Composition for enhancing memory and preventing, ameliorating or treating cognitive impairment comprising complex of atractylodes macrocephala and rumex acetosa extracts as active ingredient

Abstract

The present invention relates to a composition for enhancing memory and preventing, ameliorating or treating cognitive impairment, comprising, as an active ingredient, a complex of Atractylodes macrocephala and Rumex acetosa extracts. The complex of Atractylodes macrocephala and Rumex acetosa extracts not only promotes CREB phosphorylation in hippocampal neurons, but also has a cytoprotective effect in brain neuronal cell lines, affects synapse remodeling at neuronal terminals that occurs during long-term potentiation (LTP), thereby increasing protein expression levels of synaptic remodeling factors and neuroplastic factors, and has been confirmed to improve short-term / long-term memory ability in behavioral evaluation of animal models, and thus can be effectively used as a health functional food, a medicine, or a feed additive, for enhancing memory and preventing, ameliorating or treating cognitive impairment.

Description

Composition for improving memory and preventing, improving or treating cognitive dysfunction, comprising a complex of extracts of Baekchul and Suyeong as active ingredients

[0001] The present invention relates to a composition for enhancing memory and preventing, improving or treating cognitive dysfunction, comprising a complex of extracts of Baekchul and Suyeong as active ingredients.

[0002] Cognitive impairment (Cognitive Impairment) is a comprehensive term for mental and behavioral disorders caused by brain diseases, brain injuries, addictions, aging, etc., as well as endocrine and metabolic nutritional disorders, and mental illnesses caused by drugs, etc. Depending on the severity of the symptoms, it is a type of brain disease that appears not only in amnesia, which is diagnosed as temporary short-term memory impairment, but also in cognitive dysfunction and dementia, which causes judgment impairment, and neurodegenerative diseases. Among them, mild cognitive impairment (MCI) refers to a state in which cognitive ability is lowered compared to that of people of the same age. It is a state in which cognitive areas such as memory, language ability, and judgment are reduced. MCI is a syndrome with various causes and symptoms, and is known to be caused by various causes such as degeneration due to aging, vascular causes due to vascular causes, psychiatric factors such as depression or anxiety, and other internal medicine factors.

[0003] Meanwhile, as the population of the elderly has rapidly increased due to the aging society, dementia and Parkinson's disease are recognized as degenerative central nervous system diseases in this social environment, and as the number of patients increases day by day, social issues are increasing, and the importance of preventing, improving, and treating cognitive dysfunction is increasingly highlighted.

[0004] Alzheimer's disease affects virtually all nerves, including cholinergic, adrenergic, GABAergic, and glutamatergic neurons, but research suggests that damage and loss of cholinergic neurons are particularly severe. Senile dementia, a disease characterized by a gradual loss of cognitive ability, is also associated with the activity of cholinergic neurons in the central nervous system, primarily due to a marked decline in acetylcholine and choline acetyltransferase activity in the brain. While active efforts are underway to develop treatments aimed at restoring or improving the function of cholinergic neurons, these efforts remain inadequate.

[0005] Meanwhile, Atractylodes macrocepala (also known as San-gae or Cheon-gae) is the dried root of the Atractylodes macrocepala plant, which is also called San-gae or Cheon-gae. It is very bitter, with a slightly sweet flavor. Its white flesh and full of seeds are preferred, and those harvested in the fall are said to be particularly effective.

[0006] The genus Rumex, which includes Rumex acetosa, is the largest genus in the Polygonaceae family, with approximately 200 species distributed worldwide, primarily in temperate regions of the Northern Hemisphere. About 10 species, including Rumex acetosa and water strider, are found in Korea. They are perennials found in mountains and fields throughout the country. Adult plants grow to 30–80 cm tall. Their stems are ridged, and they, along with their leaves, have a sour taste. The leaves on the stem are alternate and oblong, with short petioles at the base that disappear as they ascend. At the ends of the main stem branches, panicles 10–30 cm long and bear short flower stalks. The tender, sessile leaves are edible, and the roots are used as a scab remedy in the home market. Rumex leaves are known to resemble spinach, a highly sour plant found in Europe and the United States. Extracts of R. acetosa are known to have antipyretic, diuretic, insecticidal, antibacterial, and anticancer properties. Furthermore, water boiled in R. acetosa has been reported to be used as a folk remedy for arthritis, gastritis, and gastric ulcers. The leaves of R. acetosa contain vitexin and quercitrin, the roots contain anthraquinones and their glycosides, including chrysophanol, and the aerial parts contain flavonoids and their glycosides, including orientin.

[0007] As for technologies related to cognitive dysfunction or memory, Korean Patent Publication No. 2023-0174777 discloses a composition for enhancing memory and preventing, improving, or treating cognitive dysfunction containing Gungunjatang as an active ingredient, and Korean Patent Registration No. 1753057 discloses a pharmaceutical composition for preventing or treating cognitive dysfunction or degenerative brain disease. However, to date, no composition for enhancing memory and preventing, improving, or treating cognitive dysfunction has been disclosed, containing the complex of Baekchul and Suyeong extracts of the present invention as an active ingredient.

[0008] The present invention was derived from the above-mentioned needs, and the present invention provides a composition for enhancing memory and preventing, improving, or treating cognitive dysfunction, which contains a complex of extracts of Atractylodes Rhizome and Sophia japonica as an active ingredient, and the complex of extracts of Atractylodes Rhizome and Sophia japonica not only promotes CREB phosphorylation in hippocampal neurons, but also has a cytoprotective effect in a brain neuron cell line, and affects synapse remodeling of neuronal terminals that occurs when LTP (Long-term potentiation) is formed, thereby increasing the protein expression amount of synaptic remodeling factors and neuroplasticity factors, and improving short-term / long-term memory ability in a behavioral evaluation of an animal model, thereby completing the present invention.

[0009] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating cognitive dysfunction, which comprises a complex of extracts of Baekchul and Suyeong as an active ingredient.

[0010] In addition, the present invention provides a health functional food composition for improving memory and preventing or improving cognitive dysfunction, which comprises a complex of extracts of Baekchul and Suyeong as an active ingredient.

[0011] In addition, the present invention provides a veterinary composition for preventing or treating cognitive dysfunction, comprising a complex of extracts of white atractylodes and soybean as an active ingredient.

[0012] In addition, the present invention provides a feed additive for improving memory and preventing or improving cognitive dysfunction, which comprises a complex of extracts of white atractylodes and soybean sprouts as an effective ingredient.

[0013] The present invention relates to a composition for enhancing memory and preventing, improving or treating cognitive dysfunction, comprising a complex of extracts of Atractylodes Rhizome and Soybean Sprout as an active ingredient. The complex of extracts of Atractylodes Rhizome and Soybean Sprout not only promotes CREB phosphorylation in hippocampal neurons, but also has a cytoprotective effect in a brain neuron cell line, and affects synapse remodeling that occurs when LTP (Long-term potentiation) is formed, thereby increasing the protein expression levels of synaptic remodeling factors and neuroplasticity factors, and has the effect of improving short-term / long-term memory ability in behavioral assessments of animal models.

[0014] Figure 1 shows the results of confirming the cytotoxicity of the extract of Baekchul, Sooyoung, and the combined extract of Baekchul and Sooyoung in SH-SY5Y cells.

[0015] Figure 2 shows the results of confirming the cytoprotective effects of Atractylodes Rhizome extract, Soybean sprout extract, and Atractylodes Rhizome + Soybean sprout complex extract on cell death induced by scopolamine (SCO) treatment in SH-SY5Y cells. ## indicates that the cytoprotective effect of the scopolamine-treated group was statistically significantly decreased compared to the untreated control group, and p<0.01; *, **, and *** indicate that the cytoprotective effects of the Atractylodes Rhizome extract, Soybean sprout extract, and Atractylodes Rhizome + Soybean sprout complex extract treated groups were statistically significantly increased compared to the scopolamine-treated group, and * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0016] Figure 3 shows the results of a Western blot confirming changes in the protein expression levels of PSD95, AMPA receptor (subunits: GluA1), and NMDA 2A receptor (NR2A) after treating primary cultured brain cells from the hippocampus of a fetal rat with extracts of Baekchul Extract, Sooyoung Extract, and Baekchul Extract + Sooyoung Complex Extract.

[0017] Figure 4 shows the results of a Western blot that confirmed changes in the protein expression levels of PSD95, AMPA receptor (subunits: GluA1), and NMDA 2A receptor (NR2A) after treating primary cultured brain cells of the hippocampus of a fetal rat with a mixture of Baekchul and Suyeong extracts at different concentrations in a weight ratio of 2:1.

[0018] Figure 5 shows the results of the Morris water maze test, which confirmed the effects on long-term (A) and short-term (B) memory after administering a combination of extracts of Atractylodes Rhizome and Sophia japonica to SD rats. *, *** indicate that the long-term or short-term memory of the group administered the combination of extracts of Atractylodes Rhizome and Sophia japonica of the present invention statistically significantly increased compared to the control group. * indicates p<0.05, and *** indicates p<0.001.

[0019] Figure 6 shows the results of confirming the effect on long-term memory in the Morris water maze test probe test after administering a combination of extracts of Atractylodes rhizome and Sooyoung to SD rats. **, *** indicate that the swimming time in the quadrant where the escape zone was located was statistically significantly increased in the group administered the combination of extracts of Atractylodes rhizome and Sooyoung of the present invention compared to the control group. ** is p<0.01, and *** is p<0.001.

[0020] Figure 7 shows the results of confirming the changes in the expression levels of NMDA receptors (Subunits: NR2A, NR2B), AMPA receptors (subunits: GluA1), and PSD95 proteins in the hippocampal tissues extracted from SD rats after administering a combination of extracts of Baekchul and Suyeong. *, **, and *** indicate that the expression levels of NMDA receptors (Subunits: NR2A, NR2B), AMPA receptors (subunits: GluA1), and PSD95 proteins in the group administered a combination of extracts of Baekchul and Suyeong were statistically significantly increased compared to the control group. * is p<0.05, ** is p<0.01, and *** is p<0.001.

[0021] Figure 8 shows the results of confirming the change in the expression level of BDNF protein (A) and the phosphorylation of ERK and CaMKII (B) in the hippocampal tissue extracted after administering a complex of extracts of Baekchul and Suyeong to SD rats. **, *** indicate that the expression level of BDNF protein and the phosphorylation of ERK and CaMKII in the group administered the complex of extracts of Baekchul and Suyeong were statistically significantly increased compared to the control group. ** is p<0.01, and *** is p<0.001.

[0022] The present invention relates to a pharmaceutical composition for preventing or treating cognitive dysfunction, comprising a complex of extracts of Baekchul and Suyeong as an active ingredient.

[0023] The above extract of Baekchul is preferably a root extract of Saposhnikovia japonica, and the extract of Suyeong is preferably a leaf extract of Suyeong, but is not limited thereto.

[0024] The above complex of white and swimming extracts can be prepared by a method including, but not limited to, the following steps:

[0025] (1) A step of extracting by adding an extraction solvent to each of dried or dried white radish;

[0026] (2) A step of filtering the white or swimming extract of step (1) above;

[0027] (3) A step of drying the filtered extract of the above step (2) to prepare extracts of the white atractylodes and the water parsley extract, respectively; and

[0028] (4) A step of obtaining a complex of white atractylodes and water parsley extracts by mixing the white atractylodes and water parsley extracts prepared in the above step (3).

[0029] In the above step (1), the extraction solvent is preferably selected from water, C1-C4 lower alcohols, or mixtures thereof, more preferably water or ethanol, but is not limited thereto. In the above manufacturing method, the extraction method may use any conventional method known in the art, such as hot water extraction, immersion extraction, reflux cooling extraction, and ultrasonic extraction. It is preferable to extract the extraction solvent by adding 10 to 40 times the weight of the white or green tea extract. The extraction temperature is preferably 20 to 110°C, but is not limited thereto. In addition, the extraction time is preferably 1 to 5 hours, and more preferably 2 to 4 hours, but is not limited thereto. In the above step (3), the drying is preferably reduced pressure drying, vacuum drying, boiling drying, spray drying, or freeze drying, and is more preferably freeze drying, but is not limited thereto.

[0030] The complex of the extracts of Baekchul and Suyeong obtained in the above step (4) is a mixture of the extracts of Baekchul and Suyeong in a weight ratio of 0.5 to 2.5:1, more preferably 1.5 to 2.5:1, and most preferably 2:1, but is not limited thereto.

[0031] The above cognitive dysfunction is preferably, but not limited to, any one selected from dementia, learning disorder, agnosia, amnesia, aphasia, apraxia or delirium, mild cognitive impairment, and Binswanger disease, and the above dementia is preferably, but not limited to, any one selected from Alzheimer's dementia, vascular dementia, Lewy body dementia, frontal lobe dementia, semantic dementia, and multi-infarct dementia.

[0032] In addition to the above active ingredients, pharmaceutically acceptable carriers, excipients or diluents may be included, but are not limited thereto.

[0033] In addition, the present invention relates to a health functional food composition for improving memory and preventing or improving cognitive dysfunction, which comprises a complex of extracts of Baekchul and Suyeong as an active ingredient.

[0034] The above health functional food composition is preferably manufactured in any one dosage form selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto. The health functional food composition of the present invention can be manufactured by adding the complex of Atractylodes japonica and Solanum lucidum extracts as is or mixing them with other foods or food ingredients, and can be manufactured appropriately according to a conventional method. Examples of foods to which the complex of Atractylodes japonica and Solanum lucidum extracts can be added include dairy products including caramel, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and includes all health functional foods in the conventional sense. That is, there is no particular limitation on the type of the above food. The above health functional food composition may contain various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavorings, coloring agents and enhancers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain fruit pulp for the production of natural fruit juice and vegetable beverages. The above components may be used independently or in combination.

[0035] The health functional food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, wherein the natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. The proportion of the natural carbohydrate is not particularly important, but is preferably 0.01 to 0.04 g, and more preferably 0.02 to 0.03 g, per 100 g of the composition of the present invention, but is not limited thereto. As the sweetener, a natural sweetener such as thaumatin and stevia extract, or a synthetic sweetener such as saccharin and aspartame, may be used.

[0036] In addition, the present invention relates to a veterinary composition for preventing or treating cognitive dysfunction, comprising a complex of extracts of Baekchul and Suyeong as an active ingredient.

[0037] The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using a method well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile topical preparations, etc. The effective amount of the veterinary composition according to the present invention may be appropriately selected depending on the individual animal. It may be determined according to factors including the severity of the disease or condition, the sensitivity to the active ingredient of the present invention depending on the age, weight, health condition or sex of the individual, the route of administration, the period of administration, other compositions combined with or used simultaneously with the composition, and other factors well known in the field of physiology or veterinary medicine.

[0038] In addition, the present invention relates to a feed additive for improving memory and preventing or improving cognitive dysfunction, which comprises a complex of extracts of Baekchul and Suyeong as an effective ingredient.

[0039] The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. The term "feed" in the present invention may mean any natural or artificial diet, meal, etc., or ingredients of the meal, which are intended for or suitable for animals to eat, ingest, and digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feeds such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, meal, or grain by-products; and animal feeds such as proteins, inorganic substances, oils and fats, mineral substances, oils and fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0040]

[0041] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0042]

[0043] Example 1. Preparation of a complex of extracts of Baekchul and Suyeong

[0044] 300 g of dried sophora japonica leaves were heated at 100°C for 3 hours in 9 L of distilled water to obtain hot water extraction. The extracted sophora japonica leaf hot water extract was concentrated under reduced pressure and freeze-dried to obtain 64.45 g of freeze-dried product.

[0045] The dried powder of Baekchul (Root of Saposhnikovia japonica) was extracted using the same method as described above. Afterwards, the extract of Suyeong and Baekchul were mixed in the weight ratios shown in Table 1, and their efficacy was confirmed.

[0046] Mixing ratio of white chrysanthemum and water extracts Dry weight ratio of white chrysanthemum and water extracts Manufacturing example 1-11:1 Manufacturing example 1-21:2 Manufacturing example 1-32:1

[0047]

[0048] Example 2. Primary culture of hippocampal neurons from fetal rats. All cells used in the examples of the present invention are primary cultured hippocampal neurons. Hippocampal neurons were isolated from fetuses of SD rats on days 18 to 19 of pregnancy. The isolated hippocampal neural tissue was placed in HBSS (Hank's Balanced Salt Solution) containing 0.25% trypsin and reacted at 37°C for 10 minutes. After washing several times with HBSS, single cells were separated by careful pipetting using a 1 ml pipette. The isolated cells were cultured in Neuro basal / B27 medium (0.5 mM L-glutamine, 25 μM glutamate, 25 μM 2-mercaptoethanol, 100 U / mL penicillin, 100 μg / mL streptomycin) in a culture dish pre-coated with poly-L-lysine (0.5 mg / mL) at 37°C and 5% CO2. The medium was changed on days 4 and 11 of cell culture, and cells on day 14 of culture were used for the experiment.

[0049]

[0050] Example 3. Determination of CREB phosphorylation levels in hippocampal neurons of fetal rats.

[0051] Primary cultured hippocampal neurons cultured for 14 days were replaced with B-27-free medium 1 hour before sample treatment. As a positive control, 100 μM AMPA, an agonist of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA), was used. After treating the cells with 1 μg / mL of Atractylodes japonica extract, Sooyoung extract, and their (Atractylodes japonica + Sooyoung) complex extract (weight ratio 1:1) for 10 minutes, the cells were washed with ice-cold PBS, and lysed. The lysed cell fluid was quantified according to the BCA protein quantification method, and the same amount was measured using a phospho-CREB (Ser133) sandwich ELISA kit (Cell signaling) according to the manufacturer's instructions.

[0052] As a result, as disclosed in Table 2, the CREB phosphorylation level was significantly increased in the complex of the extracts of Atractylodes japonica and Sooyoung (weight ratio 1:1) according to the present invention compared to each of the extracts of Atractylodes japonica and Sooyoung (1 ㎍ / ㎖) alone.

[0053] Relative CREB phosphorylation degree of Baekchul extract, Sooyoung extract, and complex of Baekchul and Sooyoung extracts mixed in a weight ratio of 1:1 Concentration 1 ㎍ / ㎖ pCREB Absorbance (450 nm) % of Control Weight ratio Baekchul Sooyoung No treatment (Control) 0.12 10 Positive control 0.48 17 8.87*** 10 0.16 5 9.24* 0 10.14 3 4.63 110.21 7 2 0.10**

[0054] *, **, *** indicate that the content of phosphorylated CREB in the positive control group, the group treated with the extract of Atractylodes japonica and the group treated with the complex of Atractylodes japonica and Atractylodes spp. of the present invention significantly increased compared to the untreated group. * indicates p<0.05, ** indicates p<0.01, and *** indicates p<0.001.

[0055] Additionally, in order to determine the optimal combination ratio for increasing CREB phosphorylation of the complex of extracts of Baekchul and Suyeong, an experiment was conducted on the ability of complexes of extracts of Baekchul and Suyeong at various mixing ratios as disclosed in Table 1 above to increase CREB phosphorylation.

[0056] As a result, compared to the untreated control group, the experimental group treated with a complex of extracts of Atractylodes japonica and Sooyoung mixed at a weight ratio of 1:2 at a concentration of 1 μg / ml showed a 28.81% increase in CREB phosphorylation, and the experimental group treated with a complex of extracts of Atractylodes japonica and Sooyoung mixed at a weight ratio of 2:1 at a concentration of 1 μg / ml showed a 39.55% increase in CREB phosphorylation (Table 3).

[0057] CREB phosphorylation degree according to the mixing ratio of the complex of Baekchul and Suyeong extractsConcentration 1 (㎍ / ㎖)p-CREB absorbance (450 nm)% of ControlWeight ratioBaekchul and Suyeong untreated (Control)0.1210Positive control (Positive control)0.48174.87***110.21720.10**120.25928.81**210.31139.55***

[0058] **, *** indicate that the phosphorylated CREB content of the positive control group and the group treated with the complex of the extracts of Atractylodes japonica and Sophia japonica of the present invention significantly increased compared to the untreated group, ** is p<0.01, and *** is p<0.001. From the above experimental results, it was found that the complex extract has a synergistic effect of promoting CREB phosphorylation compared to the extracts of Atractylodes japonica and Sophia japonica alone, and in particular, the CREB phosphorylation effect of the complex of Atractylodes japonica and Sophia japonica extracts mixed at a weight ratio of 2:1 is more remarkable than when mixed at other weight ratios.

[0059]

[0060] Example 4. Confirmation of cytoprotective effect against scopolamine-induced apoptosis in a brain neuronal cell line (SH-SY5Y)

[0061] SH-SY5Y cells (neuroblastoma, human dopaminergic neuronal cells) were cultured in MEM medium containing 1% antimycotics / antibiotics and 10% FBS and seeded in 96-well plates at a cell number of 1 × 10 5 After dividing the cells into 100 cells / ml, the cells were cultured for 24 hours. To determine the cytotoxicity of the nerve cells, 50 μg / ml of the extract of Atractylodes japonica, the extract of Atractylodes japonica, and the combined extract of Atractylodes japonica and Atractylodes japonica were treated for 24 hours, and the cell viability was determined using the MTT assay.

[0062] As a result, it was confirmed that the extracts of Baekchul, Suyeong, and Baekchul + Suyeong combined extracts did not exhibit cytotoxicity toward brain neural cell lines (Fig. 1).

[0063] Meanwhile, in order to confirm the effect on cell death induced by memory impairment by scopolamine (SCO), 3 hours before treatment with SCO, 30 and 50 ㎍ / ㎖ of extracts of Atractylodes japonica and Sooyoung and their combined extracts were treated, and then 5 mM SCO was treated, and cell viability was measured 24 hours later.

[0064] As a result, as shown in Fig. 2, in the treatment groups of 30 and 50 ㎍ / ㎖ concentrations of the complex extracts of Atractylodes Rhizome and Sooyoung mixed in a 1:1 weight ratio, the cytoprotective effects were 82.84±0.59% and 97.47±0.54%, respectively, in the complex extracts of Atractylodes Rhizome and Sooyoung mixed in a 1:2 weight ratio, the cytoprotective effect was confirmed to be 98.92±0.57% at a concentration of 50 ㎍ / ㎖, and in the complex extracts of Atractylodes Rhizome and Sooyoung mixed in a 2:1 weight ratio, the cytoprotective effect was confirmed to be the best, with 90.65±0.52% and 109.38±0.62% at a concentration of 30 and 50 ㎍ / ㎖, respectively.

[0065]

[0066] Example 5. Identification of factors involved in synapse remodeling in hippocampal neurons of fetal rats.

[0067] Primary cultured hippocampal neurons cultured for 14 days were treated with 1 μg / ml of extracts of Baekchul and Suyeong alone and their combined extracts for 1 minute, then washed with ice-cold PBS and Syn-PER TM Cells were lysed using Synaptic Protein Extraction reagent, and Western blotting was performed to compare the protein expression levels of PSD-95 (Post Synaptic Density 95 kDa), AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid) receptors (subunits: GluA1), and NMDA (N-methyl-D-aspartate) 2A receptors, which are factors involved in synapse reorganization.

[0068] As a result, as shown in Fig. 3, it was confirmed that the protein expression of PSD95, AMPA receptor (subunits: GluA1), and NMDA 2A receptor increased in the complex of Atractylodes japonica and Sooyoung extracts compared to the case where the extracts of Atractylodes japonica and Sooyoung extracts were treated at a concentration of 1 ㎍ / ㎖ alone, and in particular, it was confirmed that the protein expression amount increased the most in the complex of Atractylodes japonica and Sooyoung extracts mixed at a weight ratio of 2:1.

[0069] In addition, as shown in Fig. 4, the complex of the extracts of Baekchul and Suyeong mixed at a weight ratio of 2:1, which showed the highest activity, was treated for 1 minute at various concentrations (1, 3, and 10 μg / ml) to determine the changes in the protein expression levels of PSD95, AMPA receptor (subunits: GluA1), and NMDA 2A receptors. As a result, it was confirmed that the protein expression levels of PSD95, AMPA receptor, and NMDA 2A receptor were significantly increased in all experimental groups treated at concentrations of 1, 3, and 10 μg / ml.

[0070]

[0071] Example 6. Morris water maze test

[0072] [Experimental Animals and Breeding]

[0073] Six-week-old male SD rats were purchased from SAMTACO (Korea) and acclimated for one week in an animal breeding room maintained under constant conditions (temperature: 22±2℃, humidity: 50±5%, light / dark cycle: 12 hours) before use.

[0074] Male SD rats were orally administered the present invention's complex extract of Baekchul and Suyeong (10, 50, and 100 mg / kg) once daily for 21 days for the evaluation of laboratory animal behavior. An untreated group that was not administered the sample was used as a control group. For the Morris water maze test, a circular pool (diameter: 180 cm, height: 75 cm) was filled with water to a height of 25 ± 1 cm, and an escape platform (24 cm) was hidden 1 cm below the water surface. To identify the location, spatial cues were installed on all four sides of the walls, and skim milk powder was dissolved in the water to prevent the escape platform from being visible to the naked eye. The day before the experiment, all experimental groups were allowed to swim freely for 1 minute in a pool without an escape platform to acclimate them to swimming. On the first day of the experiment, the animals were randomly placed in two of three quadrants, excluding the quadrant with the escape platform, and were allowed to find the escape platform for 60 seconds. Once the animal found the escape platform, it was instructed to remain on it for 20 seconds. If the animal failed to find the platform within 45 seconds, the experimenter guided the animal to the platform by hand and had the animal remain there for 20 seconds. After 20 seconds of location learning, the animal was immediately started in a random quadrant, and the time it took to find the platform and the distance traveled were measured. Once the animal found and climbed onto the platform, it was given 20 seconds, and the experiment was repeated three times a day for three consecutive days. However, on the second and third days, the initial learning to lead the animal to the platform was omitted to assess the long-term memory acquired the previous day. All data were captured by a camera installed above the swimming pool and analyzed using a video tracking device (Panlab, Barcelona, ​​Spain).

[0075] As a result, as shown in Figure 5A, the first trial of each experimental day is an indicator of long-term memory, and it was confirmed that the time spent exploring the escape platform in the first trials of the groups administered 10, 50, and 100 mg / kg of the complex of white atractylodes and soybean extracts was significantly reduced compared to the first trial of the control group (CTL).

[0076] In addition, as disclosed in Figure 5B, the last trial of each experimental day is an indicator of short-term memory, and it was confirmed that the time spent exploring the escape platform in the last trials of the groups administered 10, 50, and 100 mg / kg of the combined extract of Baekchul and Suyeong was significantly reduced compared to the last trial of the control group (CTL).

[0077] Meanwhile, a probe test was conducted on the fourth day, 24 hours after the end of the three-day experiment. The subjects were allowed to swim freely for 90 seconds without an escape platform, to test whether they retained memory of the location of the previous escape platform (the time spent swimming in the quadrant where the escape platform was located among the four quadrants).

[0078] As a result, as disclosed in Fig. 6, the swimming time in the quadrant where the escape zone was present was significantly increased in the group administered the complex of white and swimming extracts compared to the control group (CTL).

[0079]

[0080] Example 7. Confirmation of changes in the expression of synaptic reorganization factors and neuroplasticity factors in the hippocampus isolated from SD rats.

[0081] On the day all behavioral assessments were completed, the hippocampus was removed from the SD rats, and the expression levels of NMDA (N-methyl-D-aspartate) receptors (subunits: NR2A, NR2B), AMPA receptors (subunits: GluA1), and PSD95 proteins, which are synaptic remodeling factors, and the expression levels of ERK, CaMKII, and BDNF proteins, which are neuroplasticity factors, were confirmed by Western blot.

[0082] As a result, as disclosed in Fig. 7, it was confirmed that the protein expression levels of NR2A, NR2B, GluA1, and PSD95 increased in the groups administered the combined extracts of Baekchul and Suyeong (10, 50, and 100 mg / kg) compared to the control group.

[0083] In addition, as disclosed in Fig. 8A, compared to the control group, the expression level of brain-derived neurotrophic factor (BDNF) protein involved in synapse formation was significantly increased in the group administered the complex extract of Baekchul and Suyeong (10, 50, and 100 mg / kg), and as disclosed in Fig. 8B, it was confirmed that the expression of ERK and CaMKII phosphorylation, signaling pathways involved in synapse plasticity regulation, was significantly increased in the group administered the complex extract of Baekchul and Suyeong (10, 50, and 100 mg / kg).

Claims

1. A pharmaceutical composition for preventing or treating cognitive dysfunction, comprising a complex of extracts of Baekchul and Suyeong as active ingredients.

2. A composition for preventing or treating cognitive dysfunction, characterized in that in paragraph 1, the extraction solvent of the extracts of Baekchul and Suyeong is water, a lower alcohol of C1 to C4, or a mixture thereof.

3. A pharmaceutical composition for preventing or treating cognitive dysfunction, characterized in that the complex of the extracts of Baekchool and Suyeong is a mixture of the extracts of Baekchool and Suyeong in a weight ratio of 1.5 to 2.5:

1.

4. A pharmaceutical composition for preventing, improving or treating cognitive dysfunction, characterized in that the cognitive dysfunction in paragraph 1 is any one selected from dementia, learning disorder, agnosia, amnesia, aphasia, apraxia or delirium, mild cognitive impairment and Binswanger disease.

5. A pharmaceutical composition for preventing or treating cognitive dysfunction, characterized in that in paragraph 4, the dementia is any one selected from Alzheimer's dementia, vascular dementia, Lewy body dementia, frontal lobe dementia, semantic dementia, and multi-infarct dementia.

6. A pharmaceutical composition for preventing, improving or treating cognitive dysfunction, characterized in that in paragraph 1, in addition to the effective ingredient, it further comprises a pharmaceutically acceptable carrier, excipient or diluent.

7. A health functional food composition for improving memory and preventing or improving cognitive dysfunction, comprising a complex of extracts of Baekchul and Suyeong as active ingredients.

8. A health functional food composition for improving memory and preventing or improving cognitive dysfunction, characterized in that the composition in the 7th paragraph is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage.

9. A veterinary composition for preventing or treating cognitive dysfunction, comprising a complex of extracts of white chrysanthemum and swimming pool as active ingredients.

10. A feed additive for improving memory and preventing or improving cognitive dysfunction, containing a complex of extracts of white chrysanthemum and soybean as active ingredients.

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