KR20130039561A - Pharmaceutical composition comprising acer tegmentosum max, alnus japonica cortex, taraxaci herba, cirsii japonici herba, xanthii fructus, puerariae flos, hovenia dulcis thunb and puerariae radix as an active ingredient for prevention or treatment of brain diseases - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing medicinal herb materials as active ingredients for preventing and treating brain diseases is provided to protect cells from cytotoxicity due to hippocampus H_2O_2, to recover reduced memory, and to block hippocampus injury due to alcohol. CONSTITUTION: A pharmaceutical composition for preventing and treating brain diseases contains Acer tegmentosum Max, Alnus japonica Cortex, Taraxaci Herba, Cirsii Japonici Herba, Xanthii Fructus, Puerariae Flos, Hovenia Dulcis Thunb., and Puerariae Radix as active ingredients. A health food for preventing and treating brain diseases contains one or more medicinal herb materials selected from the group consisting of Aucklandiae radix, Amomi fructus, Phragmitis rhizoma, and Raphani semen. A method for manufacturing a pill formulation with brain cell protection activity comprises: a step of pulverizing Acer tegmentosum Max, Alnus japonica Cortex, Taraxaci Herba, Cirsii Japonici Herba, Xanthii Fructus, Puerariae Flos, Hovenia Dulcis Thunb., and Puerariae Radix and mixing; a step of mixing with 500-800 parts by weight of honey and kneading; a step of making the dough in a pill form with a diameter of 1-2 cm; and a step of drying the pill. [Reference numerals] (AA) Mixed medicinal herb(mg/kg);

Description

Pharmaceutical composition comprising Acer tegmentosum Max, Alnus japonica Cortex, Taraxaci Herba, Cirsii Japonici Herba, Xanthii Fructus, Puerariae Flos, Hovenia Dulcis Thunb and Puerariae Radix as an active ingredient for prevention or treatment of brain diseases}

The present invention relates to a mixed herbal medicine, and more particularly, to a composition for preventing or treating brain diseases, which contains Sancheongmu, Alder, Pogongyoung, Daegye, Changja, Galli, Sediment and Root as active ingredients.

Alcohol is one of the most commonly abused substances in the modern world, with about 30-45% of the population experiencing alcohol-related problems, causing economic, physical and mental losses. In particular, as the consumption of alcohol increases, alcoholic diseases such as alcohol dependence, addiction, and schizophrenia are rapidly increasing along with alcoholic liver lesions. Alcoholic liver diseases such as hepatitis, fatty liver, hepatic fibrosis, cirrhosis, and liver failure due to toxic stress caused by oxidative stress and proinflammatory cytokine caused by acetaldehyde, a by-product produced during the decomposition of alcohol. (Alcoholic liver disease) has been known as a general effect of alcohol (Hoek JB, Pastorino JG. Ethanol, oxidative stress, and cytokine-induced liver cell injury. Alcohol. 2002; 27: 63-8 .; Zima T, Fialova L, Mestek O, Janebova M, Crkovska J, Malbohan I, Stipek S, Mikulıkova L, Popov P. Oxidative stress, metabolism of ethanol and alcohol-related diseases.Journal of Biomedical Science. 2001; 8: 59-70.) .

Until now, alcohol has been primarily focused on the study of alcohol and liver damage because it adversely affects the liver, but alcohol causes diseases not only in the liver, but also in the body, including the brain, heart, and peripheral blood vessels. In particular, alcohol dependence and intoxication not only increase the incidence of Acute respiratory distress syndrome and reduce the function of various organs (Moss M, Burnham EL. Choronic alcohol abuse, acute respiratoey distress syndrome, and multiple organs). dysfunction.Critical Care Medicine. 2003; 31 (4): 207-12.) Influences the central nervous system and decreases cerebral degeneration, brain white and gray doses, as well as Korsakoff's syndrome, memory loss, and depression. The study of alcohol-induced brain damage is being actively conducted with reports of causing mental disorders (Harper C. The neuropathology of alcohol-related brain damage. Alcohol & Alcoholism. 2009; 44 (2): 136-40.) . In addition, alcoholic memory loss and memory impairment is known to be caused by the damage of neuronal cells of the brain involved in memory and learning due to alcohol and its cell function is lost, and attention to alcoholic brain damage with the increase of diseases caused by alcohol Because of this increase, there is a need for research on preparations that can prevent and treat it.

H ₂ O ₂ is a toxic substance that induces oxidative stress. It generates free radicals and radicals, which leads to oxidative stress, leading to oxidative damage of cells (Cowan KJ, Storey KB. Mitogen-activated protein kinases: new signaling pathways functioning in cellular responses to environmental stress.Journal of Experimental Biology. 2003; 206: 1107-15.), oxidative stress balances the production of free radicals such as hydroxy radicals and the antioxidant mechanisms that defend them. This breakdown occurs due to the destruction of mitochondria in cells, protein and DNA modification, and lipid peroxidation due to the increase in free radicals (Sayre ML, Perry G, Smith AM. Oxidative stress and neurotoxicity.Chemical Research in Toxicology 2008; 21: 172-88.). Oxidative stress mechanisms are responsible for the pathogenesis of major diseases including brain and cardiovascular diseases (Madamanchi NR, Vendrov A, Runge MS. Oxidative stress and vascular disease.Arterioscler Thrombosis, and Vascular Biology. 2005; 25: 29-38 ;; MacNee W. Oxidative stress and lung inflammation in airways disease.European Journal of Pharmacology. 2001; 429: 195-207 .; Mosley RL, Benner EJ, Kadiu I, Thomas M, Boska MD, Hasan K, Laurie C, Gendelman. HE. Neuroinflammation, oxidative stress and the pathogenesis of Parkinson's disease.Clinical Neuroscience Research. 2006; 6: 261-81.). Specifically, free radicals and reactive nitrogen species (RNS) may cause stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis ), etc. have been thought to play an important role in many neurological diseases (Beal MF. Mitochondrial dysfunction in neurodegenerative diseases. Biochem Biophys Acta 1998; 1366: 211-23.).

To date, studies on alcohol-induced liver damage in Chinese medicine have been actively conducted, but studies on alcohol-induced brain injury have been conducted only with a few drugs. For example, curcumin, a well-known component of turmeric and turmeric, has been shown to inhibit alcohol-induced cytotoxicity in HT22 hippocampal cells (mitogen-activated protein kinase phosphatase-1). Reported reduction through the activity of (Pae HO, Jeong SO, Zheng M, Ha HY, Lee KM, Kim EC, Kim DH, Hwang SY, Chung HT.Curcumin attenuates ethanol-induced toxicity in HT22 hippocampal cells by activating mitogen-activated protein kinase phosphatase-1. Neuroscience Letters. 2009; 453: 186-9.), and Daegeum-eum Pharmacopuncture, a representative prescription of periodontal diet, was used for the treatment of cerebral nerve cells in the hippocampus of alcohol-toxic rats. Kim HJ, Kim EH, LEY. Effect of Daekumeumja Herb-acupuncture on alcohol-induced suppressed cell proliferation and expression of nitric oxide synthase in hippocampus of rats. The Journal of Korean Acupuncture & Moxibustio n Society. 2006; 23 (5): 187-98.), The effect of improving the memory on the alcohol-induced learning, cognitive impairment, and scopolamine-induced memory loss by brown water extract (Yamazaki T, Yaguchi M, Nakajima Y, Hosonoa T, Niiho Y, Hibi Y, Kinjo J, Nohara T. Effects of an aqueous extract of Puerariae flos (Thomsonide) on impairment of passive avoidance behavior in mice. Journal of Ethnopharmacology. 2005; 100: 244-8.), And the antioxidant effect of ginkgo biloba extract on oxidative stress of rat brain tissues by alcohol and acetaldehyde (Park SU, Kim JB, Heo Y, Lee SD, Kim HJ, Lee IS, Han JH, Park YC, Effects of Ginkgo Biloba extracts on ethanol and acetaldehyde-induced oxidative stress in rat brain.The Korean Journal of Oriental Preventive Medicine. 2004; 8 (1): 109-14.). However, studies on the mechanisms and candidates for treating alcoholic brain injury are still insignificant.

Accordingly, the present inventors have made efforts on substances having a neuroprotective effect on neurons, and an alcohol-induced memory decay inhibitory effect, and a hippocampal neuron protective effect. Mixed herbal medicines containing in vitro ( in vitro) hippocampal neurons in the HT22 cells and show a cytoprotective effect on the cytotoxicity of each of H ₂ O ₂ on the PC12 pheochromocytoma cells, in vivo (in In vivo ) shows a recovery effect against the memory loss caused by alcohol, and has a protective effect against hippocampal nerve cell damage caused by alcohol, it can be useful as an active ingredient for preventing or treating brain diseases or health foods. The present invention has been completed by revealing that there is.

It is an object of the present invention to provide a pharmaceutical composition for preventing and treating brain diseases, which contains mountain bark, alder tree, pogongyoung, dagyeong, changja, browning, earth tree and roots as active ingredients.

Still another object of the present invention is to provide a healthy food for preventing and improving brain diseases, which contains mountain bark, alder tree, pogongyoung, daegye, changja, hwahwa, earthwood and reed as an active ingredient.

Another object of the present invention

1) 20 ~ 30 parts by weight of mountain cheongmok, 20 ~ 30 parts by weight of duck wood, 20 ~ 30 parts by weight of tack, 20 ~ 30 parts by weight of leach, 20 ~ 30 parts by weight of malt, 20 ~ 30 parts by weight of dermis, 10 ~ 15 parts by weight of dermis , 10-15 parts by weight, 10-15 parts by weight, 10-15 parts by weight of plum, 5-10 parts by weight of phosphorus, 5-10 parts by weight of gardenia, 5-10 parts by weight, 5-10 parts by weight of nabokjag, 5 to 10 parts by weight of fruit room, 5 to 10 parts by weight of celestial organs, 5 to 10 parts by weight of safflower, 5 to 10 parts by weight of cheongpi, 5 to 10 parts by weight of gingko, 5 to 10 parts by weight, licorice 5 to 10 parts by weight, Pogongyoung 5-10 parts by weight, total 5-10 parts by weight, Changjaja 5-10 parts by weight, earthwood 5-10 parts by weight, 5-10 parts by weight, browning 5-10 parts by weight, 5-10 parts by weight, respectively Pulverizing and mixing;

2) kneading the mixed mixture of step 1) with 500 to 800 parts by weight of honey;

3) formulating the dough of step 2) in the form of a ring of 1 to 2 cm in diameter; And

4) It provides a method for producing a pill formulation having a neuronal cell protective activity, comprising the step of drying the ring of step 3).

Still another object of the present invention is to provide a pill formulation having brain neuronal cell protective activity prepared by the above method.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating cerebral disease, which contains mountain bark, alder tree, pogongyoung, daejak, changja, grinding, earth tree and roots as an active ingredient.

In addition, the present invention is selected from the group consisting of Sancheongmok, Alder, Pogongyoung, Daegye, Changja, Galli, Sediment, Malt, Gumpi, Baekbokryeong, Plum, Injin, Jisil, Dangmokyang, Sinsa, Root and Nabok It provides a pharmaceutical composition for the prevention and treatment of brain diseases comprising any one or more of the medicines.

In addition, the present invention is any one or more medicinal herbs selected from the group consisting of cheonsaek, dermis, pogongyoung, daegye, Changja, Galhwa, earthwood and roots in addition to taeksa, dermis, groceries, kwakyang, gardenia, cheonggung, safflower, green skin and licorice It provides a pharmaceutical composition for preventing and treating brain diseases.

In another aspect, the present invention provides a health food for preventing and improving brain diseases containing mountain bark, alder wood, pogongyoung, Daegye, Changja, browning, earthwood and roots as an active ingredient.

In addition, the present invention is selected from the group consisting of Sancheongmok, Alder, Pogongyoung, Daegye, Changja, Galli, Sediment, Malt, Gumpi, Baekbokryeong, Plum, Injin, Jisil, Dangmokyang, Sinsa, Root and Nabok It provides a healthy food for preventing and improving brain diseases comprising any one or more of the medicines.

In addition, the present invention is any one or more medicinal herbs selected from the group consisting of cheonsaek, dermis, pogongyoung, daegye, Changja, Galhwa, earthwood and roots in addition to taeksa, dermis, groceries, kwakyang, gardenia, cheonggung, safflower, green skin and licorice It provides a health food for preventing and improving brain diseases comprising.

In addition,

1) 20 ~ 30 parts by weight of mountain cheongmok, 20 ~ 30 parts by weight of duck wood, 20 ~ 30 parts by weight of tack, 20 ~ 30 parts by weight of leach, 20 ~ 30 parts by weight of malt, 20 ~ 30 parts by weight of dermis, 10 ~ 15 parts by weight of dermis , 10-15 parts by weight, 10-15 parts by weight, 10-15 parts by weight of plum, 5-10 parts by weight of phosphorus, 5-10 parts by weight of gardenia, 5-10 parts by weight, 5-10 parts by weight of nabokjag, 5 to 10 parts by weight of fruit room, 5 to 10 parts by weight of celestial organs, 5 to 10 parts by weight of safflower, 5 to 10 parts by weight of cheongpi, 5 to 10 parts by weight of gingko, 5 to 10 parts by weight, licorice 5 to 10 parts by weight, Pogongyoung 5-10 parts by weight, total 5-10 parts by weight, Changjaja 5-10 parts by weight, earthwood 5-10 parts by weight, 5-10 parts by weight, browning 5-10 parts by weight, 5-10 parts by weight, respectively Pulverizing and mixing;

2) kneading the mixed mixture of step 1) with 500 to 800 parts by weight of honey;

3) formulating the dough of step 2) in the form of a ring of 1 to 2 cm in diameter; And

4) It provides a method for producing a pill formulation having a neuroprotective activity, comprising the step of drying the ring of step 3).

In another aspect, the present invention provides a pill formulation having a neuroprotective activity, prepared by the above method.

The mixed herbal medicines containing wild blue, alder, pogongyoung, dagyeok, changja, browning, earth, and root of the present invention as active ingredients are cytotoxicity by H ₂ O ₂ in HT22 cells, which are hippocampal neurons and PC12 cells, which are pheochromocytomas, respectively. It shows a cytoprotective effect against, shows a recovery effect against the memory loss by alcohol, and a cytoprotective effect against hippocampal nerve cell damage caused by alcohol, thereby preventing or treating a brain disease pharmaceutical composition or health food It can be usefully used as an active ingredient of.

1 is a graph showing the effect of the mixed herbal medicine on H ₂ O ₂ induced cytotoxicity in HT22 cells.
(A) A graph showing treatment of mixed herbal medicines for 7 hours. (B) a graph showing that the cells were treated with mixed herbal medicine for one hour and then treated with 300 μM H ₂ O ₂ for six hours. Cell viability was measured using MTT assay and expressed as a percentage relative to the control. The measured values are expressed as mean ± SEM. *** p <0.001 compared to control. ## p <0.01 compared to the group treated with H ₂ O ₂ only.
Figure 2 is a graph showing the effect of the mixed herbal medicines on H ₂ O ₂ induced cytotoxicity in PC12 cells.
(A) A graph showing treatment of mixed herbal medicines for 7 hours. (B) A graph showing treatment of 50 μM H ₂ O ₂ for 6 hours after treatment with mixed herbal medicines for one hour. Cell viability was measured using MTT assay and expressed as a percentage relative to the control. The measured values are expressed as mean ± SEM. *** p <0.001 compared to control. ## p <0.01 compared to the group treated with H ₂ O ₂ only.
Figure 3 is a graph showing the effect of mixed herbal medicine on alcohol-induced memory impairment in ICR mice. The measured values are expressed as mean ± SEM. ** p <0.01 compared to control. ## p <0.01 compared to the group treated with EtOH only.
4 is a diagram showing the effect of mixed herbal medicine on alcohol-induced hippocampal neuronal damage in ICR mice. (A to C) Representative photomicrographs of hippocampus (hippocampus) in each group, (D to F) representative micrographs of CA3, and (G to I). (A, D, G) controls; (B, E, H) 5 g / kg EtOH treatment group, (C, F, I) 5 g / kg EtOH + 100 mg / kg mixed herbal treatment group. Scale bar = 200 μm.
5 is a diagram confirming the effect of the mixed herbal medicine on NeuN-immunological cells against alcohol in the hippocampus of ICR mice. (A to C) Representative photomicrographs of hippocampus (hippocampus) in each group, (D to F) representative micrographs of CA3, and (G to I). (A, D, G) controls; (B, E, H) 5 g / kg EtOH treatment group, (C, F, I) 5 g / kg EtOH + 100 mg / kg mixed herbal treatment group. Scale bar = 200 μm.
Figure 6 shows the effect of mixed herbal medicine on alcohol-induced memory impairment in ICR mice. (A) The result of Western blotting is a graph showing the ratio with the control group beta-actin (β-actin). (B) The figure shows the result of western blot. The measured values are expressed as mean ± SEM. * P <0.05 compared to control. ## p <0.01 compared to the group treated with EtOH only.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for the prevention and treatment of brain diseases, containing alum, oleander, pogongyoung, Daegye, Changja, browning, earthwood and roots as active ingredients.

The pharmaceutical composition is specifically the group consisting of Sancheongmok, Alderwood, Pogongyoung, Daegye, Changja, Gallium, Earthwood and Brown root, White baekchul, Malt, Gampi, Baekbokryeong, Plum, Injin, Jisil, Dangmok-scent, Sine, Root and Nabok It includes, but is not limited to any one or more medicinal herbs selected from.

Specifically, the pharmaceutical composition is any one selected from the group consisting of sapphire, dermis, groin, gwakyang, gardenia, cheonggung, safflower, blue skin and licorice, in addition to Sancheongmok, Alder, Pogongyoung, Daegye, Changja, Gallium, Tert. It includes, but is not limited to, the above medicines.

The pharmaceutical composition is specifically 20 ~ 30 parts by weight of the mountain cheongmok, 20 ~ 30 parts by weight of alderwood, 20 ~ 30 parts by weight, 20 to 30 parts by weight, 20 to 30 parts by weight, malt 20 to 30 parts by weight, 20 to 30 parts by weight of dermis, dermis 10 to 15 parts by weight, 10 to 15 parts by weight of Baekbokryeong, 10 to 15 parts by weight thick, 10 to 15 parts by weight of plum, 5 to 10 parts by weight, gardenia 5 to 10 parts by weight, 5 to 10 parts by weight, nabokjak 5 ~ 10 parts by weight, 5 to 10 parts by weight, 5 to 10 parts by weight Cheongung, 5 to 10 parts by weight, safflower 5 to 10 parts by weight, 5 to 10 parts by weight of cheongmok, 5 to 10 parts by weight, sine 5 to 10 parts by weight, licorice 5 ~ 10 parts by weight, poongyoung 5-10 parts by weight, total 5-10 parts by weight, Changzai 5-10 parts by weight, earthwood 5-10 parts by weight, brown roots 5-10 parts by weight, browning 5-10 parts by weight, labor root 5 It contains but is not limited to ~ 10 parts by weight as an active ingredient.

More specifically, 20 ~ 24 parts by weight of mountain cherub, 20 ~ 24 parts by weight of duck, 20 to 24 parts by weight, 20 to 24 parts by weight, 20 to 24 parts by weight of malt, 20 to 24 parts by weight of malt, 10 to 12 parts by weight of dermis Part, 10-12 parts by weight, 10-12 parts by weight, 10-12 parts by weight of plum, 5-6 parts by weight, 5-6 parts by weight of gardenia, 5-6 parts by weight, 5-6 parts by weight of moths , 5-6 parts by weight, 5-6 parts by weight Cheongung, 5-6 parts by weight of safflower, 5-6 parts by weight of cheongpi, 5-6 parts by weight of Mokmok, 5-6 parts by weight, licorice 5-6 parts by weight , 5-6 parts by weight, 5-6 parts by weight, 5-6 parts by weight, Changji 5-6 parts by weight, 5-6 parts by weight, 5-6 parts by weight, 5-6 parts by weight, 5-6 parts by weight It contains, but is not limited to, an active ingredient.

Most specifically, 24 parts by weight of mountain cheongsam, 24 parts by weight of duckwood, 24 parts by weight of tack, 24 parts by weight of malt, 24 parts by weight of malt, 24 parts by weight of dermis, 12 parts by weight of dermis, 12 parts by weight of Baekbokyeong, 12 parts by weight of plum, 12 plums Weight part, 6 weight part of Jinjin, 6 weight part of gardenia, 6 weight part of Gwakyang, 6 weight part of nagyeokja, 6 weight part of fibrosis, 6 weight part of safflower, 6 weight part of safflower, 6 weight part of Dangmok flavor, 6 parts by weight of Dangmok 6 parts by weight, licorice 6 parts by weight, pogongyoung 6 parts by weight, total 6 parts by weight, Changja 6 parts by weight, earthwood 6 parts by weight, brown root 6 parts by weight, browning 6 parts by weight, old roots 6 parts by weight It is not limited to this.

The pharmaceutical composition is specifically Sancheongmok (山 靑 木) (Acer tegmentosum Max., Jecheon, 24 g), the taxi company (澤瀉)Alismatis Rhizoma, Suncheon, 24 g), Baekchul (白 朮)Atractylodis Rhizoma Alba, Youngcheon, 24 g), malt (麥芽)Hordei Fructus Germinatus, Yeosu, 24 g), Gumpi (柑 皮)Persimmon Peels, Qingdao, 24 g), Alderwood (赤 陽 皮)Alnus japonica Cortex, Army, 24 g), dermis (陳皮)Citri Unshii Pericarpium, Jeju, 12 g)Hoelen, Cheongsong, 12 g)Magnoliae Cortex, China, 12 g), Plum (Mume Fructus, China, 12 g), Injin (茵 蔯)Artemisiae Capillaris Herba, Racing, 6 g), gardenia (梔子)Gardeniae Fructus, Jecheon, 6 g), Kwakhyang (藿香)Agastachis Herba, Cheongsong, 6 g)Raphani Semen, China, 6 g)Ponciri Fructus, Lord, 6 g)Cnidii Rhizoma, Andong, 6 g), safflower (紅花)Carthami Flos, China, 6 g), Cheongpi (靑 皮)Citrii Unshiu Immaturi Pericarpium, Jeju, 6 g), Mokhyang (木香)Aucklandiae Radix, China, 6 g), sine (砂仁)Amomi Fructus, China, 6 g), licorice (甘草)Glycyrrhizae Radix meat Rhizoma, China, 6 g), Pogongyoung ((公 英)Taraxaci Herba, Gyeongbuk Yeongcheon, 6 g)Cirsii Japonici Herba, China, 6 g), Changsha ((子)Xanthii Fructus, China, 6 g), Earth (Hovenia Dulcis Thunb, Chungbuk Jecheon, 6 g), Brown root (葛根)Puerariae Radix, Gangwon Wonju, 6 g), Gall flower (葛花)Puerariae Flos, China, 6 g), Root (Phragmitis Rhizoma, China, 6 g), and the like, which are prepared by grinding and mixing the same, but not limited thereto. For uniform sample processing and dosing between individuals, 20 times of distilled water is added to the mixed herbal medicine and sonicated at room temperature for 2 hours. After sonication, vacuum filtration was performed (Whatman No. 2). The filtrate was lyophilized by freezing below -60 ℃ (FDU-550R; Eyela Co, Japan), and used after storage at -20 ℃.

The brain diseases include stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, alcoholic cranial nerve disease, alcoholic dementia and berber. Any one selected from the group consisting of Wernicke-Korsakoff's syndrome is not limited thereto.

The pharmaceutical composition specifically protects nerve cells from oxidative stress or damage caused by alcohol, but is not limited thereto.

The inventors of the present invention to investigate the effect of the mixed herbal medicine according to the present invention on H ₂ O ₂ induced cytotoxicity in HT22 cells, which are mouse hippocampal neuronal cells, cells according to the drug treatment by concentration of the mixed herbal medicine As a result of measuring the survival rate and the cytoprotective effect on H ₂ O ₂ by MTT assay, when HT22 cells were treated with only 1 ~ 100 ㎍ / ml of the mixed herbal medicine, it did not affect the cell viability (Fig. 1). A), 300 μM H ₂ O ₂ reduced the cell viability to 60.16% compared to the control group, while pretreatment of the mixed herbal medicine (1 ~ 100 ㎍ / ㎖) showed a survival rate of 73.72 ~ 72.35%, respectively, H ₂ O _It was confirmed that the protective effect against the cytotoxicity induced by ₂ (see FIG. 1B).

In addition, the present inventors, in order to determine the effect of the mixed herbal medicine according to the present invention on H ₂ O ₂ induced cytotoxicity in PC12 cells of rat pheochromocytoma, the drug treatment by concentration of the mixed herbal medicine According to the MTT assay of the cell survival rate and the cytoprotective effect on H ₂ O ₂ , PC12 cells did not affect the cell survival rate when only 1-100 μg / ml of the mixed herbal medicine was treated ( ₂ , the cell viability was reduced to 64.87% compared to the control group by 50 μM H ₂ O ₂ , while the survival rate of 78.14% and 78.35%, respectively, by pretreatment of the mixed herbal medicines (1 and 10 μg / ml), respectively. It was confirmed that it showed a protective effect against H ₂ O ₂ induced cytotoxicity (see B of FIG. 2).

In addition, the present inventors, in order to confirm the weight change according to the administration of the mixed herbal medicine and alcohol according to the present invention in the male ICR mouse, every two days after the administration of the mixed herbal medicine 100 mg / kg and 5 g / kg for 4 weeks As a result of measuring the weight, the control group without alcohol toxicity showed a tendency to increase steadily, whereas the alcohol alone group and the alcohol and mixed medicament group administered the alcohol body weight from 2nd to 4th day It was reduced and began to recover from day 6, and on day 10 there was no weight difference between the three groups. In particular, the group administered with alcohol and the mixed herbal medicine showed more weight loss than the alcohol alone group, but showed a rapid recovery rate, and showed no statistically significant weight difference between the control group and the alcohol alone group (see Table 1). .

In addition, the present inventors conducted an object recognition experiment 24 hours after the administration of the mixed herbal medicine and alcohol to evaluate the effect of the mixed herbal medicine according to the present invention on alcohol-induced memory loss, and had two similar objects 24 hours before the experiment. When training was performed, each group showed no statistically significant difference (64.29%, 55.54%, and 48.96%), whereas in the study conducted 24 hours after training, the alcohol alone group was 37.91% (62.62%). Compared to), the new object was searched for less, and the group administered with alcohol and the mixed herbal medicine showed a statistically significant recovery effect at 66.63%. Therefore, in the object recognition behavior experiment, it was confirmed that the mixed herbal medicine had a protective effect against the memory loss caused by alcohol (see Fig. 3).

In addition, the present inventors stained the nuclei of cells in order to confirm that the decrease in memory was induced by alcohol through the object recognition experiments and to examine the effects of alcohol and the mixed herbal medicine according to the present invention on the hippocampal part of the brain involved in memory. By expressing the nucleus of cresyl violet (cresyl violet) and neurons to determine the apoptosis was stained NeuN factor that can determine the damage of cells. As a result, it was confirmed that the apoptosis was induced by alcohol as the cell number was decreased and lightly colored in the CA3 portion of the hippocampal cells in the alcohol alone group. While showing a tendency to occur, it was confirmed that the apoptosis was inhibited by administration of the mixed herbal medicine in the group administered with alcohol and mixed herbal medicine (see FIG. 4). In addition, in the immunostaining results of the NeuN protein, less NeuN-positive cells were expressed by alcohol, resulting in neuronal damage caused by alcohol toxicity, and the mixed herbal medicines inhibited neuronal cell death by alcohol. (See Figure 5).

In addition, after confirming the hippocampal neuronal cell protective effect of the mixed herbal medicine according to the present invention by NeuN immunohistochemistry, the results of Western blot analysis to determine the difference in protein expression through the quantification of NeuN protein, the results confirmed by histological picture As shown in the alcohol-only group, the expression of NeuN protein was decreased in the hippocampal tissues to which alcohol was administered. In the group to which alcohol and the mixed herbal medicine were administered, the decrease in the expression of NeuN protein was suppressed by the mixed herbal medicine. Therefore, it was confirmed that there is a hippocampal neuronal protective effect against alcoholic toxicity of the mixed herbal medicine (see Fig. 6).

Therefore, the mixed herbal medicine according to the present invention shows a cytoprotective effect against cytotoxicity by H ₂ O ₂ in HT22 and PC12 cells, respectively, shows a recovery effect against memory loss by alcohol, and damage to hippocampal neurons by alcohol. Since it has a cytoprotective effect against it can be usefully used as an active ingredient of the pharmaceutical composition for preventing and treating brain diseases.

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

The composition of the present invention may be administered orally or parenterally, and when parenteral administration, it is preferable to select external or intraperitoneal injection, rectal injection, subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection method. It is not limited thereto.

The composition of the present invention can be used in the form of oral formulations, external preparations, suppositories, and sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, respectively, according to conventional methods. have. Carriers, excipients and diluents that may be included in the composition 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose in mixed herbal medicines. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at 0.0001 to 1 g / kg, preferably 0.001 to 200 mg / kg, but is not limited thereto. The above administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Pill formulation of the mixed herbal medicine according to the present invention is prepared in the following steps, but is not limited thereto.

1) 20 ~ 30 parts by weight of mountain cheongmok, 20 ~ 30 parts by weight of duck wood, 20 ~ 30 parts by weight of tack, 20 ~ 30 parts by weight of leach, 20 ~ 30 parts by weight of malt, 20 ~ 30 parts by weight of dermis, 10 ~ 15 parts by weight of dermis , 10-15 parts by weight, 10-15 parts by weight, 10-15 parts by weight of plum, 5-10 parts by weight of phosphorus, 5-10 parts by weight of gardenia, 5-10 parts by weight, 5-10 parts by weight of nabokjag, 5 to 10 parts by weight of fruit room, 5 to 10 parts by weight of celestial organs, 5 to 10 parts by weight of safflower, 5 to 10 parts by weight of cheongpi, 5 to 10 parts by weight of gingko, 5 to 10 parts by weight, licorice 5 to 10 parts by weight, Pogongyoung 5-10 parts by weight, total 5-10 parts by weight, Changjaja 5-10 parts by weight, earthwood 5-10 parts by weight, 5-10 parts by weight, browning 5-10 parts by weight, 5-10 parts by weight, respectively Pulverizing and mixing;

2) blending and kneading the ground mixture of step 1);

3) reclaiming the dough of step 2);

4) repurposing the redeemed ring of step 3); And

5) drying the packaged ring of step 4) and packaging.

In the above method, step 1) is specifically 20 ~ 24 parts by weight of acid wood, 20 ~ 24 parts by weight of alder wood, 20 ~ 24 parts by weight of tack, 20 ~ 24 parts by weight, 20 to 24 parts by weight of malt, 20 ~ 24 parts of gampi 10 parts by weight, dermis 10-12 parts by weight, 10-12 parts by weight of baekbokyeong, 10-12 parts by weight, plums 10-12 parts by weight, 5-6 parts by weight, gardenia 5-6 parts, weight 5-6 parts by weight 5-6 parts by weight, 5-6 parts by weight of lichens, 5-6 parts by weight, 5-6 parts by weight of celery, 5-6 parts by weight of safflower, 5-6 parts by weight of cheongpi, 5-6 parts by weight of Dangmok, 5-6 parts by weight Parts, licorice 5-6 parts by weight, poongyoung 5-6 parts by weight, total 5-6 parts by weight, Changzai 5-6 parts by weight, earth tree 5-6 parts by weight, brown root 5-6 parts by weight, browning 5-6 parts by weight Part 5, 6 parts by weight of the furnace roots are mixed, but not limited thereto.

More specifically, 24 parts by weight of mountain cheongmok, 24 parts by weight of duckwood, 24 parts by weight of tack, 24 parts by weight of malt, 24 parts by weight of malt, 24 parts by weight of dermis, 12 parts by weight of dermis, 12 parts by weight of Baekbokyeong, 12 parts by weight of plum, 12 plums Weight part, 6 weight part of Jinjin, 6 weight part of gardenia, 6 weight part of Gwakyang, 6 weight part of nagyeokja, 6 weight part of fibrosis, 6 weight part of safflower, 6 weight part of safflower, 6 weight part of Dangmok flavor, 6 parts by weight of Dangmok 6 parts by weight, licorice 6 parts by weight, pogongyoung 6 parts by weight, total 6 parts by weight, Changja 6 parts by weight, earthwood 6 parts by weight, brown root 6 parts by weight, grinding 6 parts by weight, 6 parts by weight of ground roots and then mixed It is not limited thereto.

The present invention specifically provides a method of preparing a pill formulation comprising the following steps.

1) 20 ~ 30 parts by weight of mountain cheongmok, 20 ~ 30 parts by weight of duck wood, 20 ~ 30 parts by weight of tack, 20 ~ 30 parts by weight of leach, 20 ~ 30 parts by weight of malt, 20 ~ 30 parts by weight of dermis, 10 ~ 15 parts by weight of dermis , 10-15 parts by weight, 10-15 parts by weight, 10-15 parts by weight of plum, 5-10 parts by weight of phosphorus, 5-10 parts by weight of gardenia, 5-10 parts by weight, 5-10 parts by weight of nabokjag, 5 to 10 parts by weight of fruit room, 5 to 10 parts by weight of celestial organs, 5 to 10 parts by weight of safflower, 5 to 10 parts by weight of cheongpi, 5 to 10 parts by weight of gingko, 5 to 10 parts by weight, licorice 5 to 10 parts by weight, Pogongyoung 5-10 parts by weight, total 5-10 parts by weight, Changjaja 5-10 parts by weight, earthwood 5-10 parts by weight, 5-10 parts by weight, browning 5-10 parts by weight, 5-10 parts by weight, respectively Pulverizing and mixing;

2) kneading the mixed mixture of step 1) with 500 to 800 parts by weight of honey;

3) formulating the dough of step 2) in the form of a ring of 1 to 2 cm in diameter; And

4) It provides a method for producing a pill formulation having a neuroprotective activity, comprising the step of drying the ring of step 3).

In the above method, step 1) is specifically 20 ~ 24 parts by weight of acid wood, 20 ~ 24 parts by weight of alder wood, 20 ~ 24 parts by weight of tack, 20 ~ 24 parts by weight, 20 to 24 parts by weight of malt, 20 ~ 24 parts of gampi 10 parts by weight, dermis 10-12 parts by weight, 10-12 parts by weight of baekbokyeong, 10-12 parts by weight, plums 10-12 parts by weight, 5-6 parts by weight, gardenia 5-6 parts, weight 5-6 parts by weight 5-6 parts by weight, 5-6 parts by weight of lichens, 5-6 parts by weight, 5-6 parts by weight of celery, 5-6 parts by weight of safflower, 5-6 parts by weight of cheongpi, 5-6 parts by weight of Dangmok, 5-6 parts by weight Parts, licorice 5-6 parts by weight, poongyoung 5-6 parts by weight, total 5-6 parts by weight, Changzai 5-6 parts by weight, earth tree 5-6 parts by weight, brown root 5-6 parts by weight, browning 5-6 parts by weight Part 5, 6 parts by weight of the furnace roots are mixed, but not limited thereto.

More specifically, 24 parts by weight of mountain cheongmok, 24 parts by weight of duckwood, 24 parts by weight of tack, 24 parts by weight of malt, 24 parts by weight of malt, 24 parts by weight of dermis, 12 parts by weight of dermis, 12 parts by weight of Baekbokyeong, 12 parts by weight of plum, 12 plums Weight part, 6 weight part of Jinjin, 6 weight part of gardenia, 6 weight part of Gwakyang, 6 weight part of nagyeokja, 6 weight part of fibrosis, 6 weight part of safflower, 6 weight part of safflower, 6 weight part of Dangmok flavor, 6 parts by weight of Dangmok 6 parts by weight, licorice 6 parts by weight, pogongyoung 6 parts by weight, total 6 parts by weight, Changja 6 parts by weight, earthwood 6 parts by weight, brown root 6 parts by weight, grinding 6 parts by weight, 6 parts by weight of ground roots and then mixed It is not limited thereto.

In the above method, the honey of step 2) is specifically 600 to 700 parts by weight, more specifically 600 parts by weight, but not limited thereto, and may be replaced with any one selected from the group consisting of oligosaccharides, natural juices and xylitol, but It is not limited.

In another aspect, the present invention provides a pill formulation having a neuroprotective activity, prepared by the above method.

The pill formulation specifically protects brain neurons from oxidative stress or alcohol damage, but is not limited thereto.

In another aspect, the present invention provides a health food for preventing and improving brain diseases containing mountain bark, alder wood, pogongyoung, Daegye, Changja, browning, earthwood and roots as an active ingredient.

The health food is specifically from the group consisting of Sancheongmok, Alderwood, Pogongyoung, Daegye, Changja, Gallium, Earthwood and Brown root, Baekchul, Malt, Gumpi, Baekbokryeong, Plum, Injin, Jisil, Dangmok, Sine, Root and Nabok It includes but is not limited to any one or more selected medicines.

Specifically, the health food is one or more selected from the group consisting of Sancheongmok, Alder, Pogongyoung, Daegye, Changja, Galhwa, Earthwood, and Root, in addition to Taeksa, Dermis, Hubak, Gwakhyang, Gardenia, Cheongung, Safflower, Cheongpi and Licorice. It includes but is not limited to medicinal herbs.

The brain diseases include stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis, alcoholic cranial nerve disease, alcoholic dementia and berber. Any one selected from the group consisting of Wernicke-Korsakoff's syndrome is not limited thereto.

The health food is specifically, but not limited to protecting brain neurons from oxidative stress or damage caused by alcohol.

The mixed herbal medicine according to the present invention shows a cytoprotective effect against cytotoxicity by H ₂ O ₂ in HT22 and PC12 cells, respectively, shows a recovery effect against memory loss by alcohol, and against hippocampal nerve cell damage by alcohol. Since it has a cytoprotective effect, it can be usefully used as an active ingredient in healthy foods for preventing and improving brain diseases.

There is no particular limitation on the kind of the food. Examples of the foods include drinks, meat, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, alcoholic beverages and vitamins. Combinations and the like, and include all of the health foods in the conventional sense.

The mixed herbal medicine according to the present invention may be added to food as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). In general, the amount of the mixed herbal medicine in the health food can be added to 0.01 to 15% by weight of the total food weight, the health beverage composition is added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the mixed herbal medicines as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. . Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the mixed herbal medicine of the present invention may contain a flesh for preparing natural fruit juice and fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The ratio of such additives is not so important but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the mixed herbal medicine of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples and Production Examples.

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

< Example  1> Preparation of Mixed Herbal Medicines

<1-1> Preparation of Mixed Herbal Medicines

The inventors have sancheongmok (山靑木) (Acer tegmentosum Max , Jecheon, 24 g), Tajik ( Alismatis Rhizoma , Suncheon, 24 g), Atractylodis Rhizoma Alba , Yeongcheon, 24 g), Malde Fructus Germinatus, Yeosu, 24 g), gampi (柑皮) (Persimmon Peels , Qingdao, 24 g), Alnus japonica Cortex , Army, 24 g), Dermis ( Citri Unshii Pericarpium , Jeju, 12 g), Hokbokyeong ( Hoelen , Cheongsong, 12 g), Magnoliae Cortex , China, 12 g), Mume Fructus , China, 12 g), Artemisiae Capillaris Herba , Gyeongju, 6 g), Gardenia ( Gardeniae Fructus , Jecheon, 6 g), Agastachis Herba , Cheongsong, 6 g), Raphani Semen , China, 6 g), Ponciri Fructus , lord, 6 g), Cnidii Rhizoma , Andong, 6 g), Safflower ( Carthami Flos , China, 6 g), Chengrii Unshiu Immaturi Pericarpium, Jeju, 6 g), per elecampane (Aucklandiae Radix, China, 6 g), sign (砂仁) (Amomi Fructus , China, 6 g), Licorice ( Glycyrrhizae Radix meat Rhizoma , China, 6 g), Pogongyoung (Taraxaci Herba , Gyeongbuk Yeongcheon, 6 g), Dagye ( Cirsii Japonici Herba , China, 6 g), Changi ( Xanthii Fructus , China, 6 g) , Earth tree ( Hovenia Dulcis Thunb , Chungbuk Jecheon, 6 g), Brown root ( Puerariae ) Radix , Wonju Kang, 6 g), Puerariae Flos , China, 6 g), Phragmitis Rhizoma , China, 6 g) and the like were ground and mixed to manufacture. For uniform sample treatment and dosing between subjects, 20 times of distilled water was added to the mixed herbal medicines, followed by sonication for 2 hours at room temperature, followed by vacuum filtration (Whatman No. 2). The filtrate was lyophilized by freezing at −60 ° C. or lower (FDU-550R; Eyela Co, Japan), and the yield after lyophilization was 40.42%. After storage at -20 ℃ was used in each solvent dissolved in the experiment.

<1-2> Manufacture of medicinal herbs

Sancheongmok 24 parts by weight, alder 24 parts by weight, talc 24 parts by weight, white extract 24 parts by weight, malt 24 parts by weight, gampi 24 parts by weight, dermis 12 parts by weight, Baekbokyeong 12 parts by weight, thick 12 parts by weight, plum 12 parts by weight, 6 parts by weight of jinjin, 6 parts by weight of gardenia, 6 parts by weight of hors d'oeuvre, 6 parts by weight of nachos, 6 parts by weight of fruit room, 6 parts by weight of safflower, 6 parts by weight of safflower, 6 parts by weight of cheongmok, 6 parts by weight of saengmok , 6 parts by weight of licorice, 6 parts by weight of Pogongyoung, 6 parts by weight of ginseng, 6 parts by weight of Changjaja, 6 parts by weight of earthwood, 6 parts by weight of brown root, 6 parts by weight of grind, and 6 parts by weight of labor root in powder Prepared.

600 g of the powder and honey prepared above were mixed and kneaded.

The dough prepared above was put into Jang Wan-gi (Suin Industry), and then put into a recirculator (Suin Industry) to make a ring.

The above-prepared ring was put into a sorting machine (suin industry), sorted, and then put into a dryer (Duritec) to dry to finally prepare a ring.

< Example  2> Confirmation of Cytotoxicity of Mixed Herbal Medicines

<2-1> Cell Culture

HT22 hippocampal neuronal cells and rat pheochromocytoma PC12 cells were used by the Korea Cell Line Bank (Seoul, Korea). HT22 cell cultures were high-glucose containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin (P / S) at 37 ° C, 5% CO ₂ , 95% air. A high-glucose Dulbecco's modified Eagle's medium (DMEM) medium was used and PC12 cell culture was performed under the same conditions, with high-glucose containing 10% horse serum, 5% FBS and 1% P / S. glucose) Roswell Park Memorial Institute (RPMI) 1640 medium was used. Each cell was passaged and cultured at a ratio of 1: 3 every 2-3 days.

<2-2> Mixed Herbal Medicine  Determine impact on cell viability

In a 96-well plate, HT22 cells were dispensed at 0.5 × 10 ⁴ / well and PC12 cells at 2 × 10 ⁴ / well in a 96-well plate coated with 50 μg / ml collagen. After culturing for 24 hours, the mixed herbal medicine of Example <1-1> was treated for 7 hours at concentrations of 0.1-100 μg / ml. After treatment with the mixed herbal medicine, the supernatant was removed and treated with MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) 1 mg / ml for 2 hours, followed by DMSO (Dimethyl). Crystallization with sulfoxide was performed (decrystalize) and the absorbance was measured at 570 nm with a spectrophotometer (Versamax microplate reader; Molecular Device, Sunnyvale, Calif., USA). Cell viability was expressed as a percentage of the control.

As a result, as shown in FIG. 1A, when the mixed medicinal herbs were treated in HT22 cells, it was confirmed that they did not affect the cell viability compared to the control group without the mixed medicinal herbs (FIG. 1A), and FIG. 2. As shown in A, when the mixed herbal medicine was treated to PC12 cells, it was confirmed that there was no effect on cell viability compared to the control without the mixed herbal medicine (FIG. 2A).

< Example  3> Mixed Herbal Medicines H ₂ O ₂ Confirmation of cytoprotective effect on toxicity

<3-1> H ₂ O ₂ Toxic For mixed herbal medicines  Confirmation of cytoprotective effect

In the same manner as in Example <2-2>, HT22 and PC12 cells were dispensed into 96-well plates, respectively, and treated with 0.1-100 μg / ml of the mixed herbal medicine of Example <1-1> after 1 hour, respectively. 300 μM H ₂ O ₂ in cells And 50 μM of H ₂ O ₂ were further treated for 6 hours. After completion of the reaction of the mixed herbal medicine and H ₂ O ₂ , the supernatant was removed and treated with MTT 1 mg / ml and reacted for 2 hours. After crystallization using DMSO, crystallization was carried out using a spectrophotometer at 570 nm. Absorbance was measured at. Cell viability was expressed as a percentage of the control.

As a result, as shown in FIG. 1B, the cell viability was reduced to 60.16% by 300 μM H ₂ O ₂ compared with the mixed herbal and H ₂ O ₂ treated groups, and the mixed herbal medicine (1 to 100 μg / ml). ) Showed that the survival rate of 73.72 ~ 72.35% by pretreatment respectively (Fig. 1B), as shown in B of FIG. ₂ , the cell viability was mixed by 50 μM H ₂ O ₂ as mixed herbal medicine and H ₂ O ₂ Was reduced to 64.87% compared to the untreated control group, it was confirmed that the survival rate of 78.14% and 78.35% by pretreatment of the mixed herbal medicine (1 and 10 ㎍ / ㎖) respectively (Fig. 2B).

< Example  4> ICR Confirmation of Protective Effect of Mixed Herbal Medicine against Alcohol Toxicity in Mice

<4-1> ICR Effects of Mixed Herbal Medicines on Mouse Weight

Experimental animals and feed were purchased from Daewoo Biolink (Eumseong, Korea). Six-week-old ICR-based magnetic mice (25-28 g) were acclimated for 5 days under constant conditions (temperature: 22 ± 1 ° C, humidity: 55 ± 3%, 12 hour contrast cycle), and 8 mice were used in each group. Human dose (3 g) was applied to the metabolic rate and body surface area of the mouse (Reagan-Shaw S, Nihal M, Ahmad N. Dose translation from animal to human studies revisited.The FASEB Journal. 2007; 22: 659- 61.), referring to the doses of herbal extracts administered to animals in previous studies (Kim JJ, Seo BI, Choi HS, Kim SM, Woo CH, Koo JS, Park GY. Preventive effect of Daekumeumja on fatty degeneration of liver and immunosuppression induced by alcohol.The Journal of Korean Oriental Medicine.2010; 16 (2): 167-79 .; Ahn TK, Shin JW, Cho CK, Cho JH, Yoo HS, Lee YW, Lee NH, Yun DH, Son CG Effect of Galhwahyejung-tang (GHT) on protection for alcohol-induced liver injury.The Journal of Korean Oriental Medicine.2005; 26 (1): 76-84.), The mixed herbal medicine of Example <1-1> It was dissolved in physiological saline and administered orally once a day at 100 mg / kg for 28 days. After one hour of administration of the mixed herbal medicine, 5 g / kg of 30% alcohol (EtOH) was orally administered twice at 4 hour intervals (denoted as 'EtOH + mixed herbal medicine'), and the same dose of physiological saline was used as a control group. The same dose of alcohol alone was used as the alcohol alone administration group (denoted as 'EtOH'). In order to observe the weight change of the control group, the alcohol alone group (labeled 'EtOH') and the alcohol and the mixed herbal drug (labeled 'EtOH + mixed herbal medicine'), the body weights of the mice were measured at two-day intervals.

As a result, as shown in Table 1, the control group that did not add alcohol toxicity showed a tendency to increase steadily, whereas the alcohol alone group and the alcohol and mixed medicament group that administered alcohol were 2 days to 4 days after the start of administration. The body weight decreased until 6 days and began to recover, and on day 10 there was no difference in weight between the three groups. In particular, the group administered with alcohol and mixed medicinal herbs showed more weight loss than the alcohol alone group, but showed a rapid recovery rate (Table 1).

Weight Table of TCR Mouse date Control group EtOH EtOH  + Mixed Herbal Medicine 0 25.86 ± 0.22 25.78 ± 0.75 24.75 ± 0.26 2 25.26 ± 0.21 25.18 ± 0.69 22.95 ± 0.24 ** ## 4 26.51 ± 0.43 24.76 ± 1.28 20.73 ± 0.25 ** # 6 29.24 ± 0.48 27.88 ± 0.74 22.25 ± 0.30 *** ### 8 30.75 ± 0.38 30.08 ± 0.47 28.32 ± 0.32 ** # 10 29.15 ± 0.35 29.35 ± 0.39 29.50 ± 0.41 12 30.69 ± 0.27 30.67 ± 0.47 30.86 ± 0.49 14 29.30 ± 0.30 29.40 ± 0.32 30.24 ± 0.69 16 30.46 ± 0.41 30.02 ± 0.37 30.28 ± 0.45 18 31.58 ± 0.31 30.89 ± 0.46 31.18 ± 0.53 20 32.34 ± 0.38 30.60 ± 0.50 31.37 ± 0.47 22 32.33 ± 0.51 30.53 ± 0.42 31.42 ± 0.62 24 32.24 ± 0.51 30.71 ± 0.36 31.54 ± 0.58 26 31.28 ± 0.40 29.21 ± 0.42 29.95 ± 0.50 28 32.02 ± 0.47 29.90 ± 0.35 31.21 ± 0.70

The measured values are expressed as mean ± SEM of each group. *** p <0.001, ** p <0.01 compared to control. ### p <0.001, ## p <0.01, #p <0.05 compared with EtOH only group.

< Example  5> Object recognition experiment Novel object recognition test )through Mixed Herbal Medicine  Identify the effects on alcohol-induced memory impairment

The day after the last administration of Example <4-1>, the object recognition behavior experiment was performed. After adapting the experimental animals for 1 hour in the environment where the experiment is going to proceed, they were placed in an experimental acrylic box for 5 minutes in order to proceed with the training (training) prior to this experiment. After 5 minutes of adaptation, two similar objects were inserted to measure the time for which the mouse was searching for each object for 3 minutes. After 24 hours, experiment in the same way, but measure the time to find each object by inserting a new object into one of the two objects.Recognition index (%) = new object search time / (new object search time + other The object search time) x 100 is shown as a graph.

As a result, when the training was performed as shown in Figure 3, each group did not show a statistically significant difference of 64.29%, 55.54% and 48.96%, whereas the group administered only alcohol after 24 hours of training 37.91% of the subjects searched for a new object less than the control group (62.62%), and the alcohol and mixed herbal medicine group was 66.63%, which showed a statistically significant recovery effect (FIG. 3). .

< Example  6> Mixed Herbal Medicine  Determination of Alcohol-induced Hippocampal Neuronal Damage

<6-1> Brain Tissue Separation

After the administration was completed by the method of Example <4-1>, the rats of each group after the administration were killed and the hippocampal tissues of the brain were examined for the effect on the hippocampal cells by Western blot method. Were separated and stored at -20 ° C. For immunohistochemistry, mice were perfused with PBS (phosphate buffer saline) and 4% PFA to extract the brain and stored in 4% paraformaldehyde (PFA) for 1 day to prevent tissue damage from freezing. And refrigerated in 30% sucrose. Brain tissue was cryostated (cryostat) (CM3000, Leica, Wetzlar, Germany) after 30 μm lyophilization and stored in a storage solution (storing solution) at 4 ℃.

<6-2> Cresyl  Violet Dyeing Cresyl violet staining ) And Immunohistochemistry

Select the hippocampus of the brain tissue fixed for Cresyl violet staining, wash it three times with PBS, and mount it on a gelatin coated slide to cresyl violet (Sigma-Aldrich). (St. Louis, CA, USA) solution and reacted for 3 minutes. After washing with distilled water and dehydration and clearing process of 70% to 100% alcohol and xylene, the tissue was covered with a cover slide and stored. After washing the hippocampal tissue three times with PBS for the antibody reaction of NeuN, treatment with hydrogen peroxide to remove endogenous peroxidase, and the primary antibody mouse anti-NeuN (1: 1000 dilution) ( Chemicon (Temecula, CA, USA) was reacted overnight. Secondary antibody biotinylated anti-mouse (1: 200 dilution, 1 hour) (Vector company (Burlingame, CA, USA)), ABC (avidin-biotin peroxidase complex) (Vector company (Burlingame, CA, USA) The reaction was carried out for 3 minutes using diaminobenzidine (DAB) after 1 hour reaction. Three washes were performed with PBS between each procedure. Brain tissue was stored on a gelatin-coated slide and covered with a cover slide after dehydration and clarification of 70-100% alcohol and xylene. The effect of the mixed herbal medicine on the cells of the hippocampus was observed using a microscope (Olympus Microscope System BX51; Olympus, Tokyo, Japan).

As a result, as shown in FIG. 4, when the alcohol alone administration group was examined, the cell number was decreased and lightly colored in the CA3 portion of the hippocampal cells, indicating that cell death was induced by alcohol. In particular, cell death by alcohol While CA1 showed a tendency to occur more sensitively than CA1, it was confirmed that apoptosis was suppressed by administration of the mixed herbal medicine in the group administered with alcohol and the mixed herbal medicine (FIG. 4). As shown in FIG. 5, even in the immunostaining result of NeuN protein, in the alcohol-only group, NeuN-positive cells were less expressed by alcohol, resulting in nerve cell damage caused by alcohol toxicity, and alcohol and mixed medicinal herbs were administered. In confirmed that the mixed herbal medicines inhibited neuronal cell death by alcohol (Fig. 5).

<6-3> Western  Blot Western blotting )

After separating the mouse hippocampal tissue from the protein quantitative protein and loaded on a 12% SDS (sodium dodesyl sulfate) -acrylamide gel (electron amide gel) by using an electrophoresis device and transferred to the membrane (membrane) and then NeuN (1: 1000) (Chemicon (Temecula, Calif., USA)), β-actin (1: 10000) (Assay Designs Inc. (Ann Arbor, MI, USA)), anti-mouse / rabbit HRP-conjugated (secondary antibody) 1: 2000) (Assay Designs Inc. (Ann Arbor, MI, USA)). Membranes were washed four times for 15 minutes with TBST for each procedure. After the antibody reaction, the membrane was exposed through the LAS-4000 mini system (Fujifilm Corporation, Tokyo, Japan) using an ECL detection kit (Bio-Rad Laboratories (Hercules, Calif., USA)). Beta-actin was included as a control.

As a result, as shown in FIG. 6, when the alcohol alone was administered, the expression of NeuN protein was decreased in the hippocampal tissues to which alcohol was administered, and the decrease of NeuN protein expression was suppressed by the mixed herbal medicines to the alcohol and mixed herbs. It was confirmed (Fig. 6).

Hereinafter, the preparation example of the mixed herbal medicine of the present invention is as follows.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> Sanje  Produce

2 g of mixed herbal medicines of the present invention

1 g lactose

The above components were mixed and packed in airtight bags to prepare powders.

<1-2> Preparation of tablets

100 mg mixed herbal medicine of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

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

&Lt; 1-3 > Preparation of capsules

100 mg mixed herbal medicine of the present invention

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

<1-4> Preparation of Granules

150 mg mixed herbal medicine of the present invention

Soybean Extract 50mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added and the mixture was dried at 60 캜 to form granules, which were then filled in a capsule.

< Manufacturing example  2> Manufacturing of food

Food containing the mixed herbal medicine of the present invention was prepared as follows.

<2-1> Production of flour food

0.5 to 5.0 parts by weight of the mixed herbal medicine of the present invention was added to flour, and bread, cake, cookies, crackers and noodles were prepared using the mixture.

<2-2> soup  And juicy ( gravies Manufacturing

0.1 to 5.0 parts by weight of the mixed herbal medicine of the present invention was added to soups and broths to prepare meat products for health promotion, soups of noodles and broths.

<2-3> Ground Beef  Produce

10 parts by weight of the mixed herbal medicine of the present invention was added to the ground beef to prepare a ground beef for health promotion.

<2-4> Dairy products ( dairy products Manufacturing

5 to 10 parts by weight of the mixed herbal medicine of the present invention was added to milk, and various milk products such as butter and ice cream were prepared using the milk.

<2-5> Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and the mixture was granulated to a powder having a particle size of 60 mesh.

Black soybeans, black sesame seeds, and perilla seeds were steamed and dried by a conventional method, and then they were prepared into powder having a particle size of 60 mesh by a pulverizer.

The mixed herbal medicine of the present invention was concentrated under reduced pressure in a vacuum concentrator, and the dried product obtained by drying with a spray and a hot air dryer was pulverized with a particle size of 60 mesh to obtain a dry powder.

The grains, seeds and the mixed herbal medicines of the present invention prepared above were formulated in the following ratios:

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley)

Seeds (7 parts by weight of perilla, 8 parts by weight of black beans, 7 parts by weight of black sesame seeds)

Mixed herbal medicine of the present invention (3 parts by weight),

(0.5 parts by weight), and

Rhubarb (0.5 parts by weight).

< Manufacturing example  3> Manufacturing of beverage

<3-1> Health drink  Produce

Instant sterilization by homogeneously mixing 5 g of the mixed herbal medicine of the present invention with subsidiary materials such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5%) and water (75%). Then it was prepared by packaging in a small packaging container such as glass bottles, plastic bottles.

<3-2> Preparation of vegetable juice

5 g of the mixed herbal medicine of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<3-3> Production of fruit juice

1 g of the mixed herbal medicine of the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice.

As seen above, the mixed herbal medicine of the present invention has excellent neuronal cell damage protection effect and no cytotoxicity by alcohol, and various formulations including circumcision to treat brain disease, herbal medicine for nerve damage protection by alcohol or It can be useful for the development of dietary supplements.

Claims (13)

Cortex Acer tegmentosum Max ), Alder japonica Cortex ), Taraxaci Herba ), Cirsii Japonici Herba ), Xanthii Fructus ), brown hair ( Puerariae Flos ), Earth ( Hovenia Dulcis A pharmaceutical composition for preventing and treating brain diseases containing Thunb ) and Puerariae Radix as an active ingredient.
The method of claim 1, wherein Atractylodis Rhizoma Alba ), malt ( Hordei Fructus Germinatus), gampi (Persimmon Peels ), Hoelen , Mume Fructus ), Artemisiae Capillaris Herba ), Ponciri Fructus), elecampane Party (Aucklandiae Radix), sign (Amomi Fructus ), Phragmitis Rhizoma ) and Raphani Semen ( Raphani Semen ) A pharmaceutical composition for the prevention and treatment of brain diseases, further comprising any one or more selected from the group consisting of.
According to claim 1, Alismataceae (Alismatis Rhizoma ), the dermis ( Citri) Unshii Pericarpium ), Magnoliae Cortex ), Agastachis Herba), Gardenia (Gardeniae Fructus), Firmament (Cnidii Rhizoma ), safflower ( Carthami Flos ), Citrii Unshiu Immaturi Pericarpium ) and Licorice ( Glycyrrhizae Radix meat Rhizoma ) pharmaceutical composition for preventing and treating brain diseases, further comprising any one or more selected from the group consisting of.
The brain disease according to any one of claims 1 to 3, wherein the brain disease is Stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis), alcoholic cerebral neuropathy, alcoholic dementia, and Wernicke-Korsakoff's syndrome (Wernicke-Korsakoff's syndrome) is a pharmaceutical composition for the prevention and treatment of brain diseases, characterized in that any one selected from the group consisting of.
The pharmaceutical composition according to any one of claims 1 to 3, wherein the composition protects nerve cells from oxidative stress or damage caused by alcohol.
Cortex Acer tegmentosum Max ), Alder japonica Cortex ), Taraxaci Herba ), Cirsii Japonici Herba ), Xanthii Fructus ), brown hair ( Puerariae Flos ), Earth ( Hovenia Dulcis Health food for preventing and improving brain diseases, containing Thunb ) and Puerariae Radix as active ingredients.
According to claim 6, Atractylodis Rhizoma Alba , Malt ( Hordei Fructus Germinatus), Persimmon Peels ), Hoelen , Mume Fructus ), Artemisiae Capillaris Herba ), Ponciri Fructus), elecampane Party (Aucklandiae Radix), sign (Amomi Fructus ), Phragmitis Rhizoma ) and Raphani Semen ( Raphani Semen ) A health food for the prevention and improvement of brain diseases, characterized in that it further comprises any one or more selected from the group consisting of.
According to claim 6, Alismatis Rhizoma ), the dermis ( Citri) Unshii Pericarpium ), Magnoliae Cortex ), Agastachis Herba), Gardenia (Gardeniae Fructus), Firmament (Cnidii Rhizoma ), safflower ( Carthami Flos ), Citrii Unshiu Immaturi Pericarpium ) and Licorice ( Glycyrrhizae Radix meat Rhizoma ) health foods for the prevention and improvement of brain diseases, further comprising any one or more selected from the group consisting of.
The method according to any one of claims 6 to 8, wherein the brain disease is Stroke, Parkinson's disease, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis), alcoholic cerebral neurological disease, alcoholic dementia and Wernicke-Korsakoff's syndrome (Heernicke-Korsakoff's syndrome) is a health food for preventing and improving brain diseases, characterized in that any one selected from the group.
1) 20 ~ 30 parts by weight of mountain cheongmok, 20 ~ 30 parts by weight of duck wood, 20 ~ 30 parts by weight of tack, 20 ~ 30 parts by weight of leach, 20 ~ 30 parts by weight of malt, 20 ~ 30 parts by weight of dermis, 10 ~ 15 parts by weight of dermis , 10-15 parts by weight, 10-15 parts by weight, 10-15 parts by weight of plum, 5-10 parts by weight of phosphorus, 5-10 parts by weight of gardenia, 5-10 parts by weight, 5-10 parts by weight of nabokjag, 5 to 10 parts by weight of fruit room, 5 to 10 parts by weight of celestial organs, 5 to 10 parts by weight of safflower, 5 to 10 parts by weight of cheongpi, 5 to 10 parts by weight of gingko, 5 to 10 parts by weight, licorice 5 to 10 parts by weight, Pogongyoung 5 to 10 parts by weight, total 5 to 10 parts by weight, Changjaja 5 to 10 parts by weight, earthwood 5 to 10 parts by weight, 5 to 10 parts by weight, 5 to 10 parts by weight and 5 to 10 parts by weight Pulverizing and mixing;
2) kneading the mixed mixture of step 1) with 500 to 800 parts by weight of honey;
3) formulating the dough of step 2) in the form of a ring of 1 to 2 cm in diameter; And
4) A method of producing a pill formulation having a neuronal cell protective activity, comprising the step of drying the ring of step 3).
The method according to claim 10, step 1) is 20 to 24 parts by weight of acid wood, 20 to 24 parts by weight of alder, 20 to 24 parts by weight, 20 to 24 parts by weight, 20 to 24 parts by weight, 20 to 24 parts by weight of malt, 20 to 24 parts by weight 10 to 12 parts by weight, 10 to 12 parts by weight of Baekbokyeong, 10 to 12 parts by weight thick, 10 to 12 parts by weight of plum, 5 to 6 parts by weight, 5 to 6 parts by weight of gardenia, 5 to 6 parts by weight , 5 to 6 parts by weight of the moths, 5 to 6 parts by weight, 5 to 6 parts by weight of celestial organs, 5 to 6 parts by weight of safflower, 5 to 6 parts by weight of cheongpi, 5 to 6 parts by weight of Dangmok, 5 to 6 parts by weight , Licorice 5-6 parts by weight, poongyoung 5-6 parts by weight, total 5-6 parts by weight, Changzai 5-6 parts by weight, earth tree 5-6 parts by weight, brown root 5-6 parts by weight, browning 5-6 parts by weight And 5 to 6 parts by weight of the roots of the preparation method of a pill formulation having a neuronal cell protective activity, characterized in that after mixing.
12. The method of claim 11, wherein the honey of step 2) is 600 to 700 parts by weight of the preparation of a pill formulation having a neuroprotective activity, characterized in that the neurons.
A pharmaceutical formulation having a cranial nerve cell protective activity, prepared by the method of claim 10.

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