KR100575229B1 - Food Compositions for Preventing or Treating Brain Neuronal Diseases - Google Patents

Abstract

The present invention relates to a composition for preventing or treating cerebral neurological diseases, and more particularly, (a) a pharmaceutically effective amount of a silk peptide produced by degradation of silk protein; And (b) relates to a composition for the prevention or treatment of cerebral neurological diseases comprising a pharmaceutical carrier.

Silk, silk peptide, fibroin, apoptosis, ischemia, reperfusion, stroke, acetylcholine, cognitive function, depression, neurodegenerative diseases, functional food

Description

Food Compositions for Preventing or Treating Brain Neuronal Diseases

Figure 1a is a graph showing the results of the MTT reduction experiment to confirm the effect of one type of silk peptide of the present invention on the death of neurons;

Figure 1b is a graph showing the results of the MTT reduction experiment to confirm the effect of the other type of silk peptide of the present invention on the death of neurons;

FIG. 2A is a picture of pest staining results showing that one type of silk peptide of the present invention inhibits apoptosis of neurons;

FIG. 2B is a picture of results of pest staining showing that another type of silk peptide of the present invention inhibits apoptosis of neurons;

3A is a graph showing that one type of silk peptide of the present invention inhibits caspase activity;

3B is a graph showing that another kind of silk peptide of the present invention inhibits caspase activity;

Figure 4a is a photograph showing that one kind of silk peptide of the present invention inhibits the production of activator oxygen in the cell;

4b is a photograph showing that another type of silk peptide of the present invention inhibits the generation of activator oxygen in cells;

Figure 5a is a photograph showing the effect of the silk peptide of the present invention on the site of cerebral infarction by ischemia;

Figure 5b is a graph showing the effect of the silk peptides of the present invention on the site of cerebral infarction by ischemia;

Figure 5c is a graph showing the effect of the silk peptide of the present invention on the cerebral infarction site by ischemia;

Figure 5d is a photograph showing the effect of the silk peptide of the present invention on the site of cerebral infarction by ischemia;

FIG. 5E is a graph of passive avoidance test results showing the efficacy of the silk peptides of the present invention for preventing cognitive impairment by ischemia-reperfusion.

FIG. 5F is a graph of results for an eight-way maze measurement showing the efficacy of the silk peptides of the present invention for preventing cognitive impairment by ischemia-reperfusion.

Figure 5g is a photograph of the H & E staining results of the hippocampus showing the efficacy of the silk peptide of the present invention to prevent cognitive impairment caused by ischemia;

Figure 6 is a graph showing the results of unilateral rotational reactions with apomorphine using Parkinson's disease animal model;

7 is a graph showing the protective effect of the silk peptide of the present invention on dopamine neurons;

8 is a graph of malondialdehyde experimental results showing that the silk peptide of the present invention has an anti-oxidative effect on Parkinson's disease;

Figure 9a is a photograph showing the tyrosine hydroxylase staining results showing that the silk peptide of the present invention is effective in Parkinson's disease;

9B is a graph showing the results of tyrosine hydroxylase staining showing that the silk peptides of the present invention are efficacious in Parkinson's disease;

10 is a graph showing that the silk peptide of the present invention inhibits the decrease in the concentration of acetylcholine in the brain;

Figure 11a is a graph showing the antidepressant effect when a single acute treatment of the silk peptide of the present invention; And

Figure 11b is a graph showing the antidepressant effect in the long-term repeated treatment of the silk peptide of the present invention.

The present invention relates to a composition for preventing or treating cerebral neurological diseases, and more particularly, to a composition for preventing or treating cerebral neurological diseases, comprising as an active ingredient a silk peptide derived from silk protein.

Stroke is a cerebrovascular disease that causes brain vessels to burst or become blocked, resulting in abnormal function of local brain tissue. It is often called stroke, and is leading the cause of death in Korea. Moreover, the incidence rate is gradually increasing due to the extension of the average life expectancy caused by industrialization and medical development. Strokes can occur anywhere in the brain and can therefore impair nearly every function of the body. Medically, stroke can be categorized into 'ischemic stroke' due to large blockage of the blood vessels of the brain and 'hemorrhagic stroke' caused by cerebral hemorrhage, and is closely related to hypertension and atherosclerosis, which are known as the cause of adult disease. The incidence of ischemic stroke is higher.

Ischemic stroke is caused by blockage of blood vessels anywhere from the carotid artery in the neck, the vertebral base artery to the slender arteries in the brain, resulting in the death of the brain tissue controlled by the blood vessels, or 'infarction'. This will occur. Once developed, the site of cerebral infarction cannot restore its function, so the central nervous system disorder caused by cerebral infarction does not recover and remains permanent. Therefore, the most important thing in the treatment of stroke is prevention of cerebral ischemia, as well as preventive measures such as hypertension, diabetes, hyperlipidemia, which are known as risk factors for stroke. In addition, the focus is on reducing brain edema when stroke is caused by stroke, and reducing secondary brain damage by allowing the ischemic state to circulate properly.

Current substances used for the protection of nerve cells include excitatory amino acid antagonists ganglioside, nimodipine, GABA agonist clomethiazole, and magnesium sulfate and glycine antagonists. A phase clinical study is underway and piracetam is currently undergoing a large scale clinical study. However, current neuroprotective agents are agents that work at different stages of the ischemic process and require the development of combination therapies that work simultaneously on these different processes, but the challenges of side effects and drug interference are addressed. Is holding.

In addition, the symptoms of ischemic stroke come suddenly without any special prognosis, so it is more effective to consume functional foods for continuous ischemia prevention and suppression of apoptosis of neurons after ischemia, rather than expecting treatment through drugs administered at the time of cerebral ischemia. It is judged.

US Pat. No. 6,245,757 discloses progestin for use in treating cell damage by ischemia and US Pat. No. 6,380,193 describes strokes comprising poly (adenosine 5'-diphospho-ribose) polymerase inhibitors. Therapeutic compositions are disclosed. In addition, US Pat. No. 6,288,041 discloses a composition for treating stroke comprising sialic acid derivatives, and US Pat. No. 5,580,866 discloses a composition for treating stroke comprising 1,4-thiazetin as an active ingredient.

Parkinson's disease (PD), a neurodegenerative disorder, was first reported by James Parkinson in 1817 as a neurodegenerative brain disorder that causes motor and cognitive impairment. The incidence of the disease is about 750,000 to 100 million patients in the US, ranging from 100 to 150 per 100,000 population, and 60,000 new cases are diagnosed each year. Prevalence is expected to continue to increase. Histopathologically, the loss of dopamine neurons located in the substantia nigra is characteristic, and dopamine in the caudate nucleus and putamen, which are the projection sites of the nerve fibers, is reduced. Motor and cognitive dysfunctions include tremor, bradykinesia, rigidity, and disturbance of posture.

The main therapeutic drugs used in Parkinson's disease are drugs that compensate for the lack of dopamine in the brain, or other drugs to prevent or delay the destruction of nerve cells or to control other symptoms such as depression. Etc. Representative drugs include dopamin precursors, levodopa (L-dopa), madadopar, dopamine receptor agonists, bromidine, lisuride, and anti-acetylcholinergic drugs altan. Various neuropharmacologically compensatory therapies have been developed, such as artane and cogentin. Levodopa, a precursor of dopamine, is most effectively used to ameliorate symptoms of Parkinson's disease by replenishing insufficient levels of dopamine in the brain, but when administered for longer than 3-5 years, the drug effect time becomes shorter (wearing-off), or drugs Side effects such as severe fluctuations in motor control function (on-off) and diskinesia (Freed et. Al., N. Engl. J. Med. 327: 1549-) 55 (1992).

In addition, surgical treatment of Parkinson's disease includes thalamotomy, pallidotomy, deep brain stimulation, and neuronal cell transplantation. However, the duration of treatment effect varies greatly from patient to patient, and rarely is accompanied by side effects such as hypoplasia, dysarthria, and memory attenuation following surgery (Ondo et. Al., Neurology) . 50: 266-270 (1998); Shannon et. Al., Neurology 50: 434-438 (1998).

Recent treatment for Alzheimer's disease is centered on the possibility that Alzheimer's disease is derived from impaired cholinergic signaling and transmission in the cerebral cortex and hippocampus (Bartus et al. , Science. 217 (4558): 408-14 (1982)); And Coyle et al., Science. 219 (4589): 1118-90 (1983). Since these areas of the brain are linked to memory and intelligence, functional deficiencies in these parts of the brain damage certain memories and judgments and cause loss of intellectual ability. While the exact process by which neuronal signaling impairs damage is controversial, senile plaques and neurofibrillary tangles (NFTs) are believed to be the main causes of nerve damage. Sensitive plaques due to the accumulation of amyloid beta (Aβ) are the most hallmark of the disease, and Alzheimer's disease can be confirmed by post mortem examination (Khachaturian, Arch. Neurol. 42 (11): 1097-105 (1985)).

In Alzheimer's disease, drugs and therapies are suggested to increase the amount of acetylcholine to suppress the damage to cholinergic signaling, to allow acetylcholine to be present for a long time, or to make acetylcholine work more effectively in neuronal delivery. Many compounds are being used to increase the acetylcholine activity of Alzheimer's disease patients. Currently, the most effective approach is to inhibit the activity of acetylcholinesterase, which rapidly breaks down acetylcholine at synapses, preventing neuronal signal transduction. Indeed, these inhibitors (eg tacrine, donepezil and rivastigmine) are currently on the market as FDA-approved Alzheimer's disease drugs. In many cases these drugs are effective in preventing the destructive progression of the disease, but are not well applied to the recovery of the nervous system to its pre-disease state.

Some compounds aim to improve the general state of health of the nerves so that the cells function normally as they age. For example, some drugs, such as NGF and estrogen, play a neuroprotective role in slowing neuronal degeneration, while others, such as antioxidants, reduce the oxidation of cells, reducing the increase in harmful cell damage that results from normal aging. Let's do it. If the amount of amyloid beta peptide accumulates, Alzheimer's disease becomes severe, and it is thought that lowering the accumulation of amyloid beta in the neuritic space will slow the progression of Alzheimer's disease. Amyloid precursor protein (APP) is thought to proceed in many different forms in combination with intracellular proteases such as α-, β- and γ-secretases. However, since the formation process of amyloid beta has not been fully scientifically identified, it is not yet possible to control the formation of amyloid beta.

The process by which amyloid beta accumulates abnormal neuronal signal transmission is not clear. Plaque formation is caused when APP is abnormally cleaved and a lot of amyloid beta is generated and accumulated in neuritis. Thus, many other factors involved in this cleavage reaction (such as inflammatory reactions) increase the phosphorylation of tau protein, increase the accumulation of NFTs and paired helical filaments (PHFs) and eventually nerves. Increases the damage. All of these factors cause nerve dysfunction and ultimately accelerate progression to Alzheimer's type of dementia.

Although there are many developments in treatment to reduce the effects of Alzheimer's disease, it is currently providing temporary improvement of symptoms. In conclusion, current treatment for Alzheimer's disease focuses on improving symptoms rather than reversing the course of the disease. We know more about the biological knowledge of the disease, but the results of the application to the clinic are not yet successful.

US Patent No. 5,532,219 discloses a composition for treating Alzheimer's disease, including 4,4'-diaminodiphenylsulfone, and the like, US Patent No. 5,506,097 discloses para-amidinophenylmethanesulfonyl fluoride or everlactone A Disclosed is a composition for treating Alzheimer's disease, comprising US Patent No. 6,136,861, which discloses a composition for treating Alzheimer's disease comprising bicyclo [2.2.1] heptane.

On the other hand, stress has emerged as an important health problem for modern society, and more than one-third of Korean adults over 20 years of age are under normal stress. I feel a lot of stress. Stress varies in intensity depending on the individual's personality, hobbies, resolution, surroundings, and control, but most of the time comes with depression. Depression is a mental illness that is also caused by stress, with extreme consequences such as suicide, and is recognized as a very important disease due to high recurrence rate and rapid patient incidence. The cause of depression has been found to be a disorder of neurotransmitters such as adrenaline, dopamine or serotonin, and also involves brain damage such as atrophy of the hippocampus and inhibition of adult neurogenesis. Tricyclic antidepressant (TCA) is a representative antidepressant known at present. In particular, amitriptyline is widely prescribed in Korea, but has many problems such as various side effects. Fluoxetine, a selective serotonin re-uptake inhibitor (SSRI), was developed in the United States in the 80's to overcome the side effects of TCAs, significantly increasing drug compliance and reducing treatment failure rates. It was ranked seventh among the medicines sold. However, SSRI does not show a big difference compared to TCA in terms of effects, and there is a problem that drug interaction is severe.

In addition, the nervous system disturbance caused by stress is continuously induced, and since the current treatment method does not have any special measures for preventing recurrence after prescription of antidepressant drugs, the level of the administered drug is continuously lowered. Therefore, it is important to develop a substance that has an excellent effect on the correction of stress-induced neuronal apoptosis or neurotransmitter system. In addition, there is a tendency to avoid visiting a treatment institution for the treatment of depression due to stress, or to overlook the occurrence of serotonin nervous system disturbance and brain damage caused by stress, so that a functional food having a function to treat stress depression is actually used. The need is very large.

Already, many countries in the world are devoting their efforts to prevent and treat neurodegenerative diseases. For example, US Pat. No. 6,020,127 discloses a gene encoding a neuronal apoptosis inhibitory protein on human chromosome 5q13, and US Pat. No. 6,288,089 No. discloses pyridyl imidazoles that can treat neurodegenerative diseases by inhibiting apoptosis of dopamine neurons.

Throughout this specification, numerous papers and patent documents are referenced and their citations are indicated in parentheses. The disclosures of cited papers and patent documents are incorporated herein by reference in their entirety, so that the level of the technical field to which the present invention belongs and the gist of the present invention are more clearly explained.

The present inventors have completed the present invention by confirming that silk peptides derived from natural products exert the above-described effects as a result of earnest research efforts to develop substances effective for the prevention or treatment of cerebral neurological diseases.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cerebral neurological diseases.

Another object of the present invention to provide a functional food composition for the prevention or treatment of cerebral neurological diseases.

Other objects and advantages of the present invention will be more clearly understood through the following detailed description and drawings.

According to one aspect of the invention, the invention provides a pharmaceutical composition comprising (a) a pharmaceutically effective amount of a silk peptide produced by the degradation of a silk protein; And (b) provides a pharmaceutical composition for the prevention or treatment of cerebral neurological diseases comprising a pharmaceutically acceptable carrier.

According to another aspect of the invention, the present invention provides a functional food composition for the prevention or treatment of cerebral neurological diseases, comprising as an active ingredient a silk peptide produced by the degradation of silk protein.

Silk peptides used as active ingredients in the compositions of the present invention are derived from silk. As used herein, the term "silk" refers to a fiber made by silkworm insects, and preferably means a silkworm sanded by a silkworm (Bombyx Mori).

The silk protein used as a raw material of the peptide of the present invention is silk sericin or silk fibroin. Silk protein is composed of fibroin and sericin, fibroin and sericin are present on average 75% and 25%, respectively. Fibroin proteins have exceptionally high glycine and alanine contents, while sericin has a completely different amino acid composition from fibroin, with serine accounting for more than one third of the total constituent amino acids. According to recent studies, silk fibroin has a structure in which H-chain (350 kDa) and L-chain (26 kDa) are SS-bonded, and glycoprotein P25 (30 kDa) is non-covalently linked to the two chains. It was identified as a large protein of 2.3 BG101a each having a mole composition of 6: 6: 1, and this structure results in a block-type polymer property in which crystalline and amorphous regions are continuously exchanged.

According to a preferred embodiment of the invention, the silk protein used is silk fibroin.

According to a preferred embodiment of the invention, the degradation of the silk protein is achieved by (a) degradation in calcium salt solution, (b) acid hydrolysis, (c) hydrolysis by enzymes, or (d) a combination of the above methods. It may be implemented, but is not limited thereto. The weight average molecular weight of the silk peptides produced by decomposition by the above-described method shows a value of about 200-100,000.

The decomposition in the (a) calcium salt solution is usually used calcium chloride, CaCl ₂ ethanol. By the decomposition in the calcium salt solution, silk fibroin is decomposed into a relatively high molecular peptide having a molecular weight of about 25,000-50,000. Such high molecular weight peptides are suitable for cosmetic ingredients because of their high molecular weight, but are not suitable for food and pharmaceuticals because of their low absorption in vivo.

The (b) acid hydrolysis is usually carried out using an inorganic acid (eg hydrochloric acid), the strong acid is usually used, and the weight average molecular weight of the obtained peptide is about 200-10,000. This decomposition method has the advantage of forming a peptide having a small molecular weight, but has a problem that it is difficult to obtain a peptide having a suitable molecular weight. According to a more preferred embodiment of the invention, the weight average molecular weight of the silk peptide obtained by acid hydrolysis is 200-3,000, most preferably 300-1,500. Peptides of the invention obtained by acid hydrolysis exhibit substantially 100% solubility in water and very low solubility in organic solvents such as ethanol, methanol, acetone and dimethylformamide.

Hydrolysis by the enzyme (c) is carried out using a proteolytic enzyme. Preferably, the silk average so that the weight average molecular weight of the finally obtained silk peptide is in the range of 200-15,000, more preferably 200-4,000, even more preferably 300-2,000, and most preferably 300-1,500. It is carried out using enzymes or combinations of enzymes that can degrade proteins.

The degrading enzymes act specifically or nonspecifically on amino acid sequences to catalyze hydrolysis. The degrading enzymes are trypsin, pepsin, alkalase, thermoase, flavozyme, sumyzyme, protamex and protein (protamex). protin) and the like, but is not limited thereto. According to a more preferred embodiment of the present invention, the enzyme used to obtain the silk peptide of the present invention is an enzyme that specifically decomposes an amino acid sequence, and according to a still more preferred embodiment, a thermoase, flavozyme, sumizyme and Combination of these.

In the case of decomposition by the combination (d), the decomposition is first performed by calcium salt or by hydrolysis by weak acid or alkali, and then secondly hydrolyzed by enzyme. More preferably, silk peptides are obtained by firstly decomposing by calcium salt and secondly by thermoase, flavozyme, sumizyme or a combination thereof. The weight average molecular weight of the silk peptide obtained by such a combinatorial method is 200-15,000, preferably 200-4,000, more preferably 300-2,000, and most preferably 400-1,200. By this method the peptide of the present invention exhibits substantially 100% solubility in water and very low solubility in organic solvents such as ethanol, methanol, acetone and dimethylformamide.

Preferred peptides in the present invention include (a) peptides obtained through acid hydrolysis with silk fibroin as a substrate; And (b) Calcium salts are primarily degraded using silk fibroin as a substrate, and trypsin, pepsin, alcalase, thermoase, flavozyme, sumime or combinations thereof. It is a peptide obtained by digestion, and more preferred peptides are (b) calcium salt degradation with silk fibroin as a substrate, trypsin, pepsin, alcalase, thermooa And peptides obtained by digestion with flavozyme, sumizyme or a combination thereof.

The composition of the present invention comprising such a silk peptide shows efficacy in the prevention or treatment of neurological diseases. This action of the present invention is generally shown through the protective action of nerve cells. As used herein, the term "nerve cell" includes neurons, nerve support cells, glia, Schumann cells, and the like, which form structures such as the central nervous system, the brain, the brain stem, the spinal cord, the junction of the central nervous system and the peripheral nervous system, and the like. As used herein, the term "protection of nerve cells" refers to the action of reducing or ameliorating neuronal insults, or of protecting or restoring nerve cells damaged by neuronal insults. In addition, the term "neural insult" herein means damage to nerve cells or nerve tissues caused by various causes (eg, metabolic causes, toxic causes, neurotoxic causes and chemical causes, etc.).

In the present specification, the neurological disease means all diseases caused by the above-described damage of nerve cells or nerve tissues, and particularly the neurological disease means all diseases caused by the damage of nerve cells, glial cells or nerve tissues of the brain. .

Specific examples of diseases to which the composition of the present invention may be applied include, but are not limited to, neurodegenerative diseases, diseases caused by ischemia or reperfusion, and mental diseases. More specifically, neurodegenerative diseases such as dementia, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis; Diseases caused by damage to nerve cells following ischemia or reperfusion, such as stroke (particularly ischemic stroke); And (c) can be used for the prevention or treatment of mental disorders such as schizophrenia, depression, and post-traumatic stress disorder.

The compositions of the present invention are particularly useful for the prevention or treatment of diseases (eg stroke) caused by damage to nerve cells following ischemia or reperfusion.

Such efficacy of the compositions of the invention is largely shown through the neuronal protective action of the silk peptides of the invention. The protective action of nerve cells by the silk peptide of the present invention can be exerted through a variety of mechanisms, for example, by suppressing the death of nerve cells, the death of these neurons necrosis (necrosis) and apoptosis of nerve cells (apotosis). In the case of inhibiting apoptosis of nerve cells, one of the targets of the silk peptide of the present invention is caspase (caspase, Guy et. Al., Cell 91: 443-446 (1987)), which inhibits the activity of the enzyme to apoptosis Will be suppressed.

The silk peptide of the present invention shows excellent efficacy in improving cognitive function. Preferably, the silk peptides of the present invention exhibit excellent efficacy in ameliorating or preventing deterioration of cognitive function accompanying the neurological diseases described above. In particular, the silk peptides of the present invention inhibit the decrease in the amount of acetylcholine in the brain to achieve an improvement in cognitive function. In addition, the silk peptide of the present invention inhibits the death of nerve cells caused by stroke to prevent impairment of cognitive function. According to a preferred embodiment of the present invention, the improvement of the cognitive function exerted by the silk peptide of the present invention is the improvement of learning ability and / or memory ability.

Acetylcholine is a neurotransmitter that is projected from the basal ganglia of the brain to the cerebral cortex and hippocampus and is very important for normal knowledge function (Richter et. Al., Life Sci. 19; 26 (20): 1683-9 (1980) ). In particular, it is known that learning and memory can be altered by drugs acting on the acetylcholine system. Examination of death from Alzheimer's dementia has found that in all cases, acetylcholinergic neurons of the basal ganglia projecting into the cerebral cortex are largely damaged. Recently, it has been known to increase the concentration of acetylcholine to prevent the improvement of cognitive ability and progression of dementia, which is excellent in the treatment and prevention of dementia, and has been using choline agonists or choline esterase inhibitors in patients. Drugs developed to date include lecithin as an acetylcholine precursor, RS-86 and nicotine as a receptor agonist, and are approved by the FDA as an acetylcholinesterase inhibitor. Tacrine and Aricept, which are recently used in the market, are recently used in Korea, but the effects are still controversial due to their temporary, weak and severe toxicity. However, the silk peptide of the present invention is extremely useful as a medicament or a functional food for improving cognitive function since the toxicity to the human body is extremely weak.

As used herein, the term "prevention" means that it has not been diagnosed as having a disease or condition but inhibits the occurrence of the disease or condition in an animal that tends to be prone to such disease or condition. As used herein, the term “treatment” means (a) inhibiting the development of a disease or condition; (b) alleviation of the disease or condition; And (c) elimination of the disease or condition.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation of carbohydrate compounds, such as lactose, amylose, dextrose, sucrose, sorbitol, mannitol, starch, cellulose, and the like. ), Acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, salt solution, alcohol, gum arabic, vegetable oil (e.g. corn oil, cotton Seed oil, soy milk, olive oil, coconut oil), polyethylene glycol, methyl cellulose, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention further includes, but is not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, or the like.

Suitable dosages of the pharmaceutical compositions of the invention vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to reaction, Usually a skilled practitioner can easily determine and prescribe a dosage effective for the desired treatment or prophylaxis. According to a preferred embodiment of the invention, a suitable dosage is 50 mg-10 g once daily on an adult basis.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

On the other hand, the functional food composition of the present invention includes components that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients and seasonings. For example, when prepared with a drink, it may further include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, tofu extract, jujube extract, licorice extract, etc., in addition to the silk peptide of the present invention. In view of easy access to food, the food of the present invention is very useful for the treatment or prevention of neurological diseases, for the treatment or prevention of diseases caused by oxidative stress, and for improving cognitive function.

The composition of the present invention not only exhibits various effects as described above, but also contains a natural silk peptide as an active ingredient, so that the side effects on the human body are extremely less than that of chemical synthetic medicines.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it is to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. Will be self-evident.

Example I Preparation of Functional Silk Peptides BG201 and BG101

Example I-1 Purification of Pure Silk Fibroin

The silk protein used in the present invention was used by refining the silkworm cocoon obtained by breeding Gajam ( Bombyx mori ) into the messenger upper lobe. First, the pupa remaining in the cocoon was removed to remove sericin, and 150 g of silkworm cocoon was added to a solution containing 8 L of water, 0.45 g of sodium carbonate, and 0.75 g of Marcel soap, followed by boiling treatment twice for 40 minutes. Thereafter, the mixture is boiled again twice with an appropriate amount of water for about 20 minutes, washed three times with water, and dried to dissolve the water-soluble sericin. The reduced weight at this time was 37 g (about 25% reduction). After washing with water, the product was dried naturally to obtain pure fibroin protein.

Example I-2 Preparation of Silk Peptide BG101

Based on 35 g of fibroin obtained by the method of Example I-1, calcium chloride (CaCl ₂ , primary, Mw = 110.99) -H ₂ O-ethanol (1: 8: 2 molar content) was added and 90 ° C. After reacting for 5 hours and dissolving, the foreign material was completely filtered with gauze and nonwoven fabric, and then diluted twice with distilled water. Neutral salts were removed by gel filtration using a GradiFac system (Pharmacia Biotech, Sephadex G-25 Media, HiLoad P-50 Pump UV-1 Monitor, Sweden) 10 cm in diameter and 1 m in length. Subsequently, 1% of proteolytic enzyme mixed with Flavorzyme (Sumizyme; NOVA, United States) (1: 1) was added to the obtained fibroin solution, and hydrolysis was performed at 55 ° C for 5 hours. . Then, heat treatment was performed at 100 ° C. for 5-10 minutes to remove the activity of the enzyme and prepare a powder by lyophilization, which was named “BG101”.

Example I-3 Preparation of Silk Peptide BG201

Based on 113 g of fibroin obtained by the method of Example I-1, 3,400 ml of 25% aqueous hydrochloric acid solution was added to perform hydrolysis at 110 ° C. for 12 hours. It was adjusted to pH 5.0-5.5 with 4 M aqueous sodium hydroxide solution. Subsequently, after removing the residue by filtration of paper, passing through a filter filled with activated charcoal, and putting 1,000 ml into the sample bath in the electrodialysis apparatus, an electric desalination was performed using a device in which voltage and current of 15 V and 20 mA were automatically adjusted. Was carried out. The powder was then produced by a spray dryer, which was named "BG201".

Example II Analysis of Silk Peptides

Example II-1 Molecular Weight Analysis

Absolute molecular weight was calculated by gel permeation chromatography on BG101 and BG201 prepared in Example I above. Absolute molecular weight distribution was automatically determined from the refractive index (RI), light scattering (LS), diffraction pressure, and ultraviolet (280 nm) absorbance values of 0.2 N NaNO ₃ as a buffer solution. A GPC system (Viscoteck, United States) device was used. Reproducibility was confirmed using polyethylene oxide (PEO, Mw = 110,000) as a standard sample. As a result, the weight average molecular weight (Mw) of BG101 was 1070, and BG201 was found to be 850.

Example II-2: Solubility Analysis According to Solvent

The solubility of BG101 and BG201 prepared in Example I was dissolved in ton and dimethylformamide using several solvents and the solubility (unit%) was measured, and the results are shown in Table 1. As can be seen from Table 1, the solubility of BG101 and BG201 powder was completely dissolved in distilled water, but solubility was remarkably inferior in organic solvents such as ethanol and methanol.

menstruum Distilled water ethanol Methanol Acetone Dimethylformamide BG101 100 20 45 30 50 BG201 100 30 50 32 50

Example II-3 Solubility Analysis According to pH

The solubility of BG101 and BG201 prepared in Example I was measured while changing the pH. 0.1 g of silk peptide powder was dissolved in 10 ml of distilled water and the solubility was measured. The pH of distilled water was adjusted to pH 3, 5, 7, 9 and 11 with 0.1 N hydrochloric acid and sodium hydroxide. As a result, as shown in Table 2, the silk peptide was well dissolved regardless of the pH of the solvent.

pH 3 5 7 9 11 BG101 100 100 100 100 100 BG201 100 100 100 100 100

Example II-4: Amino Acid Composition Analysis

The amino acid composition of BG101 and BG201 manufactured in Example I was analyzed. After 0.05% of each sample was taken, 1 mL of 6N aqueous hydrochloric acid solution was added to the mixture, followed by nitrogen treatment. After complete evaporation of hydrochloric acid, amino acid analysis was performed using a fully automatic amino acid machinery (Amersham Pharmacia Biotech, Model: Biochrom 20 Plus, Sweden) after dilution in pH 2.2 loading buffer. The obtained amino acid composition (unit: mol%) is shown in Table 3.

 amino acid BG201 BG101 Gly 43.2 42.46 Ala 27.2 26.64 Ser 15.94 11.24 Tyr 2.00 4.41 Val - 2.02 Asp 1.45 2.11 Glu 1.61 1.68 Thr 0.6 - Met 0.15 0.11 Ile 0.98 0.65 Leu 0.71 0.57 Phe 1.00 0.76 His 0.52 0.52 Lys 0.38 1.04 Arg 1.01 1.04 Sum 96.21 95.25

As can be seen from the results in Table 3, both samples do not show a big difference in the content of amino acids. However, as described below, BG201 and BG101 have some differences in the effects and efficacy of in vivo or in vitro , and these results indicate that the structure of the finally formed silk peptide differs from each other according to the method of preparing the silk peptide. Means that.

Experimental Example I: Confirmation of brain neuron cell protective action and antioxidant activity

In order to determine the effect of the silk peptide prepared in the above example on brain neurons, the following experiment was conducted:

Experimental Example I-1: Selection of Neurons and Cell Culture

Primary cultures of neurons were collected using Okuda et al. (Okuda S. et al., Neuroscience 63 (3): 691-9 (1994)) to extract the striatum of mouse embryos at 15 days of gestation (E15). Treated with 0.25% trypsin and 0.01% DNase I enzyme, separated into individual cells, followed by further pipetting, spreading on a PEI-coated plate at a density of 1 × 10 ⁵ cells / cm 2, and after 3 days, glial cells with ara-C. The culture of these was inhibited to obtain pure neurons. In addition, neural origin cell lines SK-N-SH or SHS-Y5Y (ATCC, United States of America) were spread on a PEI-coated plate at a density of 80% and cultured in a medium supplemented with 10% FBS in DMEM or RPMI. Amyloid β (Aβ), 6-hydroxydopamine (6-hydroxydopamine, 6-OHDA), ceramide, H ₂ O ₂ or 3-hydroxykynurenine (3-hydroxykynurenine, 3-HK) as in the following experimental example When treated, pretreatment with low serum medium (containing 1% FBS) for 2 hours followed by a suitable time.

Experimental Example I-2: MTT Reduction Experiment (Checking Cell Viability)

Previously reported methods for examining the effects of BG101 and BG201 on neuronal cell death (Shearman et al., Proc. Natl. Acad. Sci. 91 (4): 1470-4 (1994), Shearman et al. , J. Neurochem. 65 (1): 218-27 (1995), and Kaneko et al., J. Neurochem. 65 (6): 2585-93 (1995)) with a slight modification to the MTT reduction experiment as follows: 10 pM of BG101 or BG201 was treated for 6 hours in the selected and cultured neurons in the above experiments, followed by 10 μM Aβ, 10 μM 6-OHDA, 10 μM ceramide, 300 μM H ₂ O ₂ , or 250 μM 3-HK was added. The added material induces cell death by cytotoxicity, which induces about 50% cell death within 36 hours at the indicated concentration.

Then incubated for 48 hours at 37 ° C. and 5% CO ₂ incubator, followed by 3- (4,5-dimailthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT: Sigma, US) ) Solution was added to each vessel to a final concentration of 0.5 mg / ml, followed by further incubation for 4 and a half hours.

Formazan precipitate formed by the reduction of MTT was dissolved in a dissolution solution (0.1 N HCl in anhydrous isopropanol) and then absorbance at 570 nm was measured using an ELISA reader (Molecular Devices, United States). The values of each sample were expressed as relative values with the control value added with only the solvent used as 100% viability and the value when the cell was completely destroyed by 0.9% Triton X-100 as 0% viability (see 1a and 1b). In FIG. 1A and FIG. 1B, each measured value is expressed as mean ± standard deviation (SD). 1A and 1B, it could be seen that BG101 and BG201 of the present invention effectively inhibit neuronal cell death (P <0.05).

On the other hand, in order to further confirm the above-described results, cell counts and total cell numbers in which apoptosis occurred through trypan blue or crystal violet staining were measured by blood cell counters, and thus cell survival as the number of viable cells for the total cell number. Sex was measured, and as a result, the effect of inhibiting apoptosis of BG101 and BG201 almost the same level as the results of the accompanying Figures 1a and 1b was confirmed. In addition, this apoptosis had apoptosis type, which is a cell suicide process, which observed DNA shape using Hoechst staining, which observes the firmness and cell morphology of the following example By reconfirming it.

Experimental Example Ⅰ-3 Hoechst staining

BG101 or BG201 was treated for 6 hours by the same method as in Example I-2, and Aβ, ceramide, H ₂ O ₂ , 6-OHDA or 3-HK at the same concentration as in Example I-2 was cultured in the cultured cells. Was treated. Then, fixed with 4% paraformaldehyde (in PBS, pH 7.4) for 15 minutes, washed with PBS, and then stained each treated cell for 5 minutes with 8 μg / ml paste die 33258 solution (Sigma, USA). It was. It was then washed twice with distilled water, mounted with glycerol / PBS (9: 1) and observed with a fluorescence microscope (Olympus Microscope, Japan) (see Figures 2A and 2B). As a result, the cells treated only with the apoptosis-inducing substance in FIGS. 2A and 2B were able to confirm typical apoptosis morphology such as condensation or nucleic acid fragmentation of chromosomes. On the other hand, when BG101 and BG201 were pretreated and treated with insult, it was confirmed that chromosome condensation and nucleic acid fragmentation were effectively inhibited compared to the experimental group treated with insult alone. Marked as mock is a control, showing the appearance of normal nuclei. As can be seen in Figure 2, BG101 and BG201 of the present invention was found to effectively suppress apoptosis, it was possible to reconfirm the reliability of the results of Experimental Example I-2.

Experimental Example I-4: Caspase Activity Experiment

In order to determine whether the effect of inhibiting apoptosis by BG101 or BG201 in the results of Experimental Examples I-2 and I-3 was due to the inhibition of caspase activity in the molecular mechanism of its operation, the following experiment was performed. First, BG101 or BG201 was treated for 6 hours by the same method as in Experimental Example I-2 above, and amyloid β, 6-OHDA, ceramide at the same concentration as described in Experimental Example I-2 was applied to the cultured cells. , H ₂ O ₂ , FeSO ₄ or 3-HK. 10 × 10 ⁶ cells per P100 plate were disrupted with 1 ml of cell lysis buffer (50 mM Tris-Hcl, 0.03% IGEPAL, 1 mM DTT, pH 7.5) to obtain cell lysate. Then, 50 μl of cell lysate was loaded with Ac-DEVD-AMC (Enzyme Systems Products, Canada), a fluorogenic substrate of caspase in HEPES buffer (40 mM HEPES, pH 7.5, 20% glycerol, 4 mM DTT). 500 μM was added and reacted at 37 ° C. for 1 hour. In addition, a sample prepared with 10 μM pretreatment of zVAD-FMK (Enzyme Systems Products, ESP, Canada), which is a broad-range (Pan) -caspase inhibitor, was prepared to serve as a positive control for caspase activity inhibition.

The degree of fluorescence produced when the substrate was cleaved by caspase was then measured by a fluorimeter (Perkin-Elmer Luminometer: excitation wavelength 380 nm, emission wavelength 420-460 nm). In addition, the cleaved FMK was relatively quantified by obtaining a standard curve using the degree of fluorescence (see FIGS. 3A to 3B). In FIG. 3A and FIG. 3B, each measured value represents the mean ± standard deviation (P <0.05). As can be seen from the figure, it can be seen that BG101 or BG201 of the present invention inhibits caspase activity induced by a substance causing apoptosis. In conclusion, it can be seen that the apoptosis inhibitory effect of BG101 or BG201 of the present invention is related to the inhibition of caspase activity.

Experimental Example I-5: Quantitative Detection of Active Group Oxygen in Cells

Active group oxygen is not only the main cause of aging but also the direct and indirect causes of various diseases. Thus, the effect of the silk peptide of the present invention on the active group oxygen was investigated as follows:

Treatment with BG101 or BG201 for 6 hours in the same manner as in Example I-2, and Aβ, 6-OHDA, ceramide, H ₂ O ₂ , FeSO at the same concentration as described in Experimental Example I-2 ₄ or 3-HK was treated. Then, 10 μM of DCFDA (6-carboxy) dissolved in cultured cells in HCSS buffer (20 mM HEPES, 2.3 mM CaCl ₂ , 120 mM NaCl, 10 mM NaOH, 5 mM KCl, 1.6 mM MgCl ₂ , 15 mM glucose) -2 ', 7'-dichloro-dihydrofluoresceine diacetate, dicarboxym-ethylester) and suspension aid 2% Pluronic F-127 were treated for 30 minutes at 37 ℃. DCF fluorescence by intracellular activator oxygen was observed on an Olympus IX70 inverted microscope equipped with a mercury lamp fluorescence accessory at room temperature (excitation wavelength 488 nm, emission wavelength 510 nm), captured with a CCD camera, and then using the NIH Image 1.65 program. Image analysis was performed (see FIGS. 4A and 4B).

In FIG. 4A and FIG. 4B, the moiety shows only the solvent used. As can be seen in Figure 4, it can be seen that the active group oxygen is generated by Aβ, 6-OHDA, ceramide, H ₂ O ₂ , FeSO ₄ or 3-HK. On the other hand, when BG101 or BG201 is pretreated, it can be seen that it significantly inhibits the activator oxygen increased by the respective apoptosis-inducing substances.

Therefore, BG101 or ED of the present invention may act as an antioxidant in vivo by inhibiting the formation of activator oxygen caused by amyloid β, 6-OHDA, ceramide, H ₂ O ₂ , FeSO ₄ or 3-HK. It can be seen that.

Experimental Example II: Ischemic Animal Model Experiment

In order to determine the effect of the silk peptide BG101 or BG201 of the present invention prepared in the above example on the cerebral infarction area due to ischemia, the following experiment was performed:

Experimental Example II-1: Local Ischemia Induction Animal Model

A. Construction of Drug Treatment and Local Ischemia-Induced Animal Models

Local ischemia-induced animal models were constructed using Sprague-Dawley rats weighing 200-250 g. The drug was orally administered 1 g / kg of BG101 once 1 hour before ischemia induction or once 1 hour after ischemia induction (oral administration group, n = 6), and the ischemia control group (n = 6) was administered at the same dose as the drug treatment group. Physiological saline was administered. The animals were anesthetized by intramuscular injection of ketamine 30-40 mg / kg, and then a skin incision was made at the neck in the supine position, followed by a common carotid artery, an external carotid artery, and an internal carotid. artery) and isolated from surrounding tissues. First, the upper upper thyroid artery, the larynx and the artery, which is the branch of the external carotid artery, are electrocauterized. Was inserted into the internal carotid artery through the external carotid artery, and then placed in a carotid artery with a nylon thread entering 16-18 mm to occlude the middle cerebral artery starter.

B. Experimental effect of reducing cerebral infarction by ischemia

Six hours after induction of ischemia, the animals were guillotine and brains were extracted. The extracted brain was sectioned at 2 mm intervals from the anterior pole, reacted with 2% triphenyl tetrazolium chloride (TTC, Sigma, United States) solution at 37 ° C. for 30 minutes, and then 4% paraform. Fixed in aldehyde (Sigma, United States) solution. Photographs of the stained tissue sections (see FIGS. 5A and 5D) were used in the MCID image processing system (Imaging Research Inc., Canada) to show that cerebral infarction developed and turned white, unlike the normal region stained in red. The area of was measured, and the average value was calculated after calculating the ratio of cerebral infarction to the area of the whole brain slices (see FIG. 5B). In addition, the ratio of the volume occupied by the cerebral infarction area to the total volume in each brain section was measured (see FIG. 5C).

As can be seen in Figure 5a, the silk peptide of the present invention is reducing the cerebral infarction site due to ischemia to a significant level relative to the control. In the comparison of each brain section in Figure 5b it was observed that the effect of reducing cerebral infarction is not centered on a specific site but appeared overall (P <0.05). In addition, as a result of measuring the volume ratio of cerebral infarction to the volume of the entire brain section in Figure 5c, BG101 pretreatment showed a significant effect on the reduction of cerebral infarction area by local ischemia. In FIG. 5B and FIG. 5C, each measured value represents an average ± standard error. In addition, the silk peptide of the present invention treated after ischemia induction as shown in Figure 5d significantly reduced the damage site by cerebral infarction compared to the control.

  Therefore, it can be seen that the silk peptide of the present invention can be usefully used in functional foods and drugs related to the prevention and treatment of ischemic stroke.

Experimental Example II-2: Local Ischemia-Reperfusion Induced Animal Model

A. Drug Treatment and Local Ischemia-Reperfusion Induction Animal Model Construction

Anesthetized by intramuscular injection of ketamine 30-40 mg / kg in male rats of 200-250 g, and then cut the skin in the neck at the supine position and pull the SCM muscles to find carotid, internal carotid and external carotid arteries I was. First, the upper upper thyroid artery, the larynx and the artery, which is the branch of the external carotid artery, are electrocatheterized. The catheter, which is the branch of the internal carotid artery, is electrocauterized. After insertion into the carotid artery, a nylon thread was placed in the carotid artery with 16-18 mm of entry. The skin incision was reconstructed and recovered naturally under anesthesia. After 1 hour, the inserted nylon thread was removed to reperfusion blood. Dosage of BG101 or BG201 was orally administered 7 days and 7 times a day for 6 days from the next day after ischemia-reperfusion at a dose of 1 g / kg, respectively. As an experimental control group, an ischemic control group administered with the same number of times as a normal control group and a drug treatment group and a dose of physiological saline was used. Passive avoidance test and 8-arm maze test were performed 7 days after induction of local ischemia-reperfusion.

B. Learning and Memory Tests

end. Passive avoidance test

A manual avoidance test was conducted and the experimental method was as follows: An automated shuttle box (Model PACS-30, Columbus Instruments International Company) was used as a test instrument. The shuttle box is divided into two rooms of the same size (19 "L x 9" W x 10.875 "H) by a partition door (3" L x 2.625 "W), and the floor is provided with a current flow. Each room was illuminated by a 20W bulb placed on a hinged plexiglass lid. White paper can enter a dark room through a partition door. The experiment was conducted in a room with less than 60 dB noise and low light. If you put the white paper into the room where the light is initially turned on and open the partition door, you will search the room around and move to the dark room, which will automatically close and turn off the light. This training process was continued until the white paper entered the dark room within 20 seconds. After 24 hours of training, the white paper was put back into the lighted room, and when it entered the dark room, the partition door was closed and the current (1 mA) flowed for 3 seconds at the bottom of the shuttle box. Seven days after local ischemia-reperfusion induction, the white paper was placed in the lit room of the shuttle box and the time to crossing over to the dark room was measured. The time which does not enter the dark room at most is performed as 5 minutes (refer FIG. 5E).

In FIG. 5E, the change in latency time indicates the decline and recovery of memory, which means that the memory capacity is increased as the time increases. There was no change in latency in the sham operated control group, and latency significantly decreased in the ischemic control group administered with solvent only (P <0.05). On the other hand, in the group repeatedly administered BG101 or BG201, the incubation period showed a tendency to recover significantly to the normal level (P <0.05). In particular, the recovery of the incubation time in the BG101 administration group was greater than that in the BG201 administration group, indicating that the efficacy of BG101 was better. In FIG. 5E, each measured value represents an average ± standard error.

I. 8-arm radial maze test

An eight-way radial maze (Etho Vision, The Netherlands) was used as the experimental instrument. Eight arms (60 cm long and 12 cm wide) emerge from the octagonal center (34 cm radius), 45 cm from the floor, and the arm and center area are walls (40 cm high). At the end of each arm is provided a food cup. Sunflower seeds were used as a reward. It was darkened around the maze, illuminated with 50W light and monitored by a video camera. Learning training was provided by reducing feed to 80% one week before. After placing the manoes in the labyrinth three times a day for 30 minutes, they were allowed to acclimate for three days, and then each dog was placed in the labyrinth and measured the number and time of visits to each cancer until the sunflower seeds in the food cup were eaten. I trained until I reached it within two times. The number of times of entering the cancer which had already entered was made into the number of errors.

The trained man was made with an animal model of cerebral ischemia by the following method, and orally administered BG101 or BG201 at a dose of 1g / kg once a day for 7 days and the number of errors and latency time from 5 days thereafter were compared with the control group. .

8-arm using a cerebral ischemic animal model created by temporarily blocking the middle cerebral artery to detect whether BG101 or BG201 protects the spatial memory damage caused by cerebral ischemia that cannot be detected by manual avoidance measurement. Maze test was performed. As a result of the maze test for 5 days from 1 week after administration, the number of errors from 1 day to 5 days after administration showed little difference from the control group (FIG. 5f). From day 3, the number of errors in the BG101 or BG201 administration group significantly decreased compared to the control group in which only the solvent was administered. In particular, the number of errors in the BG101 administration group tended to decrease more than the error in the BG201 administration group. By the last day of the measurement, the number of errors in the BG101 or BG201-administered group decreased to the error level in the control group, while the number of errors in the solvent-only group did not recover (*, P <0.05). As a result, spatial memory is decreased due to the temporary obstruction of the middle cerebral artery, but this loss of spatial memory is suppressed by repeated administration of BG101 or BG201, so that BG101 or BG201 is effective in suppressing learning and memory loss due to cerebral ischemia. This was confirmed. In FIG. 5F, each measured value represents a mean ± standard deviation (SD).

MCA was closed for 1 hour to cause ischemia. After 7 days, learning and memory were measured using passive avoidance test and 8-arm maze. It was also confirmed that ischemia causes impaired cognitive function. After seven doses, the researchers found for the first time that there was an effect of restoring lost memory.

C. Hematoxylin & Eosin (H & E) staining in hippocampus

Many studies have reported that memory ischemia-induced memory loss and learning ability in experimental animals are closely related to hippocampal damage (Hodges et al., Neuroscience, 72 (4), 959-88, 1996). Therefore, the efficacy of the present invention was measured in specific regions of the hippocampus (CA1, CA2, DG).

end. Organization Preparation

Experimental Example II-2B After the measurement of the experimental animal with chloral hydrate (chloral hydrate: 400 mg / ㎖) and anesthetized 200 M of 0.1 M phosphate buffered saline (PBS, pH 7.4) through the heart through blood vessels The components were removed and 250-300 mL of fixative (4% paraformaldehyde / PBS) was perfused. The brains were extracted and post-fixed at 4 ° C. for 15-24 hours with the same fixative. After fixation of the fixative solution with PBS, 10%, 20%, and 30% sucrose solutions were sequentially infiltrated to prevent ice crystals from freezing. After embedding the brain tissue into embedding liquid and rapidly cooling in liquid isopentane, which was pre-cooled with liquid nitrogen, and then subjected to continuous coronal section with a thickness of 10 μm using a cryostat (Cryostat; Reichert Frigocut model 2000) coated with gelatin Immediately attached to the slide glass and dried at room temperature for 1 hour and stored at -70 ℃.

I. H & E Dyeing

The stored slide glass was taken out, washed with distilled water, and reacted with hematoxylin for 10 minutes. After the reaction, the slide glass was treated with 70% ethanol containing 1% Hcl to remove excess hematoxylin and fixed with ammonia. Again, the tissue is dyed for about 20 seconds using eosin, and then washed with distilled water. After dehydration using 70%, 90% and 100% ethanol, treated with xylene and sealed with a cover glass and Canadian balsam and observed with an optical microscope.

All. H & E staining analysis result

 As a result of H & E staining in the hippocampus by BG101, the eosin deposited on the nucleus, which is typical of apoptosis in the ischemia-induced control group, is expressed in the whole part of the hippocampus (CA1, CA2, DG). (Figure 5g, arrow). On the other hand, in the experimental group (BG101 treatment group) to which the silk peptide of the present invention was administered, it was observed that the cells of the normal form were significantly increased than the cells of the eosinophil type compared to the experimental control group.

These results suggest that the exact mechanism of action should be further studied, but BG101 or BG201 has new effects to prevent neuronal necrosis and cognitive impairment that may be caused by stroke. Available as

Experimental Example III: Parkinsonism Animal Model Experiment

Dopamine hydroxide (6-hydroxydopamine, 6-OHDA), widely used as a Parkinson's disease-inducing drug, is absorbed through the cell membrane of catecholaminergic neurons and selectively toxic to catecholaminergic neurons. Animal models in which dopamine neurons are destroyed by injecting dopamine hydroxide into the unilateral brain can be used as a control, and can be compared to spontaneous movement and drug-induced rotational movement.

In order to investigate the effects of silkworm silk peptides BG101 and BG201 of the present invention on Parkinson's disease, a type of degenerative brain disease, the following experiments were conducted:

Experimental Example III-1 Construction of an Animal Parkinson's Disease Animal Model

The method of Joo et al. (Joo WS et al., Neuroreport, 9 (18), 4123-4126, 1998) that injects dopamine hydroxide into the unilateral striatum of the white paper brain to cause progressive degeneration of dopamine neurons. Animal models were constructed as follows:

First, anesthesia was injected by intraperitoneally injecting 3 ml / kg of equithesin into male white Sprague-Dawley rats weighing 200-250 g (Daehan Biolink, South Korea). Anesthetized animals were perforated with a skull using a stereotaxic frame (Deotrotaxic frame, David Kopf, United States of America), 0.2 mg / ml ascorbic acid in the sham group, and the lesioned group and BG101 or BG201 The oral treated group was injected with dopamine hydroxide (20 μg / 5 μl free base in 0.2 mg / ml ascorbic acid) into the right striatum using a Hamilton syringe (10 μl, 26G needle) at a rate of 1 μl per minute. (Paxinos et al., J. Neurosci. Methods. 3 (2), 129-149, 1980). The needle was left in place for 5 minutes after drug injection, the needle was removed at a rate of 1 mm per minute, and the incision site was closed. On the other hand, BG101 or BG201 was orally administered at two doses of 1 g / kg and 5 g / kg.

To determine the effects of dopamine hydroxide in the advanced Parkinson's disease animal model, 14 days after the lesion was produced, behavioral analysis was confirmed through behavioral analysis using apomorphine, the next day the animal was cervical distal, and the brain was extracted and used for the experiment. It was. To determine the protective effect of BG101 and BG201 on dopamine neurons, we measured lipid peroxidation, dopamine concentration in striatum using HPLC and immunohistochemical staining for tyrosine hydroxylase.

Experimental Example III-2: Unilateral Rotational Reaction with Apomorphine

In the Parkinson's disease animal model prepared with dopamine hydroxide, behavioral changes over time were subcutaneously injected with apomorphine (0.5 mg / kg) in the back neck at 14 days after lesion preparation and unilateral rotational response was measured for 60 minutes. (See FIG. 6).

In this experiment, when dopamine neurons are killed and the concentration of dopamine in the striatum is reduced, the hypersensitivity of the dopamine receptors in the striatum is induced. Animals use the principle of rotational movement in the opposite direction to the damaged side (Ungerstedt, Brain Res. 24, 485-493, 1970). Rotational motion was used by the automated rotometer described in the above paper by Ungerstedt, where each rotational speed is [net turns = non-lesional rotational-ipsilateral ].

As can be seen in Figure 6, the unidirectional net rotation of the normal control group administered 0.2 mg / ml ascorbic acid to the striatum did not show a significant change, but the unilateral net rotational speed was significantly increased in the lesion control group administered only hydroxide dopamine. (P <0.05). On the other hand, in the experimental group to which the silk peptide of the present invention was administered, the unilateral net rotational speed was significantly decreased compared to the control group, and the effect was also increased as the dose was increased (P <0.05). In FIG. 6, each measured value represents an average ± standard error.

Experimental Example III-3 Measurement of Dopamine and its Metabolites in Striatum

In order to determine the effect of BG101 and BG201 of the present invention on the concentration of dopamine neurotransmitter which is an important cause of Parkinson's disease, after administration of BG101 or BG201 in the same manner as in Experimental Example III-2, the lesion was made two weeks later. Later, dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) concentrations in the striatum were measured using high performance liquid chromatography (HPLC, Gilson, France).

A. Organization Preparation

Brain tissue was extracted immediately after sacrifice by cervical dislocation, and a tissue punch was obtained by taking a slice of 2 mm intervals based on the optic chiasma with a brain slicer (ZIVIC MILLER, United States of America). ), The black matter was separated. After separating the left and right cerebral hemispheres along the cerebral median fever on the cooled glass plate, the anterior part of the brain left after separation was separated and cut off only the striatum from the thalamus and cerebral cortex with the border of the corpus callosum. . This was rapidly frozen using dry ice and then stored at -70 ° C.

B. Analysis Method

To the frozen sample, cooled 0.1 M perchloric acid and 1 mM EDTA were added, and tissue homogenate was prepared by an ultrasonic crusher, and the supernatant was taken by centrifugation at 12,500 g for 20 minutes. The supernatant was filtered through nitrocellulose membrane filter paper (pup size 0.2 μm) and injected into HPLC. Separation conditions were used with a WATERS uBondapak ^TM C ₁₈ 3.9 x 300 mm column (particle size 10 μm), mobile phase 0.07 M sodium phosphate monobasic, 1 mM sodium octanesulfonic acid, 0.1 mM EDTA and 8% acetonitrile (pH 4.0) The solution was used at a flow rate of 0.7 ml / min. In order to measure the concentration of each substance, a certain amount of dihydroxybenzylamine was added as an internal standard to prepare a tissue equalizer, and the separated substances were detected by an electrochemical detector.

C. HPLC Measurement Results

The change in dopamine ratio (lesion side / non-lesion side,%) in striatum is shown in FIG. 7.

As shown in FIG. 7, the dopamine concentration in the striatum was significantly decreased in the control group administered only dopamine hydroxide compared to the Siamese control group (P <0.05). On the other hand, in the experimental group administered the silk peptide of the present invention it can be seen that the dopamine concentration significantly increased compared to the control group treated only with dopamine hydroxide, it can be seen that the effect is increased as the dose is increased (P <0.05). In FIG. 7, each measured value represents an average ± standard error.

Parkinson's disease is caused by a decrease in dopamine concentration in the striatum where these neurons are projected due to the loss of dopamine neurons located in the black matter. It is an important measure of the loss of dopamine nerve fibers in the striatum and the striatum. In fact, in patients with late Parkinson's disease, dopamine content in striatum is only about 10% of normal people. Therefore, the results of this experiment in which the reduction of the dopamine concentration is suppressed by the silk peptide of the present invention demonstrate physiological and biochemical protection effects on dopamine neurons.

Experimental Example III-4: Determination of Lipid Peroxidation in Striatum (Malondialdehyde Experiment)

In order to determine whether BG101 or BG201 of the present invention orally administered in Experimental Example III-2 inhibits lipid peroxidation production by antioxidant, BG101 or BG201 was administered in the same manner as in Experimental Example III-2 and the lesions were treated. After preparation, two weeks later, the amount of malondialdehyde (MDA) in the striatum was measured.

A. Organization Preparation

Take the striatum of experimental animals in the same manner used in Experimental Example III-3C-ga, Krebs-Ringer Buffer, NaCl 120 mM, KCl 4.8 mM, CaCl ₂ 1.3 mM, MgSO ₄ 1.2 mM, NaHCO ₃ 25 mM, Glucose 6 mM, pH 7.6) was added to prepare a tissue equalizer using an ultrasonic sonicator and used in the experiment.

B. Lipid Peroxidation Measurement

The concentration of thibarbituric acid reactive substances (TBARS), a measure of the degree of lipid peroxidation, was measured according to the method of Ohkawa et al. (Anal Biochem. 95 (2): 351-8 (1979)). Add 8.1% SDS, 20% acetic acid (pH 2.5), 0.8% Thiobarbituric acid (TBA) to a volume of tissue equalizer, and add BHT (2,6-di-t-butyl-p-) to inhibit autooxidation. cresol, 200 uM) was added and then reacted at 100 ° C for 30 minutes. After the reaction, the vessel was immersed in iced water and the temperature was rapidly lowered to stop the reaction. Then, the sample was measured at a wavelength of 532 nm with an absorbance photometer to calculate the concentration of TBARS.

The concentration of the MDA-TBA (Malonaldehyde-thiobarbituric acid) complex was determined by Lazzarino et al. (Lazzarino et al., Free. Radic. Biol. Med. 13 (5), 489-98, 1992) and Chirico (1987). Was measured. Inject the sample into a 5 uM Lichrosper 100 RP18 column (4.6 x 250 mm) and elution with a 65% KH ₂ PO ₄ (50 mM) / 15% methanol / 20% acetonitrile at a rate of 0.9 ml / min for UV / visible detector (Hewlett-Packard, Series 1050), and 1,1,3,3-tetramethoxypropane (prepared in ethanol / water 40:60, v / v) was used as a standard sample to identify peaks of the BG101A-TBA complex. .

C. Measurement Results

The ratio of MDA production in the striatum (lesional / non-lesional,%) is expressed as the relative concentration (%) of the MDA-TBA complex to the concentration of TBARS and the values are shown in FIG. 8. As can be seen in Figure 8, the degree of lipid peroxidation in the striatum was significantly increased in the control group administered only dopamine hydroxide compared to the Siamese control group (P <0.05). On the other hand, in the experimental group administered the silk peptide of the present invention it can be seen that the degree of lipid peroxidation is significantly reduced compared to the control group, the effect is also increased as the dose is increased (P <0.05). In FIG. 8, each measured value represents an average ± standard error.

The main cause of aging and degenerative diseases is active oxygen, which causes the peroxidation of proteins and lipids, and the peroxidized lipids and proteins lose their normal function, resulting in cell weakening and aging. It also causes oxidative damage to DNA, leading to mutations. Recently, reports of abnormal increase in the amount of hydroxyl radicals due to the decrease of antioxidant enzymes such as glutathione peroxidase and catalase and the increase of ferrous ions in Parkinson's disease patients indicate that oxidative stress is an important cause of Parkinson's disease. (Ogawa, Eur. Neurol. 34 (suppl), 20-28, 1994). Therefore, in the present experiment to determine the degree of damage caused by aging-causing agents such as active group oxygen by measuring the degree of lipid peroxidation, the result that the silk peptide of the present invention reduced the degree of lipid peroxidation was anti-aging of the silk peptide of the present invention. It indicates that it has an effect.

Experimental Example III-5: Measurement of TH Immunopositive Cell Proportion in Black Matter (TH Immunohistochemistry Experiment)

In order to confirm the effect of BG101 or BG201 of the present invention on Parkinson's disease animal model by histological change, TH immunohistochemistry was performed. After administration of BG101 or BG201 in the same manner as in Experimental Example III-2, the lesions were prepared, and two weeks later, the black sections were observed at high magnification by immunohistochemical staining for tyrosine hydroxylase (TH). Stained TH immune positive cells were counted.

A. Organization Preparation

BG101 or BG201 was administered in the same manner as in Experimental Example II-2, and the anesthetized animal was anesthetized with chloral hydrate (400 mg / mL), and 200 mL of 0.1 M PBS (pH 7.4) was injected through the heart. Perfusion removes the vascular blood components, and 250-300 ml of fixative (4% paraformaldehyde / PBS) was perfused. Then, the brain was extracted and post-fixed at 4 ° C. for 15-24 hours with the same fixative. Then, the fixative was washed thoroughly with PBS and then infiltrated with 10%, 20%, and 30% sucrose solutions in order to prevent ice crystals from freezing. Then, the brain tissue was embedded in embedding liquid, and rapidly frozen in isopentane, which was previously cooled with liquid nitrogen, followed by continuous coronal section with a thickness of 40 μm using a cryostat (Reryert Frigocut model 2000). It was stored in a stock solution containing% glycerol, 30% ethylene glycol and 10% phosphate buffer (PB).

B. Immunohistochemical Analysis

The tissue contained in the stock solution was taken out, washed three times with PBS for 10 minutes, and reacted with 5% hydrogen peroxide for 10 minutes. Subsequently, the reaction was performed with 10% normal goat serum (NGS), 3% bovine serum albumin (BSA, Sigma) and 0.3% Triton X-100 for 30 minutes. The first antibody was reacted overnight using mouse α TH (dilution factor 1: 500, Boehringer Mannheim), and the next day was reacted with biotinylated secondary antibody (Vector laboratories, USA) for 1 hour at room temperature. The avidin-biotin-peroxidase complex (ABC, Vector laboratories, United States), which was diluted 1: 100 beforehand 30 minutes before, was reacted at room temperature for 1 hour. Then, it was added to DAB (diaminobenzidine 0.05%, H ₂ O ₂ 0.003%) and developed for 5 minutes. The tissue was attached to gelatin-coated slides and dried. Then, dehydrated with 70%, 90% and 100% ethanol, treated with xylene and then sealed with lid glass and Canadian balsam and observed under an optical microscope. In each culture step, the excess reagent was washed three times with PBS three times for 10 minutes, and normal serum was added instead of the first antibody or the second antibody for control staining and treated with other tissues.

C. Immunohistochemical Assay

 The results of TH immunohistochemical staining in the striatum and melanoma by BG101 or BG201 and the change of the proportion of TH immunopositive cells (lesion / non-lesion side,%) in the melanoma are shown in Figs. 9A and 9B, respectively.

As can be seen in Figure 9a, TH immunostaining of the normal control group is clearly expressed in the entire striatum, it was confirmed that most of the dopamine neurons located in the black matter. On the other hand, the striatum's striatum did not stain the doso-lateral area, which is known as the upper limb locomotor region, and the par compacta, which dominates the area, did not stain. Most of the dopamine neurons were lost. On the other hand, in the experimental group administered with the silk peptide of the present invention (BG1015) it can be seen that the dopamine nerve fiber terminal and nerve cell body in the striatum and black matter significantly compared to the lesion control group.

As can be seen in Figure 9b, counting to measure the protective effect of dopamine neurons in the black matter (count), the percentage of TH immune positive cells was significantly reduced in the control group administered only dopamine hydroxide compared to the normal control (P <0.05). On the other hand, in the experimental group administered the silk peptide of the present invention it can be seen that the ratio of TH immune positive cells significantly increased compared to the control group, it can be seen that the effect is also increased as the dose is increased (P <0.05). In FIG. 9B, each measured value represents an average ± standard error.

Tyrosine hydroxylase (TH) is a key enzyme in metabolic processes that produce dopamine from tyrosine. That is, the dopamine neurons and nerve endings stained by TH can produce dopamine and can construct a dopaminergic neural network. To demonstrate the protective effect of cells.

The experimental results indicate that the silk peptide of the present invention is effective in Parkinson's disease through behavioral and histological changes in the Parkinson's disease animal model, and the biochemical results indicate that the silk peptide of the present invention specifically protects dopamine neurons. Has proved to be an excellent effect. Therefore, it can be seen that the silk peptide of the present invention can be usefully used in functional foods and drugs related to the prevention and treatment of Parkinson's disease, a degenerative brain disease.

Experimental Example IV: Effect on Acetylcholine Concentration

Amyloid β-induced brain damage caused by brain damage reduced the amount of acetylcholine (acetylcholine) in experimental rats of the present invention BG101 or BG201 inhibits the decrease in the amount of acetylcholine can improve cognitive function and depressive brain disease Was examined.

Experimental Example IV-1: Experimental Animal Preparation

The experimental animals were male white papers (Sprague Dawley, 140-180 g, Korea Experimental Animal Center), which were placed in a single box for four weeks in a given environment (room temperature 25 ± 1 ℃, relative humidity 60 ± 10%). Adapted to the environment by ingesting water and feed without limit, it was used in the experiment.

Experimental Example IV-2 Determination of Acetylcholine Concentration

To determine the effect of acetylcholine concentration, the concentration of acetylcholine was measured by chemiluminescence using Islael and Lesbats methods ( J Neurochem. 37 (6): 1475-83 (1981)). The measuring method is a method using a reaction in which acetylcholine is hydrolyzed to choline by acetylcholine esterase, and then changed into bestaine and hydrogen peroxide by choline oxidase. Hydrogen peroxide chemically emits light when reacted with luminol (5-amino-1,2,3,4-tetrahydro-1,4-phthalazinedione, luminol; Merck, Darmstadt, Germany) and peroxidase (Sigma, USA) Therefore, by measuring the amount of light using chemiluminescence can indirectly determine the concentration of acetylcholine.

As a result of analyzing the amount of acetylcholine due to Aβ in the experimental control group treated with Aβ and the drug test group administered BG101 or BG201 at 1 g / kg for more than a week, the experimental control group treated with Aβ was found to be a normal control group. In comparison, the rats in the group continuously receiving BG101 significantly inhibited the decrease of acetylcholine (P <0.05). In FIG. 10, each measured value represents a mean ± standard deviation (SD).

Acetylcholine is a neurotransmitter that is very important for normal knowledge function as it is projected from the basal ganglia to the cerebral cortex and hippocampus. In particular, it is known that learning and memory can be altered by drugs acting on the Ach system. Examination of death from Alzheimer's dementia has found that in all cases, acetylcholinergic neurons in the basal ganglia projecting into the cerebral cortex are largely damaged. Recently, it has been known to increase the concentration of acetylcholine to prevent cognitive improvement and dementia progression, and to have an excellent effect on the treatment and prevention of dementia, and a choline agonist or choline esterase inhibitor is used in patients. Drugs developed to date include Lecithin as an acetylcholine precursor, RS-86 as a receptor agonist, and nicotine as commercially available in Korea with FDA approval as an acetylcholinesterase inhibitor. Tacrine and Aricept, which have recently been approved, are still controversial due to their temporary, mild and severe toxicity. The above experimental results reveal that the silk peptide of the present invention has an effect of improving cognitive function and inhibiting aging and degenerative diseases by inhibiting the decrease of acetylcholine amount from aging and degenerative diseases which decrease the amount of acetylcholine in the brain.

Experimental Example Ⅴ: Depression animal model experiment

Experimental Example Ⅴ-1: Preparation of experimental animals

The experimental animals were male white papers (Sprague Dawley, 140-180 g, Korea Experimental Animal Center), which were placed in a single box for four weeks in a given environment (room temperature 25 ± 1 ℃, relative humidity 60 ± 10%). Adapted to the environment by ingesting water and feed without limit, it was used in the experiment. A total of 50 animals were used in this experiment, except for the lack of movement, developmental development, abnormal homologous behavior in forced breeding, and a significant decrease in swimming capacity.

Experimental Example Ⅴ-2: Animal Model Experiment for Depression

In this experiment, a forced swimming test (FST), a standardized test called the behavioral despair test, was used. Ecologically, white papers do not like water, so when exposed to water they exhibit characteristic behaviors to escape this environment. In other words, the experimental animals are forced to swim to get out of the water and do not swim anymore after a certain period of time. Using this principle, the forced swimming test is known as a basic experiment to detect antidepressant effects in drug development. have. The process of FST was carried out according to the method of the original proponent Porsolt et al. (Porsolt et al., Eur. J. Pharmacol. 51 (3), 291-294, 1978):

First, a transparent acrylic cylindrical water tank having a height of 40 cm and a diameter of 18 cm was filled with water at 25 ° C. to a height of 15 cm, and the white paper of Experimental Example V-1 was forcibly omitted and left for 15 minutes. During the first few minutes, the white paper was very resistant to get out of it, but as time passed, the time for immobilization increased, and the body remained almost immobile for the last few minutes. A typical floating state is a white paper that floats on water with only a small amount of movement to balance the body with only a part of the face on the surface. After the forced swimming, the white paper was wiped dry and then dried in a 37 ° C dryer for 30 minutes and returned to the breeding box.

Secondary forced swimming was performed after 24 hours. In this experiment, the secondary forced swimming was kept in the tank for 5 minutes under the same conditions as the primary forced swimming, and the total dead time was measured during this period. In secondary forced swimming, the white paper shows that most of the results of learned helplessness are inactive for more time than primary forced swimming. Since the increase in immobility, which is considered a symptom indicator of depression in the forced swimming model, is again reduced by treatment with antidepressants, this decrease in immobility is thought to be related to the action of antidepressants. In the forced swimming test, some drugs did not show the effect during the acute treatment, but the long-term treatment showed that the drug was treated for 7 days before the second stress swimming. Since the antidepressant effects of antidepressants are generally obtained after drug administration for at least two weeks, this method is widely used in forced swimming tests. For the measurement of immobility, the whole process of secondary forced swimming was taken by video and the immobility time was measured to compare and evaluate the immobility time between the control group and the experimental group. Through the video screen analysis, three trained evaluators measured the dead time with a stopwatch and calculated the average value between evaluators.

A. Methods of Administration

BG101 or BG201 of the present invention was prepared as a 100 mg / ml solution and orally administered. All samples were dissolved in distilled water and orally administered. One dose was administered 1 hour before the second forced swimming, and in the case of long-term repeated administration for 7 days, 30 minutes after the first forced swimming was repeated and repeated for 7 days.

B. Antidepressant Effect in One Acute Treatment

The antidepressant effect of one acute treatment (1 g / kg) of BG101 or BG201 of the present invention was confirmed. Existing antidepressant as a positive control was used imipramine (Sigma, United States), which is the basic drug among tricyclic antidepressants, and the dosage was based on previous studies (Eur. J. Pharmacol. 138 (3), 413-416, 1987; Neuropharmacology 28 (3), 229-233, 1989), 20 mg / kg in a single dose. The experimental results are shown in Figure 11a.

As can be seen in Figure 11a, the mean dead time by acute treatment was significantly shortened by imipramine administration compared to the control (*, p <0.05). In addition, it can be seen that the dead time was significantly shortened in the experimental group to which the BG101 or BG201 of the present invention was administered (**, P <0.05). In FIG. 10A, each measured value represents an average ± standard deviation.

C. Antidepressant effect on long-term repeated treatment (7 days)

The antidepressant effect of long-term repeated treatment of BG101 or BG201 of the present invention was confirmed. BG101 or BG201 was orally administered at 50 mg / kg once a day for 7 days, and the same experiment as in the single treatment was performed. The experimental results are shown in Figure 11b.

As can be seen in Figure 11b, the mean dead time by long-term treatment was significantly shortened by imipramine administration compared to the control (P <0.05). In addition, it was found that the dead time was significantly shortened in the experimental group administered BG101 or BG201 (P <0.05). In FIG. 11B, each measured value represents an average ± standard deviation.

Based on the above results, immipramine administration significantly reduced immobility time compared to the control group, and acute and long-term treatment groups of BG101 or BG201 did not reduce immobility time as imipramine, but showed statistically significant antidepressant effect. It can be seen that it has. Although imipramine may have side effects and is difficult to take for a long time, BG101 or BG201 of the present invention is somewhat lower than imipramine in terms of effects, but has a merit of taking a large amount in the long term, which is excellent as a preventive and therapeutic agent for depression. You can expect.

As described in detail above, the present invention provides a composition for preventing or treating cerebral neurological diseases and a composition for improving cognitive function. The composition of the present invention not only exhibits the efficacy described in the detailed description of the present invention, but also includes a natural natural silk peptide as an active ingredient, so that side effects on the human body are extremely less than that of chemical synthetic medicines.

Claims (20)

Silk fibroin, a silk protein, is first decomposed by calcium salt and secondly to an enzyme selected from the group consisting of trypsin, pepsin, alkalase, thermoase, flavozyme, sumizime and combinations thereof. Silk peptides having a weight average molecular weight of 200-15,000 produced by the hydrolysis of as an active ingredient, dementia, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis, ischemic stroke, depression, schizophrenia and post-traumatic stress Functional food composition for the prevention of cerebral nerve disease selected from the group consisting of disorders. delete delete delete delete delete 2. The composition of claim 1, wherein the silk protein is first degraded by calcium salt and secondly hydrolyzed by a mixed enzyme of flavozyme and sumizime. 8. The composition of claim 7, wherein the calcium salt is calcium chloride. The silk protein silk fibroin, which has a weight average molecular weight of 200-10,000 produced by acid hydrolysis as an active ingredient, includes dementia, Huntington's disease, Parkinson's disease and amyotrophic lateral sclerosis, ischemic stroke, depression, Functional food composition for the prevention of cerebral neurological diseases selected from the group consisting of schizophrenia and post-traumatic stress disorder. 10. The composition of claim 9, wherein the weight average molecular weight of the silk peptide is 200-3,000. The composition of claim 10, wherein the weight average molecular weight of the silk peptide is 300-1,500. delete The composition of claim 1 wherein the weight average molecular weight of the silk peptide is 300-4,000. The composition of claim 13, wherein the weight average molecular weight of the silk peptide is 300-2,000. 15. The composition of claim 14, wherein the weight average molecular weight of the silk peptide is 400-1,200. delete delete delete delete delete

Images