KR100746592B1 - Functional food for preventing and ameliorating neurodegenerative disease comprising oleic acid having neuronal cell-protecting activity - Google Patents

Abstract

The present invention relates to a composition containing an oleic acid having neuroprotective activity as an active ingredient, the oleic acid of the present invention has an effect of protecting the brain neuronal cell damage induced by cerebral ischemia, and neurobehavioral recovery effect Since there is an excellent recovery effect in the experiment, the composition containing oleic acid can be used as a pharmaceutical and health functional food for the prevention and treatment of degenerative brain diseases caused by the death of nerve cells.

Oleic acid, nerve cell protection, cerebral ischemia, degenerative brain disease

Description

Functional food for preventing and ameliorating neurodegenerative disease comprising oleic acid having neuronal cell-protecting activity

Figure 1a is a diagram illustrating the brain tissue sections of rats by TTC staining in order to observe the protective effect of neuronal cell damage in the rat brain tissue according to the concentration of oleic acid (oleic acid),

FIG. 1B is a diagram comparing brain tissue damage rate (or cerebral infarction rate) between a control group administered with 5% Tween 20 and an oleic acid group. FIG.

Figure 2 is a diagram showing the neurobehavioral recovery effect of the administration of oleic acid (oleic acid).

The present invention relates to a health functional food for the prevention and improvement of degenerative brain disease containing oleic acid (oleic acid) having a neuroprotective activity as an active ingredient.

Looking at the proportion of Korea's aging population, announced by the National Statistical Office in October 2003, Korea entered the aging society as the proportion of the population aged 65 or over reached 7.2% in 2000, and this ratio is 14% in 2019. It is expected to enter aging society beyond. As the aging problem becomes a social issue in recent years, the public's interest in the characteristics of the elderly population, the elderly, such as housing, health, culture, leisure, etc. is increasing, and the demand for statistics is increasing. The key to these changes is that chronic degenerative diseases are becoming more of an issue than acute infectious diseases, which have been the leading cause of death for the past 50 years, due to the aging population. In particular, the death from cerebrovascular disease among chronic degenerative diseases is a very important disease that ranks second in the mortality rate from a single disease.

Cerebrovascular diseases can be classified into two types. One is hemorrhagic brain disease seen in cerebral hemorrhage and the like, and the other is ischemic brain disease caused by occlusion of cerebrovascular vessels. Hemorrhagic brain disease is mainly caused by traffic accidents, and ischemic brain disease is a disease that frequently occurs in elderly people.

In the case of transient cerebral ischemia in the cerebrum, oxygen and glucose supply are blocked, resulting in a decrease in ATP and edema in neurons, resulting in extensive brain damage. Neuronal death occurs after a significant period of time after cerebral ischemia, which is called delayed neuronal death. Delayed neuronal death was observed in a transient forebrain ischemic model using Mongolian gerbil, and neuronal cell death was observed in the CA1 region of the hippocampus (hippcampus) 4 days after induction of cerebral ischemia for 5 minutes. It is reported (Kirino T, Sano K. Acta Neuropathol ., 62: 201-208, 1984; Kirino T. Brain Res ., 239: 57-69, 1982).

There are two mechanisms of neuronal death caused by cerebral ischemia that most people accept so far. One is the excitatory neuronal cell death mechanism (Kang TC, et al . . J. Neurocytol, 30: 945-955, 2001) and ischemia-due to the sudden supply of oxygen at the time of reperfusion in the oxidative neuronal death, there are two mechanisms that cause wear damage to the DNA and the cytoplasm due to the increase in vivo radical (Won MH, et al ., Brain Res ., 836: 70-78, 1999; Sun AY, Chen YM. J. Biomed . Sci ., 5: 401-414, 1998; Flowers F, Zimmerman J. New Horiz . 6: 169-180, 1998).

Based on these mechanisms, many researches have been conducted to search for substances that effectively inhibit neuronal cell death in cerebral ischemia or to reveal the mechanism of the substances. However, there are few substances that effectively inhibit neuronal cell death caused by cerebral ischemia.

Tissue plasminogen activator (Tissue plasminogen activator), the only commercially available drug for treating cerebral ischemia, is a substance that induces rapid supply of oxygen and glucose by melting blood clots that cause cerebral ischemia with thrombolytics. Therefore, it is not necessary to directly protect nerve cells, so it is necessary to use it quickly. Due to the characteristics of thrombolysis, the overuse or frequent use causes thinning of the blood vessel wall, which eventually causes hemorrhagic cerebrovascular disease.

MK-801, a calcium channel blocker to effectively inhibit early calcium influx, has been clinically tested, but the drug has been discarded due to its side effects.

On the other hand, in Korea, many natural substances are marketed as health foods that are effective in preventing stroke, but most of them are not scientifically verified, and they are often social problems because they cause health food abuse.

Therefore, there is an urgent need for an effort to objectively verify natural resources that have been used for a long time and to prove their safety, and to develop natural materials having the effect of treating and preventing brain diseases.

Oleic acid (oleic acid) is a kind of unsaturated fatty acid widely present in living body, molecular weight is 282.5, melting point 13.3 ℃, boiling point 223 ℃ / 10 mmg Hg, pure colorless, odorless oil-like liquid. In nature, it exists as a main component such as olive oil, camellia oil, and major components such as iron oil and pork oil. In vivo, it is synthesized through palmitic acid through stearic acid. It is also converted to linoleic acid in plants. Phospholipids are present in the biofilm as constituents, in addition to being present in adipose tissue in the form of glycerol and esters (glycerides) rather than in the form of free acids. It is manufactured from resin as a raw material for industrial use, and is applied to the raw material of special soaps, such as soft soap and monopole soap, and the waterproofing material of textiles. Oleic acid has one double bond in one molecule and is a geometric isomer of ellidic acid which is a trans form. That is, oleic acid is the same substituent (cis) on the same side with respect to the double bond, and transversely opposite (trans) is ellide acid.

To date, studies on oleic acid have shown anticancer effects (Menendez et al., Annals of Oncology, p . 10, 2005), lowering blood viscosity to facilitate blood flow ( Alberts et al, Gerland Publishing, Inc., New York). , 1994), regeneration of nerve cells and myelination of nerve fibers (Medina and Tabernero, J Physiology, 96: 265-271, 2002). However, no studies on neuroprotective effects have been reported. In particular, studies on neuroprotective effects using animal models are not known.

The present inventors completed the present invention by confirming the inhibitory activity against cerebral ischemic neuronal cell death of oleic acid and the protective effect against ataxia due to neuronal damage.

An object of the present invention is to provide a composition for the prevention and treatment of degenerative brain diseases and neurological functional foods for the prevention and improvement of degenerative brain diseases.

In accordance with the above object, the present invention provides a pharmaceutical composition for the treatment and prevention of degenerative brain diseases containing oleic acid (oleic acid) having a neuroprotective activity as an active ingredient.

Furthermore, the present invention provides a dietary supplement for the prevention and improvement of degenerative brain diseases containing oleic acid as an active ingredient.

Hereinafter, the present invention will be described in detail.

Oleic acid of the present invention is octadisenoid acid ( Cis- 9-Octadecenoic acid), and is represented by the following formula (1).

The degenerative brain diseases include stroke, stroke, dementia, Alzheimer's disease, Huntington's disease, Pick's disease or Creutzfeld-Jakob's disease caused by neuronal cell death.

In addition, the oleic acid is first pulverized the raw material from the vegetable extract of the olive oil containing oleic acid and the animal fat, and then shaken and extracted by adding nucleic acid or chloroform and filtered to concentrate the nucleic acid layer in a vacuum concentrator to obtain a dried product, dried matter The silica gel column chromatography was used to separate and purify the oleic acid, wherein the mobile phase was separated by acetone and a nucleic acid 1: 9 mixed solvent, and separated by thin membrane chromatography to confirm the purity of the obtained oleic acid. It features.

The pharmaceutical composition for the treatment and prevention of degenerative brain diseases by neuronal cell death of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

The pharmaceutical composition comprising the compound of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

Pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the compound 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. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food for the prevention and improvement of degenerative brain disease, comprising the compound having a neuroprotective activity and a food supplement acceptable food supplement. Examples of the food to which the compound of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of protecting neurons and preventing degenerative brain diseases. At this time, the amount of the compound in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml. have.

Health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, 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 proportion of said natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Oleic acid of the present invention has little toxicity and side effects, so can be used with confidence even for prolonged use for prophylactic purposes.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Reference Example  1: Preparation of Laboratory Animals

Eight weeks-old Sprague-dawly male rats of about 300 g as experimental animals were purchased from Samtakosa (Seoul, South Korea) and were adapted to the experimental environment for one week with sufficient feed and water.

Experimental Example 1: Oleic Acid Nerve cells  Protective effect

To measure the neuroprotective effect of oleic acid on neuronal damage caused by focal cerebral ischemia, an intraluminal suture method was used (Zea Longa, et al. , Strok e, 20: 84-91). , 1989).

First, at the end of the 25 mm 4-0 nylon suture, about 5-8 mm length was coated with silicon, but the diameter was 0.30 mm and used for the experiment. Rats were subjected to general anesthesia with 5% isoflurane in a mixed gas of 70% N ₂ O and 30% O ₂ , and then a syringe for blood collection and one for a blood pressure probe were used. ), And blood pressure was observed, and blood glucose and blood gas were analyzed and observed. The skin in the center of the anterior neck was dissected and the right carotid and external carotid arteries (ECA) were carefully separated from the surrounding tissues and nerves. The upper thyroid artery and the laryngeal artery, which are the branches of the external carotid artery, are cauterized with electrocautery. The pterygoid palatal artery, which is the branch of the internal carotid artery, is cauterized. After inserting about 20 mm was fixed with a thread. The skin incision was resealed and then recovered naturally under anesthesia. Immediately and at 120 minutes oleic acid was intraperitoneally injected at a concentration of 10 and 100 mg / kg in 5 ml Tween 20 at a volume of 0.1 ml per 100 g of rat body weight, respectively. Tween 20 was administered. All experimental groups produced the same number of animals on the same day, and surgery was observed while maintaining the body temperature at 37 ± 0.5 ° C. 120 minutes after the operation, the anesthesia was re-analyzed in the same manner as above, and the probe was retreated to reperfusion. After 24 hours after reperfusion, the cervical spine was dissected, and the brain was removed within 2 minutes to obtain 6 sections with a thickness of 2 mm. The tissues were immersed in a 16 well plate containing 2% triphenyltetrazolium chloride (TTC), left at 37 ° C. for 30 minutes, and fixed with 4% paraformaldehyde to observe the tissues. All surgical procedures were performed under a surgical microscope. Anesthesia was performed to keep the isoflurane at 2% while keeping the temperature below 37 ° C. Tissues were taken one by one with a digital camera and then transferred to a computer, using the image analysis program Optimas 6.5 (Optimas 6.5, Bioscan) to calculate the cerebral infarction (%) by the following equation (1). At this time, the volume of the corrected cerebral infarction (mm ³ ) was calculated by the following equation (2).

Cerebral infarction rate (%) = (A-B / A) × 100

A: volume of normal left hemisphere (mm ³ )

B: volume of corrected cerebral infarction (mm ³ )

Volume of corrected cerebral infarction (mm ³ )

= (Volume of normal left hemisphere)-(normal site volume of damaged hemisphere)

As a result of measuring the protective effect of oleic acid on the brain tissue and neuronal cells, as shown in FIG. 1A, the group to which oleic acid was administered compared to the control group had a small area of the area where neurons were killed and not stained with TTC, and the nerve cells were damaged. As a result, it was found that the area of the black-red region stained with TTC was small. For reference, when brain tissue is stained with TTC, the areas where tissues are killed due to damage are not stained and appear white, and the tissues of normal areas are dyed and appear black.

In addition, as shown in FIG. 1B, the control group administered with 5% Tween 20 had a cerebral infarction rate (%) of 37.1 ± 4.34%, and in the group administered with oleic acid at 10 and 100 mg / kg, 20.9 ± 3.48 and 13.0 ± 2.79% showed concentration-dependent neuroprotective effect. They showed neuroprotective effects of 32.9 and 64.9%, respectively, compared to the control group, showing the highest efficacy at the dose of 100 mg / kg.

Experimental Example 2: Verification of neurobehavioral recovery effect of oleic acid

For Example 1 and neurobehavioural recovery of paper treated in the same way the effect was determined according to Longa (Longa) Neurobehavioral scores (Neurological score) measurements, such as (Stroke, 20, pp.84-91, 1989 ) . When the rats were raised about 50 cm from the floor, the rats scored 5 points of normal condition with both forelimbs pointing to the floor, 4 points for bending the forelimbs of the damaged area without any other symptoms, and completely bent the forelimbs. 3 points in the absence state, 2 points to the left, or 2 points, and rounded to the left was 1 point. 2 shows the final neurobehavioral score of each administration group by adding up the scores of each test item given according to the above evaluation score method. The control group showed a 2.86 ± 0.21 score, whereas the group injected with 100 mg / kg oleic acid showed a 3.95 ± 0.15 score (p <0.001), showing a neuroprotective effect at each dose.

Experimental Example 3: Acute Toxicity  Experiment

3-1: Oral administration

ICR mice (weight 25 ± 5 g) and Sprague-dawly rats were divided into four groups of 10 rats each to divide the composition of the present invention into doses of 100, 250, 500 and 1000 mg / kg, respectively. Two weeks after oral administration, toxicity was not observed in all animals treated with the test substance. There were no clinical signs or deaths. There were no changes in toxicity in weight changes, blood tests, blood biochemical tests, and autopsy findings. Not observed. As a result, the compound of the present invention did not show a change in toxicity even in rats up to 1000 mg / kg, respectively, the minimum lethal dose (LD ₅₀ ) was determined to be a safe substance more than 100 mg / kg.

3-2. Intraperitoneal administration

       ICR mice (weight 25 ± 5 g) and Sprague-dawly rats were divided into four groups of 10 rats each to divide the composition of the present invention into doses of 25, 50, 100 and 200 mg / kg, respectively. After 24 hours of intraperitoneal administration, no toxicity was observed in all animals treated with the test substance. There were no specific clinical symptoms or dead animals. Toxicity changes were observed in weight changes, blood tests, blood biochemistry tests, and autopsy findings. Was not observed. As a result, the compound of the present invention did not show toxicity change even in rats up to 200 mg / kg, respectively, and was determined to be a safe substance.

Hereinafter, an example of the preparation of a pharmaceutical composition comprising the oleic acid of the present invention will be described, but the present invention is not intended to be limited thereto but only to be described in detail.

Formulation example  One: Powder  Produce

Oleic acid 300 mg

Lactose 100 mg

Talc 10 mg

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

Formulation example  2: preparation of tablets

50 mg oleic acid

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation example  3: Manufacture of capsule

50 mg oleic acid

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  4: Preparation of Injection

50 mg oleic acid

Appropriate sterile distilled water for injection

pH adjuster

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation example  5: Liquid  Produce

Oleic acid 1000 mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water was added to adjust the total volume to 1000 ml. According to the conventional method for preparing a liquid, the above components were mixed, and then filled into a brown bottle and sterilized to prepare a liquid.

Formulation example  6. Manufacture of healthy food

Oleic acid 1000 mg

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.

Formulation example  7: manufacture of health drinks

Oleic acid 1000 mg

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components according to the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and then refrigerated and stored Used to prepare the healthy beverage composition of the invention.

As described above, the composition containing the oleic acid (oleic acid) having a neuronal protective activity of the present invention as an active ingredient not only has an excellent effect of inhibiting neuronal cell death generated in cerebral ischemia, but also has a sensory motor function due to nerve damage. By confirming the protective effect against ataxia, it can be used as a pharmaceutical composition or health functional food for the prevention or treatment of degenerative brain diseases caused by necrosis of neurons and for the improvement of sequelae.

Claims (3)

Selected from the group consisting of stroke, stroke, dementia, Alzheimer's disease, Huntington's disease, Pick's disease and Creutzfeld-Jakob's disease containing oleic acid of formula (I) as an active ingredient Health functional foods for the prevention and improvement of degenerative brain diseases caused by neuronal cell death:

(I). The dietary supplement according to claim 1, which has a formulation of tablets, capsules, pills or liquids. The dietary supplement according to claim 1, wherein the oleic acid is separated and obtained from olive oil, plant extracts or animal and vegetable fats and oils.

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