KR100600695B1 - Method for producing silk peptide having neuronal cell protective activity - Google Patents

Abstract

The present invention relates to a method for producing silk peptides having neuronal cell protective activity, and more particularly, to silk proteins, preferably silk fibroin, by weight hydrolysis of silk peptides having a weight average molecular weight of 300-100,000 It relates to a manufacturing method of.

Silk, silk peptide, fibroin, hydrolysis, nerve cell, ischemia

Description

Method for Preparing Silk Peptide with Neuronal Cellular Protective Activity {Method for Preparing Silk Peptide with Protecting Nerve Cell}             

Figure 1a is a graph showing the results of the MTT reduction experiment to confirm the effect of silk peptides produced by the method of the present invention on the death of nerve cells;

Figure 1b is a graph showing the results of the MTT reduction experiment for confirming the effect of silk peptides produced by another method of the present invention on the death of nerve cells;

FIG. 2A is a picture of pest staining results showing that silk peptides prepared by the method of the present invention inhibit apoptosis of nerve cells; FIG.

FIG. 2B is a picture of pest staining results showing that silk peptides prepared by another method of the present invention inhibit apoptosis of neurons; FIG.

3A is a graph showing that silk peptides prepared by the method of the present invention inhibit caspase activity;

3B is a graph showing that silk peptides prepared by another method of the present invention inhibit caspase activity;

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

4b is a photograph showing that silk peptides prepared by another method of the present invention inhibit the production of activator oxygen in cells;

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

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

Figure 5c is a graph showing the effect of silk peptides prepared by the method of the present invention on the site of cerebral infarction by ischemia.

The present invention relates to a method for producing a silk peptide having neuronal protective activity, and more particularly, to a method for producing a silk peptide having a specific weight average molecular weight by hydrolyzing silk protein and exhibiting neuroprotective activity. It is about.

In order to use silk protein as an active ingredient of a functional food or pharmaceutical, various methods for obtaining silk peptides from silk protein have been proposed. For example, Japanese Patent Application Laid-Open No. 59-31520 discloses a method for preparing a silk pibroin peptide solution, and Korean Patent Application Publication No. 1999-74787 discloses a method for preparing silk powder peptide by enzymatic degradation.

However, according to the conventional manufacturing methods, there is a problem in that absorption into the body is difficult because the molecular weight of the finally produced silk peptide is large. In addition, conventional methods for producing silk peptides have been developed with a focus on simply obtaining peptides having a low molecular weight, and there is almost no development of methods for preparing silk peptides exhibiting specific physiological activities. Moreover, a technique for producing a silk peptide capable of protecting neurons has not been developed yet.

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 made intensive studies to develop a method for producing silk peptides with neuronal cell protective activity. As a result, the present inventors have confirmed that silk peptides having a specific weight average molecular weight obtained through hydrolysis have excellent neuronal cell protective activity. The invention was completed.                         

Accordingly, it is an object of the present invention to provide a method for producing silk peptides having neuronal protective activity.

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

According to an aspect of the present invention, the present invention provides a method for producing silk peptides having a weight average molecular weight of 300-100,000 and having neuronal cell protective activity by hydrolyzing silk proteins.

Silk proteins are used as starting materials in the process of the invention. 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).

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 research results, 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 present invention, the hydrolysis of silk fibroin is carried out by (a) decomposition in calcium salt solution, (b) acid hydrolysis, (c) hydrolysis by enzymes or (d) a combination of the above methods. It can be carried out through. The weight average molecular weight of the silk peptide produced by decomposition by the above-described method may be in the range of about 300-100,000, and serves to protect neurons.

Decomposition in the calcium salt solution is usually using calcium chloride, preferably 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. Since this method produces a polymer peptide, it is not preferable compared with other hydrolysis methods.

Hydrolysis by the enzyme is carried out using a proteolytic enzyme. Preferably, the degrading enzyme acts nonspecifically on the amino acid sequence to catalyze random hydrolysis. Suitable degradation enzymes for the present invention are thermoase, flavozyme, sumyzyme, protamex or protein. According to a more preferred embodiment of the present invention, the enzymes used to obtain the silk peptides of the present invention are thermoases, flavozymes, sumizymes and combinations thereof, most preferably including flavozymes and sumimimes It is a mixed enzyme.

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. Even more preferably, the decomposition of silk fibroin is carried out primarily by decomposition with calcium salt and secondly by hydrolysis with a mixed enzyme of flavozyme and sumizime. According to a most preferred embodiment, (a) dissolving silk fibroin in a solution containing calcium salt; (b) removing the calcium salt contained in the silk fibroin solution; And (c) treating the silk fibroin solution with a proteolytic enzyme in which flavozyme and sumyzyme are mixed to prepare silk peptides.

The calcium salt that can be used in the method of the present invention may be in various forms, most preferably calcium chloride. The step (a) is preferably carried out by dissolving silk fibroin in a solution containing calcium chloride, water and ethanol at 60-95 ℃, more preferably 70-95 ℃, most preferably 85-90 Dissolve at ° C. Step (b) may be carried out by various methods known in the art, such as gel filtration chromatography. The step (c) is preferably carried out at a high temperature of 45-60 ℃, more preferably 50-60 ℃, most preferably at about 55 ℃.

The weight average molecular weight of the silk peptide obtained by such a combination method is 400-15,000, Preferably it is 400-4,000, More preferably, it is 400-2,000, Most preferably, it is 400-1,200. Peptides prepared by this method exhibit substantially 100% solubility in water and very low solubility in organic solvents such as ethanol, methanol, acetone and dimethylformamide.

The weight average molecular weight of the silk peptide produced by acid hydrolysis in the method of the present invention may have a range of 300-10,000. The acid that can be used varies, but is preferably hydrolyzed using an inorganic acid solution of a strong acid, most preferably hydrochloric acid. Preferred temperatures for acid hydrolysis are 70-120 ° C., more preferably 90-110 ° C., and most preferably about 110 ° C.

According to a specific embodiment of the acid hydrolysis method of the present invention, (a) adding hydrochloric acid solution to silk fibroin to perform hydrolysis at 70-120 ℃; (b) adding an alkaline solution to the hydrolysis solution to raise its pH; And (c) removing the salt formed in step (b), wherein the weight average molecular weight of the finally produced silk peptide is 300-3,000. Most preferably, the weight average molecular weight of the silk peptide produced by acid hydrolysis is 300-1,500. The most suitable alkaline solution used in step (b) is sodium hydroxide solution. In addition, the method of removing the salt in the step (c) can be carried out through various methods known in the art, for example, gel filtration chromatography or electric desalting method.

Silk peptides obtained by acid hydrolysis exhibit substantially 100% solubility in water and very low solubility in organic solvents such as ethanol, methanol, acetone and dimethylformamide.

Silk peptides prepared by the method of the present invention has a low molecular weight, so it is well absorbed into the body and exhibits 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.). As illustrated in the examples below, silk peptides prepared by the methods of the present invention can be used for the treatment and prevention of various neurological diseases through neuronal protective action.

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 according to 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 for 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 measured using several solvents. 0.1 g of silk peptide powder was dissolved in 10 ml of distilled water, ethanol, methanol, acetone and dimethylformamide 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 the shape of DNA using Hoechst staining, which observes the firmness and morphology of the cell membrane. 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 obtained a standard curve using the degree of fluorescence to relatively quantify the fluorescence measurement value (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

Activator 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 an CCD camera, and the NIH Image 1.65 program was used. 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: Preparation of Drug Treatment and Local Ischemia Induction Animal Model

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.

Experimental Example II-2: Experiment of the 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. After taking the stained tissue sections (Fig. 5a), the area of the area where the cerebral infarction occurred and turned white was different from the normal area stained in red using the MCID image processing system (Imaging Research Inc., Canada). Was calculated, 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.

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

The present invention provides a method for producing a silk peptide having neuronal protective activity. The method of the present invention not only efficiently prepares silk peptides having neuronal protective activity from silk proteins, but also provides low molecular weight silk peptides that can exhibit good body absorption.

Claims (19)

In the method for producing silk peptide by hydrolyzing silk fibroin, the hydrolysis is carried out primarily by decomposition with calcium salt and secondly by hydrolysis with a mixed enzyme of flavozyme and sumizime, The weight average molecular weight of the finally produced silk peptide is 400-4,000, and the resulting silk peptide is characterized in that it has nerve cell protective activity, a method for producing a silk peptide. delete delete delete delete delete delete delete The method of claim 1, wherein the method comprises: (a) dissolving silk fibroin in a solution containing calcium salt; (b) removing the calcium salt contained in the silk fibroin solution; And (c) preparing a silk peptide having a weight average molecular weight of 400-2,000 by treating proteolytic enzymes in which the flavozyme and sumyzyme are mixed in the silk fibroin solution. Method for producing a silk peptide having a protective activity. 10. The method of claim 9, wherein the calcium salt is calcium chloride method of producing a silk peptide having neuronal cell protective activity, characterized in that. The method of claim 10, wherein the weight average molecular weight of the silk peptide is 400-1,200 method for producing a silk peptide. 10. The method according to claim 9, wherein the step (a) of the silk fibroin is prepared by reacting and dissolving silk fibroin in a solution containing calcium chloride, water and ethanol at 60-95 ℃ to have a silk peptide with neuronal cell protective activity Way. The method of claim 9, wherein step (c) is a method for producing a silk peptide having neuronal cell protective activity, characterized in that carried out at a high temperature of 45-60 ℃. delete delete In the method of producing a silk peptide by acid hydrolysis of silk fibroin, the hydrolysis is carried out for 12 hours at a temperature of 70-120 ℃ with hydrochloric acid solution, the weight average molecular weight of the resulting silk peptide is 300-1,500 And, the generated silk peptide is characterized in that it has nerve cell protective activity, silk peptide production method. delete The method of claim 16, wherein the acid hydrolysis method comprises the steps of: (a) adding hydrochloric acid solution to silk fibroin to perform hydrolysis at 70-120 ° C. for 12 hours; (b) adding an alkaline solution to the hydrolysis solution to raise its pH; And (c) removing the salt formed in step (b). delete

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