Method for preparing ethanol extract of ecklonia cava, and composition for preventing, alleviating or treating cognitive dysfunction, comprising same as active ingredient

Abstract

The present invention relates to a method for preparing an ethanol extract of Ecklonia cava, and a composition for preventing, alleviating or treating cognitive dysfunction, comprising same as an active ingredient. The ethanol extract of Ecklonia cava, of the present invention, contains phlorotannin oligomer, eckol, 7-phloroeckol, 6,6'-bieckol, 6,8'-bieckol, dibenzodioxin-fucodiphlorethol, diekcol, phlorofuroeckol A and 2,7''- phloroglucinol-6,6'-bieckol in ratios of 1-2 : 5-6 : 6.5-7.5 : 7-8 : 20-21 : 3-4 : 47-48 : 4.5-5.5 : 1.5-2.5, respectively, has excellent effects of improving behavioral function, improving antioxidant system, improving mitochondrial function, improving cholinergic system, alleviating toxicity caused by Aβ aggregation and Tau hyperphosphorylation, improving inflammation and inhibiting cell death, and thus can be effectively used as a pharmaceutical composition or health functional food composition for preventing, alleviating, or treating cognitive dysfunction.

Description

Method for producing ethanol extract of perilla leaves and composition for preventing, improving or treating cognitive dysfunction containing the same as an effective ingredient

[0001] The present invention relates to a method for producing a perilla leaf ethanol extract and a composition for preventing, improving or treating cognitive dysfunction comprising the same as an effective ingredient.

[0002] The human brain is an important part that controls learning, memory, movement, etc., and nerve cells are connected to each other to form a huge neural network, which becomes the nervous system foundation that includes learning and memory.

[0003] However, when harmful reactive oxygen species (ROS) are excessively produced due to various causes and accumulate in the body, they cause oxidative damage to cells, leading to cell degeneration and cell necrosis. Normally, they are removed by the body's removal mechanisms such as superoxide dismutase (SOD), catalase, carotenoids, and glutathione, but ROS remaining in the body induces degeneration and death of brain nerve cells, negatively affecting the formation of proper neural networks, reducing learning ability and memory, and in severe cases, causing degenerative brain diseases such as Parkinson's disease and Alzheimer's disease.

[0004] Furthermore, cognitive abilities in the brain, such as learning and memory, are manifested by neurotransmitters such as acetylcholine (ACh). Secreted from nerve cell terminals, ACh normally transmits signals through synaptic receptors in response to external stimuli, such as learning. It is then broken down into acetate and choline by acetylcholinesterase (AChE), and partially reabsorbed. This process is called the cholinergic system, and it is known that most Alzheimer's patients experience serious problems during this process.

[0005] While a fundamental cure for Alzheimer's disease has not yet been developed, the majority of treatments used worldwide are acetylcholinesterase inhibitors. These cannot completely halt the progression of the disease, but rather alleviate some symptoms or slow its progression. Examples of this class of drugs include donepezil, rivastigmine, galantamine, and tacrine. Following the global trend of adopting acetylcholinesterase as the most common pharmacological target in therapeutic development, there is a need to develop acetylcholinesterase inhibitors and functional materials with fewer side effects and superior efficacy, utilizing various natural products.

[0006] Accordingly, the inventors of the present invention completed the present invention by confirming that the ethanol extract of Gamtae extracted using ethanol has excellent effects in improving behavioral function, improving antioxidant system, improving mitochondrial function, improving cholinergic system, improving toxicity due to Aβ aggregation and Tau hyperphosphorylation, improving inflammation, and inhibiting cell death.

[0007] The object of the present invention is to provide a composition comprising phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA and 2,7′′-Phloroglucinol-6,6′-bieckol in a ratio of 1~2 : 5~6 : 6.5~7.5 : 7~8 : 20~21 : 3~4 : 47~48 : 4.5~5.5 : The purpose is to provide an ethanol extract of Ecklonia cava contained in a ratio of 1.5 to 2.5.

[0008] In addition, another object of the present invention is to provide a method for producing a composition comprising a step of mixing Ecklonia cava and ethanol, followed by extraction, wherein the phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA and 2,7′′-Phloroglucinol-6,6′-bieckol are mixed in a ratio of 1 to 2: 5 to 6: 6.5 to 7.5: 7 to 8: The present invention provides a method for producing a perilla leaf ethanol extract in a ratio of 20~21: 3~4: 47~48: 4.5~5.5: 1.5~2.5.

[0009] In addition, another object of the present invention is to provide a pharmaceutical composition for preventing or treating cognitive dysfunction, which comprises the Ecklonia cava ethanol extract as an active ingredient.

[0010] In addition, another object of the present invention is to provide a health functional food composition for preventing or improving cognitive dysfunction, which contains the Ecklonia cava ethanol extract as an active ingredient.

[0011] In addition, another object of the present invention is to provide a method for increasing the dieccol content of a kelp extract, which comprises the step of mixing kelp (Ecklonia cava) and ethanol and then extracting it.

[0012] In addition, another object of the present invention is to provide a method for preventing or treating cognitive dysfunction, which comprises administering the pharmaceutical composition to a subject.

[0013] In order to achieve the above object, the present invention provides a composition comprising phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA and 2,7′′-Phloroglucinol-6,6′-bieckol in a ratio of 1~2 : 5~6 : 6.5~7.5 : 7~8 : 20~21 : 3~4 : 47~48 : Provides Ecklonia cava ethanol extract in a ratio of 4.5~5.5 : 1.5~2.5.

[0014] Furthermore, the present invention comprises a step of mixing Ecklonia cava and ethanol, and then extracting; Phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA and 2,7′′-Phloroglucinol-6,6′-bieckol are mixed in a ratio of 1 to 2: 5 to 6: 6.5 to 7.5: 7 to 8: A method for producing a perilla leaf ethanol extract is provided, wherein the extract is contained in a ratio of 20~21: 3~4: 47~48: 4.5~5.5: 1.5~2.5.

[0015] Next, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction comprising the Ecklonia cava ethanol extract as an active ingredient.

[0016] In addition, the present invention provides a health functional food composition for preventing or improving cognitive dysfunction, which comprises the Ecklonia cava ethanol extract as an active ingredient.

[0017] In addition, the present invention provides a method for increasing the dieccol content of a kelp extract, comprising the step of mixing kelp (Ecklonia cava) and ethanol and then extracting it.

[0018] Finally, the present invention provides a method for preventing or treating cognitive dysfunction, comprising administering the pharmaceutical composition to a subject.

[0019] Phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA and 2,7′′-Phloroglucinol-6,6′-bieckol of the present invention are in the ratio of 1~2 : 5~6 : 6.5~7.5 : 7~8 : 20~21 : 3~4 : 47~48 : 4.5~5.5 : The ethanol extract of perilla leaves contained in a ratio of 1.5 to 2.5 has excellent effects of improving behavioral function, improving antioxidant system, improving mitochondrial function, improving cholinergic system, improving toxicity caused by Aβ aggregation and Tau hyperphosphorylation, improving inflammation, and inhibiting cell death, and can be usefully used as a pharmaceutical composition or health functional food composition for preventing, improving, or treating cognitive dysfunction.

[0020] FIG. 1 is a diagram showing the results of evaluating spatial cognitive ability in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: total number of times entered into the maze, B: degree of crossing progress in the maze, C: path tracing).

[0021] FIG. 2 is a diagram showing the results of evaluating short-term memory ability in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: time to enter the dark room on the first day, B: time to enter the dark room on the second day).

[0022] FIG. 3 is a diagram showing the results of evaluating long-term memory ability in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: escape delay time, B: time spent in the W area, C: path tracing).

[0023] FIG. 4 is a diagram showing the results of confirming the effect of improving the antioxidant system in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: Malondialdehyde (MDA) content, B: Reduced gluathione (GSH) level, C: Superoxide dismutase (SOD) level).

[0024] FIG. 5 is a diagram showing the results of confirming the effect of improving mitochondrial function in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: reactive oxygen species (ROS) level, B: mitochondrial membrane potential (MMP) level, C: adenosine triphosphate (ATP) concentration).

[0025] FIG. 6 is a diagram showing the results of confirming the effect of improving the cholinergic system in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: Acetylcholine (ACh) content, B: Acetylcholinesterase (AChE) activity).

[0026] FIG. 7 is a diagram showing the results of analyzing the expression level of proteins related to the cholinergic system and synapse function in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: expression level of proteins related to the cholinergic system and synapse function, B: AChE expression level, C: ChAT expression level, D: SYP expression level, E: PSD-95 expression level).

[0027] FIG. 8 is a diagram showing the results of analyzing the expression level of Aβ / Tau pathway-related proteins in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: Aβ / Tau pathway-related protein expression level, B: Aβ expression level, C: IDE expression level, D: p-tau expression level, E: p-GSK-3β expression level).

[0028] FIG. 9 is a diagram showing the results of analyzing the expression level of neuroinflammation-related proteins in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: neuroinflammation-related protein expression level, B: TLR-4 expression level, C: MyD88 expression level, D: p-NF-κB expression level, E: p-IκB-α expression level, F: IL-1β expression level, G: TNF-α expression level).

[0029] FIG. 10 is a diagram showing the results of analyzing the expression level of apoptosis-related proteins in an Aβ-induced cognitive dysfunction model according to one embodiment of the present invention (A: Apoptosis-related protein expression level, B: p-JNK expression level, C: p-Akt expression level, D: BAX expression level, E: BCl-2 expression level, F: BAX / BCl-2 ratio level, G: caspase 3 expression level).

[0030] FIG. 11 is a diagram showing the results of analyzing the indicator components contained in the perilla extract using UPLC-Q-TOF / MS in one embodiment of the present invention (A: indicator components contained in 70% perilla ethanol, B: indicator components contained in perilla water extract).

[0031] FIG. 12 is a diagram showing the results of quantitative analysis of the content of dieckol contained in a persimmon extract using HPLC-DAD in one embodiment of the present invention (A: standard curve of dieckol, B: content of dieckol contained in a 70% ethanol extract of persimmon, C: content of dieckol contained in a water extract of persimmon).

[0032] Hereinafter, the present invention will be described in detail with reference to the attached drawings and exemplary embodiments thereof. However, the following exemplary embodiments are provided as illustrative examples of the present invention. If a detailed description of a technology or configuration well known to those skilled in the art is judged to unnecessarily obscure the gist of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the following claims and equivalents interpreted therefrom.

[0033] In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and this may vary depending on the intention of the user or operator, or the customs of the field to which the present invention belongs. Therefore, the definition of these terms should be determined based on the contents throughout this specification. Throughout the specification, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be included, unless specifically stated otherwise.

[0034] Throughout this specification, '%' used to indicate the concentration of a particular substance means solid / solid (w / w) %, solid / liquid (w / v) %, and liquid / liquid (v / v) %, unless otherwise stated.

[0035] In one aspect, the present invention provides a composition comprising phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA and 2,7′′-Phloroglucinol-6,6′-bieckol in a ratio of 1~2 : 5~6 : 6.5~7.5 : 7~8 : 20~21 : 3~4 : 47~48 : 4.5~5.5 : Provides an ethanol extract of Ecklonia cava contained in a ratio of 1.5 to 2.5, preferably 1.35 : 5.32 : 7.06 : 7.63 : 20.79 : 3.71 : 47.25 : 4.81 : 2.08.

[0036] In one embodiment of the present invention, the phlorotannin oligomer is a polyphenol compound found in brown algae, and is formed by combining phloroglucinol (C6H6O3) units.

[0037] In one embodiment of the present invention, the molecular formula of Eckol is C 18 H 12 Its chemical formula is O9, its IUPAC name is 4-(3,5-Dihydroxyphenoxy)oxanthrene-1,3,6,8-tetrol, and its molecular weight is 372.285 g / mol. In addition, this compound has the chemical structure shown in Chemical Formula 1 below.

[0038]

[0039] In one embodiment of the present invention, the molecular formula of the 7-Phloroeckol is C 24 H 16 O 12, and its IUPAC name is 4-(3,5-dihydroxyphenoxy)-8-(2,4,6-trihydroxyphenoxy)dibenzo-p-dioxin-1,3,6-triol, and it has a molecular weight of 496.4 g / mol. In addition, this compound has a chemical structure as shown in Chemical Formula 2 below.

[0040]

[0041] In one embodiment of the present invention, the molecular formula of the 6,6′-Bieckol is C 36 H 22 O 18 , and its IUPAC name is 6,6′-Bis(3,5-dihydroxyphenoxy)[1,1′-bioxanthrene]-2,2′,4,4′,7,7′,9,9′-octol, and it has a molecular weight of 742.55 g / mol. In addition, this compound has a chemical structure as shown in Chemical Formula 3 below.

[0042]

[0043] In one embodiment of the present invention, the molecular formula of the 6,8′-Bieckol is C 36 H 22 O 18 , and has a molecular weight of 742.55 g / mol. In addition, this compound has a chemical structure as shown in Chemical Formula 4 below.

[0044]

[0045] In one embodiment of the present invention, the molecular formula of the dibenzodioxin-fucodiphloroethol is C 36 H 24, and its IUPAC name is 4-({2'-[2,4-Dihydroxy-6-(2,4,6-trihydroxyphenoxy)phenoxy]-4,4',6,6'-tetrahydroxy-2-biphenylyl}oxy)-1,3,6,8-oxanthrenetetrol, and it has a molecular weight of 744.565 g / mol. In addition, this compound has a chemical structure as shown in Chemical Formula 5 below.

[0046]

[0047] In one embodiment of the present invention, the molecular formula of the diechol is C 36 H 22 O 18 , and its IUPAC name is 2,4,6-Trioxa-1(1),5(7,1)-dioxanthrena-3(1,4),7(1)-dibenzenaheptaphane-12,14,17,19,33,35,52,54,59,73,75-undecol, and it has a molecular weight of 742.52 g / mol. In addition, this compound has a chemical structure as shown in Chemical Formula 6 below.

[0048]

[0049] In one embodiment of the present invention, the molecular formula of the phlorofuroeckolA is C 30 H 18 O 14 , and its IUPAC name is 4,9-Bis(3,5-dihydroxyphenoxy)[1]benzofuro[3,2-a]oxanthrene-1,3,6,10,12-pentol, and it has a molecular weight of 602.45 g / mol. In addition, this compound has a chemical structure as shown in Chemical Formula 7 below.

[0050]

[0051] In one embodiment of the present invention, the molecular formula of the 2,7''-phloroglucinol-6,6'-bieckol is C 48 H 30 O 23 , and its IUPAC name is 4-[2,6-bis(3,5-dihydroxyphenoxy)-4,7,9-trihydroxydibenzo-p-dioxin-1-yl]-9-(3,5-dihydroxyphenoxy)-8-(2,4,6-trihydroxyphenoxy)dibenzo-p-dioxin-1,3,6-triol, and it has a molecular weight of 974.7 g / mol. In addition, this compound has a chemical structure as shown in Chemical Formula 8 below.

[0052]

[0053] The extract according to the present invention can be obtained by extracting and separating from nature using extraction and separation methods known in the art, and the "extract" defined in the present invention is extracted from kelp using an appropriate solvent, and includes, for example, a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract. Any pharmaceutically acceptable organic solvent may be used as a suitable solvent for extracting the extract from the above-mentioned seaweed, and water or an organic solvent may be used, and is not limited thereto, but for example, purified water, alcohols having 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane, etc., may be used alone or in mixtures, and preferably ethanol, and more preferably 70% ethanol, is used. Any one of the following extraction methods may be selected and used: hot water extraction, cold immersion extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, dissolution, and pressing. Furthermore, the desired extract may be subjected to additional conventional fractionation processes and purified using conventional purification methods.

[0054] There is no limitation on the method for preparing the extract of the present invention, and any known method can be used. For example, the extract included in the composition of the present invention can be prepared in a powder form by additional processes such as reduced pressure distillation and freeze drying or spray drying of the primary extract extracted by the above-mentioned hot water extraction or solvent extraction method. In addition, the primary extract can be further purified to obtain a fraction using various chromatography methods such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. Therefore, in the present invention, the extract is a concept that includes all extracts, fractions, and purified products obtained at each stage of extraction, fractionation, or purification, as well as their dilutions, concentrates, or dried products.

[0055] In one embodiment of the present invention, the persimmon ethanol extract may contain, but is not limited to, 80 to 100 μg of diechol per 1 mg of the persimmon ethanol extract, preferably 85 to 95 μg, more preferably 90 to 92 μg, and even more preferably 90.89 μg.

[0056] In one embodiment of the present invention, the persimmon ethanol extract may contain, but is not limited to, 1.5 to 3.5 μg, preferably 2 to 3 μg, more preferably 2.5 to 2.7 μg, and even more preferably 2.60 μg of phlorotannin oligomers per 1 mg of the persimmon ethanol extract.

[0057] In one embodiment of the present invention, the persimmon ethanol extract may contain, but is not limited to, 7 to 14 μg of ecoll per 1 mg of persimmon ethanol extract, preferably 9 to 12 μg, more preferably 10 to 11 μg, and even more preferably 10.23 μg.

[0058] In one embodiment of the present invention, the persimmon ethanol extract may contain 10 to 17 μg, preferably 12 to 15 μg, more preferably 13 to 14 μg, and even more preferably 13.58 μg of 7-fluoroecol per 1 mg of the persimmon ethanol extract, but is not limited thereto.

[0059] In one embodiment of the present invention, the persimmon ethanol extract may contain 11 to 18 μg, preferably 13 to 16 μg, more preferably 14 to 15 μg, and even more preferably 14.68 μg of 6,6′-biecol per 1 mg of the persimmon ethanol extract, but is not limited thereto.

[0060] In one embodiment of the present invention, the persimmon ethanol extract may contain 36.5 to 43.5 μg of 6,8′-biecol per 1 mg of persimmon ethanol extract, preferably 38.5 to 41.5 μg, more preferably 39.5 to 40.5 μg, and even more preferably 39.99 μg, but is not limited thereto.

[0061] In one embodiment of the present invention, the persimmon ethanol extract may contain, but is not limited to, 3.5 to 10.5 μg, preferably 5.5 to 8.5 μg, more preferably 6.5 to 7.5 μg, and even more preferably 7.14 μg of dibenzodioxin-fucodifluoroethol per 1 mg of persimmon ethanol extract.

[0062] In one embodiment of the present invention, the persimmon ethanol extract may contain 6 to 13 μg, preferably 8 to 11 μg, more preferably 9 to 10 μg, and even more preferably 9.25 μg of phloropucol A per 1 mg of the persimmon ethanol extract, but is not limited thereto.

[0063] In one embodiment of the present invention, the persimmon ethanol extract may contain 1 to 7 μg, preferably 2 to 6 μg, more preferably 3 to 5 μg, and even more preferably 4.00 μg of 2,7''-phloroglucinol-6,6'-biecol per 1 mg of the persimmon ethanol extract, but is not limited thereto.

[0064] In one embodiment of the present invention, the ethanol extract of the perilla leaf may improve, but is not limited to, impaired spatial cognitive ability, impaired short-term memory, and impaired long-term memory ability.

[0065] In one embodiment of the present invention, the ethanol extract of Gamjatda may reduce the content of malondialdehyde (MDA) in brain tissue and increase the level of reduced gluathione (GSH) and superoxide dismutase (SOD), but is not limited thereto.

[0066] In one embodiment of the present invention, the ethanol extract of Gamjatda can increase the content of Acetylcholine (ACh) in brain tissue and decrease Acetylcholinesterase (AChE) activity, but is not limited thereto.

[0067] In one embodiment of the present invention, the ethanol extract of the perilla leaf may increase the expression of AChE, Akt and BCl-2 in brain tissue, but is not limited thereto.

[0068] In one embodiment of the present invention, the ethanol extract of perilla leaves may reduce the expression of ChAT, SYP and PSD-95, Aβ, tau, JNK and BAX in brain tissue, but is not limited thereto.

[0069] In one embodiment of the present invention, the ethanol extract of the perilla leaf may reduce the expression of a neuroinflammation-related protein in brain tissue, but is not limited thereto.

[0070] In one embodiment of the present invention, the neuroinflammation-related protein may be at least one selected from the group consisting of TLR-4, MyD88, NF-κB, IκB-α, IL-1β, and TNF-α, but is not limited thereto.

[0071] In one aspect, the present invention comprises a step of mixing Ecklonia cava and ethanol, and then extracting; Phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA and 2,7′′-Phloroglucinol-6,6′-bieckol are mixed in a ratio of 1 to 2: 5 to 6: 6.5 to 7.5: 7 to 8: A method for producing a perilla leaf ethanol extract is provided, wherein the extract is contained in a ratio of 20~21 : 3~4 : 47~48 : 4.5~5.5 : 1.5~2.5, preferably 1.35 : 5.32 : 7.06 : 7.63 : 20.79 : 3.71 : 47.25 : 4.81 : 2.08.

[0072] In one aspect, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction, comprising the Ecklonia cava ethanol extract as an active ingredient.

[0073] In one embodiment of the present invention, the cognitive impairment may be at least one selected from the group consisting of dementia, Alzheimer's disease, ischemic stroke, traumatic brain injury, amnesia, Parkinson's disease, Pick's disease, Creutzfeldt-Jakob disease, and mild cognitive impairment, but is not limited thereto.

[0074] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient, the ethanol extract of Gamjatang. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete or incomplete adjuvant may be further included to increase its immunity.

[0075] The pharmaceutical composition according to the present invention can be prepared in a form in which the ethanol extract of Gamjatang can be mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0076] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0077] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0078] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0079] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0080] The above pharmaceutical composition can be formulated into various oral or parenteral dosage forms.

[0081] Oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsions, syrups, and granules. These dosage forms may further contain, in addition to the active ingredient, diluents (e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, and / or glycine), lubricants (e.g., silica, talc, stearic acid and its magnesium or calcium salts, and / or polyethylene glycol). In addition, the tablets may contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, and / or polyvinylpyrrolidine, and, if desired, disintegrants or effervescent mixtures such as starch, agar, alginic acid or its sodium salt, and / or absorbents, coloring agents, flavoring agents, and sweetening agents. The above formulation can be prepared by conventional mixing, granulating or coating methods.

[0082] In addition, representative parenteral administration formulations include injectable preparations, and solvents for injectable preparations include water, Ringer's solution, isotonic saline solution, or suspensions. Sterile fixed oils for the injectable preparations can be used as solvents or suspension media, and any non-irritating fixed oil, including mono- and di-glycerides, can be used for this purpose.

[0083] Additionally, the above injectable formulation may use a fatty acid such as oleic acid.

[0084] In one aspect, the present invention provides a health functional food composition for preventing or improving cognitive dysfunction, comprising the Ecklonia cava ethanol extract as an active ingredient.

[0085] The term "health functional food" used in the present invention is the same as food for special health use (FoSHU), and refers to a food with high medical and healthcare effects that has been processed to efficiently exhibit a bioregulatory function in addition to providing nutrition. Here, "function" means regulating nutrients for the structure and function of the human body or obtaining a useful effect for health purposes such as physiological action. The health functional food of the present invention can be manufactured by a method commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a food. The health functional food of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material, and is highly portable, so the health functional food of the present invention can be taken as a supplement to enhance the effect of improving blood circulation.

[0086] The above health functional foods refer to foods that have a more active health maintenance or promotion effect than regular foods, while health supplement foods refer to foods intended for health supplementation. In some cases, the terms health functional foods, health foods, and health supplements are used interchangeably.

[0087] Specifically, the health functional food means a food product manufactured by adding the composition of the present invention to food materials such as beverages, teas, spices, gums, and confectionery, or by manufacturing it in the form of encapsulation, powder, suspension, etc., and which brings about a specific health effect when consumed, but unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs made from food as a raw material.

[0088] The above health functional food may additionally include a physiologically acceptable carrier. The type of carrier is not particularly limited, and any carrier commonly used in the relevant technical field may be used.

[0089] In addition, the health functional food may include additional ingredients commonly used in foods to improve odor, taste, appearance, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and amino acids such as lysine, tryptophan, cysteine, and valine.

[0090] In addition, the above health functional food may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), sterilizers (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glucose), film agents, gum bases, foam suppressants, solvents, and improvers. The above additives can be selected according to the type of food and used in an appropriate amount.

[0091] As an example of the health functional food of the present invention, it can be used as a health beverage composition, and in this case, it can contain various flavoring agents or natural carbohydrates as additional ingredients like a regular beverage. The above-mentioned natural carbohydrates can be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol and erythritol. The sweetener can be a natural sweetener such as thaumatin and stevia extract; a synthetic sweetener such as saccharin and aspartame, etc. The proportion of the natural carbohydrate can be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

[0092] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, it may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly important, but is typically selected within the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

[0093] In one aspect, the present invention provides a method for increasing the dieccol content of an extract of Ecklonia cava, comprising the steps of mixing Ecklonia cava with ethanol and then extracting it.

[0094] In one aspect, the present invention provides a method for preventing or treating cognitive dysfunction comprising administering the pharmaceutical composition to a subject.

[0095] As described above, specific embodiments of the present invention have been described in detail. However, those skilled in the art who understand the spirit of the present invention will be able to easily suggest other inventions that are backwards or other embodiments included within the scope of the spirit of the present invention by adding, changing, or deleting other components within the scope of the same spirit. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention is indicated by the scope of the claims described below rather than the detailed description described above, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

[0096] <Example 1> Preparation of perilla extract and Angelica gigas Nakai extract powder

[0097] The Ecklonia cava extract used in the present invention was provided by Three Brooks Therapeutics Co., Ltd. (Pohang, Korea) and used in the experiment. It was extracted by adding 70% ethanol to Ecklonia cava.

[0098] The extract powder of Angelica gigas Nakai used as a positive control (PC) in the present invention was purchased from Nutragen Co., Ltd. (Eumseong-gun, Chungcheongbuk-do, Republic of Korea).

[0099] The extracts of Gamtae and Angelica gigas extract powders were stored at -20℃ and used.

[0100] <Example 2> Preparation of Aβ-induced cognitive dysfunction model

[0101] The experimental animals used in the present invention were 4-week-old ICR male mice purchased from a laboratory animal supplier, and all experimental animals were raised under constant temperature (22±2℃) and constant humidity (50-55%) conditions, with sufficient food and drinking water supplied under shady conditions with day and night alternating at 12-hour intervals. After acclimation for one week, the mice were divided into a normal control (NC), an Aβ (Amyloid beta) treatment group, a positive control group (a positive control group treated with 100 mg / kg of body weight (BW) of Angelica gigas Nakai extract powder), and a sample group (ethanol extract of Ecklonia cava (EE)). The sample group was divided into low concentration (EE25, 25 mg / kg of BW), medium concentration (EE50, 50 mg / kg of BW), and high concentration (EE100 mg / kg of BW). All animal experiment procedures were performed under the approval of the Animal Experiment Ethics Committee of Gyeongsang National University (Approval Number: GNU-231005-M0192).

[0102] After adaptation, the control and Aβ treatment groups were administered drinking water, and the sample group was administered the sample dissolved in drinking water orally once a day for 3 weeks. In order to induce cognitive dysfunction, 410 pM Aβ was administered to the bregma of the mice using a Hamilton micro-syringe in the remaining groups, except for the normal control group. 1-42 was injected intracerebroventricularly (icv).

[0103] <Experimental Example 1> Confirmation of behavioral improvement in Aβ-induced cognitive dysfunction model.

[0104] 1-1. Spatial Cognitive Ability Assessment (Y-maze Test)

[0105] To evaluate spatial cognitive ability in the Aβ-induced cognitive dysfunction model of the extract of Gamtae, a Y-maze experiment was performed.

[0106] Specifically, a Y-shaped maze device consisting of three arms made of black plastic was used. Each arm was randomly designated as A, B, and C. A mouse was placed on one arm, and its movement path was recorded for 8 minutes using a video tracking system. One point was awarded for entering a different arm in succession, and alternation behavior was calculated using the total number of arm entries.

[0107] As a result, as shown in Fig. 1, the mobility of the mice was measured by the number of times they entered each arm of the Y-maze, and no significant difference was observed in any group (Fig. 1(A)). The level of crossing behavior of the mice was significantly reduced in the Aβ group (27.64%) compared to the NC group (44.60%) (Fig. 1(B) and (C)). On the other hand, compared to the Aβ group, the PC, EE25, EE50, and EE100 groups (33.86%, 32.38%, 33.65%, and 40.21%) all showed significant improvement, and the EE100 group showed the greatest improvement in particular.

[0108] Through this, the effect of the extract of Gamtae on improving spatial cognitive ability was confirmed.

[0109] 1-2. Short-term memory ability assessment (Passive avoidance test)

[0110] To evaluate short-term memory ability in the Aβ-induced cognitive dysfunction model of perilla extract, a passive avoidance experiment was performed.

[0111] Specifically, a device was used, divided into two zones (light and dark chambers) and constructed with a stainless steel floor. First, mice were acclimated to a dark environment for 1 minute in the light chamber with the lights off, and then exposed to light for 2 minutes. Then, the door between the two chambers was opened, and when the mouse moved all four paws into the dark chamber, a 0.5 mA electric shock was delivered for 3 seconds. Twenty-four hours later, the time it took for the mouse to move into the dark chamber was measured using the same method, up to a maximum of 300 seconds.

[0112] As a result, as shown in Fig. 2, on the first day of the passive avoidance test, there was no significant difference in the time it took for the mice to enter the dark chamber among all groups (Fig. 2(A)). On the second day of the passive avoidance test, the time it took to enter the dark chamber decreased in the Aβ group (103.00 s) compared to the NC group (298.57 s), but the time it took to enter the dark chamber significantly increased in the PC, EE25, EE50, and EE100 groups (185.14 s, 178.71 s, 187.14 s, 227.29 s) (Fig. 2(B)). In particular, the time it took to enter the dark chamber was the longest in the EE100 group compared to the other groups that were treated with the sample.

[0113] Through this, the effect of the extract of perilla leaves on improving short-term memory was confirmed.

[0114] 1-3. Long-term memory assessment (Morris water maze test)

[0115] To evaluate long-term memory ability in the Aβ-induced cognitive dysfunction model of the extract of perilla leaves, a Morris water maze experiment was performed.

[0116] Specifically, a circular tank (diameter 150 cm, height 60 cm) was filled with water to a height of 30 cm (23±2℃), and the tank was divided into four quadrants to display visual cues. An escape platform was installed in the central area of ​​one quadrant, and non-toxic squid ink was dissolved. On the first day of the experiment, the platform was exposed, and the experimental animals were allowed to swim for 60 seconds to find it. If they could not find it, they were guided to the platform and kept there for 20 seconds to learn its location. Thereafter, for four days of training, the tank was filled with water so that the platform was not visible 1 cm below the water surface, and the experimental animals were allowed to swim four times a day, each time entering the water (N, S, E, W zone), conducting repeated training. The delay time it took for the mice to escape from the water to the platform was recorded using a video tracking system. If the experimental animals reached the platform within 60 seconds, they were allowed to stay there for 15 seconds. If they could not find it, they were guided onto the platform and kept there for 20 seconds to conduct training. On the 6th day of the experiment, in the probe test, the platform was removed and the time spent in the area where the platform was (W zone) for 60 seconds was measured.

[0117] As a result, as shown in Fig. 3, the time it took for the mice to escape from the tank to the platform was measured for a total of 4 days, and no significant difference was observed in any group on the 1st day (Fig. 3(A)). On the 4th day, the escape time increased in the Aβ group (39.92 s) compared to the NC group (7.83 s), but the escape time significantly decreased in all of the PC, EE25, EE50, and EE100 groups (20.91 s, 19.52 s, 18.03 s, 12.83 s). In particular, the escape time was reduced most significantly in the E100 group compared to the other groups treated with the sample. Afterwards, when the platform was removed and the percentage of mice staying in the area where the platform was was measured, it was found to significantly decrease in the Aβ group (28.33%) compared to the NC group (48.14%) (Fig. 3(B) and (C)). However, the proportion of people staying in the area where the platform was located increased in the PC, EE25, EE50, and EE100 groups (35.05%, 38.69%, 40.41%, and 41.24%), and the EE100 group showed the highest proportion.

[0118] Through this, the effect of the perilla extract on improving long-term memory ability was confirmed.

[0119] <Experimental Example 2> Confirmation of the effect of improving the antioxidant system in an Aβ-induced cognitive dysfunction model.

[0120] 2-1. Malondialdehyde (MDA) content analysis

[0121] To confirm the antioxidant system improvement effect of the extract of Gamjatdae in the Aβ-induced cognitive dysfunction model, the malondialdehyde (MDA) content was measured in the brain tissue of mice.

[0122] Specifically, mouse brain tissue was extracted using PBS, thiobarbituric acid was added, and the mixture was reacted at 95°C for 1 hour, and the absorbance of the supernatant was measured at 532 nm.

[0123] As a result, as shown in Fig. 4(A), the MDA content significantly increased in the Aβ group (6.06 nmole / mg of protein) compared to the NC group (4.08 nmole / mg of protein). On the other hand, the content decreased in the PC, EE25, EE50, and EE100 groups (4.88 nmole / mg of protein, 4.86 nmole / mg of protein, 4.79 nmole / mg of protein, 4.03 nmole / mg of protein), showing significant improvement, and in particular, the EE100 group showed the best improvement effect.

[0124] Through this, it was confirmed that the extract of perilla leaves has an effect of improving the antioxidant system by reducing the content of malondialdehyde (MDA).

[0125] 2-2. Analysis of reduced gluathione (GSH) levels

[0126] To confirm the antioxidant system improvement effect of the extract of Gamjatdae in the Aβ-induced cognitive dysfunction model, the level of reduced gluathione (GSH) was measured in the brain tissue of mice.

[0127] Specifically, mouse brain tissue was extracted using a phosphate buffer, metaphosphoric acid was added to remove interfering proteins, and tris-HCl (pH 7.8), 0.65 N-NaOH, and OPT (in methanol) were added to measure fluorescence.

[0128] As a result, as shown in Fig. 4(B), the reduced GSH level was significantly decreased in the Aβ group (75.23% of control) compared to the NC group (100%). On the other hand, the level increased in the PC, EE25, EE50, and EE100 groups (94.57% of control, 89.07% of control, 98.24% of control, 104.09% of control), and in particular, the EE100 group showed the greatest improvement effect.

[0129] Through this, it was confirmed that the extract of perilla leaves has an effect of improving the antioxidant system by increasing the level of reduced gluathione (GSH).

[0130] 2-3. Superoxide dismutase (SOD) level analysis

[0131] To confirm the effect of perilla extract on improving the antioxidant system in the Aβ-induced cognitive dysfunction model, superoxide dismutase (SOD) levels were measured in mouse brain tissue.

[0132] Specifically, mouse brain tissue was extracted using PBS, 1X cell extraction buffer was added, and the resulting supernatant was subjected to an experiment using an SOD determination kit (Sigma-Aldrich Chemical Co.).

[0133] As a result, as shown in Fig. 4(C), the SOD level was significantly decreased in the Aβ group (1.91 U / mg of protein) compared to the NC group (2.93 U / mg of protein). On the other hand, the level increased in the PC, EE25, EE50, and EE100 groups (2.20 U / mg of protein, 2.27 U / mg of protein, 2.40 U / mg of protein, 2.53 U / mg of protein), and in particular, the EE100 group showed the greatest improvement effect.

[0134] Through this, it was confirmed that the extract of perilla leaves has an effect of improving the antioxidant system by increasing the level of superoxide dismutase (SOD).

[0135] <Experimental Example 3> Confirmation of the effect of improving mitochondrial function in an Aβ-induced cognitive dysfunction model.

[0136] 3-1. Analysis of reactive oxygen species (ROS) levels

[0137] To confirm the effect of perilla extract on improving mitochondrial function in the Aβ-induced cognitive dysfunction model, reactive oxygen species (ROS) levels were measured in mouse brain tissue.

[0138] Specifically, mouse brain tissue was homogenized in mitochondrial isolation (MI) buffer [215 mM mannnitol, 75 mM sucrose, 0.1% BSA, 20 mM HEPES sodium salt (pH 7.2)] containing 10 times 1 mM EGTA. The homogenate was centrifuged at 1,300 g for 10 min at 4 °C to remove intact cells and nuclei, and the supernatant was centrifuged again at 13,000 g for 10 min at 4 °C. The supernatant was removed to remove synaptosomes, and the remaining mitochondrial pellet was mixed with MI buffer containing 0.1% digitonin and reacted for 5 min.

[0139] To measure the ROS content in mitochondria extracted through the above process, mitochondria were reacted with KCl-based respiration buffer [125 mM potassium chloride, 2 mM potassium phosphate monobabic, 20 mM HEPES, 1 mM magnesium chloride, 500 μM EGTA, 2.5 mM malate, and 5 mM pyruvate] and 25 μM DCF-DA for 20 minutes, and then fluorescence was measured.

[0140] As a result, as shown in Fig. 5(A), there was a significant increase in the Aβ group (132.87% of control) compared to the NC group (100%). On the other hand, improvements were observed in the PC, EE25, EE50, and EE100 groups (113.39% of control, 95.47% of control, 91.03% of control, 87.03% of control), and the perilla extract showed a superior improvement effect compared to the PC group. In particular, the EE100 group showed the greatest improvement effect.

[0141] Through this, it was confirmed that the extract of perilla leaves has the effect of improving mitochondrial function by reducing the level of reactive oxygen species (ROS).

[0142] 3-2. Analysis of Mitochondrial Membrane Potential (MMP) Levels

[0143] To confirm the effect of the extract of Gamtae on improving mitochondrial function in the Aβ-induced cognitive dysfunction model, the level of mitochondrial membrane potential (MMP) was measured in mouse brain tissue.

[0144] Specifically, mouse brain tissue was extracted using a phosphate buffer, metaphosphoric acid was added to remove interfering proteins, and tris-HCl (pH 7.8), 0.65 N-NaOH, and OPT (in methanol) were added to measure fluorescence.

[0145] As a result, as shown in Fig. 5(B), there was a significant decrease in the Aβ group (72.86% of control) compared to the NC group (100%). On the other hand, there was improvement in the PC, EE25, EE50, and EE100 groups (86.19% of control, 87.21% of control, 91.54% of control, 95.12% of control), and in particular, the EE100 group showed the greatest improvement effect.

[0146] Through this, it was confirmed that the extract of perilla leaves has an effect of improving mitochondrial function by increasing the level of mitochondrial membrane potential (MMP).

[0147] 3-3. Adenosine triphosphate (ATP) content analysis

[0148] To confirm the effect of perilla extract on improving mitochondrial function in the Aβ-induced cognitive dysfunction model, the adenosine triphosphate (ATP) content was measured in mouse brain tissue.

[0149] Specifically, mouse brain tissue was extracted using a phosphate buffer, metaphosphoric acid was added to remove interfering proteins, and tris-HCl (pH 7.8), 0.65 N-NaOH, and OPT (in methanol) were added to measure fluorescence.

[0150] As a result, as shown in Fig. 5(C), there was a significant decrease in the Aβ group (2.69 nmole / mg of protein) compared to the NC group (7.34 nmole / mg of protein). On the other hand, there was improvement in the PC, EE25, EE50, and EE100 groups (3.55 nmole / mg of protein, 3.51 nmole / mg of protein, 4.36 nmole / mg of protein, 4.56 nmole / mg of protein), and in particular, the EE100 group showed the greatest improvement effect.

[0151] Through this, it was confirmed that the extract of perilla leaves has the effect of improving mitochondrial function by increasing the content of adenosine triphosphate (ATP).

[0152] <Experimental Example 4> Confirmation of the effect of improving the cholinergic system in an Aβ-induced cognitive dysfunction model.

[0153] 4-1. Analysis of Acetylcholine (ACh) content

[0154] To confirm the effect of the extract of Gamtae on improving the cholinergic system in the Aβ-induced cognitive dysfunction model, the content of acetylcholine (ACh) in the brain tissue of mice was measured.

[0155] Specifically, after extracting the mouse brain tissue using PBS, alkaline hydroxylamine reagent (3.5 N sodium hydroxide and 2 M hydroxylamine in HCl) was added and reacted for 1 minute, and then 0.5 N HCl (pH 1.2) and 0.37 M FeCl3 in 0.1 N HCl were added and the absorbance was measured at 540 nm.

[0156] As a result, as shown in Fig. 6(A), the content was significantly decreased in the Aβ group (0.64 mmole / mg of protein) compared to the NC group (1.16 mmole / mg of protein). On the other hand, the content increased in the PC, EE25, EE50, and EE100 groups (0.86 mmole / mg of protein, 0.80 mmole / mg of protein, 0.81 mmole / mg of protein, 1.13 mmole / mg of protein), and in particular, the EE100 group showed the best improvement effect.

[0157] Through this, it was confirmed that the extract of perilla leaves has an effect of improving the cholinergic system by increasing the content of acetylcholine (ACh).

[0158] 4-2. Acetylcholinesterase (AChE) activity analysis

[0159] To confirm the effect of the extract of Gamtae on improving the cholinergic system in the Aβ-induced cognitive dysfunction model, Acetylcholinesterase (AChE) activity was measured in the brain tissue of mice.

[0160] Specifically, after extracting the mouse brain tissue using PBS, 65 ㎕ of 50 mM sodium phosphate buffer was added to 5 ㎕ of enzyme, reacted at 37 ℃ for 15 minutes, and 70 ㎕ of Ellman's reaction mixture was added, and the absorbance was measured at 405 nm.

[0161] As a result, as shown in Fig. 6(B), activity significantly increased in the Aβ group (128.11% of control) compared to the NC group (100%). On the other hand, activity decreased in the PC, EE25, EE50, and EE100 groups (104.79% of control, 111.45% of control, 110.65% of control, 98.79% of control), and in particular, the EE100 group showed the greatest improvement effect.

[0162] Through this, it was confirmed that the extract of perilla leaves has an effect of improving the cholinergic system by reducing acetylcholinesterase (AChE) activity.

[0163] 4-3. Analysis of protein expression levels related to the cholinergic system and synaptic function

[0164] To confirm the effect of the extract of Gamtae on improving the cholinergic system in the Aβ-induced cognitive dysfunction model, the expression levels of proteins related to the cholinergic system and synaptic function were measured in the brain tissue of mice.

[0165] Specifically, mouse brain tissue was homogenized with protein extraction buffer containing 1% protase inhibitor 10 times the amount, and then centrifuged at 13,000 rpm to obtain the supernatant. The supernatant was subjected to western blot analysis to measure protein expression levels, and the expression levels of proteins related to the cholinergic system and synaptic function (AChE, ChAT, SYP, and PSD-95) were measured.

[0166] As a result, as shown in Fig. 7(B), the AChE expression level results showed that the Aβ group (1.50) showed a significantly increased expression level compared to the NC group, while the EE25 and EE100 groups (1.19, 1.04) showed a significant improvement.

[0167] In addition, as shown in Figures 7(C), (D), and (E), the protein expression levels of ChAT, SYP, and PSD-95 were significantly reduced in the Aβ group (0.83, 0.42, 0.63) compared to the NC group, and the EE100 group (1.00, 0.76, 1.15) significantly improved them.

[0168] Through this, it was confirmed that the extract of Gamtae significantly improved proteins related to the cholinergic system and synaptic function, thereby having an effect of improving the cholinergic system.

[0169] <Experimental Example 5> Analysis of protein expression levels in an Aβ-induced cognitive dysfunction model

[0170] 5-1. Analysis of protein expression levels related to the Aβ / Tau pathway

[0171] To confirm the improvement effect of the extract of perilla frutescens on toxicity caused by Aβ aggregation and Tau hyperphosphorylation in the Aβ-induced cognitive dysfunction model, the expression level of proteins related to the Aβ / Tau pathway was measured in mouse brain tissue.

[0172] Specifically, mouse brain tissue was homogenized with protein extraction buffer containing 1% protase inhibitor 10 times the amount, and then centrifuged at 13,000 rpm to obtain the supernatant. The supernatant was subjected to western blot analysis to measure protein expression levels, and the expression levels of Aβ / Tau pathway-related proteins (Aβ, IDE, p-tau, p-GSK-3β) were measured.

[0173] As a result, as shown in Figs. 8(B) and (D), the expression levels of Aβ and Tau were significantly increased in the Aβ group (1.39, 1.91) compared to the NC group, and the EE100 group (0.93, 1.14) significantly improved them. In addition, as shown in Figs. 8(C) and (E), the expression levels of IDE and p-GSK-3β, known to regulate the expression levels of Aβ and Tau, were significantly decreased in the Aβ group (0.39, 0.48) compared to the NC group, whereas the EE100 group (0.69, 0.69) significantly increased them.

[0174] Through this, it was confirmed that the extract of perilla leaves had an improving effect on toxicity caused by Aβ aggregation and Tau hyperphosphorylation by reducing the expression level of proteins related to the Aβ / Tau pathway.

[0175] 5-2. Analysis of protein expression levels related to neuroinflammation

[0176] To confirm the neuroinflammation-improving effect of the extract of Gamchaetae in the Aβ-induced cognitive dysfunction model, the expression levels of neuroinflammation-related proteins were measured in mouse brain tissue.

[0177] Specifically, the mouse brain tissue was homogenized with protein extraction buffer containing 1% protase inhibitor 10 times that of the mouse brain tissue, and then centrifuged at 13,000 rpm to obtain the supernatant. The supernatant was subjected to western blot analysis to measure protein expression levels, and the expression levels of neuroinflammation-related proteins (TLR-4, MyD88, p-NF-κB, p-IκB-α, IL-1β, TNF-α) were measured.

[0178] As a result, as shown in Figures 9(B) and (C), the protein expression levels of TLR-4 and MyD88 significantly increased in the Aβ group (1.57, 1.64) compared to the NC group, and significantly decreased in the EE 100 group (0.77, 0.66). In addition, as shown in Figures 9(D) and (E), the protein expression levels of p-NF-κB and p-IκB-α significantly increased in the Aβ group (1.57, 1.35) compared to the NC group. The EE100 group (0.90, 0.60) significantly reduced the increased expression levels in the Aβ group. As shown in Figures 9(F) and (G), the expression levels of IL-1β and TNF-α significantly increased in the Aβ group (2.01, 1.48) compared to the NC group. In contrast, the EE25 group (1.09, 0.96) and the EE100 group (0.78, 0.98) showed significant improvement.

[0179] Through this, it was confirmed that the extract of perilla leaves has an effect of improving neuroinflammation by reducing the expression level of proteins related to neuroinflammation.

[0180] 5-3. Analysis of Apoptosis-Related Protein Expression Levels

[0181] To confirm the protective effect of the extract of Gamjatdae against apoptosis in the Aβ-induced cognitive dysfunction model, the expression levels of apoptosis-related proteins were measured in mouse brain tissue.

[0182] Specifically, mouse brain tissue was homogenized with protein extraction buffer containing 1% protase inhibitor 10 times the amount, and then centrifuged at 13,000 rpm to obtain the supernatant. The supernatant was subjected to western blot analysis to measure protein expression levels, and the expression levels of apoptosis-related proteins (p-JNK, p-Akt, BAX, BCl-2, caspase 3) were measured.

[0183] As a result, as shown in Fig. 10 (B) and (D), the expression levels of p-JNK and BAX in the Aβ group (1.85, 2.34) were significantly increased compared to the NC group, and the EE100 group (1.09, 0.98) significantly decreased them. In addition, as shown in Fig. 10 (C) and (E), the expression levels of p-Akt and BCl-2 in the Aβ group (0.43, 0.42) were significantly decreased compared to the NC group, and the EE100 group (0.79, 0.99) significantly increased them. As shown in Fig. 10 (F), the BAX / BCl-2 ratio level was significantly increased in the Aβ group (3.23) compared to the NC group, and the EE100 group (1.12) significantly improved the increased level in the Aβ group. As shown in Fig. 10(G), the caspase 3 expression level was measured and significantly increased in the Aβ group (1.27) compared to the NC group. On the other hand, the EE100 group (0.68) significantly reduced the increased caspase 3 expression level in the Aβ group.

[0184] Through this, it was confirmed that the extract of perilla leaves has a protective effect against apoptosis by improving the expression level of apoptosis-related proteins.

[0185] <Experimental Example 6> Detection and analysis of indicator components in perilla extract

[0186] To analyze the bioactive (marker) substances contained in the extract of Kelvin (Germany) kelp, Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF / MS) and high-performance liquid chromatography with photodiode-array detection (HPLC-DAD) systems were used. The quantitative analysis of the marker component (dieckol) contained in the water extract and 70% ethanol extract of Kelvin (Germany) kelp was performed using the standard calibration curve method using HPLC.

[0187] As a result of analysis using UPLC-Q-TOF / MS, as shown in Fig. 11(A) and Table 1, it was confirmed that the 70% ethanol extract of Gam-tae contained 9 compounds. As shown in Fig. 11(B) and Table 2, it was confirmed that the water extract of Gam-tae contained 8 compounds. In addition, it was confirmed that the peak height and area of ​​dieckol, an indicator component of Gam-tae, were higher in the 70% ethanol extract of Gam-tae than in the water extract of Gam-tae.

[0188] In addition, the nine compounds listed in Table 1 below, Phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA, and 2,7′′-Phloroglucinol-6,6′-bieckol, were 1.35 : 5.32 : 7.06 : 7.63 : 20.79 : 3.71 : 47.25 : It was confirmed that it was included in the ethanol extract of perilla leaves at a ratio of 4.81:2.08.

[0189] No.RT(min)m / z[M-H]-FragmentsPropose compounds13.24373233, 247, 229, 124, 189Phlorotannin oligomer24.55371149, 201, 245, 263, 217Eckol34.64495154, 263, 297, 3877-Phloroeckol44.82741201, 229, 371, 4776,6′-Bieckol54.88741260, 371, 479, 6156,8′-Bieckol65.30743125, 139, 231, 353Dibenzodioxin-fucodiphloroethol76.02741201, 229, 261, 369, 371, 493, 615Diekcol86.89601244, 299, 385, 492, 493PhlorofuroeckolA96.96973229, 493, 601, 602, 741, 707, 9732,7′′-Phloroglucinol-6,6′-bieckol (PHB)

[0190] No.RT(min)m / z[M-H]-FragmentsPropose compounds13.24373233, 247, 229Phlorotannin oligomer24.69495263, 387, 4597-Phloroeckol34.82741201, 229, 371, 4776,6′-Bieckol44.91741260, 371, 479, 6156,8′-Bieckol55.32743125, 139, 231, 353Dibenzodioxin-fucodiphloroethol66.03741201, 229, 261, 369, 371, 493, 615Diekcol76.90601299, 366, 492, 493PhlorofuroeckolA86.99973229, 493, 353, 7072,7′′-Phloroglucinol-6,6′-bieckol (PHB)

[0191] For more precise analysis, HPLC-DAD was used for quantitative analysis through the standard curve of dieckol (Fig. 12(A)). As a result, 90.89 ± 0.20 μg / mg of dried weight was quantified in the 70% ethanol extract of Gam-tae, as shown in Fig. 12(B) and Table 3. In addition, as shown in Fig. 12(C) and Table 3, 14.08 ± 0.35 μg / mg of dried weight was quantified in the water extract of Gam-tae.

[0192] Dieckol concentration (Unit: μg / mg of dried weight)70% ethanol extract ofE. cava90.89 ± 0.20Water extract ofE. cava14.08 ± 0.35

[0193] Through this, it was confirmed that the content of dieckol, an indicator component of the perilla extract, was approximately 6 times higher in the 70% ethanol extract of perilla leaves compared to the water extract.

[0194] The present invention has been described above, focusing on preferred embodiments and experimental examples. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. Phlorotannin oligomer, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA and 2,7′′-Phloroglucinol-6,6′-bieckol were 1~2 : 5~6 : 6.5~7.5 : 7~8 : 20~21 : 3~4 : 47~48 : 4.5~5.5 : Ecklonia cava ethanol extract, contained in a ratio of 1.5 to 2.

5.

2. In paragraph 1, The above-mentioned ethanol extract of perilla leaves is characterized in that it contains 80 to 100 μg of diechol per 1 mg of the ethanol extract of perilla leaves.

3. In paragraph 1, A perilla leaf ethanol extract, characterized in that the ethanol is 70% ethanol.

4. In paragraph 1, The above-mentioned ethanol extract of persimmon is characterized by improving impaired spatial cognitive ability, impaired short-term memory, and impaired long-term memory ability.

5. In paragraph 1, The above-mentioned ethanol extract of Gamjatda is characterized by reducing the content of malondialdehyde (MDA) in brain tissue and increasing the level of reduced gluathione (GSH) and superoxide dismutase (SOD).

6. In paragraph 1, The above-mentioned ethanol extract of Gamjatda is characterized by reducing the level of reactive oxygen species (ROS) in brain tissue and increasing the level of mitochondrial membrane potential (MMP) and adenosine triphosphate (ATP) content.

7. In paragraph 1, The above-mentioned ethanol extract of Gamjatda is characterized by increasing the content of Acetylcholine (ACh) in brain tissue and decreasing the activity of Acetylcholinesterase (AChE).

8. In paragraph 1, The above-mentioned ethanol extract of Gamjatda is characterized by increasing the expression of AChE, Akt and BCl-2 in brain tissue.

9. In paragraph 1, The above-mentioned ethanol extract of Gamjatda is characterized by reducing the expression of ChAT, SYP and PSD-95, Aβ, tau, JNK and BAX in brain tissue.

10. In paragraph 1, The above-mentioned ethanol extract of Gamjatda is characterized by reducing the expression of proteins related to neuroinflammation in brain tissue.

11. In paragraph 10, An ethanol extract of perilla leaves, characterized in that the neuroinflammation-related protein is at least one selected from the group consisting of TLR-4, MyD88, NF-κB, IκB-α, IL-1β, and TNF-α.

12. A method for producing a mixture of Ecklonia cava and ethanol, comprising: mixing ethanol and extracting phenols; comprising: preparing phosphorotannin oligomers, Eckol, 7-Phloroeckol, 6,6′-Bieckol, 6,8′-Bieckol, Dibenzodioxin-fucodiphloroethol, Diekcol, PhlorofuroeckolA, and 2,7′′-Phloroglucinol-6,6′-bieckol in a ratio of 1 to 2: 5 to 6: 6.5 to 7.5: 7 to 8: A method for producing a perilla leaf ethanol extract, comprising the ingredients in a ratio of 20~21 : 3~4 : 47~48 : 4.5~5.5 : 1.5~2.

5.

13. A pharmaceutical composition for preventing or treating cognitive dysfunction, comprising the ethanol extract of Ecklonia cava of paragraph 1 as an active ingredient.

14. In paragraph 13, A pharmaceutical composition for preventing or treating cognitive dysfunction, characterized in that the cognitive dysfunction is at least one selected from the group consisting of dementia, Alzheimer's disease, ischemic stroke, traumatic brain injury, amnesia, Parkinson's disease, Pick's disease, Creutzfeldt-Jakob disease, and mild cognitive dysfunction.

15. A health functional food composition for preventing or improving cognitive dysfunction, comprising the ethanol extract of Ecklonia cava of Article 1 as an active ingredient.

16. A method for increasing the dieccol content of a kelp extract, comprising the steps of mixing kelp (Ecklonia cava) and ethanol and then extracting.

17. A method for preventing or treating cognitive dysfunction, comprising administering to a subject the pharmaceutical composition of claim 13.

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