KR20210054684A - Composition for preventing the ischemia stroke disease containing laminarin - Google Patents

Abstract

The present invention relates to a composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient, wherein the laminarin is a polysaccharide abundant in brown algae that has been taken as food or a folk medicine for medicinal purposes, and is not only a substance which has been proven to be safe without problems such as toxicity, tolerance or the like, but also a substance that can be administered before the occurrence of cerebral ischemia as a result of experiments with experimental animals. In this case, the neuroprotective ability and neuronal apoptosis inhibitory ability are remarkably excellent, and thus the effect of preventing the cerebral ischemia is remarkably excellent, and it is possible to effectively prevent nerve damage in the CA1 region of the hippocampal tissue of the brain, which is known to be sensitive to the cerebral ischemia. Accordingly, the composition can be advantageously used to effectively prevent ischemic cerebrovascular disease.

Description

Composition for preventing ischemic cerebrovascular disease containing laminarin as an active ingredient {COMPOSITION FOR PREVENTING THE ISCHEMIA STROKE DISEASE CONTAINING LAMINARIN}

The present invention relates to a pharmaceutical composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient.

Due to the recent improvement in the economic level and the development of medicine and life science, the number of elderly people around the world is increasing rapidly. According to the '2017 Korea's Social Indicators' released by the National Statistical Office in 2018, more than half of the total population in Korea by 2040 will enter an'ultra-aging society' over 52 years old, and the population 65 years or older determines the aging society. As for the ratio, of the total population of Koreans (51,466 thousand) in 2017, the 65-year-old population recorded 13.8% (7,076 thousand), and is expected to increase continuously to 24.5% in 2030, 32.8% in 2040, and 40.1% in 2060. . In addition, it was confirmed that the life expectancy of Koreans was 82.4 years, a significant increase compared to 1970 when they recorded 61.9 years. However, in terms of the quality of health, it is estimated that Koreans will suffer from various diseases for 17.5 years, which is 64.9 years old in 2016, and 21.2% of their life expectancy.

In addition, according to the statistics of the recent National Statistical Portal, as of 2019, the proportion of the aged 65 or older to the total population of Korea is 14.9%, which means that Korea is already in an aging society. The proportion of the elderly population is expected to increase steadily to 15.7% in 2020, 25% in 2030, 33.9% in 2040, and 43.9% in 2060. Due to the increase in the incidence and prevalence of various senile diseases caused by physical and mental aging. The resulting social cost is expected to be enormous.

As such, with the increase in life expectancy of Koreans and the aging trend of Korean society, the number of patients with senile diseases is increasing year by year, and suffering and social costs are expected to increase as well.

As described above, with the aging of the population, the incidence and prevalence of various senile diseases caused by aging are increasing, and in particular, central nervous system (CNS) diseases including damage to the brain or spine continue to increase. It is a trend. The CNS disease, which has the highest proportion of these, is cerebrovascular disease, which ranks first in the single disease mortality rate, and there is a trend of 60,000 patients in Korea every year.

The cerebrovascular disease is a typical senile disease, commonly referred to as stroke, and is a disease caused by clogging or bursting of blood vessels in the brain. The cerebrovascular disease is characterized in that the blood vessels in the brain are blocked or burst, and the circulation of a certain part of the brain suddenly stops due to abnormal cerebral blood flow, resulting in loss of the function of the nerve corresponding to that part. Induces nerve cell damage and causes serious dysfunction.

The cerebrovascular disease is regarded as the first cause of permanent disorders such as muscle paralysis, consciousness disorder, visual impairment, and cognitive decline, unlike other diseases in which the defect is recovered upon survival. Although the demand for development is increasing, the situation is causing enormous social and economic losses because it is known that a large number of them recur even after treatment.

The cerebrovascular disease is largely due to a hemorrhagic stroke caused by the burst of blood vessels that supply blood to the brain due to an impact such as a traffic accident, blood clots, and decreased perfusion, thereby temporarily blocking the blood flow to the brain. It can be divided into two types: ischemic stroke, which occurs due to a lack of supply. The hemorrhagic stroke is an important cause of primary cerebral hemorrhage due to high blood pressure and subarachnoid hemorrhage due to arteriovenous malformations or aneurysms.

The stroke may cause hemiplegia, speech impairment, loss of memory and reasoning ability, coma, and even death in severe cases, depending on the area of the brain to be damaged.

Recently, in Korea, the incidence of ischemic cerebrovascular disease tends to increase steadily compared to hemorrhagic cerebrovascular disease due to the westernization of diet and lifestyle. According to one study, ischemic cerebrovascular disease accounts for about 80% of all cerebrovascular disease types.

As described above, in ischemic cerebrovascular disease, which has a high incidence among cerebrovascular diseases, blood clots and other waste products generated in the cardiovascular system cause blockages in the cerebrovascular system, or blood is not supplied to the brain in pathological conditions such as cardiac arrest. It refers to a disease caused by neuronal cell death in a specific area of the brain. The neuronal cell death is called ischemic neuronal cell death, and ischemic cerebrovascular disease can be subdivided into thrombosis, embolism, and transient ischemic attack (TIA).

As an example, looking at an experiment with a Mongolian gerbil, it is known that neuronal cell death was observed in the CA1 (conus ammonis 1) region of the hippocampal formation 4 days after inducing cerebral ischemia for 5 minutes. , This is called'delayed neuronal death' (Kirino, Brain Res ., 239, pp 57-69, 1982).

Until now, many mechanisms have been suggested through research on neuronal cell death following ischemic stroke. Specific mechanisms include ^{excitotoxcity due to excessive increase in glutamate and Ca 2+} , oxidative stress due to increased reactive oxygen species, and neuroblastoma. The inflammatory response due to activation of glial cells and invasion of peripheral inflammatory cells is known (Mitani et al ., Neuroscience., 42, pp661-670, 1991; Chu et al ., J Neuroinflammation., 9, pp 69 , 2012; Wang et al ., Free Radic Biol Med., 43, 43-47 2016-05-04 pp 1048-1060, 2007), Currently, a new mechanism of neuronal apoptosis following cerebral ischemia is unknown. The hypothesis about this is continuously being presented.

Accordingly, the current research for the treatment of ischemic cerebrovascular disease is proceeding in the direction of effectively suppressing neuronal cell death caused by cerebral ischemia based on the above-mentioned mechanism, and in this regard, the relationship with whether ischemia or cerebrovascular occlusion Without, it is distinguished from the treatment of degenerative cerebrovascular disease, which treats the loss or extinction of nerve cells in a specific area or the inhibition of neuronal differentiation.

Currently, the calcium channel antagonist nimodipine, flunarizine, and glutamate antagonist MK-801, dextraphan, and free radical scavenger, tirilazad, are nerve cells. Although it was developed as a protective agent and proved its neuroprotective efficacy through preclinical studies, it is difficult to see it as a drug for clinical use as a treatment for brain ischemia due to lack of efficacy and side effects in large-scale clinical trials.

Due to such side effects, in the case of drug administration for the prevention of ischemic stroke, as with drugs for general diseases, substances derived from natural products extracted from natural resources are preferred rather than single compounds that may have side effects. In the case of a single compound by a chemical method, in order to develop a drug that guarantees the safety and efficacy of effectively inhibiting neuronal death due to cerebral ischemia, it is necessary to start from the search for candidate substances and go through the preclinical and final clinical trial stages. However, it is because enormous consumption is required in terms of time and cost.

Therefore, there is an increasing need for research on a substance capable of inhibiting or delaying neuronal cell death due to cerebral ischemia among natural substances or substances derived from natural substances recognized as safe to date.

KR 10-1316085B KR 10-1900970B KR 10-2017-0118870A KR 10-2019-0038873A

In order to solve the above problems, an object of the present invention is to provide a pharmaceutical composition or health food containing as an active ingredient a substance effective in preventing ischemic cerebrovascular disease by inhibiting or delaying ischemic neuronal cell death. .

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing ischemic cerebrovascular disease. Specifically, the pharmaceutical composition for preventing ischemic cerebrovascular disease may include laminarin as an active ingredient. The laminarin may be extracted from brown algae. In addition, the laminarin may be a polysaccharide represented by the following formula (1). In the following Formula 1, n may be 2 to 1,000.

[Formula 1]

The pharmaceutical composition for preventing ischemic cerebrovascular disease may be a composition for preventing ischemic cerebrovascular disease, which is any one selected from the group consisting of thrombosis, embolism, transient ischemic attack, subarachnoid hemorrhage, and small infarction.

The laminarin, which is an active ingredient of the pharmaceutical composition for preventing ischemic cerebrovascular disease, may be characterized in that it protects neurons in the CA1 region of the brain hippocampal tissue.

In addition, the pharmaceutical composition for preventing ischemic cerebrovascular disease may contain 0.001 to 99.999% by weight of the laminarin, which is an active ingredient, based on the total weight of the composition.

In addition, the present invention may be a food composition for improving or preventing ischemic cerebrovascular disease using laminarin as an active ingredient. The laminarin may be extracted from brown algae. In addition, the laminarin may be a polysaccharide represented by the following formula (1). In the following Formula 1, n may be 2 to 10,000.

[Formula 1]

The food composition for improving or preventing ischemic cerebrovascular disease may be a health functional food.

In addition, the food composition for improving or preventing ischemic cerebrovascular disease may contain 0.001 to 49.999% by weight of the laminarin, which is an active ingredient, based on the total weight of the composition.

Laminarin, an active ingredient of the present invention, is a polysaccharide derived from brown algae, a natural product, and has been consumed for a long time as well as recently used for manufacturing artificial joints or as an excipient for food or medicine, and has no side effects or tolerance. It was confirmed that the safety was not only recognized, but as a result confirmed by the present inventors, the effect of the laminarin in preventing ischemic cerebrovascular disease, specifically, the ischemic brain, which is remarkably excellent due to the protective effect of cranial nerve cells and the delaying effect of death in ischemic conditions. Since the effect of preventing or improving vascular diseases is recognized, the laminarin can be widely used to prevent death or disorder due to ischemic cerebrovascular disease in an aging society and in a modern society in which circulatory diseases due to nutritional excess rapidly increase.

1 is a hippocampal tissue and hippocampus of an experimental animal to which laminarin was administered before cerebral ischemia in order to investigate the therapeutic or preventive effect of ischemic brain disease through the protection and damage improvement of nerve cells of laminarin according to an embodiment of the present invention. This is a photograph of the result of staining the CA1 area of the tissue with cresyl violet.
2 is a CA1 of the hippocampal tissue of an experimental animal to which laminarin was administered before cerebral ischemia in order to investigate the treatment or prevention effect of ischemic brain disease through the protection and damage improvement of neurons of laminarin according to an embodiment of the present invention. This is a photograph of the result of staining the region by immunohistochemistry staining using a mouse anti-NeuN (NeuN, Neuronal Nuclei) antibody, which is a marker for neurons.
3 is a view showing the effect of laminarin in preventing ischemic brain disease through neuronal protection and damage improvement according to an embodiment of the present invention, in order to investigate the effect of laminarin in the hippocampal tissue CA1 region of the experimental animal administered with laminarin after induction of cerebral ischemia. This is a graph showing the results of counting neurons that showed immunoreactivity to NeuN, a marker for cells.
FIG. 4 is a view in the CA1 region of the hippocampal tissue of an experimental animal administered with laminarin after induction of cerebral ischemia in order to investigate the preventive effect of ischemic brain disease by protecting and improving damage to nerve cells of laminarin according to an embodiment of the present invention. This is a photograph of the results of tissue fluorescence staining using Fluoro-Jade B (FJ B, Fluoro-Jade B), which is a marker for nerve cells that are damaged, that is, apoptosis or apoptosis.
FIG. 5 is a view in the CA1 region of the hippocampal tissue of an experimental animal administered with laminarin after induction of cerebral ischemia in order to investigate the preventive effect of ischemic brain disease by protecting and improving damage to nerve cells of laminarin according to an embodiment of the present invention. This is a graph showing the counting of neurons labeled on fluorine-jade B (FJ B).

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Unless otherwise specified herein, a composition is meant to include a product comprising the specified ingredients in the specified amounts, as well as products resulting directly or indirectly from the combination of the specified ingredients in the specified amounts.

Unless otherwise specified in the present specification, an active ingredient is a composition, specifically a pharmaceutical composition or a food composition, in the treatment, amelioration or prevention effect of ischemic cerebrovascular disease, specifically ischemic or delayed neuronal cell death or activation of glial cells. It means an ingredient that can be effectively suppressed.

Unless otherwise specified herein, an effective amount means an amount of a compound or composition of the present invention that is effective to produce a desired therapeutic, ameliorating, inhibiting or prophylactic effect.

In the present invention, ischemic brain disease refers to a generic term for a disease that occurs when blood vessels in the brain are blocked for some reason and blood required for the brain is not supplied, and is also referred to as cerebral ischemic disease. The ischemic brain disease is a disease caused by delayed neuronal cell death in neurons in the CA1 region of the hippocampal tissue, which is known to be sensitive to cerebral ischemia, because oxygen and glucose are not supplied normally due to reduced blood flow to the brain. And, examples of ischemic cerebrovascular disease may include ischemic cerebrovascular disease, ischemic cerebral infarction, and the like.

The inventors of the present invention believe that, in the case of a substance having excellent neuronal cell protection against cerebral ischemia, it has excellent therapeutic or preventive effect against damage to nerve cells, and thus has a therapeutic or preventive effect on related diseases, specifically ischemic brain diseases such as ischemic cerebrovascular disease. In view of the fact that there is no problem of side effects or resistance when using natural resources that have secured human safety unlike active ingredients of drugs, which have previously been a problem of safety and side effects, it is possible to prevent neuronal cell death due to ischemic stroke from substances derived from natural substances. While researching on possible substances, laminarin derived from brown algae, a natural product that can be extracted from brown algae through animal experiments, is known to be sensitive to cerebral ischemia caused by cerebral ischemia, delayed neuronal cell death in the CA1 area of the brain hippocampus tissue. It was confirmed that the nerve cell protective effect through inhibition was very excellent, and it was confirmed that nerve cell damage caused by cerebral ischemia can be prevented, and the present invention was completed based on this.

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

The present invention relates to a pharmaceutical composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient. The laminarin may be extracted from brown algae.

In addition, the laminarin may be a polysaccharide represented by the following formula (1). In the following Formula 1, n may be 2 to 1,000 or 5 to 500.

[Formula 1]

The laminarin may be 100 Da to 100,000 Da, preferably 500 Da to 50,000 Da, more preferably 1,000 Da to 10,000 Da, that is, 1 kDa to 10 kDa.

The laminarin is a reserve polysaccharide composed of D-glucose contained in the cell wall of brown algae, specifically, brown algae belonging to the genus Limanaria sp., β-1,3 -It is composed of glucan bonds and is also called Laminaran.

The laminarin includes laminaribose in which D-glucose is bonded by a β-1,3-glucan bond as a constituent unit, and the degree of polymerization may be 2 to 1,000 or 5 to 500, but it is mainly 18 to 22. In this aspect, n in Formula 1 may be 2 to 1,000 or 5 to 500 or 10 to 100 or 15 to 30 or 18 to 22. The laminarin may be a soluble laminarin or an insoluble laminarin, and preferably a water-soluble laminarin having a small number of β-1,6-glycosidic bonds.

In addition, the laminarin may be 100 Da to 100,000 Da, preferably 500 Da to 50,000 Da, more preferably 1,000 Da to 10,000 Da, that is, 1 kDa to 10 kDa, even more preferably 4 kDa to 6 kDa. have.

The laminarin has been confirmed to have antioxidant, anticancer, anticoagulant and anti-inflammatory effects, and is also used as a material for health functional foods. In addition, it is also used as an excipient such as a thickener, which is a use of polysaccharides. It is sometimes used as a material for artificial joints, artificial bones, artificial teeth or artificial gums.

In the present invention, after ligating the warm carotid artery to induce cerebral ischemia by anesthetizing a gerbil administered with laminarin for a certain period of time, the effect of protecting nerve cells is obtained through staining of surviving neurons and staining of dead neurons in hippocampal tissue. As a result of conducting an experiment to measure, it was confirmed that the laminarin had a remarkably excellent neuronal protective effect when administered before the occurrence of cerebral ischemia. From the above results, the laminarin was evaluated to be able to effectively prevent ischemic cerebrovascular disease. In this aspect, the laminarin may be used as an active ingredient of a composition for preventing ischemic stroke.

The composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient may be applied directly to animals including humans.

The composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient may further contain the laminarin as an active ingredient or a substance having an effect of treating or preventing ischemic cerebrovascular disease in the laminarin as an active ingredient. In addition, depending on the formulation, method of use and purpose of use, additional ingredients, that is, a pharmaceutically acceptable or nutritionally acceptable carrier, excipient, diluent, or auxiliary ingredient may be additionally included.

More specifically, the composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient is in addition to the active ingredient, in addition to nutrients, vitamins, electrolytes, flavoring agents, coloring agents, heavy agents, pectic acid and salts thereof, alginic acid, and It may further contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. In addition, the carrier, excipient, or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, achysia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline 1 selected from the group consisting of cellulose, polyvinyl pyrrolidone, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline. Although it may be above, it is not limited thereto, and all of a conventional carrier, excipient, or diluent may be used. The ingredients may be added independently or in combination with the active ingredient, that is, laminarin.

The composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient includes 0.001% to 99.999% by weight, preferably 0.1% to 99% by weight of the active ingredient, specifically, the laminarin based on the total weight of the composition. %, more preferably 1% to 50% by weight. In addition, the content of the additional component may preferably be added in the range of 0.1% to 20% by weight based on the total weight of the composition for preventing ischemic cerebrovascular disease, but is not limited thereto.

In addition, when a pharmaceutical composition for preventing ischemic cerebrovascular disease containing laminarin as an active ingredient is formulated, a conventional filler, extender, binder, disintegrant, surfactant, anti-coagulant, lubricant, wetting agent, fragrance, emulsifier or preservative It may further include, and can be used both oral or parenteral.

Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient, such as starch, calcium carbonate ( calcium carbonate), sucrose or lactose, and gelatin. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

The formulation of the composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient may be in a preferred form depending on the method of use, and in particular, to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated by employing a method known in the art. Examples of specific formulations include granules, powders, syrups, solutions, suspensions, tablets, injections, spirits, cataplasma, capsules, soft or hard gelatin capsules.

Furthermore, the composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient may be prepared by using an appropriate method known in the art or by Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA). It can be preferably formulated using the method disclosed in.

The dosage of the composition for preventing ischemic cerebrovascular disease comprising laminarin according to the present invention as an active ingredient may be appropriately selected by those skilled in the art in consideration of the administration method, the age, sex and weight of the user, and the severity of the disease. I can. For example, the composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient is 0.0001 mg/kg to 1000 mg/kg per body weight of the administered individual, and 50 to be more effective. It can be administered at mg/kg or more, preferably 50 mg/kg to 200 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

In addition, the composition for preventing ischemic cerebrovascular disease comprising laminarin of the present invention as an active ingredient may further include an extract for a compound or natural product having an effect of treating or preventing ischemic cerebrovascular disease in addition to the active ingredient, , It may be included in 5 parts by weight to 200 parts by weight, respectively, based on 100 parts by weight of the active ingredient.

A food composition for improving or preventing ischemic cerebrovascular disease according to another embodiment of the present invention includes laminarin as an active ingredient.

In this aspect, the present invention relates to a food composition for improving or preventing ischemic brain disease comprising laminarin as an active ingredient.

The food refers to a natural product or processed product containing one or more nutrients, and preferably refers to a state that can be eaten directly through a certain amount of processing process, and as a general meaning, health functional food, It is intended to include all beverages, food additives and beverage additives. In this aspect, the food composition for improving or preventing ischemic brain disease may be a health functional food for preventing ischemic brain disease.

The foods include, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, and the like. In addition, the food in the present invention includes special nutritional foods (e.g., formula, infants, baby food, etc.), processed meat products, fish meat products, tofu, muk, noodles (e.g., ramen, noodles, etc.), health supplement food, seasoning food ( Examples, soy sauce, miso, red pepper paste, mixed sauce, etc.), 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, fruit, vegetable beverages, soy milk, fermented beverages, etc.), and natural seasonings (eg, ramen soup, etc.).

The foods, health functional foods, beverages, food additives, and beverage additives may be prepared by conventional manufacturing methods.

The health functional food refers to a food group or food composition that has added value to act and express the function of the food for a specific purpose by using physical, biochemical, biotechnological techniques, etc. to the food, controlling the biological defense rhythm of the food composition, preventing and recovering from diseases, etc. It refers to food that is designed and processed to sufficiently express the function of gymnastics for the living body.

The health functional food may include food additives acceptable for food, and may further include suitable carriers, excipients, and diluents commonly used in the manufacture of health functional foods.

The beverage refers to a generic term for drinking to quench thirst or to enjoy the taste, and is intended to include health functional beverages. The beverage is an essential component in the indicated ratio, and there is no particular limitation on other components other than containing the laminarin as an active ingredient, and may contain various flavoring agents or natural carbohydrates as an additional component, like a normal beverage. .

Examples of the above natural carbohydrates include monosaccharides, such as disaccharides such as glucose and fructose, such as maltose, sucrose, and the like, and polysaccharides, such as dextrin, cyclodextrin, and the like, and conventional sugars, and These are sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, 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 may be generally about 1 to 20 g, preferably 5 to 12 g, per 100 ml of the food composition of the present invention. It may further contain pulp for the manufacture of beverages.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid, and It may contain salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. These components may be used independently or in combination. The proportion of these additives is not so important, but may be selected in the range of 0 to 2,000 parts by weight per 100 parts by weight of laminarin of the present invention.

The health functional drink refers to a group of beverages or beverages that have added value to effect and express the function of the beverage for a specific purpose by using physical, biochemical, and biotechnological techniques, etc., to control the biological defense rhythm of the beverage composition, and to prevent and recover diseases. It refers to a beverage that is designed and processed to sufficiently express the function of gymnastics on the back to the living body.

The health functional drink is not particularly limited to other ingredients other than containing the laminarin of the present invention as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as an additional ingredient, like a normal drink.

Examples of the natural carbohydrates include monosaccharides, such as disaccharides such as glucose and fructose, 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 described above, natural flavoring agents (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 g to 20 g, preferably 5 g to 12 g per 100 ml of the composition of the present invention.

In addition, in the food composition for improving or preventing ischemic cerebrovascular disease comprising the laminarin as an active ingredient, the active ingredient is 0.001 to 49.999% by weight or 0.01% to 25% by weight or 0.1% by weight of the total weight of the total food composition. It may be included in% to 10% by weight, and the beverage composition may be included in a ratio of 0.02 g to 5 g, or 0.3 g to 1 g based on 100 ml.

It was confirmed that laminarin of the present invention has the effect of improving or preventing ischemic cerebrovascular disease, in particular, significantly inhibiting neuronal cell death in the CA1 region of the hippocampus caused by cerebral ischemia through pre-administration before induction of cerebral ischemia. It was experimentally confirmed that the effect of treating, improving or preventing ischemic cerebrovascular disease is remarkably excellent because it can suppress or prevent nerve cell damage caused by cerebral ischemia.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms, and the following examples are for illustrative purposes only, and the scope of the present invention is not limited by the following examples.

Example 1: Measurement of neuronal protective efficacy of laminarin during cerebral ischemia through experimental animals 1

In order to induce cerebral ischemia, 56 male Mongolian gerbils (Harlan, USA) with a weight of 75 to 80 g of 24 months old were used as experimental animals. Ischemic cerebrovascular disease is mainly invented in the elderly or the elderly and is recognized as a senile disease, and therefore, experimental animals of 24 months of age were also used for experimental animals. The Mongolian Gerbil administered laminarin before inducing cerebral ischemia, and confirmed the effect of preventing cerebral ischemia depending on whether or not laminarin was administered. The induction of cerebral ischemia of the Mongolian gerbil was performed by anesthetizing the Mongolian gerbil to induce cerebral ischemia by exposing the common carotid artery and ligating it. After inducing cerebral ischemia, the effect of inhibiting neuronal cell death due to cerebral ischemia of laminarin was confirmed through a method of staining and observing the hippocampal tissue of the Mongolian gerbil.

Example 1-1. Breeding of experimental animals

A constant contrast cycle of applying light from 7 a.m. to 7 p.m. of 56 aged male Mongolian gerbil (Meriones unguiculatus Harlan, USA) of a weight of 75 g to 80 g of 24 months old, temperature conditions of 22° C. to 24° C., and 45 It was reared under conditions of relative humidity of% to 55%. For the experimental animal feed, a general dry pellet feed (Korea Experimental Animal, Korea) was used, and the feed and water were allowed to be consumed at all times.

Example 1-2. Measurement of the protective efficacy of neurons according to cerebral ischemia of laminarin 1

In order to confirm the neuroprotective effect of laminarin, more specifically, to prevent or improve neuronal death caused by cerebral ischemia, laminarin (5 kDa, Sigma-Aldrich, cat No. L9634, USA) was dissolved in physiological saline so as to be 10 mg or 50 mg per 1 kg of the body weight of the experimental animal, and was prepared as an administration drug and administered. The administration period of the drug was administered orally using an oral feeding needle for 3 days before induction of cerebral ischemia.

Before the induction of cerebral ischemia, laminarin was administered for 3 days, and then the experimental animals were systemically treated using a gas mixed with nitrogen and oxygen at 7:3 and 3% isoflurane (Baxtor, USA). Was anesthetized. Using a gas in which 2.5% isoflurane was mixed with the nitrogen and oxygen mixture gas, the operation to induce cerebral ischemia was performed on the experimental animal while maintaining the anesthesia state of the experimental animal.

For the operation on the experimental animal, the neck area was depilated, disinfected, and then incision was made to expose both warm carotid arteries, and ligated for 5 minutes using a non-traumatic aneurysm clip (Staelting, USA). After inducing cerebral ischemia, the clip was removed to perform reperfusion. At this time, each experimental group observed whether blood circulation in the central artery of retina was observed using an ophthalmoscope to confirm complete occlusion of the warm carotid artery.

The body temperature was measured by inserting a thermometer in the rectum during the operation of the experimental animal, and the body temperature was kept constant to be 36.7°C to 37.3°C, which is the normal body temperature, using a heating pad that is automatically adjusted according to the body temperature of the experimental animal. .

The experimental animals were anesthetized by intraperitoneal injection of thiopental sodium (thiopental sodium, Yuhan Corporation, Republic of Korea) intraperitoneally at a dose of 30 mg per 1 kg of body weight 5 days after induction of cerebral ischemia, and 1,000 IU of heparin per 1000 ml was included. A physiological saline solution at 4°C was injected into the left ventricle, followed by perfusion washing. Perfusion fixation was performed using 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at 4° C. for the animal on which the perfusion washing was completed.

The brain was removed by removing the skull of the experimental animal that had been fixed perfusion using a bone cutter. The brains of the extracted experimental animals were post-fixed for 4 hours using 4% paraformaldehyde (in 0.1 M phosphate buffer, pH 7.4) at 4°C. For cryoprotecion, the brain after the post-fixation is put in a 30% sucrose solution (in 0.1 M phosphate buffer, pH 7.4) and settled until it sinks to the bottom, and then slide microtome (Reicert- Jung, Germany) cut the brain tissue to a thickness of 30 µm to make a tissue section, and the tissue section was placed in a 6-well plate containing a storage solution and stored at 4°C until staining was performed. .

In order to confirm the neuronal protective effect of laminarin in the CA1 region of the hippocampus after the ischemia-reperfusion, cresyl violet (CV) for staining the Nissl body and NeuN, a marker for neurons, respectively. Immunohistochemical staining was performed using an antibody against (a neuronal specific nuclear protein), and tissue fluorescence staining was performed using Fluor-Jade B, a marker for neurons in the process of death or death.

In addition, the control group (solvent-administered group, vehicle) was prepared in the same manner as above, except that only physiological saline used to dissolve the laminarin was administered orally, and the normal group (Sham) did not perform ischemia-reperfusion surgery. Group, and the experimental group (Ischemia) was an experimental group that performed ischemia-reperfusion surgery, and tissue sections of the normal group and the experimental group were prepared in the same manner as described above.

More specifically, a tissue that showed well hippocampal formation was selected from among the made tissue sections and used in the experiment, and 0.01M PBS (phosphate-buffer saline, pH 7.4) to remove the preservatives buried in the selected tissue sections. ) Was washed three times for 10 minutes each. The washed tissue sections were spread on a slide glass coated with gelatin and sufficiently dried at 43°C. The sufficiently dried tissue sections were briefly immersed in distilled water and then dipped in a 2% cresyl violet acetate (Sigma, USA) solution for 1 minute to perform staining of the tissue sections. Subsequently, the stained tissue sections are sufficiently washed with running water to remove excess dye from the slide, and then immersed in distilled water for a while, and then with 50%, 70%, 80%, 90%, 95% and 100% ethanol solutions. Dehydration and excess cresyl violet washing were performed by sequential treatment. After confirming that the Nissl body is visible in the tissue section, it is immersed in xylene (Junsei, Japan) to make it transparent, and then Canadian Balsam (Kanto, Japan) is used as an encapsulant, and the tissue section is covered with a cover glass. Covered.

Each tissue section of each of the experimental groups (Sham, Ischemia, Vehicle, m LA) was performed using an AxioM1 microscope (Carl Zeiss, Germany) equipped with a digital camera (Axiocam, Cal Zeiss, Germany). The tissue sections were each photographed by enlarging the area by 40 times and the CA1 area at a magnification of 200 times. It is shown in a to f.

As shown in FIG. 1, neurons were stained dark purple in the entire area of the hippocampus in the normal group (Sham) (FIG. 1A), and in particular, in the CA1 area, it can be observed that neurons are concentrated in the pyramidal layer. There was (a in Fig. 1). However, in the case of the control group in which only the solvent was administered and cerebral ischemia was induced, neuronal cell death occurred in the CA1 area of the hippocampus due to delayed neuronal death due to cerebral ischemia, so that almost no staining of neurons was observed (B in FIG. 1). ), it was confirmed that most of the neurons were destroyed in the pyramidal layer among the CA1 region (FIG. 1 b ).

On the other hand, even in the case of the normal group (10 mg/kg LA-sham, 50 mg/kg LA-sham) administered with 10 mg/kg or 50 mg/kg of laminarin before inducing cerebral ischemia, the entire area of the hippocampus Neurons were stained dark purple (C and E in FIG. 1), and in particular, in the CA1 region, it could be observed that neurons were concentrated in the pyramidal layer (c and e in FIG. 1). In addition, in the case of the experimental group (10 mg/kg LA-ischemia) in which 10 mg/kg of laminarin was administered before inducing cerebral ischemia and then ischemia was performed, neuronal cell death occurred in the CA1 area of the hippocampus. Almost no staining was observed (Fig. 1D), and it was confirmed that most of the neurons were lost in the pyramidal layer of the CA1 region of the hippocampus (Fig. 1D). On the other hand, in the experimental group (50 mg/kg LA-ischemia) in which 50 mg/kg of laminarin was administered and then ischemia was performed, a large number of neurons in the entire area of the hippocampus were stained dark purple (Fig. 1). F) In the CA1 region of the hippocampus, it was observed that neurons were also concentrated in the pyramidal layer (FIG. 1F). Based on the above results, it was confirmed that 50 mg/kg of laminarin inhibited neuronal cell death caused by cerebral ischemia.

Example 1-3. Measurement of neuronal protective efficacy of laminarin in experimental animals during brain ischemia 2

To further confirm the neuronal protection effect of laminarin, which was confirmed to have an excellent neuronal protection effect in Example 1-2. Tissue fluorescence staining was performed using a degenerative/death neuron marker, that is, Fluoro-Jade B (FJ B), a marker of a neuron in the process of apoptosis or apoptosis. NeuN, a neuronal specific nuclear protein) was used to perform immunohistochemical staining.

First, tissue fluorescence staining was performed using a fluorine-jade ratio (F-J B, Histochem, Jefferson, AR, USA), which is a marker of neurons in the process of death or death. The tissue sections prepared in Example 1-2 were washed three times for 5 minutes each with distilled water, and then reacted in a 0.06% potassium permanganate solution for 20 minutes. Thereafter, it was washed with distilled water for 2 minutes and dyed by reacting for 40 minutes in a 0.001% fluorine-jade solution containing 0.1% acetic acid (Histochem, Jefferson, AR, USA). The dyed tissues are washed with distilled water 3 times for 1 minute, dried for 20 minutes in a 50°C slide warmer, transparent to xylene (xylene, Junsei, Japan), and then covered with DPX (Sigma, USA) as an encapsulant. The glass was covered and sealed. Using a fluorescence microscope (Ex: 385nm, Em: 425nm) of the encapsulated tissue, the CA1 region was enlarged 200 times to take pictures of each tissue section, and a photograph of the CA1 region of the hippocampus was shown in FIG. 2. In addition, nerve cells of the tissue section stained with fluorine-jade B were counted using an image analyzer (Optimas 6.5, USA) program for the photographed picture, and binary variance analysis was performed to verify the significance of each group. The (two-way ANOVA test) was performed, and the counting results are shown in FIG. 3.

As shown in Figs. 2 and 3, tissue fluorescence staining using fluorine-jade B, which is a marker of neurons undergoing apoptosis or apoptosis, is used to confirm the effect of protecting nerve cells or improving nerve damage according to the administration of laminarin during cerebral ischemia. As a result of performing, no neurons stained by the fluoride ratio were observed in the CA1 region of the hippocampus of the normal group (Fig. 2A). In addition, even in the experimental group (10 mg/kg and 50 mg/kg LA-sham) that performed false surgery after administering 10 mg/kg and 50 mg/kg of laminarin, neurons stained by fluorine-jade B It was not observed (C and E in Fig. 2). However, in the case of the experimental group (vehicle-ischemia) in which ischemia was performed after administration of only the solvent, neurons stained by a number of fluorine-jades B were observed (FIG. 2B), and 10 mg/kg of laminarin In the case of the experimental group (10 mg/kg LA-ischemia) in which ischemia was performed after administering the drug, a number of neurons stained with fluorine-jade B were observed (Fig. 2D), but stained with fluorine-jade B. It was confirmed that the number of neurons was significantly reduced compared to the experimental group (vehicle-ischemia) undergoing ischemia surgery (FIG. 3). On the other hand, in the case of the experimental group (50 mg/kg LA-ischemia) that underwent ischemia surgery after administration of 50 mg/kg of laminarin, the number of neurons stained by fluorine-jade B was hardly observed (Fig. 2F), it was confirmed that the number of neurons stained by fluorine-jade B was significantly reduced compared to other ischemia experimental groups (vehicle-ischemia and 10 mg/kg LA-ischemia) (FIG. 3). Based on the above results, it was confirmed that 50 mg/kg of laminarin has an excellent effect of preventing nerve cell damage against cerebral ischemia.

In addition, immunohistochemical staining was performed by the following method using an antibody against NeuN, a neuronal marker. Specifically, from the tissue sections prepared in Example 1-2, a tissue section showing the hippocampal complex was selected and used in the experiment, and the selected tissue section removed endogenous peroxidase present in the tissue. In order to do this, it was reacted with 0.3% hydrogen peroxide (H ₂ O ₂ , in 0.01M PBS, pH 7.4) for 20 minutes. In addition, in order to prevent a non-specific immune response, the tissue sections reacted with the hydrogen peroxide were reacted with 5% normal horse serum in 0.05M PBS, pH 7.4 for 30 minutes.

The tissue sections that reacted with the 5% normal horse serum were reacted with mouse anti-NeuN (mouse anti-NeuN, Chemicon, USA) diluted 1:1000 for 24 hours at 4°C. The tissue section after the reaction was reacted with anti-mouse IgG (anti-mouse IgG, Vector), a secondary antibody diluted 1:250, for 2 hours at room temperature, and then diluted 1:250 avidin-biotin (ABC). complex, Vector) solution was reacted for 1 hour, and 3, 3'-DAB (diaminobenzidine) was used as a substrate. In performing each step of the above process, the tissue sections were washed three times for 10 minutes each using 0.01M PBS (pH 7.4) for each step.

The 3, 3'-DAB was used as a substrate, and the tissue sections were coated on a gelatin-coated slide glass, and then dried for 12 hours at room temperature, and the dried tissue sections were 50%, 70 %, 80%, 90%, 95% and 100% ethanol were dehydrated. After the dehydration process was completed, the tissue section was immersed in xylene (Junsei, Japan) to make it transparent, and covered with a cover glass on the tissue section using Canada Balsam (Japan) as an inclusion agent.

Each tissue section was photographed by magnifying the CA1 area 200 times each with an AxioM1 microscope (Carl Zeiss, Germany) attached to the enclosed tissue with a digital camera (Axiocam, Cal Zeiss, Germany). A picture of the CA1 area is shown in FIG. 4. In addition, neurons having an immune response to NeuN antibodies were counted using an image analyzer (Optimas 6.5, USA) program for the photographed picture, and a two-way ANOVA test was performed to verify the significance of each group. ) Is performed, and the counting results are shown in FIG. 5.

As shown in FIGS. 4 and 5, immunohistochemical staining was performed using NeuN, a marker of nerve cells, in order to confirm the effect of protecting nerve cells or improving nerve damage according to the administration of laminarin during cerebral ischemia. In addition to the experimental group (Vehicle-Sham) that was administered and performed false surgery, as well as the experimental group (10 mg/kg and 50 mg/kg LA-) that performed false surgery after administration of 10 mg/kg and 50 mg/kg of laminarin. Sham) also observed neurons exhibiting immunoreactivity to a number of NeuNs in the CA1 region of the hippocampus (Fig. 4A, C, and E). However, in the case of the experimental group (Vehicle-Ischemia) in which only the solvent was administered and the ischemia operation was performed, neurons exhibiting immunoreactivity to NeuN in the CA1 region of the hippocampus were hardly observed (FIG. 4B). In addition, in the case of the experimental group (10 mg/kg LA-Ischemia) that underwent ischemia surgery after administering 10 mg/kg of laminarin, few neurons exhibiting immunoreactivity to NeuN in the CA1 region of the hippocampus were observed ( 4D), in the experimental group (50 mg/kg LA-Ischemia) in which ischemia was performed after administering 50 mg/kg of laminarin, a number of neurons were observed in the CA1 region of the hippocampus (F in FIG. 4 ), it was confirmed that the number of neurons exhibiting immunoreactivity to NeuN was significantly increased as well as significantly compared to the vehicle and 10 mg/kg LA-ischemia group (FIG. 5). From the above results, it was confirmed that when 50 mg/kg of laminarin was administered before the induction of cerebral ischemia, it effectively prevented neuronal cell death.

As described above, laminarin, a polysaccharide derived from brown algae, which is a natural product, not only guarantees safety, but also has an excellent effect on nerve cell protection, and when laminarin is administered before cerebral ischemia, all of them kill neurons in the CA1 region of the hippocampus. By inhibiting, laminarin has been proven to have an effect of preventing or inhibiting nerve cell damage caused by cerebral ischemia, and was evaluated to have an effect of preventing or improving cerebral ischemia.

Claims (9)

A pharmaceutical composition for preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient. The method of claim 1,
The laminarin is a pharmaceutical composition for the prevention of ischemic cerebrovascular disease, characterized in that the polysaccharide of the following formula (1) extracted from brown algae.
[Formula 1]

The method of claim 1,
The pharmaceutical composition for preventing ischemic cerebrovascular disease is a pharmaceutical composition for preventing ischemic cerebrovascular disease, characterized in that the laminarin contains 0.001 to 99.999% by weight based on the total weight of the composition. The method of claim 1,
The ischemic cerebrovascular disease is a pharmaceutical composition for preventing ischemic cerebrovascular disease, characterized in that any one selected from the group consisting of cerebral infarction, thrombosis, embolism, transient ischemic attack, subarachnoid hemorrhage and small infarction. The method of claim 1,
The laminarin is a pharmaceutical composition for the prevention of ischemic cerebrovascular disease, characterized in that to protect the nerve cells of the CA1 region of the brain hippocampal tissue. A food composition for improving or preventing ischemic cerebrovascular disease comprising laminarin as an active ingredient. The method of claim 6,
The laminarin is a food composition for improving or preventing ischemic cerebrovascular disease, characterized in that it is a polysaccharide of the following formula (1) extracted from brown algae.
[Formula 1]

The method of claim 6,
The food composition for improving or preventing ischemic cerebrovascular disease is a food composition for improving or preventing ischemic cerebrovascular disease, characterized in that the health functional food. The method of claim 6,
The food composition for improving or preventing ischemic cerebrovascular disease is a food composition for improving or preventing ischemic cerebrovascular disease, characterized in that the laminarin contains 0.001 to 49.999% by weight based on the total weight of the composition.

Images