KR100452555B1 - A composition comprising purified extract of wild ginseng having neuronal cell-protecting activity - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing the raw ginseng extract protecting nerve cells induced by brain ischemia is provided. It can be used as drugs for prevention and treatment of degenerative brain disease caused by neural cell death, namely stroke, cerebral apoplexy, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick's disease and Creutzfeldt-jakob. CONSTITUTION: The pharmaceutical composition contains 0.1 to 50% by weight of the raw ginseng extract, based on the total weight of the composition. The raw ginseng extract is obtained by the steps of: extracting raw ginseng by distillation in the first stage; re-distilling the extract in the second stage; freezing the extract to give frozen fraction repeatedly; thawing and filtering the frozen fraction; heating the filtrate slowly in a double boiler; freezing and thawing the frozen filtrate; and sterilizing the thawed filtrate.

Description

A composition comprising purified extract of wild ginseng having neuronal cell-protecting activity}

The present invention relates to a composition containing wild ginseng extract tablets having neuronal protective 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 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 for 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. (Kirino T, Sano K. Acta Neuropathol., 62 , pp201-208, 1984; Kirino T. Brain Res ., 239 , pp57-69, 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 the accumulation of excess intracellular calcium (Kang TC, et al., J. Neurocytol., 30 , pp945-955, 2001) and two mechanisms of oxidative neuronal death, which are caused by damage to DNA and cytoplasm due to an increase in radicals in vivo due to sudden oxygen supply during ischemia-reperfusion. (Won MH, et al., Brain Res ., 836 , pp 70-78, 1999; Sun AY, Chen Y M. J. Biomed. Sci ., 5 , pp401-414, 1998; Flowers F, Zimmerman JJ. New Horiz. 6 , pp 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) 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 is not necessary to directly protect nerve cells, so it is necessary to use it quickly. Due to the characteristics of thrombolysis, the blood vessel wall becomes thinner 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 efforts to develop natural materials that have a long-term use and proven safety by objectively verifying natural diseases with the effect of treating and preventing brain diseases.

Recently, in order to overcome the side effects of cancer and brain disease treatment by chemotherapy of western medicine, alternative medicines are being developed to treat diseases with all other medicine except western medicine, the most representative of which is to enhance immune function Chinese medicine treatment based on.

In oriental medicine, we try to extract herbal medicines with less side effects while improving immunity and use them as therapeutics. Medicinal acupuncture is being used as one of the methods to enhance the effects among oriental medical treatments. Herbal acupuncture is a method of extracting an effective ingredient by selecting a drug that can be most effectively treated for each disease appearing in the human body, and then injecting it into acupuncture points or acupuncture points that can most effectively treat the disease. It is possible to maximize the effectiveness of treatment because it extracts and refines pure herbal medicine and injects a very small amount of drug into the place where saliva is placed.

However, the conventional herbal medicine extracts are not sufficiently purified, so the side effects of the herbal medicines remain, which cannot be used for acupuncture or injectables, or there is a problem that side effects such as fever, pain and edema occur when used.

In addition, conventional drugs for treating brain diseases have been difficult to use for a long time due to side effects such as weight loss and immunity lowering, and also extracts by extracting substances from natural ingredients such as herbs using organic solvents, or There was a problem that could not be used for injection or herbal medicine other than oral administration because it is not properly purified.

Therefore, the present inventors use an active ingredient from wild ginseng extract tablets, and using a purified liquid that does not contain impurities, the inhibitory activity against cerebral ischemic neuronal cell death and learning ability due to neuronal cell damage in an animal model inducing whole cerebral ischemia. This invention was completed by confirming the improvement effect to a fall.

It is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of degenerative brain diseases, which contains wild ginseng extract and tablet as an active ingredient.

1 is a graph showing the results of measurement of the number of mainly correct choices in a radial maze learning experiment after induction of whole cerebral ischemia in rats,

Figure 2 is a graph showing the measurement results of the number of error rate mainly in the radial maze learning experiment after the rat is induced whole cerebral ischemia,

3 is a photograph showing the degree of nerve cell damage in the hippocampus of the brain tissue using the Cresyl violet staining method,

Figure 4 is a graph showing the degree of nerve cell damage in the hippocampus of the brain tissue using Cresyl violet staining method,

5 is a diagram illustrating the degree of AchE expression in the CA1 region of the brain hippocampus using acetylcholinesterase (AchE) staining method,

6 is a diagram illustrating the degree of AchE expression in the CA3 region of the brain hippocampus using acetylcholinesterase (AchE) staining method,

7 is a diagram illustrating the degree of ChaT expression in the CA1 region of the brain hippocampus using choline acetyltransferase (ChAT) immunohistochemical staining (Immunohistochemistry),

8 is a diagram illustrating the degree of ChaT expression in the CA3 region of the brain tissue hippocampus using choline acetyltransferase (ChAT) immunohistochemical staining (Immunohistochemistry).

In accordance with the above object, the present invention is carried out by repeated distillation method by adding water or lower alcohol to wild ginseng raw material, and contains the extracted ginseng extract purified liquid obtained by performing a freezing treatment and filtration process as an active ingredient The present invention provides a pharmaceutical composition for preventing and treating degenerative brain disease, including a pharmaceutically acceptable carrier, diluent or excipient.

The extractive distillate comprises an extractive distillate soluble in water, preferably a polar solvent selected from lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof.

Specifically, the wild ginseng extract purification solution of the present invention is the first step of producing a primary extract by extracting and distilling wild ginseng raw material; A second step of preparing a second distillate by re-distilling the primary extract; A third step of repeatedly performing a process of freezing the secondary distillate to obtain only a frozen fraction; A fourth step of obtaining a filtrate by thawing and filtering the frozen fraction obtained from the third step; A fifth step of freezing the filtrate of the fourth step again after heating the bath; And a sixth step of obtaining a wild ginseng extract tablet having a neuronal cell protective activity by thawing the frozen matter of the fifth step and then sterilizing it.

The wild ginseng extract tablets may be used wild, cultivated or cultured wild ginseng.

Referring to the method of purifying the wild ginseng extract tablets in more detail,

A polar solvent selected from water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof is added to the wild ginseng at a rate of 1.0 to 3.0 kg per liter of water, preferably for 1 to 4 hours, preferably for 3 hours. After leaving as it is, the first step of heating for 60 to 120 ℃, preferably 80 to 100 ℃ for 6 to 24 hours, preferably 8 to 10 hours in a distillation apparatus to obtain a primary extract;

The primary extract of the first step is placed in a flask containing boiling stone, and the distillation apparatus at 60 to 120 ℃, preferably 80 to 100 ℃, more preferably 100 ℃ for 7 to 24 hours, preferably 8 to Obtaining a second distillate by performing a second process of heating and distilling gradually increasing the temperature until it becomes 70 to 90% by volume relative to the total volume of the primary extract for 12 hours;

The second distillate of the second process is frozen at -5 to -15 ° C, preferably -8 to -12 ° C until the non-frozen portion is less than 5% of the total volume, and then the non-frozen portion is After removing the remaining distillate slowly at room temperature, the fourth step of repeating the above-mentioned freezing treatment 1 to 6 times, preferably 2 to 5 times, to obtain a frozen fraction from which the toxic fraction was removed. step;

Obtaining a filtrate by performing a fourth process of completely dissolving the frozen fraction of the third process at room temperature and then filtering the filter using 0.1 to 0.6 μm filter paper;

The filtrate of the fourth step is heated at 60 to 120 ℃, preferably 80 to 100 ℃ 20 to 40 minutes, preferably 30 minutes, and then -30 to -10 ℃, preferably -15 ℃ Performing a fifth process of completely freezing in the water;

The frozen product of the fifth step is thawed completely at room temperature, and then sterilized to obtain a wild ginseng extract tablet having neuroprotective activity.

In the purification method of the extraction tablet, the concentration of the primary extract can be adjusted by adjusting the addition ratio of wild ginseng in the first step of preparing the primary extract.

In addition, in the purification method of the extracted tablet liquid, the freezing portion and the non-frozen portion according to the difference in freezing point of the specific components of the distillate during the freezing treatment, among the components of the wild ginseng extract tablet liquid having a high toxicity is a low freezing point Due to the freezing of the pure material portion of the lower density sequentially, it is possible to remove the non-frozen portion containing the toxic components, and by repeating such a method, the extraction of the toxic components that may cause side effects of wild ginseng itself is removed. Purified liquid can be prepared.

In addition, the present invention for the prevention and treatment of degenerative brain diseases containing wild ginseng extract tablets having a neuroprotective activity purified by the above purification method as an active ingredient, and containing a pharmaceutically acceptable carrier, diluent or excipient. Provide a pharmaceutical composition.

The wild ginseng extract tablet of the present invention has a neuronal protective action against neuronal cell damage induced by whole cerebral ischemia in experimental animals, thereby improving the impaired learning ability.

Therefore, the present invention shows that wild ginseng extract tablets with neuronal cell protective activity can be used as a medicament for the prevention or treatment of degenerative brain diseases caused by neuronal cell death.

In addition, the wild ginseng extract tablets are components that have been separated from natural products that have been used as herbal medicine for a long time, so there is almost no toxicity and side effects, so they can be used with confidence even for long-term use.

The composition of the present invention comprises 0.1 to 50% by weight of the wild ginseng extract tablet based on the total weight of the composition.

The degenerative brain disease is characterized by 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 or Creutzfeldt- Creutzfeld-Jakob disease may be included.

The composition comprising a wild ginseng extract tablet having a neuroprotective activity of the present invention may further comprise a suitable carrier, excipient or diluent according to a conventional method.

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

The composition comprising the extract tablet according to the present invention, powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations, such as aerosols, external preparations, suppositories, sterile injectable solutions or herbal needles, respectively, according to a conventional method It can be used in the form of a formulation.

In detail, when formulated, it may be prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. which are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, and the like in the extractive tablet solution. It can be prepared by mixing sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and 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.

Extracted tablet solution of the present invention may vary depending on the age, sex, and weight of the patient, but generally in an amount of 0.01 to 500 mg / kg, preferably 0.1 to 100 mg / kg, divided once or several times daily May be administered. In addition, the dosage of the extract can be increased or decreased depending on the route of administration, the degree of disease, sex, weight, age and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example, can be administered by acupuncture points, oral, rectal or muscle, subcutaneous, intrauterine dural or medicinal acupuncture, preferably in the preparation of acupuncture needles administered to acupuncture points. have.

Wild ginseng extract tablets of the present invention has little toxicity and side effects, so can be used with confidence even for long-term use for the purpose of prevention.

Hereinafter, the present invention will be described in detail by the following reference examples, examples and experimental examples.

However, the following reference examples, examples and experimental examples are merely to illustrate the invention, the content of the present invention is not limited by the following reference examples, examples and experimental examples.

Reference Example 1. Preparation of Laboratory Animals

Sprague-dawly male rats (260-300 g) were purchased from the Tertiary Animal Center for one week in laboratory conditions (temperature: 22 ± 3 ° C, humidity: 50 ± 10%. Illumination: 12 hours / Adapted from During the adaptation period, water and feed (Samyang Oil, Seoul, Korea) were freely ingested, and the rat was used in the following experimental example.

Example 1 Preparation of Purified Goat Extract

1 L of water was added to 2.5 kg of Sanyangsan ginseng, which was purchased from Gyeongsan, Gyeongsangbuk-do, and washed to a size of 3 mm. The flask containing goat ginseng was connected to a distillation apparatus, and heated to 90 ° C. for 8 hours to obtain a primary extract. 5 g of boiling stone was added to the flask, and then the primary extract was added thereto, and the flask was connected to a distillation apparatus. The primary extract was heated and distilled at 90 ° C. for 10 hours while gradually increasing the temperature to obtain a secondary distillate. The second distillate is frozen at -10 ° C until the portion that is not frozen is less than 5% of the total volume, then the portion that is not frozen is removed, thawed slowly at room temperature, and then again the above procedure twice Repeated. The frozen frozen fractions were completely dissolved at room temperature, filtered using a 0.4 μm filter paper, and the filtrate was bathed at 90 ° C. for 30 minutes and then completely frozen to −15 ° C. The frozen extract tablet solution was completely dissolved at room temperature, and then, 100 ml each of the sterilized bottles were stored at 4 ° C. or lower and used as a sample of the following experimental example.

Example 2 Preparation of Purified Cultured Ginseng Extract Tablet

1 liter of water was added to 2 kg of cultured ginseng (400 g for dried cultured ginseng), washed and shredded to 3 mm size, supplied from the Department of Oriental Resource Science, Kyung Hee University (Seoul, Korea) for 3 hours at room temperature at 20 ℃. After standing, cultured ginseng extract tablet solution was prepared in the same manner as in Example 1, and used as a sample of the following experimental example.

Comparative Example 1. Preparation of unpurified Sansam ginseng extract tablet

Purified and washed in Gyeongsangbuk-do, Gyeongsangbuk-do, 2.5 g of Sanyangsan ginseng sliced to 3 mm in size, add 1 L of water, boil at 100 ° C, filter, cool to room temperature, and store under refrigeration at 4 ° C or below. It was.

Comparative Example 2. Preparation of crude purified ginseng extract tablet

1 ℓ of water was added to 2 ㎏ of cultured ginseng, which was provided from Kyung Hee University, Department of Oriental Resource Science (Seoul, South Korea), cut into 3 ㎜ sizes, boiled, filtered, cooled to room temperature, and refrigerated at 4 ℃ or below. It used as the sample of an experiment example.

Experimental Example 1. Observation of body temperature and spot formation after administration of wild ginseng extract

The experimental animal of Reference Example 1 was used to observe the change in body temperature and the generation of spots, and the back hairs were removed to observe the generation of spots on the back, and the experimental animals of Example 1, Example 2 and Comparative Example 2 The test group was divided into groups, and each experimental group was five. In order to observe the change in body temperature and the formation of spots on the back after administration of each sample, the samples of Example 1, Example 2 and Comparative Example 2 were intraperitoneally administered by 20 ㎖ / kg, respectively, 0.5, 1, The body temperature was measured every 2 hours, and as a result of observing spot formation, the group treated with Example 1 and Example 2 showed little change in body temperature and maintained a normal body temperature. It was confirmed that the body temperature is increased in the group administered (see Table 1). In addition, it was confirmed that some abnormal spots were generated in the group administered with Comparative Example 2, but the side effects as described above were not observed in the groups administered with Examples 1 and 2.

Elapsed time Example 1 Example 2 Comparative Example 2 Before dosing 36 ± 0.7 36 ± 0.7 36 ± 0.7 0.5 hours after dosing 36 ± 0.8 36 ± 0.8 38 ± 0.2 1 hour after dosing 36 ± 0.9 36 ± 0.7 39 ± 0.2 2 hours after dosing 36 ± 0.8 36 ± 0.7 39 ± 0.1 4 hours after dosing 36 ± 0.7 36 ± 0.7 38 ± 0.9

Experimental Example 2. Confirmation of memory damage recovery effect using Morris underwater maze

2-1. Memory damage animal model production

Rats anesthetized with sodium pentobarbital (50 mg / kg, ip) were subjected to stereotaxic technique using the medial septum (AP: -0.2, L: ± 0.3, H) of the rat cerebrum. : 1 g of acetylcholine-type 192 saporin (192 saporin, ATS, San Diego, Calif., USA), which only damages nerve cells, was injected at the -6.2) position. Microinjection was connected to a 1 ml gas-tight glass syringe (Hamilton, Reno, NV, USA) using a polyethylene tube to connect a perfusion pump (Pump 22, Harvard Apparatus, South Natick, MA). 5 minutes after injecting at a flow rate of 0.2 ml / min, and then removing the syringe.

2-2. Memory effect recovery using Morris underwater maze

The water tank used for the underwater maze is a cylindrical cylinder with a diameter of 180 cm and a height of 50 cm. The water is filled with water of 22 ± 2 ℃ to 30 cm. An adjusting device and the like were installed and the position was kept constant. The sheath was attached to a round transparent acrylic with a diameter of 12 cm and placed 1.5 cm lower than the water surface. The underwater labyrinth is divided into four identical quadrants, divided into northeast, northwest, southeast, and southwest, with the shelters placed in the center of the northeast quadrant, and one of them is used as the starting position.

The administration of the sample was performed from the day after the memory damage model was constructed. The Sham group was a cerebrospinal fluid made of 145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl ₂ , and 1.0 mM MgCl ₂ instead of the damaging drug (192 saporin). Was injected in the same manner as the administration of the damaging drug, the control group was saline, the experimental group was the sample of Example 2 and Comparative Example 1 1 / 2cc, respectively, U-100 insulin 30 gauge needle (U-100 insuline gauge needle , Becton Dicken and Company, USA) were injected with acupuncture 0.05cc in both limbs. The animals were trained four times a day for six days, and at the end of the seventh day, a free swim test was performed, where the animals were allowed to swim for 60 seconds with the escape pads removed. All animal behaviors were recorded with a video camera, which measured the time taken from the start to climb into the shelter.

As a result of the Morris water maze experiment, a significant learning disability was found in the control group, and the acupuncture treatment groups of Example 2 and Comparative Example 1 showed an improvement in learning ability. However, it can be seen that the improvement of learning ability of the herbal treatment group of Example 2 is more enhanced than the herbal treatment group of Comparative Example 1 (see Table 2).

Experimental group 1 day 2 days 3 days 4 days 5 days 6 days Sham 120 ± 1 second 88 ± 2 seconds 73 ± 2 seconds 61 ± 2 seconds 39 ± 1 seconds 30 ± 2 seconds Control 170 ± 2 seconds 141 ± 1 second 133 ± 2 seconds 117 ± 2 seconds 98 ± 1 second 85 ± 2 seconds Example 2 128 ± 1 second 105 ± 2 seconds 80 ± 2 seconds 50 ± 1 second 35 ± 1 seconds 31 ± 1 seconds Comparative Example 1 155 ± 2 seconds 122 ± 1 second 100 ± 1 second 85 ± 2 seconds 63 ± 2 seconds 50 ± 1 second Comparative Example 2 161 ± 2 seconds 131 ± 2 seconds 112 ± 2 seconds 93 ± 2 seconds 81 ± 2 seconds 62 ± 2 seconds

Experimental Example 3. Confirmation of the recovery effect of memory damage through the radial maze experiment

3-1. Production of Whole Cerebral Ischemia Animal Model by Occlusion

An animal model of transient whole cerebral ischemia was constructed by occlusion of the middle cerebral artery using the applied intraluminal suture method (Zea LE., Et al., Strok e, 20 , pp84-91, 1989).

After inducing anesthesia using 5% isoflurane mixed with 70% N ₂ O and 30% O ₂ , the experimental animal of Reference Example 1 was maintained with anesthesia with 2% isoflurane. I was. A thermometer probe was inserted into the rectum of the rat and the body temperature was maintained at 38 ° C. for the duration of the experiment using a warming lamp and a heated mattress. To occlude the middle cerebral artery, the skin is incised along the cervical midline, the total carotid artery is exposed between the sternal and sternal roots and an intraluminal filament (￠ 0.28 mm, rounded tip) is inserted into the internal carotid artery. It reached the proximal part of the forearm cerebral artery after the base of the cerebral artery. The common carotid artery and the external carotid artery were ligated, and after 2 hours of blockage, the filament was removed and the right common carotid artery was ligated and reperfused through the lateral circulation.

The experimental animals were divided into Singer group (Sham, n = 6), whole cerebral ischemia inducing group (n = 7), and physiological saline after the whole cerebral ischemia induction (n = 6). After induction of cerebral ischemia, Example 1 was divided into acupuncture-treated group (n = 4), and Example 2 after induction of whole cerebral ischemia was divided into acupuncture-treated group (n = 5). The acupuncture treatment of the control group and the experimental group was treated with both acupuncture needles (100 mg / kg) once a day at 10 am, a fixed time once a day before the ischemic induction, and acupuncture for two weeks after the induction.

3-2. Confirmation of recovery from memory damage through radial maze experiment

The radial labyrinth used an apparatus in which eight pathways made of wood extend at an angle of 45 ° (radial) about a central central platform. The central starting area was a square octagonal box inscribed in a circle having a diameter of 50 cm, and the height was 25 cm. Primarily connected to a 10 × 25 cm passageway on each side of the starting box, 70 cm in length and preventing animals from going out. At the end of the main, a 7 × 5 × 3 cm container (food dish) was installed to accommodate food or water provided as a reward. A video camera was installed to record the behavior of the test animal in and out of the main. The experimental animals underwent a radial maze for one week after two weeks of acupuncture treatment. The animal's entry and exit behavior was recorded with a video camera to calculate the number of rat visits and errors. For 36 hours before entering the experiment, rats that had been deprived of food in the breeding box and were hungry were transferred to the behavior observation room and allowed to acclimate to the environment for 30 minutes. The rats were placed in the starting box of the labyrinth and allowed to acclimate to the situation. After one minute, the passage to each province was opened to allow the rats to move freely through the labyrinth. The rats visited the main and ran all the way to feed in the reward vessels. However, if the same week was visited repeatedly, the second visit did not provide food and the reaction was recorded as an error. If the rat failed to visit all eight provinces within five minutes, the trial was discontinued and the trial was considered a failure. After 5 days of learning, rats who reached the learning criteria were examined for memory 24 hours later. The procedure before the memory test and the rat's entry into the fourth week were performed in the same manner as in the learning experiment. When the rat selects and enters the fourth mainland and blocks its main passage, delays it for 30 seconds, and then opens the main passage again to let the rats go to the other four main states and receive food as a reward. I took it out of the box and returned it to the nursery. The number of errors that rats make until they visit the remaining four provinces and feed them is recorded as an indicator of memory.

All measurements were expressed as mean ± standard error (mean ± SE), statistical analysis between each experimental group was used for the SPSS for the window (Window). The comparison of behavioral measures among the groups was performed by repeated ANOVA tests, and the statistical significance of the experiments was meaningful at the confidence interval P <0.05.

As a result of the radial labyrinth learning experiment, the results obtained in the acquisition trial to accurately measure the number of weeks in 300 seconds for 5 days showed the results shown in Table 3 (F (4,23) = 11.035, P <0.001). As a result of the post-mortem test, on the fourth day, the treatment group of Example 2 was significantly increased in the whole cerebral ischemia-induced group (P <0.01) and saline-treated group (P <0.05).

In the radial maze learning, the memory test for the cognitive space on the 6th day of the last day showed significant differences between the groups as shown in Table 3 (F (4,27) = 5.248, P <0.01). As a result of the post-mortem examination, the Example 2 treatment group was statistically significantly increased with respect to the physiological saline treatment group (P <0.05) (see Fig. 2).

Experimental group 1 day 5 days 6 days Sham 3.53 ± 0.30 seconds 6.38 ± 0.42 seconds 2.57 ± 0.29 seconds Whole cerebral ischemia group 2.54 ± 0.30 seconds 4.61 ± 0.11 seconds 1.57 ± 0.15 seconds Physiological saline treatment group after induction of whole cerebral ischemia 2.77 ± 0.74 seconds 4.62 ± 0.91 seconds 1.70 ± 0.17 seconds Example 1 treatment group after induction of whole cerebral ischemia 2.05 ± 0.21 seconds 5.95 ± 0.57 seconds 2.10 ± 0.22 seconds Example 2 treatment group after whole cerebral ischemia induction 1.74 ± 0.27 seconds 5.82 ± 0.26 seconds 2.54 ± 0.20 seconds

In addition, in the radial maze learning experiment, the acquisition results of measuring the number of error rates of the main roads, which were visited once within 300 seconds for 5 days, were shown in Table 4 (F (4,23)). = 7.363, P <0.01), and as a result of the post hoc test by group, at Day 4, the Example 2 treatment group was statistically significantly decreased in the whole cerebral ischemia-induced group (P <0.01) and saline-treated group (P <0.05). On the 5th day, the Example 1 treatment group (P <0.01) and the Example 2 treatment group (P <0.05) were significantly decreased in the whole cerebral ischemia-induced group, and the Example 2 treatment group was the saline treatment group. There was a statistically significant decrease for (P <0.05). In the radial maze learning, the memory test of the spatial cognition on the 6th day of the last day showed significant differences between the groups as shown in Table 4 (F (4,27) = 0.435, P <0.01). As a result of the post-mortem examination, the Example 2 treatment group was statistically significantly reduced compared to the whole cerebral ischemia-induced group (P <0.05) (see FIG.

Experimental group 1 day 5 days 6 days Sham 4.55 ± 0.17 seconds 2.03 ± 0.36 seconds 2.57 ± 0.29 seconds Whole cerebral ischemia group 5.63 ± 0.83 seconds 4.51 ± 0.56 seconds 1.57 ± 0.15 seconds Physiological saline treatment group after induction of whole cerebral ischemia 5.45 ± 0.86 seconds 4.00 ± 0.34 seconds 1.70 ± 0.17 seconds Example 1 treatment group after induction of whole cerebral ischemia 5.93 ± 0.68 seconds 2.13 ± 0.50 seconds 2.10 ± 0.22 seconds Example 2 treatment group after whole cerebral ischemia induction 6.30 ± 0.50 seconds 2.08 ± 0.27 seconds 2.54 ± 0.20 seconds

Experimental Example 4. Confirmation of the protective effect of the brain neurons in brain tissue using histochemical analysis

4-1. Organization preparation

The experimental animal immediately after completion of the experiment and measurement of memory through the radial labyrinth experiment of Experimental Example 3 was anesthetized with sodium pentobarbital (80 mg / kg, ip), followed by 200 ml of 0.9% saline solution followed by phosphate buffer (phosphate). perfusion through the heart with 800 ml of 4% formalin fixative prepared in buffer). The first 200 ml of fixative was slowly perfused for 2 minutes at high flow rate and the remaining 800 ml for 25 minutes. After fixing the brains of the fixed rats, fix them with the same fixative for 2 hours, put them in PBS containing 20% sucrose and store them at 4 ° C. for one day. (hippocampus) site was prepared by cutting to a thickness of 30 ㎛.

4-2. Brain Tissue Observation Using Cresyl Violet Staining

After washing the brain tissue prepared in Experimental Example 4-1 several times with PBS, 5 minutes in xylene, 2 minutes in 100% alcohol, 1 minute in 95% alcohol, 1 minute in 70% alcohol, distilled water After dipping for 2 minutes in (DW) in order to degrease, dehydration, it was stained for 5 minutes with cresyl violet buffer (cresyl violet buffer). The stained tissue was observed under an optical microscope at high magnification (× 200), and the position of the probe was determined by referring to the coordinates given in the brain tissue atlas of Paxinos and Watson. The neuronal cell number was observed. One-way ANOVA was used for the analysis of the histological analysis, and the Turkish test (Tukey test) was applied for the post hoc test, and the statistical significance of the experiment was given meaning at the confidence interval P <0.05.

As a result of the analysis, the neuronal damage in the hippocampus of each group is as shown in Figures 4 and 5. The degree of neuronal staining in the hippocampal region is shown in Table 5 and showed significant differences among the groups (F (4,83) = 5.310, P <0.05). The number of cells increased statistically significantly for the cerebral ischemia-induced group (P <0.05). This shows that the herbal acupuncture extract of cultured ginseng extract, which is a sample of the herbal acupuncture, prevented the damage of nerve cells in the hippocampus of the brain due to the transient induction of whole cerebral ischemia.

Experimental group Dyeing degree (%) Sham 100 Whole cerebral ischemia group 71.1 ± 5.2 Physiological saline treatment group after induction of whole cerebral ischemia 72.1 ± 5.2 Example 1 treatment group after induction of whole cerebral ischemia 74.3 ± 7.4 Example 2 treatment group after whole cerebral ischemia induction 95.9 ± 7.9

4-3. Observation of Cerebral Neuronal Cell Damage Protection Using Acetylcholinesterase (AchE) Staining

The brain tissue prepared in Experimental Example 4-1 was washed three times in PBS and then acetylcholine iodide in 325 ml of 0.1 M sodium hydrogen phosphate buffer (NaH ₂ PO ₄ H ₂ O, pH 6.0). (acetylcholine iodide) 25 ml of 0.1 M sodium citrate, 50 ml of 30 mM copper sulfate, 50 ml of 5 mM potassium ferricyanide, 50 ml of distilled water in a solution of 250 mg of acetylcholine iodide. After mixing and waiting for a few seconds to give a pale green, the brain tissue was added and incubated for 1 to 2 hours at room temperature. All treated brain tissues were observed by light microscopy. The density of AchE neurons in the hippocampus was increased by 100 times using a 200 × 200 um microscope rectangle grid, using a Scion image program (Sciion image program, Scion Corp. MD, USA). Measured.

As a result of the measurement, the expression level of AchE in hippocampus of each group is as shown in FIGS. 6 and 7. AchE expression in CA1 of hippocampus is shown in Table 6 and showed significant difference between groups (F (4,80) = 6.747, P <0.001). There was a statistically significant increase in the induction group (P <0.05) and the saline treatment group (P <0.05). In addition, AchE expression in CA3 of hippocampus is shown in Table 7 and there was no significant difference among the groups (F (4,80) = 0.395, P = 0.811). It showed protection against neuronal loss in the hippocampus.

Experimental group CA1 site staining degree (%) Sham 100 Whole cerebral ischemia group 84.55 ± 2.29 Physiological saline treatment group after induction of whole cerebral ischemia 86.05 ± 2.60 Example 1 treatment group after induction of whole cerebral ischemia 84.71 ± 3.40 Example 2 treatment group after whole cerebral ischemia induction 95.44 ± 6.17

Experimental group CA3 site staining degree (%) Sham 100 Whole cerebral ischemia group 97.54 ± 2.44 Physiological saline treatment group after induction of whole cerebral ischemia 95.08 ± 3.03 Example 1 treatment group after induction of whole cerebral ischemia 99.95 ± 3.51 Example 2 treatment group after whole cerebral ischemia induction 102.27 ± 10.80

4-4. Observation of Cerebral Neuronal Cell Damage Protection Using Choline Acetyltransferase (ChAT) Immunohistochemical Staining (Immunohistochemistry)

After washing brain tissue prepared in Experiment 4-1 about 3 times in PBS, the primary sheep polyclonal ChAT antibody, Cambridge Research Biochemicals, Wilmington, DE, USA). Primary antibody was prepared by diluting 2000-fold with 2% rabbit serum and 0.1% sodium azide (sodium azid, Sigma, St. Louis, MO, USA) in PBST with 0.3% Triton X-100 in PBS. It was. Brain tissues were incubated in primary antiserum with continuous shaking at 4 ° C. for 72 hours, after which the tissues were washed three times or more in PBST and then diluted 200-fold in PBST containing 2% rabbit serum at room temperature for 2 hours. Biotinylated anti-sheep serum (VectorLaboratories, Burlingame, Calif., USA) was reacted. After washing three times with PBS, brain tissues were soaked in avidin-biotin-peroxidase complex (Vectastain-Elite ABC kit, Vector Laboratories. Inc., USA) for 2 hours at room temperature, then PBS. After several rinses, the tissues were fortified with nickel chloride and expressed using 3,3'-diamino benzidine tetrahydrochloride (DAB) as colorant. All treated brain tissues were fixed on gelatine-coated slides, covered with cover glass while removing air, and observed under an optical microscope. The density of ChAT neurons in the hippocampus was measured using a Zion image program (Scion image program, Scion Corp. MD, USA) at a magnification of 100 times using a 200 × 200 um microscopic rectangular grid.

As a result of the measurement, the expression level of ChAT in hippocampus of each group is as shown in FIGS. 8 and 9. ChAT expression in the CA1 region of the hippocampus was shown in Table 8, and there was a significant difference between the groups (F (4,86) = 6.252, P <0.001). ChAT expression in the CA3 region of the hippocampus was shown in Table 9. There was a significant difference between the groups (4,86) = 5.262, P <0.01). As a result of the post hoc test in each group, the Example 2 treatment group was statistically compared with the precerebro ischemia-induced group (P <0.05). Significantly increased. This shows that the purified wild ginseng extract tablet, which is a sample of medicinal needles, protected nerve cell damage in the hippocampal region of the brain due to induction of whole cerebral ischemia.

Experimental group CA1 site staining degree (%) Sham 100 Whole cerebral ischemia group 83.03 ± 3.07 Physiological saline treatment group after induction of whole cerebral ischemia 70.23 ± 4.42 Example 1 treatment group after induction of whole cerebral ischemia 93.54 ± 10.41 Example 2 treatment group after whole cerebral ischemia induction 84.87 ± 8.55

Experimental group CA3 site staining degree (%) Sham 100 Whole cerebral ischemia group 83.08 ± 3.07 Physiological saline treatment group after induction of whole cerebral ischemia 76.29 ± 5.30 Example 1 treatment group after induction of whole cerebral ischemia 88.20 ± 8.93 Example 2 treatment group after whole cerebral ischemia induction 93.10 ± 8.47

Experimental Example 5. Acute Toxicity Test

5-1. Oral administration

ICR-based mice (weight 25 ± 5 g) and Sprague-dawly rats each divided into four groups of 10 rats each of the composition of Example 1 of the 100, 250, 500 and 1000 mg / kg After two weeks of oral administration, no toxicities were observed in all four groups and no symptoms were observed.

5-2. 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 Example 2 of the present invention was 25, 50, 100 and 200 mg / kg, respectively. Toxicity was observed for 24 hours after intraperitoneal administration at. There were no deaths in all 4 groups and no symptoms were apparent in the control group.

As a result, it was confirmed that the composition of the present invention has little acute toxicity.

Wild ginseng extract tablets of the present invention can be prepared in the following formulation, the formulation examples below are merely to illustrate the invention, whereby the content of the present invention is not limited.

Formulation Example 1 Preparation of Powder

300 mg of extracted tablet solution of Example 1

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 Tablet

50 mg of extracted tablet solution of Example 1

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 Preparation of Capsule

50 mg of extracted tablet solution of Example 1

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 of extracted tablet solution of Example 2

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 Preparation of Liquid

1000 mg of extracted tablet solution of Example 2

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.

As described above, the composition containing the wild ginseng extract tablets having a neuronal protective activity according to the present invention as an active ingredient is a degenerative brain caused by the death of nerve cells because the effect of protecting the nerve cells damage induced by cerebral ischemia It can be used as a medicament for the prevention and treatment of diseases, such as stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Pick disease and Creutzfeld-Jakob disease.

Claims (4)

A first step of extracting and distilling wild ginseng raw material to prepare a primary extract; A second step of preparing a second distillate by re-distilling the primary extract; A third step of repeatedly performing a process of freezing the secondary distillate to obtain only a frozen fraction; A fourth step of obtaining a filtrate by thawing and filtering the frozen fraction obtained from the third step; A fifth step of freezing the filtrate of the fourth step again after heating the bath; And a wild ginseng extract tablet having a neuroprotective activity obtained by performing a sixth step of thawing the frozen product of the fifth step and sterilizing, and a pharmaceutically acceptable carrier, diluent or excipient. Pharmaceutical composition for the prevention and treatment of degenerative brain diseases, including. The pharmaceutical composition according to claim 1, wherein the wild ginseng is natural ginseng, cultivated or cultured wild ginseng. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is an oral dosage form comprising a powder, granule, tablet, capsule, suspension, emulsion, syrup or aerosol form. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is in the form of an external preparation, a suppository, a sterile injectable solution or a weak acupuncture agent.