KR20070000112A - Grape seed composition as a β-secretase inhibitor - Google Patents

Abstract

A composition for inhibiting activity of beta-secretase(beta-site amyloid precursor protein cleaving enzyme, BACE) by using grape seeds is provided to prevent dementia without side effects by inhibiting activity of beta-secretase. The composition for preventing dementia comprises the aqueous extract or alcohol extract of grape seeds as an effective ingredient, wherein the aqueous extract or alcohol extract of grape seeds specifically inhibits activity of beta-secretase relating to beta-amyloid peptide production; the aqueous extract or alcohol extract of grape seeds contains proanthocyanidin as an oligomer and polymer of polyhydroxy flavan-3-ol units; and the composition is formulated as fermentation oil, beverage or health food.

Description

Grape seed composition as a β-secretase inhibitor}

The present invention relates to an Alzheimer's dementia prevention composition, fermented milk, health foods and beverages comprising an aqueous extract of grape seed or an alcohol extract of grape seed as an active ingredient, and more specifically, beta amyloid peptide which is a causative agent of dementia. Characterized in that it comprises an aqueous extract of grape seed or alcohol extract of grape seed as an active ingredient that specifically inhibits β-secretase activity, one of the enzymes involved in the production of β-amyloid peptide. It relates to an Alzheimer's dementia prevention composition, fermented milk, health food and beverage.

The rapid growth of the aged population in modern society has led to the development of age-related dementia (senile dementia) as a serious social problem. Despite the high incidence of this disease, there is currently no clear treatment or preventive agent, causing huge socioeconomic losses. Alzheimer's disease (AD) is the most common form of dementia and is now the third leading cause of death in the United States after cardiovascular disease and cancer. Alzheimer's disease is a progressive neurodegenerative disorder characterized by memory loss and cognitive impairment. Alzheimer's disease is a pathological feature of plaque accumulation, nerve fiber entanglement, amyloid deposition, synaptic damage and neuronal death in nerve tissues and blood vessels. The cost of Alzheimer's disease is huge (more than $ 100 billion annually in the United States), resulting in patient pain, patient family pain, and loss of patient and caregiver productivity. The incidence of Alzheimer's disease will increase significantly as the life span of human society increases, and if no method of preventing and treating Alzheimer's disease is developed, it is estimated that by 2020, 10 million people will suffer from Alzheimer's disease in the United States alone. do. Currently, 10% of the population over 65 and 50% of the population over 85 are believed to suffer from Alzheimer's disease. Nevertheless, there is currently no treatment available to effectively prevent Alzheimer's disease or to reverse its clinical symptoms and existing pathophysiology. The treatment of senile dementia currently marketed under the approval of the US Food and Drug Administration (FDA) includes tacrine (Cognex) and donepezil (donepezil, Aricept), but these are all drugs of acetylcholine, a neurotransmitter. Increasingly active substances only slightly slow the progression of dementia in about 20-40% of patients. To date, one of the biggest reasons for the lack of treatment for Alzheimer's disease has been that the cause of the disease is unclear. However, according to recent clinical, genetic, molecular and cytobiological studies, the accumulation of beta amyloid in the brain and its neurotoxicity have been found to be a very important cause of the onset. Therefore, it is considered that development of a substance having low side effects or having inhibitory effects on the production of beta amyloid should be suitable for the development of a senile dementia treatment. There are many other theories related to the etiology and onset of Alzheimer's disease. These theories were based on similarities with other diseases and symptoms (eg, latent virus and aluminum theory), or based on pathological observations (eg, cholinergic, amyloid, or entanglement theory). Genetic analysis can potentially differentiate competitive theories. The identification of mutations in the beta amyloid precursor protein (β-APP) in individuals prone to early onset forms of Alzheimer's disease and related diseases strongly supports amyloid production theory. Beta-amyloid (Aβ) peptides, a major component of the senile plaques that are common in people with Alzheimer's dementia, are derived from the amyloid precursor protein (APP) 695, 751, 770. The amyloid precursor protein is in most cases cleaved by proteases, called α-secretase and γ-secretase, and is a soluble protein called sAPPα out of P3 peptides and cells. Will emit. Specifically, in Alzheimer's dementia patients, alpha secretase and gamma secretase are activated to release beta amyloid peptides and proteins called secreted form of β-secretase derived APP (sAPPβ). Among Alzheimer's disease patients, mutations in the beta amyloid precursor protein (APP) gene are mostly due to mutations in the cognitive part of α, β and γ-secretases. These mutations have been shown to alter the structure of proteins, resulting in better exposure to β and γ-secretase, resulting in higher levels of beta amyloid peptides than normal individuals. The beta amyloid peptide has insoluble properties consisting of 42 amino acid residues, and is known to be deposited in brain regions related to memory and recognition, leading to apoptosis of brain cells. Recently, mutations in the beta amyloid precursor protein (APP) gene are cleaved by acting as a substrate for the apoptotic protease called caspase-6 and caspase-3. The cleaved beta amyloid precursor protein (APP) is known to be easily formed beta amyloid by β-, γ-secretase (β-, γ-secretase). Beta-amyloid precursor protein (APP) overexpressing transgenic mice that overexpress beta amyloid precursor protein (APP) mutation genes in people with Alzheimer's dementia. It is reported that this deterioration result was shown. Taken together, these reports suggest that mutations in beta amyloid precursor protein (APP) genes in Alzheimer's disease are closely related to the production of beta amyloid peptides and play an important role in the development of Alzheimer's disease. Known. As such, many studies have been conducted to investigate the cause of Alzheimer's disease and to develop a treatment method, but the development of therapeutic materials with a clear effect until now is insufficient.

On the other hand, grape seeds are rich in antioxidants such as catechin (cathechin), epicatechin (epicathechin), tocopherol (tocopherol), procyanin (procyanin). In particular, grape seeds contain oligomers of polyhydroxy flavan-3-ol units and polyphenol compounds called proanthocyanidins, which are polymers, as useful substances. It is. The useful substance of grape seed is known to have an effective effect on free radical scavenging, atherosclerosis suppression, senile dementia, diabetes, colon cancer prevention, and the like.

The catechin of green tea which is well-known about the function of such polyphenols is mentioned. This catechin component lowers cholesterol, inhibits hypertension or arteriosclerosis, inhibits the production of lipid peroxide, prevents aging, decreases the lipid concentration in serum, inhibits the formation of neutral lipids, prevents obesity, It is known to increase resistance. However, compared to green tea, polyphenols of grape seed have a structure of polymer which is polymerized structure of catechin of green tea.

In addition, procyanidin contained in grape seeds is catechin, epicatechin, catechin gallate, epicatechin gallate, gallocatechin gallate or epigallocatechin. It is based on gallate (Epigallocatechin gallate) and is a generic name for oligomers and polymers of a dimer or more, and is a non-hydrolyzable tannin contained in a wide range of plants. Procyanidins are known to have the ability to bind proteins, and Dimer is reported to have anti-inflammatory effects. In addition, procyanidins have been registered patents (US Patent No. 5,494,661, US Patent No. 4,797,421, WO 9,324,106, etc.) with respect to the antioxidant effect and antibacterial effect, in addition to pharmaceutical products (Europe Patent No. 812,592, Japanese Patent No. 6,183,958). Etc.), cosmetics (European Patent No. 694,305, Japanese Patent No. 6,336,420, etc.), Food Additives (Japanese Patent No. 10,004,923, Japanese Patent No. 9,549,333, etc.) and Procyanidin production methods (Japanese Patent No. 63,267,774, ZA 8,205,023, etc.) There is also a related patent. Products extracted from grape seeds are also available in the United States.

The inventors of the present invention have been researching to develop a dementia prevention composition, health food, dairy products and beverages that are excellent in treating dementia but have no side effects and are capable of causing the treatment of dementia. The present invention was completed by confirming that it has a specific inhibitory activity on β-secretase, which plays an important role in production, and thus can be used as an agent for preventing and treating Alzheimer's dementia.

Therefore, an object of the present invention is to specifically inhibit the activity of β-secretase (β-secretase), which plays a most important role in the production of beta amyloid peptides causing Alzheimer's disease. It is to provide an Alzheimer's dementia prevention composition, health food, dairy products and beverages comprising an aqueous extract or an alcohol extract of grape seed as an active ingredient.

In order to achieve the above object, the present invention is characterized by providing a dementia prevention composition, health food, dairy products and beverages comprising an aqueous extract of grape seed or an alcohol extract of grape seed as an active ingredient.

Hereinafter, the present invention will be described in detail.

Looking at the process of efficiently separating the aqueous or alcoholic extract of grape seed, characterized in that it specifically inhibits the activity against β-secretase (β-secretase) from grape seeds as follows.

In the case of the aqueous extract of grape seed, Vinphenol Grapephenon of Polyphenolics (USA) was purchased and used. After crushing the grape seed presented above finely put the crushed grape seed in water and extracted 2-3 times while reflux cooling for 3 hours at 90 ℃ ~ 100 ℃. At this time, the amount of the aqueous solution is suitable about 10 times the volume of the grape seed.

In addition, grape seed alcohol extract, Tianjin Jianfeng Natural Product R & D Co.Ltd (China) purchased and used Grape seed E. After crushing the grape seed presented above finely pulverized grape seed into ethanol and extracted for 2 hours at 35 ℃. At this time, the amount of the aqueous solution is suitable about 10 times the volume of the grape seed.

Then, the aqueous or alcoholic extract of grape seed isolated above was evaporated and concentrated under the conditions of 35 ° C. to 40 ° C., frozen at −10 ° C. for 24 hours, and freeze-dried at −80 ° C. for 24 hours to make the extract powder. . 10 mg of the extract powder is dissolved in 1 ml of water or dimethyl sulfoxide (DMSO) and used as a β-secretase activity inhibitor.

Among the extracts, grape seed alcohol extract has a higher content of proanthocyanin than grape seed aqueous extract and a lot of hydrophobic components are extracted.

On the other hand, the aqueous extract of grape seed is water as an extraction solvent, the content of proanthocyanidin is 65% by weight or more, pH is 2.0 ~ 5.5, Arsenic is 2ppm or less, heavy metal is 10ppm or less and the appearance is Rose -Beige to reddish brown powder (rose-beige to reddish brown powder), specific gravity 0.3 ~ 0.5 is water-soluble.

In addition, the alcohol extract of grape seed is water and ethanol as an extraction solvent, the content of proanthocyanidin is 95% by weight or more, pH is 2.0 ~ 5.5, Arsenic is 2ppm or less, heavy metal is 10ppm or less and the appearance is reddish Reddish brown powder with a density of 0.25-0.5 (g / ml), insoluble in water, less than 5%.

Alzheimer's dementia prevention composition comprising an aqueous or alcoholic extract of grape seed according to the present invention as an active ingredient may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. In addition, the composition for preventing Alzheimer's dementia, comprising the aqueous or alcoholic extract of grape seed of the present invention as an active ingredient, may be formulated into various formulations, and when formulated, fillers, extenders, binders, wetting agents, disintegrating agents, and interfaces commonly used It may be formulated using diluents or excipients such as active agents. Solid preparations for oral administration include tablets, pills, powders, granules and capsules, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, or the like in hot water, methanol or ethanol extract of Alder , Lactose and gelatin are mixed and prepared. Lubricants may also be used in addition to simple excipients. As a liquid preparation for oral administration, suspensions. Solvents, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are commonly used simple diluents. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene gulchol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. Glycerol and gelatin may be used as the base of the suppository.

For humans, typical daily dosages may range from 1 to 30 mg / kg body weight and may be administered once or in divided doses. However, the actual dosage should be determined in light of several relevant factors such as the route of administration, the age, sex and weight of the patient, health condition and severity of the disease.

Since the aqueous or alcoholic extract of grape seed of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

On the other hand, the present invention can be used as a health food for preventing Alzheimer's dementia, dairy products and beverages containing an aqueous or alcoholic extract of grape seeds obtained by the extraction method as an active ingredient.

The fermented milk for preventing dementia may be a concentrate of juice, dietary fiber, glucose, oligosaccharide, calcium, vitamin, grape seed inoculated with lactobacillus casei HY 2782 (Lactobacillus casei HY 2782) in a crude oil whose nonfat milk content is adjusted with skim milk powder. To prepare a fermented milk by dissolving the aqueous or alcoholic extracts of the syrup prepared by mixing, stirring and homogenizing to a certain ratio.

In addition, the beverage for preventing dementia is added with an aqueous or alcoholic extract of grape seed as an active ingredient, in addition to jujube extract, Angelica concentrate, liquid fructose, purified water, mixed with a drink bottle and sterilized and cooled to room temperature To prepare.

In addition, the dementia preventive health foods may contain vitamin B1, B2, B5, B6, E and acetate esters, nicotinic acid amides, oligosaccharides, etc. in addition to the aqueous or alcoholic extract of grape seeds, and other foods. Additives may be added.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Example 1>

Separation and Purification of Aqueous or Alcoholic Extracts of Grape Seeds

Separation and Purification of Aqueous Extract of Grape Seed

Distilled water was added to a dry weight of 100 g of finely pulverized grape seeds in a volume of about 10 times the dry weight of grape seeds, and extracted three times with reflux cooling at 100 ° C. for 3 hours and 35 ° C. for 2 hours. The extracts obtained here were concentrated by evaporation at 40 ° C., and some of the concentrates were lyophilized and powdered.

Separation Tablets of Alcohol Extract of Grape Seed

40% ethanol was added to a dry weight of 100 g of finely ground grape seeds in a volume of about 10 times the dry weight of grape seeds, and extracted three times with reflux cooling at 100 ° C. for 3 hours and 35 ° C. for 2 hours, respectively. The extracts obtained here were concentrated by evaporation at 40 ° C., and some of the concentrates were lyophilized and powdered.

<Example 2>

Determination of β-Secretase Activity from Grape Seed Aqueous or Alcoholic Extracts

β-secretase activity inhibition was measured using a fluorescence resonance energy transfer assay method (FRET assay method), panvera BACE1 FRET Assay Kit was used. The measuring principle is as follows.

The peptide substrate consists of two fluorophores, a fluoroscent donor and a quenching acceptor, and when light excitation occurs by light, the fluorescence of the donor energy is quenched by the acceptor. However, when protease cleavage occurs, the two chromophore groups are separated to generate fluorescence energy. Eventually, enzymatic cleavage by the protease can be known by measuring the fluorescence energy generated at this time.

Measuring method using grape seed aqueous or alcohol extract is as follows.

Determination of Activity of β-Secretase from Aqueous Extracts of Grape Seeds

110 μl of the assay buffer (50 mM sodium acetate, pH4.5) was added to 10 μl of the aqueous extract β-secretase activity inhibitor of the grape seed, followed by mixing at room temperature for 10 minutes. 20 μl of this was added and 20 μl of a peptide substrate (Rh-EVNLDAEFK-Quencher, in 50 mM ammonium bicarbonate) was added and mixed at room temperature for 5 minutes. Fluorescence of the mixed samples was measured using an ELISA reader (ex: 530 nm / em: 590 nm, 12 nm bw, sensitivity: 75) to obtain a blank measurement value of the aqueous extract sample of grape seed.

In addition, 20 μl of β-secretase enzyme (50mM Tris (pH 7.5), 10% glycerol) was added to the mixed sample of the grape seed aqueous extract and assay buffer and incubated at room temperature for 60 minutes. After completion of the culture, add 20 µl of stop solution (2.5M sodium acetate) to terminate the reaction, and measure the fluorescence of the culture sample using an ELISA reader (ex: 530nm / em: 590nm, 12nmbw, sensitivity: 75). It was set as the measured value of the extract sample.

As a control, the aqueous extract of grape seed β-secretase activity inhibitor was measured using the same method as above using dimethyl sulfoxide without mixing.

Determination of Activity of β-Secretase from Alcohol Extracts of Grape Seed

The alcohol extract of the grape seed β-secretase (β-secretase) activity inhibitory substance was added to 10μl assay buffer (50mM sodium acetate, pH4.5) 110μL was mixed at room temperature for 10 minutes. 20 μl of this was added and 20 μl of a peptide substrate (Rh-EVNLDAEFK-Quencher, in 50 mM ammonium bicarbonate) was added and mixed at room temperature for 5 minutes. Fluorescence of the mixed samples was measured using an ELISA reader (ex: 530nm / em: 590nm, 12nm bw, sensitivity: 75) to obtain a blank measurement value of the alcohol extract sample of the grape seed.

In addition, 20 μl of β-secretase enzyme (50mM Tris (pH 7.5), 10% glycerol) was added to the mixed sample of the alcohol extract of the grape seed and the assay buffer and incubated at room temperature for 60 minutes. After the incubation, 20 µl of stop solution (2.5M sodium acetate) was added to terminate the reaction, and the fluorescence of the culture sample was measured using an ELISA reader (ex: 530nm / em: 590nm, 12nmbw, sensitivity: 75). It was set as the measured value of the extract sample.

As a control, the aqueous extract of grape seed β-secretase activity inhibitor was measured using the same method as above using dimethyl sulfoxide without mixing.

Inhibition of activity (%) was calculated by the following equation.

Deactivation (%) = {1- (sample measurement-Blank measurement) / (control measurement-Blank measurement)} × 100

Experimental results of inhibiting β-secretase activity of aqueous or alcoholic extracts of grape seeds are shown in Table 1 and FIG. 1.

As shown in Fig. 1, grape seed aqueous or alcohol extracts specifically inhibit the activity of β-secretase, which plays an important role in the production of beta amyloid peptides that cause Alzheimer's disease. It was found to inhibit.

Table 1. Results of β-secretase activity inhibition of grape seed extract

Sample Name Inhibition Rate (%) Vinox Grapephenon (Aqueous Extract) 81.7 ± 3.9 Grape seed E (alcoholic extract) 79.9 ± 5.4

<Example 3>

Inhibition of β-secretase activity in the positive control group

A positive control test was performed to verify the β-secretase activity inhibition assay (FRET assay method).

STA-200 (Sequence: Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn- (Statine) -Val-Ala-Glu-Phe-OH) from Enzyme systems products was used as a positive control.

1 mg of STA-200 (Sequence: Lys-Thr-Glu-Glu-Ile-Ser-Glu-Val-Asn- (Statine) -Val-Ala-Glu-Phe-OH) 1 ml of dimethylsulfoxide (DMSO) Dissolve in to prepare 0.61mM stock solution. The prepared stock solution was diluted with assay buffer (50 mM sodium acetate, pH4.5) to prepare concentrations of 0.61, 0.061, and 0.0061 μM, respectively, to make positive controls 1, 2, and 3. 20 μl of each was added, and 20 μl of the peptide substrate (Rh-EVNLDAEFK-Quencher, in 50 mM ammonium bicarbonate) was added and mixed at room temperature for 5 minutes. Fluorescence of the mixed samples was measured using an ELISA reader (ex: 530 nm / em: 590 nm, 12 nm bw, sensitivity: 75) to give blank control values of positive controls 1, 2, and 3.

In addition, 20 μl of β-secretase enzyme (50 mM Tris (pH 7.5), 10% glycerol) was added to each of the mixed samples of the positive controls 1,2,3 and incubated at room temperature for 60 minutes. After the incubation, 20 μl of stop solution (2.5M sodium acetate) was added to terminate the reaction, and the fluorescence of the culture sample was measured using an ELISA reader (ex: 530nm / em: 590nm, 12nmbw, sensitivity: 75). It was set as the sample measured value of 2,3. Negative control was measured using the same method as the gastric positive control using dimethylsulfoxide (DMSO).

Activity inhibition (%) was calculated by the following equation.

Deactivation (%) = {1- (sample measurement-Blank measurement) / (control measurement-Blank measurement)} × 100

Experimental results of inhibiting β-secretase activity of the positive control group are shown in Table 2 and FIG. 2.

As shown in FIG. 2, it can be seen that the grape seed aqueous or alcohol extract specifically inhibits the activity of β-secretase, which plays an important role in the production of beta amyloid peptides causing Alzheimer's disease. there was.

Table 2. Results of β-secretase activity inhibition of positive control group

Positive control group STA200 concentration (μM) % Inhibition One 0.61 91 2 0.061 37 3 0.0061 7

<Example 4>

Determination of β-secretase Activity by Grape Seed Aqueous or Alcohol Extract Concentration

To 10 μl of the grape seed aqueous extract β-secretase activity inhibitor prepared in Example 1, 110 μl of assay buffer (50 mM sodium acetate, pH4.5) was added and mixed for 10 minutes at room temperature. Sample 1 (0.83 mg / ml) It was set as. 10 μl of sample 1 was taken and mixed with 90 μl of assay buffer (50 mM sodium acetate, pH4.5) to obtain sample 2 (0.083 mg / ml). 10 μl of sample 2 was taken and mixed with 90 μl of assay buffer (50 mM sodium acetate, pH4.5) to obtain sample 3 (0.0083 mg / ml). 20 μl of each of Samples 1, 2, and 3 was added, and 20 μl of peptide substrate (Rh-EVNLDAEFK-Quencher, in 50 mM ammonium bicarbonate) was added and mixed at room temperature for 5 minutes. Fluorescence of each sample was measured using an ELISA reader (ex: 530 nm / em: 590 nm, 12 nm bw, sensitivity: 75) to obtain blank measurements of samples 1, 2, and 3.

In addition, 20 μl of β-secretase enzyme {50mM Tris (pH 7.5), 10% glycerol} was added to each of the mixed samples of Samples 1, 2, and 3 and incubated at room temperature for 60 minutes. After incubation, 20 µl of stop solution (2.5M sodium acetate) was added to terminate the reaction, and the fluorescence of the cultured samples was measured using an ELISA reader (ex: 530nm / em: 590nm, 12nmbw, sensitivity: 75). It was set as the sample measured value of 2,3.

In addition, the grape seed alcohol extract β-secretase activity inhibitors prepared in Example 1 was treated as in the samples 1, 2, 3 and measured to make samples 4, 5, 6 and measured the inhibition rate thereof.

The control group was measured using the same method as above using dimethyl sulfoxide.

Inhibition of activity (%) was calculated by the following equation.

Inhibition (%) = {1- (sample measurement-blank measurement) / (Control measurement-blank measurement)} × 100

Β-secretase activity inhibition test results by the concentration of the aqueous or alcoholic extract of grape seed are shown in Table 3 and FIG.

As shown in FIG. 3, the aqueous or alcoholic extract of grape seeds specifically inhibits the activity against β-secretase, which plays the most important role in the production of beta amyloid peptides causing Alzheimer's disease. Could.

Table 3. Results of β-secretase activity inhibition by concentration

Sample Name % Inhibition Sample 1 91 Sample 2 50 Sample 3 5 Sample 4 92 Sample 5 55 Sample 6 One

Example 5

Acute Toxicity Test

Examples 2 and 4 of the present invention were divided into three groups of 25 ± 5g of ICR mice (Korean experimental animals) and 235 ± 10g of Sprague Dawley (Bio genomics) mice each of which were three. The hydrothermal, methanol or ethanol extracts of the algae were administered intraperitoneally at doses of 20 mg / kg, 10 mg / kg and 1 mg / kg, respectively, and observed for 24 hours. As a result, no deaths were observed in all 5 groups, and no symptoms were found in weight gain and feed intake. Therefore, the alder extract of the present invention was confirmed to be a safe drug.

<Example 6>

Preparation of pharmaceutical composition using extract of Alder

The preparation example of the pharmaceutical composition comprising the alder extract of the present invention as an active ingredient, but the present invention is not intended to limit it, it is intended to explain in detail only.

Preparation of Capsules

One of hot water, alder, methanol, and 40% ethanol of Alderwood was completely mixed with 100 mg, corn starch 100 mg, lactose 100 mg, and magnesium stearate 2 mg, and then filled into hard gelatin capsules according to a conventional method for preparing capsules. .

Manufacture of powder

One of hot water of alder, methanol and 40% ethanol was completely mixed with 2 g of lactose and 1 g of lactose, followed by filling into an airtight cloth to prepare a powder.

Example 7

Preparation of Fermented Milk Using Grape Seed Aqueous or Alcohol Extract

Raw milk which adjusted the nonfat milk solid content to 8-20 weight% with skim milk powder was sterilized for 15 second at 72-75 degreeC. The sterilized crude oil was cooled to 37 ° C., and then inoculated with Lactobacillus casei HY 2782 at a concentration of 10 ⁶ cfu / ml. Syrup contains 0.1-50% by weight of juice concentrate, 0.1-20% by weight of dietary fiber, 0.5-30% by weight of glucose, 0.1-15% by weight of oligosaccharide, 0.001-10% by weight of calcium, 0.0001-5% by weight of vitamin and aqueous or alcoholic seeds. 0.1 to 5% by weight of the extract was dissolved, prepared, sterilized and cooled. The syrup and the culture solution thus prepared were mixed and stirred at a volume ratio of 1: 3, homogenized, and then packaged in a container to prepare fermented milk. The produced fermented milk showed good results in flavor, physical properties and overall taste.

<Example 8>

Beverage preparation using grape seed aqueous or alcohol extract

14% by weight of aqueous or alcoholic extract of grape seed, 1.5% by weight of jujube extract and 7% by weight of Angelica Concentrate were mixed with 7.4% by weight of liquid fructose, and purified water was added and mixed into the drink bottle and sterilized at 90-95 ° C After cooling to room temperature to prepare a beverage.

Example 9

Preparation of Dietary Supplement Using Grape Seed Aqueous or Alcohol Extract

0.01 to 30% by weight of grape seed aqueous or alcohol extracts (vitamin B1, B2, B5, B6, E and acetic acid ester, nicotinic acid amide), oligosaccharides, 50% ethanol to 100% by weight of the aqueous grape seed or alcohol extract 5 to 10% by weight of the mixture was added and mixed in a high speed rotary mixer. 10 wt% of sterile purified water was added to the mixture, mixed, and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum dryer at 40 to 50 ° C., and then passed through 12 to 14 meshes to uniformly prepare granules. The granules prepared as described above may be extruded by appropriate amounts into tablets or powders or filled into hard capsules to produce hard capsule products.

As described above, the grape seed aqueous or alcohol extract of the present invention is specific for the activity of β-secretase (β-secretase), which plays the most important role in the production of beta amyloid peptides causing Alzheimer's disease. It can be used as dairy products, health foods and beverages to prevent Alzheimer's dementia.

1 is a graph showing the results of β-secretase activity inhibition of grape seed aqueous or alcohol extract.

Figure 2 is a graph showing the results of β-secretase activity inhibition of the positive control group.

3 is a graph showing the results of β-secretase activity inhibition experiments by the concentration of aqueous grape seed or alcohol extract.

Claims (5)

Alzheimer's dementia prevention composition comprising an aqueous extract of grape seeds or an alcohol extract of grape seeds as an active ingredient. The method of claim 1, The extract is Alzheimer's dementia prevention composition, characterized in that specifically inhibit the β-secretase activity involved in the production of beta amyloid peptide. A fermented milk for preventing Alzheimer's dementia, comprising an aqueous extract of grape seed or an alcohol extract of grape seed as an active ingredient. A beverage for preventing Alzheimer's dementia, comprising an aqueous extract of grape seeds or an alcohol extract of grape seeds as an active ingredient. Alzheimer's dementia preventive health supplement comprising an aqueous extract of grape seed or an alcohol extract of grape seed as an active ingredient.