WO2012020991A2 - Composition for preventing and alleviating brain disease comprising silk amino acid and tyrosine as active ingredients - Google Patents

Abstract

The present invention relates to a composition for preventing and alleviating brain disease comprising silk amino acid and tyrosine as active ingredients and relates to a production method therefor. The composition of the present invention is outstandingly efficacious in Parkinson's disease, cerebral infarction, stroke or dementia.

Description

Composition for preventing and improving brain diseases comprising silk amino acids and tyrosine as active ingredients

The present invention relates to a composition for preventing and improving brain disease and a method for producing the same using silk amino acids and tyrosine as active ingredients.

Parkinsonism (Parkinsonism or Parkinson's disease, PD) is a serious brain disease estimated to have more than half a million patients in the United States alone, resulting in the loss of dopamine neurons in the substantia nigra pars compacta (SN) region. It is characterized by the loss of exercise ability due to the reduced dopamine secretion in the striatum, and the current standard treatment for Parkinson's disease is dopamine precursor L-3,4-dihydroxyphenylalanine ( _L -DOPA or Levo-DOPA). In addition, the drug significantly alleviates the symptoms, but long-term use is known to have serious side effects.

Silk is a protein polymer, the composition, structure and composition of which are completely different depending on the type of insect. The most commonly characterized silks are from silkworms (Bombyx mori) and spiders (Nephila clavipes and Araneus diadematus). . Silk consists of two polypeptides, sericin and fibroin, and as its chemical composition is found to be pure natural amino acids, various effects according to the physiological activity of the composition amino acids have been suggested.

The inventors of the present invention while studying a composition for preventing / treating brain diseases that can be administered for a long time without serious side effects, the composition of silk amino acid with tyrosine inhibits exercise ability, dopamine increase, nerve cell protection due to dopamine and dopamine neuronal abnormalities It confirmed that the effect was excellent, and completed this invention.

An object of the present invention is to provide a composition for preventing and improving brain diseases.

Another object of the present invention to provide a method for producing a composition for preventing and improving brain diseases.

Still another object of the present invention is to provide a method for preventing and / or treating a brain disease, which comprises administering the composition.

In order to achieve the above object, the present invention provides a composition for preventing and improving brain diseases, including silk amino acids and tyrosine as an active ingredient.

The present invention also provides a method for producing a composition for preventing and improving brain diseases comprising silk amino acids and tyrosine as an active ingredient, and a method for preventing and / or treating brain diseases comprising administering the composition.

The composition of the present invention has the effect of preventing, ameliorating, and treating brain diseases by protecting dopaminergic neurons or increasing the concentration of dopamine and / or its metabolites.

1 shows TH-positive neurons in the area of black matter (SN) in the brain 15 days after 6-OHDA administration.

Figure 2 shows TH-positive neurons in the area of black matter (SN) in the brain 30 days after 6-OHDA administration.

The present invention relates to a composition for preventing, treating and improving brain diseases, including silk amino acid and tyrosine as active ingredients.

In another aspect, the present invention provides a method for producing a composition for preventing, treating and improving cerebral disease, comprising mixing silk amino acid and tyrosine.

In addition, the present invention provides a method for preventing and / or treating a brain disease comprising administering a composition comprising silk amino acid and tyrosine as an active ingredient.

In another aspect, the present invention provides a brain disease prevention, treatment and improvement of the composition comprising a silk amino acid (typical amino acid) and tyrosine (tyrosine) as an active ingredient.

The present invention also provides the use of a composition comprising silk amino acids and tyrosine as an active ingredient for the manufacture of a medicament for the prevention, improvement and treatment of brain diseases.

Hereinafter, the present invention will be described in detail.

Silk amino acid of the present invention can be prepared using silkworm or silkworm pupa. In addition, the silk amino acid of the present invention can be prepared using the silk protein. The silk protein may be fibroin and / or sericin. The silk amino acid may be prepared by acid hydrolysis of silkworm, silkworm pupae or silk protein, but is not limited thereto.

The tyrosine may be synthetic tyrosine or natural tyrosine. The tyrosine of the present invention may also be derived from silk, cocoon and the like. However, if it is tyrosine, there will be no problem in implementing this invention, and it will be said that it corresponds to the tyrosine of this invention regardless of its origin.

The brain disease is not limited to Parkinson's disease, cerebral infarction, stroke, dementia, but will be applicable if the brain disease associated with abnormalities of dopamine and / or dopaminergic neurons.

The composition for preventing and improving brain diseases of the present invention can prevent, treat, and improve brain diseases by increasing the concentration of dopamine or preventing, delaying, and protecting nerve cells from being destroyed. In addition, the composition of the present invention can restore and improve the motor function of the body inhibited by brain diseases.

The composition for preventing and improving brain diseases of the present invention may include 0.01 to 50 parts by weight of tyrosine, preferably 0.02 to 20 parts by weight, and most preferably 0.02 to 12 parts by weight, based on 100 parts by weight of the composition. We can include vice. With respect to 100 parts by weight of the composition, when the tyrosine exceeds 50 parts by weight, the prevention / improvement effect of the brain disease is increased, and when mixing tyrosine and silk amino acids, it is difficult to homogeneously mix and excessively increase the mixing time. The efficiency of the process is lowered. In addition, when tyrosine is less than 0.01 parts by weight based on 100 parts by weight of the composition, it is difficult to expect an increase in the prevention / improvement of brain diseases due to the addition of tyrosine.

The composition for preventing and improving brain diseases of the present invention may be a pharmaceutical composition or a food composition.

The pharmaceutical composition for preventing and improving brain diseases of the present invention can be administered orally or parenterally and can be used in the form of a general pharmaceutical preparation. Preferred pharmaceutical preparations include oral preparations such as tablets, hard or soft capsules, solutions, suspensions and the like, which can be used in the form of excipients in conventional pharmaceutically acceptable carriers such as oral preparations, Binders, disintegrants, lubricants, solubilizers, suspending agents, preservatives or extenders can be used.

The dosage of the pharmaceutical composition for preventing and improving brain diseases of the present invention may be determined by a specialist depending on various factors such as the patient's condition, age, sex, and complications, but generally 0.1 mg to 10 g per kg of adult, preferably Preferably from a dose of 10 mg to 5 g. In addition, it is intended to contain a daily dose of the pharmaceutical composition or a dose of 1/2, 1/3 or 1/4 thereof per unit dosage form, and may be administered 1 to 6 times a day. However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety.

The patient of the present invention is a brain disease patient. The patient of the present invention may be a patient having a disease such as Parkinson's disease, cerebral infarction, stroke, dementia, but is not limited thereto. Patients with brain diseases associated with abnormalities of dopamine and / or dopaminergic neurons correspond to patients of the invention.

The present invention also provides a food composition for preventing and improving brain diseases. The food is not limited to, but not limited to, health supplements, health functional foods, functional foods, and the like, and natural foods, processed foods, and general food materials, including the addition of the composition for preventing and improving brain diseases of the present invention.

The food composition for preventing and improving brain diseases of the present invention may be added as it is or used with other food or food compositions, and may be appropriately used according to a conventional method. The blending amount of the active ingredient can be suitably determined according to the purpose of its use (prevention, health or therapeutic treatment). Generally, 0.1 to 70 parts by weight, preferably 2 to 50 parts by weight, of the brain disease prevention and improvement composition of the present invention may be added to 100 parts by weight of the raw material of the food or beverage during the preparation of the food or beverage. The effective dose of the composition for preventing and improving brain diseases can be used in accordance with the effective dose of the pharmaceutical composition, but may be less than the above range in the case of long-term intake for the purpose of health and hygiene or health control. In addition, since the active ingredient has no problem in terms of safety, it may be used in an amount above the above range.

 There is no particular limitation on the kind of food. The food composition for preventing and improving brain diseases can be used in the form of oral preparations, such as tablets, hard or soft capsules, liquids, suspensions, etc., these preparations are acceptable conventional carriers, such as oral administration In the case of formulations, excipients, binders, disintegrants, lubricants, solubilizers, suspending agents, preservatives or extenders can be used.

Examples of foods to which the composition for preventing and improving brain diseases can be added include meat, sausages, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, Beverages, teas, drinks, alcoholic beverages and vitamin complexes, but are not limited to these kinds of foods.

Hereinafter, the present invention will be described in more detail by the following examples and experimental examples. However, the following Examples and Experimental Examples are only illustrative of the contents of the present invention and the scope of the invention is not limited by the Examples and Experimental Examples.

<Material>

Silk amino acids

Silk amino acid was obtained from World Way, Inc. as a pale yellow powder obtained by acid hydrolysis of silk protein, which was used while stored at 4 ° C. Prior to administration, the required amount by concentration was dissolved in sterile purified water and administered orally, and the solvent control group was administered the same amount of sterile purified water.

in vitro  test

Experimental Example 1

 Dissolve silk amino acids in phosphate buffer (pH 6.8) to final concentrations of 200, 500, 1,000, 1,500 and 2,000 μg / mL, add 70 μL to each 96-well plate, add 20 μL of mushroom tyrosinase (2,000 U / mg) and 10 μL of 10 mM L-DOPA was added. After the reaction for 30 minutes at 37 ℃ using a microplate reader (Infinite 200, Tecan Group Ltd., Switzerland) was measured the absorbance at 475 nm to compare the activity of tyrosine nase.

As a result, as the concentration of silk amino acid increased, it was found to inhibit the activity of tyrosinease. In other words, there was no significant change in tyrosinase activity in the silk amino acid concentration in the range of 200-500 μg / mL, but the tyrosinase activity was about 5% and 1,500 μg when the silk amino acid concentration was 1000 μg / mL. about 10% at / mL and about 14% at 1,500 μg / mL (Table 1). Therefore, silk amino acids were found not to promote tyrosinase activity.

Table 1

Experimental Example 2

Preparation of Examples 1-6

A composition was prepared in which silk amino acids were mixed with tyrosine. Specifically, a composition containing 2 parts by weight of tyrosine (Example 1), a composition containing 4 parts by weight of tyrosine (Example 2), and a composition containing 8 parts by weight of tyrosine (executed) based on 100 parts by weight of the silk amino acid Example 3), the composition which mixed 12 weight part tyrosine (Example 4), the composition which mixed 16 weight part tyrosine (Example 5), and the composition which mixed 20 weight part tyrosine (Example 6).

Dopamine Neuron Culture and MPP + Treatment

SD rats of 14-15 days of gestation bred under the same conditions were killed by cervical dislocation, and the abdomen was excised to separate fetuses under aseptic conditions. The ventral midbrain containing black matter was excised from the brains of the isolated fetuses, and neurons were cultured. The brain tissues from which the meninges were completely removed were washed with DMEM / F12 1: 1 culture medium to remove blood, and then isolated cells were obtained using a small diameter Paster pipette and transferred to a centrifuge tube, followed by centrifugation at 700 rpm for 8 minutes. The precipitated cells were then resuspended in culture and the same procedure repeated twice, followed by DMEM / F12 containing 10% fetal calf serum and 2 mM glutamine, 50 U / ml penicillin, 50 μg / ml streptomycin. After floating in a 1: 1 culture medium, the number of cells was measured under an optical microscope, and 6 × 10 ⁶ cells were cultured in a 60 mm cell culture plate, and 6 × 10 ⁴ cells were cultured in each well in a 96 well plate. Serum-free cultures were used for culturing neural neurons. 18-20 hours after incubation, 20 μg / ml insulin, 40 μg / ml transferrin, 60 mM selenium, 20 mM progesterone, 100 μM putrescin, 1 mM pyruvate, 2 mM L-glutamine instead of fetal bovine serum , 50 U / ml penicillin, 50 μg / ml streptomycin, 0.25% glucose-added DMEM / F12 1: 1 cultures were exchanged and incubated in an incubator at 37 ° C., 5% CO ₂ , 95% air. The culture vessel was coated with poly-L-lysine (10 ㎍ / mL), washed three times with distilled water, and then used after removing water. After treatment for 24 hours in the MPP + culture medium of day four a concentration started to culture investigate toxicity to dopamine neurons silk amino acid, tyrosine, Examples 1 to 6 and _L -DOPA (positive control) to a pre-treatment prior to one MPP + for 1 Until the end of the experiment.

result

After 24 hours of MPP + treatment, dopamine, dihydroxyphenyl acetic acid (DOPAC) and Homovanillic acid (HVA) contents of cultured neurons were measured (Table 2). After inducing Parkinson's disease, dopamine and DOIPAC decreased, and HVA decreased to an undetectable level. The group treated with MPP + and silk amino acids increased dopamine and DOPAC content, but it was not significant. HVA was significantly increased. However, it was confirmed that dopamine, DOPAC, and HVA were all increased to a significant level in the silk amino acid and tyrosine treated groups (Examples 1 to 6). In the tyrosine only group, dopamine and HVA were significantly increased, but DOPAC was decreased rather than the Parkinson's group.

TABLE 2

Tyrosine Daily recommended amount: 840mg / 60kg

Dopamine, DOPAC, HVA content in normal cells = 100%

In vivo  test

As a result of combining the results of Experimental Example 2, it was confirmed that the composition of Example 2 added 4% tyrosine significantly increased the content of dopamine, DOPAC, HVA, and the most suitable concentration of tyrosine does not aggregate with each other, Hereinafter, the in vivo test was conducted using the composition of Example 2.

Experimental Animal

The experimental animals were supplied with 7-week-old male Sprague-Dawley (SD) rats from Daehan Biolink (Neongbuk-neum) and used for about 200-250 g of body weight after approximately 1 week of acclimatization. Animals were housed in rats two by two. The environment of the animal laboratory was adjusted to a temperature of 23 ± 2 ° C, a relative humidity of 55 ± 10%, a ventilation frequency of 12 times / hour, a lighting cycle of 12 hours, and an illuminance of 150-300 Lux. Purina Rat Chow ^® biopia (Gunpo, Gyeonggi-do, Korea) was used as a pellet-type solid feed for experimental animals. The experiment was conducted in accordance with the Institute's Standard Operation Procedures (SOP) with the approval of the Institutional Animal Care and Use Committee (IACUC) of the Chungbuk National University.

<Preparation of model animal>

All surgical procedures were performed while maintaining aseptic status. Anesthetize the rat with enfluorane and calibrate it in a stereotaxic instrument, then use a Hamilton syringe (26 gauge needle) to add 6-OHDA (8 μg) + ascorbic acid (0.6 μg) / 3 μl saline to the right medial forebrain bundle [target coordinates : anteroposterior from bregma (AP), -4.0; mediolateral (ML), 0.8; Parkinson's disease was induced by injection into dorsoventral (DV), 8.0 mm].

Drug Administration

In Parkinson's disease model rats, the silk amino acid, silk amino acid, and tyrosine compositions of the present invention were dissolved in sterile purified water (5 mL / kg) at doses (50, 160 or 500 mg / kg) for 30 days from the induction of Parkinson's disease for 31 days. By oral administration. Tyrosine was also administered orally (40 mg / kg) and _L- DOPA (positive control) was injected intraperitoneally (25 mg / kg).

Experimental Example 3 Pole Test

On the 15th and 30th day of the test, 30 minutes after the administration of the test substance, the time until the animal's head is turned upward with the head of the animal facing up on the top of the rod with a rough surface of 10 mm diameter and 55 cm in length, ie The turning time (T _turn ) and the time to the bottom, ie landing time (T _LA ), were measured. The percentage of animals falling above physical function during landing was recorded, in which case the T _LA was 90 seconds.

On the 15th day, normal animals were not dropped, but 33% of 6-OHDA-treated Parkinson's disease animals showed motor dysfunction due to brain injury. The fall from Pole was presumed to be due to the fall of the forearm's body support due to brain injury by falling on the ground rather than head down. The composition-administered group of Example 2 had no effect at the administration of 50 mg / kg and 500 mg / kg, but only 17% of animals fell at the intermediate dose of 160 mg / kg. On the other hand, no improvement was found in all doses in the silk amino acid group, but only 17% of the animals in the 40 mg / kg tyrosine group dropped. In particular, there were no falling animals in the _L- DOPA (positive control) 25 mg / kg group (Table 3).

At 30 days after 6-OHDA administration, 86% of brain-induced animals fell during the pole test, indicating that the brain injury was further developed. However, the improvement effect of the composition of Example 2 was further enhanced than day 15, only 15% and 17% dropped at 50 and 160 mg / kg, respectively, and showed a similar effect to L-DOPA (positive control) at 500 mg / kg There was no animal falling. In addition, only 15% of animals fell in the tyrosine-treated group, and no falling animals were observed in the _L- DOPA (positive control) -treated group as in the 15th day. In the silk amino acid group, 50 mg / kg to 500 mg / kg was ineffective at 15 days compared to the Parkinson's group. On the 30th, the Parkinson's group had a 53% increase in drop rate, whereas the silk amino acid group was 50 mg / kg to 500 mg / kg. When kg was administered, the drop rate was increased by up to 16%, indicating that the drop rate increase was significantly reduced compared to the Parkinson's group.

TABLE 3

On the other hand, on the 15th day, the T _turn , which is the time the animal heads down on the pole, and the T _LA , which is the time to reach the bottom, was measured. In comparison, Parkinson's disease-causing animals were significantly delayed by 34 and 48 seconds, approximately 2 and 1.5 times that of normal animals, respectively. This dysfunction was significantly improved when the composition of Example 2 was administered. T _turn and T _LA were 14 and 46 seconds at 50 mg / kg, 14 and 33 seconds at 500 mg / kg, and 500 mg, respectively. At / kg, dose-dependently decreased to 15 and 31 seconds, respectively, and returned to normal at high doses. Interestingly, in the tyrosine-administered group, there was little effect at day 15, but at 30 days, both were significantly effective at 17 and 43 seconds. T _turn and T _LA decreased in the silk amino acid treatment group than in the Parkinson's group, but not in the high dose group. _{In the L-} DOPA (positive control) group, the T _turn and T _LA were significantly improved to about 8 and 30 seconds, respectively, showing the best effect.

At 30 days after Parkinson's disease, T _turn and T _LA were delayed to 46 and 81 seconds, respectively, indicating more severe physical dysfunction than day 15. This severe bodily dysfunction was significantly improved overall with administration of the composition of Example 2, with a significant decrease in T _{turn in the} 160 mg / kg dose group and T _LA in the 50 mg / kg and 500 mg / kg dose groups. Interestingly, tyrosine, which had little effect on day 15, significantly reduced both T _turn and T _LA to 17 and 43 seconds on day 30. The silk amino acid administration group shortened T _turn and T _LA , but the effect was lower than that of the tyrosine administration group and the composition administration group of Example 2 in which tyrosine and silk amino acid were mixed. On the other hand, in the _L- DOPA (positive control) group, T _turn and T _LA were significantly improved to 18 seconds and 40 seconds, respectively (Table 4).

Table 4

Example 4 Cylinder Test

When the animal is placed in a glass cyclider (21 x 34 cm) equipped with CCTV and stands up 10 times, the ipsilateral forelimb (the left and right brains) is responsible for the other side of the body, and the left forefoot is damaged when the right brain is damaged. Loss of function), i.e., the ratio of force to the right forefoot.

On day 15, 38% of normal animals used the right forefoot during standing up 10 times because normal animals used the left and right forelimbs at about the same frequency. In contrast, Parkinson's disease-induced animals that injured the right brain with 6-OHDA and reduced the function of the left foot increased the use of intact right paw to 68%. When the composition of Example 2 was administered to the right foot biased use, the dose-dependent frequency of bias was improved. In the 500 mg / kg-administered group, the concentration was restored to 50%. In addition, tyrosine or _L- DOPA (positive control) group also significantly improved the use of the right forefoot. In contrast, 50 mg / kg and 160 mg / kg did not show a significant effect in the silk amino acid group, but improved to a significant level in the 500 mg / kg group (Table 5).

On the other hand, on the 30th day, the effect of the composition of Example 2 was further enhanced, and improved to the normal control level at the high dose as well as the 160 mg / kg administration group. The tyrosine-treated group was slightly weaker than the 15th day, and the _L- DOPA (positive control) -treated group showed the same effect as the normal control group. In the silk amino acid group, the levels were similar at both 15 and 30 days (Table 5).

Table 5

Experimental Example 5 Apomorphine turning test

Apomorphine (0.05 mg / kg) was injected subcutaneously and the number of times the animal was contralateral circling for 1 hour using CCTV was recorded. At this time, the rotation was recorded once when the 360-degree full rotation using CCTV.

On the 15th day of the experiment, subcutaneous injection of 0.05 mg / kg of apomorphine showed two rotations per minute in normal animals, whereas the rotational speed was four times higher in eight times per minute in Parkinson's disease-causing animals. This rotational phenomenon was dose-dependently improved by administration of silk amino acids, tyrosine, _L- DOPA (positive control), Example 2 composition (Table 6).

On the 30th day, the number of rotations in the Parkinson's disease-induced group was more than 7 times greater than that of the normal animals 5 times per minute, indicating severe brain damage. This phenomenon was significantly reduced to half level in the composition 500 mg / kg administration group, _L- DOPA (positive control) administration group, tyrosine administration group of Example 2 (Table 6).

Table 6

Experimental Example 6 Measurement of Brain Cell Death Rate

The animals were anesthetized by anesthesia 30 minutes after the administration of the test substance on the following day, i.e., on days 16 and 31. Brain tissues were perfused with cold saline, fixed in 10% neutral formalin solution for 24 hours, and stored in 30% sucrose solution for 1 week. 40 μm frozen sections were prepared so that the fixed brain tissue showed black matter (AP -3.00 ~ -3.16).

In order to determine the degree of death of dopamine neurons by 6-OHDA, immunostaining was performed on TH to measure the number of neurons containing TH. In order to remove endogenous peroxidase present in the tissues, the cells were treated with 3% hydrogen peroxide (H ₂ O ₂ ) and then pretreated with 3% bovine serum albumin (BSA) for 120 minutes at room temperature. Tissue slides were reacted with anti-TH primary antibody (rabbit polyclonal IgG, 1: 100, Millipore, Temecula, USA) overnight at 4 ° C and washed several times with phosphate-buffered saline (PBS). Again reacted with a secondary antibody, green Alexa Fluor 488- or red Alexa Fluor 594 (red) -conjugated goat anti-rabbit IgG (1: 200, Molecular probes, Eugene, USA) for 1 hour at room temperature, washed with PBS and then sealed It was.

Immunostaining to observe the protective effect of TH-positive neurons, or dopamine neurons, on the area of the brain's chromosomal nitric oxide (SN) area caused by 6-OHDA administration resulted in a clear dense cell body in normal animals. In addition, a large amount of TH was confirmed to be positive in the cell bodies as well as fibers composed of axons and dendrites (FIG. 1A). On the other hand, most dopamine neurons were killed at day 15 in Parkinson's disease-causing animals, and the remaining cells also showed a shape of axons and dendritic densities (FIG. 1B). For the composition of Example 2, the dopamine neurons remained almost normal in the 500 mg / kg administration group (FIG. 1D) compared to the 50 mg / kg administration group (not shown) and the 160 mg / kg administration group (FIG. 1C). This was confirmed. On the other hand, tyrosine and _L- DOPA (positive control) 25 mg / kg administration group also showed a good effect (Fig. 1E and 1F, respectively). The silk amino acid group did not have a significant effect on the degree of dopamine neuron killing at low doses, but the cell protective effect was increased in a concentration-dependent manner, but was not as effective as the other groups (not shown).

Most dopamine neurons were killed in Parkinson's disease-induced animals even after 30 days of 6-OHDA administration, compared to normal animals (FIG. 2A). The remaining cells also showed axonal and dendritic densities (FIG. 2B). Significant cytoprotective effect was observed in the administration group of 160 mg / kg of the composition of Example 2 on the 30th day compared with the 15th day (FIG. 2C), and remained almost normal in the high dose 500 mg / kg administration group (FIG. 2D). Meanwhile, the composition of Example 2 was lower than the group administered with 500 mg / kg, but the tyrosine-administered group showed excellent cytoprotective effect (FIG. 2E), and a good effect was also confirmed in the _L- DOPA (positive control) -administered group (FIG. 2F). Silk amino acid treatment group also increased cytoprotective effect compared to day 15, but was not significant (not shown).

TH-positive cells were counted to quantify the effect of 6-OHDA on dopamine neuron killing and test substance protection. In the Parkinson's disease-inducing group, the number was significantly reduced to about 20. Example 2 In the administration group of the composition, the low dose (50 mg / kg) did not recover to about 30, but the medium dose (160 mg / kg) recovered to 55, especially the high dose (500 mg / kg). Esau excels at about 145. In comparison, the tyrosine (40 mg / kg) group recovered to about 115 and showed a significant cytoprotective effect. The _L- DOPA (positive control) 25 mg / kg group also did not meet the effect of the high dose group of the Example 2 composition. Significant improvement was shown. On the other hand, the silk amino acid administration group had no significant effect at low dose (50 mg / kg), but significantly recovered to 53 at medium dose (160 mg / kg) and improved to about 83 at high dose (500 mg / kg). (Table 7).

On the 30th day, the number of dopamine neurons in Parkinson's disease-causing animals was about 50, which was significantly reduced compared to 250 in normal animals. With respect to the neuronal cytotoxicity of 6-OHDA, the composition of Example 2 was restored to about 70 at low doses, significantly improved to about 125 at medium doses, and particularly to about 190 at high doses for excellent cytoprotective efficacy. Showed. Tyrosine also showed excellent protection against about 145. On the other hand, _L- DOPA (positive control group) showed a good effect of about 120, but showed a relatively weak efficacy compared to the high dose or tyrosine administration of the composition of Example 2, silk amino acid in the high dose administration group is about 102 cells protection The effect was the lowest.

TABLE 7

<Experiment 7> Dopamine and its degradation product concentration analysis

Dopamine and its metabolite 3,4-dehydroxyphenylacetic acid (DOPAC) and 3-methoxy-4-hydroxyphenylacetic acid (homovanillic acid (HVA)) were analyzed from the brain tissues of some animals. Perfusion of the cerebral tissue with cold saline, rapid extraction and weighing was performed, followed by adding 0.1 M perchloric acid (500 μL) and 10-5 M isoproterenol (50 μL) containing 0.05% disodium ethylenediamine tetraacetic acid (EDTA). Homogenized.

  The homogenate was centrifuged at 12,000 rpm (0 ° C.) for 20 minutes, the supernatant was diluted 30 times, and 10 μL was analyzed by injection into a high performance liquid chromatograph (HPLC). The HPLC system consists of a solvent delivery pump (Waters Model 1525), an electrochemical detector (Waters Model 2465), and a Watchers 120 ODS-BP column (5 μm, 150 x 4.6 mm), with 0.1 M citric acid monohydrate, 0.1 M for the mobile phase. Sodium acetate, 100 μM EDTA, 0.01% sodium octane sulfonate (SOS) and 7% methanol were used, and the flow rate was 1 mL / min.

The concentrations of dopamine and its metabolites, DOPAC and HVA, in the brain tissues of 6-OHDA were reduced by 45% and about 25%, respectively. Fell below an analytical level. The degradation of this dopamine and its metabolites by 6-OHDA was all significantly recovered by administering the composition of Example 2 (500 mg / kg). Tyrosine also effectively restored the concentrations of dopamine and its metabolites. In particular, 25 mg / kg of _L- DOPA (positive control) restored dopamine and HVA above normal levels. Silk amino acids, however, effectively recovered HVA, but dopamine and DOPAC increases were not significant (Table 8).

Table 8

The composition for preventing and improving cerebral disease including silk amino acid and tyrosine of the present invention as an active ingredient has an excellent effect in preventing and treating Parkinson's disease, cerebral infarction, stroke or dementia.

Claims (10)

Composition for preventing, treating and improving brain diseases comprising silk amino acid and tyrosine as active ingredients. The method of claim 1, The brain disease is Parkinson's disease, cerebral infarction, stroke or dementia, characterized in that the composition for preventing, treating and improving brain disease. The method of claim 1, A composition for preventing, treating and improving brain diseases, characterized by protecting dopamine neurons or increasing the concentration of dopamine. The method of claim 1, The composition is a composition for preventing, treating and improving brain diseases, characterized in that the pharmaceutical composition or food composition. The method of claim 1, The silk amino acid is a product for preventing, treating and improving brain diseases, characterized in that the product of the acid hydrolysis of the silk protein. The method of claim 1, The composition is a composition for preventing, treating and improving brain diseases, characterized in that it comprises 0.01 to 50 parts by weight of tyrosine, based on 100 parts by weight of the composition. A method for preventing and treating brain diseases, comprising administering to a patient a composition for preventing and improving brain diseases according to any one of claims 1 to 6. Method for producing a composition for preventing, treating and improving brain disease, comprising mixing silk amino acid and tyrosine. Use of the prevention, treatment and improvement of brain diseases of the composition comprising a silk amino acid (typical amino acid) and tyrosine (tyrosine) as an active ingredient. Use of a composition comprising silk amino acid and tyrosine as an active ingredient for the manufacture of a medicament for the prevention, improvement and treatment of brain diseases.

Images