KR20160101732A - Composition for preventing, improving or treating brain diseases comprising extract of vitis labruscana or quercetin-3-o-glucuronide isolated from the extract - Google Patents
Composition for preventing, improving or treating brain diseases comprising extract of vitis labruscana or quercetin-3-o-glucuronide isolated from the extract Download PDFInfo
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- KR20160101732A KR20160101732A KR1020150023787A KR20150023787A KR20160101732A KR 20160101732 A KR20160101732 A KR 20160101732A KR 1020150023787 A KR1020150023787 A KR 1020150023787A KR 20150023787 A KR20150023787 A KR 20150023787A KR 20160101732 A KR20160101732 A KR 20160101732A
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- quercetin
- composition
- leaf extract
- glucuronide
- preventing
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
- A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/87—Vitaceae or Ampelidaceae (Vine or Grape family), e.g. wine grapes, muscadine or peppervine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/322—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the nervous system or on mental function
Abstract
Description
The present invention relates to a composition for preventing, ameliorating or treating a cerebrospinal fluid containing grape leaf extract or quercetin-3-O-glucuronide isolated therefrom as an active ingredient.
More specifically, the grape leaves a 30 to 50% ethanol and 1-3% quercetin obtained by the grape leaf extract or grape leaf extract obtained by extraction with acetic acid with n-hexane, CH 2 CL 2 , EtOAc BuOH pure fractions 3 O-glucuronide as an active ingredient. The present invention also relates to a composition for preventing, ameliorating or treating a brain disease.
The composition according to the present invention contains 10 to 80 μM of quercetin-3-O-glucuronide on the basis of mouse BV2 microglia (5 × 10 5 cells / well) and contains iNOS, COX-2 , Decrease the production of NO, PGE 2 , and decrease the expression of the inflammatory cytokines IL-1 [beta] and TNF- [alpha].
The composition according to the present invention also has the effect of improving short-term working memory capacity and space working memory ability.
Brain diseases include cerebral hemorrhage caused by cerebral blood vessel breakdown, cerebral infarction due to blockage of blood vessels, loss of cerebral tissue, brain tumor with tumor, and degenerative brain disease with decreased brain function or brain function.
Brain inflammation is a common occurrence in degenerative brain diseases such as Alzheimer's disease and Parkinson's disease, as peripheral neuronal cells die due to an increase in toxic substances caused by nerve inflammation.
Neurons in the brain and spinal cord function in a variety of ways, and microglial cells in the central nervous system (CNS) play an important role in inflammation, immunity and degeneration of the brain, It is possible to prevent degenerative brain diseases by controlling the inflammatory reaction that occurs in these cells.
When microglial cells are activated, they remove foreign substances caused by drugs or toxins, secrete nerve growth factor, and play an important role in protecting neurons and maintaining CNS homeostasis. However, it has been shown that nitric oxide (NO), which is toxic to the nervous system when activated continuously due to brain damage or is overactivated by stimuli such as lipopolysaccharide (LPS), interferon-gamma (IFN-γ) and tumor necrosis factor- (NO), proinflammatory mediators such as prostaglandin E2 (PGE 2 ), cytokines, and reactive oxygen species (ROS), which cause neurotoxicity and destroy brain tissue Thereby causing degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, and Cropert's Jakob disease.
In the case of NO and PGE 2 secreted by the inflammatory reaction, a small amount of secretion promotes blood circulation and enhances immunity. However, when it is secreted, it promotes inflammatory reaction and causes various inflammatory diseases.
Among these, NO and PGE 2 Are produced by inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressed by inflammatory markers TNF-α, IL-1β and LPS. .
As the society ages, the number of degenerative brain diseases is increasing, and the cause of diseases and the development of drugs for them are rapidly developing.
Accordingly, the present applicant has accomplished the present invention by devising a composition for preventing, ameliorating or treating a cerebrospinal fluid containing grape leaf extract or quercetin-3-O-glucuronide isolated therefrom.
The composition according to the present invention is effective for degenerative brain diseases such as Alzheimer's disease, Parkinson's disease and Croplet's Jakob disease caused by brain inflammation, memory disorder and cognitive ability disorder.
On the other hand, the grape is a woody vine plant belonging to the Rhamnales vitaceae, and there are 11 genera and 700 kinds of grapes in the grape. Grapes are mostly European species (Vitis belongs to Asia, the Mediterranean group vinifera L.), American species ( Vitis labrusca L.) and their mutual hybrids ( Vitis labruscana B.) can be divided into three types.
Grape leaves are not used for food because they are tasted and tasted. Recently, interest in well-being has increased and there have been attempts to be used as food, and antioxidant and antidiabetic effects have also been announced.
In the Middle East, Eastern Europe and Western Europe, grape leaves are prepared for pickling or canning, and in Greece, traditional foods using grape leaves have been developed and consumed. In Korea, however, research on physiologically active ingredients in grape leaves is insufficient and people are not aware of them. Therefore, most of them are dismissed through net harvesting to increase yield and sugar content in grape cultivation.
Grape contains a large amount of sugar and minerals. It contains a polyphenol component such as anthocyanin type, tannin type, quercetin type including resveratrol which is well known as a typical ingredient. ≪ / RTI >
The various polyphenols in the grapes are known to be effective for anti-cancer, antioxidant, and anti-aging. Resveratrol has been reported to have antioxidant, anti-inflammatory, vascular and anti-cancer effects. Quercetin-3- O -glucuronide (QG), which has been reported to be isolated from red wine, has been shown to be effective in improving Alzheimer's effect on the brain (Non-Patent Document 1), anti- inflammatory effects in RAW264.7 cells and human macrophages Literature 2), and is effective in improving vascular function through eNOS activity through the pathway of AMPK (Non-Patent Document 3). However, no studies have been reported on the improvement of brain inflammation in QG.
Patent Documents relating to the present invention disclose a food composition and a pharmaceutical composition for preventing or ameliorating thrombotic diseases containing grape leaf extract as an effective ingredient in Patent Document 10-2014-0142765, -1049041 discloses that an extract of quercetin-3-O-glucuronide-containing methanol extract has anti-inflammatory and immunosuppressive properties.
However, as in the present invention, a composition for preventing, ameliorating or treating a cerebrospinal fluid containing grape leaf extract or quercetin-3-O-glucuronide isolated therefrom has not been disclosed.
It is an object of the present invention to provide a composition for preventing, ameliorating or treating brain diseases containing grape leaf extract or quercetin-3-O-glucuronide isolated therefrom as an active ingredient.
The problem to be solved in the present invention are obtained by fractionation the grape leaf extract or grape leaf extract obtained by extracting grape leaves from 30 to 50% ethanol, and acetic acid 1-3% in n-hexane, CH 2 CL 2 , EtOAc BuOH order Which comprises quercetin-3-O-glucuronide as an active ingredient. The present invention also provides a composition for preventing, ameliorating or treating a brain disease.
A problem to be solved by the present invention is to provide a kit for the treatment of ovarian cancer, which contains 10 to 80 μM of quercetin-3-O-glucuronide on the basis of mouse BV2 microglia (5 × 10 5 cells / well) 2 or a composition for the prevention, amelioration or treatment of brain diseases having an effect of reducing the production of NO, PGE 2 , and reducing the expression of inflammatory cytokines IL-1 [beta] and TNF- [alpha] To provide.
The object of the present invention is to provide a composition for preventing, ameliorating or treating a brain disease having an effect of improving short-term working memory ability and space working memory ability.
The present invention aims at solving the technical problem by providing a composition for preventing, ameliorating or treating brain diseases containing grape leaf extract or quercetin-3-O-glucuronide isolated therefrom as an active ingredient.
The present invention relates to quercetin-3-O-glucuronic acid obtained by fractionating grape leaf extract or grape leaf extract obtained from 50% ethanol and 3% acetic acid in the order of n-hexane, CH 2 CL 2 and EtOAc BuOH The present invention aims to solve the technical problem by providing a composition for prevention, improvement or treatment of brain diseases, which comprises the active ingredient as an active ingredient.
The present invention relates to a method for inhibiting the expression of iNOS and COX-2 without cytotoxicity, comprising 10 to 80 μM of quercetin-3-O-glucuronide on the basis of mouse BV2 microglia (5 × 10 5 cells / well) decrease, reduce the production of NO, PGE 2, by providing a composition for preventing, improving or treating a brain disease having an effect of reducing inflammatory cytokine expression of IL-1β and TNF-α the technical problem .
The present invention aims at solving the technical problem by providing a composition for prevention, improvement or treatment of brain diseases having an effect of improving short-term working memory ability and spatial working memory ability.
The composition for preventing, ameliorating or treating brain diseases according to the present invention has the effect of reducing the expression of iNOS and COX-2 without cytotoxicity.
The composition for preventing, ameliorating or treating brain diseases according to the present invention has an effect of reducing the production of NO, PGE 2 .
The composition for the prevention, amelioration or treatment of brain diseases according to the present invention has the effect of reducing the expression of inflammatory cytokines IL-1 [beta] and TNF- [alpha].
The composition for preventing, ameliorating or treating brain diseases according to the present invention is useful for preventing, ameliorating or treating degenerative brain diseases such as Alzheimer's disease, Parkinson's disease and Cropert's Jakob disease caused by brain inflammation, memory disorder and cognitive ability disorder .
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the molecular formula of quercetin-3-O-glucuronide (QG) according to the present invention.
FIG. 2 shows the results of measuring the survival rate of BV2 cells in order to confirm the cytotoxicity of quercetin-3-O-glucuronide (QG) according to the present invention.
FIG. 3 shows the results of measuring the expression levels of iNOS and COX-2 protein in BV2 cells induced by LPS in order to confirm the anti-inflammatory effect of quercetin-3-O-glucuronide (QG) .
FIG. 4 shows the results of measurement of NO and PGE 2 expression in BV2 cells induced by LPS in order to confirm the anti-inflammatory effect of quercetin-3-O-glucuronide (QG) according to the present invention.
FIG. 5 shows the results of measuring the expression levels of TNF-α and IL-1β in BV2 cells induced by LPS in order to confirm the anti-inflammatory effect of quercetin-3-O-glucuronide (QG) to be.
FIG. 6 is a result of performing a manual avoidance experiment to confirm the short-term memory improving effect of the grape leaf extract according to the present invention.
FIG. 7 shows the results of the Y-maze experiment to confirm the effect of improving the spatial working memory ability of the grape leaf extract according to the present invention.
The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may properly define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.
Therefore, the embodiments and the examples described herein are merely the most preferred embodiments of the present invention, and are not intended to represent all of the technical ideas of the present invention. Therefore, various equivalents and variations Examples should be understood.
Example
1. Preparation of grape leaf extract
The grape leaf extract is prepared by grooming and extracting the grape leaves.
Grape leaves may be collected and then dried, frozen or the like, preferably dried ones.
The extraction method for preparing the grape leaf extract may be a solvent extraction method, a reflux extraction method, a percolation extraction method, a steam distillation method, a supercritical fluid extraction method, a compression extraction method, etc. Preferably, a solvent extraction method using ethanol and acetic acid can be used .
Add 10 to 30 times of 30 to 50% ethanol and 1 to 3% of acetic acid to 100 g of selected grape leaves and extract at 85 to 95 ° C for 1 to 5 hours.
The extract can then be filtered. Filtration methods include filter filtration, filtration using a pressurization or decompression apparatus, centrifugal filtration and vacuum filtration. Preferably, the extract can be filtered using a microfilter of 1 mu m size.
Further, the residue after filtration can be extracted twice or more under the same conditions, and it can be extracted by filtration and concentrated.
Concentration can be carried out by methods such as normal pressure concentration, reduced pressure concentration, vacuum concentration, etc., preferably at a reduced pressure of 60 캜 or lower.
Thus, grape leaf extract can be obtained, and the prepared grape leaf extract can be used after being dried and pulverized.
Example
2.
Quercetin
-3-O-
Glucuronide
detach
The grape leaf extract can be fractionated to isolate quercetin-3-O-glucuronide.
The fraction may be separated by a solvent fractionation method, a precipitation method or a dialysis membrane, and a solvent fractionation method may be preferably used.
The solvent used in the solvent fractionation method may also be variously used such as hexane, dichloromethane (CH 2 Cl 2 ), ethylacetate (EtOAc), butanol (BuOH) and the like.
Preferably, the grape leaf extract is suspended in distilled water and fractionated in the order of n- hexane, CH 2 Cl 2 , EtOAc and BuOH.
The BuOH fractions can then be separated into four fractions by sequential fractionation with 80% and 100% MeOH solvent using sephadex-LH20 column chromatography.
The fractions can be concentrated and quercetin-3- O- glucuronide can be obtained by subjecting the third fraction to reverse phase (RP-C 18 ) column chromatography twice with 50% MeOH as the solvent.
Experimental Example
1. Cytotoxicity experiment
1-1. Preparation for experiment
It should be noted that the preparation of the experiment of all the experimental examples is performed in the same manner as the preparation of this experiment.
1) Preparation of grape leaf extract
The grape leaves used in the experiment were cultivated in grape cultivated farms in Baegu - myeon, Gimje - si, Jeonbuk, Korea.
Selected grape leaves (
2) Isolation of quercetin-3-O-glucuronide (QG)
Fourteen grams of water extract of grape leaves was suspended in distilled water and fractionated with n- hexane, CH 2 Cl 2 , EtOAc and BuOH. Each fraction was concentrated under reduced pressure to volatilize the solvent and then weighed.
n- Hexane (1.1 g), CH 2 Cl 2 (0.7 g), EtOAc (0.9 g), BuOH (2.1 g), H 2 O was obtained a (9.3 g), solvent conditions of the BuOH fractions, 2.1 g sephadex-LH20 column chromatography 80 % and 100% MeOH using the Sequential fractions were divided into four fractions and concentrated under reduced pressure. The third fraction, 0.27 g, was quercetin-3- O -glucuronide (41 mg) twice with reverse phase (RP-C 18 ) column chromatography using 50% MeOH as the solvent.
1 is a diagram showing the molecular formula of the Need (quercetin-3- O -glucuronide) as a quercetin -3-O- glucuronic separated from the grape leaf extract according to the invention.
3) BV2 microglial cell culture
BV2 microglia (5 × 10 5 cells / well) were cultured in DMEM medium containing 10% heat-inactivated FBS, penicillin G (100 IU / ml), streptomycin (100 μg / ml) and L- And 5% CO 2 Lt; RTI ID = 0.0 > 37 C. < / RTI >
1-2. Experimental course
In this experiment, MTT assay was used to determine the cytotoxicity of BV2 microglia and the concentration in the experiment and cytotoxicity of QG.
1) MTT assay
First, the cells were equally divided into 96 well plates at 1 × 10 4 cells / well and cultured for 24 hours. After removing the existing medium and adding new medium, samples dissolved in DMSO were diluted in DMEM medium at various concentrations (10, 20, 40, and 80 μM). The treatment concentration of DMSO was adjusted to be 0.1% or less of the medium. After incubation for 24 hours, the medium was removed and the MTT reagent (5 mg / ml) was added. After 4 hours, the supernatant was removed. 20 μl of DMSO was added to each well of the formed formazan, and the resultant was dissolved by using an orbital shaker. After 30 minutes, the absorbance was measured at 595 nm. Experiments were repeated three times to obtain the average value, and the cell viability was compared based on the absorbance value of the control.
2) Statistical processing
Statistical analysis was performed using GraphPad Prism, version 3.03 (GraphPad Software Inc., San Diego, Calif.).
The results between the experimental groups were expressed as mean and standard error. The results between the experimental groups were analyzed using the ANOVA test and only the items with significant differences were tested. The difference between the experimental groups was significant at 95% level (p <0.05).
In all the experiments below, we show that the statistical processing is performed as above.
1-3. Experiment result
FIG. 2 shows the results of measuring the survival rate of BV2 cells in order to confirm the cytotoxicity of quercetin-3-O-glucuronide (QG) according to the present invention.
As a result, no cytotoxicity was observed at a concentration of quercetin-3-O-glucuronide (QG) of 5 to 80 μM. The cell viability was found to decrease at 100 μM, and the concentration of quercetin-3-O-glucuronide (QG) was set within the range of 80 μM which does not cause cytotoxicity in the experimental examples.
Experimental Example
2.
iNOS
And
COX
-2 expression analysis
2-1. Experimental course
Western blot analysis was performed to analyze the expression of iNOS and COX-2.
BV2 cells were cultured in a 60 mm dish at a density of 3 × 10 5 cells / well for 24 hours, and each sample was treated by concentration. BV2 cells were added with RIPA buffer, followed by centrifugation at 14,000 x g for 25 minutes at 4 DEG C, and the supernatant was transferred to a tube.
Protein quantification was performed using a BSA protein kit. Each sample was run on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (NC membrane).
The transferred NC membrane was blocked with fresh blocking buffer (0.1
After incubation for 1 hour with 2: 1 anti-mouse IgG (anti-mouse IgG), ECL solution was added to the NC membrane at 1: 1 and light was emitted. Respectively.
In the same manner, actin was measured using actin antibody.
2-2. Experiment result
FIG. 3 shows the results of measuring the expression levels of iNOS and COX-2 protein in BV2 cells induced by LPS in order to confirm the anti-inflammatory effect of quercetin-3-O-glucuronide (QG) .
As a result, it was confirmed that the expression of COX-2 and iNOS decreased as the concentration of QG increased.
Experimental Example
3.
NO
And
PGE2
Expression analysis
3-1. Experimental course
1) nitrite assay
The cultured cells were inoculated at a density of 5 × 10 5 cells / well in a 96-well plate and incubated for 24 hours. After 24 hours, the medium was removed, and LPS (1 μg / ml), the amount of NO released from the cells in the media after 24 hours was measured using Griess reagent (0.1% (w / v) N- (1-naphathyl) -ethylenediamine and 1% (w / v) sulfanilamide in % (v / v) phosphoric acid).
The reaction was then measured at 540 nm using an ELISA microplate reader (Bio Rad Laboratories Inc., California, USA, Model 550).
2) PGE 2 Measure
PGE 2 was measured using a prostaglandin E2 enzyme immunoassay system (EIA, Amersham Pharmacia Biotech, Inc., Piscataway, NJ) using an antibody (lyophilized prostaglandin E2 conjugate to horseradish peroxidase).
3-2. Experiment result
FIG. 4 shows the results of measurement of NO and PGE 2 expression in BV2 cells induced by LPS in order to confirm the anti-inflammatory effect of quercetin-3-O-glucuronide (QG) according to the present invention.
As a result of the experiment, it was confirmed that the expression amount of NO and PGE 2 decreased as the concentration of QG increased, and it was confirmed that the concentration decreased more than 20 μM.
Experimental Example
4. Inflammatory cytokine (
IL
-1β and
TNF
-α) expression analysis
4-1. Experimental course
TNF-α and IL-1β were measured using an enzyme immunoassay system kit (R & D Systems, Abingdon, UK) using an antibody (lyophilized TNF-α or IL-1β conjugate to horseradish peroxidase).
4-2. Experiment result
FIG. 5 shows the results of measuring the expression levels of TNF-α and IL-1β in BV2 cells induced by LPS in order to confirm the anti-inflammatory effect of quercetin-3-O-glucuronide (QG) to be.
As a result, it was confirmed that the expression level of TNF-α and IL-1β decreased with increasing QG concentration.
The results of Experimental Examples 1 to 4 above show that the composition containing the grape leaf extract according to the present invention or quercetin-3-O-glucuronide isolated therefrom as an active ingredient It can be seen that brain diseases are effective against brain diseases based on brain inflammation.
Experimental Example
5. Passive avoidance experiment
Passive
Avoidance
Test
: Short-term working memory test)
5-1. Preparation for experiment
1) Experimental animals
Six-week-old male ICR mice and SD rats obtained from Orient Co. were used.
After 1 week of adaptation period, group separation was performed by random method according to the weight range. The animals were used in this experiment. No abnormal symptoms were observed during the acquisition and purification period of the animals used in the experiment.
The incubation environment was set at 23 ± 2 ℃, 55 ± 5% relative humidity, 12 hours of illumination and 150 ~ 300 lux of light intensity. The sterilized solid feed for laboratory animals supplied by Orient Bio Co., And tap water were freely ingested.
5-2. Experimental course
A passive avoidance memory test was performed using the Gemini avoidance system (avoidance learning box).
On the first day of training, mice were acclimated for 10 seconds by placing them in a light box and then automatically moved to the dark box with the door open. When moving to the dark box, an electrical stimulation of 0.25 mA was applied for 3 seconds Respectively.
In the test after 24 hours, the mouse was lightly packed in a light box for 10 seconds, and then the door was opened to the dark box. At this time, the time taken to move to the dark box was measured and the electric stimulation was not given on the second day. If the mouse does not move to a dark box for 300 seconds, the maximum score is 300 seconds.
- Latency time in Acquisition trial: change in activity
- Latency time in retention trial on the second day of experiment.
5-3. Experiment result
FIG. 6 is a result of performing a manual avoidance experiment to confirm the short-term memory improving effect of the grape leaf extract according to the present invention.
In the experiment, scopolamine was administered at a dose of 3 mg / kg to induce memory decline. In mice administered with 125 mg / kg of grape leaf extract, 24-hour short-term memory Improvement was confirmed.
Experimental Example
6. Y-Maze Experiment (Y-
maze
Test
: Spatial Working Memory Test)
6-1. Preparation for experiment
5-1. ≪ / RTI >
6-2. Experimental course
A Y-maze consisting of three branches (length 42 cm, width 3 cm, height 12 cm, angle 120 °) was used.
After setting each branch to A, B, C, carefully place the mouse on one of the branches and let it move freely for 8 minutes, and then record the branch containing the mouse (only if it completely enters the tail, record).
One point (actual change) was given to each of the three different branches in turn.
The alternation behavior was calculated by the following equation.
Change Actual Power (%) = actual alternation / maximum alternation X 100 (highest change = total number of entries - 2)
- Measuring spontaneous alternation: Measuring short-term memory recovery
- Total entry count measurement: observation of activity change
6-3. Experiment result
FIG. 7 shows the results of the Y-maze experiment to confirm the effect of improving the spatial working memory ability of the grape leaf extract according to the present invention.
Showed a marked improvement in the spatial working memory ability, which was reduced by scopolamine when administered 125 mg / kg of grape leaf extract.
The above Experimental Examples 5 and 6 show that a composition containing the grape leaf extract according to the present invention or quercetin-3-O-glucuronide isolated therefrom as an active ingredient is effective for brain inflammation It is understood that not only the improvement of the brain diseases caused by the hypertension, but also the improvement of the memory ability and the improvement of the cognitive ability are possessed.
Therefore, the composition according to the present invention can predict the efficacy of the present invention for diseases such as Alzheimer's dementia, vascular dementia, dementia of frontal lobe, Parkinson's disease, and hardness cognitive disorder in which memory impairment or cognitive impairment occurs.
Claims (5)
The composition quercetin -3-O obtained from the fraction grape leaf extract or grape leaf extract obtained by extracting grape leaves from 30 to 50% ethanol, and acetic acid 1-3% in n-hexane, CH 2 CL 2 , EtOAc BuOH order A composition for preventing, ameliorating or treating a brain disorder, which comprises glucuronide as an active ingredient.
The composition for preventing, improving or treating a brain disease, which comprises containing BV2 mouse microglial cells (5 × 10 5 cells / well ) into, the Need 10 ~ 80 μM as quercetin -3-O- glucuronic reference / RTI >
The composition reduces the expression of iNOS (Inducible Nitric Oxide Synthase), COX-2, reduces the production of NO (Nitric oxide), PGE 2 (Prostaglandin E2), and inhibits the inflammatory cytokine IL- (Interleukin-1 beta) and TNF-alpha (Tumor necrosis factor alpha), and improve short-term working memory ability and spatial working memory ability.
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| WO2019045286A3 (en) * | 2017-08-30 | 2019-04-18 | 한국식품연구원 | Composition comprising miquelianin as effective ingredient for preventing or treating th2-mediated immune disease |
| KR20200135621A (en) | 2019-05-23 | 2020-12-03 | 주식회사 하람 | Obesity prevention and treatment materials with Quercetin-3-O-glucuronide as a valid ingredient |
| US11504349B2 (en) | 2017-10-19 | 2022-11-22 | Ilab | Composition for preventing or treating TNF-related diseases, containing novel derivative as active ingredient, and method for inhibiting TNF activity by using same |
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| KR20140142765A (en) | 2013-06-04 | 2014-12-15 | 한경수 | Food and pharmaceutical composition for preventing or improving thrombotic disease comprising extract of grape leaf as effective component |
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| US11504349B2 (en) | 2017-10-19 | 2022-11-22 | Ilab | Composition for preventing or treating TNF-related diseases, containing novel derivative as active ingredient, and method for inhibiting TNF activity by using same |
| US11850230B2 (en) | 2017-10-19 | 2023-12-26 | Ilab | Composition for preventing or treating TNF-related diseases, containing novel derivative as active ingredient, and method for inhibiting TNF activity by using same |
| KR20200135621A (en) | 2019-05-23 | 2020-12-03 | 주식회사 하람 | Obesity prevention and treatment materials with Quercetin-3-O-glucuronide as a valid ingredient |
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