WO2015020241A1 - Composition containing galla rhois extract as active ingredient for improving cognition and preventing or treating alzheimer's disease - Google Patents

Composition containing galla rhois extract as active ingredient for improving cognition and preventing or treating alzheimer's disease Download PDF

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WO2015020241A1
WO2015020241A1 PCT/KR2013/007093 KR2013007093W WO2015020241A1 WO 2015020241 A1 WO2015020241 A1 WO 2015020241A1 KR 2013007093 W KR2013007093 W KR 2013007093W WO 2015020241 A1 WO2015020241 A1 WO 2015020241A1
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extract
test
mice
amyloid
active ingredient
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PCT/KR2013/007093
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French (fr)
Korean (ko)
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김현기
김진우
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Kim Hyun Kee
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    • 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)
    • A61K36/22—Anacardiaceae (Sumac family), e.g. smoketree, sumac or poison oak
    • 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
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • the present invention relates to a cognitive improvement composition comprising a gall bladder extract as an active ingredient, and more particularly, to a composition for preventing and treating Alzheimer's disease (AD) comprising the gall bladder extract as an active ingredient.
  • AD Alzheimer's disease
  • Cognitive, or memory, deterioration caused by dementia is a "syndrome,” which causes remarkable difficulties in maintaining social and daily life due to temporary or persistent damage to the cranial nerve, mainly due to medical and neurological causes.
  • Representative organic disorders with changes in cognitive function and personality are characterized by short-term and long-term memory impairments, and are highly cortical disorders of high brain cortical function including thinking ability, mental ability, comprehension, computational ability, language and judgment (F, H. and Kurz, A, Eur Arch Psychiatry Clin Neurosci, 249 (6): 288-290 (1999); Jaworski, T. et al., Biochim Biophys Acta, 1802 (10): 808-818 (2010)) .
  • Alzheimer's dementia accounts for the majority of people with dementia and 10% of people over 70 are known to suffer. It can occur at any age, not only in the elderly, but in reality, symptoms rarely occur before the age of 50, and since 60 years of age, the incidence increases gradually as the age increases. Causes significant medical, social and economic problems, and the prevalence is 10% for ages 65-74, 19% for ages 75-84, and 47% for ages 85 and older (Reitz, C., et al., Nat Rev Neurol). , 7 (3): 137-152 (2011).
  • Cerebrovascular dementia caused by Alzheimer's dementia and widespread brain lesions after stroke or by cerebral atherosclerosis accounts for about 90% of dementia diseases, and other Pick and Creutzfeldt-jakob diseases. , Head injury, dementia, and Parkinson's disease.
  • Dementia diseases are fundamentally observed as short-term and long-term memory disorders, and are considered to be a core symptom, and are thought to consist of memory disorders, disorientation resulting from them, and high brain dysfunction.
  • AD Alzheimer's disease
  • the onset mechanism of AD due to the elimination of hippocampal neurons through the accumulation of old spots outside the cells of ⁇ -amyloid peptide is also important. It is assumed to be one of the causes.
  • therapeutic agents that improve dementia symptoms by activating ACh have been developed based on the finding that a marked decrease in neurological function related to ACh is the cause of memory disorders in dementia.
  • Therapeutic agent by activating NMDA receptor, noting the reduction of N-methyl-D-aspartic acid (NMDA) receptor, one of glutamic acid neuron and glutamic acid receptors Is also being developed.
  • NMDA N-methyl-D-aspartic acid
  • benzodiazepine hereinafter abbreviated as 'BZ ⁇ 3'
  • 'BZ ⁇ 3' peripheral benzodiazepine
  • N, N-di-n-hexyl-2- (4-fluorophenyl) indole-3-acetamide N, N-di-n-hexyl-2- (4), an agonist for the BZ ⁇ 3 receptor -fluorophenyl) indol-3-aceteamide (hereinafter abbreviated as 'FGIN-1-27')
  • some types of neurosteroids e.g., pregnenolone sulfate, allopregnanolone
  • Acetylcholinesterase is an enzyme that degrades the neurotransmitter ACh.
  • AChE inhibitors acetylcholinesterase inhibitors
  • ACh neurotransmitter in the neural cleft for an increased amount of time. Enhances action by enhancing chemical and functional effects.
  • AChE inhibitors developed to date include 1,2,3,4-tetrahydro-9-acridineamine (1,2,3,4-tetrahydro-9-acridine amine; tacrine, THA; 'cog NEXO (COGNEX), Done Pezyl (E2020; ARICEPT) and Rivastigmine (ENA713; EXELON).
  • These drugs are known as drugs to prevent and treat Alzheimer's disease by increasing the concentration of ACh, a neurotransmitter, through the inhibition of AChE activity, which plays a central role in the central nervous system.
  • AChE inhibitory drugs are most effective when the cholinergic nerves are not functionally impaired, and the efficacy of these drugs decreases with the decline of cholinergic nerve function, so they are effective only in the early stages of mild or moderate senile dementia. It has cholinergic side effects of peripheral nerves, its half-life is too short, it has serious side effects such as hepatotoxicity and especially 9-amino-1,2,3,4-tetrahydroacridine (THA), an active ingredient of Cognex ) Shows oral improvement in perceptual administration, but it is not widely used due to serious side effects such as tremor, dizziness, and liver toxicity.
  • TAA 9-amino-1,2,3,4-tetrahydroacridine
  • the ginseng component is composed of the main component in the case of hypertension patients may cause another side effect such as palpitations or the homeostasis imbalance may be another problem.
  • Ginkgo biloba is a treatment for organic brain disorders involving dementia such as tinnitus, headache, memory loss, concentration disorders, depression, dizziness, etc.
  • Ginkgo leaf preparations such as Succuran are causing serious problems in questionnaire surveys in commercialization.
  • Other antioxidants include ginkgo biloba, chitosan, omega 3, SK's saponin fraction, and KD-501's dementia treatment using Guangdong Pharmaceutical's Hyunsam.
  • the side effect is the biggest problem, and especially in the case of herbal medicine, the side effect is shown to be significantly less than other drugs, but in some cases another side effect is concerned, The therapeutic effect is not enough yet, so the need for improvement of effective clinical efficacy is urgently required for improvement.
  • the present invention has been made in view of the above-mentioned problems and the need for the above-described problem, and an object of the present invention is to provide a composition for preventing or treating dementia (AD).
  • AD dementia
  • the present invention provides a pharmaceutical composition for preventing and treating Alzheimer's (AD), which contains an extract of Galla Rhois as an active ingredient.
  • the effective amount of the extract is preferably 0.001 to 500mg / kg body weight is not limited thereto.
  • the present invention also provides a pharmaceutical composition for preventing and treating dementia (AD) comprising gallic acid or methyl gallate as an active ingredient.
  • AD dementia
  • the present invention provides a health food composition for preventing and improving dementia (AD) containing a gall (Galla Rhois) extract as an active ingredient.
  • AD dementia
  • Ga Rhois gall
  • the present invention provides a pharmaceutical composition for improving the cognitive ability containing the extract (Galla Rhois) as an active ingredient.
  • the present invention also provides a pharmaceutical composition for improving cognitive ability comprising gallic acid (gallic acid) or methyl gallate as an active ingredient.
  • the present invention provides a health food composition for improving cognitive ability containing a gall (Galla Rhois) extract as an active ingredient.
  • the "germ extract” is obtained by extracting the active ingredient from the gall is a natural product includes all without particular limitation.
  • the resultant obtained by putting a ligne in water or an organic solvent and eluting an effective component through means, such as standing, stirring, pressurization, or heating is mentioned.
  • It also includes a lyophilized product obtained by lyophilizing the liquid extract thus obtained.
  • It also includes a powder obtained by grinding such lyophilisate.
  • any possible means for extracting the active ingredient includes all the extracted extracts, including all the processed after extraction and freeze-drying.
  • Others include extracts obtained by conventional extraction methods such as extraction by baths or room temperature, or by conventional extraction methods described in Chinese medicine or textbooks.
  • the pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.
  • the salt is not particularly limited as long as it is pharmaceutically acceptable.
  • hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid , Benzene sulfonic acid, toluene sulfonic acid, naphthalene sulfonic acid and the like can be used.
  • compositions according to the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, ointments, creams, external preparations, suppositories, and sterile injectable solutions. It may be used in formulated in any form suitable for pharmaceutical formulations.
  • the preferred dosage of the extract or compound of the invention depends on the subject's age, sex, weight, symptoms, extent of disease, drug form, route of administration and duration, but is appropriately selected by those skilled in the art. Can be.
  • the composition of the present invention is preferably administered at 0.001 to 500 mg / kg body weight per day. Administration may be administered once a day or may be divided several times. In addition, the dosage may be increased or decreased depending on age, sex, weight, degree of disease, route of administration, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
  • composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes, such as parenteral, oral, and all modes of administration can be expected, for example oral, rectal or It can be administered by intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
  • composition according to the present invention there is no serious toxicity and side effects can be used with confidence even for long-term use for the purpose of prevention.
  • composition according to the present invention may contain 0.001 to 99.9% by weight of the extract as an active ingredient in the total weight of the composition. This is because the effect can be expected to be 0.001% by weight or more, it is difficult to exceed 99.9% by weight due to the presence of impurities.
  • an extract or compound of the present invention into tablets, capsules, chewing tablets, small bags, granules, powders, liquid solutions, suspensions, dispersions, emulsions, syrups, etc.
  • Arabia Rubber, corn starch, binders such as microcrystalline cellulose or gelatin, excipients such as dicalcium phosphate or lactose, disintegrants such as alginic acid, corn starch or potato starch, lubricants such as magnesium stearate, sucrose or saccharin Sweetening agents and flavoring agents such as peppermint, methyl salicylate or fruit flavor may be included
  • liquid carriers such as polyethylene glycol or fatty oil may be included in addition to the above components.
  • the present invention also provides a health functional food composition
  • a health functional food composition comprising an extract or compound and a food acceptable additive.
  • Food composition according to the present invention for example, chewing gum, caramel products, candy, ice cream, various foods such as confectionery, soft drinks, mineral water, beverage products such as alcoholic beverages, health functional foods including vitamins and minerals, etc. Can be.
  • the amount of the extract or compound in the food may be added to 0.001 to 99.9% by weight of the total food weight, in the beverage in the ratio of 0.001 to 0.1 g, more preferably 0.05 to 0.1 g based on 100 ml Can be.
  • Beverages containing the extracts or compound derivatives of the present invention are not particularly limited to other ingredients except for containing the extracts or compound derivatives as essential ingredients in the indicated ratios, and various flavors or natural carbohydrates are added as in the usual beverages. It may contain as a component.
  • the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors such as synthetic and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
  • the functional food compositions of the present invention may contain fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
  • It may contain a gallate compound or a derivative thereof or a gall bladder extract comprising the same according to the present invention, and may be prepared into a pharmaceutical composition by adding a pharmaceutically acceptable carrier according to the method for preparing a pharmaceutical.
  • the present inventors have investigated whether the gall bladder extract has a significant pharmacological effect on the decrease in cognitive memory and accumulation of A ⁇ in an in vivo animal model of Alzheimer's disease.
  • the gall bladder extract significantly improved cognitive memory and inhibited the production of amyloid plaque in the Alzheimer's dementia model induced by the administration of A ⁇ (1-42) and the APPswe / PS1dE9 double-expressed Alzheimer's dementia model.
  • the results suggest the usefulness of the gall bladder extract as a therapeutic candidate for Alzheimer's disease.
  • the gall bladder extract has an immunomodulatory ability and has an effect of improving immune function.
  • FIG. 1 is a diagram showing an experimental schedule for evaluating the pharmacological efficacy of the gall bladder ethanol extract ( Galla rhois ) in the Alzheimer's disease model established by the central administration of A ⁇ .
  • Figure 2 is a diagram showing the experimental schedule for evaluating the pharmacological efficacy of UDCA and gall bladder ethanol extract ( Gala rhois ) in APPswe / PS1dE9 double-expressing mice.
  • Figure 3 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by ⁇ -amyloid (1-42). Cognitive memory was measured by the alteration behavior of the Y-maze test. Sixteen mice in each group were tested, with each mean mean ⁇ standard error. * P ⁇ 0.01 vs. Saline + ⁇ -amyloid (42-1), # P ⁇ 0.05 vs. Saline + ⁇ -amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
  • Figure 4 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by ⁇ -amyloid (1-42). Cognitive memory was measured using a novel object recognition test. Sixteen mice in each group were tested, with each mean mean ⁇ standard error. * P ⁇ 0.01 vs. Saline + ⁇ -amyloid (42-1), # P ⁇ 0.05 vs. Saline + ⁇ -amyloid (1-42) (using Fisher's PLSD test as one-way ANOVA and post test).
  • Figure 5 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by ⁇ -amyloid (1-42). Cognitive memory was measured by observing the finding latency of the water finding test. Sixteen mice in each group were tested, with each mean mean ⁇ standard error. * P ⁇ 0.01 vs. Saline + ⁇ -amyloid (42-1), # P ⁇ 0.05, ## P ⁇ 0.01 vs. Saline + ⁇ -amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
  • Figure 6 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by ⁇ -amyloid (1-42). Cognitive memory was measured by observing the step-through latency of the passive avoidance test. Sixteen mice in each group were tested, with each mean mean ⁇ standard error. * P ⁇ 0.01 vs. Saline + ⁇ -amyloid (42-1), # P ⁇ 0.05 vs. Saline + ⁇ -amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
  • Figure 7 is a diagram showing the effect of Galla rhois ( Galla rhois ) on cognitive memory degradation by ⁇ -amyloid (1-42).
  • Cognitive memory was measured using the reference memory test of Morris water maze. Eight mice per group were measured four times daily, with each mean being ⁇ standard error. * P ⁇ 0.01 vs. Saline + ⁇ -amyloid (42-1), # P ⁇ 0.05 vs. Saline + ⁇ -amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post-test for repeat measurements).
  • Figure 8 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by ⁇ -amyloid (1-42).
  • Cognitive memory was measured using a probe test of Morris water maze. Eight mice in each group were measured twice, each mean ⁇ standard error. * P ⁇ 0.01 vs. Saline + ⁇ -amyloid (42-1), # P ⁇ 0.05 vs. Saline + ⁇ -amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
  • FIG. 9 is a diagram showing the effect of Galla rhois extract ( Galla rhois ) on cognitive memory degradation by ⁇ -amyloid (1-42).
  • Galla rhois ⁇ -amyloid
  • Eight mice in each group were measured four times a day for three days, with each mean being ⁇ standard error.
  • P ⁇ 0.01 vs. Saline + ⁇ -amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
  • FIG. 10 is a diagram showing the pharmacological effect of Galla rhois on the cognitive memory of APPswe / PS1dE9 double-expressing mice (evaluated by the Y-maze test). Each figure represents the mean ⁇ standard error of 10 animals. # P ⁇ 0.05 vs. Saline (One-way ANOVA. Fisher's assessment by post-test).
  • Figure 11 shows the pharmacological effect of Gallia rhois extracts on cognitive memory (evaluated by Novel object recognition test) of APPswe / PS1dE9 double-expressing mice. Each figure represents the mean ⁇ standard error of 10 animals. # P ⁇ 0.05 vs. Saline (One-way ANOVA. Fisher's assessment by post-test).
  • FIG. 12 is a diagram showing the pharmacological effect of Galla rhois on the cognitive memory (evaluated by the finding latency of the water finding test) of APPswe / PS1dE9 double-expressing mice. Each figure represents the mean ⁇ standard error of 10 animals. # P ⁇ 0.05, ## P ⁇ 0.01 vs. Saline (One-way ANOVA. Fisher's assessment by post-test).
  • FIG. 13 shows the pharmacological effect of Gallia rhois extract on the production of amyloid plaque in hippocampus and cortical tissue of APPswe / PS1dE9 double-expressing mice.
  • FIG. 14 is a diagram showing the effect of the gall bladder and the standard on cell proliferation. Observation of cell proliferation of splenocytes obtained after injection of mice into the cell using Cell Proliferation Assay Kit (MTS).
  • MTS Cell Proliferation Assay Kit
  • FIG. 15 is a diagram showing the effect of the gall bladder and the standard on cell proliferation. After isolation of splenocytes, inject the test substance and observe cell proliferation using Cell Proliferation Assay Kit (MTS).
  • MTS Cell Proliferation Assay Kit
  • FIG. 16 is a diagram showing the effect of the gall on Nitric Oxide (NO) production. Determination of NO with time after macrophage isolation from mice and treatment with experimental material.
  • FIG. 17 is a diagram showing the effect of the gall on Nitric Oxide (NO) production. Determination of NO according to test substance concentration after macrophage isolation from mice.
  • FIG. 18 is a diagram showing the effect of the gall bladder and the standard on the production of Nitric Oxide (NO). Determination of NO after treatment of experimental material after macrophage isolation from mice.
  • FIG. 19 is a diagram showing the effect of the gall bladder and the standard on the production of Nitric Oxide (NO). NO measurement after blood isolation from mice.
  • Figure 20 is a diagram showing the cytotoxicity (cytotoxicity) effect over time after the gall bladder treatment.
  • Figure 21 is a diagram showing the cytotoxicity (cytotoxicity) effect of the concentration after the gall bladder treatment.
  • Figure 22 is a diagram showing the cytotoxicity (cytotoxicity) effect over time after treatment with the gall and standard.
  • Figure 23 is a diagram showing the cytokine expression experiments of splenocytes harvested after injection of the gall bladder and standard in mice.
  • 24 is a diagram showing an experiment on the cytokine secretion of macrophages over time after the embryonic treatment in mouse macrophages.
  • Figure 25 is a diagram showing an experiment (24 hours treatment) on the cytokine secretion of macrophages according to the gall bladder treatment concentration in mouse macrophages.
  • Fig. 26 is a diagram showing an experiment on cytokine secretion of macrophages with mouse embryonic and standard treatment concentrations and time in mouse macrophages.
  • Figure 27 is a diagram showing an experiment on the expression of factors related to cell survival of splenocytes after administration of gallocytes and standards.
  • FIG. 28 shows several measurements from gall bladder extract and active compounds treated mice.
  • FIG. 29 is a diagram showing the profile of RBCs and platelets from gall bladder extract and its active compound treated mice.
  • FIG. 35 is a diagram showing the effect of a gall on immobility time in a forced swimming test.
  • FIG. Substances were administered orally to mice and values were mean ⁇ SEM for 3 mice.
  • APPswe / PS1dE9 double-expressing mice (The Jackson Laboratory, Bar Harbor, MA, USA) of 6 months of age were used.
  • APPSwe / PS1dE9 double-expressing mice were administered orally with a gall bladder ethanol extract (250 or 500 mg / kg, p.o.) once a day for 3 months (Fig. 2).
  • the drug was administered after the behavioral and cognitive function evaluation to prevent the drugs from directly affecting the behavior.
  • One day after the end of the cognitive memory evaluation the animals were sacrificed to evaluate the accumulation of A ⁇ .
  • the Y-maze consists of a Y-shaped arm and a black plastic box 25 cm long and 14 cm high and 5 cm wide.
  • the mouse was placed at the end of one arm and allowed to move arm freely for 8 minutes. The number of times the mouse enters each arm was recorded with a video camera.
  • the evaluation index of alternation behavior was compared with the control group by obtaining one point when all three arms were included. The alternation score of the mouse was converted to 100% by dividing the actual score obtained by the mouse with the possible alternation score.
  • mice On the first day the mice were placed in a 40 cm x 40 cm x 30 cm box and allowed to move freely for 10 minutes. On day 2, two objects were placed in a box and the reaction time for each object was recorded. After 24 hours, one of the two objects was replaced with a novel object and the reaction time for the novel object was recorded.
  • the water finding test is a method of evaluating latent learning and attentional spatial memory that allows the mice to recognize the nozzle position of the water bottle in the apparatus on day 1, and then place it in the apparatus again after 24 hours of water saving to find the nozzle of the water bottle.
  • the time was measured (Finding latency).
  • the position of the water bottle and the length of the nozzle were slightly changed, and based on the information acquired on the first day, attention was paid to find a new position.
  • the device consists of two rooms with a guillotine door between them.
  • the floor is equipped with a shock generator.
  • the rat On the 1st day, the rat was placed in one of the two rooms as an acquisition trial and allowed to acclimate for 15 seconds. Then, the rat's room was turned on and the guillotine door was opened. As the mouse moved from the bright room to the dark room, the guillotine door was closed and electric shock (0.5mA, 5s) was applied to the floor of the dark room.
  • the mice were placed in the same device and the time to move from the light room to the dark room was measured (step-through latency).
  • a cylindrical tank having a diameter of 97 cm and a height of 60 cm was used.
  • powdered milk was used, filled with 23 ⁇ 2 ° C diluted water.
  • the tank was equipped with a transparent platform 2 cm below the surface of the water and four covers on the outside of the tank.
  • the motion trajectories of the mice were analyzed by video tracking system (EthoVision, Noldus, The Netherlands).
  • mice were placed in tanks facing the platform at the randomly selected five start points. Each trial records the escape latency after the mouse is placed in the tank, and when the mouse is found, the mouse stays on the platform for 10 seconds before being transferred to the home cage. When the mouse could not find a platform within 60 seconds, the escape latency was recorded as 60 seconds. Each trial was performed four times a day for four days (3 days to 6 days) from 3 days after ⁇ -amyloid administration.
  • Probe test was performed 7 days after ⁇ -amyloid administration. After removing the platform, the rats were allowed to swim in the tank for 60 seconds and the number of times they passed the platform was recorded.
  • Working memory test was performed 8 to 10 days after ⁇ -amyloid administration. It is similar to the reference memory test, but the escape latency was measured by changing the platform location every day. Five trials were performed per day, and the escape latency of the second to fifth trials was used.
  • a ⁇ plaque in hippocampus and cortex of APPswe / PS1dE9 double-expressing mice was evaluated by immunohistochemical staining. Perfusion was used to cut brain tissue fixed with 4% p-formaldehyde at 40 ⁇ m intervals, and then sectioned with 0.2% Triton X-100 before incubation overnight with A ⁇ (1-42) antibody (Invitrogen, Carlsbad, CA, USA). It was exposed for a minute and incubated in 4% normal goat serum. After 1 night, incubate with secondary antibody for 1 hour and immunostain with 3,3-diaminobenzidine as chromogen. Each step was washed with PBS (pH 7.4).
  • the statistical significance between groups was one-way ANOVA or one-way ANOVA for repeated measures. Fisher's PLSD or Bonferroni's evaluation was performed as a post test.
  • the animal efficacy test results are as follows.
  • ⁇ -amyloid 1-42 significantly reduced alteration behavior in Y-maze.
  • the gall bladder extract significantly inhibited the reduction of alteration behavior by ⁇ -amyloid (1-42) (FIG. 3).
  • ⁇ -amyloid 1-42
  • the gall bladder extract significantly inhibited the reduction of exploration of novel objects by ⁇ -amyloid (1-42) (FIG. 4).
  • ⁇ -amyloid (1-42) significantly increased the finding latency in the water finding test to assess latent learning.
  • the gall bladder extract significantly inhibited the increase in finding latency caused by ⁇ -amyloid (1-42) (FIG. 5).
  • ⁇ -amyloid (1-42) significantly reduced step-through latency in passive avoidance tests evaluating fear learning and memory.
  • the gall bladder extract significantly inhibited the decrease of step-through latency by ⁇ -amyloid (1-42) (FIG. 6).
  • mice that received ⁇ -amyloid (1-42) significantly reduced the time to find a safe platform in the tank compared to mice that received ⁇ -amyloid (42-1).
  • the test also significantly reduced the time to stay on the quadrant where the platform was.
  • Methanol ethanol extract significantly inhibited the increase in escape latency by ⁇ -amyloid (1-42) and decrease in residence time in the quadrant with the platform (FIGS. 7 and 8).
  • ⁇ -amyloid (1-42) significantly induced a decrease in spatial working memory in Morris water maze's working memory test.
  • the gall bladder extract significantly inhibited the increase in escape latency by ⁇ -amyloid (1-42) (FIG. 9).
  • the Y-maze test which evaluates short-term spatial memory in APPswe / PS1dE9 double-expressing mice, showed significantly increased alteration behavior (Fig. 10).
  • Amyloid plaque production was evaluated by immunohistochemical staining in hippocampus and cerebral cortex tissues of APPswe / PS1dE9 double-expressing mice, and the gall extract showed pharmacological effects of inhibiting amyloid plaque formation (FIG. 13).
  • mice SPF-grade BALB / c and C5BL / 6 male mice, 7-8 weeks old, were purchased from Orient Bio (Seongnam, Korea) and were tested at Yonsei University's Wonju College of Animal Experimental Laboratory, temperature 22 °C, relative humidity 56 ⁇ 5%, 12 hours Breeding under cycles.
  • the feed was free to eat the mouse feed of Samyang oil feed (Gangwon-do, Wonju-si, Korea) and the water was supplied by tap water in the animal room. After one week of acclimation, mice were sacrificed to separate splenocytes and peritoneal macrophages. Experiments were conducted in accordance with the rules of the animal laboratory of the university and in accordance with the regulations of the Animal Ethics Committee.
  • SP Mouse Splenocytes
  • the mouse was sacrificed by cervical dislocation to remove the spleen.
  • Splenocytes were isolated using a sterile glass slide with a stainless steel 200-mesh and one side rough.
  • the separated cell suspension was centrifuged at 1000 rpm for 10 minutes at room temperature, and the remaining erythrocytes were lysed with 0.83% NH 4 Cl for 5 minutes at room temperature and washed with PBS.
  • the cell precipitate was suspended in DMEM or RPMI medium containing 10% fetal bovine serum and then cultured in a 37 ° C. incubator containing 5% CO 2 .
  • mice were intraperitoneally injected with 3 ml of sterile 3% thioglycolate broth (Difico Laboratories, Detroit, MI, USA), and sacrificed mice three days later to collect abdominal cavity 3 times with 5 ml of PBS. Macrophages were obtained by centrifugation at 2000 rpm for 20 minutes. The obtained cell precipitate was suspended in DMEM, divided into 24 well plates at 5 ⁇ 10 5 cells / well, and cultured in a 37 ° C. incubator containing 5% CO 2 . After incubation for 24 hours, the samples were treated with macrophages, 1 ⁇ 10 7 cells / well.
  • Splenocyte proliferation was performed by colorimetric method, and LPS and B cell mitogen Con A were used for the experiment of weakening.
  • Splenocytes were cultured on RPMI-1640 medium with 10% FBS and 50ul of 2 x 10 6 cells / ml were added to a 96 well plate containing 50ul of LPS and Con A for experiment.
  • MTS / PMS was added and analyzed at a wavelength of 490 nm after 72 hours of incubation.
  • NO was measured for nitrogen in splenocyte culture medium using Griess assay. 600ul of Griess reagent was mixed in 100ul of culture solution, incubated at room temperature for 30 minutes, and measured at 540nm wavelength. NaNO 2 was used as a standard control.
  • RNA precipitate was dried in air, and the RNA was quantified after dissolving the RNA with distilled water containing no RNase and DNase.
  • a reaction solution was prepared by mixing 1 mg RNA, 500 ng oligo-dT primer, 15 U avian myeloblastosis virus reverse transcriptase, 20 U RNase inhibitor, 1 mM dNTP, and 5 mM MgCl 2 .
  • the cDNA produced as a result of the reverse transcription reaction was amplified using primers for IL-10, IL-6, and beta-actin for each experimental purpose.
  • 1 ml of cDNA, 10 'buffer solution 100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl 2
  • 0.2 mM dNTP 1.25 U Taqpolymerase (Takara Biochemicals, Shiga, Japan)
  • 25 ml of the reaction solution was added to the primers, and then placed in GeneAmp PCR system 2400 (Applied Biosystems).
  • the polymerase chain reaction was performed under appropriate conditions.
  • the product of the polymerase chain reaction was observed by electrophoresis at 50 mV for 40 minutes on 1.5% agarose gel.
  • Real-time PCR was performed for quantitative analysis of mRNA levels and 50ng / ul of cDNA was prepared from mRNA as a template and quantitatively analyzed using Universal SYBR Green PCR Master Mix 8. (Sigma. USA).
  • Primers for real-time PCR are IL-6, forward; 5'-ACAACCACGGCCTTCCCTACTT-3 ', IL-6 reverse; 5'-CACGATTTCCCAGAGAACATGTG-3 '; TNF-alpha, forward; 5'-CCCTCAGCAAGGACAGCAGA-3 'and TNF-alpha reverse; 5'-AGCCGTGGGTCAGTATGTGA-3 '; Beta-actin forward; 5'-CCAAGGCCAACCGCGAGAAGATGAC-3 'and beta-actin reverse; 5'-AGGGTACATGGTGGTGCCGCCAGAC-3 'was analyzed by MX3000P PCR Instrument (Stratagene, USA).
  • Yac-1 cells a mouse T lymphoma-derived cell line, are suspended in a 25 cm 2 plastic flask in 10% FBS fetal calf serum RPMI 1640 medium.
  • Agonist cells should be prepared as follows: 1) Float in complete RPMI 1640 medium to remove plastic adherent cells contained in prepared cells as follows.
  • Effector / target cell ratio (E / T ratio) shall be 200: 1.
  • A) Monoclonal Antibodies The monoclonal antibodies and the optimal dilution factor used to investigate the distribution of splenic lymphocyte subpopulations using cell surface antigen specific antibodies are as follows.
  • Table 1 is a table of the characteristics and optimal dilutions of each monoclonal antibody used in the assay for splenic lymphocyte cell surface marker expression.
  • Lymphocyte suspensions prepared in the same manner as the method described in the investigation of lymphocyte proliferation were filtered through a nylon network, and then centrifuged (1,500g, 20min) in Lympholyte-M (Cedarlane). Separated lymphocytes were extracted and centrifuged and washed with HBSS. Suspend with PBS containing 1% bovine serum albumin (BSA) and 0.1% NaN 3 and adjust to a concentration of 1x10 7 cells / ml. 100 ul of cell fluid and 10 ul of each monoclonal antibody a. Control, Fc block b. Fc block + CD3 (FITC) + CD45 (PE) c.
  • BSA bovine serum albumin
  • Test Principle How to measure the degree of reducing chemical mediators that cause inflammation and itching in the skin such as atopic dermatitis
  • Inhibition Rate (%) (Histamine amount without drug administration-amount of histamine after drug administration) x 100 / Histamine amount without drug administration
  • Nitric Oxide (NO) production a potent antioxidant component, was confirmed to have an increase in production or a similar pattern compared to the standard material, confirming the antioxidant activity of the gall bladder extract (Figs. 16-19).
  • NF-kB which is related to inflammatory responses such as COX-2
  • signaling pathways related to MAPkinase were related. It could be confirmed (Fig. 27).
  • SA and GA of five gall extracts and active compounds tended to increase the fractional cells (WBC, NE, LY, MO, EO, BA), and SA (syringic acid) was the most prominent among the administration groups, and MG (methyl gallate) ) was not significantly different from the control group.
  • erythrocyte and platelets (platelet) were increased in the number of erythrocytes, HCT, platelets in each administration group, hemoglobin (Hb), MCH, MCHC was increased except in the MG group.
  • the gall bladder extract and the active compound regulate the distribution of blood cells and blood cell-derived immune cells in mice.
  • splenocytes were isolated, RNA was isolated, and expression of IL-6 and TNF-alpha was measured using real time PCR.
  • MRNA expression of IL-6 and TNF-alpha cytokine was increased when the gall extract was treated, but TNF-alpha showed no significant increase compared with gallic acid or metyl gallate.
  • the splenocytes were isolated and cultured to determine NO in the supernatant.
  • Other active active compounds also tended to increase NO production.
  • mice (20 ⁇ 2 g) or mature male rats (200 ⁇ 10 g) were allowed to stabilize the animals for one week of the experiment, and the mice were administered the test substance after two abandonment reactions in a bathing device 3-7 days before the experiment. After a minute or 1 hour, the immobilization time was measured in a water bath for 5 to 10 minutes.
  • Immobility time was decreased when the gall extract was treated by concentration (100mg / kg-10mg / kg).

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Abstract

The present invention relates to a composition containing a Galla rhois extract as an active ingredient for improving cognition, and more specifically, to a composition containing a Galla rhois extract as an active ingredient, for preventing and treating Alzheimer's disease (AD).

Description

오배자 추출물을 유효성분으로 포함하는 인지력 개선 및 치매 예방 또는 치료용 조성물A composition for improving cognition and preventing or treating dementia comprising a gall extract as an active ingredient
본 발명은 오배자 추출물을 유효성분으로 포함하는 인지력 개선 조성물에 관한 것으로 더욱 상세하게는 오배자 추출물을 유효성분으로 포함하는 치매(alzheimer's disease; AD) 예방 및 치료용 조성물에 관한 발명이다.The present invention relates to a cognitive improvement composition comprising a gall bladder extract as an active ingredient, and more particularly, to a composition for preventing and treating Alzheimer's disease (AD) comprising the gall bladder extract as an active ingredient.
치매에 의한 인지력 즉 기억력 저하는 주로 내과적, 신경과적 원인으로 인하여 뇌신경의 일시적 혹은 지속적인 손상이 발생함으로써 사회생활과 일상생활 유지에 현저한 곤란이 발생하는 "증후군"이라 할 수 있으며, 의학적으로는 광범위한 인지기능과 인격의 변화가 따르는 대표적인 기질성 장애로 단기 및 장기 기억장애가 특징으로 나타나며, 사고력, 지남력, 이해력, 계산능력, 언어 및 판단력 등을 포함하는 고도의 뇌피질기능의 다발성 장애이다(F, H. and Kurz, A,. Eur Arch Psychiatry Clin Neurosci, 249(6):288-290 (1999); Jaworski, T. et al., Biochim Biophys Acta, 1802(10): 808-818 (2010)).Cognitive, or memory, deterioration caused by dementia is a "syndrome," which causes remarkable difficulties in maintaining social and daily life due to temporary or persistent damage to the cranial nerve, mainly due to medical and neurological causes. Representative organic disorders with changes in cognitive function and personality are characterized by short-term and long-term memory impairments, and are highly cortical disorders of high brain cortical function including thinking ability, mental ability, comprehension, computational ability, language and judgment (F, H. and Kurz, A, Eur Arch Psychiatry Clin Neurosci, 249 (6): 288-290 (1999); Jaworski, T. et al., Biochim Biophys Acta, 1802 (10): 808-818 (2010)) .
알쯔하이머형 치매는 치매 환자의 과반수를 차지하고 있으며, 70세 이상 인구의 10%가 고통받고 있는 것으로 알려져 있다. 노인에서만 나타나는 것이 아니라 어느 연령대에서도 발병할 수 있으나, 실제로 50세 이전에 증상이 나타나는 경우는 드물고, 60세 이후로는 나이가 들어감에 따라 발생빈도가 점진적으로 증가하기 때문에 노인 인구가 증가하고 있는 나라에서는 중요한 의료, 사회 및 경제적 문제를 야기하고 있고 유병율은 65~74세는 10%, 75~84세는 19%, 85세 이상에서는 47%로 나타나고 있다(Reitz, C., et al., Nat Rev Neurol, 7(3): 137-152 (2011). Alzheimer's dementia accounts for the majority of people with dementia and 10% of people over 70 are known to suffer. It can occur at any age, not only in the elderly, but in reality, symptoms rarely occur before the age of 50, and since 60 years of age, the incidence increases gradually as the age increases. Causes significant medical, social and economic problems, and the prevalence is 10% for ages 65-74, 19% for ages 75-84, and 47% for ages 85 and older (Reitz, C., et al., Nat Rev Neurol). , 7 (3): 137-152 (2011).
알쯔하이머형 치매의 발병기전은 아직까지 명확히 밝혀져 있지 않고 여러 가지 가설이 제시되어 왔으나 최근에는 뇌병변에 침착하는 신경독성단백질(β-amyloid protein)의 독성이 가장 중요한 원인으로 제시되고 있다. 이 물질은 아밀로이드 전구단백질(APP: amyloid precursor protein)의 잘못된 대사로 인해 생성되는 것으로 알려져 있고, APP의 정상 대사물은 신경세포보호작용이 있는 것으로 보고되고 있다. 최근 유전공학적 방법을 이용한 연구결과에서는 베타아밀로이드(β-amyloid)나 타우(Tau) 등 독성 단백질이 세포와 혈관에 쌓여 신경세포에 독성을 미침으로써 결과적으로 뇌기능에 장애가 초래된다는 사실이 밝혀졌다(Koric, L., et al., Rev Neurol (Paris), 167(6-7): 474-484 (2011); Prvulovic, D. and Hampel, H., Clin Chem Lab Med., 49(3):367-374 (2011)).The pathogenesis of Alzheimer's dementia has not been elucidated and various hypotheses have been suggested. However, the toxicity of β-amyloid protein deposited on brain lesions has been suggested as the most important cause. This substance is known to be produced due to incorrect metabolism of amyloid precursor protein (APP), and normal metabolites of APP have been reported to have neuroprotective effects. Recent genetic engineering studies have shown that toxic proteins such as beta-amyloid and tau accumulate in cells and blood vessels, toxic to nerve cells, resulting in impaired brain function. Koric, L., et al., Rev Neurol (Paris), 167 (6-7): 474-484 (2011); Prvulovic, D. and Hampel, H., Clin Chem Lab Med., 49 (3): 367-374 (2011)).
알츠하이머형 치매증 및 뇌졸중 후나 혹은 뇌동맥경화에 의한 광범위한 뇌병변이 발생함으로써 유발되는 뇌혈관성 치매증이 치매질환의 약 90%를 차지하며, 이외 픽(Pick)병, 크로이츠펠트-야곱 (Creutzfeldt-jakob)병, 두부손상에 의한 치매, 파킨슨(Parkinson)병 등이 있다.Cerebrovascular dementia caused by Alzheimer's dementia and widespread brain lesions after stroke or by cerebral atherosclerosis accounts for about 90% of dementia diseases, and other Pick and Creutzfeldt-jakob diseases. , Head injury, dementia, and Parkinson's disease.
치매질환은 단기간 및 장기간 기억장해가 기본적이며 또한 중핵증상으로 관찰되며, 기억장해와 이것에 기인한 부위감각상실 (disorientation) 및 이것에 대한 고도의 뇌기능 장해로 이루어지는 것으로 생각된다.Dementia diseases are fundamentally observed as short-term and long-term memory disorders, and are considered to be a core symptom, and are thought to consist of memory disorders, disorientation resulting from them, and high brain dysfunction.
치매 환자는 주로 대뇌 피질 및 대뇌 변연계에서 다양한 신경전달물질계의 현저한 기능 저하 및 대뇌 에너지 물질 대사의 기능저하를 나타내며 특히 알츠하이머형 치매증 (Alzheimer's Disease, 이하 'AD'로 약칭함)에 있어서는, 환자의 아세틸콜린 (acetylcholine, 이하 'ACh'로 약칭함)계, 글루타민 산 (glutamic acid)계, 신경펩티드 (neuropeptides)계 및 모노아민 (monoamine)계에서의 신경전달의 기능저하가 나타나, 이들 신경전달계의 기능 장해가 AD의 주된 원인인 것으로 추정되고 있다.Patients with dementia show significant decreases in various neurotransmitter systems and decreases in cerebral energy metabolism, mainly in the cerebral cortex and cerebral limbic system, especially in Alzheimer's disease (AD). Deterioration of neurotransmission in choline (acetylcholine, abbreviated as 'ACh'), glutamic acid, neuropeptides and monoamines has been shown to function. Disability is believed to be the leading cause of AD.
또한, β-아밀로이드 펩티드 (β-amyloid peptide)에 의해 유발된 신경세포 독성작용의 관점으로부터 β-아밀로이드 펩티드의 세포 밖으로의 노인 반점의 집적을 통한 해마 신경세포의 탈락에 의한 발증 기전도 AD의 주요 원인 중 하나로 추정되고 있다.Also, from the viewpoint of neuronal cytotoxicity induced by β-amyloid peptide, the onset mechanism of AD due to the elimination of hippocampal neurons through the accumulation of old spots outside the cells of β-amyloid peptide is also important. It is assumed to be one of the causes.
치매 질환의 치료를 위한 의약품이나 기능성 식품을 개발하기 위하여 여러 가지 연구가 진행되고 있기는 하지만, 극히 일부 어느 정도의 효능을 보이는 정도의 치료제가 있을 뿐이지, 치료효과가 뚜렷한 명약이 아직까지 개발되고 있지 못하다.Although various studies are being conducted to develop medicines or functional foods for the treatment of dementia diseases, there are only a few therapeutic drugs that show only a certain degree of efficacy, and clear medicines with clear therapeutic effects have not yet been developed. Can not do it.
현재 치매의 치료는 증상을 조기에 발견해 치료함으로써 진행을 늦추는 데 목표를 두고 있으며, 현단계에서 치매의 진행을 억제하거나 예방할 수 있는 약물은 없다. 미국 FDA의 허가를 받고 시장에 발매된 제품은 Cogne(Parke-Davis), Aricept(Eisai & Pfizer), Exellon(Novartis) 및 Ebixa(Lundbeck)이 있지만, Ebixa를 제외한 약물들은 단지 알쯔하이머에 의해 나타나는 증상의 일부를 완화(신경전달 물질인 Acetylcholine 농도를 높이기 위해 이의 분해효소인 Acetylcholine exterase를 억제)시키고, 환자 및 보호자를 안심시키는데 기여하고 있을 뿐이다 (McShane, R., et a., Cochrane Database Syst Rev, Apr 19;(2):CD003154 (2006); Ji, W. and Ha, I., Exp Neurobiol, 19(3):120-131 (2010); Bandyopadhyay, S., et al., Expert Opin Ther Targets, 14(11):1177-1197 (2010)); Olivares, D. et al., Curr Alzheimer Res, 2011 Aug 30. [Epub ahead of print])).Currently, the treatment of dementia aims to slow the progression by early detection and treatment of symptoms. At this stage, there are no drugs that can prevent or prevent the progression of dementia. Products released on the market with the approval of the U.S. FDA are Cogne (Parke-Davis), Aricept (Eisai & Pfizer), Exellon (Novartis), and Ebixa (Lundbeck), except for Ebixa. It only contributes to relieving some (inhibiting its degrading enzyme Acetylcholine exterase to increase the concentration of the neurotransmitter Acetylcholine) and contributing to reassuring patients and caregivers (McShane, R., et a., Cochrane Database Syst Rev, Apr. 19; (2): CD003154 (2006); Ji, W. and Ha, I., Exp Neurobiol, 19 (3): 120-131 (2010); Bandyopadhyay, S., et al., Expert Opin Ther Targets, 14 (11): 1177-1197 (2010)); Olivares, D. et al., Curr Alzheimer Res, 2011 Aug 30. [Epub ahead of print]).
주로 치료제 개발의 예로, ACh와 관련한 신경기능의 현저한 저하가 치매증에 있어서 기억장해의 원인이라는 발견을 기초로, ACh를 활성시킴으로써 치매증상을 개선하는 치료제가 개발되고 있다.As an example of the development of therapeutic agents, therapeutic agents that improve dementia symptoms by activating ACh have been developed based on the finding that a marked decrease in neurological function related to ACh is the cause of memory disorders in dementia.
글루타민산 신경단위 및 글루타민산의 수용체 중 하나인 N-메틸-D-아스파르트산(N-methyl-D-aspartic acid, 이하, 'NMDA'로 약칭함) 수용체의 감소에 주목한 NMDA 수용체의 활성화에 의한 치료제의 개발도 행해지고 있다.Therapeutic agent by activating NMDA receptor, noting the reduction of N-methyl-D-aspartic acid (NMDA) receptor, one of glutamic acid neuron and glutamic acid receptors Is also being developed.
또한, 최근에는 치매증상의 개선을 목적으로 하는 치료제로서 말초형 벤조디아제핀(benzodiazepine, 이하, 임의로 'BZω3'로 약칭함) 수용체 작용약 (agonist)을 개발하려는 시도가 있음. 예를 들어, BZω3 수용체에 대한 작용약인 N, N-디-n-헥실-2-(4-플루오로페닐)인돌-3-아세트아미드(N,N-di-n-hexyl-2-(4-fluorophenyl)indol-3-aceteamide,이하, 'FGIN-1-27'로 약칭함) 및 어떤 종류의 뉴로스테로이드 (neurosteroids) (예를 들어, 프레그네놀론 설페이트, 알로프레그나놀론)은 동물의 학습장해모델 (능동적조건 회피반응 시험, 방사상 미로시험 및 물 미로 시험)에서 약한 개선 작용을 갖는 것이 보고되어 있다.Recently, there has been an attempt to develop a peripheral benzodiazepine (benzodiazepine, hereinafter abbreviated as 'BZω3') receptor agonist as a therapeutic agent aimed at improving dementia symptoms. For example, N, N-di-n-hexyl-2- (4-fluorophenyl) indole-3-acetamide (N, N-di-n-hexyl-2- (4), an agonist for the BZω3 receptor -fluorophenyl) indol-3-aceteamide (hereinafter abbreviated as 'FGIN-1-27') and some types of neurosteroids (e.g., pregnenolone sulfate, allopregnanolone) It has been reported that the solution model (active condition avoidance test, radial maze test and water maze test) has a slight improvement.
Acetylcholinesterase는 신경전달물질 ACh을 퇴화시키는 효소로, AChE 억제제(acetylcholinesterase inhibitor)는 이 퇴화 과정을 억제함으로써, ACh 신경전달물질은 증가된 시간 동안 신경 클레프트(cleft)에 남게 되고, 따라서 신경전달물질의 화학적 및 기능적 효과를 상승시켜 작용을 향상시킨다.Acetylcholinesterase is an enzyme that degrades the neurotransmitter ACh. AChE inhibitors (acetylcholinesterase inhibitors) inhibit this degradation process, leaving the ACh neurotransmitter in the neural cleft for an increased amount of time. Enhances action by enhancing chemical and functional effects.
지금까지 개발된 AChE 억제제로 1,2,3,4-테트라히드로-9-아크리딘아민(1,2,3,4-tetrahydro- 9-acridine amine; 타크린(tacrine), THA; '코그넥스' (COGNEX)), 도네페질(Done Pezyl, E2020; '아리셉트' (ARICEPT)) 및 리바스티그민(Rivastigmine, ENA713; '엑셀론' (EXELON))이 있다.AChE inhibitors developed to date include 1,2,3,4-tetrahydro-9-acridineamine (1,2,3,4-tetrahydro-9-acridine amine; tacrine, THA; 'cog NEXO (COGNEX), Done Pezyl (E2020; ARICEPT) and Rivastigmine (ENA713; EXELON).
이들 약품은 치매를 다스리는 작용기전이 중추신경전달계에 중심적인 역할을 하는 AChE의 활성억제를 통해 신경전달물질인 ACh의 농도를 증가시킴으로써 알츠하이머 질환을 예방 및 치료하는 약제로 알려지고 있다.These drugs are known as drugs to prevent and treat Alzheimer's disease by increasing the concentration of ACh, a neurotransmitter, through the inhibition of AChE activity, which plays a central role in the central nervous system.
그러나 이러한 AChE 억제 약물들은 콜린성 신경이 기능적으로 손상되지 않았을 때 가장 효과적이고 이들 약물의 효능은 콜린성 신경 기능의 감퇴와 함께 감소되기 때문에, 경증이나 중등도의 노인성 치매의 초기 단계에만 효과가 있으며, 대부분이 말초신경의 콜린성 부작용을 나타내며, 반감기가 너무 짧고, 간 독성 등의 심각한 부작용이 있고 특히 코그넥스 (Cognex)의 유효성분인 9-아미노-1,2,3,4-테트라히드로아크리딘 (THA)은 경구 투여시, 지각능력 향상의 소견을 보이기는 하지만 떨림증, 어지럼증, 간 독성 등의 심각한 부작용을 수반하고 있어 널리 활용되지 못하고 있는 실정이다.However, these AChE inhibitory drugs are most effective when the cholinergic nerves are not functionally impaired, and the efficacy of these drugs decreases with the decline of cholinergic nerve function, so they are effective only in the early stages of mild or moderate senile dementia. It has cholinergic side effects of peripheral nerves, its half-life is too short, it has serious side effects such as hepatotoxicity and especially 9-amino-1,2,3,4-tetrahydroacridine (THA), an active ingredient of Cognex ) Shows oral improvement in perceptual administration, but it is not widely used due to serious side effects such as tremor, dizziness, and liver toxicity.
한편, 이러한 AChE 억제 약물의 단점을 발현하지 아니하는 생약으로서 인삼을 주성분으로 하여 수종의 생약을 혼합 추출한 제품이 한국특허공개 제99-85202호와 미국특허 제 6,010,702호에 개시되어 있으나 이러한 생약 혼합 추출물의 경우 노인성 치매에 어느 정도의 약효를 보이고 있기는 하지만, 부작용 등 아직도 확실하고 안정적인 효과를 보장하지 못하며, 종래의 다른 치매 치료제와는 작용기전이 다른 것으로 추측되고 있을 뿐이지 확실한 처방이라고 볼 수 없는 불완전한 조성물로 나타나고 있다.On the other hand, as a herbal medicine that does not express the disadvantages of the AChE inhibitory drug, a product obtained by mixing and extracting several herbal medicines based on ginseng is disclosed in Korean Patent Publication Nos. 99-85202 and 6,010,702, but such herbal extracts Although it shows some medicinal effects on senile dementia, it still does not guarantee a definite and stable effect such as side effects, and it is assumed that the mechanism of action is different from other conventional dementia treatment agents. It is shown as a composition.
특히, 인삼성분이 주성분으로 구성되어 있어서 고혈압 환자의 경우는 심계항진과 같은 부작용을 초래하거나 신체의 항상성 불균형을 초래할 수 있는 경향이 있어서 또 다른 문제가 될 수 있다.In particular, the ginseng component is composed of the main component in the case of hypertension patients may cause another side effect such as palpitations or the homeostasis imbalance may be another problem.
우리나라도 노인인구가 급증하여 2030년에는 노인인구비중이 19.3%로 예상되며, 이에 대한 의료비가 증가될 것으로 보임. 특히 치매와 같은 퇴행성 질환은 효과적인 약물이 적고, 환자의 생존기간이 길어 국가적 부담이 크며, 따라서, 이에 대한 효과적인 예방 및 진행 억제를 위한 약물 개발이 시급하며, 이는 고부가가치 아이템이 될 것으로 사료된다.In Korea, the elderly population has soared, and the proportion of the elderly population is expected to be 19.3% by 2030, and medical expenses are expected to increase. In particular, degenerative diseases such as dementia have a high national burden due to low effective drugs and long life span of patients. Therefore, it is urgent to develop drugs for effective prevention and suppression of progression, which is expected to be a high value-added item.
국내에서는 천연물제재 즉 은행잎제제(Ginkgo biloba)는 은행잎 성분을 함유한 이명, 두통, 기억력감퇴, 집중력장애, 우울감, 어지러움 등의 치매증상을 수반하는 기질성 뇌장애 치료제로 말초 및 뇌혈관의 혈액순환을 개선시키는 작용을 한다고 소개되고 있으나, 현재 서큐란과 같은 은행잎제제의 경우 제품화에 있어 앙케이트조사문답에서 심각한 문제가 발생하고 있다. 그 외에도 항산화제로서 다른 은행엽제제, 키토산, 오메가3, SK사의 백두옹 saponin 분획, 광동제약의 현삼을 이용한 KD-501의 치매치료제등이 소개되고 있다. 이와 같이 종래의 치매 치료용 조성물의 경우, 그 부작용이 가장 커다란 문제가 되고 있으며, 특히 생약제의 경우 다른 약물에 비하여 부작용이 크게 적은 특징으로 보여주고는 있으나 경우에 따라서는 또 다른 부작용이 우려되고, 치료효과도 아직까지는 충분하지 않아서 효과적인 임상적 효능의 증진이 필요한 실정이므로 이에 관한 개선이 절실히 요구되고 있다.In Korea, Ginkgo biloba (Ginkgo biloba) is a treatment for organic brain disorders involving dementia such as tinnitus, headache, memory loss, concentration disorders, depression, dizziness, etc. Ginkgo leaf preparations such as Succuran are causing serious problems in questionnaire surveys in commercialization. Other antioxidants include ginkgo biloba, chitosan, omega 3, SK's saponin fraction, and KD-501's dementia treatment using Guangdong Pharmaceutical's Hyunsam. As described above, in the case of the conventional composition for treating dementia, the side effect is the biggest problem, and especially in the case of herbal medicine, the side effect is shown to be significantly less than other drugs, but in some cases another side effect is concerned, The therapeutic effect is not enough yet, so the need for improvement of effective clinical efficacy is urgently required for improvement.
본 발명은 상기의 문제점을 해결하고 상기의 필요성에 의하여 안출된 것으로서 본 발명의 목적은 치매 (AD) 예방 또는 치료용 조성물을 제공하는 것이다.SUMMARY OF THE INVENTION The present invention has been made in view of the above-mentioned problems and the need for the above-described problem, and an object of the present invention is to provide a composition for preventing or treating dementia (AD).
상기의 목적을 달성하기 위하여 본 발명은 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 치매 (AD) 예방 및 치료용 약학 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating Alzheimer's (AD), which contains an extract of Galla Rhois as an active ingredient.
본 발명의 일 구현예에 있어서, 상기 오배자(Galla Rhois) 추출물의 유효량은 0.001∼500mg/kg 체중인 것이 바람직하나 이에 한정되지 아니한다.In one embodiment of the present invention, the effective amount of the extract (Galla Rhois) is preferably 0.001 to 500mg / kg body weight is not limited thereto.
또 본 발명은 갈릭에시드(gallic acid) 또는 메틸갈레이트를 유효성분으로 포함하는 치매 (AD) 예방 및 치료용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing and treating dementia (AD) comprising gallic acid or methyl gallate as an active ingredient.
또한 본 발명은 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 치매 (AD) 예방 및 개선용 건강식품 조성물을 제공한다.In another aspect, the present invention provides a health food composition for preventing and improving dementia (AD) containing a gall (Galla Rhois) extract as an active ingredient.
또한 본 발명은 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 인지능력 개선용 약학 조성물을 제공한다.In another aspect, the present invention provides a pharmaceutical composition for improving the cognitive ability containing the extract (Galla Rhois) as an active ingredient.
또한 본 발명은 갈릭에시드(gallic acid) 또는 메틸갈레이트를 유효성분으로 포함하는 인지 능력 개선용 약학 조성물을 제공한다.The present invention also provides a pharmaceutical composition for improving cognitive ability comprising gallic acid (gallic acid) or methyl gallate as an active ingredient.
또 본 발명은 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 인지능력 개선용 건강 식품 조성물을 제공한다.In another aspect, the present invention provides a health food composition for improving cognitive ability containing a gall (Galla Rhois) extract as an active ingredient.
본 명세서에서 "오배자 추출물"이란 천연물인 오배자로부터 유효성 분을 추출하여 얻어진 것이라면 특별히 한정하지 않고 모두 포함한다. 예컨대, 오배자를 물이나 유기용매에 넣고 정치, 교반, 가압 또는 가열 등의 수단을 통해 유효성분을 용출함으로써 얻어진 결과물을 들 수 있다. 또한, 그와 같이 하여 얻어진 액상 추출물을 동결건조함으로써 얻어진 동결건조물을 포함한다. 또한, 그러한 동결건조물을 분쇄한 분말을 포함한다. 그 외에도 유효성분을 추출하기 위해 가능한 수단이 무엇이든지 가리지 않고 추출된 모든 추출물을 포함하며, 추출된 후 동결 건조 등의 가공을 거친 것까지도 모두 포함된다. 기타, 중탕이나 상온에 의한 추출법과 같이 예로부터 전해 내려오거나, 한의서 또는 교과서에 기재되어 있는 통상적인 추출법에 의한 추출물을 포함한다. 또한, 특정 활성 화합물들을 분리하기 위한 특별한 추출방법인 Stass Otto 추출법이나 각종 칼럼 크로마토그래피 방법 등을 통하여 얻어진 분획 추출물을 포함한다.In the present specification, if the "germ extract" is obtained by extracting the active ingredient from the gall is a natural product includes all without particular limitation. For example, the resultant obtained by putting a ligne in water or an organic solvent and eluting an effective component through means, such as standing, stirring, pressurization, or heating, is mentioned. It also includes a lyophilized product obtained by lyophilizing the liquid extract thus obtained. It also includes a powder obtained by grinding such lyophilisate. In addition, any possible means for extracting the active ingredient includes all the extracted extracts, including all the processed after extraction and freeze-drying. Others include extracts obtained by conventional extraction methods such as extraction by baths or room temperature, or by conventional extraction methods described in Chinese medicine or textbooks. In addition, it includes a fraction extract obtained through the Stass Otto extraction method, various column chromatography methods, etc., which is a special extraction method for separating specific active compounds.
본 발명에 따른 약제학적 조성물은 약제학적 조성물의 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더 포함할 수 있다.The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
본 발명의 조성물의 약학적 투여 형태는 이들의 약학적 허용가능한 염의 형태로도 사용될 수 있고, 또한 단독으로 또는 타 약학적 활성 화합물과 결합뿐만 아니라 적당한 집합으로 사용될 수 있다. 상기 염으로는 약학적으로 허용되는 것이면 특별히 한정되지 않으며, 예를 들어 염산, 황산, 질산, 인산, 불화수소산, 브롬화수소산, 포름산 아세트산, 타르타르산, 젖산, 시트르산, 푸마르산, 말레산, 숙신산, 메탄술폰산, 벤젠술폰산, 톨루엔술폰산,나프탈렌술폰산 등을 사용할 수 있다.The pharmaceutical dosage forms of the compositions of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection. The salt is not particularly limited as long as it is pharmaceutically acceptable. For example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid , Benzene sulfonic acid, toluene sulfonic acid, naphthalene sulfonic acid and the like can be used.
본 발명에 따른 약제학적 조성물은, 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 경구형 제형, 연고, 크림 등의 외용제, 좌제 및 멸균 주사용액 등을 비롯하여 약제학적 제제에 적합한 어떠한 형태로든 제형화하여 사용될 수 있다.The pharmaceutical compositions according to the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, ointments, creams, external preparations, suppositories, and sterile injectable solutions. It may be used in formulated in any form suitable for pharmaceutical formulations.
본 발명에 따른 조성물에 있어서, 본 발명의 추출물 또는 화합물의 바람직한 투여량은 대상자의 연령, 성별, 체중, 증상, 질병의 정도, 약물형태, 투여경로 및 기간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.In the compositions according to the invention, the preferred dosage of the extract or compound of the invention depends on the subject's age, sex, weight, symptoms, extent of disease, drug form, route of administration and duration, but is appropriately selected by those skilled in the art. Can be.
그러나, 바람직한 효과를 위해서, 본 발명의 조성물은 1일 0.001 내지 500 mg/kg 체중으로 투여하는 것이 좋다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 또한, 그 투여량은 연령, 성별, 체중, 질병의 정도, 투여경로등에 따라서 증감될 수 있다. 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.However, for the desired effect, the composition of the present invention is preferably administered at 0.001 to 500 mg / kg body weight per day. Administration may be administered once a day or may be divided several times. In addition, the dosage may be increased or decreased depending on age, sex, weight, degree of disease, route of administration, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
본 발명에 따른 조성물은, 쥐, 생쥐, 가축, 인간 등의 포유동물에 비경구, 경구 등의 다양한 경로로 투여될 수 있으며, 투여의 모든 방식은 예상될수 있는데, 예를 들면, 경구, 직장 또는 정맥, 근육, 피하, 자궁내 경막 또는 뇌혈관내(intracerebroventricular) 주사에 의해 투여될 수 있다.The composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans by various routes, such as parenteral, oral, and all modes of administration can be expected, for example oral, rectal or It can be administered by intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.
한편, 본 발명에 따른 조성물은, 심각한 독성 및 부작용은 없으므로 예방 목적으로 장기간 사용 시에도 안심하고 사용할 수 있다.On the other hand, the composition according to the present invention, there is no serious toxicity and side effects can be used with confidence even for long-term use for the purpose of prevention.
본 발명에 의한 조성물은, 유효성분으로서 추출물을 전체 조성물 중량 중 0.001 내지 99.9중량% 함유할 수 있다. 0.001중량% 이상이어야 효과를 기대할 수 있고, 불순물의 존재 등으로 인해 99.9중량%를 넘기 어렵기 때문이다.The composition according to the present invention may contain 0.001 to 99.9% by weight of the extract as an active ingredient in the total weight of the composition. This is because the effect can be expected to be 0.001% by weight or more, it is difficult to exceed 99.9% by weight due to the presence of impurities.
한편, 경구 투여의 목적으로 본 발명의 추출물 또는 화합물을 정제, 캡슐, 츄잉정, 작은 봉지, 과립, 분말, 액체 용액, 현탁액, 분산액, 에멀젼, 시럽등의 제제로 제형화하는 경우에 있어서는, 아라비아 고무, 옥수수 전분, 미세 결정질 셀룰로오스 또는 젤라틴과 같은 결합제, 인산 이칼슘 또는 락토오즈와 같은 부형제, 알긴산, 옥수수 전분 또는 감자 전분과 같은 붕해제, 스테아르산 마그네슘과같은 윤활제, 슈크로오즈 또는 사카린과 같은 감미제 및 페퍼민트, 메틸 살리실산염 또는 과일향과 같은 향미제가 포함될 수 있으며, 투여 단위 형이 캡슐제인 경우에는 상기 성분 외에도 폴리에틸렌 글리콜 또는 지방유와 같은 액상 담체가 포함될수 있다.On the other hand, in the case of formulating an extract or compound of the present invention into tablets, capsules, chewing tablets, small bags, granules, powders, liquid solutions, suspensions, dispersions, emulsions, syrups, etc., for the purpose of oral administration, Arabia Rubber, corn starch, binders such as microcrystalline cellulose or gelatin, excipients such as dicalcium phosphate or lactose, disintegrants such as alginic acid, corn starch or potato starch, lubricants such as magnesium stearate, sucrose or saccharin Sweetening agents and flavoring agents such as peppermint, methyl salicylate or fruit flavor may be included, and in the case of the dosage unit form of capsules, liquid carriers such as polyethylene glycol or fatty oil may be included in addition to the above components.
또한, 본 발명은 추출물 또는 화합물 및 식품학적으로 허용 가능한 식품보조 첨가제를 포함하는 건강 기능성 식품 조성물을 제공한다. 본 발명에 따른 식품 조성물은, 예를 들어, 츄잉껌, 캐러멜 제품, 캔디류, 빙과류,과자류 등의 각종 식품류, 청량 음료, 미네랄 워터, 알코올 음료 등의 음료 제품, 비타민이나 미네랄 등을 포함한 건강기능성 식품류일 수 있다.The present invention also provides a health functional food composition comprising an extract or compound and a food acceptable additive. Food composition according to the present invention, for example, chewing gum, caramel products, candy, ice cream, various foods such as confectionery, soft drinks, mineral water, beverage products such as alcoholic beverages, health functional foods including vitamins and minerals, etc. Can be.
이 때, 상기 식품 중의 상기 추출물 또는 화합물의 양은 전체 식품중량의 0.001 내지 99.9 중량%로 가할 수 있으며, 음료 중에는 100 ㎖를 기준으로 0.001 내지 0.1 g, 더 바람직하게는 0.05 내지 0.1 g의 비율로 가할 수 있다.At this time, the amount of the extract or compound in the food may be added to 0.001 to 99.9% by weight of the total food weight, in the beverage in the ratio of 0.001 to 0.1 g, more preferably 0.05 to 0.1 g based on 100 ml Can be.
본 발명의 추출물 또는 화합물 유도체를 함유하는 음료는 지시된 비율로 필수 성분으로서 상기 추출물 또는 화합물 유도체를 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.Beverages containing the extracts or compound derivatives of the present invention are not particularly limited to other ingredients except for containing the extracts or compound derivatives as essential ingredients in the indicated ratios, and various flavors or natural carbohydrates are added as in the usual beverages. It may contain as a component.
상기 외에 본 발명의 건강기능성 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 증진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 기능성 식품 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료 의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0 내지 약 20 중량부의 범위에서 선택 되는 것이 일반적이다.In addition to the above, the functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavors such as synthetic and natural flavors, colorants and enhancers (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the functional food compositions of the present invention may contain fruit flesh for the production of natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
본 발명에 따른 갈레이트 화합물 또는 그 유도체 또는 이들을 포함하는 오배자 추출물을 함유할 수 있으며 의약품 제조 방법에 따라 약학적으로 허용가능한 담체가 첨가되어 약학적 조성물로 제조될 수도 있다.It may contain a gallate compound or a derivative thereof or a gall bladder extract comprising the same according to the present invention, and may be prepared into a pharmaceutical composition by adding a pharmaceutically acceptable carrier according to the method for preparing a pharmaceutical.
이하 본 발명을 설명한다.Hereinafter, the present invention will be described.
본 발명자들은 알츠하이머병의 in vivo 동물모델에서 나타나는 인지기억능 저하 및 Aβ의 축적에 오배자 추출물이 유의한 약리효능을 나타내는지 규명하였다. The present inventors have investigated whether the gall bladder extract has a significant pharmacological effect on the decrease in cognitive memory and accumulation of Aβ in an in vivo animal model of Alzheimer's disease.
본 발명에서는 오배자 추출물이 Aβ (1-42)의 투여로 유도된 알츠하이머 치매 모델 및 APPswe/PS1dE9 이중발현 알츠하이머 치매 모델에서 유의하게 인지기억능을 향상시키고 amyloid plaque의 생성을 억제함을 규명하였고,그러한 결과는 알츠하이머병에 대한 오배자 추출물의 치료 후보물질로서의 유용성을 제시한다. In the present invention, it was confirmed that the gall bladder extract significantly improved cognitive memory and inhibited the production of amyloid plaque in the Alzheimer's dementia model induced by the administration of Aβ (1-42) and the APPswe / PS1dE9 double-expressed Alzheimer's dementia model. The results suggest the usefulness of the gall bladder extract as a therapeutic candidate for Alzheimer's disease.
또한 오배자 추출물은 면역조절능력이 있으며 면역기능을 개선하는 효과를 가진다.In addition, the gall bladder extract has an immunomodulatory ability and has an effect of improving immune function.
도 1은 Aβ의 중추투여로 성립하는 알츠하이머병 모델에서 오배자 에탄올 추출물 (Galla rhois)의 약리효능을 평가하기 위한 실험일정을 나타낸 그림. 1 is a diagram showing an experimental schedule for evaluating the pharmacological efficacy of the gall bladder ethanol extract ( Galla rhois ) in the Alzheimer's disease model established by the central administration of Aβ.
도 2는 APPswe/PS1dE9 이중발현 생쥐에서 UDCA와 오배자 에탄올 추출물 (Galla rhois)의 약리효능을 평가하기 위한 실험일정을 나타낸 그림. Figure 2 is a diagram showing the experimental schedule for evaluating the pharmacological efficacy of UDCA and gall bladder ethanol extract ( Gala rhois ) in APPswe / PS1dE9 double-expressing mice.
도 3은 β-amyloid (1-42)에 의한 인지기억능 저하에 대한 오배자 에탄올추출물 (Galla rhois)의 효과를 나타낸 그림. 인지기억능은 Y-maze test의 alteration behavior로 측정하였음. 각 군당 16 마리의 생쥐로 실험하였으며, 각 수치는 평균 ± 표준오차임. * P < 0.01 vs. Saline + β-amyloid (42-1), # P < 0.05vs. Saline + β-amyloid (1-42) (one-way ANOVA 및 사후검정으로서 Fisher's PLSD test를 사용).Figure 3 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by β-amyloid (1-42). Cognitive memory was measured by the alteration behavior of the Y-maze test. Sixteen mice in each group were tested, with each mean mean ± standard error. * P <0.01 vs. Saline + β-amyloid (42-1), # P <0.05 vs. Saline + β-amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
도 4는 β-amyloid (1-42)에 의한 인지기억능 저하에 대한 오배자 에탄올추출물 (Galla rhois)의 효과를 나타낸 그림. 인지기억능은 novel object recognition test를 이용하여 측정하였음. 각 군당 16 마리의 생쥐로 실험하였으며, 각 수치는 평균 ± 표준오차임. * P < 0.01 vs. Saline + β-amyloid (42-1), # P < 0.05vs. Saline + β-amyloid (1-42)(one-way ANOVA 및 사후검정으로서 Fisher's PLSD test를 사용).Figure 4 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by β-amyloid (1-42). Cognitive memory was measured using a novel object recognition test. Sixteen mice in each group were tested, with each mean mean ± standard error. * P <0.01 vs. Saline + β-amyloid (42-1), # P <0.05 vs. Saline + β-amyloid (1-42) (using Fisher's PLSD test as one-way ANOVA and post test).
도 5는 β-amyloid (1-42)에 의한 인지기억능 저하에 대한 오배자 에탄올추출물 (Galla rhois)의 효과를 나타낸 그림. 인지기억능은 water finding test의 finding latency를 관찰하여 측정하였음. 각 군당 16 마리의 생쥐로 실험하였으며, 각 수치는 평균 ± 표준오차임. * P < 0.01 vs. Saline + β-amyloid (42-1), # P < 0.05, ## P < 0.01 vs. Saline + β-amyloid (1-42) (one-way ANOVA 및 사후검정으로서 Fisher's PLSD test를 사용).Figure 5 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by β-amyloid (1-42). Cognitive memory was measured by observing the finding latency of the water finding test. Sixteen mice in each group were tested, with each mean mean ± standard error. * P <0.01 vs. Saline + β-amyloid (42-1), # P <0.05, ## P <0.01 vs. Saline + β-amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
도 6은 β-amyloid (1-42)에 의한 인지기억능 저하에 대한 오배자 에탄올추출물 (Galla rhois)의 효과를 나타낸 그림. 인지기억능은 passive avoidance test의 step-through latency를 관찰하여 측정하였음. 각 군당 16 마리의 생쥐로 실험하였으며, 각 수치는 평균 ± 표준오차임. * P < 0.01 vs. Saline + β-amyloid (42-1), # P < 0.05 vs. Saline + β-amyloid (1-42) (one-way ANOVA 및 사후검정으로서 Fisher's PLSD test를 사용).Figure 6 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by β-amyloid (1-42). Cognitive memory was measured by observing the step-through latency of the passive avoidance test. Sixteen mice in each group were tested, with each mean mean ± standard error. * P <0.01 vs. Saline + β-amyloid (42-1), # P <0.05 vs. Saline + β-amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
도 7은 β-amyloid (1-42)에 의한 인지기억능 저하에 대한 오배자 에탄올추출물 (Galla rhois)의 효과를 나타낸 그림. 인지기억능은 Morris water maze의 reference memory test를 이용하여 측정하였음. 각 군당 8마리의 생쥐를 매일 4번씩 측정하였으며, 각 수치는 평균 ± 표준오차임. * P < 0.01 vs. Saline + β-amyloid (42-1), # P < 0.05 vs. Saline + β-amyloid (1-42) (반복측정에 대한 one-way ANOVA 및 사후검정으로서 Fisher's PLSD test를 사용).Figure 7 is a diagram showing the effect of Galla rhois ( Galla rhois ) on cognitive memory degradation by β-amyloid (1-42). Cognitive memory was measured using the reference memory test of Morris water maze. Eight mice per group were measured four times daily, with each mean being ± standard error. * P <0.01 vs. Saline + β-amyloid (42-1), # P <0.05 vs. Saline + β-amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post-test for repeat measurements).
도 8은 β-amyloid (1-42)에 의한 인지기억능 저하에 대한 오배자 에탄올추출물 (Galla rhois)의 효과를 나타낸 그림. 인지기억능은 Morris water maze의 probe test를 이용하여 측정하였음. 각 군당 8마리의 생쥐를 2회 측정하였으며, 각 수치는 평균 ± 표준오차임. * P < 0.01 vs. Saline + β-amyloid (42-1), # P < 0.05 vs. Saline + β-amyloid (1-42) (one-way ANOVA 및 사후검정으로서 Fisher's PLSD test를 사용).Figure 8 is a diagram showing the effect of the gall ethanol extract ( Gala rhois ) on cognitive memory degradation by β-amyloid (1-42). Cognitive memory was measured using a probe test of Morris water maze. Eight mice in each group were measured twice, each mean ± standard error. * P <0.01 vs. Saline + β-amyloid (42-1), # P <0.05 vs. Saline + β-amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
도 9는 β-amyloid (1-42)에 의한 인지기억능 저하에 대한 오배자에탄올 추출물 (Galla rhois)의 효과를 나타낸 그림. 각 군당 8마리의 생쥐를 하루 4회씩 3일간 측정하였으며, 각 수치는 평균 ± 표준오차임. * P < 0.01 vs. Saline + β-amyloid (42-1), # P < 0.05, ## P < 0.01 vs. Saline + β-amyloid (1-42) (one-way ANOVA 및 사후검정으로서 Fisher's PLSD test를 사용).9 is a diagram showing the effect of Galla rhois extract ( Galla rhois ) on cognitive memory degradation by β-amyloid (1-42). Eight mice in each group were measured four times a day for three days, with each mean being ±± standard error. * P <0.01 vs. Saline + β-amyloid (42-1), # P <0.05, ## P <0.01 vs. Saline + β-amyloid (1-42) (using Fisher's PLSD test as a one-way ANOVA and post test).
도 10은 APPswe/PS1dE9 이중발현 생쥐의 인지기억능 (Y-maze test로 평가)에 대한 오배자 에탄올추출물 (Galla rhois)의 약리효과를 나타낸 그림. 각 수치는 10마리의 평균 ± 표준오차를 의미함. # P < 0.05 vs. Saline (One-way ANOVA. 사후검정으로 Fisher's 평가).10 is a diagram showing the pharmacological effect of Galla rhois on the cognitive memory of APPswe / PS1dE9 double-expressing mice (evaluated by the Y-maze test). Each figure represents the mean ± standard error of 10 animals. # P <0.05 vs. Saline (One-way ANOVA. Fisher's assessment by post-test).
도 11은 APPswe/PS1dE9 이중발현 생쥐의 인지기억능 (Novel object recognition test로 평가)에 대한 오배자 추출물 (Galla rhois)의 약리효과를 나타낸 그림. 각 수치는 10마리의 평균 ± 표준오차를 의미함. # P < 0.05 vs. Saline (One-way ANOVA. 사후검정으로 Fisher's 평가).Figure 11 shows the pharmacological effect of Gallia rhois extracts on cognitive memory (evaluated by Novel object recognition test) of APPswe / PS1dE9 double-expressing mice. Each figure represents the mean ± standard error of 10 animals. # P <0.05 vs. Saline (One-way ANOVA. Fisher's assessment by post-test).
도 12는 APPswe/PS1dE9 이중발현 생쥐의 인지기억능 (water finding test의 finding latency로 평가)에 대한 오배자 에탄올추출물 (Galla rhois)의 약리효과를 나타낸 그림. 각 수치는 10마리의 평균 ± 표준오차를 의미함. # P < 0.05, ## P < 0.01 vs. Saline (One-way ANOVA. 사후검정으로 Fisher's 평가).12 is a diagram showing the pharmacological effect of Galla rhois on the cognitive memory (evaluated by the finding latency of the water finding test) of APPswe / PS1dE9 double-expressing mice. Each figure represents the mean ± standard error of 10 animals. # P <0.05, ## P <0.01 vs. Saline (One-way ANOVA. Fisher's assessment by post-test).
도 13은 APPswe/PS1dE9 이중발현 생쥐의 해마와 대뇌피질 조직에서 amyloid plaque의 생성에 미치는 오배자 추출물 (Galla rhois)의 약리효과를 나타낸 그림. FIG. 13 shows the pharmacological effect of Gallia rhois extract on the production of amyloid plaque in hippocampus and cortical tissue of APPswe / PS1dE9 double-expressing mice.
도 14는 오배자 및 표준물질이 세포증식에 미치는 영향을 나타낸 그림. 생쥐에 실험물질 주입 후 획득한 비장세포를 Cell Proliferation Assay Kit (MTS)를 이용하여 세포증식 관찰.14 is a diagram showing the effect of the gall bladder and the standard on cell proliferation. Observation of cell proliferation of splenocytes obtained after injection of mice into the cell using Cell Proliferation Assay Kit (MTS).
도 15는 오배자 및 표준물질이 세포증식에 미치는 영향을 나타낸 그림. 비장세포 분리 후 실험물질 주입 후 Cell Proliferation Assay Kit (MTS)를 이용하여 세포증식 관찰.15 is a diagram showing the effect of the gall bladder and the standard on cell proliferation. After isolation of splenocytes, inject the test substance and observe cell proliferation using Cell Proliferation Assay Kit (MTS).
도 16은 오배자가 Nitric Oxide(NO) 생성에 미치는 영향을 나타낸 그림. 생쥐에서 대식세포 분리 후 실험물질 처리 후 시간에 따른 NO 측정.16 is a diagram showing the effect of the gall on Nitric Oxide (NO) production. Determination of NO with time after macrophage isolation from mice and treatment with experimental material.
도 17은 오배자가 Nitric Oxide(NO) 생성에 미치는 영향을 나타낸 그림. 생쥐에서 대식세포 분리 후 실험물질 농도에 따른 NO 측정.17 is a diagram showing the effect of the gall on Nitric Oxide (NO) production. Determination of NO according to test substance concentration after macrophage isolation from mice.
도 18은 오배자 및 표준물질이 Nitric Oxide(NO) 생성에 미치는 영향을 나타낸 그림. 생쥐에서 대식세포 분리 후 실험물질 처리 후 NO 측정.18 is a diagram showing the effect of the gall bladder and the standard on the production of Nitric Oxide (NO). Determination of NO after treatment of experimental material after macrophage isolation from mice.
도 19는 오배자 및 표준물질이 Nitric Oxide(NO) 생성에 미치는 영향을 나타낸 그림. 생쥐에서 혈액 분리 후 NO 측정.19 is a diagram showing the effect of the gall bladder and the standard on the production of Nitric Oxide (NO). NO measurement after blood isolation from mice.
도 20은 오배자 처리 후 시간에 따른 세포독성(cytotoxicity)효과를 나타낸 그림. Figure 20 is a diagram showing the cytotoxicity (cytotoxicity) effect over time after the gall bladder treatment.
도 21은 오배자 처리 후 농도에 따른 세포독성(cytotoxicity)효과를 나타낸 그림. Figure 21 is a diagram showing the cytotoxicity (cytotoxicity) effect of the concentration after the gall bladder treatment.
도 22는 오배자와 표준물질 처리 후 시간에 따른 세포독성(cytotoxicity)효과를 나타낸 그림. Figure 22 is a diagram showing the cytotoxicity (cytotoxicity) effect over time after treatment with the gall and standard.
도 23은 마우스에 오배자 및 표준물질 주사 후 적출한 비장세포의 사이토카인 발현 실험을 나타낸 그림.Figure 23 is a diagram showing the cytokine expression experiments of splenocytes harvested after injection of the gall bladder and standard in mice.
도 24는 마우스 대식세포에 오배자 처리 후 시간에 따른 대식세포의 사이토카인 분비에 관한 실험을 나타낸 그림. 24 is a diagram showing an experiment on the cytokine secretion of macrophages over time after the embryonic treatment in mouse macrophages.
도 25는 마우스 대식세포에 오배자 처리 농도에 따른 대식세포의 사이토카인 분비에 관한 실험(24시간 처리)을 나타낸 그림. Figure 25 is a diagram showing an experiment (24 hours treatment) on the cytokine secretion of macrophages according to the gall bladder treatment concentration in mouse macrophages.
도 26은 마우스 대식세포에 오배자 및 표준물질 처리 농도와 시간에 따른 대식세포의 사이토카인 분비에 관한 실험을 나타낸 그림.Fig. 26 is a diagram showing an experiment on cytokine secretion of macrophages with mouse embryonic and standard treatment concentrations and time in mouse macrophages.
도 27은 오배자 및 표준물질 투여 후 비장세포의 세포생존 관련인자 발현에 관한 실험을 나타낸 그림.Figure 27 is a diagram showing an experiment on the expression of factors related to cell survival of splenocytes after administration of gallocytes and standards.
도 28은 오배자 추출물 및 그 활성 화합물 처리된 마우스로부터 여러 측정치를 나타낸 그림.FIG. 28 shows several measurements from gall bladder extract and active compounds treated mice.
도 29는 오배자 추출물 및 그 활성 화합물 처리된 마우스로부터 RBC 및 혈소판들의 프로파일을 나타낸 그림.FIG. 29 is a diagram showing the profile of RBCs and platelets from gall bladder extract and its active compound treated mice.
도 30은 IL-6 mRNA 발현에 대한 gallic acid 및 그 유도체의 효과를 나타낸 그림. 비장을 gallic acid 및 그 유도체로 처리된 마우스로부터 분리하고, IL-6 수준을 realtime PCR로 측정하였음. 데이터는 평균±SEM (n=3)으로 나타냄. 30 is a diagram showing the effect of gallic acid and its derivatives on IL-6 mRNA expression. Spleens were isolated from mice treated with gallic acid and derivatives thereof, and IL-6 levels were measured by realtime PCR. Data are presented as mean ± SEM (n = 3).
도 31은 TNF-알파 발현에 대한 gallic acid 및 그 유도체의 효과를 나타낸 그림. 비장을 gallic acid 및 그 유도체로 처리된 마우스로부터 분리하고, IL-6 수준을 realtime PCR로 측정하였음. 데이터는 평균±SEM (n=3)으로 나타냄.Figure 31 shows the effect of gallic acid and its derivatives on TNF-alpha expression. Spleens were isolated from mice treated with gallic acid and derivatives thereof, and IL-6 levels were measured by realtime PCR. Data are presented as mean ± SEM (n = 3).
도 32 및 33은 30일 동안 gallic acid 및 그것의 유도체 처리된 C57BL/6 마우스에서 ConA 및 LPS-자극된 비장 증식을 나타낸 그림. 데이터는 평균±SEM (n=3)으로 나타냄.32 and 33 show ConA and LPS-stimulated spleen proliferation in gallic acid and its derivative treated C57BL / 6 mice for 30 days. Data are presented as mean ± SEM (n = 3).
도 34는 오배자 추출물 및 그것의 활성 화합물 처리된 마우스 유래 비장의 배양 상등액에서 NO의 농도를 나타낸 그림.데이터는 평균±SEM (n=3)으로 나타냄.Fig. 34 shows the concentration of NO in culture supernatants of mouse gall bladder extract and mouse-derived spleens treated with the active compounds thereof. Data are shown as mean ± SEM (n = 3).
도 35는 forced swimming 테스트에서 부동 시간(immobility time)에 대한 오배자의 효과를 나타낸 그림. 물질들을 마우스에 경구 투여하였고, 값들은 3 마우스에 대한 평균±SEM임.FIG. 35 is a diagram showing the effect of a gall on immobility time in a forced swimming test. FIG. Substances were administered orally to mice and values were mean ± SEM for 3 mice.
도 36은 forced swimming 테스트에서 부동 시간(immobility time)에 대한 gallic acid 및 그 구성성분들의 효과를 나타낸 그림. 물질들을 마우스에 경구 투여하였고, 값들은 3 마우스에 대한 평균±SEM임.N: Control, S : Syringic acid,G : Gallic acid, M : Methyl gallate, E : 오배자 추출물(Extracts from Galla rhois )36 shows the effect of gallic acid and its components on immobility time in a forced swimming test. Substances were administered orally to mice, with mean ± SEM for 3 mice. N: Control, S: Syringic acid, G: Gallic acid, M: Methyl gallate, E: Extract from Galla rhois
실시예 :오배자 추출물의 제조Example: Preparation of Mellitus Extract
오배자 (K P) 5kg을 정선하여 약전통칙 절도 및 분말도에 따라 조절로 한 다음 생약을 달아 추출기에 넣고 8-10배의 95% 에탄올을 넣어 교반추출기에서 5~6시간 2회 추출하여 추출액을 1㎛ 크기의 마이크로필터를 이용하여 여과하고 여액을 50℃이하에서 감압농축한 후 건조하여 건조엑스 2.39kg을 얻었다.(수득율 47.8%)Select 5kg 5g (KP) and adjust it according to pharmacopoeia theft and powder level. Then, put the herbal medicine into the extractor, put 8-10 times 95% ethanol and extract twice in the stirring extractor for 5-6 hours to extract the extract. Filtration was carried out using a micro filter having a size of µm, and the filtrate was concentrated under reduced pressure at 50 ° C. or lower and dried to obtain 2.39 kg of dry extract. (Yield 47.8%)
실험예 1:알츠하이머병 모델에서 오배자 추출물의 약리효능평가Experimental Example 1: Evaluation of Pharmacological Efficacy of Mellitus Extract in Alzheimer's Disease Model
실험동물 및 약물투여Laboratory Animals and Drug Administration
1) Aβ의 중추투여로 성립하는 알츠하이머병 모델에서의 약물투여 및 실험일정1) Drug administration and experimental schedule in Alzheimer's disease model established by central administration of Aβ
Aβ에 의한 인지기억능의 저하는 12주령의 생쥐에서 평가되었다. ICR 생쥐 [(주)오리엔트바이오, 경기, 한국]를 구입하여 실험동물실에 1주일간 적응시키고, 약물투여 및 인지기억능의 평가는 도 1과 같은 일정으로 실시하였다. 독성의 Aβ (1-42) (American Peptide Company, Sunnyvale, CA, USA)와 대조물질인 무독성의 Aβ (42-1) (American Peptide Company, Sunnyvale, CA, USA)은 400 pmol을 제 3뇌실로 투여하였다. Aβ의 투여 3일 후부터 인지기억능 평가를 실행하였다. 오배자 에탄올추출물 (250 또는 500 mg/kg, p.o.)은 Aβ 투여 2일 전부터 인지기억능 평가가 끝날 때까지 1일 1회씩 경구투여 하였다. 인지기억능 평가가 이루어지는 기간에는 행동에 약물들이 직접적인 영향을 미치는 것을 막기 위하여, 행동 및 인지기능 평가가 끝난 후 약물을 투여하였다. Reduction of cognitive memory by Aβ was assessed in 12-week-old mice. ICR mice [Orient Bio Co., Gyeonggi, Korea] were purchased and adapted to the experimental animal room for 1 week, and drug administration and evaluation of cognitive memory were performed on the schedule as shown in FIG. 1. Toxic Aβ (1-42) (American Peptide Company, Sunnyvale, CA, USA) and the non-toxic Aβ (42-1) (American Peptide Company, Sunnyvale, CA, USA), a control, have 400 pmol Administered. Cognitive memory evaluation was performed three days after the administration of Aβ. The gall bladder ethanol extract (250 or 500 mg / kg, p.o.) was administered orally once a day from 2 days before Aβ administration until the end of cognitive memory evaluation. During the cognitive memory evaluation period, the drug was administered after the behavioral and cognitive function evaluation to prevent the drugs from directly affecting the behavior.
2) 알츠하이머병 모델인 amyloid precursor protein/presenilin-1 (APPswe/PS1dE9) 이중발현 생쥐에 대한 약물투여 및 실험일정2) Drug administration and experimental schedule for amyloid precursor protein / presenilin-1 (APPswe / PS1dE9) double-expressing mice, Alzheimer's disease model
본 실험에는 6월령의 APPswe/PS1dE9 이중발현 생쥐 (The Jackson Laboratory, Bar Harbor, MA, USA)를 사용하였다. APPswe/PS1dE9 이중발현 생쥐에 오배자 에탄올추출물 (250 또는 500 mg/kg, p.o.)을 1일 1회씩 3개월간 경구투여 한 후 인지기억능 평가를 실행하였다(도 2). 인지기억능 평가가 이루어지는 기간에는 행동에 약물들이 직접적인 영향을 미치는 것을 막기 위하여, 행동 및 인지기능 평가가 끝난 후 약물을 투여하였다. 인지기억능 평가가 끝나고 1일 후 동물을 희생시켜 Aβ의 축적 정도를 평가하였다. In this experiment, APPswe / PS1dE9 double-expressing mice (The Jackson Laboratory, Bar Harbor, MA, USA) of 6 months of age were used. APPSwe / PS1dE9 double-expressing mice were administered orally with a gall bladder ethanol extract (250 or 500 mg / kg, p.o.) once a day for 3 months (Fig. 2). During the cognitive memory evaluation period, the drug was administered after the behavioral and cognitive function evaluation to prevent the drugs from directly affecting the behavior. One day after the end of the cognitive memory evaluation, the animals were sacrificed to evaluate the accumulation of Aβ.
인지기억능 평가법Cognitive memory evaluation method
(1) Y-maze test(1) Y-maze test
Y-maze 의 구성은 Y-자 형태의 arm 으로 되어 있고 길이 25 cm 와 높이 14 cm 넓이 5 cm 의 검은 plastic 상자로 되어 있다. 마우스를 한쪽 arm의 끝에 놓고 8분 동안 자유롭게 arm을 왕래하도록 하였다. 마우스가 각각의 arm에 들어가는 횟수를 video camera로 찍었다. Alternation behavior (공간 교체행동)의 평가지표는 3개의 arm을 모두 들어가는 경우에 1점을 획득하는 것으로 하여 대조군과 비교하였다. 마우스의 alternation score는 실제 마우스가 획득한 score를 가능한 alternation score 로 나누어 100% 로 환산하였다.The Y-maze consists of a Y-shaped arm and a black plastic box 25 cm long and 14 cm high and 5 cm wide. The mouse was placed at the end of one arm and allowed to move arm freely for 8 minutes. The number of times the mouse enters each arm was recorded with a video camera. The evaluation index of alternation behavior was compared with the control group by obtaining one point when all three arms were included. The alternation score of the mouse was converted to 100% by dividing the actual score obtained by the mouse with the possible alternation score.
(2) Novel object test(2) Novel object test
첫 날에는 생쥐를 40 cm X 40 cm X 30 cm 의 상자에 넣고 10 분간 자유롭게 이동하도록 하여 적응시켰다. 2일째에는 상자에 두 개의 물체를 놓고 각각의 물체에 대한 반응시간을 기록하였다. 이로부터 24시간 후 두 개중 한 개의 물체를 새로운 물체 (novel object)로 바꾼 후 novel object에 대한 반응시간을 기록하였다. On the first day the mice were placed in a 40 cm x 40 cm x 30 cm box and allowed to move freely for 10 minutes. On day 2, two objects were placed in a box and the reaction time for each object was recorded. After 24 hours, one of the two objects was replaced with a novel object and the reaction time for the novel object was recorded.
(3) Water finding test(3) Water finding test
Water finding test는 latent learning 및 attentional spatial memory를 평가하는 방법으로, 1일에 생쥐로 하여금 apparatus 안에서 물병의 노즐 위치를 인지하게끔 한 다음, 24시간의 절수 후에 다시 apparatus 안에 위치시켜 물병의 노즐을 찾도록 하여 그 시간을 측정하였다(Finding latency). 단 2일에는 물병의 위치 및 노즐의 길이를 약간 변화시켜, 1일에 습득한 정보를 바탕으로 하여, 주의력을 기울여서 새로운 위치를 찾게끔 하였다. The water finding test is a method of evaluating latent learning and attentional spatial memory that allows the mice to recognize the nozzle position of the water bottle in the apparatus on day 1, and then place it in the apparatus again after 24 hours of water saving to find the nozzle of the water bottle. The time was measured (Finding latency). On the second day, the position of the water bottle and the length of the nozzle were slightly changed, and based on the information acquired on the first day, attention was paid to find a new position.
(4) Passive avoidance test(4) Passive avoidance test
Passive avoidance test 장치는 두 개의 방으로 이루어져 있으며, 두 방의 사이에는 guillotine door로 나뉘어져 있다. 방의 바닥에는 쇼크 발생기가 장치되어져 있다. 1일에는 acquisition trial로서 쥐를 두 개의 방 중 한 개의 방에 넣고 15초간 적응시킨 후에 쥐가 있는 방의 불을 켬과 동시에 guillotine door를 열어 줌. 쥐가 밝은 방으로부터 어두운 방으로 이동함과 동시에 guillotine door가 닫히고 어두운 방의 바닥에 electric shock (0.5mA, 5s)을 주었다. 2일에는 같은 장치에 쥐를 넣고 밝은 방에서 어두운 방으로 이동하는 시간을 측정하였다(step-through latency). Passive avoidance test The device consists of two rooms with a guillotine door between them. The floor is equipped with a shock generator. On the 1st day, the rat was placed in one of the two rooms as an acquisition trial and allowed to acclimate for 15 seconds. Then, the rat's room was turned on and the guillotine door was opened. As the mouse moved from the bright room to the dark room, the guillotine door was closed and electric shock (0.5mA, 5s) was applied to the floor of the dark room. On day 2, the mice were placed in the same device and the time to move from the light room to the dark room was measured (step-through latency).
(5) Morris water maze test (5) Morris water maze test
Water maze 평가를 위한 수조는 지름이 97cm이고 높이가 60cm인 원통형 수조를 사용하였다. 테스트 기간동안 분유를 희석한 23±2°C의 물을 채워 사용하였다. 수조 안에는 수면에서 2cm 아래에 투명한 플랫폼을 장치하였으며, 수조 바깥쪽에 네 개의 표지를 장치하였다. 마우스의 운동궤적은 비디오트래킹 시스템 (EthoVision, Noldus, The Netherlands)을 통하여 분석되었다.For the water maze evaluation, a cylindrical tank having a diameter of 97 cm and a height of 60 cm was used. During the test period, powdered milk was used, filled with 23 ± 2 ° C diluted water. The tank was equipped with a transparent platform 2 cm below the surface of the water and four covers on the outside of the tank. The motion trajectories of the mice were analyzed by video tracking system (EthoVision, Noldus, The Netherlands).
(5-1) Reference memory test(5-1) Reference memory test
각 trial 기간 동안 마우스는 다섯 군데의 시작점을 무작위로 선택한 지점에서 플랫폼을 보지 못하는 쪽을 향하여 수조안으로 넣어졌다. 각 trial에서는 마우스가 수조로 넣어진 후 플랫폼을 찾는 시간 (escape latency) 을 기록하게 되며, 마우스가 플랫폼을 찾으면 10초간 플랫폼 위에서 머무르게 한 후 home cage로 옮겨지게 된다. 마우스가 60초 내로 플랫폼을 찾지 못할 경우 escape latency는 60초로 기록하였다. 각 trial은 β-amyloid를 투여한 3일 후부터 4일간 (3일~6일), 하루 네 번씩 실행되었다.During each trial, mice were placed in tanks facing the platform at the randomly selected five start points. Each trial records the escape latency after the mouse is placed in the tank, and when the mouse is found, the mouse stays on the platform for 10 seconds before being transferred to the home cage. When the mouse could not find a platform within 60 seconds, the escape latency was recorded as 60 seconds. Each trial was performed four times a day for four days (3 days to 6 days) from 3 days after β-amyloid administration.
(5-2) Probe test(5-2) Probe test
β-amyloid 투여 7일 후에 probe test를 진행하였다. 플랫폼을 제거한 후, 60초간 쥐로 하여금 수조안을 수영하게 하면서 플랫폼이 있던 위치를 몇 번이나 지나는지 기록하였다.Probe test was performed 7 days after β-amyloid administration. After removing the platform, the rats were allowed to swim in the tank for 60 seconds and the number of times they passed the platform was recorded.
(5-3) Working memory test(5-3) Working memory test
Working memory test는 β-amyloid 투여 후 8일~10일에 진행하였다. Reference memory test와 비슷하지만, 플랫폼의 위치를 매일 바꾸어 주면서 escape latency를 측정하였다. 하루에 5번의 trial을 진행하며, 2번째~5번째 trial의 escape latency를 사용하였다.    Working memory test was performed 8 to 10 days after β-amyloid administration. It is similar to the reference memory test, but the escape latency was measured by changing the platform location every day. Five trials were performed per day, and the escape latency of the second to fifth trials was used.
면역조직화학적 염색Immunohistochemical Staining
APPswe/PS1dE9 이중발현 생쥐의 해마와 피질조직에서 Aβ plaque의 생성은 면역조직화학적 염색법을 이용하여 평가하였다. Perfusion 시켜서 4% p-formaldehyde로 고정된 뇌조직을 40μm씩으로 절단 후 Aβ (1-42) antibody (Invitrogen, Carlsbad, CA, USA)에 1일 밤 배양시키기 전에 section을 0.2% Triton X-100에 15분간 노출시키고 4% normal goat serum에 배양시켰다. 1일 밤이 지난 후 secondary antibody에 1시간 배양시키고 3,3-diaminobenzidine을 chromogen으로 하여 면역 염색함. 각 단계마다 PBS (pH 7.4)로 세척하였다. The production of Aβ plaque in hippocampus and cortex of APPswe / PS1dE9 double-expressing mice was evaluated by immunohistochemical staining. Perfusion was used to cut brain tissue fixed with 4% p-formaldehyde at 40μm intervals, and then sectioned with 0.2% Triton X-100 before incubation overnight with Aβ (1-42) antibody (Invitrogen, Carlsbad, CA, USA). It was exposed for a minute and incubated in 4% normal goat serum. After 1 night, incubate with secondary antibody for 1 hour and immunostain with 3,3-diaminobenzidine as chromogen. Each step was washed with PBS (pH 7.4).
통계statistics
군간 통계학적인 유의성은 일원분산분석 (one-way ANOVA) 혹은 중복측정 일원분산분석 (one-way ANOVA for repeated measures)을 사용하였으며, 사후검정으로서 Fisher's PLSD 평가 혹은 Bonferroni's 평가를 수행하였다. The statistical significance between groups was one-way ANOVA or one-way ANOVA for repeated measures. Fisher's PLSD or Bonferroni's evaluation was performed as a post test.
상기 동물효력시험 결과는 하기와 같다.The animal efficacy test results are as follows.
β-amyloid (1-42)의 투여로 유도된 spatial short-term memory의 저하에 대한 오배자 에탄올 추출물의 인지기억능 개선효과Cognitive Memory Enhancement Effect of Methanol Ethanol Extract on Degradation of Spatial Short-term Memory Induced by β-amyloid (1-42)
β-amyloid (1-42)의 투여는 Y-maze에서 alteration behavior를 유의하게 감소시켰다. 오배자 추출물은 β-amyloid (1-42)에 의한 alteration behavior의 감소를 유의하게 억제시켰다(도 3). Administration of β-amyloid (1-42) significantly reduced alteration behavior in Y-maze. The gall bladder extract significantly inhibited the reduction of alteration behavior by β-amyloid (1-42) (FIG. 3).
β-amyloid (1-42)의 투여로 유도된 visual recognition memory의 저하에 대한 오배자 에탄올추출물의 효과Effect of Mellitus Ethanol Extract on Degradation of Visual Recognition Memory Induced by Administration of β-amyloid (1-42)
β-amyloid (1-42)의 투여는 novel object recognition test에서 novel object에 대한 exploration을 유의하게 감소시켰다. 오배자 추출물은 β-amyloid (1-42)에 의한 novel object에 대한 exploration의 감소를 유의하게 억제시켰다 (도 4). Administration of β-amyloid (1-42) significantly reduced the exploration of novel objects in the novel object recognition test. The gall bladder extract significantly inhibited the reduction of exploration of novel objects by β-amyloid (1-42) (FIG. 4).
β-amyloid (1-42)의 투여로 유도된 latent learning의 저하에 대한 오배자 에탄올추출물의 인지기억능 개선효과Improvement of Cognitive Memory on Ginseng Ethanol Extract on Degradation of Latent Learning Induced by β-amyloid (1-42) Administration
β-amyloid (1-42)의 투여는 latent learning을 평가하는 water finding test에서 finding latency를 유의하게 증가시켰다. 오배자 추출물은 β-amyloid (1-42)에 의한 finding latency의 증가를 유의하게 억제하였다(도 5). The administration of β-amyloid (1-42) significantly increased the finding latency in the water finding test to assess latent learning. The gall bladder extract significantly inhibited the increase in finding latency caused by β-amyloid (1-42) (FIG. 5).
β-amyloid (1-42)의 투여로 유도된 fear learning 및 memory의 저하에 대한 오배자 에탄올추출물 인지기억능 개선효과Effect of β-amyloid (1-42) on Fear Learning and Memory Degradation
β-amyloid (1-42)의 투여는 fear learning 및 memory를 평가하는 passive avoidance test에서 step-through latency를 유의하게 감소시켰다. 오배자 추출물은 β-amyloid (1-42)에 의한 step-through latency의 감소를 유의하게 억제하였다 (도 6). The administration of β-amyloid (1-42) significantly reduced step-through latency in passive avoidance tests evaluating fear learning and memory. The gall bladder extract significantly inhibited the decrease of step-through latency by β-amyloid (1-42) (FIG. 6).
β-amyloid (1-42)의 투여로 유도된 spatial reference memory의 저하에 대한 오배자 에탄올추출물의 인지기억능 개선효과Effect of β-amyloid (1-42) on the Reduction of Spatial Reference Memory and Its Effect on Cognitive Memory
β-amyloid (1-42)의 투여는 Morris water maze의 hidden platform test 및 probe test를 통하여 평가한 결과, 유의하게 spatial reference memory의 저하를 유도하였다. 즉, β-amyloid (1-42)를 투여한 생쥐는 β-amyloid (42-1)을 투여한 생쥐에 비하여 수조 안의 안전한 platform을 찾는 시간이 유의하게 감소되었을 뿐 아니라, platform을 빼고 측정하는 probe test에서도 platform이 있던 quadrant에 머무는 시간도 유의하게 감소되었다. 오배자 에탄올추출물은 β-amyloid (1-42)에 의한 escape latency의 증가 및 platform이 있던 quadrant에서의 체류시간 감소를 유의하게 억제하였다(도 7과 8). The β-amyloid (1-42) was evaluated by the hidden platform test and the probe test of Morris water maze, which significantly reduced the spatial reference memory. In other words, mice that received β-amyloid (1-42) significantly reduced the time to find a safe platform in the tank compared to mice that received β-amyloid (42-1). The test also significantly reduced the time to stay on the quadrant where the platform was. Methanol ethanol extract significantly inhibited the increase in escape latency by β-amyloid (1-42) and decrease in residence time in the quadrant with the platform (FIGS. 7 and 8).
β-amyloid (1-42)의 투여로 유도된 spatial working memory의 저하에 대한 오배자 에탄올추출물의 인지기억능 개선효과Improvement of Cognitive Memory on Ginseng Ethanol Extract on Decreased Spatial Working Memory Induced by β-amyloid (1-42) Administration
β-amyloid (1-42)의 투여는 Morris water maze의 working memory test에서 유의하게 spatial working memory의 저하를 유도하였다. 오배자 추출물은 β-amyloid (1-42)에 의한 escape latency의 증가를 유의하게 억제하였다(도 9). The administration of β-amyloid (1-42) significantly induced a decrease in spatial working memory in Morris water maze's working memory test. The gall bladder extract significantly inhibited the increase in escape latency by β-amyloid (1-42) (FIG. 9).
APPswe/PS1dE9 이중발현 생쥐에서 spatial short-term memory에 대한 오배자 추출물의 인지기억능 개선효과Improving Cognitive Memory Effect of Gallia Extracts on Spatial Short-term Memory in APPswe / PS1dE9 Double-expressing Mice
APPswe/PS1dE9 이중발현 생쥐에서 단기공간기억력 (short-term spatial)을 평가하는 Y-maze test를 수행한 결과, 오배자 추출물은 alteration behavior를 유의하게 증가시켰다(도 10). The Y-maze test, which evaluates short-term spatial memory in APPswe / PS1dE9 double-expressing mice, showed significantly increased alteration behavior (Fig. 10).
*APPswe/PS1dE9 이중발현 생쥐에서 visual recognition memory에 대한 오배자 에탄올추출물의 개선효과* Improvement effect of gall bladder ethanol extract on visual recognition memory in APPswe / PS1dE9 double-expressing mice
APPswe/PS1dE9 이중발현 생쥐에서 시각재인지기억 (visual recognition memory)을 평가하는 novel object recognition test를 실시한 결과, 오배자 추출물은 novel object에 대한 exploration을 유의하게 증가시켰다(도 11). As a result of a novel object recognition test evaluating visual recognition memory in APPswe / PS1dE9 double-expressing mice, the gall bladder extract significantly increased the exploration of novel objects (FIG. 11).
APPswe/PS1dE9 이중발현 생쥐에서 latent learning에 대한 오배자 추출물의 개선효과Improved Effect of Mellitus Extracts on Latent Learning in APPswe / PS1dE9 Double-expressing Mice
APPswe/PS1dE9 이중발현 생쥐에서 잠재학습 (latent learning)을 평가하는 water finding test를 실시한 결과, 오배자 에탄올추출물은 finding latency를 유의하게 감소시켰다(도 12). As a result of performing a water finding test evaluating latent learning in APPswe / PS1dE9 double-expressing mice, the gall bladder ethanol extract significantly reduced the finding latency (FIG. 12).
APPswe/PS1dE9 이중발현 생쥐에서 amyloid plaque 생성에 대한 오배자 에탄올추출물의 억제효과Inhibitory Effect of Methanol Ethanol Extract on Amyloid Plaque Production in APPswe / PS1dE9 Double-expressing Mice
APPswe/PS1dE9 이중발현 생쥐의 해마와 대뇌피질 조직에서 면역조직화학적 염색법을 이용하여 amyloid plaque 생성은 평가한 결과, 오배자 추출물은 amyloid plaque 형성을 억제하는 약리효능을 나타내었다(도 13). Amyloid plaque production was evaluated by immunohistochemical staining in hippocampus and cerebral cortex tissues of APPswe / PS1dE9 double-expressing mice, and the gall extract showed pharmacological effects of inhibiting amyloid plaque formation (FIG. 13).
실험예 2:오배자추출물의 면역학적 활성Experimental Example 2: Immunological Activity of Gallia Extract
동물animal
생후 7-8 주령 SPF급 BALB/c 및 C5BL/6 수컷 마우스를 오리엔트바이오(Seongnam, Korea)에서 구입하여 연세대학교 원주의과대학 동물실험 사육실에서 온도 22℃, 상대습도 56±5%, 12시간 명암주기 하에서 사육하였다. 사료는 삼양유지사료(강원도, 원주시, 대한민국)의 마우스용 배합사료를 자유롭게 먹을 수 있게 하였고 물은 동물실의 수돗물을 정수하여 공급하였다. 일주일간 순화기간을 거친후 마우스를 희생시켜 비장세포와 복강대식세포를 분리하였다. 실험은 동 대학 동물실험실 규칙을 준수하였고, 동물윤리위원회 규정에 의거하여 실험하였다.   SPF-grade BALB / c and C5BL / 6 male mice, 7-8 weeks old, were purchased from Orient Bio (Seongnam, Korea) and were tested at Yonsei University's Wonju College of Animal Experimental Laboratory, temperature 22 ℃, relative humidity 56 ± 5%, 12 hours Breeding under cycles. The feed was free to eat the mouse feed of Samyang oil feed (Gangwon-do, Wonju-si, Korea) and the water was supplied by tap water in the animal room. After one week of acclimation, mice were sacrificed to separate splenocytes and peritoneal macrophages. Experiments were conducted in accordance with the rules of the animal laboratory of the university and in accordance with the regulations of the Animal Ethics Committee.
마우스 비장세포 (splenocyte, SP)의 분리 Isolation of Mouse Splenocytes (SP)
마우스를 경추 탈구법으로 희생시켜 비장을 꺼냈다. stainless 200-mesh 및 한쪽에 거친 면이 있는 무균 유리 슬라이드를 이용하여 비장세포를 분리하였다. 분리된 세포부유액을 실온에서 1000 rpm으로 10분간 원심분리한 후 남아있는 적혈구는 0.83% NH4Cl로 실온에서 5분간 용해시키고 PBS로 세척하였다. 세포침전물을 10% 우태아 혈청이 함유된 DMEM 또는 RPMI 배지에 부유시킨 후 5% CO2가 함유된 37℃ 배양기에서 배양하였다. The mouse was sacrificed by cervical dislocation to remove the spleen. Splenocytes were isolated using a sterile glass slide with a stainless steel 200-mesh and one side rough. The separated cell suspension was centrifuged at 1000 rpm for 10 minutes at room temperature, and the remaining erythrocytes were lysed with 0.83% NH 4 Cl for 5 minutes at room temperature and washed with PBS. The cell precipitate was suspended in DMEM or RPMI medium containing 10% fetal bovine serum and then cultured in a 37 ° C. incubator containing 5% CO 2 .
마우스 복강대식세포 분리Mouse Peritoneal Macrophage Isolation
BALB/c 마우스에 무균 처리한 3% thioglycolate broth (Difico Laboratories, Detroit, MI, USA) 3 ml를 복강 내 주사하고, 3 일 후 마우스를 희생시켜 복강을 5 ml의 PBS로 3번 세척하여 모은 후 2000 rpm으로 20분간 원심 분리하여 대식세포를 얻었다. 얻은 세포침전물을 DMEM에 부유시킨 후 5X105cell/well되게 24 well plate에 나누어 담고 5% CO2가 함유된 37℃ 배양기에서 배양하였다. 24시간 배양후 시료를 대식세포, 1X107cell/well에 처리하였다. BALB / c mice were intraperitoneally injected with 3 ml of sterile 3% thioglycolate broth (Difico Laboratories, Detroit, MI, USA), and sacrificed mice three days later to collect abdominal cavity 3 times with 5 ml of PBS. Macrophages were obtained by centrifugation at 2000 rpm for 20 minutes. The obtained cell precipitate was suspended in DMEM, divided into 24 well plates at 5 × 10 5 cells / well, and cultured in a 37 ° C. incubator containing 5% CO 2 . After incubation for 24 hours, the samples were treated with macrophages, 1 × 10 7 cells / well.
세포증식 및 유약화실험Cell Proliferation and Glazing Experiment
두 차례 세척한 후 얻은 마우스 비장세포를 1X106cells/ml개로 이용하여 분석하였다. 비장세포의 증식능은 비색법을 이용하였고 유약화 실험을 위하여 B 세포 분열원질(mitogen)인 LPS와 T 세포 증식제인 Con A를 이용하였다. 비장세포는 10% FBS가 첨가된 RPMI-1640 medium상에서 배양되었고 실험을 위하여 LPS와 Con A가 50ul 씩 첨가된 96 well plate에 2 x 106cells/ml의 cell을 50ul씩 분주하였으며 well마다 20 ㎕의 MTS/PMS 가하여 72시간 배양 후 490nm의 파장에서 분석하였다. Mouse splenocytes obtained after washing twice were analyzed using 1 × 10 6 cells / ml. Splenocyte proliferation was performed by colorimetric method, and LPS and B cell mitogen Con A were used for the experiment of weakening. Splenocytes were cultured on RPMI-1640 medium with 10% FBS and 50ul of 2 x 10 6 cells / ml were added to a 96 well plate containing 50ul of LPS and Con A for experiment. MTS / PMS was added and analyzed at a wavelength of 490 nm after 72 hours of incubation.
배양 상등랙의 NO 측정 NO measurement of culture upper rack
NO는 Griess assay법을 이용하여 비장세포 배양액의 질소 양을 측정하였다. 100ul의 배양액에 600ul의 Griess reagent를 혼합하였고 30분 동안 실온에서 배양 후 540nm 파장에서 측정하였으며 standard control로는 NaNO2를 사용하였다.NO was measured for nitrogen in splenocyte culture medium using Griess assay. 600ul of Griess reagent was mixed in 100ul of culture solution, incubated at room temperature for 30 minutes, and measured at 540nm wavelength. NaNO 2 was used as a standard control.
RNA분리 및 cDNA 합성RNA isolation and cDNA synthesis
TRIzol Reagent (InvitrogenLifeTechnologies,Carlsbad, CA, USA)를 이용하여 일차 분리 세포 또는 세포주의 총 RNA를 분리하였다. 그 과정으로는 TRIzol 시약으로 세포를 깬 후 chloroform을 첨가한 후 15초간 진탕하였다. 원심분리 후에 RNA가 포함된 상층액을 tube에 옮겨 담은 후 isopropanol를 첨가하고 상온에 10 분간 두었다가 4℃에서 12,000 rpm으로 15분간 원심분리하였다. 다음 RNA 침전물을 제외한 나머지 용액을 버리고 75% 에탄올을 첨가하여 혼합한 후 4℃에서 7,500 rpm으로 10 분간 원심분리하였다. 이후 상층액은 제거하고 RNA 침전물을 공기 중에서 건조시킨 후 RNase와 DNase를 포함하지 않은 증류수로 RNA를 녹이고 나서 RNA를 정량하였다. cDNA를 합성하기 위해 1 mg의 RNA, 500 ng의 oligo-dT primer, 15 U avian myeloblastosis virus 역전사효소, 20 U의 RNase inhibitor, 1 mM의 dNTP, 5 mM의 MgCl2를 혼합하여 반응액을 만든 후 (Reverse Transcription System A3500: Promega, Madison, WI, USA) GeneAmp PCR system (Applied Biosystems, Foster City, CA, USA)을 이용하여 42℃에서 1시간 반응시킨 후 95에서 5분간, 4℃에서 10분간 두었다가 실험에 사용할 때까지 -20℃에 보관하였다.Total RNA was isolated from primary isolated cells or cell lines using TRIzol Reagent (Invitrogen Life Technologies, Carlsbad, Calif., USA). As a procedure, the cells were crushed with TRIzol reagent and shaken for 15 seconds after the addition of chloroform. After centrifugation, the RNA-containing supernatant was transferred to the tube, and isopropanol was added thereto, and the mixture was placed at room temperature for 10 minutes and centrifuged at 12,000 rpm for 15 minutes at 4 ° C. The remaining solution except the RNA precipitate was discarded and mixed with 75% ethanol, followed by centrifugation at 7,500 rpm for 10 minutes at 4 ° C. The supernatant was then removed, the RNA precipitate was dried in air, and the RNA was quantified after dissolving the RNA with distilled water containing no RNase and DNase. In order to synthesize cDNA, a reaction solution was prepared by mixing 1 mg RNA, 500 ng oligo-dT primer, 15 U avian myeloblastosis virus reverse transcriptase, 20 U RNase inhibitor, 1 mM dNTP, and 5 mM MgCl 2 . (Reverse Transcription System A3500: Promega, Madison, WI, USA) Using the GeneAmp PCR system (Applied Biosystems, Foster City, Calif., USA) for 1 hour at 42 ° C and 5 minutes at 95 and 10 minutes at 4 ° C Store at −20 ° C. until used for experiment.
반 정량-중합효소연쇄반응 (semi-quantitative PCR)Semi-quantitative PCR
역전사 반응의 결과로 만들어진 cDNA는 각각의 실험목적에 맞게 IL-10, IL-6, 베타-actin에 대한 primer을 사용하여 증폭하였다. 중합효소연쇄반응을 수행하기 위하여 cDNA 1 ml, 10' 완충용액 (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl2), 0.2m M dNTP, 1.25U Taqpolymerase (Takara Biochemicals, Shiga, Japan), primer를 넣어서 25 ml의 반응액을 만든 후 GeneAmp PCR system 2400 (Applied Biosystems)에 넣고 적절한 조건으로 중합효소연쇄반응을 수행하였다. 중합효소연쇄반응의 산물은 1.5% agarose gel에 50 mV, 40분간 전기영동하여 관찰하였다. The cDNA produced as a result of the reverse transcription reaction was amplified using primers for IL-10, IL-6, and beta-actin for each experimental purpose. To perform polymerase chain reaction, 1 ml of cDNA, 10 'buffer solution (100 mM Tris-HCl, pH 8.3, 500 mM KCl, 15 mM MgCl 2 ), 0.2 mM dNTP, 1.25 U Taqpolymerase (Takara Biochemicals, Shiga, Japan), 25 ml of the reaction solution was added to the primers, and then placed in GeneAmp PCR system 2400 (Applied Biosystems). The polymerase chain reaction was performed under appropriate conditions. The product of the polymerase chain reaction was observed by electrophoresis at 50 mV for 40 minutes on 1.5% agarose gel.
실시간 중합효소연쇄반응 (Real-Time PCR)Real-Time PCR
mRNA수준의 발현의 정량분석을 위하여 Real-Time PCR을 수행하였으며 주형으로 mRNA로부터50ng/ul의 cDNA를 제조하여 Universal SYBR Green PCR Master Mix 8. (Sigma. USA)를 이용하여 정량 분석하였다. Real-time PCR을 위한 primer는 IL-6, forward; 5'-ACAACCACGGCCTTCCCTACTT-3', IL-6 reverse; 5'-CACGATTTCCCAGAGAACATGTG-3'; TNF-알파, forward; 5'-CCCTCAGCAAGGACAGCAGA-3' and TNF-알파 reverse; 5'-AGCCGTGGGTCAGTATGTGAGA-3'; 베타-actin forward; 5'-CCAAGGCCAACCGCGAGAAGATGAC-3' 및 베타-actin reverse; 5'-AGGGTACATGGTGGTGCCGCCAGAC-3'를 MX3000P PCR Instrument (Stratagene, USA)로 분석하였다.  Real-time PCR was performed for quantitative analysis of mRNA levels and 50ng / ul of cDNA was prepared from mRNA as a template and quantitatively analyzed using Universal SYBR Green PCR Master Mix 8. (Sigma. USA). Primers for real-time PCR are IL-6, forward; 5'-ACAACCACGGCCTTCCCTACTT-3 ', IL-6 reverse; 5'-CACGATTTCCCAGAGAACATGTG-3 '; TNF-alpha, forward; 5'-CCCTCAGCAAGGACAGCAGA-3 'and TNF-alpha reverse; 5'-AGCCGTGGGTCAGTATGTGAGA-3 '; Beta-actin forward; 5'-CCAAGGCCAACCGCGAGAAGATGAC-3 'and beta-actin reverse; 5'-AGGGTACATGGTGGTGCCGCCAGAC-3 'was analyzed by MX3000P PCR Instrument (Stratagene, USA).
혈구검사Blood count
마우스안와로부터 피를 채혈하여 EDTA가 함유된 Microtainer Brand tube를 이용하여 HEMAVET 950FS(Drew Scientific Inc, USA)를 이용하여 혈구검사 및 분별세포수 검사를 시행하였다.  Blood was collected from the orbits of the mice, and blood cell and fractional cell counts were performed using HEMAVET 950FS (Drew Scientific Inc, USA) using a Microtainer Brand tube containing EDTA.
강제 수영검사 (Forced Swim Test)Forced Swim Test
Forced Swim test는 전통적인 방법인 Lucki법을 변형하여 수행하였다. 실험을 위한 장치는 50cm 높이에 20cm 넓이의 투명한 실린더를 실온에서 사용하여 바닥에 mice의 발과 꼬리가 닿지 않도록 하였다. 성숙한 웅성 ICR 생쥐 (20± 2g) 또는 성숙한 웅성 흰쥐 (200±10g)을 실험 1주일간 동물을 안정화시키고 실험 전 3-7일 전에 수욕 장치에서 2회 포기 반응 시행후 생쥐에게 시험물질을 투여하고 30분 또는 1시간 후에 6분간 수욕상에서 포기반응 시간(Immobility time) 을 측정하였다.   Forced Swim test was performed by modifying the traditional Lucki method. For the experiment, a 50cm high and 20cm wide cylinder was used at room temperature to prevent the feet and tails of the mice from touching the floor. Adult male ICR mice (20 ± 2g) or mature male rats (200 ± 10g) were allowed to stabilize the animals for 1 week and the mice were treated with test substances after two abandonment reactions in a bathing device 3-7 days before the experiment. After 6 minutes or 1 hour, the immobilization time was measured in a water bath for 6 minutes.
통계statistics
모든 실험은 평균±SEM 값으로 표현하였으며 Student's t-test를 사용하여 분석하였다. P값이 0.0.5이하는 의미있는 것으로 간주하였다.  All experiments were expressed as mean ± SEM values and analyzed using Student's t-test. P values of 0.0.5 or less were considered significant.
면역실험 Immune test
시험관시험법In vitro test method
림프구 증식능 및 활성화능 측정Lymphocyte proliferation and activation
1) 시험 원리 cell-based system    1) Test principle cell-based system
2) 시험 방법 오배자추출물이 정상면역세포의 증식에 미치는 영향을 검사하기 위해 실시한다.   2) Test method To examine the effect of gall bladder extract on the proliferation of normal immune cells.
가) 2 x 105 개의 비장세포가 한 well에 들어가도록 조정한 후 flat bottom 96 well microplate에 분주한 후 메칠 칼레이트와 이의 유도체들을 농도별(10 ㎍, 20 ㎍, 50 ㎍ 그리고 100 ㎍)로 처리한 후 37℃ 5% CO2 incubator에서 48시간 배양한다. A) Adjust 2 x 10 5 splenocytes into one well and dispense into a flat bottom 96 well microplate, and then apply methyl chloride and its derivatives by concentration (10 ㎍, 20 ㎍, 50 ㎍ and 100 ㎍). After treatment, incubate for 48 hours in a 37% 5% CO 2 incubator.
나) 오배자추출물에 의한 비장세포의 증식은 MTT assay를 통하여 측정하며, positive control로서는 B cell의 mitogen인 Lipopolysaccharide(LPS, 25mcg/ml)와 T cell mitogen인 Concana-valin A(ConA, 5mcg/ml) 를 사용하며 면역억제제의 control로는 prednisolone (10mcg/ml)을 사용한다.    B) Proliferation of splenocytes by gall bladder extract was measured by MTT assay, and positive control was Lipopolysaccharide (LPS, 25mcg / ml), a B cell mitogen, and Concana-valin A (ConA, 5mcg / ml), a T cell mitogen. Use prednisolone (10mcg / ml) as an immunosuppressant control.
NK세포 활성 NK cell activity
1) 시험 원리 Cell based system    1) Test principle Cell based system
2) 시험 방법    2) test method
가) NK세포에 대한 표적 세포로서는 마우스T lymphoma 유래세포주인 Yac-1세포를 25cm2 플라스틱 프라스크에 10% FBS 우태아혈청 RPMI 1640 배지에 부유 배양한다.A) As target cells for NK cells, Yac-1 cells, a mouse T lymphoma-derived cell line, are suspended in a 25 cm 2 plastic flask in 10% FBS fetal calf serum RPMI 1640 medium.
나) 작용자 세포는 다음과 같이 준비 : 1)과같이준비된세포에포함되어있는플라스틱부착성세포를제거하기 위하여 complete RPMI 1640배지에 부유시킨 다음 배양기에 1시간 동안 정치한다.    B) Agonist cells should be prepared as follows: 1) Float in complete RPMI 1640 medium to remove plastic adherent cells contained in prepared cells as follows.
다) 시간이 경과된 후 비부착성 세포만을 수거한다.    C) Only non-adherent cells are collected after a period of time.
라) 수거된 세포를 4x106 세포/ml로 세포농도를 조절하여 사용한다. D) Collect the collected cells to 4x10 6 cells / ml and adjust the cell concentration.
마) 오배자 추출물은 배양액 ml 당3, 10, 30μg 씩 되게 적하한다.    E) 5 g extract is added dropwise to 3, 10, 30 μg per ml of culture medium.
바) Effector/target cell ratio (E/T ratio)는200：1로 한다.    F) Effector / target cell ratio (E / T ratio) shall be 200: 1.
사) 96 wells microplate를 사용하여 well당 표적 세포를 1,000개씩 분주(50μl)하고, 작용자 세포를 중첩분주(50μl)한다.    G) Dispense 1,000 target cells per well (50 μl) using 96 wells microplate, and divide the effector cells (50 μl).
아) 대조군으로는 각 세포 농도의 작용자 세포만의 배양, 표적세포만의 배양 그리고 배지만의 배양 well들을 준비한다.    H) As a control, prepare only the effector cell culture, the target cell culture, and the culture-only culture well of each cell concentration.
자) 각각의 시험은 triplicate로 시행한다.   I) Each test shall be conducted with triplicate.
차) 분주가 완료된 plate는 배양기에 4일 동안 배양한다.    C) Incubate plate for 4 days.
카) 4일 배양 후에는 MTT 시험법에 준하여 540nm에서 측정한다.    K) After 4 days of incubation, measure at 540nm according to MTT test method.
타) OD에서 나타난 수치에서 % dead cell (% D)은 다음과 같은 식으로 산출한다.   (5)% dead cell (% D) is calculated from the following equation.
OD effector+target-OD effector             OD effector + target-OD effector
% D=(1- -------------------------- ) x100     % D = (1- --------------------------) x100
(OD target-OD blank)                    (OD target-OD blank)
산화질소 생성능 시험Nitric oxide generation ability test
1) 시험 원리 Cell based system.   1) Test principle Cell based system.
2) 시험 방법 - 대식세포에 각 메칠 칼레이트와 이의 유도체들을 각 농도로 24시간 처리후, Griess reagent를 동량 혼합하여 반응시킨 후에 540 nm에서 흡광도 측정하였다. (positive control : LPS)   2) Test Method-After treating each methyl calate and its derivatives with macrophages at each concentration for 24 hours, the same amount of Griess reagent was reacted and the absorbance was measured at 540 nm. (positive control: LPS)
실험동물 시험법Laboratory Animal Test
가. 세포활성 물질 및 면역세포의 변화   end. Changes in Cellular Active Materials and Immune Cells
1) 시험 원리 Cell based system   1) Test principle Cell based system
2) 시험 방법   2) test method
가) 공시단크론항체: 세포표면항원 특이항체를 이용한 비장 임파구 아군의 분포도 변화조사에 사용된 단크론 항체와 최적희석배수는 다음과 같다.    A) Monoclonal Antibodies: The monoclonal antibodies and the optimal dilution factor used to investigate the distribution of splenic lymphocyte subpopulations using cell surface antigen specific antibodies are as follows.
표 1
항체 Isotype Fomat Application Optimaldilution(v/v)
CD32/16 Rat IgG2b 정제된 항체의 비특이적 결합에 대한 대조군(Fc block) 1:10
CD3ε Hamster IgG1 FITC pan-T lymphocyte marker 1:10
CD4/L3T4 Rat IgG2b FITC T lymphocyte subset marker 1:10
CD8a/Ly2 Rat IgG2a PE T lymphocyte subset marker 1:10
CD45R/B220 Rat IgG2a PE pan-B lymphocyte marker 1:30
Table 1
Antibodies Isotype Fomat Application Optimaldilution (v / v)
CD32 / 16 Rat IgG2b Refined Control for Nonspecific Binding of Antibodies (Fc Block) 1:10
CD3ε Hamster IgG1 FITC pan-T lymphocyte marker 1:10
CD4 / L3T4 Rat IgG2b FITC T lymphocyte subset marker 1:10
CD8a / Ly2 Rat IgG2a PE T lymphocyte subset marker 1:10
CD45R / B220 Rat IgG2a PE pan-B lymphocyte marker 1:30
표 1은 비장 림프구 세포 표면 마커 발현에 대한 분석에 사용된 각 단클론 항체의 특성 및 최적 희석 표이다.Table 1 is a table of the characteristics and optimal dilutions of each monoclonal antibody used in the assay for splenic lymphocyte cell surface marker expression.
나) 세포표면항원에 대한 면역형광염색 및 분석: 임파구증식능 조사에 공시된 방법과 동일하게 준비된 임파구 부유액을 나이론망에 걸러준 후 Lympholyte-M (Cedarlane)에 넣어 원심분리(1,500g, 20min)로 구분된 임파구층을 뽑아내 HBSS로 원심분리와 세척을 하였다. 1% bovine serum albumin(BSA)과 0.1% NaN3가 함유된 PBS로 부유시키고 1x107 cells/ml의 농도로 조절한다. 100ul의 세포액과 각각의 단크론항체 10ul를 ㄱ. Control, Fc block ㄴ. Fc block + CD3(FITC)+CD45(PE) ㄷ. Fc block +CD4(FITC)+CD8(PE)와 같은 조합으로 구성된 튜브에 넣어 가볍게 섞은 뒤 얼음위에서 빛을 피하여 30분간 반응시켰다. 최종적으로 5ml의 PBS/BSA/NaN3 용액으로 1회 세척한 뒤 500ul의 0.5% formalin용액으로 고정하고 flowcytometer로 분석하였다.B) Immunofluorescence staining and analysis of cell surface antigens: Lymphocyte suspensions prepared in the same manner as the method described in the investigation of lymphocyte proliferation were filtered through a nylon network, and then centrifuged (1,500g, 20min) in Lympholyte-M (Cedarlane). Separated lymphocytes were extracted and centrifuged and washed with HBSS. Suspend with PBS containing 1% bovine serum albumin (BSA) and 0.1% NaN 3 and adjust to a concentration of 1x10 7 cells / ml. 100 ul of cell fluid and 10 ul of each monoclonal antibody a. Control, Fc block b. Fc block + CD3 (FITC) + CD45 (PE) c. Fc block + CD4 (FITC) + CD8 (PE) in a tube composed of a combination such as a light mixture and lightly reacted on the ice for 30 minutes. Finally, the solution was washed once with 5 ml of PBS / BSA / NaN3 solution, fixed with 500ul of 0.5% formalin solution and analyzed by flowcytometer.
나. 각종 cytokine 분비능 조사 I. Various cytokine secretion
1) 시험 원리 Cell based system   1) Test principle Cell based system
2) 시험 방법 - 오배자추출물를 처리하고 48시간 배양하여 분비된 각종의 cytokine을 double sandwich ELISA technique을 이용한 제조사(BD Pharmingen)의 protocol 에 따라 측정하였다.  2) Test Method-Various cytokine secreted by treatment with the gall bladder extract and incubated for 48 hours were measured according to the manufacturer's protocol (BD Pharmingen) using the double sandwich ELISA technique.
다. Forced Swimming test (FST)  All. Forced Swimming test (FST)
1) 시험 원리 - 실험 동물 : 성숙한 웅성 ICR 생쥐 (20±2g) 또는 성숙한 웅성 흰쥐 (200±10g)  1) Test Principle-Experimental Animal: Mature male ICR mouse (20 ± 2g) or mature male rat (200 ± 10g)
2) 시험방법   2) Test method
가) 제한 공간의 수욕 구조의 실험 장치를 활용하고   A) utilizing the experimental apparatus of the bathing structure of the confined space
나) 1주일간 동물을 안정화시키고 실험 전 3-7일 전에 수욕 장치에서 1회 포기 반응 시행하였으며,   B) The animals were stabilized for 1 week and subjected to one abandonment reaction in a bathing device 3-7 days before the experiment.
다) 생쥐 또는 흰쥐에게 시험물질을 투여하고 30분 또는 1시간 후에 5～10분간 수욕상에서 포기 반응 시간, 욕조를 기어오르려는 횟수와 시간을 측정하였다.   C) 30 minutes or 1 hour after the administration of test substance to mice or rats, the aeration reaction time in the water bath and the number and time of climbing the bath were measured.
라) 효능 평가를 위한 분석 항목   D) analysis items for evaluation of efficacy;
- 포기 반응 시간     Abandonment reaction time
- 욕조를 기어오르려는 횟수     -Number of attempts to climb the tub
- 욕조를 기어오르는 시간     -Time to climb bath
라. 염증제어 측정: Histamine secretion assay실험 la. Inflammatory control assay: Histamine secretion assay
1) 시험원리: 아토피성 피부염과 같은 피부에서의 염증과 가려움증을 유발하는 화학적 매개물질을 감소시키는 정도를 측정하는 방법  1) Test Principle: How to measure the degree of reducing chemical mediators that cause inflammation and itching in the skin such as atopic dermatitis
2) 시험방법  2) Test method
가) 에펜돌프 튜브에 시료 500μl를 넣고 0.1 N-HCl에 과염소산 용액 50μl를 넣고 원심분리   A) Put 500μl of sample into the Eppendorf tube and 50μl of perchloric acid solution in 0.1 N-HCl and centrifuge
나) 상층액에 5N-NaOH 용액 500μl, 증류수 3ml, n-Butanol 10 ml 및 NaCl 1.2g을 혼합한 시험관에 넣고 원심분리   B) Centrifuge into 500 μl of 5N-NaOH solution, 3 ml of distilled water, 10 ml of n-Butanol, and 1.2 g of NaCl.
다) Butanol층 8ml에 0.1N-HCl, n-Heptane 가하여 진탕한 후 원심분리   C) Centrifugation after shaking by adding 0.1N-HCl and n-Heptane to 8 ml of butanol layer
라) 여기서 얻은 수층 2ml에 1N-NaOH와 o-Phthaldialdehyde 넣어 반응 후 3N-HCl을 넣고 spectrophotometer로 형광도 측정   D) Add 2N-NaOH and o-Phthaldialdehyde to 2 ml of the aqueous layer obtained, add 3N-HCl and measure the fluorescence with a spectrophotometer.
마) 히스타민 억제율 계산   E) Calculation of histamine inhibition rate
억제율(%)= (약물 투여하지 않았을때의 히스타민 양-약물 투여 후 히스타민 양) x 100 / 약물을 투여하지 않았을때의 히스타민 양     Inhibition Rate (%) = (Histamine amount without drug administration-amount of histamine after drug administration) x 100 / Histamine amount without drug administration
상기 실험예 2의 결과는 하기와 같다.The results of Experimental Example 2 are as follows.
오배자가 세포증식에 미치는 영향  Effect of Mellitus on Cell Proliferation
생쥐에 실험물질 투여(50mg/kg) 후 획득한 비장세포 또는 분리한 비장세포를 이용하여 오배자 추출물이 세포증식에 미치는 영향을 살펴본 결과는은 표준물질과 비교하여 세포증식이 유의적으로 증가함을 확인할 수 있었다(도 14, 및 15).  The effects of gall bladder extract on cell proliferation using splenocytes or isolated splenocytes obtained after administration of test substance (50mg / kg) to mice showed that the cell proliferation was significantly increased compared to the standard. It could be confirmed (FIG. 14, and 15).
또한 강력한 항산화 성분인 Nitric Oxide(NO) 생성의 경우 표준물질과 비교하여 다소 생성이 증가하거나 유사한 양상을 보임으로써 오배자 추출물의 항산화 작용을 확인할 수 있었다(도 16-19).   In addition, Nitric Oxide (NO) production, a potent antioxidant component, was confirmed to have an increase in production or a similar pattern compared to the standard material, confirming the antioxidant activity of the gall bladder extract (Figs. 16-19).
세포독성에 대한 실험은 오배자 추출물이 농도나 시간에 관계없이 독성이 증가하는 양상을 보이지 않았으며(도 20-21), 표준물질과 비교하여서는 유의적인 차이를 보이지는 않았다(도 22).  Experiments on cytotoxicity showed no increase in toxicity of the gall bladder extract regardless of concentration or time (Figs. 20-21), and did not show a significant difference compared to the standard (Fig. 22).
사이토카인 측정Cytokine measurement
오배자 추출물을 마우스 투여후 비장세포 및 대식세포를 분리한후 IL-6, TNF-알파 등의 분비가 증가함을 확인할 수 있었다(도 23-26).  After gall bladder extract was isolated from mice and splenocytes and macrophages, the secretion of IL-6, TNF-alpha, etc. was increased (Figs. 23-26).
염증관련 전사인자 측정Inflammation-related transcription factor measurement
오배자 추출물을 투여후 마우스 비장세포를 분리후 COX-2 등의 염증 반응과 관련이 있는 NF-kB 발현을 본 결과 유의한 차이로 발현함을 확인할 수 있었고, 또한 MAPkinase 와 관련된 신호전달기전이 관계됨을 확인할 수 있었다(도 27).    After administration of the gall bladder extract, mouse splenocytes were isolated and the expression of NF-kB, which is related to inflammatory responses such as COX-2, was found to be significantly different. Also, signaling pathways related to MAPkinase were related. It could be confirmed (Fig. 27).
세포활성 물질에 의한 혈액 변화Blood Change by Cellular Active Substances
오배자 추출물(E)과 활성 유효 화합물을 마우스(군당 3-5마리)에 50mg/kg 구강 투여후 24시간 후혈액을 분리하여 혈구 및 혈액검사를 시행하였다. 결과는 아래와 같다.   Blood cells and blood tests were performed for 24 hours after the oral administration of 50 mg / kg oral administration of the gall extract (E) and the active compound to 3-5 mice / group. The result is shown below.
오배자 추출물 및 활성화합물중 SA, GA은 각 분획 세포들 (WBC , NE, LY, MO, EO, BA )를 증가 시키는 경향이 있으며 투여군 중 SA(syringic acid) 가 가장 현저하였으며, MG (메칠 갈레이트)는 대조군과 큰 차이가 없었다.  SA and GA of five gall extracts and active compounds tended to increase the fractional cells (WBC, NE, LY, MO, EO, BA), and SA (syringic acid) was the most prominent among the administration groups, and MG (methyl gallate) ) Was not significantly different from the control group.
적혈구(RBC) 및 혈소판(platelets)의 변화로는 각 투여군에서 적혈구수, HCT, 혈소판수가 증가하였으며 혈색소(Hb), MCH, MCHC는 MG 투여군만 제외하고는 증가하였다.    The changes of erythrocyte (RBC) and platelets (platelet) were increased in the number of erythrocytes, HCT, platelets in each administration group, hemoglobin (Hb), MCH, MCHC was increased except in the MG group.
이상의 혈액소견으로 보아 오배자 추출물 및 활성화합물은 마우스 생체내에서 혈구세포 및 혈구유래 면역세포의 분포를 조절하는 양상을 보였다.    In view of the above blood findings, the gall bladder extract and the active compound regulate the distribution of blood cells and blood cell-derived immune cells in mice.
각종 cytokine 분비능 조사 Various cytokine secretion
오배자 추출물(E)과 활성 유효 화합물을 마우스에 50mg/kg 구강 투여후, 비장세포를 분리하여 RNA를 분리하여 real time PCR 을 이용하여 IL-6, TNF-알파의 발현을 측정하였다.  After administering 50 mg / kg oral administration of the gall extract (E) and the active compound to mice, splenocytes were isolated, RNA was isolated, and expression of IL-6 and TNF-alpha was measured using real time PCR.
IL-6, TNF-알파 cytokine의 mRNA 발현은 오배자 추출물 처리 시, 증가하는 양상을 보이나 특히 TNF-알파의 경우 gallic acid나 metyl gallate와 비교하여 유의한 증가를 보이지는 않았다. MRNA expression of IL-6 and TNF-alpha cytokine was increased when the gall extract was treated, but TNF-alpha showed no significant increase compared with gallic acid or metyl gallate.
T 세포 및 B 세포 유약화반응 측정Measurement of T Cell and B Cell Fragility
오배자 추출물(E)과 활성 유효 화합물을 마우스에 50mg/kg 구강 투여후, 비장세포를 분리후 오배자 추출물을 처리후 Con A와 LPS를 처리하여 유약화반응을 검사하였다. Fifty five mg / kg oral administration of the gall bladder extract (E) and the active compound to mice, the splenocytes were isolated and treated with gall bladder extract and then treated with Con A and LPS to examine the weakening reaction.
오배자 추출물과 유효화합물을 비장세포에 처리시, 농도에 따른 T, B 세포 유약화 반응을 확인할 수 있었다. When the gall extract and the active compound were treated on splenocytes, T and B cell weakening reactions were confirmed according to the concentration.
B cell의 경우, 오배자 추출물과 gallic acid, methyl gallate와 유의적인 차이를 보이지 않았다.In the case of B cells, there was no significant difference between the gallnut extract, gallic acid and methyl gallate.
NO(산화 질소)측정NO (Nitric Oxide) Measurement
오배자 추출물(E)과 활성 유효 화합물을 마우스에 50mg/kg 구강 투여후, 비장세포를 분리후 배양후에 상층액에서 NO를 측정한 결과 오배자 추출물 처리시간에 따라 NO 생성이 증가하였다. 다른 활성 유효 화합물들도 NO 생성이 증가하는 경향을 보였다.After oral administration of the gall bladder extract (E) and the active active compound to the mice at 50 mg / kg, the splenocytes were isolated and cultured to determine NO in the supernatant. Other active active compounds also tended to increase NO production.
오배자 추출물 투여군과 다른 처리군들과 비교하여 유의적인 차이를 보이지는 않았다.There was no significant difference compared with the control group and the other groups.
Forced Swimming test (FST) Forced Swimming test (FST)
성숙한 웅성 ICR 생쥐 (20 ± 2g) 또는 성숙한 웅성 흰쥐 (200±10g)을 실험 1주일간 동물을 안정화시키고 실험 전 3-7일 전에 수욕 장치에서 2회 포기 반응 시행후 생쥐에게 시험물질을 투여하고 30분 또는 1시간 후에 5～10분간 수욕상에서 포기반응 시간(Immobility time)을 측정하였다. Adult male ICR mice (20 ± 2 g) or mature male rats (200 ± 10 g) were allowed to stabilize the animals for one week of the experiment, and the mice were administered the test substance after two abandonment reactions in a bathing device 3-7 days before the experiment. After a minute or 1 hour, the immobilization time was measured in a water bath for 5 to 10 minutes.
오배자 추출물(E)과 활성 유효 화합물을 마우스에 50mg/kg 구강 투여 후, immobility time(포기반응 시간)을 관찰한 결과 대조군(정상마우스)에 비하여 immobility time이 감소하였다. After oral administration of the gall extract (E) and the active compound to the mice at 50 mg / kg, the immobility time was observed as a result of observing the immobility time.
오배자 추출물을 농도별(100mg/kg-10mg/kg) 처리 시 immobility time이 감소하는 양상을 보였다. Immobility time was decreased when the gall extract was treated by concentration (100mg / kg-10mg / kg).

Claims (7)

  1. 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 치매 (AD) 예방 및 치료용 약학 조성물.A pharmaceutical composition for preventing and treating dementia (AD), which contains an extract of Galla Rhois as an active ingredient.
  2. 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 치매 (AD) 예방 및 개선용 건강식품 조성물.Health food composition for the prevention and improvement of dementia (AD) containing five extracts (Galla Rhois) as an active ingredient.
  3. 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 인지능력 개선용 약학 조성물.A pharmaceutical composition for improving cognitive ability, which contains an extract of Galla Rhois as an active ingredient.
  4. 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 인지능력 개선용 건강 식품 조성물.Health food composition for improving the cognitive ability containing the extract (Galla Rhois) as an active ingredient.
  5. 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 면역 증강용 약학 조성물.Immunity-enhancing pharmaceutical composition containing the extract (Galla Rhois) as an active ingredient.
  6. 오배자(Galla Rhois) 추출물을 유효성분으로 함유하는 면역 증강용 건강식품 조성물.Immunity enhancement health food composition containing the extract (Galla Rhois) as an active ingredient.
  7. 갈릭에시드(gallic acid) 또는 메틸갈레이트를 유효성분으로 포함하는 치매(AD) 예방 및 치료용 약학 조성물Pharmaceutical composition for preventing and treating dementia (AD) comprising gallic acid or methyl gallate as an active ingredient
PCT/KR2013/007093 2013-08-06 2013-08-06 Composition containing galla rhois extract as active ingredient for improving cognition and preventing or treating alzheimer's disease WO2015020241A1 (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008080162A2 (en) * 2006-12-22 2008-07-03 The Johns Hopkins University Anti-cholesterolemic compounds and methods of use
WO2011060307A2 (en) * 2009-11-13 2011-05-19 Jingxuan Kang Food products having multiple health benefits
KR20110110638A (en) * 2010-04-01 2011-10-07 김현기 Composition for improving recognition comprising galla rhois extract
KR20120045591A (en) * 2010-10-29 2012-05-09 일성신약주식회사 Composition comprising an combined herb extract including rhei radix et rhizoma for treating or preventing cognitive dysfunction
KR20130053016A (en) * 2011-11-14 2013-05-23 (주) 메디플랜 Rhus chinensis mill. salt for skin aging prevention and producing method thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008080162A2 (en) * 2006-12-22 2008-07-03 The Johns Hopkins University Anti-cholesterolemic compounds and methods of use
WO2011060307A2 (en) * 2009-11-13 2011-05-19 Jingxuan Kang Food products having multiple health benefits
KR20110110638A (en) * 2010-04-01 2011-10-07 김현기 Composition for improving recognition comprising galla rhois extract
KR20120045591A (en) * 2010-10-29 2012-05-09 일성신약주식회사 Composition comprising an combined herb extract including rhei radix et rhizoma for treating or preventing cognitive dysfunction
KR20130053016A (en) * 2011-11-14 2013-05-23 (주) 메디플랜 Rhus chinensis mill. salt for skin aging prevention and producing method thereof

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