KR20030002214A - Compositions for preventing and treating brain diseases - Google Patents

Abstract

PURPOSE: A composition containing an extract of medicinal plants which has excellent effects on reduction of an ischemic area of the brain, or edema is provided which can be used in the prophylaxis and treatment of a brain disease such as cerebral apoplexy, dementia or the like. CONSTITUTION: A mixture of 3.57 to 16% by weight of Angelicae Gigantis Radix, 3.57 to 16% by weight of Achyranthis bidentatae Radix, 3.57 to 16% by weight of Rehmanniae Radix, 3.57 to 10.7% by weight of Persicae Semen, 3.57 to 9% by weight of Aurantii Fructus, 3.57 to 9% by weight of Paeoniae Radix rubra, 1.78 to 5.35% by weight of Bupleuri Radix, 1.78 to 5.35% by weight of Glycyrrhizae Radix, 2.68 to 7.14% by weight of Platycodi Radix, 2.68 to 7.14% by weight of Cnidii Rhizoma, 3.57 to 9% by weight of Lumbricus and 3.57 to 9% by weight of Panax wangianus Sun is extracted in a solvent selected from the group consisting of water, hexane, butanol, ether and ethylacetate.

Description

Compositions for preventing and treating brain diseases

The present invention relates to a herbal composition effective for the prevention and treatment of brain diseases. More specifically, the weight ratio of the composition of the whole composition, Angelica 3.57-16%, dew 3.57-16%, saenghwanghwang 3.57-16%, doin 3.57- 10.7%, crust 3.57-9%, opposition 3.57-9%, shiho 1.78-5.35%, licorice 1.78-5.35%, plantain 2.68-7.14%, celestial 2.68-7.14%, job offer 3.57-9%, and prech 3.57-9 The present invention relates to a composition for preventing and treating cerebral disease, including a solvent extract of a Chinese herbal medicine.

Fish blood is a unique etiology of the Chinese medicine, and the blood is stagnant or dirty at certain parts of the body, or it is the blood that is out of the meridians and is recognized as a pathological product and an important cause of disease. Live blood hormonal medicine is used for incurable diseases such as cancer. Recently, it is recognized that the range of fish blood is similar to various thrombosis, disseminated intravascular coagualtion (DIC), hyperviscomia (hyperviscomia), etc. These two etiologies include various cardiovascular diseases (coronary artery disease, hypertension, thrombosis). , Hyperlipidemia, etc.) and brain diseases (such as stroke and dementia) to increase mortality, so its treatment and prevention is an important task.

Research has been conducted on the premise that fish blood is similar to thrombosis, a blood clot in a blood vessel or heart. In particular, thrombosis includes obstructive thrombosis, wall thrombosis, and decayed or fungal thrombosis, which are caused by age, dehydration, malignancy, obesity, anemia, hypertension, hepatitis, diabetes, hyperlipidemia, collagen vascular disease, and endotoxin shock. This is recognized to cause damage to the inner wall of blood vessels, changes in blood flow and changes in blood components. Symptoms caused by blood clots are so diverse that symptoms can occur from no symptoms to congestion, swelling, infarction due to percutaneous necrosis, cardiac dysfunction, and sepsis, and sudden death from coronary pulmonary or abdominal aortic thrombosis. It may come early. Endotoxin and dextran are used as thrombogenic substances in blood vessels, and experimental studies have been conducted using a method of causing hematoma by injecting blood subcutaneously. In addition, the animal model of thrombosis is a probe that damages the vascular endothelium to induce thrombosis, induce vascular blood clots, evaluate thrombolysis through angioplasty, and platelet count and coagulation, which are important factors in thrombosis. Hwajin is conducting research to inhibit platelet aggregation by inhibiting TXA2 (thromboxane 2) as a feed back system by promoting the production of cAMP and cGMP in platelets. However, in the selection of anti-thrombotic drugs, there have been few studies comparing the single-term herbal medicines or herbal prescriptions of biomolecular oils in consideration of the concept of Korean blood, and in the oriental medicine system in experimental studies related to fish blood which is the etiology of oriental medicine. There is not much research using recent advanced experimental techniques. In the future, this study intends to develop various models in addition to the endotoxin fish blood model, which has already been made in oriental medicine, and tests the antithrombotic effect by using herbal prescriptions and danmi drugs, which have the possibility of antithrombotic activity, and the important theory of herbal remedies. The study considering synergism is urgently needed to develop herbal remedies that can contribute to the prevention and treatment of cardiovascular diseases by removing the blood and showing antithrombotic effects.

In particular, cerebrovascular disease is mainly due to stroke and senile dementia, and the incidence rate is increasing day by day due to the aging of the population and changes in dietary patterns due to the improvement of medical and welfare fields. Research on drugs that improve brain function deterioration caused by cerebrovascular disease is considered to be an area of concern for the development of new drugs in the future, and it is essential to establish efficacy screening methods to activate the development of such brain function improving agents.

As a study on stroke, one of the patients frequently encountered in oriental medicine, studies on the effectiveness of the stroke and the development of new prescriptions are needed by using herbal prescriptions and simple medicines that are effective in clinical trials. The pathogenesis of cerebral vascular accidents (CVA) is known as hypertension, cerebral thrombosis, cerebral embolism, cerebral infarction and transient ischemic accidents. Experimental models that induce double cerebral infarction are mainly caused by focal cerebral ischemia, such as bilateral carotid occlusion, microembolism and four vessel occlusion.

The present invention has been made in view of the above problems and the above necessity, the object of the present invention is to provide a composition that is effective in treating and preventing stroke.

1 is a photograph showing the effect of the control (right) and KHCT (left) on edema and ischemia area in MCA treated mice.

Figure 2 is a photograph showing the effect of the control (right) and KHCT hexane fraction (left) on edema and ischemia area in MCA treated mice.

Figure 3 is a photograph showing the effect of the control (right) and KHCT ether fraction (left) on edema and ischemia area in MCA treated mice.

Figure 4 is a photograph showing the effect of the control (right) and KHCT.EA fraction (left) on edema and ischemia area in MCA treated mice.

Figure 5 is a photograph showing the effect of the control (right) and KHCT. Butanol fraction (left) on edema and ischemia area in MCA treated mice.

Figure 6 is a secondary culture photograph of rat cortical stellate cells.

Figure 7 is a diagram showing the effect of Gami-Byeolbujangeotang on primary cultured astrocytes.

Figure 8 is a figure showing the effect of Gami-Bebuchukeotang on ischemic toxicity.

Figure 9 is a diagram showing the effect of Gami-Bibuchukeotang on p38 activity.

Figure 10 is a figure showing the effect of Kamyomibucchieotang on JNK activity.

11 is a diagram showing the induction of Vascular endothelial growth factor (VEGF) by ischemia. A is the result of RT-PCR and B is the result of Western blot.

12 is a diagram showing the effect of ischemia on HIF (hypoxia-inducible factor) activity.

Figure 13 shows the effect of the control and KHCT on edema and ischemia area in MCA treated mice.

Figure 14 is a diagram showing the effect on the migration activity of Gami-Bibujangeotang EA layer.

In order to achieve the above object, the present invention is a weight ratio of the composition of the total composition of Angelica 3.57-16%, wort 3.57-16%, live turmeric 3.57-16%, phosphate 3.57-10.7%, crust 3.57-9%, red medicine 3.57- Prevention of brain diseases, including 9%, shiho 1.78-5.35%, licorice 1.78-5.35%, plantain 2.68-7.14%, celery 2.68-7.14%, job offer 3.57-9%, and solvent extract of herbal medicine 3.57-9% And it provides a therapeutic composition.

If the range of each medicine of the composition exceeds its upper limit may cause indigestion, diarrhea, etc., below the lower limit there is a problem that the anti-thrombotic and cerebral infarction protective effect may be weak or weak.

The solvent used to extract the herbal medicine of the present invention was extracted using water, hexane, butanol, ether, ethyl acetate and the like.

In addition, the brain diseases referred to in the present invention include stroke and dementia.

Experimental animals used in the present invention were 5 weeks old male rat Splague Dawley produced by the National Institute of Health and Safety at 23 ± 2 ℃, 55 ± 10% humidity, 16 hours of SER under 12 hours lighting cycle conditions, Wistar The rats were reared until the weight of 300g or more, and water and feed were freely ingested. ,), Iksinhaengheoltang (益腎 活血 湯), Lee, Bo-Hi-Bang (활 心 二號 方), Lively Hyoyeongdan, Tonggyuhaengheol-tang (通 화 活血 湯), Hwa-chan Yuk-tang (化) 대), Rhubarb Jachunghwan, Gyeji Bokryeonghwan, Rhubarb Mokdanpitang, Donugunggigitang, Pangdantang, Boyanghwanotang湯), Sammyo Yongantang, Soseontongeotang (消 栓 通 구), Cheonmagu Deungeum (天 飮 藤 飮), etc. The acceleration and various hwalhyeol hwaeo (活血 化瘀) was to use experimental drugs to prescribe.

Example 1 Composition of the Drug Used in the Present Invention and Preparation of the Organic Solvent Fraction Layer

Presently, the representative rooms of Eoyeolbang, Blood-Bucked Fish Soup, Gyeok-ha-Jung-eup-tang, Lee Ji-ho-bang, Bowak Hyoyoungdan, Tonggyuhaengheol-tang, Iksin-shalhe-tang, Saenghwa-tang, Sobok-tang Fish-tang, Boyanghwan-o-tang, Rhubarb Jachung-hwan, Ginseng Star Crab Conversion, Gyeji Boknyeonghwan, Rhubark Mokdanpitang, Dokuk Seunggitang, Mordangtang, Ongyeongtang, Doin Performance, Iksintang, Samyoyongantang, Seogakjihwangtang, Cheongeumwigyeongtang, Huaechan Yuktang, Iksimtang, Cheongseoneoheoltang, etc. 8g of ginseng and 8g of Chilchi (田七) were added to make a kamibang and used as an experimental prescription.

a. Manufacture of drugs

The medicinal herbs to be used in the experiment were put in extractors (S-15000, SEA IK MEDICAL), and 1500 ml of water was added thereto, followed by heat extraction for 2 hours, to obtain respective extracts. The filtrate obtained by filtering the extract with a filter paper was concentrated under reduced pressure using a rotary vaccum evaporator (Switzerland) to obtain a viscous extract. The extract was then frozen with a deep freezer and freeze-dried with a freeze dryer, FDU 540 (EYELA co., Japan). Diluted to the concentration required for this experiment was used.

The organic solvent fraction is added to 20kg of 95% methanol in about 10kg of the plant part to be extracted first, and then heated in reflux for 3 hours, filtered and the mother liquor is concentrated to about 50ml, and then a mixed solution of chloroform and water. (100 ml each), shake well to separate the two layers, and then proceed with the procedure in the table for the chloroform layer by separating the mixture with a given solvent (each 100 ml each) to obtain the final five fractions, and then concentrate as follows. According to one method, a physiological activity test was performed.

If a fraction of the plant extract has anticancer activity, the active site is re-extracted and in particular, the separation of the active ingredient of the single herb can be carried out. That is, fractionation was carried out by mobilizing methods such as chromatography. The fractions were compared on TLC, and when the active spots were matched with the material spots, a sufficient amount of active fractions were obtained for mass separation and animal experiments.

TABLE 1 Composition of Kami-Blood Fish Cake

Medicinal name Herbal medicine Volume Donkey Angelicae gigantis radix 12g Dew Achyranthis bidentatae radix 12g Safflower Carthami flos 12g Living Rehmanniae radix 12g Doin Persicae semen 12g tardy Aurantii fructus 8 g Peony Paeonia radix rubra 8 g Shiho Bupleuri radix 4 g licorice Glycyrrhizae radix 4 g Street view Platycodi radix 6 g Cheongung Cnidii rhizoma 6 g Job Lumbricus 8 g Prepainted Notoginseng radix 8 g Sum 112 g

b. Preparation of Organic Solvent Fraction Layer

Prepare 4,640g (40 pouches) of Kami-Bubu-chae-tang (hereinafter referred to as "KHCT"), add 12 liters of methanol to these medicines, leave for 3 days, and filter the filter paper twice to secure the methanol layer. The methanol layer was evaporated using a vacuum condenser to secure methanol extract concentrate (330 g). Dissolve 1L of tertiary distilled water in this concentrate, put it in a separatory funnel, add the same amount of hexane, shake it vigorously, mix it, and repeat it for 2-3 times to separate the hexane layer, and then add the same amount of ether and ethyl to the remaining water layer. Acetate was added sequentially to separate the organic solvent layer in the same manner. The obtained organic solvent layers were each evaporated using a vacuum concentrator to secure each organic solvent extraction concentrate. Ethyl acetate concentrate (11.2244 g) was separated into four layers by silica gel chromatography, and each fraction (1 fraction-1.82 g, 2 fractions-1.55 g, 3 fractions-4.13 g, 4 fractions) was separated. -3.68g) layer was used as a test drug after securing the concentrate by evaporating all the organic solvent using a vacuum condenser.

Example 2: MCA Occlusion

a. Probe Fabrication

A 4-0 standard nylon suture (Deknatel Inc.) was cut to the appropriate length and heated to form a knot at one end. After making the total length to 18 mm, the probe was made by thinly coating the opposite side of the knot with a thickness of about 0.3 mm of silicon mixed with a curing agent.

b. Middle cerebral artery occlusion

The overnight fasted rats were lightly anesthetized with ether, and then injected intraperitoneally with ketamine chloride (100 mg / kg) and fixed on the operating table. Dissecting the center of the neck according to Nagasawa et al., Taking care not to damage the vagus nerve, separating the right common carotid artery, the internal carotid artery and the external carotid artery, and ligation the common carotid artery and the external carotid artery and the branching points of the external carotid artery and the internal carotid artery. After inserting the entire probe except the knot, the ipsilateral middle cerebral artery was closed by ligation just above the insertion site. Total operation time was less than 30 minutes, and infrared rays were prevented to reduce body temperature as much as possible and bleeding did not occur during surgery. In the sham operation, only the total and external carotid arteries were ligated.

c. Neurological examination

After 24 hours after surgery, neurologic examinations were performed to determine the degree of neurologic deficits due to the middle cerebral artery occlusion. The degree of neurological deficits was scored by dividing the degree into 4 grades by Bederson et al., And the classification according to the symptoms is shown in Table 1 and 2.

Table 2: Neurological Investigation Rating System in Forelimbs

0 ratings No defects observed 1 rating The forelimbs are deflected by lifting the tail 2 ratings Reduced forefoot resistance to side push 3 ratings Rotate to hang (body twist)

Table 3 Neurological Investigation Rating System in Hind Legs

0 ratings Immediately position the back part on the table (normal) 1 rating Missing limbs / exercise

d. Measuring ischemia area and edema

Immediately after neurological examination, the rats were single-headed and the brains were quickly taken out to obtain 2 mm thick coronal brain slices using a brain matrix (ASI Instruments, Warren, MI., USA), and only 8 pieces were selected and 3% triphenyltetrazolium was selected. A chloride (TTC) solution was added and incubated at 37 ° C. for 50 minutes. Normal tissues were stained dark red by TTC, but tissues with ischemia were not stained. After incubation, the stained tissues were fixed with 10% formalin neutral buffer solution, photographed with a color photographic film within 2-3 days, developed, and the ischemic area of each parietal fragment was measured by an image analyzer. Ischemia area and edema rate were calculated by the following equation.

Ischemia area (%) = C × 100 A + B

Edema rate (%) = A-B × 100 2 x B

A ischemia-induced cerebral hemisphere area in each parietal slice

B: the cerebral hemisphere area in each parietal piece

C ischemia area in each parietal piece

The effects on the ischemic area and edema rate following middle cerebral artery occlusion

The development of the stroke model was carried out through MCA occlusion, and an effective fraction study was carried out with the addition of roasted chicken paste in the blood bun fish tang and the larvae of interest.

As a result, it was found that Rho-bang-bang of interest was more effective than blood-rich fish tang, and especially the butanol layer of R-bang-bang-bang was the most effective.

TABLE 4 Effect of control and KHCT on edema and ischemia area in MCA treated mice:

Sample number Ischemic area (%) Edema rate (%) Sample number Ischemic area (%) Edema rate (%) C-1 9.56 15.76 KH-1 ND ND C-2 26.70 13.87 KH-2 ND 1.45 C-3 28.52 18.58 KH-3 ND 2.19 C-4 22.25 10.26 KH-4 ND 1.2 C-5 17.22 9.71 KH-5 ND 0.23 C-6 4.41 4.26 KH-6 ND ND C-7 1.56 3.27 KH-7 ND ND C-8 ND 2.89 KH-8 ND ND

In the above table, C1-8 means brain fragment number of the control group, KH1-8 means brain fragments of the KHCT-treated group, and ND is not detected.

TABLE 5 Effect of control and KHCT hexane fraction on edema and ischemia area in MCA treated mice:

Sample number Ischemic area (%) Edema rate (%) Sample number Ischemic area (%) Edema rate (%) C-1 9.56 15.76 KH.H-1 0.8 2.56 C-2 26.70 13.87 KH.H-2 12.4 9.42 C-3 28.52 18.58 KH.H-3 21.42 11.58 C-4 22.25 10.26 KH.H-4 19.30 10.42 C-5 17.22 9.71 KH.H-5 12.3 9.52 C-6 4.41 4.26 KH.H-6 4.2 3.48 C-7 1.56 3.27 KH.H-7 ND 1.42 C-8 ND 2.89 KH.H-8 ND 1.21

In the above table, C1-8 means brain fragment number of the control group, KH.H1-8 means brain slices of the group treated with hexane fraction of KHCT, and ND is not detected.

TABLE 6 Effect of control and KHCT ether fractions on edema and ischemia area in MCA treated mice

Sample number Ischemic area (%) Edema rate (%) Sample number Ischemic area (%) Edema rate (%) C-1 9.56 15.76 KH.E-1 1.2 1.87 C-2 26.70 13.87 KH.E-2 2.4 2.21 C-3 28.52 18.58 KH.E-3 8.2 3.36 C-4 22.25 10.26 KH.E-4 10.4 12.42 C-5 17.22 9.71 KH.E-5 5.4 8.7 C-6 4.41 4.26 KH.E-6 0.6 0.82 C-7 1.56 3.27 KH.E-7 ND 0.38 C-8 ND 2.89 KH.E-8 ND 0.21

In the above table, C1-8 means brain fragment number of the control group, KH.E1-8 means brain fragments of the group treated with ether fraction of KHCT, and ND is not detected.

TABLE 7 Effect of control and KHCT.EA fraction on edema and ischemia area in MCA treated mice:

Sample number Ischemic area (%) Edema rate (%) Sample number Ischemic area (%) Edema rate (%) C-1 9.56 15.76 KH.EA-1 8.2 12.4 C-2 26.70 13.87 KH.EA-2 15.4 13.2 C-3 28.52 18.58 KH.EA-3 23.21 13.8 C-4 22.25 10.26 KH.EA-4 18.21 11.48 C-5 17.22 9.71 KH.EA-5 5.84 8.42 C-6 4.41 4.26 KH.EA-6 3.42 6.23 C-7 1.56 3.27 KH.EA-7 ND 3.41 C-8 ND 2.89 KH.EA-8 ND 1.56

In the above table, C1-8 means brain fragment number of the control group, KH.EA1-8 means brain fragments of the group treated with ethyl acetate fraction of KHCT, and ND is not detected.

TABLE 8 Effect of control and KHCT.butanol fraction on edema and ischemia area in MCA treated mice:

Sample number Ischemic area (%) Edema rate (%) Sample number Ischemic area (%) Edema rate (%) C-1 9.56 15.76 KH.B-1 ND ND C-2 26.70 13.87 KH.B-2 ND ND C-3 28.52 18.58 KH.B-3 ND 2.42 C-4 22.25 10.26 KH.B-4 ND 1.78 C-5 17.22 9.71 KH.B-5 ND ND C-6 4.41 4.26 KH.B-6 ND ND C-7 1.56 3.27 KH.B-7 ND ND C-8 ND 2.89 KH.B-8 ND ND

In the above table, C1-8 means brain fragment number of the control group, KH.EA1-8 means brain fragments of the group treated with ethyl acetate fraction of KHCT, and ND is not detected.

The degree of neurological deficits of fore limb was 2.89 ± 0.11 after operation in the control group, compared with the hexane level of Gamiblood Buchaeotang in the experimental group. The grade improved to 1.52 ± 0.33 and 2.15 ± 0.25 in the butanol layer. The control group after hypoxia was 2.89 ± 0.11, compared to that of Kami-Bibujangeotang. Butanol layer was not significantly improved to 2.13 ± 0.45 and 2.45 ± 0.25. Also, the grade after recirculation was improved from 2.57 ± 0.20 to 2.0 ± 0.00.

The degree of neurological deficit in the hind limbs was 0.44 ± 0.18 in the control group, whereas KHCT: hexane. Ether, Butanol, KGSIHB: The grades were improved to 0.1 ± 0.18, 0.11 ± 0.11, 0.11 ± 0.11, 0.00 ± 0.00 degrees and 0.1 ± 0.11 in the ether and butanol layers. In the case of hypoxia, the control grade was 0.11 ± 0.11 compared to KHCT: hexane, butanol, KGSIHB ether, butanol 0.00 ± 0.000. The grade improved to 0.00 ± 0.00, 0.11 ± 0.11, and 0.00 ± 0.00. In addition, the grade of the control group after recirculation was 0.14 ± 0.14, but the grade was improved to 0.18 ± 0.44, 0.00 ± 0.00, 0.14 ± 0.28, 0.00 ± 0.00 in the KHCT: hexane, butanol, KGSIHB: ether, butanol layers.

[Table 9] Effect of neurologic deficits on middle cerebral artery occlusion (forelimbs)

Operation Hypoxia Recirculation Control 2.78 ± 0.15 2.89 ± 0.11 2.57 ± 0.20 KHCT 2.32 ± 0.21 2.53 ± 0.31 2.5 ± 0.25 KHCT. Hexane 1.52 ± 0.33 2.13 ± 0.45 2.0 ± 0.00 KHCT. ether 2.57 ± 0.2 2.45 ± 0.25 2.35 ± 0.25 KHCT. EA 2.7 ± 0.25 2.5 ± 0.75 2.75 ± 0.25 KHCT. Butanol 2.15 ± 0.25 2.45 ± 0.25 2.5 ± 0.29 KGSIHB 2.66 ± 0.33 2.5 ± 0.75 2.5 ± 0.75 KGSIHB. Hexane 2.7 ± 0.25 2.75 ± 0.25 2.5 ± 0.75 KGSIHB. ether 2.5 ± 0.75 2.5 ± 0.75 2.75 ± 0.25 KGSIHB. EA 2.75 ± 0.25 3.0 ± 0.00 2.75 ± 0.25 KGSIHB. Butanol 2.5 ± 0.45 2.5 ± 0.45 2.5 ± 0.25

In the table, the control group was treated with normal saline, and KHCT: Kami-Bubu-echi-tang, KHCT. Hexane: Kami-Bubu-echijangtang hexane layer, KHCT. Ether: Kami-Bubu-cha-e-tang, KHCT. EA; Kami Blood Bubbly Fish Soup EA, KHCT. Butanol: Kami-Bibu-Chameotang-Butanol layer, KGSIHB: Kami-Choibangbang, KGSIHB. Hexane: Kamishibangbang hexane layer, KGSIHB. ether; Flavor interest ether layer, KGSIHB. EA; Kami Interest Room EA floor, KGSIHB. Butanol; Kami attention means the butanol layer in the room.

[Table 10] Effect of neurologic deficits on middle cerebral artery occlusion (hind limbs)

Operation Hypoxia Recirculation Control 0.44 ± 0.18 0.11 ± 0.11 0.14 ± 0.14 KHCT 0.25 ± 0.25 0.25 ± 0.25 0.1 ± 0.28 KHCT. Hexane 0.1 ± 0.18 0.00 ± 0.00 0.18 ± 0.44 KHCT. ether 0.11 ± 0.11 0.25 ± 0.25 0.25 ± 0.25 KHCT. EA 1.00 ± 0.00 0.75 ± 0.5 0.75 ± 0.5 KHCT. Butanol 0.11 ± 0.11 0.00 ± 0.00 0.00 ± 0.00 KGSIHB 0.28 ± 0.12 0.25 ± 0.25 0.25 ± 0.25 KGSIHB. Hexane 0.25 ± 0.25 0.25 ± 0.25 0.25 ± 0.25 KGSIHB. ether 0.00 ± 0.00 0.11 ± 0.11 0.14 ± 0.28 KGSIHB. EA 0.25 ± 0.25 0.28 ± 0.11 0.25 ± 0.25 KGSIHB. Butanol 0.1 ± 0.11 0.00 ± 0.00 0.00 ± 0.00

In the above table, the control group means normal saline treatment group, and KHCT: Kami-Bebu-echukeo-tang, KHCT. Hexane: Kami-Bubu-echijangtang hexane layer, KHCT. Ether: Kami-Bubu-cha-e-tang, KHCT. EA; Kami Blood Bubbly Fish Soup EA, KHCT. Butanol: Kami-Bibu-Chameotang-Butanol layer, KGSIHB: Kami-Choibangbang, KGSIHB. Hexane: Kamishibangbang hexane layer, KGSIHB. ether; Flavor interest ether layer, KGSIHB. EA; Kami Interest Room EA floor, KGSIHB. Butanol; Kami attention means the butanol layer in the room.

Example 2 KCN Induced Coma Time Measurement

Ten ICR mice were used as a group 1, and Chinese medicine was orally administered at low and high concentrations using an oral zonde (Daejong equipment). The control group was orally administered with the same amount of saline used to dissolve the sample solution. Thirty minutes after oral administration, 1.2 mg / kg KCN was injected intravenously, and the time from the loss of the clove to the recovery of the clove was measured to determine KCN-induced sleep time.

a. KCN-induced sleep time

First of all, screening for brain injury was performed by KCN. The sleep time of the control group induced by KCN (1.2mg / kg / 20g) was 66.71 ± 4.84 sec, compared to 42.5 ± 6.54 sec (34.30%, P <0.01) for Gyeok-hak-eotang and 48.43 ± 3.38sec 27.41%, P <0.01), interested room is 23.83 ± 3.35sec (64.28%, P <0.001), Dokukseunggi-tang is 39.0 ± 4.23 sec (41.54%, P <0.001), and 35.85 ± 3.78sec (46.26%, P <0.001), Boyanghwanotang was 25.8 ± 5.17 sec (61.33%, P <0.001), belonging to Myeongtang 41.25 ± 2.58 sec (38.16, P <0.01), and 6 days net acid was 43.25 ± 3.58 sec (35.57 %, P <0.01) significantly reduced sleep time induced by KCN.

Table 11 Sleep Times Induced by KCN

Sample number Number of animals Volume (mg / 20g) Time (sec.) % Inhibition Control 8 HBSS 66.71 ± 4.84 Hard to eat fish soup 8 15.4 42.5 ± 6.54 34.30 Blood Shaved Fish Soup 8 24.3 48.43 ± 3.38 27.41 Interested in 8 13.33 23.83 ± 3.35 64.28 Doknuseunggitang 8 18.8 39.0 ± 4.23 41.54 Tongyubyeolheoltang 8 52.6 35.85 ± 3.78 46.26 Boyanghwanotang 8 22.67 25.8 ± 5.17 61.33 Affiliation 8 9.93 41.25 ± 2.58 38.16 6 days 8 26.4 43.43 ± 3.58 35.57

HBSS in the table is Hanks' Balanced Salt Solution.

In addition, screening for brain injury was performed by KCN, the brain damage protection effect of each prescription was as follows. Sleeping time induced by KCN (1.2mg / kg / 20g) was 48.03 ± 3.23 sec (17.3%) in the Kami-Buibu-chaetang hexane layer compared to 58.12 ± 6.72sec. ), The blood layer fish sauce EA layer is 37.03 ± 7.42 sec (36.2%), the blood layer fish sauce butanol layer 27.32 ± 4.84sec (52.9%), the room of interest 42.38 ± 3.42sec (27.1%), the room layer of hexane of interest 42.23 ± 5.25 sec (27.3%), interest ether layer 51.43 ± 5.56 sec (11.5%) interest EA layer 53.21 ± 6.24 sec (8%), interest butanol layer 36.42 ± 5.26sec (37.3%) In addition, sleep time induced by KCN was reduced.

Table 12: Sleep Times Induced by KCN in Various Prescriptions

Sample name Number of animals Volume (mg / 20g) Time (sec.) % Inhibition Control 8 HBSS 58.12 ± 6.72 KHCT 8 10 48.03 ± 3.23 17.3 KHCT.hexane 8 10 31.42 ± 2.38 45.9 KHCT. ether 8 10 42.53 ± 5.56 26.8 KHCT. EA 8 10 37.03 ± 7.42 36.2 KHCT.Butanol 8 10 27.32 ± 4.84 52.9 KGSIHB 8 10 42.38 ± 3.42 27.1 KGSIHB. Hexane 8 10 42.23 ± 5.25 27.3 KGSIHB .ether 8 10 51.43 ± 5.56 11.5 KGSHB .EA 8 10 53.21 ± 6.24 8 KGSIHB.Butanol 8 10 36.42 ± 5.26 37.3

In the table, the control group was treated with KCN (1.2 mg / kg / 20 g), and KHCT: Kami-Blood Bok-eotang, KHCT. Hexane: Kami-Bubu-echijangtang hexane layer, KHCT. Ether: Kami-Bubu-cha-e-tang, KHCT. EA; Kami Blood Bubbly Fish Soup EA, KHCT. Butanol: Kami-Bibu-Chameotang-Butanol layer, KGSIHB: Kami-Choibangbang, KGSIHB. Hexane: Kamishibangbang hexane layer, KGSIHB. ether; Flavor interest ether layer, KGSIHB. EA; Kami Interest Room EA floor, KGSIHB. Butanol; Kami attention means the butanol layer in the room.

Example 3: KCN-induced survival rate measurement

Ten ICR mice were used as a group 1, and Chinese medicine was orally administered at low and high concentrations using an oral zonde, and the control group was orally administered with normal saline used to dissolve the sample solution. Thirty minutes after oral administration, a lethal dose of 3.0 mg / kg KCN was injected intravenously and the survival rate of the test animals was measured.

The mortality rate of mice caused by lethal dose of KCN (3.0 mg / kg / 20 g i.v.) indicates a mortality rate of 100% in the control group. Compared to the control group, the hexane and butanol fractio of KHCT showed a significantly increased survival rate compared to the KGSIHB fractio.

TABLE 13 Survival Number of Mice Induced by KCN After KHCT and KGSIHB Administration

Sample name The number of animals Volume (mg / 20g) Can survive % Inhibition Control 10 HBSS 10 KHCT 10 10 8 20 KHCT.hexane 10 10 2 80 KHCT .ether 10 10 4 60 KHCT .EA 10 10 4 60 KHCT.Butanol 10 10 2 80 KGSIHB 10 10 6 40 KGSIHB. Hexane 10 10 6 40 KGSIHB .ether 10 10 8 20 KGSHB. EA 10 10 8 20 KGSIHB.Butanol 10 10 6 40

In the table, the control group was KCN (3.0 mg / kg / 20 g), KHCT: Kami-Bebu-echukeotang, KHCT. Hexane: Kami-Bubu-echijangtang hexane layer, KHCT. Ether: Kami-Bubu-cha-e-tang, KHCT. EA; Kami Blood Bubbly Fish Soup EA, KHCT. Butanol: Kami-Bibu-Chameotang-Butanol layer, KGSIHB: Kami-Choibangbang, KGSIHB. Hexane: Kamishibangbang hexane layer, KGSIHB. ether; Flavor interest ether layer, KGSIHB. EA; Kami Interest Room EA floor, KGSIHB. Butanol; The taste is treated by the protective butanol layer.

Example 4: PC12 Brain Cell Culture and Cytotoxicity Study

a. PC12 Cell Culture

PC12 cells are brain cells derived from rat phechromocytoma. 5% FCS was added to RPMI1640, 10% horse serum serum and 50 ng NGF (Nerve growth factor) inactivated by heat, followed by differentiation by collagen coated dish. It was.

b. Ischemic Brain Cell Production

Cultured cells were cultured for 6 hours or more under hypoxic conditions (N ₂ 95% + O ₂ 5%) to induce ischemic brain cells.

c. Lactic Dehydrogenase (LDH) Measurement

24 hours after induction of hypoxia, the activity of LDH released into the medium by cell damage was measured. LDH was measured to change the amount of NADH produced when Pyruvate and NADH were reacted with lactate and NAD.

d. Protein Quantitation

It measured using the method of Lowry et al.

Table 14 Number of Neurons in Hippocampus After MCA Occlusion

Experimental group Normal part (%) Ischemic site (%) Control 100 37.1 KHCT Hexane 100 80.0 ether 100 36.7 EA 100 37.2 Butanol 100 81.3 KGSIHB Hexane 100 24.3 ether 100 27.9 EA 100 38.8 Butanol 100 45.6

The survival rate of PC12 cells was reduced to 42.95% by β-amyloid protein 25-35 (50μM), while the sample treatment group did not show a significant protective effect. , 38.74% 39.8% 40.19%

TABLE 15 Cytoprotective effect of PC12 cells induced by β-amyloid protein

Sample name Amount (mg / ml) Control group (%) normal 100.01 ± 4.90 Control 50μmol 57.05 ± 1.63 KHCT + Aβ (25-35) One 46.07 ± 1.35 0.5 53.23 ± 0.64 KHCT. Hexane + Aβ (25-35) One 52.36 ± 1.69 0.5 50.93 ± 2.68 KHCT. Ether + Aβ (25-35) One 56.32 ± 3.11 0.5 61.21 ± 2.25 KHCT. EA + Aβ (25-35) One 57.21 ± 4.91 0.5 57.12 ± 3.93 KH5CT. Butanol + Aβ (25-35) One 61.26 ± 1.95 0.5 58.21 ± 5.20 KGSIHG + 0Aβ (25-35) One 52.68 ± 1.21 0.5 54.32 ± 3.05 KGSIHG. Hexane + Aβ (25-35) One 53.19 ± 2.37 0.5 56.49 ± 2.66 KGSIHG. Ether + Aβ (25-35) One 54.17 ± 5.32 0.5 58.34 ± 2.41 KGSIHG. EA + Aβ (25-35) One 60.21 ± 1.51 0.5 58.91 ± 2.41 KGSIHG. Butanol + Aβ (25-35) One 59.81 ± 2.14 0.5 57.49 ± 2.33

In the table, the control group was Aβ-amyloid protein (25-35), KHCT: Kami-Byeolbucchieotang, KHCT. Hexane: Kami-Bubu-echijangtang hexane layer, KHCT. Ether: Kami-Bubu-cha-e-tang, KHCT. EA; Kami Blood Bubbly Fish Soup EA, KHCT. Butanol: Kami-Bibu-Chameotang-Butanol layer, KGSIHB: Kami-Choibangbang, KGSIHB. Hexane: Kamishibangbang hexane layer, KGSIHB. ether; Flavor interest ether layer, KGSIHB. EA; Kami Interest Room EA floor, KGSIHB. Butanol; The taste interest indicates the protective butanol layer.

The survival rate of B103 cells was reduced to 37.77% by β-amyloid protein 25-35 (50μM).

Table 16. Cytoprotective effect of B103 cells induced by β-amyloid protein

Sample name Amount (mg / ml) Control group (%) normal 100 ± 0.2 Control 50μmol 62.23 ± 1.52 HCT + Aβ (25-35) One 56.12 ± 2.71 0.5 58.92 ± 2.61 HCT. Hexane + Aβ (25-35) One 57.46 ± 2.55 0.5 54.26 ± 2.17 HCT. Ether + Aβ (25-35) One 59.51 ± 3.42 0.5 51.23 ± 3.86 HCT. EA + Aβ (25-35) One 61.24 ± 1.80 0.5 60.21 ± 4.35 HCT. Butanol + Aβ (25-35) One 62.89 ± 1.20 0.5 61.24 ± 2.42 GSIHG + Aβ (25-35) One 60.24 ± 2.16 0.5 59.64 ± 1.53 GSIHG. Hexane + Aβ (25-35) One 61.42 ± 2.48 0.5 59.10 ± 1.49 GSIHG. Ether + Aβ (25-35) One 62.49 ± 2.10 0.5 61.46 ± 2.43 GSIHG. EA + Aβ (25-35) One 60.89 ± 2.45 0.5 60.25 ± 2.50 GSIHG. Butanol + Aβ (25-35) One 62.31 ± 2.21 0.5 61.34 ± 1.51

In the table, the control group was Aβ-amyloid protein (25-35) KHCT: Kami-Byeolbucchieotang, KHCT. Hexane: Kami-Bubu-Chameotang-Han-hexane, KHCT. Ether: Kami-Bubu-e-Chan-Tang Ether, KHCT. EA; Kami Blood Bukkake Fish EA, KHCT. Butanol: Kami-Bibu-Chameotang Butanol; Hexane: Kamikaze hexane, KGSIHB. ether; Kamchatzia Ethe, KGSIHB. EA; Kami Interest Room EA floor, KGSIHB. Butanol; Kami attention means a protective butanol.

Reduction of LDH release by 12.54% 18.35% 12.32% 17.82% in KHCT: ether, butanol KGSIHB: EA, butanol in LDH amount of PC-12 cells released by β-amyloid protein (50 μM), which is an indicator of cytotoxicity The other fractions had no significant effect on LDH release.

Table 17 LDH Amount of PC-12 Cells

Sample name Amount (mg / ml) Control group (%) Control 0.05 100 ± 2.65 KHCT + Aβ (25-35) One 89.52 ± 3.50 KHCT. Hexane + Aβ (25-35) One 89.34 ± 2.74 KHCT. Ether + Aβ (25-35) One 87.46 ± 3.95 KHCT. EA + Aβ (25-35) One 91.68 ± 2.36 KHCT. Butanol + Aβ (25-35) One 81.65 ± 1.50 KGSIHB + Aβ (25-35) One 88.54 ± 2.61 KGSIHB .. Hexane + Aβ (25-35) One 95.65 ± 2.44 KGSIHB .. ether + Aβ (25-35) One 92.69 ± 1.91 KGSIHB .. EA + Aβ (25-35) One 87.68 ± 3.84 KGSIHB .. Butanol + Aβ (25-35) One 82.18 ± 2.51

In the table, the control group was Aβ-amyloid protein (25-35), and KHCT: Kami-Bubu-Ae-tang, KHCT. Hexane: Kami-Bubu-echijangtang hexane layer, KHCT. Ether: Kami-Bubu-cha-e-tang, KHCT. EA; Kami Blood Bubbly Fish Soup EA, KHCT. Butanol: Kami-Bibu-Chameotang-Butanol layer, KGSIHB: Kami-Choibangbang, KGSIHB. Hexane: Kamishibangbang hexane layer, KGSIHB. ether; Flavor interest ether layer, KGSIHB. EA; Kami Interest Room EA floor, KGSIHB. Butanol; The taste interest indicates the protective butanol layer.

Example 5: Protein kinase C inhibition test

The activity inhibition of protein kinase C was determined by Hwang et al., And the activity inhibition of the herbal medicine was calculated by comparing the activity of the control sample without the addition of the herbal sample to the PKC enzyme reaction solution. PKC enzyme reactions were performed in Tris-HCL buffer 30 mM (pH 7.5), 6 mM Mg acetate, 0.12 mM [γ- ³² P] ATP, cpm / μ), 0.25 mM EGTA, 0.4 mM CaCl ² , 0.04% Nonidet P = 40, 100 μg / ml phosphatidyl serine, 20 μg / ml diolein, and 1 mg / ml histon IIIS were prepared. The final protein concentration of PKC was 1.75 mg / ml by the addition of PKC enzyme solution. After 25 µl of enzyme reaction solution and 5 µl of crude drug sample were mixed and reacted at 30 ° C. for 15 minutes, 10 µl of the reaction solution was added to the cellulose-Pi paper strip soaked with 20 μl of 20% TCA and 0.2M ATP solution. After stopping and drying, TLC was performed using 5% TCA and 0.2M KCl solution as a developing solvent, and radioactivity at the starting point was measured by a liquid scintillation counter. Phosphorylation of histone caused by protein kinase other than PKC was measured by cpm after reaction without addition of diolein and phosphatidyl serine to enzyme reaction solution. The PKC activity inferiority of the crude herb samples was calculated by the following equation: cpm was D ⁺ , D ^- of the reaction solution without diolein and phosphatidyl serine, and cpm of the enzyme reaction solution with the crude herb sample. It was.

(D ⁺ - sample ^{cpm) × 100 / (D +} -D -) = inhibition rate

After MCA occlusion, the brains of the left and right hemispheres were cut and protein quantified. As shown in Table 5, the left hemisphere decreases the amount of protein compared to the right hemisphere, which seems to be related to the area of brain injury.

Table 18 Protein quantification by cleavage of the left and right hemispheres after MCA occlusion

Sample brain Absorbance Protein (ug / ml) Control Right hemisphere 0.3237 ± 0.0228 7.75 ± 0.5923 Left hemisphere 0.2936 ± 0.0042 6.9750 ± 0.1031 KHCT. Butanol Right hemisphere 0.3155 ± 0.005 7.525 ± 0.1377 Left hemisphere 0.2896 ± 0.0059 6.8750 ± 0.1652 KGSIHB. Butanol Right hemisphere 0.2809 ± 0.0164 6.633 ± 0.433 Left hemisphere 0.2498 ± 0.0280 5.833 ± 0.7219

The control group in the above table is the untreated group, KHCT. Butanol: Kami-Bibu-Chameotang Butanol layer, KGSIHB. Butanol; The taste interest indicates the protective butanol layer.

Reduced PKC activity tended to be increased in the right hemisphere by KHCT butanol (P <0.08) and the left hemisphere increased (P <0.05) PKC activity (P <0.05). But KGSIHB. Butanol administration did not affect PKC activity

TABLE 19 PKC activity in brain hemispheres (unit: pmole / min)

Sample Right brain Left brain Control 23.1 ± 3.4 25.3 ± 6.1 KHCT. Butanol 28.3 ± 3.6 32.2 ± 5.3 KGSIHB. Butanol 23.9 ± 2.8 27.5 ± 2.0

The control group in the above table represents the untreated group, KHCT. Butanol: Kami-Bibu-Chameotang Butanol layer, KGSIHB. Hexane; The taste interest represents the hexane layer of the protective layer.

Example 6 Measurement of Viability of Ischemic Brain Cells

In order to investigate the effect of Gami-Bubu-Ae-Chan-Ae EA on the viability of ischemic brain cells, we used established astrocyte culture and in vitro ischemic mimic environment. Cytotoxicity was measured immediately (0 hours), 2 hours, and 24 hours after adding EA layer and hexane layer of Kami-Bebujangeotang at concentrations of 0, 0.1 and 0.2mg / ml during ischemia for 4 hours. . Cytotoxicity measurements were determined using the method of Flick and Gifford (Flick and Gifford, 1984). Astrocytes were seeded in 96-well microtiter plates at 3-4 × 10 ⁴ cells per well and incubated at 37 ° C., 5% CO ₂ conditions for 24 hours. Discard the upper medium of the 96-well plate, mix the already sterilized test sample with the growth medium containing 2 ㎍ / ㎖ actinomycin D in a ratio of 1: 1 and put 200 ㎕ per well, 37 ℃, 5 Incubated for 18 hours in% CO ₂ . After incubation, the plate medium was shaken off and stained for 15 minutes in a crystal biot stain solution (0.1% crystal violet, 10% calcium carbonate saturated formalin, 11.1% ethanol). The stained 96-well plates were washed well with tap water, then shaken and dried and the absorbance was measured at 540 nm using a microplate reader. The cytotoxicity of the test sample was determined as follows.

% Cytotoxicity = × 100 (%)

Here, the control group was determined as the absorbance at 540 nm of the wells in which only the growth medium was added instead of the test sample.

The biggest change in the brain ischemia is to stop the supply of oxygen and glucose, so we established the brain cell primary culture system. Astrocytes were first cultured from the prefrontal cortex of mouse pups 1-2 days old. Macarthy's brain cell culture method was mainly used, but slightly modified. After removing the newborn mouse cerebrum, the meninges and blood vessels were quickly removed under an anatomical microscope (Olympus, Japen) and the frontal cortex was extracted. Lightly trituratoin using a syringe and Pastero pipette followed by cerebral hemispheres medium (DMEM; GibcoBRL, USA), 26.2 mM sodium bicarbonate, 21 mM glucose, 2 mM glutamin, 0.7 mM HEPES, in 100 mm dish (Corning, USA). 200 mg penicillin, 19000 unit streptomycine G, 5% fetal bovine serum (FBS; GibcoBRL, USA)] was added to 10 ml and incubated at 37 ° C. for 2 weeks in a 5% CO ₂ -95% air incubator (Forma, USA). The culture was exchanged with fresh medium 2-3 times a week, and gently shaken twice a week from the first week of culture to remove microglia so that only pure astrocytes remained. In order to confirm that the stellate cells were successfully cultured, they were stained with an anti-glial fibrillary acidic protein (GFAP) antibody. The results of fluorescence microscopy are shown in Figure 1.

Cultured astrocytes lack oxygen and glucose to mimic ischemia in cell culture systems ( in vitro ). For this purpose, all manipulations were performed using glucose-free media in hypoxia chamber (Forma, USA) and washed three times with degassed, glucose-free media before all experiments to remove residual oxygen in the media. . The optimal ischemic dose was determined by the time to reduce protein synthesis rate to 50% using metabolic labeling with S ³⁵ -Methionine. When the astrocytes were cultured by oxygen and glucose deficiency for 4 hours before and after 4 hours, the synthesis rate of protein was reduced by about 50% compared to the normal state. After 4 hours, morphological changes of astrocytes began to appear, and after 8 hours, significant cell death was observed.

The established astrocyte culture and in vitro ischemic mimic environment were used to investigate the effect of the EA layer of Kami-Bubu-Ache-tang on the viability of ischemic brain cells. During 4 hours of ischemia treatment, Flick and Gifford methods were used immediately (0 hours), 2 hours, and 24 hours after adding EA layer and hexane layer of Kamiblooded fish tang at 0, 0.1, 0.2mg / ml concentration. Flick and Gifford, 1984) were used to measure cytotoxicity. First, in order to examine the cytotoxicity of the EA layer of Kami-Blood Buche-tang, we measured the cytotoxic activity by varying the concentration of Kami-Blood Bu-Eup-tang EA layer without ischemia, as shown in Figure 3. Increasing the concentration of (EA layer) did not increase cytotoxicity compared to the control. Therefore, it is considered that there is no self-toxicity of Gami-Blood Fish Cake. However, Kami-Bubu-chae-tang extracted from hexane was highly toxic. Therefore, all experiments used Kami-Blood Buche-tang, separated from EA layer. Cytotoxicity was investigated after treatment with Kami-Bubu-Ae-Tang for 4 hours of ischemia. This suggests that Gami-Bubu-Ae-Chan-Tang protects cells from cerebral ischemic toxicity.

Example 7: Mitogen activated protein kinase (MAP kinase) activity measurement

MAP kinase activity was investigated by using antibodies against the activated form of MAP kinase to determine whether the cytoprotective effect of Gami-Buchukeo-tang on cerebral ischemic toxicity was shown through the regulation of MAP kinase activity. While the astrocytes were ischemed for 4 hours, the scavenger was added at 0.1 mg / ml concentration immediately (0 hours), and 0.5 hours later, the cells were placed on ice and washed three times with 4 ° C. PBS. 1 ml of SDS-gel sample buffer (0.1% bromophenol blue, 20% glycerol, 4% sodium dodecil sulfate (SDS), 10% 2-mercapto ethanol) per 100 mm dish was dissolved and boiled for 5 minutes. . Total protein of the collected cells was quantified according to Lowry method (Lowry, 1951) and stored at -20 ℃ until the experiment after quantification. The same amount of protein (40 μg) was electrophoresed on 10% polyacrylamide gel using the method of Laemmli et al . (Laemmli et. Al ., 1970). During electrophoresis, the current was maintained at 50 mA in the stacking gel and 70 mA in the separation gel. After electrophoresis, the electrotransfer kit (Hoeffer, USA) was used to transfer current to nitrocellulose (NC) membrane (BioRad, USA), maintaining the current at 400 mA for 4 hours. After each experiment, the remaining gel was stained with coomasie brilliant blue R250 to confirm that the protein in the gel was completely transferred to the membrane.

The NC membrane was lightly washed with PBS and blocked at room temperature for 2 hours or more with 5% skim milk powder to prevent nonspecific binding of the antibody. Blocking membranes are antibodies against MAP kinase [ERK1; 1: 1000, Santa Cruz, Swiss: phospho ERK (pERK, activated form); 1: 1000, SantaCruz, Swiss), (p38; 1: 2000, NEB, Canada: phospho p38 (pp38, activated form); 1; 2000, NEB, Canada), (JNK1; 1: 1000, SantaCruz, Swiss: phosphoJNK (pJNK, activated form); 1: 1000, SantaCruz, Swiss)] were reacted at room temperature for at least 5 hours and washed three times with PBS added with 0.1% tween 20 for 20 minutes each. After reacting the secondary antibody with Horse radish peroxidase (HRP) to NC membrane for 1 hour, washed 3 times with PBS with 0.1% tween 20 for 20 minutes, and then enhanced chemiluminece (ECL) Analyzes were made using the system (Amersham, USA).

a. ERK activity was confirmed by Western blot using an anti-rabbit polyclonal ERK antibody against exrtacellular signal-regulated kinase (ERK). The experimental results showed that there was no significant difference in the activated form of pERK when treated only with ischemia and treated with ischemia. There was no change in ERK, an inactive form.

b. p38 activity was confirmed by Western blot using an antibody against p38 (anti mouse monoclonal p38 antibody). The experimental results showed that there was no significant difference between p38, an activated form of ischemia alone and both ischemia and ischemia. Inactive form p38 also did not change.

c. The activity of JNK was confirmed by Western blot using an antibody against c-jun N-terminal Kinase (JNK) (anti mouse monoclonal JNK1 antibody). Experimental results show that the active form of pJNK decreases when both the drug and ischemia are treated compared to the treatment with ischemia alone. There was no change in the inactive form of JNK.

Example 8 Expression of Angiogenesis Factor by Ischemia

After 4 hours of ischemia in astrocytes, immediately after (0 hours), 30 minutes, 1 hour and 3 hours, the cells were placed on ice and washed three times with PBS at 4 ° C. RNA isolation from the treated astrocytes was extracted using a single step guanidium isothiocyanate lysis method and quantified using a spectrometer (Kontron, USA). 1 μg of RNA, random hexamer, and moloney murine leukemia virus (MMLV; Promega, USA) were mixed from the total RNA collected and reverse transcription (RT) was performed at 37 ° C. for 1 hour. Various primers (20 pmole, GenoTech, Korea), dNTP (1.25 mM, Promega, USA), 10X PCR buffer (10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl ₂ ), AmpliTag DNA polymerase PerkinElmer, USA) and 5 μl of RT solution (1 μg cDNA) were mixed and PCR was performed. The PCR machine used was from PerkinElmer, USA. The base sequences of the primers used in the experiment are summarized in Table 1. HPRT was used as an internal control. As a result of RT-PCR, DNA was electrophoresed on 1.5% agarose gel and stained with ethidium bromide. Then, the band was checked by UV illuminator (Spectronics, USA) and photographed using a polaroid camera.

Protein separation was performed using the same method as in Experiment 4, and the antibodies used were as follows. [VEGF; 1: 1000, Santa Cruz, Swiss: EPO; 1: 1000, Santa Cruz, Swiss, TGF-β1; 1; 1000, Santa Cruz, Swiss]

Table 20 Location and sequence of PCR pairs for neovascular genes

gene Nucleotide sequence Product size (bps) location EPO 5'-AGCCCTGCGTCTAATGTTTC-3'5'-CGACCACCAGAGACCCTTCA-3 ' 537 2317-23362854-2835 VEGF 5'-TGCACTGGACCCTGGCTTTA-3'5'-TTTGCAGGAACATTTACACG-3 ' 472 109-128581-562 TGF-β 1 5'-CCCGCGTGCTAATGGTGGAC-35'-AGCGCCCGGGTTGTGTTGGT-3 ' 724 678-6921402-1383 hprt 5'-GTTGGATACAGGCCAGACTTTGTTG-3'5'-GATTCAACTTGCGCTCATCTTAGGC-3 ' 162 601-629763-739

Angiogenesis factors are known to play an important role in the production of new blood vessels by increasing the amount of tissue by maintaining the oxygenation of the tissue and regulating the O ₂ pressure of the cells, or when the pathophysiological environment is established. In this experiment, ischemia induced vascular endothelial growth factor (VEGF, Stein et al ., 1995), erythropoietin (EPO, Wang and Semenza, 1993), and transforming growth factor-β1 (TGF-β1, Brown et al ., 1997). Expression patterns were investigated. Ischemia in astrocytes increased the genes and proteins of VEGF. However, EPO and TGF-β1 showed no significant change.

Example 9: Protective effect of cerebral ischemia in the EA layer subdivisions

a. Effect on KCN-induced Survival Rate

Screening for brain injury was carried out using KCN, and the brain injury protection effect was examined using EA fraction subdivisions I, II, III, IV of Gami-Byeolchukeo-tang.

The results are shown in the table below. The sleep time induced by KCN (3.0mg / kg) showed 37.5% inhibition of KHCT fr.1, and the remaining KHCT fr.2,3,4 inhibited 12.5%. The effect of KHCT fr.1 was relatively good.

[Table 21] Effect of KHCT subfraction on KCN-induced survival rate

Sample name The number of animals Volume (mg / 20g) survival % Inhibition Control 8 2.5mg 0 0% KHCT fr.1 8 2.5mg 3 37.50% KHCT fr.2 8 2.5mg One 12.5% KHCT fr.3 8 2.5mg One 12.5% KHCT fr.4 8 2.5mg One 12.5%

In the table, the control group was KCN (3.0 mg / kg), and KHCT fr.

b. KCN-induced sleep time

Screening for brain injury was carried out using KCN, and the brain injury protection effect was examined using EA fraction subdivisions I, II, III, IV of Gami-Byeolchukeo-tang.

Sleep time induced by KCN (1.2m / kg) was KHCT fr.1 55.38 ± 5.78, KHCT fr.2 65.63 ± 6.63, KHCT fr.3 63.63 ± 4.03, KHCT fr.4 64.13 KHCT fr.1 reduced KCN-induced sleep time by ± 3.60.

[Table 22] KHCT subdivision for KCN-induced sleep time

Sample name Number of animals Volume (mg / 20g) Time (sec) Control 8 2.5mg 66.38 ± 4.90 KHCT fr.1 8 2.5mg 55.38 ± 5.78 KHCT fr.2 8 2.5mg 65.63 ± 6.63 KHCT fr.3 8 2.5mg 63.63 ± 4.03 KHCT fr.4 8 2.5mg 64.13 ± 3.60

In the table, the control group was KCN (3.0 mg / kg), and KHCT fr.1, fr.2, fr.3, and fr4: Kami-Bubu-chaetang subdivisions 1,2,3,4.

Example 10: Effect on the ischemic area and edema rate of the middle cerebral artery occlusion of the Gami-Blook-Jung-Ae-Chan EA layer

The experiments were carried out with subfractions 1,2,3,4 of the EA layer of Kami-Blood-Shaped Fish-tang, which had a good effect on the middle cerebral artery ischemia.

The subfraction 1 of the subdivided fractions 1,2,3, and 4 of Kami-Bubu-chae-tang was more effective than the other fractions.

[Table 23] Effect of ischemia area and edema rate of middle cerebral artery occlusion

Sample number Ischemic area (%) Sample number Ischemic area (%) C-1 32.46 KHCT-1 5.11 C-2 43.68 KHCT-2 13.78 C-3 43.40 KHCT-3 21.67 C-4 42.30 KHCT-4 33.45 C-5 48.12 KHCT-5 33.15 C-6 49.10 KHCT-6 29.66

In the above table, C-1 to 6 represent brain fragment numbers of the control group, KHCT fr.

Example 11 Effect of Neurodegenerative Deficits on Middle Cerebral Artery Occlusion

The neurological deficits of the forelimbs were grade 2.75 ± 0.46 after control operation, grade 2.88 ± 0.35 after hypoxia treatment, and recirculation 2.75 ± 0.46.For the KHCT fr.1 treated group, grade 2.67 ± 0.52 after hypoxia treatment The grade of 2.67 ± 0.52 and recycling 2.67 ± 0.52 were shown. The experimental group treated with KHCT fr.1 showed a tendency to improve compared with the control group, but not significantly improved.

The degree of neurological deficits of the hind limbs was 0.38 ± 0.52 after operation in the control group, 0.38 ± 0.52 after hypoxia treatment, and 0.5 ± 0.53 in the recirculation treatment. 0.46, hypoxia after treatment was 0.25 ± 0.46, recycle 0.38 ± 0.52, KHCT fr.1 treated group showed a tendency to improve compared to the control group, but did not improve significantly.

Table 24: Effect of Fraction 1 on the Changes in Neurologic Defects due to Closing of the Middle Cerebral Artery in the EA Layer of Kami-Blood-Shaped Fish (Forelimbs)

Operation Hypoxia Recirculation Control 2.75 ± 0.46 2.88 ± 0.35 2.75 ± 0.46 Fraction 1 2.67 ± 0.52 2.67 ± 0.52 2.67 ± 0.52

In the table, the control group is a 10% alcohol treatment group, and KHCT fraction 1 is EA fraction subdivision 1 of Kami-Buchukeotang.

[Table 25] Effect of Fraction 1 on Changes in Neurologic Deficits of the Middle Cerebral Artery Occlusion in Gami-Blood-Shark Fish-Ea EA Layer (hind Leg)

Operation Hypoxia Recirculation Control 0.38 ± 0.52 0.38 ± 0.52 0.5 ± 0.53 Fraction 1 0.25 ± 0.46 0.25 ± 0.46 0.38 ± 0.52

In the table, the control group is a 10% alcohol treatment group, and KHCT fraction 1 is EA fraction subdivision 1 of Kami-Buchukeotang.

Example 12: Influence on the Mobility Activity of Kami-Blood-Shake Fish-Ea EA Layer

In rats that induced ischemia through MCA occlusion, an experiment was conducted to determine the behavioral change and its drug effects by ischemia by measuring the activity of the normal group and the group treated with KHCT fraction 1.

As a result, there were many differences between the control group and the KHCT fraction 1 group and the normal group.

There was little difference between the control group and the KHCT fraction 1 group. Therefore, the effect of the medicinal herb is evaluated to have little effect on the behavior.

[Table 26] Effect on the migration activity of Gami-Blood Fish Extract EA layer

normal Control KHCT Fraction 1 1h 6045 3899.33 3222.5 2h 4055.67 3305.67 2240 3h 3186.33 3221 2576 sum 13,287 10426 8035.5

In the table above the unit is cm and n = 3.

Example 13: Cytoprotective effect of PC-12 cells induced by β-amyloid protein in EA layer

Table 27: Cytoprotective effect of PC-12 cells by β-amyloid protein

KHCT Fraction 1 KHCT Fraction 4 Control group (%) 500 ug / ml + β-amyloid 10 uM 6.63 6.56 250 ug / ml + β-amyloid 10 uM 3.66 3.81 125 ug / ml + β-amyloid 10 uM 1.37 1.69 62.5 ug / ml + β-amyloid 10 uM 0 0.42 β-amyloid 10 uM 0.70

In KHCT fr.1, the β-amyloid-treated group showed a low survival rate of 0.9%, but the drug-treated group showed a high survival rate depending on the concentration. KHCT fr.4 also showed the same trend as KHCT fr.1.

As described above, when the composition of the present invention is treated, it has an excellent effect in reducing ischemic area of the brain or reduction of edema and can be used for treating and preventing brain diseases such as stroke and dementia.

Claims (4)

By weight ratio of the composition of the whole composition, Angelica 3.57-16%, Uls 3.57-16%, Saenghwanghwang 3.57-16%, Doin 3.57-10.7%, Crust 3.57-9%, Red-eye 3.57-9%, Seahawk 1.78-5.35%, Licorice 1.78-5.35%, plantain 2.68-7.14%, horoscope 2.68-7.14%, job offer 3.57-9%, and a composition for the prevention and treatment of brain diseases comprising a solvent extract of Chinese medicine 3.57-9%. The method of claim 1, The solvent is a composition for preventing and treating brain diseases, characterized in that the solvent selected from the group consisting of water, hexane, butanol, ether, and ethyl acetate. The method according to claim 1 or 2, The brain disease is a composition for preventing and treating brain diseases, characterized in that the stroke or dementia. The method according to claim 1 or 2, The composition of the composition for preventing and treating brain diseases, characterized in that the anti-thrombotic and cerebral infarction protective effect.