Peptide for improving memory and preventing or improving cognitive dysfunction, composition containing the same, and method for preparing the same

Abstract

This invention relates to a method for preparing silk-derived peptides. The method for preparing silk-derived peptides according to the invention includes: a dissolution step of dissolving silk fibroin; a step of treating and hydrolyzing the silk fibroin lysate with a mixed enzyme comprising a first proteolytic enzyme and a second proteolytic enzyme, wherein the first proteolytic enzyme comprises bromelain, and the second proteolytic enzyme comprises one selected from the group consisting of flavorzyme, protamex, neutral protease, Veron, Sumizyme, zingibain, Calpain, Protease NP, Validase, and mixtures thereof; and an enzyme removal step of removing the proteolytic enzyme.

Description

Technical Field

[0001] This invention relates to peptides for improving memory and preventing or improving cognitive impairment, compositions comprising the peptides, and methods for preparing the same. More specifically, it provides peptides for improving memory and preventing or improving cognitive impairment, and compositions comprising the peptides. Furthermore, it provides a method for providing peptide compositions for improving memory and preventing or improving cognitive impairment by enzymatically hydrolyzing silk fibroin. Background Technology

[0002] In modern society, in the field of functional materials, in addition to finding new materials, existing materials are also provided in a form that meets the needs by specifying the required effective components and purifying and separating them.

[0003] On the other hand, silkworm cocoons are composed of fibroin, a fibrous protein, and sericin, a gelatinous protein, surrounding the fibroin. The amino acids in fibroin include glycine, alanine, sericin, and tyrosine, with major amino acids accounting for over 90%. Recently, there has been active development into using this protein in functional foods or beverages. However, current technologies involve either directly using fibroin collected from silkworm cocoons without further processing, or purifying the dissolved fibroin aqueous solution through dialysis. Therefore, in large-scale production, this presents uneconomical aspects such as reduced production yield. Furthermore, due to the fibrous structure and amino acid arrangement of fibroin, it is slow to decompose or difficult to decompose in the human body, reducing its absorption rate and thus diminishing its value as a functional food ingredient.

[0004] The known techniques for preparing silk amino acids generally include acid hydrolysis and enzymatic hydrolysis. In Japan, silk protein is broken down into low-molecular-weight peptides or amino acids through acid, alkali, or enzymatic hydrolysis, allowing for easy digestion and absorption. Enzymatic hydrolysis, based on the specific binding of enzymes to proteins and the cleavage of binding rings, results in a very low rate of silk protein breakdown into amino acids. Acid hydrolysis, however, breaks down most of the silk protein into amino acids or peptides. In actual mass production, while enzymatic hydrolysis is simpler, its low breakdown rate leads to low yield and low absorption rate by the human body. In contrast, silk amino acids produced through acid hydrolysis have an effective form with nearly 90% human absorption rate and contain more functional amino acids with beneficial physiological activities.

[0005] The acid hydrolysis method for silk protein proposed in this invention is a novel method that can produce products with a greater advantage in terms of the amino acid composition and the content of physiologically active functional components in the acid hydrolysate.

[0006] Currently, Japan holds a leading position in the market and research and development of functional peptides produced through protein hydrolysis. However, the application scope of peptide foods remains limited, and the market is still in its formative stage. Currently, commercially available functional peptide foods in Japan are under continuous research in the following areas: promoting digestion and absorption; the molecular weight distribution and digestion-related studies of proteins in silk hydrolysates; the serum cholesterol-inhibiting effect of silk fibroin; the importance of sulfur-containing amino acids in cholesterol metabolism; promoting calcium absorption and preventing osteoporosis; inhibiting alcohol absorption; preventing hypertension; antioxidant function; and anti-allergic function.

[0007] Furthermore, as another functional study of silk fibroin, research results related to its utilization in foamed food processing and the application of silk residue as a food material have also been reported. However, systematic studies on the physiological activities of these proteins have not yet been conducted. Instead, the hydrolysates have been administered to animals to confirm their activities (anti-diabetic, alcohol metabolism-promoting, cholesterol-lowering effects, etc.), or to estimate that the activities exhibited by the major amino acids are also present in the hydrolysates. The functionalities of the product proposed in this invention include anti-cancer and anti-genotoxic effects for inhibiting gene damage.

[0008] On the other hand, dementia is a complex clinical syndrome, generally characterized by significant impairment in cognitive function, intelligence, mood, and behavior, including cortical dysfunction in areas such as memory, attention, language, and spatial awareness, leading to difficulties in daily and social life. When defined as dementia, it refers to a condition involving impairment in one or more cognitive functions besides memory; memory loss alone does not constitute dementia.

[0009] Causes of dementia include Alzheimer's disease or Parkinson's disease (degenerative brain diseases), cerebral hemorrhage, hepatic encephalopathy and Wilson's disease (metabolic diseases), neurosyphilis (infectious diseases), acquired immunodeficiency diseases, drug poisoning such as alcohol, and traumatic brain injury. In some forms, diseases that cause structural or functional abnormalities of the central nervous system can lead to dementia. Among these, Alzheimer's disease causes the most dementia, accounting for 50 to 60%, followed by dementia caused by cerebrovascular diseases.

[0010] Memory and cognitive impairment is the initial and most common symptom of Alzheimer's disease. In the early stages of Alzheimer's, patients experience recent memory impairment, making it difficult to recall details of recent conversations or events. This is due to damage to hippocampal neurons, which reduces the ability to store recent memories. However, during this period, remote long-term memory is relatively well preserved. But as the disease progresses, damage to the cerebral cortex associated with long-term memory storage gradually impairs the storage of these past memories as well. Summary of the Invention

[0011] The problem the invention aims to solve

[0012] The purpose of this invention is to provide a peptide, a composition containing the peptide, and a method for preparing the peptide, wherein the peptide has excellent cognitive function improvement effects, especially in the early stages.

[0013] The purpose of this invention is to provide a peptide composition for improving memory and preventing or improving cognitive impairment, wherein the peptide composition has excellent activity in improving memory and cognitive impairment, especially in the early stages.

[0014] The purpose of this invention is to provide a method for preparing silk-derived peptides, which can use hydrolytic enzymes to produce silk-derived peptides more efficiently.

[0015] With the same final production volume, the present invention can miniaturize existing process equipment and produce silk-derived peptides with high yields in a short time through a small process.

[0016] means for solving problems

[0017] To achieve the above objectives, a peptide according to an embodiment of the present invention may consist of an amino acid sequence represented by any one of the following general formulas 1 to 5:

[0018] General Formula 1

[0019] Tyr-Gly-X1-X2

[0020] (In the general formula, X1 is Ala, Tyr, Thr, Val, Gly, Ile or Gln, and X2 is Ala, Tyr, Ile, Val or Gly);

[0021] General Formula 2

[0022] Ile-Gly-Z1-Z2

[0023] (In the general formula, Z1 is Ala, Tyr, Val or Gln, and Z2 is Ala, Tyr, Val or Gly);

[0024] Formula 3

[0025] Ile-Gly-Val-a1-a2

[0026] (In the general formula, a1 is Ala, Tyr or Val, and a2 is Ala, Tyr, Thr, Val, Gly or Gln);

[0027] Formula 4

[0028] a1-Gly-Gly-a1

[0029] (In the general formula, a1 is Ala, Tyr or Val);

[0030] General Formula 5

[0031] a1-Gly-Gly-a1-Gly-a2

[0032] (In the general formula, a1 is Ala, Tyr or Val, and a2 is Ala, Tyr or Ile or Val).

[0033] Another embodiment of the present invention provides a peptide composition for improving memory and preventing or improving cognitive impairment, which may contain the aforementioned peptides.

[0034] The composition may be derived from silk fibroin.

[0035] Another embodiment of the present invention provides a method for preparing silk-derived peptides for improving memory and preventing or improving cognitive impairment, which may include: a dissolution step of dissolving silk fibroin; a step of treating and hydrolyzing the silk fibroin lysate with a mixed enzyme comprising a first proteolytic enzyme and a second proteolytic enzyme, wherein the first proteolytic enzyme comprises bromelain, and the second proteolytic enzyme comprises any one selected from the group consisting of flavorzyme, protamex, neutral protease, Veron, Sumizyme, zingibain, calpain, protease NP, validase, and mixtures thereof; and an enzyme removal step of removing the proteolytic enzyme.

[0036] Another embodiment of the present invention provides a method for preparing silk-derived peptides for improving memory and preventing or improving cognitive impairment, which may include: a dissolution step of dissolving silk fibroin; a step of treating and hydrolyzing the silk fibroin lysate with a proteolytic enzyme, wherein the proteolytic enzyme is selected from any one of the group consisting of flavorzyme, papain, protamex, neutral protease, Veron, Sumizyme, zingibain, calpain, protease NP, Validase, and mixtures thereof; and an enzyme removal step of removing the proteolytic enzyme.

[0037] Another embodiment of the present invention provides a method for preparing silk-derived peptides for improving memory and preventing or improving cognitive impairment, which may include: a dissolution step of dissolving silk fibroin; a step of treating and hydrolyzing the silk fibroin lysate with a proteolytic enzyme, wherein the proteolytic enzyme is selected from any one of the group consisting of delvorase, chymotrypsin, subtilisin, actinidin, lactoferrin, Kokulase P, keratinase, cathepsin K, and mixtures thereof; and an enzyme removal step of removing the proteolytic enzyme.

[0038] The hydrolyzing enzymes also include umamizymes.

[0039] The hydrolytic enzymes also include ficin.

[0040] In the method for preparing silk-derived peptides for improving memory and preventing or improving cognitive impairment, the dissolution step of dissolving silk fibroin is a step of dissolving silk fibroin powder. The silk fibroin powder is prepared by mixing silk fibroin and glycerol, pulverizing, washing with water, filtering, and drying.

[0041] The cognitive impairment can be selected from the group consisting of dementia, learning disability, agnosia, amnesia, aphasia, apraxia, delirium, AIDS-induced dementia, Binswanger disease, Lewy body dementia, frontotemporal dementia, mild cognitive impairment, multiple infarct dementia, Pick's disease, semantic dementia, Alzheimer's disease, or vascular dementia.

[0042] The present invention will now be described in more detail.

[0043] Silk refers to the fiber extracted from silkworm cocoons, and is a general term for products made from spun silk. In a broader sense, it can also refer to silk products made from natural silk such as tussah silk or spun silk. The cocoon of the domesticated silkworm, commonly known as "silk," is also called cultivated or domestic silk. However, the definition of silkworm cocoon in this invention includes the concept of silk.

[0044] Silk fibers are composed of fibroin and sericin, and also contain small amounts of ceramides, lipids, and inorganic salts. The components of silk are fibroin and sericin, which are fibrous proteins. In their natural state, they are composed of biological macromolecules. Typically, compounds with a molecular weight of 10,000 or higher exist as fibers, membranes, or blocks. Silk is a high-molecular-weight protein; proteins are composed of amino acids, which form simple α-amino acids by binding amino (-NH2) and carboxyl (-COOH) groups to carbon.

[0045] Furthermore, a polymer consisting of many amino acids linked in a ring-like manner is called a polypeptide. Ultimately, the -NH2 and -COOH groups of two amino acids react, dehydrate (H2O), and align in parallel to form fibrous structures.

[0046] The amino acids that make up silk fibroin are mostly glycine (side chain -H) and alanine (-CH3), characterized by their short side chains. Furthermore, serine (side chain -CH2OH) and tyrosine (side chain -CH(OH)CH2) together account for over 90% of the composition. Unlike sericin, silk fibroin contains less acidic amino acids (aspartic acid, glutamic acid) and basic amino acids (lysine, alanine, histidine). When these amino acids are linked by different bond combinations, polypeptides are formed. When silk fibroin is hydrolyzed, it yields amino acids with a molecular weight of 300,000 to 500,000, each molecule consisting of 3,000 to 3,500 α-amino acids.

[0047] The silk-derived peptides mentioned in this invention refer to products derived from silk fibroin that are hydrolyzed and processed into a size that can be absorbed by the body and have a certain amino acid sequence.

[0048] In addition, the silk-derived peptides described in this invention can also be expressed as silk proteins, and can be used with the same meaning.

[0049] Preferably, prior to the dissolution step of dissolving silk fibroin, the preparation method of silk peptides may further include a sericin removal step, which involves mixing silkworm cocoons and purified water and heat-treating them to remove sericin and obtain silk fibroin.

[0050] The sericin removal step refers to the process of removing the sericin that encapsulates silk fibroin. Since sericin contains polar amino acids, it is typically removed during heat treatment in purified water. In particular, the efficiency of the sericin removal process increases with higher temperatures; however, temperatures above 97°C result in the loss of silk fibroin, thus limiting the ability to raise the temperature indefinitely.

[0051] In the preparation method of the silk-derived peptide, the sericin removal step includes thickeners and additives, and can be heat-treated at 30 to 70°C.

[0052] In the sericin removal step, when thickeners and additives are included and heat treatment is performed at 30 to 70°C, the sericin removal process can be much faster than when heat treatment is performed at the same temperature without increasing viscosity. In particular, when the viscosity is increased, there is the advantage that no loss of silk fibroin occurs.

[0053] In the preparation method of the silk-derived peptide, the viscosity of the mixture of silkworm cocoon and purified water in the sericin removal step can be from 1.2 to 120 cP.

[0054] Within the specified viscosity range, the sericin removal process can not only remove sericin quickly but also with almost no loss of silk fibroin.

[0055] Preferably, in the method for preparing the silk-derived peptide, the viscosity of the mixture of silkworm cocoon and purified water in the sericin removal step is 1.3 to 40 cP, and may include a primary heat treatment step of heat treatment at 98 to 100°C for 2 to 10 minutes and a secondary heat treatment step of heat treatment at 30 to 40°C.

[0056] Although the heat treatment step is performed at the temperature at which silk fibroin dissolves, no loss of silk fibroin will occur if the viscosity of the mixture of silkworm cocoons and purified water is adjusted to 1.3 to 40 cP and the heat treatment is started.

[0057] Subsequently, a secondary heat treatment at a relatively low temperature can quickly remove the sericin.

[0058] If the viscosity of the mixture of silkworm cocoons and purified water in the first heat treatment step is less than 1.3 cP, a sharp loss of silk fibroin will occur. Conversely, if the viscosity is greater than 40 cP, even if a heat treatment step is performed, there will be no improvement in the process for removing sericin.

[0059] Therefore, by performing a single heat treatment and a double heat treatment process while maintaining the viscosity range, sericin can be removed more effectively without loss of silk fibroin, thus improving the efficiency of the sericin removal process.

[0060] When the additive is included, not only can the loss of silk fibroin be minimized, but the yield of silk fibroin hydrolysate, i.e. silk peptide, which exhibits improved memory and cognitive function, can be increased in subsequent steps.

[0061] Preferably, the additive comprises xylanase, and relative to 100 parts by weight of the xylanase, it comprises 10 to 100 parts by weight of sorbitol; 1 to 10 parts by weight of mannitol; and 0.1 to 5 parts by weight of galactose.

[0062] Within the specified range, the loss of silk fibroin in the sericin removal step can be reduced, and the yield of silk-derived peptides with cognitive function-improving effects can be increased in the hydrolysis step, thereby improving the efficiency of cognitive function and memory improvement.

[0063] The dissolution step refers to preparing the silk fibroin into a state that is mixed with purified water, organic solvents, or other liquids.

[0064] The hydrolysis step refers to the process of treating and hydrolyzing silk fibroin lysate with a mixed enzyme including a first proteolytic enzyme and a second proteolytic enzyme, wherein the first proteolytic enzyme includes bromelain, and the second proteolytic enzyme includes any one selected from the group consisting of flavorzyme, protamex, neutral protease, Veron, Sumizyme, zingibain, calpain, protease NP, Validase, and mixtures thereof.

[0065] Preferably, the hydrolytic enzyme can be any one selected from the group consisting of bromelain, gingerase, calpain, and mixtures thereof. When applicable within the above range, silk fibroin peptides with high cognitive function and memory improvement effects can be produced.

[0066] More preferably, the hydrolase comprises bromelain, and the hydrolase may be a mixed enzyme comprising 10 to 50 parts by weight of ginger protease and 5 to 30 parts by weight of calpain, relative to 100 parts by weight of bromelain. When applied within the above range, it not only exhibits excellent cognitive function and memory improvement effects, but also rapidly enhances cognitive function and memory improvement effects within a short period of time. Therefore, silk fibroin peptides that exhibit cognitive function and memory improvement effects in a relatively short time can be produced.

[0067] Preferably, the hydrolytic enzyme comprises bromelain, and the hydrolytic enzyme may be a mixed enzyme comprising 10 to 50 parts by weight of ginger protease, 5 to 30 parts by weight of calpain, and 1 to 20 parts by weight of umami enzyme relative to 100 parts by weight of bromelain. When the above range is applied, silk-derived peptides with significantly enhanced activity of active ingredients for improving the absorption rate of hydrolysates and cognitive function can be prepared.

[0068] Preferably, the hydrolytic enzyme can be any one selected from the group consisting of papain, ginger protease, calpain, and mixtures thereof. When applicable within the above range, silk fibroin peptides with high cognitive function and memory improvement effects can be produced.

[0069] More preferably, the hydrolytic enzyme comprises papain, and the hydrolytic enzyme may be a mixed enzyme using 10 to 50 parts by weight of ginger protease and 5 to 30 parts by weight of calpain, relative to 100 parts by weight of papain. When applied within the above range, it can not only exhibit excellent cognitive function and memory improvement effects, but also rapidly improve cognitive function and memory improvement effects in a short period of time. Therefore, silk fibroin peptides that exhibit cognitive function and memory improvement effects in a relatively short period of time can be produced.

[0070] Preferably, the hydrolytic enzyme comprises papain, and the hydrolytic enzyme may be a mixed enzyme comprising 10 to 50 parts by weight of ginger protease, 5 to 30 parts by weight of calpain, and 1 to 20 parts by weight of umami enzyme relative to 100 parts by weight of papain. When the above range is applied, silk-derived peptides with significantly enhanced activity of active ingredients for improving the absorption rate of hydrolysates and cognitive function can be prepared.

[0071] Preferably, the hydrolytic enzyme can be any one selected from the group consisting of actinidin, coculase P, keratinase, and mixtures thereof. When applicable within the above range, silk fibroin peptides with high cognitive function and memory improvement effects can be produced.

[0072] More preferably, the hydrolase comprises actinidin protease, and the hydrolase may be a mixed enzyme comprising 5 to 20 parts by weight of coculase P and 5 to 20 parts by weight of keratinase per 100 parts by weight of the actinidin protease. When applied within the above range, not only can excellent cognitive function and memory improvement effects be exhibited, but these effects can also be rapidly improved within a short period of time. Therefore, silk fibroin peptides that exhibit cognitive function and memory improvement effects in a relatively short time can be produced.

[0073] Preferably, the hydrolytic enzyme comprises actinidin, and the hydrolytic enzyme may be a mixed enzyme comprising 5 to 20 parts by weight of coculase P, 5 to 20 parts by weight of keratinase, and 1 to 5 parts by weight of cathepsin K, relative to 100 parts by weight of the actinidin. Within the above scope, silk-derived peptides with significantly enhanced activity of active ingredients for improving the absorption rate of hydrolysates and cognitive function can be prepared.

[0074] More preferably, the hydrolytic enzyme comprises fig protease, and the hydrolytic enzyme may be a mixed enzyme using 5 to 20 parts by weight of coculase P and 5 to 20 parts by weight of keratinase mixed with 100 parts by weight of the fig protease. When the above range is applied, not only can excellent cognitive function and memory improvement effects be exhibited, but also cognitive function and memory improvement effects can be rapidly enhanced in a short period of time. Therefore, silk fibroin peptides that exhibit cognitive function and memory improvement effects in a relatively short period of time can be produced.

[0075] Preferably, the hydrolytic enzyme comprises fig protease, and the hydrolytic enzyme may be a mixed enzyme comprising 5 to 20 parts by weight of cocopusase P, 5 to 20 parts by weight of keratinase, and 1 to 5 parts by weight of cathepsin K, relative to 100 parts by weight of the fig protease. When the above range is applied, silk-derived peptides with significantly enhanced activity of active ingredients for improving the absorption rate of hydrolysates and cognitive function can be prepared.

[0076] The enzyme removal step for removing proteolytic enzymes refers to the step of removing the enzyme or removing the enzyme activity after the hydrolysis step, and is defined to include all forms that can be used by those skilled in the art.

[0077] In addition, the enzyme removal step may be a step of removing enzyme activity by heat treatment.

[0078] The enzyme removal step may be a step of removing enzyme activity through heat treatment.

[0079] In the preparation method of the silk-derived peptide, the silk-derived peptide can be used to improve memory.

[0080] The silk fibroin powder can be made by mixing silk fibroin and glycerin, pulverizing, washing with water, filtering, and drying.

[0081] The silk fibroin may be treated with low-temperature plasma.

[0082] When using the aforementioned silk fibroin powder, compared to existing technologies, the silk fibroin dissolving step can utilize less calcium chloride and water, and the silk fibroin can be dissolved at a relatively low temperature. Therefore, not only can the processes required when using silk-derived peptides be minimized, but the amount of water used can also be reduced.

[0083] Specifically, in existing technologies, silk fibroin is dissolved in a calcium chloride solution and then hydrolyzed. Therefore, the calcium chloride solution used to dissolve silk fibroin typically uses approximately 5 to 10 times the volume of the silk fibroin as solvent. Furthermore, the reaction temperature used for dissolution is also a relatively high temperature of 80 to 110°C.

[0084] However, when using the silk fibroin powder, a calcium chloride solvent amount of 0.8 to 1.3 times the mass of the silk fibroin powder is mixed to dissolve the powder. Furthermore, the reaction can be carried out at a relatively low temperature of 40 to 60°C. Therefore, due to the reduced amount of reactants, the process can be miniaturized. In addition, the reduced amount of calcium chloride solution used reduces the amount of water used for desalination and shortens the time required for the desalination process. Therefore, silk-derived peptides can be prepared in a shorter time using a smaller reactor to obtain the same amount of final product, resulting in excellent process efficiency.

[0085] Specifically, the silk fibroin powder is obtained by mixing silk fibroin (after removing sericin) with glycerin, followed by washing, filtering, and drying the pulverized mixture. The silk fibroin powder prepared by the above method has a certain degree of solubility.

[0086] In addition, the silk fibroin after removing sericin is subjected to low-temperature plasma treatment under atmospheric pressure to modify the surface of the silk fibroin material. After mixing with glycerol, it is washed, filtered and dried, which results in better solubility.

[0087] Typical silk fibroin is insoluble in water, while the silk fibroin powder prepared by the above method has a water solubility of over 30%. Furthermore, unlike the case with typical silk fibroin, the silk fibroin can be completely dissolved even with a smaller amount of calcium chloride solution.

[0088] In the preparation method of the silk-derived peptide, the silk-derived peptide can be used to prevent or improve cognitive dysfunction.

[0089] The cognitive impairment can be selected from the following groups: dementia, learning disability, agnosia, amnesia, aphasia, apraxia, delirium, AIDS-induced dementia, Binswanger disease, Lewy body dementia, frontotemporal dementia, mild cognitive impairment, multiple infarct dementia, Pick's disease, semantic dementia, Alzheimer's disease, or vascular dementia.

[0090] Another embodiment of the present invention provides a food for improving cognitive function and memory, which may contain silk-derived peptides prepared according to the method for preparing the silk-derived peptides.

[0091] The improvement of cognitive function and memory described in this invention not only refers to simply restoring and further improving cognitive function and memory impairment, but also includes preventing or improving cognitive function and memory decline caused by neurological diseases.

[0092] In another embodiment of the present invention, a medicament for the prevention and improvement of neurological diseases may comprise a silk-derived peptide prepared according to the method for preparing the silk-derived peptide.

[0093] The neurological diseases described in this invention include: neurodegenerative diseases such as dementia, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis; diseases caused by ischemia or reperfusion, such as ischemic stroke; and mental illnesses such as depression, schizophrenia, and post-traumatic stress disorder.

[0094] A peptide according to an embodiment of the present invention can be represented by the following general formula 1.

[0095] General Formula 1

[0096] Tyr-Gly-X1-X2

[0097] (In the general formula, X1 is Ala, Tyr, Thr, Val, Gly, Ile or Gln, and X2 is Ala, Tyr, Ile, Val or Gly).

[0098] It has been confirmed that when silk fibroin is hydrolyzed by a combination of specific proteolytic enzymes containing amino acids with specific sequences, not only are cognitive and memory functions improved, but these improvements also occur in a very short time. In order to identify the amino acid sequence that has a rapid effect and is different from that of general hydrolysates, a specific analysis was conducted.

[0099] Therefore, by analyzing the hydrolysate, it is inferred that the applicable peptide or peptide composition can exhibit the effects described above, wherein the peptide contains the amino acid sequence of general formula 1 that can exhibit the effects described above, and the peptide composition is a mixture of a peptide having the amino acid sequence shown in general formula 1 and a peptide having the amino acid sequence shown in general formula 2. Experimental results confirm that when using the above-mentioned peptide or peptide composition, not only is there a high degree of improvement in cognitive function and memory, but also an improvement in cognitive function and memory in a very short time.

[0100] Preferably, the peptide containing the amino acid sequence of general formula 1 can be any one of P1-1 to P1-6 as shown in Table 1 below.

[0101] Table 1

[0102] peptides amino acid sequence P1-1 Tyr-Gly-Gln-Gly P1-2 Tyr-Gly-Gly-Ala P1-3 Tyr-Gly-Ala-Ala P1-4 Tyr-Gly-Val-Gly P1-5 Tyr-Gly-Val-Ile P1-6 Tyr-Gly-Ile-Ala

[0103] More preferably, the peptide of general formula 1 can be peptides P1-2, P1-3, P1-5, and P1-6 from Table 1. When these peptides are used, not only are the effects of improving cognitive function and memory excellent, but the initial effects are also very good, thus the effects of improving cognitive function and memory can appear in a short period of time.

[0104] In particular, when the peptides P1-5 and P1-6 are applied, not only are there excellent effects in improving cognitive function and memory, but also excellent effects in improving cognitive function can be achieved in a shorter time.

[0105] According to another embodiment of the present invention, a peptide can be represented by the following general formula 2.

[0106] General Formula 2

[0107] Ile-Gly-Z1-Z2

[0108] (In the general formula, Z1 is Ala, Tyr, Val or Gln, and Z2 is Ala, Tyr, Val or Gly).

[0109] According to another embodiment of the present invention, a peptide composition may comprise a peptide having an amino acid sequence represented by said general formula 1 and a peptide having an amino acid sequence represented by said general formula 2.

[0110] Preferably, the peptide represented by the general formula 2 can be any one of P2-1 to P2-4 shown in Table 2.

[0111] Table 2

[0112] peptides amino acid sequence P2-1 Ile-Gly-Tyr-Val P2-2 Ile-Gly-Val-Ala P2-3 Ile-Gly-Ala-Tyr P2-4 Ile-Gly-Gln-Gly

[0113] When the peptides in Table 2 are mixed to form a peptide composition, not only is there an excellent effect on improving cognitive function and memory, but it can also take effect in a short period of time.

[0114] On the other hand, the peptide composition may contain 50 to 80 parts by weight of peptide represented by general formula 2, relative to 100 parts by weight of peptide represented by general formula 1.

[0115] According to an embodiment of the present invention, a peptide can be represented by the following general formula 3.

[0116] Formula 3

[0117] Ile-Gly-Val-a1-a2

[0118] (In the general formula, a1 is Ala, Tyr or Val, and a2 is Ala, Tyr, Thr, Val, Gly or Gln).

[0119] Preferably, the peptide comprising the amino acid sequence represented by the general formula 1 can be any one of F1 to F3 in Table 3 below.

[0120] Table 3

[0121] peptides amino acid sequence F1 Ile-Gly-Val-Ala-Thr F2 Ile-Gly-Val-Ala-Tyr F3 Ile-Gly-Val-Ala-Gly

[0122] When applied to Table 3, not only are there excellent improvements in cognitive function and memory, but also, due to its superior activity, the initial improvement effect can be rapidly enhanced.

[0123] The peptide for improving memory and cognitive function prepared in another experimental step of the present invention comprises amino acids represented by general formula 3 and amino acids represented by general formula 1 above.

[0124] It has been confirmed that when silk fibroin is hydrolyzed by a combination of specific proteolytic enzymes containing amino acids with specific sequences, not only are cognitive and memory functions improved, but these improvements also occur in a very short time. In order to identify the amino acid sequence that has a rapid effect and is different from that of general hydrolysates, a specific analysis was conducted.

[0125] Therefore, by analyzing the hydrolysate, it is inferred that the applicable peptide or peptide composition can exhibit the effects described above, wherein the peptide contains the amino acid sequence of general formula 1-1 that can exhibit the effects described above, and the peptide composition is a mixture of a peptide having the amino acid sequence shown in general formula 1-1 and a peptide having the amino acid sequence shown in general formula 1-2. Experimental results confirm that when using the above-mentioned peptide or peptide composition, not only is there a high degree of improvement in cognitive function and memory, but also an improvement in cognitive function and memory in a very short time.

[0126] The peptides according to the present invention may contain an amino acid sequence represented by the following general formula 1a or general formula 1b.

[0127] Formula 4

[0128] a1-Gly-Gly-a1

[0129] (In the general formula, a1 is Ala, Tyr or Val).

[0130] General Formula 5

[0131] a1-Gly-Gly-a1-Gly-a2

[0132] (In the general formula, a1 is Ala, Tyr or Val, and a2 is Ala, Tyr or Ile or Val).

[0133] It has been confirmed that when silk fibroin is hydrolyzed by a combination of specific proteolytic enzymes containing amino acids with specific sequences, not only are cognitive and memory functions improved, but these improvements also occur in a very short time. In order to identify the amino acid sequence that has a rapid effect and is different from that of general hydrolysates, a specific analysis was conducted.

[0134] Therefore, by analyzing the hydrolysate, it is inferred that the applicable peptide or peptide composition can exhibit the effects described above, wherein the peptide contains the amino acid sequence of general formula 4 or general formula 5 that can exhibit the effects described above, and the peptide composition is a mixture of a peptide having the amino acid sequence shown in general formula 4 or general formula 5 and a peptide having the amino acid sequence shown in general formula 2. Experimental results confirm that when using the above-mentioned peptide or peptide composition, not only is there a high degree of improvement in cognitive function and memory, but also an improvement in cognitive function and memory in a very short time.

[0135] Preferably, the peptide having the amino acid sequence of general formula 4 can be any one of P4-1 to P4-3 shown in Table 4 below. Furthermore, the peptide having the amino acid sequence of general formula 5 can be any one of P5-1 to P5-4 shown in Table 4 below.

[0136] Table 4

[0137] peptides amino acid sequence P4-1 Ala-Gly-Gly-Ala P4-2 Tyr-Gly-Gly-Tyr P4-3 Val-Gly-Gly-Val P5-1 Tyr-Gly-Gly-Tyr-Gly-Ile P5-2 Tyr-Gly-Gly-Tyr-Gly-Val P5-3 Tyr-Gly-Gly-Tyr-Gly-Ala P5-4 Tyr-Gly-Gly-Tyr-Gly-Tyr

[0138] More preferably, the peptide of general formula 4 can be peptides P4-2 and P4-3 in Table 4. When these peptides are used, not only are the effects of improving cognitive function and memory excellent, but the initial effects are also very good, so the effects of improving cognitive function and memory can appear in a short period of time.

[0139] In particular, when the peptide P4-2 is applied, not only are there excellent effects in improving cognitive function and memory, but also excellent effects in improving cognitive function can be achieved in a shorter time.

[0140] More preferably, the peptide of general formula 5 may be peptide P5-1 or P5-4 from Table 4.

[0141] When applied within the above-mentioned range, it has excellent effects on improving cognitive function and memory, and the improvement effect can be rapidly enhanced in the early stages of administration.

[0142] Preferably, the peptide composition according to another embodiment of the present invention may comprise a peptide having an amino acid sequence represented by the above general formula 4, and may also comprise a peptide having an amino acid sequence represented by the above general formula 2.

[0143] Furthermore, the peptide composition according to the present invention comprises a peptide having an amino acid sequence represented by the above general formula 5, and may also comprise a peptide having an amino acid sequence represented by the above general formula 2.

[0144] The peptides represented by general formulas 1 to 5 can be synthesized and added, and the preparation method of the present invention can prepare compositions containing said peptides.

[0145] When applied within the above scope, the synergistic effect caused by the interaction results in rapid improvement of cognitive function and memory.

[0146] According to another embodiment of the present invention, a food product may contain the peptide or the peptide composition.

[0147] A medicament for preventing or improving cognitive impairment according to another embodiment of the present invention may contain the peptide or the peptide composition.

[0148] The cognitive impairment can be selected from the following groups: free dementia, learning disability, agnosia, amnesia, aphasia, apraxia, delirium, AIDS-induced dementia, Binswanger disease, Lewy body dementia, frontotemporal dementia, mild cognitive impairment, multiple infarct dementia, Pick's disease, semantic dementia, Alzheimer's disease, or vascular dementia.

[0149] The composition may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers contained in the composition are commonly used in formulations and include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine cellulose, polyvinylpyrrolidone, cellulose, water, syrup, oil, etc. In addition to the above-mentioned components, the pharmaceutical composition may also contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0150] The pharmaceutical composition can be administered orally or non-gastrointestinally.

[0151] When administered orally, proteins or peptides are digested, therefore oral compositions should be coated with the active pharmaceutical ingredient or formulated to prevent degradation in the stomach. Furthermore, the composition can be administered via any device capable of moving the active substance to target cells.

[0152] An appropriate dose of the pharmaceutical composition may be a pharmaceutically effective amount, meaning an amount sufficient to prevent or treat memory impairment, cognitive impairment, or learning impairment.

[0153] The effects of the invention

[0154] The present invention provides peptides, compositions containing such peptides, and methods for their preparation, wherein the peptides have excellent cognitive function improvement effects, especially in the early stages.

[0155] This invention provides peptide compositions for improving memory and preventing or improving cognitive impairment, wherein the compositions exhibit excellent activity in improving memory and cognitive impairment, especially in the early stages of improvement.

[0156] This invention provides a method for preparing silk-derived peptides, which can use hydrolytic enzymes to produce silk-derived peptides more efficiently.

[0157] With the same final production volume, the present invention can miniaturize existing process equipment and produce silk-derived peptides with high yields in a short time with fewer processes. Attached Figure Description

[0158] Figure 1 This is a flowchart of a method for preparing silk-derived peptides according to an embodiment of the present invention.

[0159] Figure 2 , 4 6 and 8 are the results of a cognitive memory evaluation experiment of silk peptides prepared according to an embodiment of the present invention.

[0160] Figure 3 , 5 7 and 9 are experimental results showing the effect of the prepared silk peptides according to an embodiment of the present invention on ischemic cerebral infarction.

[0161] Figures 10 to 15 An experiment relating to the cognitive memory improvement effect of peptides according to an embodiment of the present invention. Detailed Implementation

[0162] This invention relates to peptides for improving memory and preventing or improving cognitive impairment, compositions containing the peptides, and methods for preparing the peptides. It provides peptides for improving memory and preventing or improving cognitive impairment, compositions containing the peptides, and a method for providing peptide compositions for improving memory and preventing or improving cognitive impairment by enzymatic hydrolysis of silk fibroin.

[0163] The embodiments of the present invention will be described in detail below to enable those skilled in the art to readily implement the invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein.

[0164] Preparation Example 1: Preparation of Silk-Derived Peptides

[0165] The dissolved silk fibroin was mixed with purified water and treated with 1 wt% of proteolytic enzymes T1 to T11 according to the composition shown in Table 5 below, and hydrolyzed for 5 hours. Afterwards, it was heat-treated for 10 minutes to remove enzyme activity, and then lyophilized to prepare the powder shown in Table 6.

[0166] Table 5

[0167] T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11 Complex flavor protease 100 - - - - - - - - - - Complex protease - 100 - - - - - - - - - Bromelain - - 100 100 100 100 100 100 100 100 100 ginger protease - - - 5 10 30 50 60 30 30 30 Caloplasm - - - 5 10 20 30 40 20 20 20 Umami enzymes - - - - - - - - 0.5 10 25

[0168] (Unit: parts by weight)

[0169] Table 6

[0170] NF1 NF2 TF1 TF2 TF3 TF4 TF5 TF6 TF7 TF8 TF9 Processing enzymes T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11

[0171] Experimental Example 1: Experiment on the effect of improving memory and cognitive function

[0172] Experimental Example 1-1: Animal model experiment related to improving cognitive function

[0173] A. Preparation of experimental animals

[0174] Male white rats (Sprague Dawley, 140 to 180 g, from the Korea Laboratory Animal Center) were used as experimental animals. They were raised in a regulated environment (room temperature 25 ± 1°C, relative humidity 60 ± 10%) for one week. Four rats were placed in each cage, with unrestricted access to water. At this time, feeding was restricted to maintain 80% of their body weight. A total of 50 rats were used in the experiment. Three days before the training, the rats were allowed to adapt to the maze for 5 minutes and were examined.

[0175] B. Delayed alternation test

[0176] A "T-shaped" maze was prepared, which had three channels (left, right, and center). Dividers were set at the entrance of each channel to allow the experimenter to control the entry and exit of the animals.

[0177] In the first trial, the rats were allowed to adapt to the starting box for 1 minute. The first trial was a "forced-choice trial". One side of the channel was blocked with a divider. When the animal entered the opposite side channel, the rear was blocked with a divider. If the animal's four limbs passed through the channel entrance, it was considered that the animal had entered. The animal was allowed to eat in this channel for 60 seconds.

[0178] Subsequent trials were "free-choice trials". The animals were allowed to start from the starting box and freely choose the channel direction (left or right). Food was only rewarded when the animal entered the channel opposite to the one it entered in the previous trial.

[0179] Delay times of 5 seconds and 20 seconds were set between trials. The cut-off time for each trial was set at 60 seconds. The number of times the animal correctly entered the reinforced channel and the time taken to enter the channel were measured. Ten trials were conducted each day, and the same procedure was carried out for 9 days for evaluation.

[0180] C. Administration method

[0181] The silk peptides NF1, 2, and TF1 to TF9 prepared according to the method of the present invention were prepared into 100 mg / ml solutions and administered orally. All samples were dissolved in distilled water and administered orally.

[0182] D. Cognitive memory assessment results

[0183] The results of the T-maze experiment on the groups treated with silk peptides NF1, 2 and TF1 to TF9 are shown below. Figure 2 . Figure 2 The experiment was conducted with a test interval of 5 seconds, and the performance gradually improved during the 9-day training period.

[0184] In other experiments conducted simultaneously, the inventors observed a similar tendency to that observed when using a mixed enzyme primarily composed of papain, as described below. Figure 2 It can be confirmed that when TF3 to TF5 and TF7 to TF9 are used, significant improvements in cognitive function can be observed. Furthermore, it is known that when the above-mentioned preparations are used, the initial improvement effect is also excellent.

[0185] In particular, compared with other preparations, TF8 exhibits significantly superior cognitive function improvement in the initial stage of drug administration. This can be understood as follows: when peptides with specific sequences are mixed within a specified range in silk fibroin hydrolyzed by a mixed hydrolytic enzyme, the improvement in cognitive function is accelerated, resulting in rapid efficacy in the initial stage of drug administration.

[0186] As a result of the analysis, as described below, it can be confirmed that the hydrolysate contains peptides represented by general formulas 1 to 5, which can produce the accelerating effect described above.

[0187] Therefore, when applicable to the above scope, silk-derived peptides with cognitive and memory-improving effects can be provided, especially silk peptides that can accelerate the initial improvement of cognitive and memory functions and further enhance memory and cognitive function.

[0188] Experimental Examples 1-2: Animal Models of Local Ischemia Induction

[0189] To verify the extent to which the silk peptides prepared using the hydrolytic enzymes NF1, NF2, TF5 and TF8 improve memory and cognitive function, an experiment was conducted using a local ischemia-induced animal model to evaluate the effect on the area of ​​ischemic cerebral infarction.

[0190] A. Drug treatment and the creation of animal models of local ischemia induction

[0191] A local ischemia-induced animal model was established using male Sprague-Dawley rats weighing 200–250 g. The drug was administered orally once one hour before ischemia induction or once one hour after ischemia induction (oral administration group, n=6). Drug experimental numbers were NF1, NF2, TF5, and TF. The ischemia control group (n=6) was administered the same dose of BG101 as the drug treatment group. After anesthetizing the experimental animals with ketamine (30–40 mg / kg) intramuscularly, the skin of the neck was incised to locate and separate the common carotid artery, external carotid artery, and internal carotid artery from surrounding tissues. First, the superior thyroid artery, laryngeal artery, and larynx, which are branches of the external carotid artery, are electrocauterized. The pterygopalatine artery, which is a branch of the internal carotid artery, is electrocauterized. The external carotid artery is then severed. A 4-0 nylon suture (ETHICON, INC) is inserted into the internal carotid artery through the external carotid artery. A 16 to 18 mm nylon suture is then inserted at the branch of the common carotid artery to occlude the origin of the middle cerebral artery.

[0192] B. Experiments on the reduction effect of ischemic stroke sites

[0193] Six hours after ischemia induction, the animals were decapitated and the brains were removed. The removed brains were sliced ​​at 2 mm intervals from the anterior pole, reacted with 2% triphenyltetrazolium chloride (TTC, Sigma) solution at 37°C for 30 minutes, and then fixed in 4% paraformaldehyde (Sigma) solution. After photographing the stained tissue sections, the area of ​​the infarcted areas (whitened) in the images, which differed from the red-stained normal areas, was measured using an MCID image processing system (Imaging Research Inc.). The ratio of the total brain section area to the infarct area was calculated, and the average value was determined. Furthermore, the proportion of the infarcted area in the total area of ​​each brain section was measured. The results are presented below. Figure 3 .

[0194] Reference Figure 3 It can be confirmed that the free silk fibroin provided by this invention can reduce the number of sites of cerebral infarction.

[0195] Preparation Example 2: Preparation of Silk-Derived Peptides

[0196] The dissolved silk fibroin was mixed with purified water and treated with 1 wt% of proteolytic enzymes T1 to T11 according to the composition shown in Table 7 below, and hydrolyzed for 5 hours. Afterwards, it was heat-treated for 10 minutes to remove enzyme activity, and then lyophilized to prepare the powder shown in Table 8.

[0197] Table 7

[0198]

[0199]

[0200] (Unit: parts by weight)

[0201] Table 8

[0202] NF1 NF2 NF3 NF4 NF5 NF6 NF7 NF8 NF9 NF10 NF11 Processing enzymes T1 T2 T3 T4 T5 T6 T7 T8 T9 T10 T11

[0203] Experiment Example 2: An Experiment on the Improvement of Memory and Cognitive Function

[0204] Experiment Example 2-1: Animal Model Experiments Related to Improvement of Cognitive Function

[0205] The following experiments were conducted using the same methods as in Experiment 1-1 above: A. Animal model experiments related to cognitive function improvement; B. Delayed alternation test; C. Drug administration method; D. Cognitive memory evaluation.

[0206] The results of the T-maze test on the groups treated with silk peptides NF1 to NF11 are shown below. Figure 4 . Figure 4 The experiment was conducted with a test interval of 5 seconds, and the performance gradually improved during the 9-day training period.

[0207] Refer to the following Figure 4 It can be confirmed that when NF5 to NF7 and NF9 to NF11 are applied, significant improvements in cognitive function can be observed. Furthermore, when the above-mentioned preparations are used, the initial improvement effect is also excellent.

[0208] In particular, compared with other preparations, NF10 exhibits significantly superior cognitive function improvement in the initial stage of drug administration. This can be understood as follows: when peptides with specific sequences from silk fibroin hydrolyzed by mixed hydrolytic enzymes are mixed within a specified range, the improvement in cognitive function is accelerated, resulting in rapid efficacy in the initial stage of drug administration.

[0209] As the analysis results show, it can be confirmed that when peptides containing sequences represented by both general formula 1 and general formula 2 are present, the acceleration effect described above will also occur.

[0210] Therefore, when applicable to the above scope, silk-derived peptides with cognitive and memory-improving effects can be provided, especially silk peptides that can accelerate the initial improvement of cognitive and memory functions and further enhance memory and cognitive function.

[0211] Experimental Example 2-2: Local Ischemia Induction Animal Model

[0212] The following experiments were conducted using the same methods as in Experiments 1-2 above: A. Establishment of animal models of drug treatment and local ischemia induction; B. Experiments on the reduction effect based on ischemic cerebral infarction sites. The results are presented below. Figure 5 .

[0213] Reference Figure 5 It can be confirmed that the free silk fibroin provided by this invention can reduce the number of sites of cerebral infarction.

[0214] Preparation Example 3: Preparation of Silk-Derived Peptides

[0215] The dissolved silk fibroin was mixed with purified water and treated with 1 wt% of proteolytic enzymes S1 to S11 according to the composition shown in Table 9 below, and hydrolyzed for 5 hours. Afterwards, it was heat-treated for 10 minutes to remove enzyme activity, and then lyophilized to prepare the powder shown in Table 10.

[0216] Table 9

[0217] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 Complex Flavor Protease 100 - - - - - - - - - - Complex protease - 100 - - - - - - - - - Kiwifruit protease - - 100 100 100 100 100 100 100 100 100 Coculus P - - - 1 5 10 20 30 10 10 10 keratinase - - - 1 5 10 20 30 10 10 10 cathepsin K - - - - - - - - 0.5 3 6

[0218] (Unit: parts by weight)

[0219] Table 10

[0220] NF1 NF2 TP1 TP2 TP3 TP4 TP5 TP6 TP7 TP8 TP9 Processing enzymes S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11

[0221] Experiment Example 3: An experiment related to the improvement of memory and cognitive function

[0222] Experiment Example 3-1: Animal Model Experiments Related to Improvement of Cognitive Function

[0223] The following experiments were conducted using the same methods as in Experiment 1-1 above: A. Animal model experiments related to cognitive function improvement; B. Delayed alternation test; C. Drug administration method; D. Cognitive memory evaluation.

[0224] The results of the T-maze test on the groups treated with silk peptides NF1, 2 and TP1 through TP9 are shown below. Figure 6 . Figure 6 The experiment was conducted with a test interval of 5 seconds, and the performance gradually improved during the 9-day training period.

[0225] In other experiments conducted concurrently, the inventors were able to obtain results similar to those obtained using a mixed enzyme primarily composed of papain, as detailed below. Figure 6 It can be confirmed that applying TP3 to TP5 and TP7 to TP9 can demonstrate a significant improvement in cognitive function. Furthermore, it is evident that the initial improvement effect is also excellent when using the aforementioned preparations.

[0226] In particular, compared with other preparations, TP8 showed significantly superior cognitive function improvement in the initial stage of drug administration. This can be understood as follows: when peptides with specific sequences in silk fibroin hydrolyzed by mixed hydrolytic enzymes are mixed within a specified range, the improvement in cognitive function is accelerated, thus resulting in rapid efficacy in the initial stage of drug administration.

[0227] Experimental Example 3-2: Local Ischemia Induction Animal Model

[0228] The following experiments were conducted using the same methods as in Experiments 1-2 above: A. Establishment of animal models of drug treatment and local ischemia induction; B. Experiments on the reduction effect based on ischemic cerebral infarction sites. The results are presented below. Figure 7 .

[0229] Reference Figure 7 It can be confirmed that the free silk fibroin provided by this invention can reduce the number of sites of cerebral infarction.

[0230] Preparation Example 4: Preparation of Silk-Derived Peptides

[0231] The dissolved silk fibroin was mixed with purified water and treated with 1 wt% of proteolytic enzymes S1 to S11 according to the composition shown in Table 11 below for 5 hours. Afterwards, it was heat-treated for 10 minutes to remove enzyme activity, and then lyophilized to prepare the powder shown in Table 12.

[0232] Table 11

[0233] S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 Complex Flavor Protease 100 - - - - - - - - - - Complex protease - 100 - - - - - - - - - Fig protease - - 100 100 100 100 100 100 100 100 100 Coculus P - - - 1 5 10 20 30 10 10 10 keratinase - - - 1 5 10 20 30 10 10 10 cathepsin K - - - - - - - - 0.5 3 6

[0234] (Unit: parts by weight)

[0235] Table 12

[0236] NF1 NF2 TN1 TN2 TN3 TN4 TN5 TN6 TN7 TN8 TN9 Processing enzymes S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11

[0237] Experiment Example 4: An experiment related to the improvement of memory and cognitive function

[0238] Experiment Example 4-1: Animal Model Experiments Related to Improvement of Cognitive Function

[0239] The following experiments were conducted using the same methods as in Experiment 1-1 above: A. Animal model experiments related to cognitive function improvement; B. Delayed alternation test; C. Drug administration method; D. Cognitive memory evaluation.

[0240] The results of the T-maze test on the groups treated with silk peptides NF1, 2 and TN1 to TN9 are shown below. Figure 9 . Figure 9 The experiment was conducted with a test interval of 5 seconds, and the performance gradually improved during the 9-day training period.

[0241] In other experiments conducted concurrently, the inventors were able to obtain results similar to those obtained using a mixed enzyme primarily composed of papain, as detailed below. Figure 8 It can be confirmed that when TN3 to TN5 and TN7 to TN9 are applied, significant improvements in cognitive function can be observed. Furthermore, it is evident that the initial improvement effects are also excellent in the aforementioned preparation examples.

[0242] In particular, compared with other preparations, TN8 showed significantly superior cognitive function improvement in the initial stage of drug administration. This can be understood as follows: when peptides with specific sequences from silk fibroin hydrolyzed by mixed hydrolytic enzymes are mixed within a specified range, the improvement in cognitive function is accelerated, thus resulting in rapid efficacy in the initial stage of drug administration.

[0243] The analysis results show that when a peptide containing both an amino acid sequence represented by general formula 1a or general formula 1b and an amino acid sequence represented by general formula 2 is present, the acceleration effect described above can be produced.

[0244] Experimental Example 4-2: Local Ischemia Induction Animal Model

[0245] The following experiments were conducted using the same methods as in Experiments 1-2 above: A. Establishment of animal models of drug treatment and local ischemia induction; B. Experiments on the reduction effect based on ischemic cerebral infarction sites. The results are presented below. Figure 9 .

[0246] Reference Figure 9 It can be confirmed that the free silk fibroin provided by this invention can reduce the number of sites of cerebral infarction.

[0247] Preparation Example 5: Preparation of Silk Fibroin Powder

[0248] As shown in Table 13, silk fibroin R1 with sericin removed was processed to prepare silk fibroin powders R2 to R4.

[0249] Table 13

[0250] R1 Silk fibroin with sericin removed R2 Alcohol is mixed with silk fibroin, pulverized, and dried to form a powder. R3 Glycerin is mixed with silk fibroin, pulverized, washed with water, and dried to make a powder. R4 Silk fibroin treated with atmospheric pressure plasma was powdered using the same method as P3.

[0251] Experimental Example 5: Preparation of Silk Fibroin Powder

[0252] The silk fibroin powders R2 to R4 generated in Table 14 were maintained at 50°C, while the silk fibroin powders R1 to R4 were mixed with calcium chloride solution. The amount of calcium chloride solution required to completely dissolve the silk fibroin to be hydrolyzed was evaluated. The amount of calcium chloride solution was expressed as a ratio to the weight of the silk fibroin used as a solute.

[0253] Table 14

[0254] R1 R2 R3 Calcium chloride solution 830％ 130％ 80％

[0255] As shown in Table 14, when R2 and R3 are applied, silk fibroin can be completely dissolved even with a relatively small amount of calcium chloride solution. In particular, excellent solubility can be achieved even at reaction temperatures lower than those in the prior art.

[0256] Therefore, when using the aforementioned silk fibroin powder, the amount of calcium chloride solution used as a solvent can be significantly reduced, allowing for the miniaturization of reactors used in the silk fibroin dissolution and hydrolysis steps, thereby improving process efficiency. Furthermore, due to the reduced amount of calcium chloride used, the amount of water and time required for desalination are also reduced, thus achieving greater efficiency.

[0257] The experiments in Examples 1 to 3 were performed using the prepared silk fibroin powder, and the same results were obtained. Therefore, it can be confirmed that the efficiency of the dissolution and hydrolysis processes can be improved when using the silk fibroin powder.

[0258] Sequence analysis of silk hydrolysate

[0259] To identify peptides exhibiting the aforementioned synergistic effects, the peptides contained in the hydrolysate of the silk fibroin were analyzed, compared with known peptides, and peptides producing the aforementioned accelerating and synergistic effects were screened. The analytical results confirmed that the aforementioned initial synergistic and accelerating effects occurred when peptides represented by the following general formulas 1 to 5 were included.

[0260] In particular, in order to confirm the synergistic and accelerating effects of the peptides represented by general formulas 1 to 5, peptides composed of amino acid sequences of general formulas 1 to 5 were synthesized, and experiments related to cognitive function improvement were conducted based on these peptides.

[0261] General Formula 1

[0262] Tyr-Gly-X1-X2

[0263] (In the general formula, X1 is Ala, Tyr, Thr, Val, Gly, Ile or Gln, and X2 is Ala, Tyr, Ile, Val or Gly).

[0264] General Formula 2

[0265] Ile-Gly-Z1-Z2

[0266] (In the general formula, Z1 is Ala, Tyr, Val or Gln, and Z2 is Ala, Tyr, Val or Gly).

[0267] Formula 3

[0268] Ile-Gly-Val-a1-a2

[0269] (In the general formula, a1 is Ala, Tyr or Val, and a2 is Ala, Tyr, Thr, Val, Gly or Gln).

[0270] Formula 4

[0271] a1-Gly-Gly-a1

[0272] (In the general formula, a1 is Ala, Tyr or Val).

[0273] General Formula 5

[0274] a1-Gly-Gly-a1-Gly-a2

[0275] (In the general formula, a1 is Ala, Tyr or Val, and a2 is Ala, Tyr or Ile or Val).

[0276] Preparation Example 6: Synthesis and Mixing of Peptides

[0277] To confirm whether the predicted peptides would show improvement in cognitive function and memory in the short term, the peptides P1-1 to P1-6 were synthesized by an external organization.

[0278] In addition, to confirm the synergistic effect based on the mixing of each peptide, P2-1 to P2-4 of Table 2 were synthesized, and peptide compositions were prepared by mixing the synthetic peptides with the components shown in Table 15 below.

[0279] Table 15

[0280] P1-6 M1 M2 M3 M4 P1-6 100 100 100 100 100 P2-1 - 50 - - - P2-2 - - 50 - - P2-3 - - 50 - P2-4 - - - - 50

[0281] (Unit: parts by weight)

[0282] Experiment Example 6: An experiment related to the improvement of memory and cognitive function

[0283] Experiment Example 6-1: Animal Model Experiments Related to Improvement of Cognitive Function

[0284] The following experiments were conducted using the same methods as in Experiment 1-1 above: A. Animal model experiments related to cognitive function improvement; B. Delayed alternation test; C. Drug administration method; D. Cognitive memory evaluation.

[0285] The results of the T-maze test performed on the groups treated with P1-1 to P1-6 and M1 to M4 are shown below. Figure 10 and Figure 12 . Figure 10 and Figure 12 The experiment was conducted with a test interval of 5 seconds, and the performance gradually improved during the 9-day training period.

[0286] Reference Figure 10 When the peptides P1-1 to P1-6 disclosed in this invention are applied, overall cognitive and memory abilities are improved. Furthermore, compared to P1-1 and P1-4, the application of peptides P1-2, P1-3, P1-5, and P1-6 enhances both cognitive function and memory.

[0287] On the other hand, compared with other synthetic peptides, in the case of P1-6, the improvement in cognitive function and memory is rapidly enhanced from the initial stage of administration.

[0288] Reference Figure 12 It is understood that, as disclosed in this invention, M1 to M4, when combined with P2-1 to P2-4, not only produce a synergistic effect of improving cognitive function and memory, but also rapidly enhance the initial improvement effect. In other words, referring to... Figure 11 P2-2 showed better effects in improving cognitive function and memory, while P2-1, P2-3, and P2-4 alone had lower effects on improving cognitive function and memory. However, when P2-1 to P2-4 were mixed with peptides of general formula 1 and provided as a peptide composition, the synergistic effect caused by the interaction significantly enhanced the improvement in cognitive function and memory, as well as the initial improvement effect. Therefore, when peptides of general formula 1 are mixed with peptides of general formula 2 to form a peptide composition, a composition with rapid improvement in cognitive and memory effects and initial activity can be provided.

[0289] In particular, in the case of foods or medicines used to improve cognitive function and memory, since initial activity is very important, the present invention can provide foods or medicines that improve cognitive function and memory and the initial improvement effect.

[0290] Preparation Example 7: Synthesis and Mixing of Peptides

[0291] Based on the above experimental results, an external agency was commissioned to synthesize the peptides F1, F2, and F3 shown in Table 3.

[0292] Experiment Example 7: An experiment related to the improvement of memory and cognitive function

[0293] Experiment Example 7-1: Animal Model Experiments Related to Improvement of Cognitive Function

[0294] The cognitive function improvement experiments of synthetic peptides F1, F2, and F3 were conducted using the same method as in Experiment 6 above.

[0295] Reference Figure 13 It can be confirmed that F1, F2, and F3 as a whole showed a higher improvement effect on cognitive function and memory than P2-2, and the initial improvement activity of F1, F2, and F3 was also better than P2-2.

[0296] Preparation Example 8: Synthesis and Mixing of Peptides

[0297] We analyzed the hydrolysates of silk fibroin that showed short-term improvements in cognitive function and memory, and predicted the peptides with rapid effects.

[0298] Therefore, in order to confirm whether the predicted peptides would show improvement in cognitive function and memory in the short term, the peptides P4-1 to P4-3 and P5-1 to P5-4 were synthesized by an external organization.

[0299] In addition, to confirm the synergistic effect based on the mixing of each peptide, P2-1 to P2-4 of Table 2 were synthesized, and peptide compositions were prepared by mixing the synthetic peptides with the components shown in Table 16 below.

[0300] Table 16

[0301]

[0302]

[0303] (Unit: parts by weight)

[0304] Experiment Example 8: An experiment related to the improvement of memory and cognitive function

[0305] Experiment Example 8-1: Animal Model Experiments Related to Improvement of Cognitive Function

[0306] The following experiments were conducted using the same methods as in Experiment 1-1 above: A. Animal model experiments related to cognitive function improvement; B. Delayed alternation test; C. Drug administration method; D. Cognitive memory evaluation.

[0307] The results of the T-maze test performed on groups P1-1 to P1-6 and M1 to M4 are shown below. Figure 14 and Figure 15 . Figure 14 and Figure 15 The experiment was conducted with a test interval of 5 seconds, and the performance gradually improved during the 9-day training period.

[0308] Reference Figure 14 When using P4-1 to P4-3 disclosed in this invention, overall cognitive and memory abilities are improved. Furthermore, compared to P4-1 and P1-3, the improvement in cognitive function and memory is increased when using peptide P4-2. In particular, compared to other synthetic peptides, the improvement in cognitive function and memory is rapidly enhanced from the initial stage of administration in the case of P4-1.

[0309] Furthermore, P5-1 to P5-4 showed superior cognitive function and memory improvement effects compared to P4-1 and P4-3. In particular, P5-1 not only showed significantly better cognitive function and memory improvement effects than P4-2, but also exhibited excellent initial improvement activity.

[0310] Reference Figure 15 It is evident that further combining M1 to M4 disclosed in this invention not only generates a synergistic effect based on interaction, but also rapidly improves the initial improvement effect. In other words, referring to... Figure 11 P2-2 showed better improvement in cognitive function and memory, while P2-1, P2-3, and P2-4 alone showed lower improvement in cognitive function and memory. However, when P2-1 to P2-4 were mixed with peptides represented by general formula 4 or general formula 5 and provided as a peptide composition, the improvement in cognitive function and memory, as well as the initial improvement, were significantly enhanced due to the synergistic effect caused by the interaction.

[0311] It has been confirmed that when peptides represented by general formula 4 or general formula 5 are mixed with peptides represented by general formula 2 to form peptide compositions, cognitive memory improvement effects can be enhanced, and compositions with rapid initial activity can be provided.

[0312] Therefore, when applying the present invention to the method for preparing peptides for improving memory and preventing or improving cognitive impairment, accelerated and synergistic effects can be derived from the generation of the peptides. Furthermore, methods skilled in the art can be used to produce the specific peptides and include them in products, thereby exhibiting synergistic effects and rapid initial improvement in cognitive function compared to existing technologies.

[0313] While the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto. Various modifications and improvements made by those skilled in the art using the basic concepts of the present invention as defined in the claims also fall within the scope of the present invention.

[0314] Industrial applicability

[0315] This invention relates to peptides for improving memory and preventing or improving cognitive impairment, compositions containing the peptides, and methods for preparing the peptides. It provides peptides for improving memory and preventing or improving cognitive impairment, compositions containing the peptides, and a method for providing peptide compositions for improving memory and preventing or improving cognitive impairment by enzymatically hydrolyzing silk fibroin.

Claims

1. A peptide consisting of an amino acid sequence represented by the following general formula 1, General Formula 1 Tyr-Gly-Val-X2 (In the general formula, X2 is Ile).

2. Use of a peptide comprising an amino acid sequence represented by the following general formula 1 in the preparation of peptide compositions for improving memory and preventing or improving cognitive impairment, general formula 1 Tyr-Gly-Val-X2 (In the general formula, X2 is Ile or Gly).

3. The use according to claim 2, characterized in that, The composition is derived from silk fibroin.

4. A method for preparing a peptide according to claim 2, characterized in that, include: Dissolving steps for silk fibroin; The step of treating and hydrolyzing silk fibroin lysates with a mixed enzyme comprising a first proteolytic enzyme and a second proteolytic enzyme, wherein the first proteolytic enzyme comprises bromelain, and the second proteolytic enzyme comprises any one selected from the group consisting of complex flavor protease, complex protease, neutral protease, Belon-W, Sumex, ginger protease, calpain, protease NP, validaase, and mixtures thereof; and Enzyme removal steps for removing proteolytic enzymes.

5. A method for preparing a peptide according to claim 2, characterized in that, include: Dissolving steps for silk fibroin; The step of treating and hydrolyzing the silk fibroin lysate with a proteolytic enzyme, wherein the proteolytic enzyme is selected from any one of the group consisting of complex flavor protease, papain, complex protease, neutral protease, Belon-W, Sumex, ginger protease, calpain, protease NP, validaase, and mixtures thereof; and Enzyme removal steps for removing proteolytic enzymes.

6. A method for preparing a peptide according to claim 2, characterized in that, include: Dissolving steps for silk fibroin; The step of treating and hydrolyzing the silk fibroin lysate with a proteolytic enzyme, wherein the proteolytic enzyme is selected from any one of the group consisting of Delvase, chymotrypsin, subtilisin, actinidin, lactoferrin, coculase P, keratinase, cathepsin K, and mixtures thereof; and Enzyme removal steps for removing proteolytic enzymes.

7. The method for preparing the peptide according to claim 4 or 5, characterized in that, The hydrolytic enzymes also include umami enzymes.

8. The method for preparing the peptide according to claim 6, characterized in that, The hydrolytic enzyme also includes fig protease.

9. The method for preparing the peptide according to any one of claims 4 to 6, characterized in that, The dissolution step of silk fibroin is the step of dissolving silk fibroin powder in solution. The silk fibroin powder is made by mixing silk fibroin and glycerin, pulverizing, washing with water, filtering, and drying.

10. The method for preparing the peptide according to any one of claims 4 to 6, characterized in that, The cognitive impairment mentioned is a group consisting of dementia, learning disability, agnosia, amnesia, aphasia, apraxia, delirium, AIDS-induced dementia, Binswanger disease, Lewy body dementia, frontotemporal dementia, mild cognitive impairment, multiple infarct dementia, Pick's disease, semantic dementia, Alzheimer's disease, or vascular dementia.

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