Composition for improving memory, improving cognitive ability, or preventing or treating neurological diseases, comprising enzymatic hydrolysate of porcine brain

Abstract

The present invention relates to a composition for improving memory, improving cognitive ability, and preventing or treating brain diseases, which contains an enzymatic hydrolysate of porcine brain. The composition can protect nerve cells, produce brain-derived neurotrophic factor (BDNF), inhibit acetylcholinesterase, and inhibit reactive oxygen species (ROS).

Description

[Technical field]

[0001] This application claims priority to Korean Patent Application No. 10-2022-0004195, filed on January 11, 2022, and Korean Patent Application No. 10-2022-0143631, filed on November 1, 2022, the entire specification of which is incorporated herein by reference.

[0002] The present invention relates to a composition for improving memory, improving cognitive ability, or preventing or treating a neurological disease, comprising a porcine brain enzymatic hydrolysate. [Background technology]

[0003] The brain has several functions, but the most important are memory and cognitive abilities. If humans did not have cognitive and memory abilities, it would be difficult to carry out daily life and would be a problem for survival. Memory and cognitive impairment is the first symptom that Alzheimer's patients experience, and it is also the most common symptom. In the early stages of Alzheimer's, patients experience recent memory impairment, in which they are unable to remember the details of recent conversations or work. However, during this stage, remote long-term memory for events that occurred in the distant past is relatively well preserved. However, as the disease gradually progresses, the cerebral cortex, which is involved in the storage of long-term memory, is damaged, and these past memories also gradually become impaired.

[0004] Meanwhile, the most problematic of all geriatric diseases is dementia, a cognitive disorder. Dementia means that brain function is impaired, resulting in a continuous and overall decline in cognitive functions such as memory, language ability, time-space awareness, judgment, and abstract thinking, and in severe cases, it can cause significant disruption to daily life. Dementia is a type of brain disease, and is known to occur due to a variety of causes, including Alzheimer's disease, vascular dementia, Parkinson's disease, and other degenerative brain diseases, head trauma, and infectious diseases. Therefore, efforts are urgently needed to treat and manage dementia, which is rapidly increasing with the geometrical increase in the elderly population.

[0005] Dementia is a syndrome caused by irreversible destruction of the brain nerves due to brain atrophy, reduction in nerve cells, and the appearance of senile plaques, and is accompanied by various symptoms of acquired cognitive dysfunction such as memory, language disorder, and behavior disorder. Dementia is classified into Alzheimer's disease, vascular dementia, degenerative disease due to Parkinson's disease, metabolic disease due to hypothyroidism, and other dementias caused by brain tumors or infectious diseases. Currently, acetylcholinesterase (AChE) inhibitors such as donefidil (donepezil), galantamine, and rivastigmine are used clinically as dementia improving drugs, but the treatment efficiency is low, side effects are severe, and there is currently no effective treatment. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention provides a pork brain enzymatic hydrolysate comprising pork brain-derived peptides.

[0007] The present invention also provides a food composition comprising a pork brain enzymatic hydrolysate for improving memory, improving cognitive function, or preventing or improving a neurological disease.

[0008] The present invention also provides a functional health food composition for improving memory, improving cognitive function, or preventing or improving brain and nerve diseases.

[0009] The present invention also provides a pharmaceutical composition for improving memory, improving cognitive function, or preventing or ameliorating brain and nervous system diseases.

[0010] However, the technical problem that the present invention is to achieve is not limited to the above problem, and other problems not described will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0011] The present invention provides a peptide consisting of SEQ ID NO:1 or SEQ ID NO:2; or a pork brain enzymatic hydrolysate comprising at least one of SEQ ID NO:1 or SEQ ID NO:2.

[0012] The present invention also provides a food composition or functional health food composition for improving memory, improving cognitive function, or preventing or improving neurological diseases, which contains a porcine brain enzymatic hydrolysate as an active ingredient.

[0013] The present invention also provides a pharmaceutical composition for improving memory, improving cognitive function, or preventing or treating neurological diseases, comprising a porcine brain enzymatic hydrolysate as an active ingredient.

[0014] The above-mentioned pork brain enzymatic hydrolysate may contain peptides derived from pork brain.

[0015] The pork brain enzymatic hydrolysate may be hydrolyzed by one or more hydrolyzing agents.

[0016] The composition may be formulated into a formulation selected from the group consisting of a tablet, a capsule, a powder, a granule, a liquid phase, and a ring.

[0017] The above-mentioned enzymatic hydrolysate of pork brain may have neuroprotective activity and / or brain-derived neurotrophic factor (BDNF) producing activity. The above-mentioned enzymatic hydrolysate of pork brain may have acetylcholinesterase inhibitory activity and / or reactive oxygen species (ROS) inhibitory activity. The above-mentioned cranial nerve disease can be selected from the group consisting of Alzheimer's disease, cognitive impairment, senile dementia, Parkinson's disease, Huntington's disease, Louis body dementia, frontotemporal dementia, cerebral infarction, stroke, and brain metastasis.

[0018] The present invention also provides a method for improving memory, cognitive function, or preventing or treating neurological diseases, comprising the step of administering or taking to an individual a composition containing as an active ingredient a porcine brain enzymatic hydrolysate that contains at least one of the peptides consisting of SEQ ID NO: 1 or SEQ ID NO: 2.

[0019] The present invention also provides a use of a composition containing as an active ingredient a porcine brain enzymatic hydrolysate containing at least one peptide consisting of SEQ ID NO: 1 or SEQ ID NO: 2 for improving memory, improving cognitive function, or preventing or treating neurological diseases. Effect of the Invention

[0020] The peptide according to the present invention, or a porcine brain enzymatic hydrolysate containing the peptide, is highly effective in improving memory, improving cognitive function, and preventing or treating (or improving) neurological diseases, and is non-toxic. It can be usefully used as a functional health food composition, pharmaceutical composition, etc. [Brief description of the drawings]

[0021] FIG. 1 is a flow chart illustrating a process for producing a pork brain enzymatic hydrolysate according to one embodiment of the present invention. FIG. 2 is a graph showing the results of cytotoxicity evaluation of a peptide according to one embodiment of the present invention. 3a and 3b are graphs showing the neuroprotective ability of a peptide according to one embodiment of the present invention (*, p<0.05; **p<0.01; ***, p<0.001). 3c and 3d are graphs showing the ability of a peptide according to one embodiment of the present invention to produce brain-derived neurotrophic factor (BDNF) (*, p<0.05; **p<0.01; ***, p<0.001). FIG. 3e is a graph showing β-amyloid aggregation of a peptide (brPEP-1) according to one embodiment of the present invention. FIG. 3f is a graph showing ROS (Reactive Oxygen Species) of a peptide according to one embodiment of the present invention (*, p<0.05; **p<0.01; ***, p<0.001). FIG. 4 is a graph showing the results of cytotoxicity evaluation of a pork brain enzymatic hydrolysate according to one embodiment of the present invention. FIG. 5 is a graph showing the neuroprotective ability of a porcine brain enzymatic hydrolysate according to one embodiment of the present invention (*, p<0.05; **p<0.01; ***, p<0.001). FIG. 6 is a graph showing the BDNF producing ability of the porcine brain enzymatic hydrolysate according to one embodiment of the present invention (*, p<0.05; **p<0.01; ***, p<0.001). FIG. 7 shows protein (caspase-3, ChAT) expression profile of porcine brain enzymatic hydrolysate according to one embodiment of the present invention. FIG. 8 is a graph showing the acetylcholinesterase inhibitory potency of a porcine brain enzymatic hydrolysate according to one embodiment of the present invention (*, p<0.05; **p<0.01). FIG. 9 is a graph showing the reactive oxygen species (ROS) inhibitory ability of a porcine brain enzymatic hydrolysate according to one embodiment of the present invention (*, p<0.05; **p<0.01). FIG. 10 is a graph showing the beta-aggregation inhibitory activity of a pork brain enzymatic hydrolysate according to one embodiment of the present invention. FIG. 11 shows a schematic diagram of a passive avoidance test (a) and the results of analyzing the memory-improving effect of the porcine brain enzymatic hydrolysate through the passive avoidance test (b) (different letters or subscripts on the bar graphs indicate significant differences at p<0.05). FIG. 12 shows a schematic diagram of the Y maze test (a) and the results of analyzing the memory-improving effect of the enzymatic hydrolysate of porcine brain through the Y maze test (b) (different letters or subscripts on the bar graphs indicate significant differences from each other at p<0.05). FIG. 13 shows a schematic diagram of the Novel object test (a) and the results of analyzing the memory-improving effect of the enzymatic hydrolysate of pork brain through the Novel object test (b) (different letters or subscripts on the bar graphs are between each other at p<0.05, which means there is a significant difference). FIG. 14 shows (a) a schematic diagram of the Morris water-maze test, and (b) the results of analyzing the memory-improving effect of the enzymatic hydrolysate of porcine brain in the Morris water-maze test (different letters or subscripts on the bar graphs indicate significant differences between them at p<0.05).

[0022] The present invention will be described in detail below.

[0023] The present invention provides a peptide consisting of SEQ ID NO:1 or SEQ ID NO:2; or a pork brain enzymatic hydrolysate comprising one or more of the peptides consisting of SEQ ID NO:1 or SEQ ID NO:2.

[0024] The peptides can be used as food compositions or pharmaceutical compositions.

[0025] The above peptides may be contained in the pork brain enzymatic hydrolysate at a concentration of 0.1 ppm to 300 ppm, more preferably at a concentration of 1 to 30 ppm.

[0026] The present invention also provides a composition for improving memory, improving cognitive function, and preventing or treating (or improving) neurological diseases, which comprises a porcine brain enzymatic hydrolysate as an active ingredient.

[0027] The composition may be a food composition, a health food composition, a functional health food composition or a pharmaceutical composition.

[0028] The above-mentioned pork brain enzymatic hydrolysate can contain peptides derived from pork brain.

[0029] The pork brain enzymatic hydrolysate may be hydrolyzed by one or more hydrolyzing agents.

[0030] More preferably, the hydrolysate may be produced by primary and secondary hydrolysis.

[0031] The primary hydrolysis can be carried out by adding a protease to a solid matter dissolving solution (porcine brain sludge dissolving solution), and can be carried out at 40±20° C. for 1 to 20 hours, although the method is not limited thereto.

[0032] The secondary hydrolysis can be carried out by adding a protease to the solid matter solution after the primary hydrolysis, and can be carried out for 1 to 20 hours at 40±20° C., but is not limited thereto.

[0033] The composition may be formulated into a formulation selected from the group consisting of a tablet, a capsule, a powder, a granule, a liquid phase, and a ring.

[0034] The above-mentioned enzymatic hydrolysate of porcine brain may have neuroprotective activity and / or brain-derived neurotrophic factor (BDNF) producing activity, and may also have acetylcholinesterase inhibitory activity and / or reactive oxygen species (ROS) inhibitory activity.

[0035] The above-mentioned cranial nerve disease can be selected from the group consisting of Alzheimer's disease, cognitive impairment, senile dementia, Parkinson's disease, Huntington's disease, Louis body dementia, frontotemporal dementia, cerebral infarction, stroke, and brain metastasis.

[0036] As used herein, "prevention" refers to any action that delays the decline in memory or cognitive ability by administering a composition of the present invention, and "treatment" and "improvement" refer to any action that improves or beneficially alters the symptoms of decline in memory or cognitive ability by administering a composition of the present invention.

[0037] In the present invention, the term "health functional food" refers to food produced and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients having useful functions for the human body. Here, "functionality" means obtaining useful effects for health applications such as regulating nutrients for the structure and function of the human body or physiological actions. The health functional food of the present invention can be produced by a method commonly used in the art, and can be produced by adding raw materials and ingredients commonly added in the art during the production. In addition, the formulation of the health functional food can be produced without restrictions as long as it is a formulation recognized as a health functional food. The health functional food composition of the present invention has the advantage of being free of side effects that may occur during long-term administration of liquid or powder formulation medicines, has excellent portability, and can be taken as an adjuvant to promote the effect of preventing or improving disorders.

[0038] In the food composition or functional health food composition according to the present invention, the pork brain enzymatic hydrolysate may be contained in an amount of 1% to 20% (wt%) based on the total weight of the composition, but is not necessarily limited to this amount, and the amount of the active ingredient mixed can be appropriately determined depending on the purpose of use, such as prevention, health, or treatment.

[0039] The health functional food preparations can be in the form of an acidulant, granules, pills, tablets, capsules, as well as in the form of a general food or beverage.

[0040] There is no particular limitation on the type of the food, and examples of the food to which the substance can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, and vitamin complexes, and include all foods in the usual sense.

[0041] In general, when the food or beverage is produced in a liquid form, the active ingredient can be added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, per 100 parts by weight of the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health regulation, the amount may be less than the above range, and since the present invention utilizes a fraction from a natural product and there is no problem in terms of safety, an amount greater than the above range can also be used.

[0042] Among the functional foods according to the present invention, the beverage may contain various flavors or natural carbohydrates as additional components, similar to ordinary beverages. Examples of the natural carbohydrates include 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. Examples of sweeteners that can be used include natural sweeteners such as thaumatin and stevia extract, and synthetic sweeteners such as saccharin and aspartame. The proportion of the natural carbohydrates may be about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the beverage according to the present invention.

[0043] In addition to the above, the health functional food composition according to the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonation agents used in carbonated drinks. In addition, the health functional food composition according to the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in mixture. The proportion of these additives is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the functional food composition according to the present invention.

[0044] The pharmaceutical composition according to the present invention is not particularly limited in content as long as it contains the above-mentioned active ingredient, but preferably, the above-mentioned porcine brain enzymatic hydrolysate can be contained in an amount of 5 to 30% by weight based on the total weight of the composition. However, the amount is not limited thereto. In this case, if the active ingredient is contained in a concentration below the above-mentioned range, there is a problem that the desired preventive or therapeutic effect is difficult to be exhibited, and if the active ingredient is contained in a concentration above the above-mentioned range, the change in the expected effect may be insignificant.

[0045] The pharmaceutical compositions according to the present invention can be formulated and used in the form of oral preparations such as acids, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, and sterile injections, in accordance with conventional methods, and may contain suitable carriers, excipients, or diluents that are commonly used in the manufacture of pharmaceutical compositions for formulation.

[0046] The carrier, excipient or diluent may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl methylhydroxybenzoate, methyl propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.

[0047] When the formulation is made, it can be produced by using a diluent or excipient that is commonly used, such as a filler, weighting agent, binder, wetting agent, disintegrant, or surfactant.

[0048] A solid preparation for oral administration can be prepared by mixing at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0049] Oral liquid preparations include suspensions, liquid preparations, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. can be included.

[0050] Preparations for parenteral administration include sterilized aqueous solutions, water-insoluble agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Water-insoluble agents and suspensions can be propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases can be witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol gelatin, and the like.

[0051] The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but can be appropriately selected by those skilled in the art. However, in order to obtain the desired effect, the amount of the active ingredient contained in the composition of the present invention can be generally administered at 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg. The administration can be once a day or in several divided doses. However, the scope of the present invention is not limited by the above dosage.

[0052] The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, humans, etc., by various routes. All administration methods can be, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, intradural, or intracerebroventricular injection.

[0053] It was confirmed that short-term memory was recovered in a concentration-dependent manner in short-term memory behavioral experiments (Y-maze experiment, passive avoidance experiment, new substance exploration test) for the hydrolysate of the pig brain enzyme of the present invention (Y-maze experiment: behavioral test to confirm spatial cognitive ability, passive avoidance experiment: behavioral test to confirm the behavior of remembering and avoiding a stimulus, new substance exploration test: behavioral test to observe the cognitive behavior of exploring a new substance).

[0054] The present invention will be described in more detail with reference to the following examples. Objects, features, and advantages of the present invention will be readily understood through the following examples. The present invention is not limited to the examples described herein, and may be embodied in other forms. The examples presented herein are provided to fully convey the concept of the present invention to those skilled in the art to which the present invention pertains. Therefore, the present invention is not limited by the following examples.

[0055] Example 1. Preparation of porcine brain enzymatic hydrolysate

[0056] After thawing the pig brain with a thawing machine, the pig brain was deblooded after removing foreign matter with constant temperature water. The pig brain from which blood had been removed was crushed using a mixer to facilitate degreasing. The crushed pig brain was defatted using alcohol (ethanol, isopropyl alcohol, etc.) to remove fat and foreign matter. A protease (pepsin) was added to the degreased pig brain, and primary enzymatic hydrolysis was carried out for 1 to 20 hours. After the primary enzymatic hydrolysis was completed, filtration was carried out for solid-liquid separation. After filtration was completed, a protease (pancretin) was added to the primary pig brain hydrolysate, and secondary enzymatic hydrolysis was carried out for 1 to 20 hours. After the secondary enzymatic hydrolysis was completed, the protease was inactivated by heating, and the pig brain enzymatic hydrolysate was contacted with a filter aid, filtered, and adsorbed and purified. Then, 1 to 5 times (w / w) alcohol (ethanol) was added to the pig brain hydrolysate filtrate, left for 1 to 30 hours, and filtered through a filter. The filtered liquid was concentrated, then the sterilized filtered liquid was divided into small portions and sterilized. Finally, a high-purity enzymatic hydrolysate of porcine brain was obtained (see Figure 1).

[0057] Experimental Example 1. Identification of peptides from enzymatic hydrolysates of porcine brain and analysis of peptide efficacy

[0058] 1-1. Identification of peptides in enzymatic hydrolysates of porcine brain The porcine brain enzymatic hydrolysate obtained in Example 1 was diluted to 1 / 2 by adding 100 μl of DW to 100 μl of the sample. The diluted solution was diluted to 1 / 20 by adding 950 μl of DW to 50 μl of the diluted solution, and the solution filtered through a syringe filter was subjected to sequence analysis by ms / ms ionization analysis using an ACQUITY UPLC I-Class PLUS (Waters) HPLC system and a Xevo TQ-XS (Waters) MS system.

[0059] <Analysis conditions> - Column: ACQUITY UPLC BEH C18 (2.1 x 50 mm, 1.7 μm, Waters) - Flow rate: 200μl / min - Mobile phase A: H2O / FA=100 / 0.1(v / v) - Mobile phase B: Acetonitrile / FA=100 / 0.1(v / v) - Gradient

[0060] [Table 1]

[0061] The results obtained by analysis under the above instrument conditions are as follows.

[0062] As a result of the analysis, a total of four peptides were analyzed, and two of them were confirmed to be derived from pig brain. The following two peptides, which showed the most stable reproducibility, were selected as indicator substances.

[0063] [Table 2]

[0064] The retention times of the PSIS components in the PSIS standard solution and the porcine brain enzymatic hydrolysate test solution were confirmed by comparing the LC / MS EIC chromatograms (m / z 403.2), and it was confirmed that the retention times for the PSIS peak matched. In addition, the retention times of the GPAGPQGPR standard solution and the porcine brain enzymatic hydrolysate test solution were confirmed by comparing the LC / MS EIC chromatograms (m / z 418.7), and it was confirmed that the retention times for the GPAGPQGPR peak matched.

[0065] Subsequently, peptides with the same mass and ms / ms ionization forms as the peptides identified from the porcine brain hydrolysate were synthesized from Anigen (www.anygen.com) to confirm that the peptides identified by the porcine brain enzymes matched those present in the hydrolysate, and further experiments were performed.

[0066] 1-2. Cytotoxicity evaluation of peptides SH-SY5Y cells, a human neuronal cell line, were provided by the Korea Cell Line Bank (Cat No. 22266) and used. The cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (RPMI 1640, Cytiba) containing 10% fetal bovine serum (FBS, Cytiba), 100 IU / ml penicillin, and 100 μg / ml streptomycin in a 5% CO2, 37°C incubator for the experiments.

[0067] After culturing the human neuronal cell line SH-SY5Y, various concentrations of peptides derived from enzymatic hydrolysates of pig brain were added, and then MTT (methylthiazol tetrazolium bromide, Sigma Aldrich) was performed to confirm cell viability. An appropriate amount of cells (1X10 5 The cells were seeded at 1000 x g / well, treated with samples at each concentration, and cultured in a 37°C incubator for 48 hours to check cell viability. After reacting with MTT solution for 4 hours, 400 μl of DMSO was added to each well to dissolve insoluble formazan crystals, and the absorbance was measured at 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0068] As a result, the peptides selected as indicator components, brPEP-1 and brPEP-2, were 93.05% ± 2.23% and 108.47% ± 5.20% at 40ug / mL (w / v), respectively. At all concentrations tested (0.4-40ug / ml), the cell viability was over 90%, confirming that there were no safety issues with respect to cytotoxicity (Figure 2).

[0069] 1-3. Evaluation of the neuroprotective effect of peptides To evaluate the efficacy of two peptides (peptides of sequence numbers 1 and 2) in improving memory, a human neuronal cell line was used to confirm their cytoprotective ability against neuronal cell death caused by β-amyloid treatment at or below non-toxic concentrations.

[0070] To measure the neuroprotective effect, SH-SY5Y cells were treated with beta-amyloid, the main pathological feature of Alzheimer's disease, to induce neuronal cell death. 5 The wells were then pretreated with two peptides (peptides of SEQ ID NO: 1 and SEQ ID NO: 2) according to the present invention at different concentrations for 24 hours, and then cultured. Each well was treated with beta-amyloid at a concentration of 40 μM and cultured for an additional 24 hours. After reacting with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) for 4 hours, 400 μl of DMSO was added to each well to dissolve the insoluble formazan crystals, and the results were confirmed by measuring the absorbance at a wavelength of 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0071] As shown in Figure 3a, the cell viability of the Aβ25-35 treated group was reduced to 63.7% (p<0.001) compared to the normal control group, but when pre-treated with peptide sequence number 1 (PSIS) and then treated with 40 μM Aβ25-35 for 24 hours, it showed a neuroprotective ability of about 21 to 30%. Also, as shown in Figure 3b, the cell viability of the Aβ25-35 treated group was reduced to 63.9% (p<0.001) compared to the normal control group, but when pre-treated with peptide sequence number 2 (GPAGPQGPR) and then treated with 40 μM Aβ25-35 for 24 hours, it showed a neuroprotective ability of about 18 to 31%.

[0072] 1-4. Evaluation of BDNF production ability of brPEP-1 and brPEP-2 BDNF is a brain-derived neurotrophic factor. To confirm the production ability of BDNF, a protein involved in the signal transduction system of metabolic processes that affect cognitive function (memory and thinking ability), and to confirm the memory improvement functionality of the sample, human neuroblastoma SH-SY5Y cells were cultured in a 75T flask. When the confluency reached 80-90%, 2 × 10 5 The cells were seeded in a 6-well cell culture plate at 100°C. After one week of culture in a 5% CO2 humidity chamber at 37°C, the cells were treated with the porcine brain enzymatic hydrolysate (PBEH) according to the embodiment of the present invention at different concentrations. After 48 hours of culture in a 5% CO2 humidity chamber at 37°C, cell lysates were obtained and BDNF was measured using Bosterbio's BDNF kit (#EK0307). Then, cell number correction was performed by protein assay and corrected for protein content.

[0073] As a result of the test, it was confirmed that both the peptide of sequence number 1 (PSIS) and the peptide of sequence number 2 (GPAGPQGPR) had excellent ability to produce the nerve cell growth factor BDNF (FIGS. 3c and 3d).

[0074] 1-5. Confirmation of β-amyloid aggregation by brPEP-1 Thioflavin T assays were performed to examine the extent to which brPEP-1 according to the present invention can inhibit the aggregation of β-amyloid. Thioflavin T reacts with β-amyloid and shows fluorescence. SH-SY5Y cells were cultured, treated with brPEP-1 for 24 hours, and then treated with β-amyloid 20μM for 24 hours. After culturing, the cells were washed with PBS (pH 7.4) before harvesting, treated with 1mL of thioflavin T 5μM, and measured at 450nm excitation and 482nm emission wavelengths using an ELISA reader (TECAN, Infinite M200 pro).

[0075] By binding to amyloid aggregates, the aggregation level could be confirmed by the intensity of the fluorescence spectrum specific to Thioflavin T. By checking Thioflavin T over time, it was confirmed that the fluorescence intensity was higher in the group treated with β-amyloid (CTL) than in the group not treated with β-amyloid (N).

[0076] At 0.002ug / ml, brPEP-1 exhibited a β-amyloid aggregation inhibitory ability of approximately 62% after 18 hours, which is the concentration at which the raw material pork brain enzyme is present in a total nitrogen hydrolysate of 25ug / ml. It was confirmed that this raw material exhibited a similar β-amyloid aggregation inhibitory ability of approximately 65% ​​after 18 hours (Figure 3e).

[0077] 1-6. Confirmation of reactive oxygen species (ROS) suppression ability of brPEP-1 and brPEP-2 To measure the production of intracellular reactive oxygen species by H2O2, fluorescent DCF-DA (2',7'-dichlorodihydrofluorescein diacetate) was used to measure the intracellular reactive oxygen species level. DCF-DA is converted to DCF in cells by reactive oxygen and emits fluorescence. To measure the amount of intracellular reactive oxygen species, SH-SY5Y cells were cultured, and then treated with brPEP-1 and brPEP-2 according to the embodiment of the present invention. After 24 hours, the cells were treated with 100μM H2O2 for 24 hours and cultured. After that, the cells were treated with 20μM DCF-DA before harvesting, and cultured at 37℃ for 30 minutes. After washing with PBS (pH7.4), the cells were treated with 1% triton X-100 to lyse the cells, and then measured at 480nm excitation and 530nm emission wavelengths using an ELISA reader (TECAN, Infinite M200 pro). brPEP-1 and brPEP-2 showed 24% and 17% inhibitory activity, respectively, at 2 ug / ml, confirming that the concentration was significantly effective (Figure 3f).

[0078] Experimental Example 2. Analysis of efficacy of hydrolysates containing peptides derived from pig brain

[0079] The memory improving effect was analyzed using the porcine brain enzymatic hydrolysate (containing the peptides of SEQ ID NO: 1 and SEQ ID NO: 2) produced in Example 1. The content of each of the above peptides in the porcine brain enzymatic hydrolysate was 5 to 30 ppm.

[0080] 2-1. Cytotoxicity evaluation Human neuroblastoma SH-SY5Y cells were provided by the Korea Cell Line Bank (Cat No. 22266) and were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium (RPMI 1640, Cytiba) containing 10% fetal bovine serum (FBS, Cytiba), 100 IU / ml penicillin, and 100 μg / ml streptomycin in a 5% CO2, 37°C incubator for the experiment.

[0081] After culturing SH-SY5Y (human neuroblastoma), various concentrations of porcine brain enzymatic hydrolysate (PBEH) were added, and then MTT (methylthiazol tetrazolium bromide, Sigma Aldrich) was performed to confirm cell viability. An appropriate amount of cells (1X10 5 The cells were seeded at 1000 x g / well, treated with samples at each concentration, and cultured in a 37°C incubator for 48 hours to check cell viability. After reacting with MTT solution for 4 hours, 400 μl of DMSO was added to each well to dissolve insoluble formazan crystals, and the absorbance was measured at 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0082] As a result, it was confirmed that all the PBEH samples according to the embodiments of the present invention were non-toxic up to 50ug / ml of total nitrogen (FIG. 4).

[0083] 2-2. Neuro-protective effect To measure the neuroprotective effect, SH-SY5Y cells were treated with beta-amyloid, the main pathological feature of Alzheimer's disease, to induce neuronal cell death. 5 The wells were then pretreated with porcine brain enzymatic hydrolysate (PBEH) according to the present invention at different concentrations for 24 hours and incubated, after which each well was treated with beta-amyloid at a concentration of 40 μM and incubated for an additional 24 hours. After reacting with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide) for 4 hours, 400 μl of DMSO was added to each well to dissolve the insoluble formazan crystals, and the results were confirmed by measuring the absorbance at a wavelength of 570 nm using an ELISA reader (TECAN, Infinite M200 pro).

[0084] As a result, it was confirmed that the pork brain enzymatic hydrolysate (PBEH) according to the embodiment of the present invention exhibited 25% and 52% neuroprotective ability at 25 ug / ml and 50 ug / ml of total nitrogen (FIG. 5).

[0085] 2-3.BDNF generation ability BDNF is a brain-derived neurotrophic factor. To confirm the production ability of BDNF, a protein involved in the signal transduction system of metabolic processes that affect cognitive function (memory and thinking ability), and to confirm the memory improvement functionality of the sample, human neuroblastoma SH-SY5Y cells were cultured in a 75T flask. When the confluency reached 80-90%, 2 × 10 5The cells were seeded on a well cell culture plate at 100°C. After one week of culture in a 5% CO2 humidity chamber at 37°C, the cells were treated with the porcine brain enzymatic hydrolysate (PBEH) according to the embodiment of the present invention at different concentrations. After 48 hours of culture in a 5% CO2 humidity chamber at 37°C, cell lysates were obtained and BDNF was measured using Bosterbio's BDNF kit (#EK0307). Then, cell number correction was performed by protein assay and corrected for protein content.

[0086] The test results confirmed that PBEH showed BDNF production potential of 30%, 41%, and 47% at total nitrogen concentrations of 6.25, 25, and 50ug / ml, respectively (Figure 6).

[0087] 2-4. Protein expression (caspase-3, ChAT) Various proteins and signaling substances are involved in the process of apoptosis, and when activation of caspase-3 is promoted through this process, it induces the cleavage of PARP and causes apoptosis. The following test was conducted to confirm whether the death of β-amyloid can be suppressed by reducing the activation of caspase-3 by treating the pig brain enzymatic hydrolysate according to the embodiment of the present invention and ginkgo biloba extract, a raw material for health functional foods, as a control.

[0088] About 7 days after dispensing, when the confluency of SH-SY5Y cells reached 90%, the cells were treated with the above-mentioned porcine brain hydrolysate (PBEH) for 24 hours, then treated with 20 μM beta-amyloid and cultured. After washing with cold PBS, lysates were obtained with RIPA buffer. After confirming the protein concentration using a protein assay, the same amount of cell lysate and 2× sample buffer were mixed and heated at 100°C for 5 minutes to induce protein denaturation, and 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. After electrophoresis, the proteins in the gel were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, USA). The cells were reacted with blocking buffer (5% skim milk) at room temperature for 1 hour. The antibodies against caspase-3, ChAT, and GAPDH were diluted 1:1,000 in Tris-buffered saline (TBS-T) containing 0.1% (v / v) Tween-20 and reacted with polyvinyllidene difluoride overnight at 4°C. The secondary antibodies against each antibody and anti-rabbit IgG conjugated HRP were diluted (1:2,000) in TBS-T and reacted at room temperature for 2 hours each, and then developed using an ECL kit (Bio-Rad, USA).

[0089] The test results showed that both caspase-3 and ChAT showed effective levels with differences in band intensity, confirming the possibility of improving memory (Figure 7).

[0090] 2-5. Acetylcholinesterase inhibitory activity By inhibiting the activity of acetylcholinesterase (AChE) and suppressing the decomposition of acetylcholine, the concentration of acetylcholine in neural networks can be increased, thereby inducing improved cognitive function. The following test was conducted to confirm the AChE inhibitory ability of the enzymatic hydrolysate of porcine brain.

[0091] Culture human neuroblastoma SH-SY5Y cells in a 75T flask. When the cells reach 80-90% confluency, add 2 × 10 5The cells were seeded on a well cell culture plate (BD, Falcon) at 100°C. After one week of culture in a 5% CO2 humidity chamber at 37°C, porcine brain enzymatic hydrolysate (PBEH) and, as a positive control, ginkgo biloba extract, which is currently registered as an individually certified raw material, and the pharmaceutical drug donepezil were treated at different concentrations. After 48 hours of culture in a 5% CO2 humidity chamber at 37°C, cell lysates were obtained and AChE activity was measured using Abcam's AChE kit (#ab138871). After that, a protein assay was performed to correct for cell number, and the protein content was used to correct for cell number.

[0092] The test results showed that porcine brain enzymatic hydrolysate (PBEH) showed approximately 23% and 26% inhibitory activity at total nitrogen concentrations of 25 and 50ug / ml, while ginkgo leaf extract at 100ug / ml showed approximately 21% concentration-dependent inhibition (Figure 8).

[0093] 2-6. Reactive oxygen species (ROS) suppression ability To measure the production of intracellular reactive oxygen species by H2O2, fluorescent DCF-DA (2',7'-dichlorodihydrofluorescein diacetate) was used to measure the intracellular reactive oxygen species level. DCF-DA is converted to DCF in cells by reactive oxygen species and emits fluorescence. To measure the amount of intracellular reactive oxygen species, SH-SY5Y cells were cultured and then treated with the porcine brain enzymatic hydrolysate (PBEH) according to the embodiment of the present invention and the ginkgo leaf extract currently registered as an individually certified raw material for 24 hours, and then treated with 100 μM H2O2 for 24 hours. After that, before harvesting the cells, they were treated with 20 μM DCF-DA and cultured at 37°C for 30 minutes, washed with PBS (pH 7.4), treated with 1% triton X-100 to lyse the cells, and measured at 480 nm excitation and 530 nm emission wavelengths using an ELISA reader (TECAN, Infinite M200 pro). Pig brain enzymatic hydrolysate (PBEH) showed 43% and 60% inhibition at 25 and 50ug / ml total nitrogen, respectively, confirming that the effect varies significantly with concentration. At 100ug / ml of Ginkgo biloba leaf extract, the inhibition rate was approximately 35.9%, similar to the results for 25ug / ml of total nitrogen in pig brain enzymatic hydrolysate (Figure 9).

[0094] 2-7. Ability to inhibit β-amyloid aggregation Thioflavin T assays were performed to examine the extent to which the pig brain enzymatic hydrolysate according to the embodiment of the present invention and the ginkgo leaf extract currently registered as an individually certified raw material can inhibit the aggregation of β-amyloid. Thioflavin T reacts with β-amyloid and shows fluorescence. SH-SY5Y cells were cultured and treated with pig brain enzymatic hydrolysate (PBEH) and the ginkgo leaf extract currently registered as an individually certified raw material for 24 hours, and then treated with 20μM β-amyloid for 24 hours. The cells were washed with PBS (pH 7.4) before harvesting, and treated with 1mL of thioflavin T 5uM, and then measured at 450nm excitation and 482nm emission wavelengths using an ELISA reader (TECAN, Infinite M200 pro).

[0095] By binding to amyloid aggregates, the aggregation level could be confirmed by the intensity of the fluorescence spectrum specific to Thioflavin T. By checking Thioflavin T over time, it was confirmed that the fluorescence intensity was higher in the group treated with β-amyloid (CTL) than in the group not treated with β-amyloid (N).

[0096] It was confirmed that porcine brain enzymatic hydrolysate (PBEH) exhibited approximately 66% inhibition of beta-amyloid aggregation at 25ug / ml of total nitrogen, and ginkgo leaf extract, a raw material registered as an individually certified type, exhibited approximately 46% inhibition of beta-amyloid aggregation at 100ug / ml (Figure 10).

[0097] Experimental example 3. Animal behavior test

[0098] 3-1. Passive avoidance test In order to confirm the memory / cognitive ability improving effect of the porcine brain enzymatic hydrolysate obtained in the above example, a manual avoidance experiment was conducted (FIG. 11).

[0099] Experimental Method The passive avoidance experiment involved measuring the time it took for a mouse to move from a light room to a dark room. The first and second experiments were conducted eight hours apart, and when the mouse moved to the dark room, an electrical stimulus was administered to teach it that it should not move to the dark room. After 16 hours, the time it took to move from the light room to the dark room was measured.

[0100] Experimental Results As shown in Figure 11, in the second test, the high-PBEH group had a longer migration time, similar to the normal control group (Figure 11b). (From left to right, MD-control, positive control, low PBEH, medium-PBEH, high-PBEH, normal control.) In the third test, the normal group had a longer latency time than the induced group (MD-control group: memory decline-induced group), and the medium-PBEH (0.159ml / kg) and high-PBEH (0.478ml / kg) groups were slower to migrate to the dark room, similar to the normal group, confirming their improved memory.

[0101] 3-2.Y maze test To confirm the memory / cognitive ability improving effect of the porcine brain enzymatic hydrolysate obtained in the above example, a Y-maze test was performed ( FIG. 12 ). The Y-maze test is a spontaneous alternation and recognition memory test for spatial learning and memory research, and can test memory functions that are sensitive to hippocampal damage, genetic manipulation, and memory erasing agents.

[0102] Experimental Method The Y-maze test is a method for evaluating short-term instantaneous spatial cognition. The Y-maze is a standard maze (50.5 cm long, 20 cm wide, 20 cm high, 3 arms) with four closed Y-shaped mazes. The three branches were designated A, B, and C, and the mouse was carefully placed on one of the branches. After allowing the mouse to move freely for 8 minutes, the order in which the mouse entered the branches was recorded. Each branch was counted and 1 point (actual alternation) was given for each branch entered in order. Alternation behavior was defined as entering all three branches without overlapping.

[0103] Experimental Results The experimental results showed that the number of normal rotations was lower in the MD-control group (memory decline-induced group) than in the normal group (normal control). The percentage of normal rotations out of the total number of rotations was higher in the medium-PBEH (0.159ml / kg) and high-PBEH groups (0.478ml / kg) than in the induced group (Figure 12b, from left to right: MD-control, positive-control, low-PBEH, medium-PBEH, high-PBEH, normal control).

[0104] 3-3. New object test In order to confirm the memory / cognitive ability improving effect of the porcine brain enzymatic hydrolysate obtained in the above example, a novel object test was conducted (FIG. 13). This test investigates the recognition of novel objects, and is expressed as a percentage, with 100% meaning that all objects are recognized, and the closer the value is to 100, the better the memory.

[0105] Experimental Method Each mouse was placed in a plexiglass box on the first day of testing and allowed to explore the novel object for 5 min on the first day, 20 min on the second day, and 20 min on the third day. On the fourth day, the box was divided into four areas, and two identical objects were placed in the center of the diagonal areas. The mouse was placed next to the box and allowed to freely explore the objects for 10 min. The objects and the apparatus were washed with 75% ethanol to remove the mouse odor, and after 1 h, one of the two identical objects was replaced with an object of similar size but different shape and color.

[0106] The time it took the mice to explore new and existing objects was recorded for 5 min. Exploratory behavior was defined as the mice sniffing and touching the objects with their nose or paws, but sitting on the objects or turning around was not considered as exploratory behavior.

[0107] Experimental Results The experimental results showed that the MD-control group (memory decline-induced group) had a lower degree of novel object recognition than the normal control group, while the positive control group showed similar values ​​to the normal group. The groups that consumed the pork brain enzyme hydrolysate showed a concentration-dependent increase in novel object recognition, with the high-PBEH group showing the highest recognition rate, demonstrating improved cognitive ability (Figure 13b, from left to right: MD-control, positive-control, low-PBEH, medium-PBEH, high-PBEH, normal control).

[0108] 3-4. Morris water-maze test To confirm the memory / cognitive ability improving effect of the porcine brain enzymatic hydrolysate obtained in the above example, the Morris water maze test was carried out (Figure 14). The water maze is used to study spatial memory and learning, and animals are placed in water to check the time it takes to find a hidden escape platform.

[0109] Experimental Method Morris water-maze test was performed, and target quadrant time, number of crossing platforms, and swimming pattern were measured and analyzed by a camera operated by a computer program. Morris water-maze test was performed in a circular tank (40 cm high × 1.5 m diameter) with a 12 cm diameter target placed at the bottom of the tank, about 1 cm lower than the water surface, and water (22°C-25°C) was poured into the tank and skim milk powder was removed to make the target invisible. On the first day of the experiment, the mouse was placed in zone 1 and asked to search for the platform in zone 5. The water maze test was performed three times, and on the first and second days, the mouse was trained to find the platform in zone 5, and on the fifth day, the latency, frequency of visits, and duration of visits in zone 5 were measured to evaluate memory. The test lasted for 600 seconds.

[0110] Experimental Results The experimental results showed that the groups that consumed medium-PBEH (0.159ml / kg) and high-PBEH (0.478ml / kg) of the enzymatic hydrolysate of pork brain took less time to find the first platform compared to the MD-control group (memory decline-induced group), and the time that the group that consumed the enzymatic hydrolysate of pork brain stayed near the platform was as long as that of the normal control group, proving that the enzymatic hydrolysate of pork brain obtained in the present embodiment is effective in spatial memory (Figure 14b, from left to right: MD-control, positive-control, low PBEH, medium-PBEH, high-PBEH, normal control).

[0111] 3-5. Hippocampal tissue analysis In order to confirm the memory / cognitive ability improving effect of the porcine brain enzymatic hydrolysate obtained in the above examples, hippocampal tissue was analyzed by the following method.

[0112] Experimental Method The hippocampus was divided into two parts, one part was lysed in RIPA buffer containing protease inhibitors, and the other part was lysed in Trizol reagent (Invitrogen, Rockville, MD, USA) to extract total RNA. The hippocampal RIPA buffer lysate was centrifuged at 5000 × g for 10 min, and the supernatant was used to measure lipid peroxide and cholesterol content colorimetrically using a lipid peroxide (MDA) assay kit (Abcam, Cambridge, UK) and a cholesterol kit (Abcam, Cambridge, UK). The supernatant was deproteinized with 1.5 N perchloric acid, and glycogen content was calculated from the glucose concentration derived from glycogen hydrolyzed by α-amyloglucosidase in the acid buffer.

[0113] Glucose concentrations were measured using a glucose kit (Asan Pharmaceutics, Seoul, Korea). Triglycerides were extracted from the hippocampus with a chloroform-methanol solution (2:1, v / v) and resuspended in pure chloroform. Triglyceride content in chloroform was measured using a triglyceride colorimetric kit (Asan Pharmaceutical, Seoul, Korea). Serum TNF-α and IL-1β concentrations were measured using ELISA kits (eBioscience; San Diego, CA, USA), and serum concentrations of AST and ALT were measured using AST and ALT kits (Asan Pharmaceutics, Seoul, Korea).

[0114] Experimental Results The cholesterol content in hippocampal tissue was significantly higher in the memory decline-induced group (MD-control group) than in the normal group (normal control), while the cholesterol content was reduced in the positive control group and the group that consumed pork brain enzymatic hydrolysate. The glycogen content in the hippocampal tissue was higher in the normal group than in the induction group, and was higher in the low-PBEH (0.053 ml / kg) and medium-PBEH (0.159 ml / kg) groups when pig brain enzymatic hydrolysate was administered.

[0115] When the MDA content of peroxides in hippocampal tissue was examined, it was higher in the induction group than in the normal group, and decreased in the positive control group and the pig brain enzyme hydrolysate intake group. In particular, the medium-PBEH (0.159ml / kg) group and the Hgh-PBEH (0.478ml / kg) group showed the same effect as the normal group, showing a low MDA content. The content of TNF-α, an inflammatory cytokine, in brain tissue was similar to that of peroxide, and was higher in the induced group than in the normal group. It was confirmed that the positive control group and pig brain enzymes were decreased in the hydrolysate intake groups, medium-PBEH group, and high-PBEH group.

[0116] [Table 3]

[0117] The above description of the present invention is merely illustrative, and those skilled in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical concept or essential characteristics of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

[0118] [Problem information] Project ID: G0210300-01 Project Number: 20210210300-0 Ministry name: Ministry of Agriculture, Food and Rural Affairs Research Management Agency: Korea Food Research Institute Research project name: Food functionality evaluation support project Title of research: Study on the effect and mechanism of porcine brain enzyme hydrolysate on memory improvement Supervising agency: Unimed Pharmaceuticals Co., Ltd. Research period: 21.04~21.12

Claims

1. The present invention comprises, as an active ingredient, a porcine brain enzymatic hydrolysate containing one or more peptides consisting of SEQ ID NO: 1 or SEQ ID NO: 2; A food composition for improving memory, improving cognitive function, or preventing or improving neurological diseases.

2. The pork brain enzymatic hydrolysate is characterized in that it is hydrolyzed by one or more proteases. The food composition of claim 1.

3. The peptide is characterized in that it is derived from an enzymatic hydrolysis product of pig brain. The food composition of claim 1.

4. The enzymatic hydrolysate of the porcine brain is characterized by exhibiting neuroprotective activity and BDNF (brain-derived neurotrophic factor) producing activity. The food composition of claim 1.

5. The above-mentioned porcine brain enzymatic hydrolysate is characterized by exhibiting acetylcholinesterase inhibitory activity and reactive oxygen species (ROS) inhibitory activity. The food composition of claim 1.

6. The cranial nerve disease is selected from the group consisting of Alzheimer's disease, cognitive impairment, senile dementia, Parkinson's disease, Huntington's disease, Louis Body Dementia, Frontotemporal Dementia, cerebral infarction, stroke and brain metastasis; The food composition of claim 1.

7. The composition is formulated into a formulation selected from the group consisting of tablets, capsules, powders, granules, liquid phases and rings. The food composition of claim 1.

8. The present invention comprises, as an active ingredient, a porcine brain enzymatic hydrolysate containing one or more peptides consisting of SEQ ID NO: 1 or SEQ ID NO: 2; A pharmaceutical composition for improving memory, improving cognitive function, or preventing or treating neurological diseases.

9. The pork brain enzymatic hydrolysate is characterized in that it is hydrolyzed by one or more proteases. The pharmaceutical composition according to claim 8.

10. The peptide is characterized in that it is derived from an enzymatic hydrolysis product of pig brain. The pharmaceutical composition according to claim 8.

11. The above-mentioned porcine brain enzymatic hydrolysate is characterized by exhibiting neuroprotective activity and BDNF producing activity. The pharmaceutical composition according to claim 8.

12. The above-mentioned porcine brain enzymatic hydrolysate is characterized by exhibiting acetylcholinesterase inhibitory activity and reactive oxygen species (ROS) inhibitory activity. The pharmaceutical composition according to claim 8.

13. The cranial nerve disease is selected from the group consisting of Alzheimer's disease, cognitive impairment, senile dementia, Parkinson's disease, Huntington's disease, Louis Body Dementia, Frontotemporal Dementia, cerebral infarction, stroke and brain metastasis; The pharmaceutical composition according to claim 8.

14. The composition is formulated into a formulation selected from the group consisting of tablets, capsules, powders, granules, liquid phases and rings. The pharmaceutical composition according to claim 8.

15. Contains at least one of the peptides consisting of SEQ ID NO: 1 or SEQ ID NO: 2 Pork brain enzymatic hydrolysate.

16. The peptide is characterized in that it is purified or chemically synthesized from a porcine brain enzymatic hydrolysate. The pork brain enzymatic hydrolysate according to claim 15.

17. The peptide is characterized in that it is used for improving memory, improving cognitive ability, or preventing or treating brain and nervous system diseases. The pork brain enzymatic hydrolysate according to claim 15.

18. The cranial nerve disease is selected from the group consisting of Alzheimer's disease, cognitive impairment, senile dementia, Parkinson's disease, Huntington's disease, Louis Body Dementia, Frontotemporal Dementia, cerebral infarction, stroke and brain metastasis; The pork brain enzymatic hydrolysate according to claim 17.

19. The peptide is characterized by exhibiting neuroprotective activity and BDNF producing activity. The pork brain enzymatic hydrolysate according to claim 15.

20. The peptide is characterized in that it exhibits acetylcholinesterase inhibitory activity and reactive oxygen species (ROS) inhibitory activity. The pork brain enzymatic hydrolysate according to claim 15.

21. A peptide of SEQ ID NO:1 or SEQ ID NO:

2.

22. The peptide is characterized in that it is purified or chemically synthesized from a hydrolysate of a pig brain enzyme.

22. The peptide of claim 21.

23. The peptide is characterized in that it is used as a food composition or a pharmaceutical composition.

22. The peptide of claim 21.

24. The peptide is characterized in that it is used for improving memory, improving cognitive ability, or preventing or treating brain and nervous system diseases.

22. The peptide of claim 21.

25. The cranial nerve disease is selected from the group consisting of Alzheimer's disease, cognitive impairment, senile dementia, Parkinson's disease, Huntington's disease, Louis Body Dementia, Frontotemporal Dementia, cerebral infarction, stroke and brain metastasis; 14. The peptide of claim 13.

26. The peptide is characterized by exhibiting neuroprotective activity and BDNF producing activity.

24. The peptide of claim 23.

27. The peptide is characterized in that it exhibits acetylcholinesterase inhibitory activity and reactive oxygen species (ROS) inhibitory activity.

22. The peptide of claim 21.

28. The method includes administering or ingesting to an individual a composition containing as an active ingredient a porcine brain enzymatic hydrolysate containing one or more peptides consisting of SEQ ID NO: 1 or SEQ ID NO:

2. A method for improving memory, improving cognitive function, or preventing or treating neurological disorders.

29. The active ingredient is an enzymatic hydrolysate of porcine brain containing at least one peptide of SEQ ID NO: 1 or SEQ ID NO:

2. Use of the composition for improving memory, improving cognitive function, or preventing or treating neurological diseases.

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