KR100886948B1 - Composition comprising Nodakenin having neuronal cell-protecting activity for preventing and treating the degenerative brain disease - Google Patents

Abstract

The present invention relates to a composition containing nodakenin (Nodakenin) as an active ingredient of the Angelica extract, specifically, the nodakenin as an active ingredient of the Angelica extract exhibits the effect of protecting the brain nerve cell damage induced by cerebral ischemia Therefore, the composition containing nodakenin can be used as medicines and health functional foods for the prevention and treatment of degenerative brain diseases caused by the death of nerve cells.

Nodakeknin, nerve cell protection, cerebral ischemia, degenerative brain disease

Description

Composition comprising Nodakenin having neuronal cell-protecting activity for preventing and treating the degenerative brain disease

1 is a rat cerebral ischemia model in the cerebral ischemia model of the middle cerebral artery occlusion method (MCAo method), in order to observe the protective effect of the neuronal cell damage in the rat brain tissue according to the administration concentration of nodakenin of the present invention Tissue sections were observed by TTC staining,

2 is a diagram showing a comparison of brain tissue damage rate (or cerebral infarction rate) between the control group administered dimethyl sulfoxide (DMSO) and the nodakenin group.

      Figure 3a and b is a diagram taken with a microscope after staining the coronal section of the dorsal hippocampus (dors hip hipcampus) of the normal control rats (sham) that did not induce whole cerebral ischemia (cresyl violet),

      Figures 4a and b show the coronal sections of the dorsal hippocampus after 7 days of treatment-treated rats that induced whole cerebral ischemia for 10 minutes and then administered 20% dimethyl sulfoxide at 0 and 90 minutes. This is a diagram taken with a microscope after staining with cresyl violet.

      Figures 5a and b show the brain inertia of the dorsal hippocampus after 7 days of drug-treated rats inoculated with 30 mg / kg of the nodakenine of the present invention at 0 and 90 minutes after inducing whole cerebral ischemia for 10 minutes. The sections were stained with cresyl violet and photographed under a microscope,

      Figure 6 is a graph showing the neuroprotective effect of nodakenin in the whole cerebral ischemia experimental model. Whole cerebral ischemia was induced for 10 minutes, followed by intraperitoneal administration of 20% dimethylsulfoxide (DMSO) and nodakenin twice for 0 minutes and 90 minutes. It is a figure which shows the result of measuring the cell number of the cerebral hippocampal CA1 area | region of the control group which administered diketyl sulfoxide after 7 days, and the group which nodakenin was administered.

The present invention relates to a composition containing nodakenin (Nodakenin) as an active ingredient of the Angelica extract, specifically, the nodakenin as an active ingredient of the Angelica extract exhibits the effect of protecting the brain nerve cell damage induced by cerebral ischemia Therefore, the composition containing nodakenin can be used as medicines and health functional foods for the prevention and treatment of degenerative brain diseases caused by the death of nerve cells.

According to the percentage of Korea's aging population, released by the National Statistical Office in October 2003, we entered an aging society with a population accounting for 7.2% of the population aged 65 and over 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 this change 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, the supply of oxygen and glucose is blocked, resulting in a decrease in ATP and edema in neurons, which eventually leads to 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. (Kirino T, Sano K. Acta Neuropathol ., 1984, 62 , pp. 201-208; Kirino T. Brain Res., 1982, 239 , pp. 57-69).

There are two mechanisms of neuronal death caused by cerebral ischemia that most people accept so far. One is the excitatory neuronal death mechanism in which excess glutamate accumulates outside the cell by cerebral ischemia, and this glutamate enters the cell and eventually causes neuronal death due to the accumulation of excess intracellular calcium (Kang TC, et al., . J. Neurocytol, 30, pp.945-955, 2001) and ischemia-oxidative neuronal death that due to the sudden supply of oxygen during the reperfusion-induced wear damage to the DNA and the cytoplasm due to the increase in vivo radicals, the two mechanisms (Won MH, et al., Brain Res ., 1999, 836 , pp. 70-78 .; Sun AY, Chen YM. J. Biomed . Sci ., 1998, 5 , pp.401-414 .; Flowers F, Zimmerman J J. New Horiz ., 1998, 6 , pp. 169-180).

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) is currently the only FDA-approved drug for treating cerebral ischemia, and it is a substance that induces rapid supply of oxygen and glucose by melting blood clots that cause cerebral ischemia with thrombolytics. Therefore, it does not directly protect nerve cells, so it is necessary to use it quickly. Due to the characteristics of thrombolysis, the blood vessel wall becomes thin when used excessively or frequently, resulting in 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.

Angelica gigas, Angelica gigas Nakai, is a plant belonging to the umbelliferae family, and its roots have been used for the effects of blood and blood. The components of the Angelica gigas are coumarin derivatives decursin, decursinol, and decursinol angelate (Ryu KS et al, Kor J. pharmacology, 21,64-68). 1990), 15 essential oils and several steroid components of β-sitosterol (Yoon HR et al, Kyung Hee pharma.sci, 23, 55-71) nodakenin, n-butylidene, phthalide and the like have been reported. Pharmacological actions include inhibition against glutamate toxicity, antioxidant activity, neovascularization, anticancer, acetylcholine esterase inhibition, and serum lipid lowering action. New sense, warming, liver, heart, parenteral, blood donation, active blood circulation, yuntongtong stool Efficacy in the treatment of heart, heart, head, throat, cervical pulmonary, conjunctival congestion, etc. Also known as enema artery flow Increasing, decreased platelet aggregation (Lee YY et al Arch. Pharm . Res., 2003, 26 (9) , 723-6.), The treatment of the heart, and forgetfulness, is widely used as a summary in blood clots. : Medicinal Pharmacology, Moon Mundang, 2001, 464-467.) However, no studies have been reported on neuroprotective effects.

      Accordingly, the present inventors confirmed that nodakenin can protect neurons against neuronal cell damage caused by cerebral ischemia of experimental animals induced by middle cerebral artery occlusion and 4-vessel obstruction, thereby significantly preventing neuronal cell damage caused by cerebral ischemia. Based on this, the present invention has been completed.

It is an object of the present invention to provide a composition for the prevention and treatment of degenerative brain diseases containing nodakine, a donkey extract having neuroprotective activity, as an active ingredient.

In accordance with the above object, the present invention provides a composition for the prevention and treatment of degenerative brain diseases containing nodakine as an active ingredient isolated from the Angelica extract having neuronal protective activity.

The nodakenin is characterized in that it is separated into an extract of Angelica gigas, Angelica acutiloba or Angelica sinensis, preferably Angelica gigas.

The extract includes water, a lower alcohol having 1 to 4 carbon atoms, or a solvent selected from a mixed solvent thereof, preferably extracted with n-butanol. The degenerative brain diseases include stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease or Creutzfeld-Jakob disease caused by neuronal cell death.

Hereinafter, a method of obtaining the extract of the present invention will be described in detail.

Nodakenin of the present invention is represented by the following structural formula (A).

(A)

(A)

The extract of Angelica gigas Nakai of the present invention is dried and chopped the root of Angelica gigas Nakai, about 1 to 20 times the dry weight (kg), preferably about 3 to 10 times the water, low carbon number 1 to 4 A first step of extracting with a solvent selected from alcohol or a mixed solvent thereof, preferably 70% ethanol, and then concentrated under reduced pressure to obtain a crude extract; After the extract is suspended in distilled water, it is a nonpolar solvent such as nucleic acid, chloroform, dichloromethane (CH ₂ Cl ₂ ), preferably dichloromethane, and a polar solvent such as methanol, ethanol, propanol and butanol depending on the solvent polarity. , Preferably, two steps of sequentially fractionating n-butanol to obtain a polar solvent fraction and a nonpolar solvent fraction, respectively; Separation of the fractions obtained in the step 2, such as silica gel column chromatography, Sephadex LH-20 chromatography, and high pressure liquid chromatography, etc. Separating the active ingredient candidates from the polar solvent fractions and the non-polar fractions of Angelica gigas by using; The nonakenine compound of the present invention can be obtained from the polar solvent, preferably n-butanol fraction of the three steps.

The present invention can be significantly prevented neuronal cell damage caused by cerebral ischemia by protecting the neurons against neuronal cell damage caused by cerebral ischemia of experimental animals induced by middle cerebral artery occlusion and 4-vessel obstruction. It was confirmed.

Accordingly, the present invention provides a notakenin having a neuroprotective activity obtained by the production method, which can be used for preventing and treating degenerative brain diseases caused by neuronal cell death.

In addition, the donkey containing nodakenin is a drug that has been used for a long time as a drug extracts of the present invention extracted therefrom also have no problems such as toxicity and side effects.

Accordingly, the present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, including the nodakenin as an active ingredient having neuronal protective activity.

 The pharmaceutical composition for the treatment and prevention of degenerative brain disease by neuronal cell death of the present invention comprises 0.001 to 50% by weight of the compound based on the total weight of the composition.

The pharmaceutical composition comprising nodakenin of the present invention may further include suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The pharmaceutical dosage forms of nodakenin 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 composition comprising nodakenin according to the present invention may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external 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, propyl hydroxybenzoate, 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.

The preferred dosage of nodakenin of the present invention depends 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, and 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 100 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.

Nodakenin of the present invention can be administered to mammals such as mice, mice, livestock, humans, etc. 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 dietary supplement 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 at 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 1g based on 100 ml. have.

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.

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.

Example  One. Nodakine  Produce

Extract of Angelica gigas root (dry weight 15kg) with 70% ethanol and concentrated under reduced pressure to obtain 1.4kg of crude extract. Dichloromethane (CH ₂ Cl _2), respectively dichloride Romero burnt by fractionation successively with n- butanol _{(n-butanol) (CH 2} Cl 2) were suspended along the crude extract in distilled water to them in a polar solvent Fraction (450 g) and n-butanol fraction (130 g) were obtained. Angelica dichloromethane (CH ₂ Cl ₂ ) using various separation methods such as silica gel column chromatography, Sephadex LH-20 chromatography, and high pressure liquid chromatography The active ingredient candidates were separated from the fractions and n-butanol fractions, and the results showed that nodakenin compounds (13 g) having the following physical properties were isolated from the n-butanol fractions and compared with those described in the references. (Kim, SH, Kang SS, Kim, CM. Arch. Pharm. Res. 1992, 15 , 73-77).

Reference Example  1. Preparation of experimental animals

Samtacosa and central laboratory animals (300 g, 8-week-old Sprague-dawly male rats, and 170 g, and 5-week-old Wistar male rats, respectively) were used as experimental animals. ) Was adapted to the experimental environment for 1 week while supplying enough feed and water, and then started the experiment.

Experimental Example  One. Nodakine Nerve cells  Protective effect-middle cerebral artery occlusion model MCAo )

An intraluminal suture method was used to measure the neuroprotective effect of nodakenin against neuronal cell damage caused by focal cerebral ischemia (Zea Longa, et al, Strok e., 1989, 20 , 84-91).

First, at the end of the 25 mm 4-0 nylon suture was coated with about 5-8 mm length of silicon but 0.30 mm in diameter was used in the experiment. Rats were subjected to general anesthesia with 5% isoflurane in a mixed gas of 70% N ₂ O and 30% O ₂ , followed by incision of the skin in the center of the anterior neck and the right carotid and external carotid arteries (ECA). Carefully separated from 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 was resealed and anesthetized. Immediately and at 120 minutes, nodakenine was intraperitoneally injected at a concentration of 10 and 30 mg / kg in 20 ml DMSO (20% Dimethyl Sulfoxide) in a volume of 0.1 ml per 100 g of rats, respectively. % DMSO 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 ℃. 120 minutes after the operation, the anesthesia was re-analyzed in the same manner as described above to retreat the probe. 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 sufficiently immersed in a 16 well plate containing 2% triphenyltetrazolium chloride (TTC) and left at 37 ° C. for 30 minutes. 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, and cerebral infarction percentage (%) was calculated by Equation 1 using an image analysis program Optimas 6.5 (Optimas 6.5, Bioscan). 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 brain tissue and neuronal cell protective effect of nodakenin, as shown in FIG. 1, the group of 30 mg / kg intraperitoneally administered nodakenin compared to the control group, the area of the site where the neurons were killed and not stained with TTC It was found that the nerve cells were not damaged and the area of the black-red area 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. 2, the control group administered with 20% DMSO showed a cerebral infarction rate (%) of 30.28 ± 2.24%, and in the group administered nodakenin at 10 and 30 mg / kg, respectively, 25.5 ± 5.54 and 13.38 ± 1.67% showed neuroprotective effect at 30mg / kg. This showed a neuroprotective effect of 55.8% compared to the control.

Experimental Example  2. Nodakine Nerve cells  Protective effect-4-vascular occlusion model (4- VO )

To measure the neuroprotective effects of nodakenin on neuronal damage caused by cerebral ischemia, we used a 4-vessel occlusion model developed by Pulsinelli in 1979 to observe the effect of stroke. It was. (Pulsinelli WA, Brierley JB., Stroke., 10 , pp 267-272. 1979)

Rats were anesthetized with 5% isoflurane contained in a mixture of nitrogen and oxygen (70% nitrogen, 30% oxygen), and then the head down on the head in a horizontal plane in a stereotaxic apparatus. 30 ° C. was fixed inclined. The nose and mouth were connected to the anesthesia with a plastic cone cone and the anesthesia was maintained at 1.5% isoflurane.

The tail was fixed on the operating table and the cervical spine was extended. First of all, in order to operate the throat area, a ring was made of silicon dive in the total carotid artery to induce ischemia and reperfusion. In order to block the circulation of the microvessel during ischemia, the cervical anterior chamber is located so that trachea, esophagus, external jugular vein and common carotid arteries are located behind. (cervical) and paravertebral muscles were penetrated and then wound closed with surgical clips.

Then turn the mouse to make a midline incision and operate the cervical spine at the bottom of the occipital bone to prevent muscle injuries using a surgical magnifier, and use a small electric acupuncture needle of less than 1mm through the alar foramina of the cervical spine. The vertebral artery was cauterized by inserting it into the tunnel through which the vertebral artery passed. A surgical microscope was used to confirm that the vertebral artery present in the tunnel passing into the bone was completely cauterized and closed and then closed with a surgical clip. 24 hours after closure, the surgical clip was removed and the carotid artery was removed with an aneurysm clip. Tightening for 10 minutes caused ischemia. If there was no loss of light reflection within 1 minute, the neck was sutured tightly, and mice without loss of light reflection were excluded because no complete bilateral parallel CA1 nerve injury was induced. Excluded. 10 minutes after ischemia induction, the aneurysm clip was removed from the total carotid artery to stop ischemia and reperfusion.

One week after ischemia induction, rats were anesthetized with chloral hydrate (35.0mg / kg, ip), and then deformed.Then, the right right ventricle was incised and a needle was injected into the left ventricle. Perfusion to the heart and perfusion with 4.0% formalin fixative. After that, the brains of the rats were removed, post-fixed in 0.1M phosphate buffered formalin fixative for 2 hours, and then soaked in 30% sucrose solution for 4 hours. In the fixed brain, a coronal block in the dorsal hippocampus between -2.5 mm and -4.0 mm is incised, frozen at -70, and 30 µm using a sliding microtome. Tissue sections containing hippocampus were prepared by cutting at intervals.

Sections prepared by the above method were stained and fixed in cresyl violet and then most injured by delayed neuronal death in dorsal hippocampal CA1 (Crain BJ et al ,. Neuroscience 1988, 27 , 387-402) The number of damaged neurons present in the 1,000 μm portion of the middle zone was observed. The number of cells showed that the normal number of pyramidal cells at high magnification (X 250) was observed by three observers in two brains, two at the left and right sides of three different tissue sections in one brain tissue. Values were averaged.

As a normal control, the brain hippocampal tissues of rats that did not induce cerebral ischemia, and the brain hippocampal tissues of rats that received 20% dimethyl sulfoxide injection instead of the nodakenin of the present invention in the above-described procedure were used.

As a result, the hippocampal neurons were observed normally in the normal control (sham operated) (see FIGS. 3A and 3B), and in the non-added control neurons, the cells were released from the surrounding cells while the tissues were relaxed unlike the normal neurons. It could be observed that the cell body also lost its original pyramidal form and condensed to form a single cell. In addition, the nuclear chromatin was concentrated and the nuclear membrane collapsed was observed to confirm that apoptosis (apoptosis) was made (see Figs. 4a and 4b).

The neuronal cells of the nodakenin group administered 30 mg / kg of the present invention were similar to those of normal cells, and were easily distinguished due to their well-expanding perikaryon and the centrally located round nucleus. It was clearly distinguished from neutrophils around the furnace. In addition, it was confirmed that the majority of cells were defended from apoptosis to form a normal phramidale form and continued to maintain junctions with surrounding cells (see FIGS. 5A and 5B).

In addition, in the normal control (sham) horizontal x cells 1mm ² The mean number of sugar cells was 374 ± 34.8, whereas in the no-added control group, it was 85 ± 18.8, indicating that many neuronal apoptosis occurred.

In the experimental group administered 10, 30mg / kg of notakenine of the present invention, the average cell number was 164 ± 37.7 and 193 ± 35.7 cells / mm, respectively, indicating that 48.1 and 55.9% of nerve cells were alive compared to the control group. As a result, it was confirmed to exhibit an excellent neuronal protective effect (see FIG. 6).

Experimental Example  3. Acute Toxicity  Experiment

3 -One. Oral administration

The composition of the present invention was divided into four groups of 10 ICR mice (weight 25 g 5 g) and Sprague-dawly rats each at a dose of 100, 250, 500 and 1000 mg / kg, respectively. Toxicity was observed for 2 weeks after oral administration.

As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxicity change was observed in weight change, blood test, blood biochemistry and autopsy findings. In view of the above results, the compound of the present invention did not show a toxicity change 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, and the composition of the present invention was intraperitoneally administered at doses of 25, 50, 100 and 200 mg / kg, respectively. Toxicity was observed for 24 hours after administration.

As a result, there were no clinical symptoms or deaths in all animals treated with the test substance, and no toxic changes were observed in weight change, blood test, blood biochemical test and autopsy findings. In view of the above results, the compound of the present invention did not show a change in toxicity even in rats up to 200 mg / kg, respectively, was determined to be a safe substance.

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

Formulation example  One. Powder  Produce

Nodakenin 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

Nodakenin 50 mg

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

Nodakenin 50 mg

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 Injectables

Nodakenin 50 mg

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

Nodakine 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

Nodakine 1000 mg

Vitamin mixture proper amount

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

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 phosphate monobasic 5 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

Nodakine 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 production method, and then 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 mentioned above, the composition containing nodakenin, which is an extract of the Angelica gigas, having a neuroprotective activity according to the present invention as an active ingredient, confirms the effect of inhibiting neuronal cell death generated during cerebral ischemia, It can be used as a pharmaceutical composition or health functional food for the prevention or treatment of degenerative brain diseases caused by necrosis and for the improvement of sequelae.

Claims (5)

A pharmaceutical composition for the prevention and treatment of stroke or stroke, comprising nodakenin of the structural formula (A) as an active ingredient:

                                                 (A). delete The pharmaceutical composition according to claim 1, wherein the nodakine is separated from the root of Angelica gigas Nakai. delete delete

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