CN118541039A - Composition for improving memory, cognitive ability, or preventing or treating brain nervous system diseases comprising pig brain enzyme hydrolysate - Google Patents
Composition for improving memory, cognitive ability, or preventing or treating brain nervous system diseases comprising pig brain enzyme hydrolysate Download PDFInfo
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- CN118541039A CN118541039A CN202380016963.8A CN202380016963A CN118541039A CN 118541039 A CN118541039 A CN 118541039A CN 202380016963 A CN202380016963 A CN 202380016963A CN 118541039 A CN118541039 A CN 118541039A
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- enzyme hydrolysate
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- disease
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a composition comprising a hydrolysate of pig brain enzyme for improving memory, enhancing cognitive ability, and preventing or treating neurological disorders. The above combination can exhibit neuroprotective ability, brain-derived neurotrophic factor (BDNF) production ability, acetylcholinesterase (acetylcholinesterase) inhibition ability, and reactive oxygen species (ROS) inhibition ability.
Description
Technical Field
The present application claims priority from korean patent application No. 10-2022-0004195, filed on 1 month 11 of 2022, and korean patent application No. 10-2022-0143631, filed on 11 month 1 of 2022, which are incorporated herein by reference in their entirety.
The present invention relates to a composition for improving memory, cognitive ability, or preventing or treating a brain nervous system disease comprising a porcine brain enzyme hydrolysate.
Background
The brain has multiple functions, but most importantly memory and cognitive abilities. If the human beings do not have cognitive ability and memory ability, it is difficult to perform daily life and even survival becomes a problem.
Cognitive memory impairment is the first symptom that occurs in Alzheimer's patients and is the most common symptom. In the early stages of alzheimer's disease, patients may present a recent memory (memory) disorder that fails to memorize the contents of the latest dialog or event in detail, due to a decrease in the function of storing the recent memory caused by the damage of the nerve cells of the hippocampus. However, during this period, remote long-term memory (termmemory) of events that occurred in the distant past remains relatively good. However, as the disease progresses, the cerebral cortex associated with long-term memory storage is damaged, and this past memory is also gradually impaired.
On the other hand, dementia (dementia) is a serious cognitive disorder in senile diseases, and means that cognitive functions such as memory, language ability, space-time comprehension ability, judgment ability and abstract thinking ability are continuously and entirely reduced due to the impaired brain function, and in severe cases, many disturbances occur in daily life. Dementia is one of brain diseases, and is known to be caused not only by Alzheimer's disease and vascular dementia, but also by neurodegenerative diseases such as Parkinson's disease, head trauma, infectious diseases, and the like. Thus, it can be said that there is an urgent need for efforts to treat and manage dementia diseases that increase rapidly with the geometric growth of the elderly population. Dementia (dementia) is a syndrome of symptoms of various acquired cognitive dysfunction such as memory, language disorder, and movement disorder, which is caused by irreversible destruction of brain nerves due to brain atrophy, cytopenia, and senile plaque. Dementia is classified into degenerative diseases caused by Alzheimer's disease, vascular dementia, metabolic diseases caused by hypothyroidism, and other dementias caused by brain tumor or infectious diseases. Currently, donepezil (donepezil), galantamine (galantamine), rivastigmine (rivastigmine) and the like, which are acetylcholinesterase (acetylcholine sterase, AChE) inhibitors, are clinically used as drugs for improving dementia, but there are no effective therapeutic agents because of low therapeutic efficiency and serious side effects.
Disclosure of Invention
Technical problem
The present invention provides a pig brain enzyme hydrolysate comprising pig brain derived peptides.
The present invention provides a food composition for improving memory, improving cognitive function, or preventing or improving a brain nervous system disease comprising a pig brain enzyme hydrolysate.
Also, the present invention provides a health functional food composition for improving memory, improving cognitive function, or preventing or improving a brain nervous system disease.
Also, the present invention provides a pharmaceutical composition for improving memory, improving cognitive function, or preventing or improving a brain nervous system disease.
However, the technical problems to be solved by the present invention are not limited to the above-described problems, and other problems not mentioned can be clearly understood by those of ordinary skill in the art through the following description.
Technical proposal
The present invention provides a pig brain enzyme hydrolysate comprising a peptide consisting of sequence 1 or sequence 2 or more than one of peptides consisting of sequence 1 or sequence 2.
The present invention provides a food composition or a health functional food composition for improving memory, improving cognitive function, or preventing or improving a brain nervous system disease, comprising a pig brain enzyme hydrolysate as an active ingredient.
Also, the present invention provides a pharmaceutical composition for improving memory, improving cognitive function, or preventing or treating a brain nervous system disease, comprising a pig brain enzyme hydrolysate as an active ingredient.
The porcine brain enzyme hydrolysate may comprise a porcine brain derived peptide.
The pig brain enzyme hydrolysate can be hydrolyzed by more than one hydrolyzing agent.
The above composition may be formulated into a dosage form selected from the group consisting of tablets, capsules, powders, granules, liquid formulations and pills.
The pig brain enzyme hydrolysate can have nerve cell protecting ability and/or brain-derived neurotrophic factor (BDNF) generating ability.
The pig brain enzyme hydrolysate may have acetylcholinesterase (acetylcholine sterase) inhibiting ability and/or Reactive Oxygen Species (ROS) inhibiting ability.
The cerebral nervous system disease may be selected from the group consisting of Alzheimer's disease, mild cognitive impairment, senile dementia, parkinson's disease, huntington's disease, dementia with Lewy bodies (Lewy Body Dementia), frontotemporal dementia (Frontotemporal Dementia), cerebral infarction, cerebral apoplexy, and epilepsy.
The present invention also provides a method for improving memory, improving cognitive function, or preventing or treating a cerebral nervous system disorder, which comprises the step of administering a composition comprising, as an active ingredient, a pig brain enzyme hydrolysate containing one or more peptides consisting of sequence 1 or sequence 2 to a subject or administering the composition to the subject.
The present invention provides a use of a composition comprising a pig brain enzyme hydrolysate containing one or more peptides composed of sequence 1 or sequence 2 as an active ingredient for improving memory, improving cognitive function or preventing or treating a brain nervous system disease.
ADVANTAGEOUS EFFECTS OF INVENTION
The peptide of the present invention or a pig brain enzyme hydrolysate containing the same has excellent effects of improving memory, improving cognitive function, preventing or treating (or improving) a brain nervous system disease, and does not show toxicity, and thus can be usefully used as a health functional food composition, a pharmaceutical composition, etc.
Drawings
Fig. 1 is a flowchart schematically showing a process for producing a pig brain enzyme hydrolysate according to an embodiment of the present invention.
Fig. 2 is a graph showing the result of evaluation of cytotoxicity of a peptide according to an embodiment of the present invention.
Fig. 3a and 3b are graphs showing the neuroprotective ability of peptides according to one embodiment of the present invention (p <0.05; p <0.01; p < 0.001).
Fig. 3c and 3d are graphs showing the brain-derived neurotrophic factor (BDNF) production ability of peptides according to an embodiment of the present invention (p <0.05; p <0.01; p < 0.001).
FIG. 3e is a graph showing beta-amyloid aggregation (beta-amyloid aggregation) of peptide (brPEP-1) according to one embodiment of the invention.
Fig. 3f is a graph showing reactive oxygen species (Reactive Oxygen Species, ROS) of peptides according to one embodiment of the invention (p <0.05; p <0.01; p < 0.001).
Fig. 4 is a graph showing the cytotoxicity evaluation results of the porcine brain enzyme hydrolysate according to an embodiment of the present invention.
Fig. 5 is a graph showing the nerve cell protective ability of porcine brain enzyme hydrolysate according to an embodiment of the present invention (p <0.05, p <0.01, p < 0.001).
Fig. 6 is a graph showing the brain-derived neurotrophic factor (BDNF) production capacity of porcine brain enzyme hydrolysate according to an embodiment of the present invention (p <0.05, p <0.01, p < 0.001).
FIGS. 7a and 7b are graphs showing the expression characteristics of proteins (Caspase-3, chAT) of porcine brain enzyme hydrolysate according to an embodiment of the present invention.
Fig. 8 is a graph showing the inhibitory capacity of acetylcholinesterase (acetylcholine sterase) in porcine brain enzymatic hydrolysate according to an embodiment of the present invention (p <0.05; p < 0.01).
Fig. 9 is a graph showing the inhibitory capacity of reactive oxygen species (ROS, reactive oxygen species) of porcine brain enzyme hydrolysate according to an embodiment of the present invention (p <0.05; p < 0.01).
Fig. 10a, 10b are graphs showing the Beta-aggregation (Beta-aggregation) inhibition ability of porcine brain enzyme hydrolysate according to an embodiment of the present invention.
Fig. 11a, 11b are graphs showing a schematic view (a) of a passive avoidance experiment (passive avoidance test) and a result (b) of analysis of the memory improving effect of the porcine brain enzyme hydrolysate by the passive avoidance experiment (different letters or footnotes on the bar graph mean that there is a significant difference between each other at p < 0.05).
Fig. 12a, 12b are graphs showing a schematic diagram (a) of the Y-maze test (Y maze test) and a result (b) of analysis of the memory improving effect of the pig brain enzyme hydrolysate by the Y-maze test (different letters or footnotes on the bar chart mean that there is a significant difference between each other at p < 0.05).
Fig. 13a, 13b are graphs showing a schematic view (a) of a new object recognition experiment (Novel object test) and a result (b) of analysis of the memory improving effect of the pig brain enzyme hydrolysate by the new object recognition experiment (Novel object test) (different letters or footnotes on the bar graph mean that there is a significant difference between each other at p < 0.05).
Fig. 14a, 14b are graphs showing a schematic diagram (a) of Morris water maze test (Morris water-maze test) and a result (b) of analysis of memory improving effect of pig brain enzyme hydrolysate by Morris water maze test (different letters or footnotes on the bar chart mean that there is a significant difference between each other at p < 0.05).
Detailed Description
Hereinafter, the present invention will be described in detail.
The present invention provides a pig brain enzyme hydrolysate comprising a peptide consisting of sequence 1 or sequence 2 or more than one of peptides consisting of sequence 1 or sequence 2.
The peptides may be used as food compositions or pharmaceutical compositions.
The peptides may be contained in the pig brain enzyme hydrolysate at a concentration of 0.1ppm to 300 ppm. More preferably, it may be contained at a concentration of 1 to 30 ppm.
Also, the present invention provides a composition for improving memory, improving cognitive function, preventing or treating (or improving) a brain nervous system disease, comprising a pig brain enzyme hydrolysate as an active ingredient.
The composition can be a food composition, a health functional food composition or a pharmaceutical composition.
The porcine brain enzyme hydrolysate may comprise a porcine brain derived peptide.
The pig brain enzyme hydrolysate can be hydrolyzed by more than one hydrolyzing agent.
More preferably, the above hydrolysate can be produced by a first hydrolysis and a second hydrolysis.
The above-mentioned first hydrolysis may be carried out by adding a proteolytic enzyme to a solid solution (pig brain slurry solution), in which case it may be carried out at a temperature of 40.+ -. 20 ℃ for 1 to 20 hours. However, it is not limited thereto.
The second hydrolysis may be performed by adding a proteolytic enzyme to the solid solution in which the first hydrolysis is performed, and in this case, the second hydrolysis may be performed at a temperature of 40.+ -. 20 ℃ for 1 to 20 hours. However, it is not limited thereto.
The above composition may be formulated into a dosage form selected from the group consisting of tablets, capsules, powders, granules, liquid formulations and pills.
The porcine brain enzyme hydrolysate may have a nerve cell protective ability and/or brain-derived neurotrophic factor (BDNF) producing ability, and may have an acetylcholinesterase (acetylcholine sterase) inhibiting ability and/or Reactive Oxygen Species (ROS) inhibiting ability.
The cerebral nervous system disease may be selected from the group consisting of Alzheimer's disease, mild cognitive impairment, senile dementia, parkinson's disease, huntington's disease, dementia with Lewy bodies (Lewy Body Dementia), frontotemporal dementia (Frontotemporal Dementia), cerebral infarction, cerebral apoplexy, and epilepsy.
In the present specification, "prevention" refers to all actions of delaying a decline in memory or a decline in cognitive ability by administration of the composition of the present invention, and "treatment" and "improvement" refer to all actions of improving or beneficially altering symptoms of a decline in memory or a decline in cognitive ability by administration of the composition of the present invention.
In the present invention, the term "health functional food" means food in the form of tablets, capsules, powders, granules, liquid, pills, etc. prepared and processed using raw materials or ingredients having beneficial functions to the human body. Wherein "functional" refers to the beneficial effects of obtaining health care uses, such as regulating the nutrients or physiological actions required by the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and in the above preparation process, can be prepared by adding raw materials and ingredients commonly added in the art. The formulation of the health functional food may be prepared without limitation, as long as the food is approved as a formulation of the health functional food. The health functional food composition of the present invention has the advantage that there are no side effects or the like which may occur when a liquid or powder formulation drug is taken for a long period of time, and is convenient to carry, and thus can be taken as an auxiliary agent for enhancing the preventive or ameliorating effects of liver damage, drug intoxication or hangover caused by alcohol.
In the food composition or the health functional food composition of the present invention, the content of the pig brain enzyme hydrolysate may be 1 to 20% by weight based on the total weight of the composition, but the content is not limited thereto, and the amount of the active ingredient to be mixed may be appropriately determined depending on the respective purposes of use such as prevention, health care, and treatment.
The health functional food can be in the form of powder, granule, pill, tablet, capsule, or any common food or beverage.
Examples of foods to which the above-mentioned substances can be added include meats, sausages, breads, chocolates, candies, snacks, biscuits, pizzas, stretched noodles, other noodles, chewing gums, dairy products including ice cream, various purees, beverages, teas, drinks, alcoholic beverages, and multivitamins, etc., and may include all conventional foods.
In general, in the case of a liquid, the amount of the above-mentioned active ingredient to be added may be 15 parts by weight or less, preferably 10 parts by weight or less, based on 100 parts by weight of the raw material in the preparation of a food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the above amount may be not more than the above range, and since the present invention uses a derivative derived from a natural product without a problem in terms of safety, an amount not more than the above range may also be used.
In the functional food of the present invention, the beverage may contain various sweeteners or natural carbohydrates or the like as an additional ingredient like a general beverage. The natural carbohydrate can be monosaccharide such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, and the like; polysaccharides such as dextrin, cyclodextrin, etc.; and sugar alcohols such as xylitol, sorbitol, erythritol, and the like. As the sweetener, natural sweeteners such as thaumatin, stevia extract, or synthetic sweeteners such as saccharin, aspartame, etc. may be used. The proportion of the above natural carbohydrate may be about 0.01 to 0.04g, preferably about 0.02 to 0.03g, per 100mL of the beverage of the present invention.
In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acids and salts thereof, alginic acids and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents for carbonated beverages. In addition, the health functional food composition of the present invention may contain pulp for preparing natural juice, juice beverage and vegetable beverage. Such components may be used alone or in combination. The proportion of such an additive is not limited, but is generally selected in the range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the functional food composition of the present invention.
The pharmaceutical composition of the present invention is not particularly limited as long as it contains the above active ingredient, but preferably, the content of the pig brain enzyme hydrolysate may be 5 to 30 weight percent with respect to the total weight of the composition. However, it is not limited thereto. In this case, when the effective ingredient is below the above concentration range, there is a problem in that it is difficult to achieve an optimal preventive or therapeutic effect, and when it is above the above concentration range, a change in the effect may be expected to be weak.
The pharmaceutical composition of the present invention may be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, sterile injectable solutions, etc., respectively, according to conventional methods, and may be formulated with suitable carriers, excipients or diluents commonly used in the preparation of pharmaceutical compositions.
The carrier or excipient or diluent may be various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
In the preparation, the composition can be prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant, which are commonly used.
Solid formulations for oral administration may be prepared by mixing at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. with the above jasmonate. Also, lubricants such as magnesium stearate, talc, etc. may be used in addition to the simple excipients.
Liquid preparations for oral administration include suspensions, solutions for internal use, emulsions, syrups and the like, and may contain various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like, in addition to usual simple diluents such as water or liquid paraffin.
Formulations for parenteral administration include sterile aqueous solutions, nonaqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. can be used as nonaqueous solvents, suspensions. Semisynthetic fatty acid esters (witepsol), polyethylene glycol, tween (tween) 61, cocoa butter, glycerol laurate, glycerol gelatin, etc. can be used as a base for suppositories.
The optimal amount of the pharmaceutical composition of the present invention to be administered varies depending on the state of the patient, the body weight, the severity of the disease, the pharmaceutical form, the administration route and the period, but can be appropriately selected by one of ordinary skill in the art. However, for optimal effect, the amount of active ingredient contained in the composition of the present invention may be generally administered in an amount of 0.0001mg/kg to 2000mg/kg, preferably 0.001mg/kg to 2000mg/kg, for 1 day. The administration may be carried out 1 day for 1 time, or may be carried out in several times. However, the scope of the present invention is not limited to the above-described administration amount.
The pharmaceutical composition of the present invention can be administered to mammals such as rats, mice, livestock, humans, etc. by various routes. The administration may be in any manner, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine, intrathecal or intraventricular (intracerebroventricular) injection.
In the short-term memory behavior test (Y-maze test, passive avoidance test, new object recognition test) for the pig brain enzyme hydrolysate of the present invention, it was confirmed that short-term memory was restored in a concentration-dependent manner (Y-maze test: behavior test for confirming spatial cognitive ability, passive avoidance test: behavior test for confirming memory stimulation and avoidance, new object recognition test: behavior test for exhibiting behavior for exploring and recognizing a new object). Hereinafter, the present invention will be described in more detail by way of examples. The objects, features, and advantages of the present invention can be easily understood by the following examples. The present invention is not limited to the embodiments described herein, but may be embodied in other forms. The embodiments described herein are provided so that the spirit of the present invention may be fully conveyed to those of ordinary skill in the art to which the present invention pertains. Accordingly, the present invention should not be limited by the following examples.
EXAMPLE 1 preparation of pig brain enzyme hydrolysate
After the pig brain is thawed in a thawing machine, the pig brain with foreign matters removed by drinking water is exsanguinated. The pig brain from which blood was removed was pulverized with a stirrer so as to facilitate degreasing. The crushed pig brain is defatted with alcohol (ethanol, isopropanol, etc.) to remove fat and foreign matter. Proteolytic enzyme (pepsin) is added to the finished defatted pig brain and the first enzymatic hydrolysis is carried out for 1-20 hours. After the completion of the first enzymatic hydrolysis, filtration was performed to separate solid and liquid. After the filtration, proteolytic enzyme (pancreatin) is added to the first hydrolysate of pig brain and the second enzymatic hydrolysis is carried out for 1-20 hours. After the second enzymatic hydrolysis is completed, heating is carried out to inactivate proteolytic enzymes, and the pig brain enzymatic hydrolysate is contacted with a filter aid and then filtered to be adsorbed and purified. Then, 1 to 5 times (w/w) of alcohol (ethanol) was added to the swine brain hydrolysate filtrate and left for 1 to 30 hours, followed by filtration with a filter. After concentrating the filtered liquid, the sterile-filtered liquid is divided into small portions and sterilized. Finally, the pig brain enzyme hydrolysate with high purity is obtained (refer to figure 1).
Experimental example 1 confirmation of peptides of pig brain enzyme hydrolysate and analysis of peptide efficacy
1-1. Confirmation of peptides of pig brain enzyme hydrolysate the pig brain enzyme hydrolysate obtained in example 1 was diluted to 1/2 by adding 100. Mu.l D.W to 100. Mu.l of the sample. The filtered liquid was diluted to 1/20 by adding 950. Mu.l of deionized water (D.W) to 50. Mu.l of the dilution and sequenced by HPLC system of ACQUITY UPLC I-Class PLUS (Waters) and MS/MS ionization analysis of Xex TQ-XS (Waters)) MS system using syringe filter (SYRINGE FILTER).
< Analysis conditions >
-Column: ACQUITY UPLC BEH C18 (2.1X105 mm,1.7 μm, waters) -flow rate: 200 μl/min
Mobile phase a: h 2 O/FA=100/0.1 (v/v)
Mobile phase B: acetonitrile (Acetonitrile)/fa=100/0.1 (v/v)
Gradient (Gradient)
TABLE 1
The results obtained by the above-described instrument condition analysis are as follows.
As a result of the analysis, 4 peptides were analyzed in total, and among them, 2 peptides were confirmed to be pig brain-derived peptides. The following 2 most stable substances exhibiting reproducibility were selected as index substances.
TABLE 2 peptide sequences of index Components of pig brain enzyme hydrolysates
The results of comparing the LC/MS EIC chromatograms (m/z 403.2) to confirm the residence times of PSIS components of the PSIS standard solution and the sample solution of pig brain enzyme hydrolysate confirm that the residence times of the PSIS peaks are consistent. Further, the retention time of GPAGPQGPR components of the GPAGPQGPR standard solution and the sample solution of porcine brain enzyme hydrolysate was confirmed by comparing LC/MS EIC chromatogram (m/z 418.7), and it was confirmed that the retention time of GPAGPQGPR peaks was uniform.
Peptides were then synthesized from Anygen (www.anygen.com) with the same mass (mass) as the peptides identified by the pig brain hydrolysate and in ms/ms ionised form to identify agreement with the peptides present in the pig brain enzyme hydrolysate and follow-up experiments were performed.
1-2 Evaluation of cytotoxicity of peptides
SH-SY5Y cells used as human neural cell lines were obtained from Korean cell line bank (Cat No. 22266) and cultured in culture tank (incubator) at 5% CO 2, 37℃using 10% fetal bovine serum (Fetal bovine serum, FBS, situo (Cytiba)) and medium 1640 (Roswell Park Memorial Institute (RPMI) 1640Medium,RPMI 1640, situo (Cytiba)) containing penicillin (penicillin) and streptomycin (streptomycin) at 100IU/ml for experiments.
After the human neural cell line SH-SY5Y was cultured, pig brain enzyme hydrolysate-derived peptides were added at different concentrations, and then, a colorimetric reaction was performed with thiazole blue tetrazolium bromide (methylthiazol tetrazolium bromide, MTT, sigma Aldrich (SIGMA ALDRICH)) to confirm the cell viability. Appropriate amounts of cells (1X 10 5/well) were inoculated (seeding) into wells (well) of a 24-well plate (WELL PLATE) (BD, falcon) and treated with samples of different concentrations, respectively, and cell viability was confirmed after culturing in an incubator (incubator) at 37℃for 48 hours. After 4 hours of reaction with MTT solution, 400. Mu.l of DMSO was added to dissolve insoluble formazan crystals, and absorbance at 570nm was measured with an ELISA plate reader (Diken (TECAN), INFINITE M200 pro).
As a result, each of the peptides brPEP-1, brPEP-2 selected as the index component exhibited 93.05% + -2.23% and 108.47% + -5.20%, respectively, at a concentration of 40ug/mL (w/v). It was confirmed that the cell viability was 90% or more at all concentrations (0.4-40 ug/ml) at which experiments were performed, and that there was no safety problem with cytotoxicity (FIG. 2).
1-3 Evaluation of the neuroprotective Capacity of peptides (Neuro-protectiveeffect)
To evaluate the efficacy of 2 peptides (peptides of sequence 1 and sequence 2) to improve memory, the human neural cell line was used to confirm the cytoprotective capacity against neuronal apoptosis caused by treatment with β -amyloid (β -amyloid) at concentrations below the avirulence.
To determine the neuroprotective capacity, neuronal apoptosis was induced by treatment of SH-SY5Y with β -amyloid, a major pathological feature of alzheimer's disease, to cause toxicity. Specifically, SH-SY5Y cells (cells) as human neuroblastoma were seeded at 1X 10 5/well (well) in 24-well plates (WELL PLATE) for culture. Then, after pretreatment with 2 peptides of the present invention (peptides of sequence 1, sequence 2) at different concentrations for 24 hours for cultivation, each well (well) was treated with beta-amyloid protein (beta-amylase) at a concentration of 40uM for further cultivation for 24 hours. After 4 hours of reaction with MTT solution (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide)), insoluble formazan (formazan) crystals were dissolved by adding 400. Mu.l of DMSO, respectively, and the results were confirmed by measuring absorbance at 570nm using ELISA plate reader Diken (TECAN), INFINITE M pro.
As shown in fig. 3a, the cell viability of the group treated with aβ25-35 was reduced to 63.7% (p < 0.001) compared to the normal control group, but in the case of treatment with aβ25-35 of 40 μm for 24 hours after pretreatment with the peptide of sequence 1 (PSIS), a level of neuroprotection of about 21-30% was exhibited. Also, as shown in fig. 3b, the cell viability of the group treated with aβ25-35 was reduced to 63.9% (p < 0.001) compared to the normal control group, but in the case of pretreatment with the peptide of sequence 2 (GPAGPQGPR) followed by treatment with 40 μm aβ25-35 (amyloid beta 25-35) for 24 hours, a level of neuroprotection of about 18-31% was exhibited.
1-4 Evaluation brPEP-1, brPEP-2 of BDNF-producing ability
BDNF is Brain-derived neurotrophic factor (Brain-derived neurotrophic factor). In order to confirm the memory improving function of the test sample by confirming the ability of the protein BDNF involved in the signal transduction system to produce during the metabolic process affecting cognitive function (memory, thinking ability), SH-SY5Y cells, which are human neuroblastoma, were cultured in 75T flasks (flash) and fused (confluency) to 80-90%, and inoculated (seeding) in an amount of 2X 10≡5 into 6-well cell culture plates (well cell culture plate). After 1 week of incubation in a thermostatted, constant humidity cabinet (humidity chamber) at 37 ℃, 5% co 2, the Pig Brain Enzyme Hydrolysate (PBEH) of the present examples was treated with different concentrations. Cell lysates (CELL LYSATE) were obtained after culturing in a constant temperature and humidity cabinet (humidity chamber) at 37℃and 5% CO 2 for 48 hours, and BDNF was measured using BDNF KIT (KIT) (# EK0307) of Boschia bioengineering Co., ltd (Bosterbio). Then, the cell number was corrected with the protein content by performing protein assay.
As a result of the test, it was confirmed that the Peptide (PSIS) of sequence 1 and the peptide (GPAGPQGPR) of sequence 2 each have excellent ability to produce the nerve cell growth factor BDNF (fig. 3c, 3 d).
1-5. Confirmation brPEP-1 beta-amyloid aggregation (. Beta. -amyloidaggregation)
A thioflavin T assay (Thioflavin T Assays) was performed to see how much the brPEP-1 of the present examples can inhibit the aggregation of β -amyloid (β -amylase). Thioflavin T reacts to β -amyloid (β -amylase) and exhibits fluorescence (fluorescence). After culturing SH-SY5Y cells, each was treated with brPEP-1 and after 24 hours, with 20uM of beta-amyloid (beta-amylase) for 24 hours, then washed with PBS (pH 7.4) and treated with 5uM of thioflavin T (Thioflabin T) before the cells were collected, and then assayed using an ELISA plate (Tecan), INFINITE M pro, at an excitation wavelength (450 nm) and an emission wavelength (emission) of 482 nm.
Since thioflavin T (Thioflavin T) binds to amyloid aggregates, the aggregation level can be confirmed by the intensity of the fluorescence spectrum inherent to thioflavin T (Thioflavin T). As a result of confirming the thioflavin T (Thioflavin T) at different times, it was confirmed that the fluorescence intensity of the group (CTL) treated with beta-amyloid (beta-amylase) was stronger than that of the group not treated with beta-amyloid (beta-amylase).
It was confirmed that brPEP-1 exhibited about 62% inhibition of beta-amyloid aggregation (beta-amyloid aggregation) after 18 hours at a concentration of 0.002ug/ml, which is the concentration of the porcine brain enzyme hydrolysate present at 25ug/ml total nitrogen of the present feedstock, and the present feedstock exhibited about 65% levels of similar beta-amyloid aggregation (beta-amyloid aggregation) inhibition after 18 hours (fig. 3 e).
1-6, Activity oxygen Cluster (ROS) inhibition capability of brPEP-1, brPEP-2 was confirmed
To determine the production of intracellular reactive oxygen species by H 2O2, the intracellular reactive oxygen species levels were determined using fluorescent DCF-DA (2 ',7' -dichlorofluorescein diacetate (2 ',7' -dichlorodihydrofluorescein diacetate)). DCF-DA is converted to DCF by reactive oxygen species and fluoresces in the cell. To determine the amount of reactive oxygen species in the cells, SH-SY5Y cells were incubated, treated with brPEP-1, brPEP-2 according to the examples of the invention for 24 hours, then with 100. Mu.M H 2O2 for 24 hours, then with 20. Mu.M DCF-DA before the cells were collected, and after incubation at 37℃for 30 minutes, washed with PBS (pH 7.4) and treated with 1% triton X-100 to lyse the cells, and then assayed at 480nm excitation wavelength (extraction) and 530nm emission wavelength (emission) using an ELISA enzyme-Linked instrument (TECAN), INFINITE M pro). brPEP-1, brPEP-2 were confirmed to exhibit 24%, 17% inhibition at a concentration of 2ug/ml, respectively, and to be effective in a concentration-significant manner (FIG. 3 f).
Experimental example 2 analysis of efficacy of hydrolysates comprising pig brain-derived peptides
The pig brain enzyme hydrolysate prepared in example 1 (comprising peptides of sequence 1 and sequence 2) was used to analyze the memory improving efficacy. The content of the peptides in the pig brain enzyme hydrolysate is 5-30 ppm respectively.
2-1 Cytotoxicity evaluation
SH-SY5Y cells used as human neural cell lines were obtained from Korean cell line bank (Cat No. 22266) and cultured in culture tank (incubator) at 5% CO 2, 37℃using 10% fetal bovine serum (Fetal bovine serum, FBS, situo (Cytiba)) and medium 1640 (Roswell Park Memorial Institute (RPMI) 1640Medium,RPMI 1640, situo (Cytiba)) containing penicillin (penicillin) and streptomycin (streptomycin) at 100IU/ml for experiments.
After SH-SY5Y (human neuroblastoma) was cultured, pig Brain Enzyme Hydrolysates (PBEH) were added at different concentrations, followed by colorimetric detection of thiazole blue tetrazolium bromide (methylthiazol tetrazolium bromide, MTT, sigma Aldrich (SIGMA ALDRICH)) to confirm cell viability. Appropriate amounts of cells (1X 10 5/well) were inoculated (seeding) into wells (well) of a 24-well plate (WELL PLATE) (BD, falcon) and treated with samples of different concentrations, respectively, and cell viability was confirmed after culturing in an incubator (incubator) at 37℃for 48 hours. After 4 hours of reaction with MTT solution, 400. Mu.l of DMSO was added to dissolve insoluble formazan crystals, and absorbance at 570nm was measured with an ELISA plate reader (Diken (TECAN), INFINITE M200 pro).
As a result, it was confirmed that the sample of the Pig Brain Enzyme Hydrolysate (PBEH) of the example of the present invention was nontoxic up to 50ug/ml of total nitrogen (FIG. 4).
2-2. Nerve cell protective Capacity (Neuro-protectiveeffect)
To determine the neuroprotective capacity, neuronal apoptosis was induced by treatment of SH-SY5Y with β -amyloid, a major pathological feature of alzheimer's disease, to cause toxicity. Specifically, SH-SY5Y cells (cells) as human neuroblastoma were seeded at 1X 10 5/well (well) in 24-well plates (WELL PLATE) for culture. Then, after pretreatment with various concentrations of the Porcine Brain Enzyme Hydrolysate (PBEH) of the present example for 24 hours for cultivation, each well (well) was treated with beta-amyloid protein (beta-amylase) at a concentration of 40uM for further cultivation for 24 hours. After 4 hours of reaction with MTT solution (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (3- (4, 5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide)), insoluble formazan (formazan) crystals were dissolved by adding 400. Mu.l of DMSO, respectively, and the results were confirmed by measuring absorbance at 570nm using ELISA plate reader Diken (TECAN), INFINITE M pro.
As a result, it was confirmed that the Pig Brain Enzyme Hydrolysate (PBEH) of the example of the present invention exhibited a nerve cell protective ability of 25% and 52% at total nitrogen of 25ug/ml and 50ug/ml (FIG. 5).
BDNF production ability
BDNF is Brain-derived neurotrophic factor (Brain-derived neurotrophic factor). In order to confirm the memory improving function of the test sample by confirming the ability of the protein BDNF involved in the signal transduction system to produce during the metabolic process affecting cognitive function (memory, thinking ability), SH-SY5Y cells, which are human neuroblastoma, were cultured in 75T flasks (flash) and fused (confluency) to 80-90%, and inoculated (seeding) in an amount of 2X 10≡5 into 6-well cell culture plates (well cell culture plate). After 1 week of incubation in a thermostatted, constant humidity cabinet (humidity chamber) at 37 ℃, 5% co 2, the Pig Brain Enzyme Hydrolysate (PBEH) of the present examples was treated with different concentrations. Cell lysates (CELL LYSATE) were obtained after culturing in a constant temperature and humidity cabinet (humidity chamber) at 37℃and 5% CO 2 for 48 hours, and BDNF was measured using BDNF KIT (KIT) (# EK0307) of Boschia bioengineering Co., ltd (Bosterbio). Then, the cell number was corrected with the protein content by performing protein assay.
As a result of the test, it was confirmed that the Pig Brain Enzyme Hydrolysate (PBEH) exhibited BDNF-producing abilities of 30%, 41% and 47% at total nitrogen of 6.25, 25 and 50ug/ml, respectively (FIG. 6).
2-4 Protein expression (Proteinexpression) (Caspase-3, chAT)
Apoptosis (apotopsis) is involved in a variety of proteins and signaling substances that, when promoted by this process, promote caspase-3 activation, which results in cleavage of PARP to cause apoptosis (apotopsis). The following test was conducted to confirm whether apoptosis of beta-amyloid (beta-amylase) can be inhibited with reduced caspase-3 activation by treating with the pig brain enzyme hydrolysate of the present invention and ginkgo leaf extract as a health functional food raw material as a control group.
When SH-SY5Y cells were confluent (confluency) to 90% after about 7 days of seeding, they were treated with the above-mentioned Porcine Brain Enzyme Hydrolysate (PBEH) for 24 hours, incubated with 20uM of beta-amyloid (beta-amyoid), then washed with cold PBS and lysates (lysates) were obtained with RIPA Lysate (RIPABuffer). After confirming the concentration of the Protein by Protein assay, the same amount of cell lysate and 2X sample buffer (sample buffer) were mixed and heated at 100℃for 5 minutes to induce Protein denaturation, followed by 10% SDS-polyacrylamide gel electrophoresis (SDS-polyacrylamide gel electrophoresis, SDS-PAGE). After the electrophoresis, the proteins in the gel (gel) were transferred to a polyvinylidene fluoride (PVDF) membrane (polyvinylidene difluoride (PVDF) membrane) (Bio-Rad, USA), america. The reaction was carried out with a blocking buffer (5% skim milk (skimmilk)) at room temperature for 1 hour. Antibodies to Caspase-3, chAT, and GAPDH were diluted 1:1000 in Tris buffer salt solution (Tris-buffered saline, TBS-T) containing tween20 (tween-20) at 0.1% (v/v) and reacted with polyvinylidene fluoride (polyvinylidene difluoride) at a temperature of 4 ℃ overnight (overnight), and anti-rabbit IgG-binding HRP (anti-rabbit IgG conjugated HRP) diluted (1:2000) with TBS-T as a second antibody to each antibody and reacted separately for 2 hours at normal temperature, and then visualized using ECL kit (kit) (Bio-Rad, USA).
As a result of the test, it was confirmed that there was a difference in the intensity of bands at an effective level in both Caspase-3 and ChAT, and thus there was a possibility of improving memory (FIGS. 7a and 7 b).
2-5 Acetylcholinesterase (acetylcholinesterase) inhibitory ability
The decomposition of acetylcholine (acetylcholine) is inhibited by inhibiting the activity of acetylcholinesterase (Acetylcholinesterase, AChE), so that the concentration of acetylcholine in neuronal synapses can be increased, thereby inducing an improvement in cognitive function. The following test was performed to confirm the inhibitory capacity of AChE of porcine brain enzyme hydrolysate.
SH-SY5Y cells, which are human neuroblastomas, were cultured in 75T flasks (flash) and, when fused (confluency) to 80-90%, seeded (seeding) in 6-well cell culture plates (well cell culture plate) (BD, falcon) in an amount of 2X 10≡5 (2X 10 5). After 1 week of incubation in a constant temperature and humidity cabinet (humidity chamber) at 37 ℃ with 5% co 2, treatment was performed with different concentrations of Porcine Brain Enzyme Hydrolysate (PBEH), ginkgo leaf extract currently registered as a raw material for individual authentication as a positive control group, and Donepezil as a drug, respectively. Cell lysates (CELL LYSATE) were obtained after culturing in a constant temperature and humidity cabinet (humidity chamber) at 37℃and 5% CO 2 for 48 hours, and AChE activity was measured using an AChE antibody (KIT) (#ab 138871) from abcam. Then, the cell number was corrected with the protein content by performing protein assay.
As a result of the test, it was confirmed that the Pig Brain Enzyme Hydrolysate (PBEH) exhibited about 23% and 26% inhibition ability at 25,50 ug/ml of total nitrogen, in contrast to about 21% inhibition in a concentration-dependent manner in 100ug/ml of ginkgo leaf extract (FIG. 8).
2-6 Reactive Oxygen Species (ROS) inhibition capability
To determine the production of intracellular reactive oxygen species by H 2O2, the intracellular reactive oxygen species levels were determined using fluorescent DCF-DA (2 ',7' -dichlorofluorescein diacetate (2 ',7' -dichlorodihydrofluorescein diacetate)). DCF-DA is converted to DCF by reactive oxygen species and fluoresces in the cell. To determine the amount of intracellular reactive oxygen species, SH-SY5Y cells were cultured, and then treated with the Pig Brain Enzyme Hydrolysate (PBEH) of the present example, ginkgo leaf extract currently registered as an individual authentication material, respectively, for 24 hours, and with 100. Mu.M H 2O2 for 24 hours, and then treated with 20. Mu.M DCF-DA before collecting the cells, and after 30 minutes at 37℃the cells were washed with PBS (pH 7.4) and treated with 1% triton X-100 to lyse the cells, and then measured at 480nm excitation wavelength (encitation) and 530nm emission wavelength (emission) using an ELISA enzyme-Linkey (TECAN), INFINITE M pro). It was confirmed that Porcine Brain Enzyme Hydrolysate (PBEH) exhibited inhibition of 43% and 60% at total nitrogen of 25 and 50ug/ml, respectively, and was effective in a concentration-significant manner, whereas ginkgo leaf extract of 100ug/ml exhibited inhibition of about 35.9%, confirming a result similar to that of the porcine brain enzyme hydrolysate of 25ug/ml total nitrogen (fig. 9).
2-7. Beta. -aggregation (Beta-aggregation) inhibition ability
A thioflavin T assay (Thioflavin T Assays) was performed to see how much the pig brain enzymatic hydrolysate of the present examples and ginkgo leaf extract currently registered as an individual certified raw material can inhibit the aggregation of beta-amyloid (beta-amylase). Thioflavin T reacts to β -amyloid (β -amylase) and exhibits fluorescence (fluorescence). After culturing SH-SY5Y cells, the cells were treated with Porcine Brain Enzyme Hydrolysate (PBEH) and ginkgo leaf extract currently registered as an individual authentication type raw material, respectively, and after 24 hours, were incubated with 20uM of beta-amyloid (beta-amylase) for 24 hours, then washed with PBS (pH 7.4) and treated with 5uM of thioflavin T (Thioflabin T) for 1mL before collecting the cells, and then measured at 450nm excitation wavelength (extraction) and 482nm emission wavelength (emission) using ELISA enzyme-labeled instrument (TECAN), INFINITE M pro.
Since thioflavin T (Thioflavin T) binds to amyloid aggregates, the aggregation level can be confirmed by the intensity of the fluorescence spectrum inherent to thioflavin T (Thioflavin T). As a result, it was confirmed that the fluorescence intensity of the group (CTL) treated with beta-amyloid (beta-amylase) was higher than that of the group (N) not treated with beta-amyloid (beta-amylase) as a result of the sulfur T (Thioflavin T) at different times.
It was confirmed that the Pig Brain Enzyme Hydrolysate (PBEH) exhibited about 66% of beta-amyloid aggregation (beta-amyloid aggregation) inhibitory ability at 25ug/ml of total nitrogen, and about 46% of beta-amyloid aggregation (beta-amyloid aggregation) inhibitory ability at 100ug/ml of ginkgo leaf extract registered as an individual authentication material (fig. 10).
Experimental example 3 animal behavior test
3-1. Passive avoidance experiment (passive avoidancetest)
A passive avoidance experiment was performed to confirm the memory/cognition improving effect of the pig brain enzyme hydrolysate obtained in the above examples (fig. 11a, 11 b).
Experimental method
The passive avoidance experiment (passive avoidance test) is an experiment for determining the time required to move to the darkroom (dark room) when the white mouse is placed in the bright room (light room), the first and second times are performed at intervals of 8 hours, and the electrical stimulus is applied when the white mouse moves to the darkroom (dark room) so that learning cannot move to the darkroom (dark room), and the time required to move from the bright room (light room) to the darkroom (dark room) is measured after 16 hours.
Experimental results
As shown in fig. 11a and 11b, in the second test (real), the movement time of the High-PBEH group was similar to that of the Normal group (Normal control group) (in fig. 11b, MD control group (MD-control), positive control group (Positivel-control), low PBEH (Low-PBEH), medium PBEH (Medium-PBEH), high PBEH (High-PBEH), normal control group (Normal-control)) in this order from the left side. In the third test (real), it was confirmed that the latency (LATENCY TIME) of the normal group was longer than that of the induction group (MD control group), and the time delay for the middle PBEH (Medium-PBEH) (0.159 ml/kg) and High PBEH (High-PBEH) (0.478 ml/kg) groups to move to the dark room (dark room) was similar to those of the normal group, and thus the memory was improved.
3-2.Y maze test (Y maze test)
The Y-maze test was performed to confirm the memory/cognition improving effect of the pig brain enzyme hydrolysate obtained in the above examples (fig. 12a, 12 b). The Y-maze test can be used to study the spontaneous alternation of spatial learning and memory and to identify the memory function of the memory test sensitive to hippocampal damage, genetic engineering, memory deletion.
Experimental method
The Y-maze test (Y-maze test) is a method for evaluating instantaneous spatial cognition in a short-term memory form, the Y-maze is an omnibearing closed maze in a Y shape manufactured according to specifications (50.5 cm long, 20cm wide, 20cm high, 3 arms (arm)), three branches are respectively designated as A, B, C, a white mouse is carefully placed in one branch and allowed to freely move for 8 minutes, and then the branches into which the white mouse enters are sequentially recorded. Counting the cases of going into each branch in turn gives 1 minute (actual alternation actual alternation). The alternating behavior (alternation behavior) is defined as not repeating into three branches.
Experimental results
As a result, the number of normal cycles in the induction group (MD control group) was smaller than that in the normal group (normal control group). The Normal cycle (%) in the Medium PBEH (Medium-PBEH) (0.159 ml/kg) and High PBEH (High-PBEH) groups (0.478 ml/kg) showed higher values than the total cycle number in the induction group (in FIG. 12b, MD control (MD-control), positive control (Positivel-control), low PBEH (Low-PBEH), medium PBEH (Medium-PBEH), high PBEH (High-PBEH), normal control (Normal-control) in this order from the left).
3-3 New object identification experiment (Novelobjecttest)
A new object recognition experiment (Novel object test) was performed to confirm the memory/cognition improving effect of the pig brain enzyme hydrolysate obtained in the above examples (FIGS. 13a, 13 b). The study of identifying new objects is represented by% and 100% represents the total identification, so a value closer to 100 represents better memory.
Experimental method
Starting on the first day of the test, each white rat was allowed to explore new things in a plexiglas (plexiglass) box for 5 minutes on the first day, 20 minutes on the second day, and 20 minutes on the third day. On the fourth day, the box was divided into 4 areas and 2 identical objects were placed in the center of the diagonal area. The white mice were placed beside the box and allowed to explore freely for 10 minutes, then the objects and equipment were rinsed with 75% ethanol to remove the taste of the white mice, and after 1 hour, one of the 2 identical objects was replaced with one of similar size but different shape and color.
The time required for a 5 minute white mouse to explore a new object and an existing object was recorded. Exploring behavior is defined as white mice smelling the smell with the nose or touching with the forepaws, but sitting on an object with a fire turning over is not considered exploring behavior.
Experimental results
As a result of the experiment, it was confirmed that the level of recognition of a new object by the induction group (MD control group) was low compared with the normal group (normal control group), and the positive control group exhibited a value similar to that of the normal group. The degree of recognition of new objects in the pig brain enzyme hydrolysate intake group was increased in a concentration-dependent manner, and the recognition rate was highest in the group with a High level of PBEH (High-PBEH), and thus the cognition was improved (in fig. 13b, MD control (MD-control), positive control (Positivel-control), low PBEH (Low-PBEH), medium PBEH (Medium-PBEH), high level PBEH (High-PBEH), and Normal control (Normal-control), in this order from the left side).
Morris water maze test (Morris water-maze test)
Morris water maze experiments were performed to confirm the memory/cognition improving effect of the pig brain enzyme hydrolysate obtained in the above examples (FIGS. 14a, 14 b). The water maze is used to study spatial memory and learning, put animals into the water and confirm the time to find a hidden escape platform.
Experimental method
Morris water maze experiments (Morris water-maze test) were performed and the target quadrant time (target quadrant time), number of passes through the target (Number of crossing platform), and swimming pattern (SWIMMING PATTERN) were measured using a camera operated by a computer program for analysis. Morris water maze test (Morris water-maze test)In a circular sink (height 40 cm. Times. Diameter 1.5 m), a target (target) having a diameter of 12cm was mounted at the bottom of the circular sink so as to be 1cm below the water surface, and then, after holding water (22 ℃ C. -25 ℃ C.) in the sink and dissolving skimmed milk powder so that the target was invisible, a test was performed by placing a white rat in zone1 (zone 1) and finding a platform (platform) of zone5 (zone 5) on the first day of the experiment. The water maze test was performed 3 times in total, the first and second days training a method of finding a platform (platform) of zone5 (zone 5), and the latency, access frequency, and duration of zone5 (zone 5) were measured on the fifth day to evaluate memory. The test was performed for a total of 600 seconds.
Experimental results
As a result of the experiment, it was confirmed that the time period for which the upper limit of the plateau was first found was shorter in the middle PBEH (Medium-PBEH) (0.159 ml/kg) and upper limit PBEH (High-PBEH) (0.478 ml/kg) of the swine brain enzyme hydrolysate in comparison with the induction group (MD control group), and the time period for which the swine brain enzyme hydrolysate in the vicinity of the plateau was as long as in the Normal group (Normal control group), and therefore, the swine brain enzyme hydrolysate obtained in the example was effective in spatial memory (MD control group (MD-control), positive control group (Positivel-control), low PBEH (Low-PBEH), middle PBEH (Medium-PBEH), upper limit PBEH (High-PBEH), and Normal control group (Normal-control) in this order from the left side).
3-5 Tissue analysis of hippocampus
The tissues of hippocampus were analyzed by the following method to confirm the memory/cognition improving effect of the pig brain enzyme hydrolysate obtained in the above examples.
Experimental method
The hippocampus was divided into two parts, one part was solubilized with RIPA buffer containing protease inhibitors and the other part was solubilized with Trizol reagent (Invitrogen, rocyverer, rockville, MD, USA) to extract whole RNA. Hippocampal RIPA buffer lysates were centrifuged at 5000×g for 10 min to use supernatant, and lipid peroxide and cholesterol content were determined colorimetrically using supernatant and lipid peroxide (MDA) detection kit (assay kit) (Abcam, cambridge, UK) and cholesterol kit (Abcam, cambridge, UK). Protein in the supernatant was removed with 1.5N perchloric acid, and glycogen content was calculated from glucose concentration derived from glycogen hydrolyzed by alpha-amyloglucosidase (alpha-amyloglucosidase) in acid buffer.
Glucose concentration was measured using a glucose kit (Asan Pharmaceutics, seoul, korea). Triglycerides were extracted from hippocampus with chloroform-methanol solution (2:1, v/v) and resuspended in pure chloroform. The neutral fat content of chloroform (korea, kohlrabi pharmaceutical) was measured using a neutral fat colorimetric kit.
Serum TNF- α and IL-1β concentrations were determined using ELISA kits (eBioscience; san Diego, calif.), and serum concentrations of AST and ALT were determined using AST and ALT kits (Asan Pharmaceutics, seoul, korea).
Experimental results
The cholesterol content in the hippocampal tissue of the induction group (MD control group: memory uncline-induced group) was significantly higher than that of the normal group (normal control group), and the positive control group and the pig brain enzyme hydrolysate intake group decreased the cholesterol content.
The glycogen content in the hippocampal tissue of the normal group was higher than that of the induced group, and in the pig brain enzyme hydrolysate intake group, the glycogen content of the Low PBEH (Low-PBEH) (0.053 ml/kg) and Medium PBEH (Medium-PBEH) (0.159 ml/kg) groups was increased.
Peroxide in hippocampal tissue was confirmed by MDA content, the induced group was higher than the normal group, and was reduced in the positive control group as well as the pig brain enzyme hydrolysate uptake group. In particular, the same effect of lowering peroxide as in the normal control group was confirmed in the Medium PBEH (Medium-PBEH) (0.159 ml/kg) group and the high PBEH (Hgh-PBEH) (0.478 ml/kg) group.
It was confirmed that TNF-. Alpha.content as an inflammatory cytokine (cytokine) in brain tissue also showed similar content to that of peroxide, and that the content of the induced group was higher than that of the normal control group, and was decreased in the Medium-3565 (Medium-PBEH) group and the High-PBEH (High-PBEH) group among the positive control group and the pig brain enzyme hydrolysate intake group.
TABLE 3 Table 3
The above description of the present invention is merely for example, and it will be understood by those skilled in the art that the present invention may be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Accordingly, it should be understood that the above-described embodiments are illustrative in all respects and not restrictive.
Subject information
Topic unique number: g0210300-01
Subject number: 2021, 0210300-0
Department name: food department for agriculture, forestry and livestock
Study management specialization organization: korean food institute
Study item name: food functionality evaluation support item
Study topic name: study of influence of pig brain enzyme hydrolysate on memory improvement and mechanism
The main pipe mechanism comprises: unimed pharmaceutical Co Ltd
Study time: 21.04 to 21.12.
Claims (29)
1. A food composition for improving memory, improving cognitive function, or preventing or improving a cerebral nervous system disease, comprising a pig brain enzyme hydrolysate containing one or more peptides consisting of sequence 1 or sequence 2 as an active ingredient.
2. The food composition of claim 1, wherein the porcine brain enzyme hydrolysate is hydrolyzed by one or more proteolytic enzymes.
3. The food composition of claim 1, wherein the peptide is derived from porcine brain enzyme hydrolysate.
4. The food composition of claim 1, wherein said porcine brain enzyme hydrolysate exhibits a neuroprotective ability and a brain-derived neurotrophic factor producing ability.
5. The food composition of claim 1, wherein said porcine brain enzyme hydrolysate exhibits acetylcholinesterase inhibitory ability and active oxygen cluster inhibitory ability.
6. The food composition according to claim 1, wherein the cerebral nervous system disease is selected from the group consisting of alzheimer's disease, mild cognitive impairment, senile dementia, parkinson's disease, huntington's disease, dementia with lewy bodies, frontotemporal dementia, cerebral infarction, stroke and epilepsy.
7. The food composition of claim 1, wherein the composition is formulated into a dosage form selected from the group consisting of tablets, capsules, powders, granules, liquids, and pills.
8. A pharmaceutical composition for improving memory, improving cognitive function or preventing or treating a brain nervous system disease, comprising a pig brain enzyme hydrolysate containing one or more peptides consisting of sequence 1 or sequence 2 as an active ingredient.
9. The pharmaceutical composition of claim 8, wherein the porcine brain enzyme hydrolysate is hydrolyzed by more than one proteolytic enzyme.
10. The pharmaceutical composition of claim 8, wherein the peptide is derived from porcine brain enzyme hydrolysate.
11. The pharmaceutical composition of claim 8, wherein said porcine brain enzyme hydrolysate exhibits a neuroprotective ability and a brain-derived neurotrophic factor producing ability.
12. The pharmaceutical composition of claim 8, wherein said porcine brain enzyme hydrolysate exhibits acetylcholinesterase inhibitory ability and active oxygen cluster inhibitory ability.
13. The pharmaceutical composition of claim 8, wherein the cerebral nervous system disorder is selected from the group consisting of alzheimer's disease, mild cognitive impairment, senile dementia, parkinson's disease, huntington's disease, dementia with lewy bodies, frontotemporal dementia, cerebral infarction, stroke and epilepsy.
14. The pharmaceutical composition of claim 8, wherein the composition is formulated into a dosage form selected from the group consisting of tablets, capsules, powders, granules, liquids, and pills.
15. A pig brain enzyme hydrolysate, characterized by comprising one or more than one kind of peptide consisting of sequence 1 or sequence 2.
16. The porcine brain enzyme hydrolysate of claim 15, wherein the peptide is purified or chemically synthesized from the porcine brain enzyme hydrolysate.
17. The porcine brain enzyme hydrolysate according to claim 15 wherein the peptide is used for improving memory, improving cognitive ability or preventing or treating a brain neurological disorder.
18. The porcine brain enzyme hydrolysate according to claim 17 wherein the brain nervous system disease is selected from the group consisting of alzheimer's disease, mild cognitive impairment, senile dementia, parkinson's disease, huntington's disease, dementia with lewy bodies, frontotemporal dementia, cerebral infarction, stroke and epilepsy.
19. The porcine brain enzyme hydrolysate of claim 15 wherein the peptide exhibits neuroprotective ability and brain-derived neurotrophic factor producing ability.
20. The porcine brain enzyme hydrolysate of claim 15 wherein the peptide exhibits acetylcholinesterase inhibitory ability and active oxygen cluster inhibitory ability.
21. A peptide consisting of sequence 1 or sequence 2.
22. The peptide of claim 21, wherein the peptide is purified from porcine brain enzyme hydrolysate or chemically synthesized.
23. The peptide of claim 21, wherein the peptide is used as a food composition or a pharmaceutical composition.
24. The peptide according to claim 21, wherein the peptide is used for improving memory, improving cognitive ability or preventing or treating a cerebral nervous system disease.
25. The peptide according to claim 23, wherein the cerebral nervous system disease is selected from the group consisting of alzheimer's disease, mild cognitive impairment, senile dementia, parkinson's disease, huntington's disease, dementia with lewy bodies, frontotemporal dementia, cerebral infarction, cerebral stroke and epilepsy.
26. The peptide of claim 21, wherein said peptide exhibits neuroprotective ability and brain-derived neurotrophic factor producing ability.
27. The peptide of claim 21, wherein said peptide exhibits acetylcholinesterase inhibitory ability and active oxygen cluster inhibitory ability.
28. A method for improving memory, improving cognitive function, or preventing or treating a cerebral nervous system disease, comprising the step of administering to or administering to a subject a composition comprising as an active ingredient a porcine brain enzyme hydrolysate comprising one or more peptides consisting of sequence 1 or sequence 2.
29. Use of a composition for improving memory, improving cognitive function or preventing or treating a brain nervous system disorder, characterized in that the composition comprises a pig brain enzyme hydrolysate containing one or more peptides consisting of sequence 1 or sequence 2 as an active ingredient.
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| KR10-2022-0004195 | 2022-01-11 | ||
| KR1020220143631A KR20230109078A (en) | 2022-01-11 | 2022-11-01 | Composition for improving memory or cognition ability, or preventing or treating brain neurological diseases, comprising porcine brain enzyme hydrolysate |
| KR10-2022-0143631 | 2022-11-01 | ||
| PCT/KR2023/000392 WO2023136573A1 (en) | 2022-01-11 | 2023-01-09 | Composition for improving memory or cognition or preventing or treating cranial nerve diseases, comprising porcine brain hydrolysates |
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