USA) and corrected based on the α-tubulin band.

As a result, as shown in Figure 3, when treated with VLL 1, 10, 25, or 50㎍/㎖, the expression level of APP protein decreased compared to the negative control group (DMSO-treated group), and when treated with 50㎍/㎖ VLL, the expression of APP The amount decreased by about 49% compared to the control group. And the expression level of a-tubulin, a control protein, did not change due to VLL. In the case of sAPPa protein, the secretion amount decreased by 55% compared to the control group when treated with 50 μg/ml of VLL (Figure 3). On the other hand, the expression level of α-CTF and β-CTF proteins did not decrease compared to the negative control group, even though the expression level of APP decreased upon treatment with VLL 50㎍/㎖. Therefore, it is believed that among the activities of VLL, there is a weak inhibitory activity against γ-secretase. However, it is believed that the main mechanism by which VLL inhibits beta-amyloid production is a reduction in APP protein expression, rather than targeting β-secretase or γ-secretase, which acts on the amyloid metabolic pathway of APP.

(3) Evaluation of mRNA expression activity

Based on the results in Figure 3, in order to confirm the effect of VLL on APP mRNA expression in the APPswe cell line, RNA from the APPswe cell line treated with VLL at each concentration (1, 10, 25, or 50㎍/㎖) was isolated and RT- qPCR (Reverse transcription quantitative real-time polymerase chain reaction) was performed.

Specifically, the APPswe cell line was cultured in a 6-well plate at 1x10 ⁶ cells/well, then diluted with serum-free DMEM and treated with the VLL of Example 1 at a concentration of 1, 10, 25, 50, or 100 μg/ml. Cultured for 7 hours. After removing the medium supernatant, total RNA was isolated using the casy-spin™ total RNA extraction kit (iNtRON biotechnology, Seoul, Korea) according to the protocol manual. The absorbance of the isolated RNA was measured and quantified at 260 nm and 280 nm using Nanodrop (ND-1000 spectrophotometer, Thermo Fisher Inc., San Jose, California, USA). Each sample was diluted to 100ng/㎕. RT-qPCR was performed to determine the mRNA expression of APP. The APP primer sequence used in the experiment was TGG CCA ACA TGA TTA GTG ACC for the forward primer and AAG ATG GCA TGA GAG CAT CGT for the reverse primer. The primer sequence for GAPDH was AAC TTT GGC ATT GTG GAA GG for the forward primer and GGA TGC AGG GAT GAT GTT CT for the reverse primer. 1㎕ of primer diluted to 10 pmol, 1㎕ of Enzyme mix, 10㎕ of 2x reaction mix, 2㎕ of diluted RNA, and 7㎕ of sterile water (RNase free) were added to the PCR tube and then the analysis was performed. The reaction consisted of reverse transcription at 50°C for 30 minutes, denaturation at 95°C for 10 minutes, denaturation at 95°C for 5 seconds, annealing and elongation at 60°C for 30 seconds, and the cycle repetition was set to 40, using a Rotor-gene 3000 (Corbett). research, located in Mortlake, Sydney, Australia), and the analysis results were quantified using the Rotor-gene 6 program.

As a result, the level of APP mRNA expression decreased in a VLL concentration-dependent manner compared to the negative control group (Figure 4). Therefore, as seen in Figure 3, it can be concluded that the decrease in the expression level of APP protein depends on the amount of APP mRNA.

(4) Evaluation of acetylcholinesterase inhibitory activity

We sought to determine the effect of VLL on acetylcholinesterase (AChE), the most important enzyme for neural response and function. Acetylcholinesterase Activity Colorimetric assay kit (Dogen bio Co. Ltd, Seoul, Korea) was used. The VLL of Example 1 was diluted with a buffer solution so that the final concentration was 1, 10, 50, or 100㎍/㎖, and then 30㎕ of this dilution, 10㎕ of 0.3% acetylcholine esterase positive control, and 50㎕ of reaction mixture were added. After placing it in 96 wells and reacting at 37°C for 20 minutes, the absorbance was measured at a wavelength of 570 nm, and the degree of enzyme activity inhibition was calculated. At this time, galantamine was used as a positive control at a concentration of 10 μM.

As a result, as shown in Figure 5, VLL was found to inhibit the activity of acetylcholinesterase in a concentration-dependent manner, and the IC50 was calculated to be 53.25 ㎍/ml.

(5) Spatial cognitive ability test (water maze test, morris water maze)

A summary of animal experiments to prove the effect of VLL on improving memory and cognitive ability is shown in Table 1 below.

laboratory animals ICR mice experimental drug Campbell Early Grape Leaf Extract (VLL) Dosage 250㎎/㎏ control drug Red ginseng extract 250㎎/㎏ Causes nerve cell damage β-Amyloid(1-42) 10nM Administration method Administered based on body weight on the day so that the administered dose is included in the amount of administered liquid (200㎕)
The method of administration is to immobilize the animal using the transdorsal skin fixation method and then inject directly into the stomach using a metal strap for oral administration.

Experimental animals: 3-week-old male ICR mice were purchased from Daehan Laboratory Animal Co., Ltd. and housed in accordance with the management standards of the laboratory animal room of Woosuk University College of Pharmacy. After acclimatization for 1 week, only healthy ones were selected and used.

Administration of samples: Mice were divided into groups (n=10) so that the average body weight and variance were uniform, and placebo and VLL of Example 1 were dissolved in a solvent (water) and orally administered. A behavioral experiment was performed after administering VLL (500 mg/kg) of Example 1. The positive control group was administered red ginseng extract (100 mg/kg), and the negative control group was administered distilled water.

Preparation of Aβ infarction model by icv administration of beta-amyloid (Aβ) 42: After anesthetizing mice, they were fixed on a table and the epidermis of the bregma area was incised. 5㎕ of Aβ㎖ in saline was taken with a specially designed Hamilton syringe and injected at a depth of 2.4㎜ with an auto injector (1㎕/sec) and used as a model for behavioral experiments.

Spatial cognitive ability test (morris water maze): Prepare a circular water tank, set the water temperature to about 25℃, add milk powder or vegetable dye to the water to make the water turbid, and place a water maze at about 2cm below the water surface. A small escape support (approximately 11 cm in diameter) was placed. Mice use visual information to find clues in the laboratory and find where the supports are located. Swimming paths were recorded using a video tracking system, and path length, swimming speed, time spent in each quadrant, and several other variables were measured. The faster the experimental animal can find the support position, the shorter its swimming time (usually trained to swim for 2 minutes) will be shortened. And if the supports are removed, the swimming time in the quadrant where the supports were located will increase. Lastly, sensorimotor abilities can be evaluated by conducting an experiment using supports marked with flags.

As a result, the VLL treatment group was confirmed to have a longer distance in target zone compared to the Aβ+ group, and the number of times it passed the target also tended to increase compared to the Aβ+ group. . Summarizing the results, VLL showed an effect on improving memory and cognitive ability similar to that of red ginseng used as a positive control (Figure 6).

Claims (4)

A pharmaceutical composition for improving memory, improving cognitive ability, or preventing, delaying, or treating dementia, containing Campbell's Early grape leaf extract as an active ingredient. According to paragraph 1,
A pharmaceutical composition, wherein the extract is extracted using a 10 to 90% (v/v) ethanol aqueous solution to which 1 to 5% (v/v) acetic acid is added as a solvent. A functional food composition for improving memory, improving cognitive ability, or preventing, delaying or improving dementia, containing Campbell's Early grape leaf extract as an active ingredient. According to paragraph 3,
The extract is a functional food composition that is extracted using a 10 to 90% (v/v) ethanol aqueous solution to which 1 to 5% (v/v) acetic acid is added as a solvent.