KR100781581B1 - Pharmaceutical composition comprising gomisin-? as an effective ingredient showing neuro-protective activity - Google Patents

Abstract

The present invention relates to a composition containing Gomisin-A (gomisin-A) isolated from the fruit of Schisandra chinensis, in detail the composition containing Gomisin A of the present invention is a brain nerve cell damage induced by cerebral ischemia Health care food for preventing and improving degenerative brain disease, and for the prevention and treatment of degenerative brain disease caused by neuronal death. Can be used as

Hypermycin A, neuronal cell protection, cerebral ischemia, degenerative brain disease

Description

Pharmaceutical composition comprising gomisin A isolated from Schizandra chinensis having neuroprotective activity as an effective ingredient {pharmaceutical composition comprising gomisin-A as an effective ingredient showing neuro-protective activity}

      1A and 1B are microscopic images of coronal sections of the dorsal hippocampus of the sham rats that did not induce ischemia, followed by microscopic staining with Cresyl violet. ,

      1C and 1D stain coronal sections of the dorsal hippocampus of control rats orally administered 20% DMSO 7 days after inducing cerebral ischemia with Cresyl violet. Photo taken with a microscope,

      1E and 1F show coronal sections of the dorsal hippocampus of experimental rats 7 days after oral administration of gomisin-A (30, 10 mg / kg) after induction of whole cerebral ischemia. Was stained with Cresyl violet and photographed under a microscope,

      FIG. 2 is a graph showing the results of the measurement of the cerebral hippocampal CA1 region cell counts of the control rats treated with 20% DMSO orally at the concentration of hypermycin A after 7 days of induction of the normal group and whole cerebral ischemia. Is,

      3 is a diagram showing the neurobehavioral recovery effect of the control group, the control group that induced the whole cerebral ischemia, and the test group rats treated with hypermycin A. The sensory motor function test results at 4 days after reperfusion were normal and control group. Compared with

Figure 4 is a graph showing the change in body temperature of the experimental group rats administered with the control (control), high mycin A, which caused the whole cerebral ischemia.

    The present invention relates to a composition containing Gomisin A (gomisin-A) isolated from Schizandra chinensis having neuroprotective activity. The composition of the present invention is directed to degenerative brain diseases caused by neuronal cell death. It relates to medicines and nutraceuticals for the prevention and treatment.

    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 this change is that chronic degenerative diseases are becoming more of an issue than acute infectious diseases, which have been the main cause of death over the last 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. It is reported (Kirino T, Sano K. Acta Neuropathol ., 62 , pp 201-208, 1984; Kirino T. Brain Res ., 239 , pp 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 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 excessive accumulation of intracellular calcium. the sudden oxidation resistance being due to the supply of oxygen due to the increase in vivo radicals caused to damage the DNA, and a cytoplasmic neuronal death during the reperfusion, the two mechanisms -., J. Neurocytol, 30, pp945-955, 2001) and ischemia (Won MH, et al., Brain Res ., 836 , pp70-78, 1999; Sun AY, Chen YM. J. Biomed . Sci ., 5 , pp401-414, 1998; Flowers F, Zimmerman JJ. New Horiz . 6, pp169-180, 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) 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, because it does not directly protect nerve cells, it is necessary to use it quickly, and because of its characteristic of thrombolytic release, excessive use or frequent use of the thin wall of the blood vessels will eventually lead to 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.

Komi-Sin A is a single substance isolated from Schisandra chinensis, and Schisandra chinensis Baillon is the fruit of Schisandra chinensis Baillon, which is a major medicinal plant that uses deciduous vines and red fruits as herbal and food ingredients. Since ancient times, it has been processed and used in various kinds of food, and is widely used in the field of oriental medicine and medicine in addition to the food field.

     Consent of bodhisattva is to protect the lungs and help the kidneys, and coughs, astringents, astringents, nourishing tonics, quenching of mouth thirst, sputum sputum, or folk remedies. It is recorded that the powder (including seeds) is taken in warm water. In addition, fruit juice has been shown to have an anti-inflammatory effect when taken in gastric dyskinesia, and it is stated that the bark and leaves are steamed and dried in the shade and then eaten with tea to promote the taste and digestion (Chinese Herbs [Medicinal Herbs] Standard Collection, 2000).

     General ingredients of Schisandra chinensis consist of 80% water, 1% fat, 1.2% protein, and 14% total sugar, and sugars in fruit juice are fructose, sugar, glucose, maltose, organic acid, citric acid, tartaric acid, malic acid, and fish ( Viii) Succinate, acetic acid and lactic acid are contained. In the seed, 20-30% oil, resin, 1.6-2% essential oil and lignan compound are separated, and oil is 56-60% linoleic acid and 28-34% oleic acid.

      Lignan is identified as cyandrrin A, B, C, cyandrrol A and B, gomisin B, C, D, F, G, cyandrella A and B. It also contains 6.8% resin, about 5% saponin (bubble index 1: 200) and ascorbic acid (reduced 215 to 581 mg%) (NICS online Crop Information Center, 2003).

     The components contained in Schisandra chinensis fruit are mainly Lignan compounds, and it is reported that various components such as oil essential oils and pigments are contained. The lignan component of Schisandra chinensis was isolated for the first time in 1961 by Kochetkove et al., And was isolated and identified 30 species of lignan by several researchers (Rural Development Institute of Crop Science, 1999).

      Among the various lignan compounds contained in Schisandra chinensis, the analysis of Gomycin A has been extensively studied. In particular, hypermycin A has a hepatoprotective effect. Komishin A was found to be the same ingredient reported by Japanese and Chinese ginseng scientists as an active ingredient of Schisandra chinensis's hepatoprotective effect.

      However, none of the above teachings teaches or discloses that hypermycin A has activity to protect neurons induced by ischemia.

Accordingly, the present inventors confirmed that hypermycin A isolated from Schisandra chinensis has an activity capable of significantly inhibiting cerebral ischemic neuronal cell death, thereby completing the present invention.

      An object of the present invention is to provide a composition for the prevention and treatment of degenerative brain disease, which is harmless to the human body and contains gomisin-A as an active ingredient that can prevent neuronal cell damage caused by cerebral ischemia.

      In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, comprising as a active ingredient high mycin A represented by the following structural formula (1) having a neuroprotective activity.

(1)

(One)

In addition, the superstitious A is Schisandra ( Schisandra chinensis Baillon).

      The degenerative brain disease is characterized by induced neuronal cell death, degenerative brain disease caused by neuronal cell death, stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick (Pick) disease and Creutzfeldt- Creutzfeld-Jakob disease, preferably stroke, stroke, dementia and the like can be included.

       Hereinafter, the present invention will be described in detail.

      For example, the first step of grinding the Schizandra chinensis, ultrasonic extraction with a polar solvent such as water, methanol or ethanol and then filtration, concentrated under reduced pressure and lyophilized to obtain a crude extract soluble in a polar solvent; A second step of suspending the crude extract in water, sequentially dissolving and suspending it with a nonpolar solvent of hexane and methylene chloride, and then fractionating and filtering to obtain a nonpolar solvent soluble extract such as hexane soluble extract and methylene chloride; Using a non-polar solvent soluble extract such as hexane soluble extract as a developing solvent, hexane: acetone mixed solvent as a developing solvent, silica gel column chromatography was performed using silica gel (580 g, 70-230 mesh) as a fixed phase. And HPLC was added again (Nova-Pak C-18, Φ 2.1 × 150 mm, temperature 30 ° C., flow rate 0.2 ml / min, USA) under the conditions of mobile phase (methanol: water = 70: 30-85: 15) It is possible to separate and obtain the high mycin A of the present invention.

      The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, which contains high mycin A obtained as the active ingredient.

      The pharmaceutical composition of the present invention preferably contains 0.01 to 99% of hypermycin A, more preferably 0.1 to 90%. The above content does not limit the scope of the present invention in any aspect.

      The present invention provides a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, which comprises hypermycin A having a neuroprotective activity as an active ingredient, and includes a pharmaceutically acceptable carrier, diluent or excipient.

      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.

      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 are mixed. 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 intra-cerebroventricular injections.

     The present invention provides a dietary supplement for the prevention and improvement of degenerative diseases, including hypermycin A and neurologically acceptable food additives having neuroprotective activity. 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.

     As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

     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 is added at a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be.

      Health functional food of the present invention includes the form of tablets, capsules, pills, liquids and the like. For example, the health functional food in the form of tablets may be prepared as it is or granularly mixed with an excipient, a binder, a disintegrating agent or other additives in a suitable manner, and then compressed into a glidant, etc. It is made by directly compressing the functional food as it is or by mixing it evenly with an excipient, binder, disintegrant or other suitable additives, or mixing the health functional food as it is or evenly adding the appropriate additive to the prepared granules, The excipient, binder or other suitable additives are added to the functional food, and the powder mixed evenly is wetted with a solvent, the wet powder is molded into a mold at low pressure, and then dried and prepared by a suitable method. In addition, the nutraceutical can be added to the health functional food in the form of tablets, if necessary, can be avoided with a suitable epidermis.

      Among the health functional foods in the form of capsules, the hard capsules are usually prepared by mixing the capsules evenly with the health functional foods or excipients suitable for the health functional foods, granulated by a suitable method, or granulated with a suitable epidermal agent as it is or Soft capsules are prepared by filling them, and soft capsules are usually filled with capsules containing glycerin or sorbitol in a capsule form containing gelatin or appropriate excipients suitable for health functional foods or health functional foods. It is molded and prepared, and a coloring agent preservative etc. can be added to the said capsule base as needed.

      Circular functional foods are usually mixed with excipients, binders, disintegrants, etc., and then molded into a spherical form in a suitable manner, and the coating is carried out with white sugar or other suitable coating agent, or starch, talc or You can also be greeted with a suitable substance.

     The health functional food in the form of granules is usually made by mixing the health functional food as it is or by adding excipients, binders, disintegrating agents, etc., evenly and then granulating it in a proper way to make the particles as even as possible. , Mating agent, etc. can be added. For the health functional food in the form of granules, No. 12 (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm) were used for the next particle size test. It should be less than 5.0% and pass through No.45 to less than 15.0% of the total amount.

      The definitions of the excipients, binders, disintegrants, lubricants, copulation agents, flavoring agents, etc. of the present invention are those described in the literature known in the art and include those having the same or similar functions. , Korean College of Pharmacy, 5th Edition, p33-48, 1989).

      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.

      Gomisin A of the present invention has little toxicity and side effects, and therefore can be used with confidence even for prolonged administration 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.

Example  1. From Schisandra Superstitious  Component separation of A

The present invention carried out qualitative and quantitative analysis using HPLC to ensure standardization and stability of the composition. Methanol was added to 20 kg of Schizandra chinensis (June 15, 2005), and the mixture was extracted under reflux twice at 80 ° C. and filtered. The filtrate was concentrated under reduced pressure at 40 ~ 45 ℃ to obtain 4.5 kg of methanol. Methanol X is suspended in 2 L of distilled water, followed by hexane and ethyl acetate (EtOAc). And butanol (BuOH) sequentially. Each fraction was concentrated, and 512 g of hexane fraction was concentrated on a silica gel column and chromatographed with hexane-acetone as a developing solvent to increase polarity. Divided into small fractions (H1-6). H5 in 6 small fractions (H1 ~ 6) was suspended on a silica gel column and eluted with hexane-ethyl acetone 6: 1 to obtain H5.1 and H5.2. Hypermycin A was isolated.

      Isolated Gomicin A was subjected to HPLC (Nova-Pak C-18, Φ 2.1 × 150 mm, temperature 30 ° C., flow rate 0.2 ml / min, USA) at 254 nm UV to methanol: water (70:30-85:15 0.5 mg of the sample was dissolved in 1 mL of methanol under the conditions of) to prepare a stock solution. Purity was obtained from the chromatogram peak area obtained by precisely taking 10 L of this and injecting into HPLC. Purity is expressed as the average value obtained from three experiments. The purity of the isolated superstitis A was 95.9-99.9%.

      This isolated supermycin A was analyzed using LC / MS / MS. At 13.27 minutes, gomisin A eluted. In addition, validation of the parameters was confirmed by measurement of various parameters. As a result of the analysis of crude extracts of Schizandra chinensis, the presence of gomisin A, the main component, was confirmed in MRM mode. Physical properties of high-mycin A are shown in Table 1 below.

Properties for Komycin A origin Schisandra [ Schizandra] chinensis (Schizandraceae)] Molecular Weight C ₂₃ H ₂₈ O ₆ (416) Solubility Soluble in DMSO, ethanol, methanol, chloroform (Soluble in DMSO, EtOH, MeOH, CHCl ₃ )

Experimental Example  1. of rats Cerebral ischemia  cause

In order to confirm the neuroprotective effect of hypermycin A, a 4-vessel occlusion model developed by Pusinelli in 1979 was used (Pulsinelli WA, Brierley JB.stroke, May-Jun; 10 (3): 267-72 , 1979).

     The model is a representative animal model of neuronal death due to cerebral ischemia. When the blood vessels supplying blood to the brain of the rat are temporarily blocked and reperfused, the nerve cells of the dorsal hippocampal CA1 are mainly damaged. Using the principle, drugs that exhibit neuronal necrosis in this model are known to be effective in local ischemic animal models similar to human paralysis, and can be obtained as a model for paralytic experiments. Be in the spotlight. In addition, the forebrain ischemia model has the advantage that the blood flow of the posterior brain is not affected and does not affect respiration and body circulation.

     Five-week-old male rats of about 180 grams of test animals were imported from Japan (SLC, Japan) and adapted to the experimental environment for one week with sufficient feed and water. Anesthetize the rats, and then fix the tail 30 ° inclined downward on the head in horizontal position on a stereotaxic apparatus and plastic cone on the nose and mouth around anesthesia (Ohameda VMC / Boc Health Care, Cyprane, UK). Connected by inhaler. Anesthesia was initially maintained with 5% isoflurane contained in a mixture of nitrogen and oxygen (70% nitrogen, 30% oxygen) and subsequently maintained at 1.5% isoflurane.

     The tail was fixed on the operating table and the cervical spine was extended. First, the occipital bone was operated. The first cervical spine at the bottom of the occipital bone was operated using a surgical magnifier and the muscles were approached intact toward the alar foramina. A fine electroacupuncture needle of 1 mm or less was inserted through the alar foramina of the cervical spine 1 into the tunnel through which the vertebral artery was passed, and the vertebral artery was aborted by applying an intermittent current. Using a surgical microscope, it was confirmed that the vertebral artery present in the tunnel passing through the bone was completely cauterized and closed with a surgical clip. Then, the rats were turned to make a ring in the carotid artery with a surgical thread in order to operate the throat area, to induce ischemia and reperfusion. In order to block the circulation of microvascular during ischemic induction, trachea, esophagus, external jugular vein and common carotid arteries are located in the back and the cervix in front. The cervical and paravertebral muscles were penetrated and then wound closed with surgical clips.

     After 24 hours, the surgical clip was removed and the carotid artery was tightened for 10 minutes with an aneurysm clip to induce ischemia. If there was no loss of light reflection within 1 minute, the neck was sutured tightly, and rats without loss of light reflection were excluded from this experiment because no complete bilateral parallel CA1 nerve injury was induced. He also ruled out cramping rats. After 10 minutes, the aneurysm clip was removed from the total carotid artery and reperfused. In addition, only rats with a 20 ± 5 min loss of consciousness after reperfusion were used for the experiment.

     In the normal group, the neurons of the hippocampus were normally observed in the normal group (see FIGS. 1A and 1B), and in the case of the control group, the cells were released from the surrounding cells as the tissue was relaxed and the cell body lost its original shape and condensed. It was confirmed that it is in the form of a single cell. In addition, nuclear chromatin was concentrated and the nuclear membrane collapsed was observed to confirm that apoptosis (apoptosis) was made (see Fig. 1c, 1d).

Experimental Example  2. Superstitious  Oral administration of A

Administration of sample and setting of experimental group-In order to measure the efficacy of hypermycin on forebrain ischemia in rats, experiments were performed by dose according to the following experimental methods (Pulsinelli WA, Brierley JB. , May-Jun; 10 (3): 267-72 , 1979).

      Administration time was orally administered 20 minutes after ischemia induction. Gomisin A was dissolved in 20% DMSO and the same volume was orally administered at doses of 10 mg / kg and 30 mg / kg, respectively. The first group was the sham group with the same surgical procedure but no forebrain ischemia. In the second group, 20% of DMSO was orally administered at the same time interval as the sample administration after inducing whole cerebral ischemia as a control. Groups 3 and 4 were orally administered with high-mycin A at concentrations of 10 mg / kg and 30 mg / kg 20 minutes after ischemia induction. As a result, the control group showed a cell survival rate of 23% compared to the normal cell number, whereas the concentration of hypermycin A administered groups (10, 30mg / kg) showed a cell survival rate of 50% and 64.7%, respectively (Fig. 1E, 1F, Fig. 2).

Experimental Example  3. Superstitious  A administration Cerebral ischemia  Temperature Changes in Rats

Temperature decrease during cerebral ischemia has a neuroprotective effect by preventing nerve cell damage in the hippocampus (Busto et al, J. Cereb . Blood Flow Metab . 7 , pp720 ~ 738, 1987). The control and body temperature of the experimental group were monitored at 30 minute intervals up to 2 hours after ischemic induction, and at 1 hour intervals from 3 hours to 6 hours. In case of a decrease in body temperature, the experiment was performed while blocking the protective effect of neurons due to hypothermia while partially preventing the temperature decrease. To maintain ischemia in rats and to maintain 37 ± 0.5 ° C. during reperfusion and recovery periods, rats were fixed at 37 ° C. using a thermostat with rectal temperature. Body temperature was measured by inserting a probe at least 6 cm into the rectum to measure rectal temperature (Miyazawa T et al. J. Cereb . Blood Flow Metab , 12 , pp817-822). , 1992).

     As a result, it was observed that the body temperature did not drop in the group administered with hypermycin A (see FIG. 4).

Experimental Example  4. Tissue Observation

      After 1 week of ischemic induction, rats were anesthetized with chloral hydrate (35.0 mg / kg, ip), and the incision was made in the right ventricle and the needle was injected into the left ventricle. Heparinized 5% sodium nitrite saline was applied to the heart. Perfusion and perfusion with 4.0% paraformaldehyde fixative. Thereafter, the brains of the rats were detached and post-fixed in 0.1 M phosphate buffered paraformaldehyde fixative for 2 hours, and then immersed in 30% sucrose and fixed overnight at 4 ° C. Coronal blocks were prepared in the dorsal hippocampus region between -2.5 mm and -4.0 mm in the fixed brain. After freezing the piece, a sliding microtome was used to make tissue sections containing the hippocampus. Tissue sections for sampling were collected every 20 μm.

Observation of damaged neuronal cell counts--Sections containing Dorsal hippocampus were stained and fixed in cresyl violet and then the most susceptible to delayed neuronal death of the dorsal hippocampal CA1. (Crain BJ et al., Neuroscience , 27 , pp387-402, 1988) Neuronal cell numbers were observed at 1,000 μm length in the middle zone. In the observation of cell number, the number of pyramidal cells showing normal morphology at high magnification (× 250) was observed by three observers in two brains, 6 left and right sides of three different tissue sections in one brain tissue. Averaged (see FIG. 2).

     As a result, an average of 374 cells were observed in the normal group, whereas 85.9 in the control group showed 22.9% cell survival rate compared to the normal group. 187.3 cells and 242 cells were observed in the group of high-mycin A (10mg / kg, 30mg / kg), respectively, and the significant cell encapsulation effect was 50% in 10mg / kg oral administration and 64.7% in 30mg / kg administration compared to the control group. Was observed (p <0.05).

Experimental Example  5. Superstitious  Neural Behavioral Recovery Effect of A

Neurobehavioral recovery effect of white paper treated in the same manner as Experimental Example 1 was measured by the Y-Maze method of Helen Hodges et al. (Helen Hodges et al., Brain Research , 111 , pp213-221, 2000)

      In the Y-shaped labyrinth maze made of black acrylic plates, the three arms were designated as A, B, and C, respectively. It was. If A, B, and C travel each time in a row, it is 1 point. If A, B, and C do not travel in a row, no score is given. The measured score was divided by the total number of visits, subtracted by 2, and calculated as an actual score to obtain the actual score.

     According to the above evaluation score method, the final neurobehavioral scores of the normal group, the control group and the hypermycin A administration group are shown in FIG. 3. In the normal group, the accuracy was 68.9%, whereas in the control group, the accuracy was 7.6%, which was significantly different from the normal group, and in the experimental group administered orally with hypermycin A 30mg / kg, the accuracy was 41.8% (p <0.005). Showed a neuroprotective effect (see FIG. 3).

      Although a formulation example of a pharmaceutical composition comprising a compound of the present invention will be described, the present invention is not intended to be limiting but merely illustrative.

Formulation example  One. Powder  Produce

Gomisin A 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

Gomisin A 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

Gomisin A 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

Gomisin A 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

Gomisin A 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

Gomisin A 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 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

Gomisin A 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 described above, the composition containing gomisin-A having a neuroprotective activity of the present invention as an active ingredient is not only excellent in inhibiting neuronal cell death occurring in cerebral ischemia, but also a sense of nerve damage. By confirming the protective effect against ataxia of motor function, it can be used as a pharmaceutical composition for the prevention and treatment of degenerative brain diseases caused by necrosis of neurons or as a health functional food for the prevention and improvement of degenerative brain diseases.

Claims (5)

A pharmaceutical composition for the prevention and treatment of stroke, stroke, dementia, Alzheimer's disease or Parkinson's disease containing gomisin-A isolated from Schisandra chinensis Baillon having neuronal protective activity. delete delete delete delete

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