KR100797096B1 - Composition for preventing or improving the ischemia damage containing hydrolysates of trichiurus lepturus - Google Patents

Abstract

A composition containing hydrolysates of Trichiurus lepturus is provided to prevent or improve ischemia damage without harmful effects on human body by preventing damage of nerve cells in brain hippocampus CA1 region and inhibiting apoptosis of nerve cells by ischemia. A composition for preventing or improving ischemia damage contains 0.1-99 wt.% of hydrolysates of Trichiurus lepturus which is prepared with a protein-hydrolyzing enzyme selected from alcalase, protex, paratase and neutrase, wherein the ischemia damage is Alzheimer's disease, vascular dementia or Huntington's disease, and the composition is a food composition formulated as food, food additives or drink.

Description

Composition for preventing or ameliorating ischemic cerebrovascular disease including cutlass hydrolyzate {COMPOSITION FOR PREVENTING OR IMPROVING THE ISCHEMIA DAMAGE CONTAINING HYDROLYSATES OF TRICHIURUS LEPTURUS}

1 is a schematic diagram showing a process of obtaining a cutlas hydrolyzate according to an embodiment of the present invention,

Figure 2 is a measurement of the lactate dehydrogenase secretion in hypoxic environment of PC12 culture cells according to the dose of the saltwater hydrolyzate in order to determine the neuronal cell death of the saltwater hydrolyzate according to an embodiment of the present invention Is a graph,

FIG. 3 is a photograph showing staining of hippocampal tissue of experimental animals 4 days after the induction of cerebral ischemia with cresyl violet, in order to determine the neuronal cell death prevention effect of the cutlass hydrolyzate according to one embodiment of the present invention. ego,

Figure 4 is a graph measuring the cell number of the survival of neurons surviving neurons 4 days after the induction of cerebral ischemia in order to determine the neuronal cell death prevention effect of the cutlass hydrolyzate according to an embodiment of the present invention,

FIG. 5 is a graph showing glial cell GFAP and microprotuberant glial markers of hippocampal tissues of experimental animals 4 days after the induction of cerebral ischemia, in order to determine the neuronal cell death prevention effect of the cutlass hydrolyzate according to an embodiment of the present invention. Iba-1 is a marker of the image observed by immunohistochemistry (immunohistochemistry) method.

[Industrial use]

The present invention relates to a composition comprising a gallbladder hydrolyzate having a neuroprotective effect, and more particularly, to a composition for the prevention and improvement of cerebral ischemic disease comprising the cutlass hydrolyzate.

[Private Technology]

Of the total population released in 2005, the National Statistical Office recently made up 9.1% of the population aged 65 and over, and the proportion of young people aged 14 and younger is decreasing. The proportion of the population aged 65 and over is 7.2% in 2000. It is estimated that our society has already entered an aging society. In addition, as of 2003, the average life span of Korean people is 77.5 years old, and the elderly population is rapidly increasing. Meanwhile, Korea's GNI per capita announced by the National Statistical Office was $ 14,162 in 2004, and it is increasing by more than 10% every year. Due to such changes in social conditions, the number of deaths from infectious diseases, which are the major causes of death, has been drastically decreasing, while the number of deaths from degenerative diseases such as cancer, cerebrovascular disease, and diabetes is steadily increasing.

Cerebrovascular disease, which is the second largest cause of death in Korea, is also called stroke, and is caused by clogging or bursting blood vessels in the brain. It can be divided into two types: cerebral infarction and cerebral hemorrhage.

The cerebral infarction is also referred to as ischemic cerebrovascular disease, and it is a disease caused by brain cells dying because the brain is blocked from receiving blood and oxygen. Brain hemorrhage is also called hemorrhagic cerebrovascular disease. This is the case when the brain is damaged due to flow. When cerebral infarction or cerebral hemorrhage causes loss of brain function, physical symptoms such as apparent paraplegia, speech and consciousness disorders may appear, and in severe cases, death.

Among these, ischemic cerebrovascular disease is a disease caused by delayed neuronal death in the hippocampus CA1 region due to a decrease in blood flow in the brain and supply of oxygen and glucose (Kirino, Brain Res., 239, pp 57-69, 1982; Petito et al., Neurology, 37, pp 1281-1286, 1987; Pulsinelli et al., Ann. Neurol., 11, pp 491-498, 1982). In addition, the ischemic brain disease is known to be caused by the lack of blood supply to the brain in pathological conditions such as stroke or heart attack (Nedergaard, Acta. Neurol. Scand., 77, pp81-101, 1988; White et. al., Neurology, 43, pp 1656-1665, 1993).

The cause of delayed neuronal death in the hippocampal CA1 region caused by transient cerebral ischemia and reperfusion is caused by the influx of calcium into cells (Hara et al., Brain Res. Bull., 29, pp659-665, 1992; Nedergaard , Acta Neurol. Scand., 77, pp81-101, 1988), known as free radical-related neuronal damage and glutamate-receptor mediated neuronal damage (Won et al., Neurosci. Lett., 301). , pp 139-142, 2001). That is, when cerebral ischemia occurs, depolarization of neurons occurs, and glutamate of synapses is released, thereby increasing the concentration of extracellular glutamate.

In addition, the reversal of active transport caused by the reduction of Adenosine Triphosphate (ATP) accelerates the accumulation of extracellular glutamate. Accumulation of extracellular glutamate is characterized by N-methyl-D-aspartic acid (NMDA), ??-amino-3-hydroxy-5-methyl-4 -isoxazole-propionic acid (AMPA), and metabotropic glutamate receptor. ), Resulting in an excessive influx of calcium into cells (Olney et al., Science, 244, pp1360-1362, 1989). Intracellular influx of calcium triggers the activity of calcium-dependent proteases, lipases and modulators, and in turn leads to the generation of cytotoxic molecules, including free radicals, that break down cellular structures such as DNA and cell membranes, causing neuronal death. .

Stroke treatment includes acute treatment and preventive treatment for prevention of recurrence, and the treatment effect can be seen only if you go to the hospital within 3 hours after the stroke and receive acute stroke treatment. That is, once cerebrovascular disease is due to irreversible nerve cell damage, it is known to cause a decrease in motor skills, sexual performance, memory loss.

In addition, it has been recently reported that regeneration of neurons is possible, but the regeneration of these neurons is not easy considering the histological characteristics.

Considering the characteristics of cerebrovascular disease, cerebrovascular disease tends to be prevented rather than treated, and the necessity of preventing the cerebrovascular disease by continually ingesting a substance that can protect the nerve cells is newly introduced. It is recognized.

In response to this need, efforts are underway to find active ingredients from marine natural-derived substances that are effective in protecting neuronal cells.

The present invention effectively prevents neuronal cell damage caused by cerebral ischemia, inhibits neuronal cell death caused by cerebral ischemia, and ischemic brain comprising as an active ingredient a composition and a gallas hydrolyzate which are contained as an active ingredient. An object of the present invention is to provide a food composition for preventing or improving vascular disease.

In order to achieve the above object, the present invention provides a composition for the prevention or improvement of ischemic cerebrovascular disease comprising a cutlass hydrolyzate as an active ingredient and having a neuroprotective effect.

In addition, in order to achieve the above object, the present invention provides a food composition for preventing or improving ischemic cerebrovascular disease comprising a cutlass hydrolyzate as an active ingredient and having neuroprotective effect. Examples of the food composition of the present invention include food, food additives or beverages.

Hereinafter, the present invention will be described in more detail.

The inventors of the present invention, while studying a composition for preventing and preventing cerebrovascular disease, which ensures human safety, from substances derived from marine natural products, can effectively prevent damage to nerve cells in the CA1 region of the cerebral hippocampal tissue, which is known to be sensitive to cerebral ischemia. It was confirmed that the present invention was completed based on this.

The present invention relates to a composition for the prevention or improvement of ischemic cerebrovascular disease comprising a cutlass hydrolyzate as an active ingredient.

Galchi ( Trichiurus Lepturus ) is a fish of the sea bass, which is inhabited by the entire seabed or coastal waters of the continental shelf in the temperate or subtropical seas of the world, such as Southwestern Korea, Japan, China, Southeast Asia, Australia and Indian Ocean. The size of mature fish is about 150cm long, flat to the side, without scales, and the body is bright silver white.

The main nutrients of cutlass are protein, amino acids, unsaturated fatty acids and calcium. Proteins in the skin are mainly collagen or elastin. It also contains high amounts of essential amino acids such as lysine, phenylalanine, methionine, leucine and valine. Baked cutlass when eaten when there is no appetite has the effect of increasing appetite, is known to have a tonic effect. In addition, in oriental medicine, the skin of cutlass is known to be effective for hemostasis, and the oil of cutlass is also applied to hepatitis treatment.

Cutlass hydrolyzate of the present invention may be prepared according to a conventional manufacturing method for preparing fish hydrolyzate, and preferably may be prepared by hydrolysis using an enzyme. More preferably, the salted hydrolyzate of the present invention is added to distilled water and completely pulverized to remove the intestine, followed by reaction with the addition of a proteolytic enzyme. After removing the oil layer by centrifuging the mixed solution from which the insoluble matter is removed, the mixed solution from which the oil layer is removed may be obtained by lyophilization.

At this time, the distilled water may be preferably added to 1500ml to 300ml, more preferably 1000ml to 700ml to 1kg of cutlass, the grinding process of the cutlass can be carried out using a conventional mill, for example a domestic mixer It can be carried out by mixing twice for 3 minutes using the proteolytic enzyme is preferably one or more proteolytic enzymes selected from the group consisting of alkalase, preborim, protex, paratase and neutrase, More preferably, it may be Alcalase enzyme (Alcalase enzyme, 2%), the alkalase enzyme is preferably 0.1g to 2g, more preferably 1.0g to 1.4g per kg of cutlass before the grinding The enzyme reaction may be appropriately performed according to the type of enzyme, preferably 50 ° C to 70 ° C, more preferably 57 ° C to 63 ° C. 1 hour to 3 hours, preferably 1 hour 30 minutes to 2 hours 30 minutes, most preferably 2 hours in a shaking incubator of, the inactivation of the enzyme according to the type of enzyme 3 to 25 minutes, preferably 7 to 15 minutes, at 83 ° C to 97 ° C, preferably 87 ° C to 93 ° C, most preferably 90 ° C, Most preferably, heat treatment may be performed for 10 minutes, and the insolubles may be removed by centrifugation at 6000 rpm to 8000 rpm, preferably 6500 rpm to 7700 rpm, and more preferably 7000 rpm to 7300 rpm. The oil layer is removed from 83 ° C to 97 ° C, preferably from 87 ° C to 93 ° C, most preferably from 90 ° C to 10 minutes to 30 minutes, preferably from 15 minutes to 25 minutes, most preferably from 17 ° C. In minutes After 23 minutes of heat treatment, the oil layer may be removed by centrifugation at 7500rpm to 9300rpm, preferably 8000rpm to 8800rpm, more preferably 8400rpm to 8600rpm. The hydrolyzate of the remaining cutlass obtained by removal can be obtained, and the obtained cutlass hydrolyzate can be stored using a deep freezer.

In addition, the cutlass hydrolyzate may be one in which water is completely removed by concentrating and lyophilizing the obtained hydrolyzate.

In the present invention, the cerebrovascular disease refers to a term that refers to various diseases caused by abnormalities of blood vessels of the brain, and cerebrovascular diseases are largely classified into hemorrhagic cerebrovascular disease and ischemic cerebrovascular disease.

The ischemic cerebrovascular disease is a disease caused by delayed neuronal death in neurons of the CA1 region of the hippocampus known to be sensitive to cerebral ischemia because brain blood flow is decreased and oxygen and glucose are not normally supplied. Ischemic cerebrovascular disease may include Alzheimer's disease, dementia or Huntington's disease.

The cutlass hydrolyzate of the present invention is exposed to hypoxic environment by exposing PC12 cells, which are adrenal gland neurons, to measure the concentration of lactate dehydrogenase released from the cells. After inducing cerebral ischemia, in all experiments measuring the efficacy of staining neurons, the neuronal cell death caused by cerebral ischemia was significantly suppressed, and the neuroprotective effect was excellent.

The composition for preventing or improving cerebral ischemic disease of the present invention may include 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight of the cutlass hydrolyzate based on the total weight of the composition.

The composition is suitable for nutrients, vitamins, electrolytes, flavors, colorants, neutralizers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols And a carbonation agent used for the carbonated beverage. The composition may be added independently or in combination of the above components. The content of the additional component may be preferably added in the range of 0.1 to 20 parts by weight per 100 parts by weight of the cutlass composition.

The present invention provides a food composition or a pharmaceutical composition comprising a cutlass hydrolyzate as an active ingredient for the prevention or improvement of the cerebral ischemic disease.

The food composition or the pharmaceutical composition including the cutlass hydrolyzate as an active ingredient may further include appropriate carriers, excipients and diluents commonly used in the preparation thereof.

Carriers, excipients, and diluents that may be included in the composition comprising the hydrolases of the present invention as an active ingredient include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The food composition comprising the salt extract of the present invention as an active ingredient may be used in oral formulations such as powders, granules, tablets, suspensions, emulsions, and syrups according to conventional methods.

The oral formulation is meant to include a solid preparation and a liquid preparation for oral administration, the solid preparation for oral administration may include tablets, pills, powders, granules, capsules, etc., the solid preparation is At least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin and the like may be prepared in the digested product. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and serums.In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Preferred dosages of the compositions containing the cutlass hydrolyzate of the present invention vary depending on condition and weight, extent of disease, drug form, route of administration and duration, and may be appropriately selected by those skilled in the art. Preferably, the composition containing the saltwater hydrolyzate is 0.0001 to 1000mg / kg per day based on the amount of the saltwater hydrolyzate of the present invention, and in order to be more effective, it may be administered at 0.01 to 100mg / kg. Administration may be administered once a day or may be divided several times. The dosage and frequency of administration are not intended to limit the scope of the invention in any aspect.

The composition of the present invention can be used as food. In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all foods, food additives, health functional foods and beverages.

Examples of the food to which the cutlass hydrolyzate of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and functional foods. In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, and the like, and natural seasonings (eg, ramen soup). The food, beverage or food additives may be prepared by a conventional manufacturing method.

Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food processed and designed to fully express the gymnastics function related to the living body. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.

In the present invention, the drink is a generic term for drinking to quench thirst or to enjoy the taste and is intended to include a functional drink. The beverage is not particularly limited to other ingredients other than including the cutlass hydrolyzate as an active ingredient as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention, in addition to the composition of the present invention, the flesh for preparing natural fruit juice, fruit juice beverage, vegetable beverage It may further contain.

In addition to the above, the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0 to 20 parts by weight per 100 parts by weight of the hydrolyzate of the present invention.

Functional beverage in the present invention is a biological defense rhythm control, disease prevention and the like having a beverage group or a beverage composition that has added value to the beverage by using physical, biochemical, biotechnological techniques, etc. to function and express the function of the beverage to a specific purpose Means a beverage that is designed and processed to fully express the gymnastics function related to recovery.

The functional beverage is not particularly limited to other components except for containing the hydrolyzate of the present invention as an essential component in the indicated ratio, and may contain various flavors or natural carbohydrates, etc. as additional components, such as ordinary drinks. Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tautin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention.

In addition, in the food composition aimed at the effect of preventing or improving cerebral ischemic disease, the amount of the hydrolyzate of the cutlass may include 0.01 to 15% by weight of the total food weight, the beverage composition is 0.02 based on 100 ml To 5 g, preferably 0.3 to 1 g.

Compositions and foods comprising the salt extract of the present invention as an active ingredient have a neuroprotective effect that significantly inhibits cell death of PC12 cells, which are adrenal medulla-derived cell lines exposed to a hypoxic environment, and ischemia-induced male Mongolian gerbil ( Mongolian gerbil, Harlan, USA) has been shown to have a superior neuronal cell protective effect that significantly inhibits neuronal cell death in the hippocampus region.

Hereinafter, preferred embodiments of the present invention are described. The following examples are only for illustrating the present invention and the present invention is not limited by the following examples.

EXAMPLE

Example 1 Preparation of Cutlass Hydrolyzate

After removing the internal organs of freshly cut sprigs, add 800 ml of distilled water to 1 kg of spalls, and thoroughly grind them by mixing twice for 3 minutes using a domestic blender, followed by Alcalase enzyme (2%, Novozyme A). / S, Bagsvaerd, Denmark) was added to the reaction in a shaking incubator at 60 ℃ for 2 hours to obtain a hydrolyzate. The obtained hydrolyzate was heated at 90 ° C. for 10 minutes to inactivate the enzyme, and after centrifugation at 7200 rpm, only supernatant was obtained to remove insoluble matters such as bone, and the supernatant was further heated at 90 ° C. for 20 minutes. Then, the oil layer was removed by centrifugation at 8520 rpm, and the remaining water layer was lyophilized to obtain the hydrolyzate. The obtained hydrolyzate was stored in a deep freezer until use.

Example 2: Determination of Neuronal Protective Effect during Brain Ischemia of Cutlass Hydrolyzate

A method of measuring lactate dehydrogenase concentrations induced by the adrenal PC12 cell line derived from the adrenal gland in a hypoxic environment and then released extracellularly and the male body weight 65g to 75g Mongolian gerbil (Harlan, USA) was anesthetized by exposing the common carotid artery and inducing cerebral ischemia, and then staining the hippocampal tissue of Mongolian gerbils.

2-1: Determination of Neuroprotective Effect during Brain Ischemia of Cutlass Hydrolyzate by Measuring Lactate Dehydrogenase (in vitro)

PC12 cells were used to measure neurite growth and expression of growth factors. In addition, CoCl2 was administered to PC12 cells to induce neuronal damage through a hypoxic environment, and then the concentration of lactate dehydrogenase (LDH) secreted from the cultured cells into the extracellular culture was measured to determine whether the neurons were damaged. Confirmed.

PC12 cells were obtained from the adrenal gland of rats purchased from Korean experimental animals and cultured with Dulbecco's Modified Eagle Medium (DMEM, Mediatech, USA) added 7% fetal calf serum and 7% horse serum. 37% containing 6% CO2 concentration The cells were cultured under the conditions, and the measurement of lactate dehydrogenase was measured by taking a cell culture solution at 20 hours when almost secretion was completed from damaged and destroyed cells.

For the PC12 cell line culture broth, the distilled water and the cutlas hydrolyzate obtained in Example 1 were prepared at concentrations of 0.001 mg / ml, 0.005 mg / ml, 0.01 mg / ml, 0.05 mg / ml, 0.1 mg / ml and 0.5 mg / ml, respectively. After 30 minutes, 150 μM of CoCl 2 was treated. After culturing the PC12 cells treated with CoCl ₂ at 37 ° C. for 24 hours, a culture solution of PC12 cells was obtained, and the concentration of lactate dehydrogenase secreted from the cells was set as the Zhong Sheng Biotech standard reagent. Enzymatic mechanical method using Beckman DU-640 absorbance photometer (Hou et al. J. Neurosci. Res., 74, pp123-133, 2003; Yamakawa et al. Neurol. Res., 23, pp522-530, 2001), and the results are shown in FIG.

As shown in FIG. 2, in the group treated with the hydrolyzate, the secretion rate of lactate dehydrogenase was relatively lower than that of the control group which treated only the solvent and then caused the hypoxic environment. In the case of the treated hydrolyzate, the secretion amount of lactate dehydrogenase decreased up to 0.1 mg / ml, but the concentration of lactate dehydrogenase was higher than that. An increase in the ratio was observed. It was also confirmed that the lactate dehydrogenase secretion was reduced to about 45% in the case of the cells treated with the hydrolyzate of the cutlass at a concentration of 0.1 mg / ml.

As a result of the above, it was confirmed that the hydrolyzate of the cutlass had a neuroprotective effect and an improvement effect, and the neuroprotective effect and an improvement effect were the greatest at a concentration of 0.1 mg / ml.

2-2. Determination of Neuronal Cellular Protective Effects of Cutlass Hydrolyzate in Brain Ischemia by Animal Experiment

Animal experiments using gerbils confirmed the neuroprotective effect of hydrolyzate.

2-2-1. Breeding of Experimental Animals

40 male Mongolian gerbil (Meriones unguiculatus, Halan, USA), weighing between 65 and 75 grams, with a constant contrast cycle of light from 7 am to 7 pm, temperature from 21 ° C. to 25 ° C. and 45% to 65% It was raised under the condition of relative humidity of. For the feed of experimental animals, general dry pellet food (Korean experimental animals, Korea) was used, and feed and water were freely consumed.

2-2-2. Evaluation of Neuronal Cellular Protective Effects of Cutlass Hydrolyzate in Cerebral Ischemia

The hydrolyzate of Example 1 in distilled water was dissolved orally administered to the experimental animal to 100 mg / kg of body weight of the experimental animal using an esophageal needle every day from a week ago.

After injecting the saltwater hydrolyzate for one week, the experimental animal was anaesthetized using a gas mixed with 3% isoflurane (isoflurane, Baxtor, USA) in a gas mixed with nitrogen and oxygen at 7: 3. Surgery was performed on the experimental animals while maintaining anesthesia of the experimental animals by using the gas of 2.5% isoflurane mixed with the nitrogen and oxygen mixed gas. Surgery for the experimental animals was performed by cutting and disinfecting the hair of the neck and making an incision to expose both warm arteries and ligation for 5 minutes using an aneurysm clip (Aneurysm clip, Staelting, USA). Removal and reperfusion were carried out. At this time, each experimental group was checked for blood circulation in the central artery of retina by using an ophthalmoscope to confirm whether there was complete obstruction of the entire artery. The body temperature was measured by inserting a rectal thermometer during the induction of cerebral ischemia, and the body temperature was constantly maintained at a normal body temperature of 36.7 ° C. to 37.3 ° C. using a heating pad which is automatically adjusted according to the temperature of the experimental animal.

The experimental group was anesthetized by intraperitoneal injection of thiopental sodium at 30 mg / kg body weight 4 days after induction of cerebral ischemia, and then at 4 ° C. containing 1,000 IU per 1,000 ml. Physiological saline was injected into the left ventricle and washed perfusion. Perfusion fixation was performed using 4% 4% paraformaldehyde (in 0.1 M phosphate buffer (PB, pH 7.4)) at 4 ° C.

Using a bone cutter, the brain was extracted by opening the head bone space of the experimental animals after the perfusion fixation. The brains of the extracted experimental animals were post-fixed for 6 hours using 4% 4% paraformaldehyde (in 0.1 M phosphate buffer; PB, pH 7.4). After the post-fixed brain was placed in a 30% sucrose solution (in 0.1 M phosphate buffer) and settled to the bottom, the brain tissue was slid with a sliding microtome (Reichert-Jung, Germany). Tissue sections were cut to thickness. The tissue sections were stored in 6-well plates containing a storage solution and stored at 4 ° C. until staining was performed.

Among the tissue sections, tissues with well-formed hippocampal formation were selected, and washed three times with 0.01 M PBS for 10 minutes to remove the preservatives from the tissue sections. The washed tissue sections were plated on gelatinized slides and dried sufficiently at 37 ° C. After sufficiently immersing the above-mentioned tissue sections in distilled water for a while, the tissue sections were stained for 1 minute in 2% cresyl violet acetate (Sigma, USA) solution. The stained tissue sections were sufficiently washed with running water to remove excess dye from the slides. The tissue sections from which the excess dye was removed were briefly immersed in distilled water and then treated with 50%, 70%, 80%, 90%, 95% and 100% ethanol solution to perform dehydration and excess cresyl violet washing. After confirming that the tissue section was visible in the Nissle body (Nissle body), it was immersed in xylene (Junsei, Japan) and then transparent and encapsulated in Canada Balsam (Kanto, Japan).

The control group was prepared in the same manner as above except that only the distilled water used to dissolve the molten hydrolyzate was used, and the normal group did not perform ischemia-reperfusion surgery, nor was it administered any substance including the molten hydrolyzate. Did.

Axiphot microscope with Carl digital camera (Axiocam, Cal Zeiss, Germany) was attached to each tissue section to enlarge 25 times the total hippocampus area and 200 times the CA1 area. . FIG. 3 shows the results of photographing the control group, the normal group and the experimental group administered with 100 mg / kg of cutlass hydrolyzate.

A and C of Figure 3 represent the total hippocampal region of the normal group and the control group, respectively, B and D represent the CA1 region of the hippocampus of the normal group and the control group, respectively. In addition, E and F of Figure 3 shows the total hippocampal region and the CA1 region of the hippocampus of the experimental group administered 100mg / kg of the saltwater hydrolysis.

In order to verify the significance, Figure 3 was taken to pick the most general part of each group.

As can be seen in Figure 3, the normal group was able to observe the deep staining of nerve cells throughout the area of the hippocampus. However, it was observed that a large portion of neurons were not stained by neural cell death due to cerebral ischemia in the control group that was not administered with the saltwater hydrolyzate. On the other hand, in the experimental group administered with 100 mg / kg of the cutlass hydrolyzate, it was confirmed that the cells were deeply stained in the whole area of the hippocampus, and the cuticle hydrolyzate significantly inhibited the neuronal death.

In addition, neurons of the tissue sections stained in purple were counted using an image analyzer (Optimas 6.5, USA) program. One-way ANOVA test was performed to verify the significance of each group, and the data were expressed as mean and standard deviation.

The result of counting the nerve cells of the tissue sections is shown in Figure 4 by the relative number. As can be seen in Figure 4, the control group was observed about 11.2% of the number of CA1 region cells of the hippocampus compared to the normal group, 78% of the cells survived when compared to the control group when administered 100 mg / kg of cutlass hydrolyzate Neuronal viability was observed.

As shown in FIG. 3E and FIG. 3F, not only did the neurons survive almost all of the hippocampal CA1 region, but as shown in FIG. As it was maintained, it was confirmed that the cutlas hydrolyzate had a very excellent effect on neuronal cell protection.

Example 3. Changes of Glue Cell Activity According to Administration of Saltwater Hydrolyzate

In animal experiments using gerbils, changes in glial cell activity of cutlass hydrolyzate were confirmed by immunohistochemistry staining.

Breeding, ischemic induction, brain extraction and tissue treatment of the experimental animals were performed in the same manner as in Example 2-2-2, divided into control, normal and experimental groups.

Tissue sections showing hippocampus complexes were selected and used in the experiments. The selected tissue sections were reacted with 0.3% H 2 O 2 (in PBS) for 30 minutes to remove endogenous peroxidase. In order to prevent nonspecific immune responses, the reacted tissue sections were reacted with 3% normal goat serum for 30 minutes. The tissue sections reacted with the 3% normal goat serum were reacted with rabbit anti-GFAP (Chmicon Internationl, USA), a primary antibody diluted 1: 1000, to confirm the change of astroglia, In order to confirm the change, the tissue sections were reacted with rabbit anti-Iba-1 (Wako, USA), a primary antibody diluted 1: 1000, for 4 hours at 4 °. After completion of the reaction, the tissue sections were reacted for 2 hours with anti-rabbit IgG (Vector) or anti-mouse Ig G (Vector), which were diluted at 1: 200, and then diluted at 1,200, respectively. The reaction was carried out with ABC solution (Vector) for 2 hours, and 3,3'-diaminobezidine (DAB) was developed as a substrate.

Each step was washed three times for 10 minutes using 0.01 M PBS.

After the tertiary reaction, the tissue sections were plated on gelatin-coated slide glass and dried at room temperature for 12 hours, and the dried tissue sections were enclosed through a conventional dehydration and clearing process.

Immunostaining photographs of tissue sections that completed the reaction with the GFAP and Iba-1 are shown in FIG. 5. As shown in FIG. 5, in the experimental group administered with the cutlass hydrolyzate, the expression of the glial cells was significantly reduced compared to the control group in which the solvent was administered. In particular, the expression and microglial expression of GFAP, which are known as markers of astrocytes, were observed. It was confirmed that the expression of Iba-1, which is a marker of cells, was significantly reduced. Therefore, it was confirmed that cutlass hydrolyzate not only has a very excellent effect on neuronal cell protection, but also inhibits the expression of microglia and astroglia.

Example 4. Acute Toxicity Test Using Laboratory Rats

4-1. Toxicity test by oral administration to Mongolian gerbil

After dissolving 14 mg, 28 mg, 70 mg, 140 mg, and 700 mg of cutlass hydrolyzate of Example 1 in distilled water in the experimental animal of Example 2 daily using a esophageal needle for 0.5 week or so, the gastrointestinal tract The changes of, hemorrhagic changes, and mortality were investigated.

As a result of the investigation, no gastrointestinal and hematological changes could be observed in any group, and no fatal individuals were observed, confirming that the substance was safe.

4-2. Acute Toxicity Test by Oral Administration in Rats

In order to confirm the acute toxicity of the saltwater hydrolyzate, the saltwater hydrolyzate obtained in Example 1 was dissolved in distilled water in 6-week-old specific pathogen-free (SPF) SD rats (Sprague-Dawley, Harlan, USA). Administration was by oral administration. After the oral administration of the hydrolyzate of 14 mg, 28 mg, 70 mg, 140 mg and 700 mg in 1 ml of distilled water in 0.5 ml oral administration using an esophageal needle, the mortality, clinical symptoms and Weight changes were observed, hematological and hematological tests were performed, and the rats were examined by necropsy to observe the abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or deaths were observed in all experimental animals treated with saltwater hydrolyzate, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings. According to the above results, the cutlass hydrolyzate of the present invention did not show a toxicity change in rats up to 2 g / kg, respectively, and the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance of 2 g / kg or more.

Preparation Example: Formulation Using Cutlass Hydrolyzate

Preparation Example 1 Preparation of Powder

Cutlass Hydrolyzate Powder 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients were mixed and filled in an airtight cloth to prepare a powder.

Preparation Example 2 Preparation of Tablet

Cutlass Hydrolyzate Powder 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components was prepared by tableting according to the conventional manufacturing method of the tablet.

Preparation Example 3 Preparation of Capsule

Cutlass Hydrolyzate Powder 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients were mixed and filled into gelatin capsules to prepare capsules.

Preparation Example 4 Preparation of Liquid

Cutlass Hydrolyzate Powder 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution was prepared by sterilization.

Preparation Example 5 Preparation of Health Food

Cutlass Hydrolyzate Powder 1000mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

As described above, the cutlass hydrolyzate of the present invention effectively prevents neuronal cell damage caused by cerebral ischemia, inhibits neuronal cell death caused by cerebral ischemia, and does not cause side effects in the human body. It can be usefully used as a food composition or a medical composition for the prevention and improvement of diseases.

Claims (6)

Prevention of ischemic cerebrovascular disease comprising as an active ingredient a Trichiurus Lepturus hydrolyzate prepared using one or more proteolytic enzymes selected from the group consisting of alkalase , prevorim , protex , paratase and neutrase Improvement composition. The method of claim 1, A composition for preventing or improving ischemic cerebrovascular disease containing 0.1 to 99% by weight of the hydrolyzate. The method of claim 1, The ischemic cerebrovascular disease is a disease selected from the group consisting of Alzheimer's, vascular dementia and Huntington's disease prevention or improvement composition. delete Food composition for preventing or improving ischemic cerebrovascular disease comprising the composition according to any one of claims 1 to 4. The food composition of claim 5, wherein the food composition is a food, a food additive, or a beverage.

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