KR20040042240A - Phamaceutical use for neuroprotective effect comprising as main ingredients on the Cultured Spinal Dorsal Root Ganglion Neurons Damaged by Oxygen Free Radicals from Ramulus et Uncus Uncariae pharmaceutical preparations containing them - Google Patents

Abstract

PURPOSE: Provided is a pharmaceutical composition containing Uncariae Ramulus et Uncus as main ingredient for protection of cultured spinal dorsal root ganglion neurons damaged by oxygen free radicals. It has excellent therapeutic effect on brain diseases, such as multi-infarct dementia, Alzheimer's disease Parkinson's disease, hypothyroidism, paralysis, and the like. CONSTITUTION: A pharmaceutical composition for protection of cultured spinal dorsal root ganglion neurons damaged by oxygen free radicals is characterized by containing Uncariae Ramulus et Uncus powder or its extract as a main ingredient, and at least one selected from Aurantii nobilis Pericarpium, Hoelen, Lingusticum acutilobum, Acorus gramineus, Atractylodes Rhizoma, Angelica dahurica, Zizyphi spinosi semen, Rehmanniae radix preparata, Cornus officinalis, and Polygala tenuifolia, or the extract thereof as a side ingredient.

Description

Pharmaceutical use for neuroprotective effect comprising as main ingredients on the Cultured Spinal Dorsal Root Ganglion Neurons Damaged by Oxygen Free Radicals from Ramulus et Uncus Uncariae pharmaceutical preparations containing them}

Since allergic rhinitis, atopic dermatitis, chronic asthma and immune hypersensitivity symptomatic treatments have achieved many therapeutic results based on natural medicines based on the East and West, the present inventors have allergic rhinitis, atopic dermatitis Drugs have been developed for their excellent pharmaceutical efficacy in treating chronic asthma and immune hypersensitivity symptoms. The present invention is a stroke, cerebral infarction, hypertension, Alzheimer's disease, vascular disease (Multi-infarct dementia), Alzheimer's disease and multi-infarct dementia, Parkinson's disease, hypothyroidism, which contains Uncariae Ramulus et Uncus as a main component Hypothyrodism, alcoholic dementia The present invention relates to a pharmaceutical preparation having a neuroprotective effect, which is contained as a pharmaceutical composition for the treatment of Alzheimer's disease. Is a nervous system constituting the central nervous system with 같이 and is composed of the gods who take charge of all the movements and knowledge of the human body like 腦. Therefore, the evangelism of the human body is made by the deity from the God and the 知覺 神經 細胞. The perceptual transmission from the peripheral to the center is through the ascending road and the movement from the center to the peripheral is through the descending road. It is delivered. Since the dual perceptual conduction is connected to the olfactory nerve cells of the spinal cord through the 後 根 神 때문에 of the 脊髓, the damage of the 脊髓 後 根 神經 節 細胞 is not only a loss of sensation, but also a direct cause of 疼痛 疼痛 and movement of the quadrant. It becomes a factor. In terms of literature, it is included in the categories of 痺證, 證, 麻木 不 仁.

The term “不” is a concept of “transportation” and “阻滯”, which means that the movement of the law is reversed, and it represents the movements and feelings such as the weight of the body or the bones, as well as the weight of the body. According to the characteristics of 病邪, is classified into breeze, 寒, 濕, 熱 痺, etc., and depending on the 發病 部位 and 發病 樣 相, respectively. If you divide the groups of 位, 肌肉, 筋骨, and by division, the 나타나는, 肌肉 部 痺證 痺證 주로 mainly 주로 覺 障碍 ,, 筋骨 部 筋骨 位, 骨 重, 주로 There are many books of movement, and each of them has a number of books that correspond to the book. Indeed, it is an incompetent way of doing business, and it is an incompetent way of doing business because of the incompetence of the use of the body and the inferiority. However, all these activities involve activities and activities. In Chinese science, 麻 不知 痛痒 and 尙 覺 氣 微 流行, 木 is 氣 亦 不覺 流行, 或 風邪 走 注 皮膚 中 如 蟲 行, which means that 麻木 is the cause of feeling of whole body. I mean. Therefore, 麻木 不 仁 is a cause of unknown feelings due to the knowledge of 肌 部 knowledge.

Mul 藤 (Ramulus et Uncus Uncariae) is a 乾燥 藤, 鉤藤, 釣鉤, 弔 藤 which is a dried or stalked stem containing thorns of the 木質 藤 本, or 華 鉤藤, which belongs to Rubiaceae.釣, 嫩 釣 藤, 雙 釣 藤, 鉤 屯 has the same name. In addition, the sexual characteristics are inferior to that of 毒 微寒, 效,, 肝, 熄風, 鎭 驚 眩 兒 驚, 頭昏 目眩, 中風, 筋脈 拘攣 has been used. In fact, it is reported that 釣鉤 藤 is effective for the treatment of 高血壓 血壓, 目眩 and 神經 性 頭痛, as well as paralysis and stroke due to spinal cord and peripheral nerve damage. In addition, the old 은 藤 오래 藤 for a long time (久 煎) is weak and about 15 minutes to record the most effective, and the actual Kimi 김 藤 of 血壓 降下 作用 and experimental analysis of the hourly efficacy Corresponding results were also found in spinal cord lesions due to damage to the perceptual and motor neurons that make up the spinal cord due to factors such as trauma and immunodeficiency. In this case, the nerve fiber damage can be recovered, but once the nerve cell body is damaged, it cannot be regenerated, which causes loss of perception and loss of motor function, thereby giving up noble life as a human being. So far, known pathogens that inhibit nerve cells include deficiency of 酸 素 自由基, excitotoxic amino acids (EAAs) and neurotrophic factor (NTF). In particular, 酸 素 自由基 not only promotes lipid peroxidation in the cell membrane but also interacts with nitricoxide (NO), which is one of nitrogen free groups, to produce peroxynitrite, a highly toxic substance, which accelerates the lesions. Among these, autologous substrates such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in various physiological reactions in vivo, but when they are formed more than necessary, they can peroxidize unsaturated fatty acids in the cell membrane, resulting in lipid peroxidation. In addition to accelerating the protein, it also promotes the degeneration of proteins, such as protein kainase C (PKC), and inhibition of protein and DNA synthesis. In particular, 酸 素 自由基 affects the antioxidant system and decreases the function of antioxidant enzymes such as superoxide dismutase (SOD) and catalase, causing oxidative damage to various cells and tissues by causing unnecessary accumulation of 酸 素 自由基 in the human body. It is a well known fact. In recent years, it has been reported that 酸 素 自由基 promotes the excretion of excitatory amino acid (EAA) in cultured hippocampal neurons, revealing the phenomenon of interaction between 酸 素 自由基 and excitatory amino acid. In addition, the secreted 증가 自由基 increases the concentration of free Ca2 + in the cell and eventually leads to cell death, and acts with NO to form a toxin called peroxynitrite, which causes damage to cells. Recent evidence suggests that atrophic lateral sclerosis is a pathogenesis of neuronal cell damage in the central and peripheral nervous system, as excess SUM-1 accumulates in the patient's brain due to mutations in the SOD-1 gene. However, not only detailed mechanisms have yet to be elucidated on the toxic effects of 酸 素 自由基, and there are also very few effective treatments for various neurological lesions caused by oxidative damage of 酸 素 自由基. NMDA (N-methyl-D-aspartate) antagonists CGS 19755, MK-801, Dextrophan, Remacemide, Eliprodil, Magnesium sulfate, CNS 1102, GV 150526 have been developed as currently reported neuroprotective agents, and Nimodipine, Lifarizine , But calcium channel blockers such as SNX111 and Isradipine have been tried, but the effects have not been consistent, and protein kinase C inhibitors containing GM1 ganglioside, recently Na + channel blockers (Lubeluzole, Riluzole, Fosphenytoin) and nitric oxide (NO) synthesis Enzyme (NOS) inhibitors and various neurotrophic factors (Trofermin) have also been shown to be effective in neuroprotection, and research is being conducted to develop drugs applying them. Some types of drugs are currently at the stage of clinical trials as neuroprotective agents for stroke, and the complexities of the mechanisms related to brain damage caused by this disease, drug penetration into brain tissue, side effects, difficulty in clinical trials, etc. Due to this situation, there are many difficulties in developing a therapeutic agent.

In the neurotoxic cascade of ischemic brain injury, the extracellular cytotoxic calcium ions generate reactive oxygen species (ROS, RNS) through activation of NOS and generation of excess NO. In addition, the increased calcium ions in mitochondria further decrease energy supply and increase the production of free radicals by uncoupling oxidative phosphorylation. These free radicals damage cell membranes by lipid peroxidation in addition to DNA damage. Fortunately, natural extracts, including herbal extracts, have pharmacological activities such as antioxidant effects and cell growth factors. Results have been reported to be very effective in the treatment of diseases caused by oxidative damage. This study was conducted to investigate the effects of 釣鉤 鎭 痛 抗 痙攣 鎭 靜 비롯하여 비롯하여 생 생 생 생 생 생 생 생 생 생 생 생 생 생 생 비롯하여 비롯하여 on. The effects of 釣鉤 藤 on neuroprotective effects on the nets were measured.

Major constituents of 釣鉤 藤 were isolated rhynchophylline and several oxyindole alkaloids such as isorhynchophylline, dihydrocorynantheine, hirsutine, firsutine, 3-α-dihydrocadambine, corynoxeine, and isocorynoxeine. Studies on the composition of phytonutrients, Seoul National University, Seoul, Korea, 1995), as a physiological activity, many reports have been reported on the effect of alkaloids on blood pressure lowering or blood pressure expansion. In addition, Kim et al. Have reported on the phospholipase Cγ1 inhibitors and their anti-cancer effects of 釣鉤 藤 in the nervous system, 鎭 靜, 抗 痙攣 用 用 (, 用). And its anticancer effect, Seoul National University Graduate School Thesis 1998), and the research on the effects of the sympathetic god have not been performed yet.

As such, the neurological injury caused by reactive oxygen species has not been clearly identified, but in order to have an effect as a neuroprotective drug, the herbal medicines are first searched for the antioxidant function while having no toxicity and no brain toxicity. Results The present inventors have developed a drug useful for neuroprotection with excellent neuroprotective function and sulfated function by reactive oxygen species from a medicament derived from herbal medicines.

The present invention is a medicinal agent from a herbal medicine, vascular disease (multi-infarct dementia), a combination of Alzheimer's disease and multi-infarct dementia, Parkinson's disease, hypothyrodism, alcoholic dementia Alzheimer's, stroke, cerebral infarction It is to provide a composition having excellent pharmaceutical efficacy in treating the same brain disease symptoms.

Figure 1 shows the metabolic processes that cause neurological injury by H2O2.

Figure 2 shows a method for recovering the dry weight of the pharmaceutical composition according to the extraction process of the effective drug.

Figure 3. Cell survival rate of the dorsal muscle nerve cells by the MTT method is investigated.

Figure 4. The cell survival rate of the spinal muscle nerve cells by NR staining is investigated.

Figure 5 shows the quantitative increase by neurofilament EIA.

Figure 6 shows the activity of protein synthesis by SRB-bound protein assay.

Figure 7 shows the neuroprotective effect of REUU by LDH enzyme activity.

Figure 8 shows the protective effect of lipid peroxidation.

Figure 9 shows the protective effect of spinal muscle neurons by quantitative changes in protein

will be.

will be.

Each herbal ingredient may be powdered, or each may be extracted alone to obtain an extract, and these extracts may be mixed, or the necessary herbal ingredients may be combined and extracted together. Next, the present invention will be described in more detail with reference to Examples and Experimental Examples, which do not limit the scope of the present invention.

Example 1

1. Add and subtract such herbal medicine as secondary raw material to make the total dry weight 1kg, mix it with 3 ~ 10 times volume with 3rd distilled water (or purified water), and heat slowly for 12 hours under 85 ℃. The volume was made up to 500 ml, and then concentrated under reduced pressure to 200 ml and then frozen to obtain a freeze-dried weight of 80g (hereinafter referred to as REUU) (Fig. 2).

2. A composition that is extracted and separated from the water extract. In the present invention, 1 kg of dry weight in each extraction method was mixed with 5,000 ml of distilled water in a water bath at 85 ° C. for 12 hours, then filtered with gauze, and extracted from the filtrate under reflux for 6 hours in a boiling water bath. Extracted once more. It was concentrated under reduced pressure again and freeze dried to obtain a dry weight of about 80 g. It is another object of the present invention to provide a process for separating and purifying water extracts X.

As described later, water extracts obtained in the above-described series of drying processes include stroke, hypertension, cranial nerve disease, Alzheimer's disease, vascular disease (multi-infarct dementia), a mixture of Alzheimer's disease and multi-infarct dementia, and Parkinson. Water extract can be used as an active ingredient in the composition of the present invention because it has Alzheimer's therapeutic effect on diseases, hypothyrodism, alcoholic dementia.

Example 2

50 to 500 parts by weight of Uncariae Ramulus et Uncus is extracted with water in the same manner as in Example 1 to obtain X.

Example 3

50 to 500 parts by weight of Uncariae Ramulus et Uncus was extracted with 70% EtOH in the same manner as in Example 1 to obtain EtOH extract.

Example 4

50 to 500 parts by weight of Uncariae Ramulus et Uncus is extracted with water as in Example 2 to obtain a powder.

Example 5

Extract as 70% ethanol and remove ethanol to obtain powder as in Example 2 and Example 4.

Experimental Example 1

Herbal medicine is added or subtracted into the secondary raw material, and the total dry weight is 1 kg, and the mixture is mixed with tertiary distilled water (or purified water) at a volume of 2 to 10 times, and slowly heated for 12 hours under 85 ° C. It was made to ml, then concentrated under reduced pressure to 200 ml and then frozen to obtain a freeze-dried weight of 80g (hereinafter referred to as REUU) (Fig. 2).

Experimental Example 2

Separation of 脊髓 後 根 神經 節 細胞 was carried out according to the method of Kim et al. (1988). In other words, the neural tissues extracted from 3 days old mice were treated with phosphate buffered saline (PBS) containing 0.25% trypsin, and then washed in a thermostat controlled at 36 ° C and 5% CO2 / 95% air. After washing three times with Eagle's minimum essential medium (EMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco), cells were separated by a Pasteur pipette. The isolated cells were seeded at a density of 3 × 10 6 cells / well in 96-multiwells pretreated with poly-L-lysine (Sigma). Dispensed cells were exchanged into new cells every 3 days and used for 5 days and then cultured for a certain period of time at room temperature to investigate the effect of autologous base on the mice's 經 節 細胞 根 神經 節 細胞. After washing three times with MEM containing 0.6% -D glucose, diluting 1-100 mM hydrogen peroxide (H2O2) to the culture solution and diluting it to various concentrations. After treatment for 1 to 24 hours, the extracts were analyzed for the determination of MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (Sigma) for the determination of 酸 素 自由基 or herbal extracts. After washing three times with PBS, 50mg / ml of MTT prepared the previous day was diluted to the highest degree per well and washed in a thermostat controlled at 37 ℃ and 5% CO₂. ) Spectrophotomete The absorbance was compared with the standard spectrophotometry at 590 nm with M. The MTT assay measures the survival rate of cells in proportion to the absorption of light at 590 nm. In order to investigate the effect of hydrogen peroxide (H2O2) on 經 節 細胞 根 神經 節 細胞, the concentrations of H2O2 from 10μM to 80μM in each concentration were 5 After a period of time, the toxic effects of H2O2 were investigated by MTT assay, and the cell survival rate was 10.84% compared to the control (100%) in 10μM H2O2 treatment, but 75.4% in 20μM treatment. The cell viability of 80μM H2O2 was 50.9% (p <0.05) and 42.1% (p <0.01), respectively. In order to investigate the effect of H2O2 on 培養 根 神經 節 細胞 according to the time of REUU treatment, the cells of the cells containing 40μM H2O2 were quenched for 1 to 7 hours, respectively. Survival was compared with the control group by the MTT assay. As a result, the cell survival rate was 64.7% compared to the control group (100%) at 1 hour. In 3 hours, it was 60.0%, which was somewhat lower than the control group. In 5 hours, it was 47.1% (P <0.05), and in 7 hours, it was 33.3% (p <0.01) (Table 1, 2, FIG. 3).

Table I. Absorbance (% of control) at 590 nm wavelength for the MTT assay on hydrogen peroxide (H2O2) in cultured mouse spinal DRG neurons

H2O2 (μM) MTT absorbance (590nm) Decrease of cell viability (%) 0 0.57 ± 0.08 - 10 0.48 ± 0.04 15.8 20 0.43 ± 0.05 24.6 40 0.29 ± 0.02 * 49.1 80 0.24 ± 0.03 ** 57.9

Cultured mouse spinal DRG neurons were treated with various concentrations of H2O2 for 5 hours. The values are the mean ± SE for 6 experiments. Significant differences from the control are marked with asterisks. * p <0.05; ** p <0.01

Table 2.Time-response relationship of hydrogen peroxide (H2O2) by MTT assay in cultured mouse spinal DRG neurons

H2O2 (μM) MTT absorbance (590nm) 0hr 1hr 3hr 5hr 7hr 0 0.36 ± 0.06 0.34 ± 0.05 0.35 ± 0.03 0.34 ± 0.02 0.33 ± 0.04 40 0.35 ± 0.04 0.22 ± 0.03 0.21 ± 0.01 0.16 ± 0.02 * 0.11 ± 0.01 **

Cultured DRG neurons were treated with 40μM H2O2 for various time intervals. The values are the mean ± SE for 6 experiments. Significant differences between groups are marked with asterisks. * p <0.05; ** p <0.01

Experimental Example 3

The amount of neutral red (NR, Sigma) was followed by the method of Mosmann (1983). In other words, after washing three times with various concentrations of H2O2 with phosphate buffered saline (PBS), 5mg / ml of NR prepared the previous day was diluted to the final density per well, and then 37 ℃, 5% CO₂ for 3 hours. 정 at a thermostat controlled by. After the completion of the treatment, the plate was washed three times with PBS, fixed with 1% formalin, filtered with 1% glacial acetic acid, and absorbed at 540 nm using a spectrophotometer. NR assay is a method of measuring the survival rate of cells by light absorption at 540nm. After 시간 根 神經 節 細胞 for a certain period of time, washed three times with Ca2 +, Mg2 + -free Hank's balanced salt solution (HBSS, Gibco), and H2O2 at concentrations ranging from 1 μM to 35 μM, respectively. After survival, the cell viability was 74.4% compared to the control group (100%) and 65.1% and 48.8% (p <0.05) at 15μM and 25μM, respectively. In addition, the survival rate was 37.2% (p <0.01) in 35 μM H2O2. REUU was tested for 1-7 hours at 25μM H2O2 concentration, which is NR50 value, to investigate the effect of H2O2 on 脊髓 感覺 神經 節 細胞 over time. 62.5%, 59.3% in 3 hours culture and 52.7 (p <0.05)% and 41.5% (p <0.01) in 5 hours and 7 hours, respectively (Table 3, 4, 4).

Table 3. Absorbance (% of control) at 540 nm wavelength for the NR assay on hydrogen peroxide (H2O2) in cultured mouse DRG neurons

H2O2 (μM) NR absorbance (540nm) Decrease of cell viability (%) 0 0.43 ± 0.06 - One 0.32 ± 0.03 25.6 15 0.28 ± 0.05 34.9 25 0.21 ± 0.04 * 51.2 35 0.16 ± 0.02 ** 62.8

Cultured mouse spinal DRG neurons were grown in media containing various concentrations of hydrogen peroxide (H2O2) for 5 hours. The values represent the mean ± SE for 6 experiments. Significant differences from the control are marked with asterisks. * p <0.05; ** p <0.01

Table 4.Time-response relationship of hydrogen peroxide (H2O2) by NR assay in cultured mouse spinal DRG neurons

H2O2 (μM) NR absorbance (540nm) 0hr 1hr 3hr 5hr 7hr 0 0.57 ± 0.08 0.56 ± 0.07 0.54 ± 0.05 0.55 ± 0.06 0.53 ± 0.05 25 0.56 ± 0.07 0.35 ± 0.06 0.32 ± 0.03 0.29 ± 0.04 * 0.22 ± 0.01 **

Cultured mouse spinal DRG neurons were treated with 25μM H2O2 for various time intervals. The values are the mean ± SE for 6 experiments. Significant differences between groups are marked with asterisks. * p <0.05; ** p <0.01

Experimental Example 4

For a certain period of time, the deity was washed three times with PBS and fixed with alcohol, followed by three times with PBS containing 0.2% Triton X-100. After washing, react with NE14 (1: 100, Sigma) for 1 hour, and then use 0.04% O-phenylenediamine (OPD, Sigma) and 0.02% hydrogen peroxide, and use the ELISA reader to determine the absorbance at 490nm. It was 調査. EIA is an enzyme immunoassay that measures neuronal cell death by the amount of light absorbed at 490 nm. Neurofilament EIA was performed for the quantitative measurement of neurofilament by the change of H2O2 concentration in 經 細胞 神經 細胞. Neurofilament was treated for 5 hours after treatment with 脊髓 後 根 神經 節 細胞 for 5 hours in H2O2 concentrations of 1 ~ 70μM. The quantitative change of was compared with the control. In 1μM H2O2 treatment, the quantitative change in neurofilament was 68.5% compared to the control group (100%) and 62.0% in 15μM H2O2 treatment. In addition, neurofilament amounts of 46.7% (p <0.05) and 23.9% (p <0.01) were significantly decreased after treatment with 35μM and 70μM H2O2. In order to investigate the effect of REUU on H2O2-damaged 經 節 細胞 根 神經 節 細胞 in terms of quantitative change in neurofilament, 2 根 神經 節 細胞 for 5 hours at 35μM H2O2 concentration, the MCV value of H2O2, was investigated. Two hours before the exposure, the pretreatment was performed in the 1 ~ 60 ㎍ / ml REUU containing each, and the protective effect was investigated by the neurofilament EIA method. When 35μM H2O2 was treated only, the quantitative change in neurofilament was 42.1% compared to the control (100%). However, pretreatment with 1µg / ml REUU showed 53.8%. In addition, the treatment of 15 ㎍ / ml and 30 ㎍ / ml increased to 69.3% and 74.7%, respectively. However, it was 86.6% (p <0.05) in the case of 60 µg / ml, which showed a significant increase compared to the treatment with only H 2 O 2 (Tables 5, 6 and 5).

Table 5.Dose-response relationship of hydrogem peroxide (H2O2) byneurofilament enzymeimmuno assay (EIA) in cultured mouse spinal DRG neurons

H2O2 (μM) EI absorbance (490nm) Decrease of neurofilament (%) 0 0.92 ± 0.07 - One 0.63 ± 0.04 31.5 15 0.57 ± 0.06 38.0 35 0.43 ± 0.05 * 53.3 70 0.22 ± 0.03 ** 76.1

Cultured mouse spinal DRG neurons were exposed to various concentrations of hydrogen peroxide (H2O2) for 5 hours. Amount of neurofilament was measured by enzymeimmuno assay (EIA). The values are the mean ± SE for 6 experiments. Significant differences from the control are marked with asterisks.

* p <0.05; ** p <0.01

Table 6.Dose-response relationship of REUU for its neuroprotective effect on hydrogen peroxide (H2O2) by neuofilament EIA assay in cultured mouse DRG neurons

H2O2 (μM) EI absorbance (490nm) concentration of REUU (㎍ / ml) 0 One 15 30 60 0 0.76 ± 0.05 0.78 ± 0.08 0.75 ± 0.06 0.79 ± 0.05 0.82 ± 0.09 35 0.32 ± 0.06 0.42 ± 0.05 0.52 ± 0.04 0.59 ± 0.03 0.71 ± 0.06 *

Cultured mouse spinal DRG neurons were preincubated with various concentrations of Ramulus et Uncus Uncariae (REUU) for 2 hours before treatment with 35μM H2O2 for 5 hours. The values are the mean ± SE for 6 experiments. Significant differences between groups are marked with asterisks. * p <0.05

Experimental Example 5

For the separation of Sulforhodamine B (SRB), 200 μl of 0.4% sulforhodamine B was added to 經 節 細胞 根 神經 節 細胞, which was used for several hours as an extract of herbal or herbal medicines, and left at room temperature for 1 hour, followed by 1.0% acetic acid. Washed three times. After washing, the SRB-bound protein was dissolved using 10mM Tris base, and the absorbance was determined at 540nm by ELISA reader. Sulforhodamine B (SRB) assay measures the protein activity of cells by light absorption at 540 nm. In order to investigate the effect of H2O2 on the total protein level, the changes of protein synthesis by H2O2 after 5 hours of immersion in H2O2 containing from 10μM to 100μM, respectively As a result, the protein synthesis was 75.6% in 10μM H2O2 treatment and 68.2% in 25μM H2O2 treatment. In the case of 50μM and 100μM H2O2 treatment, protein synthesis was 47.2% (p <0.01) and 32.3% (p <0.01)%, respectively. To investigate the effect of REUU on H2O2-induced cytotoxicity on the surface of quantitative changes in total protein, 10 to 80 two hours before exposure for 5 hours at 50 μM H2O2 concentration, a midcytotoxicity value of H2O2 After the pretreatment in each containing μg / ml REUU was investigated the protective effect. As a result, when only H2O2 was treated, the quantitative change in total protein was 42.6% compared to the control (100%). On the other hand, the treatment of 10µg / ml REUU was 63.8% compared to the control, and the treatment of 20µg / ml and 40µg / ml was 71.5% and 76.7%, respectively. In addition, the treatment of 80 ㎍ / ml REUU was 81.2% (p <0.05) significantly increased compared to the control (Table 7, 8, Figure 6).

Table 7.Dose-response relationship of hydrogen peroxide (H2O2) on lipid peroxidation in cultured mouse spinal DRG neurons

H2O2 (μM) TBARS (pmol / 106 cells) Decrease rate of cell viability (%) 0 36.8 ± 4.2 - One 41.7 ± 4.8 113.3 10 46.4 ± 5.3 126.1 30 59.7 ± 6.5 ** 162.2 50 75.9 ± 8.7 ** 206.3

Cultured mouse spinal DRG neurons were exposed to various concentrations of hydrogen peroxide (H2O2) for 5 hours. Thiobarbituric acid (TBA) fluorometric assay was adopted to analyse lipid peroxidation and TBA reactive substance (TBARS) were represent as pmol / 106 cells. The values are the mean ± SE for 6 experiments. Significant differences from the control are marked with asterisks. ** p <0.01

Table 8.Dose-response relationship of REUU for its neuroprotective effect on hydrogen peroxide (H2O2) in TBARS assay in cultured mouse DRG neurons

H2O2 (μM) TBARS (pmol / 106 cells) concentration of REUU (µg / ml) 0 15 30 60 120 0 32.7 ± 5.3 32.4 ± 5.1 32.8 ± 4.2 32.1 ± 3.5 31.6 ± 4.6 30 22.7 ± 3.8 20.2 ± 2.6 17.4 ± 1.6 * 13.4 ± 2.3 ** 9.5 ± 0.7 **

Cultured mouse spinal DRG neurons were preincubated with various concentrations of Ramulus et Uncus Uncariae (REUU) for 2 hours before treatment with 30μM H2O2 for 5 hours. The values are the mean ± SE for 6 experiments. Significant differences between groups are marked with asterisks. * p <0.05; ** p <0.01

Experimental Example 6

Determination of LDH activity against the extracts of 酸 素 自由基 and medicinal herbs was carried out by the modified method of Takahashi et al. (1987). That is, 1.0 ml of the enzyme substrate solution of the LDH kit (Atron lab, Japan) was put in a tube having a diameter of 10 cm, and then the sample 培養 液 was mixed well, followed by reaction at 37 degrees for 10 minutes. After 10 minutes, 3.0 ml of the dilution stop solution was added and mixed, and then the absorbance was determined at 570 nm, which was compared with the photoluminescence. LDH activity measurement is an assay that measures the damage of the cell membrane due to the absorption of light at 570nm. In order to measure LDH activity in H2O2 培養 根 神經 節 細胞, treated with H2O2 at concentrations ranging from 1 to 40 μM for 5 hours, the sorbent was discharged into the cage. The amount of LDH was compared with the control group. As a result, 16.4M H2O2 treatment showed 116.4% (21.3 ± 2.4) compared to the control 100% (18.3 ± 1.6). In addition, the treatment of 10μM and 20μM H2O2 was 124.6% (22.8 ± 2.1) and 156.3% (28.6 ± 3.4) (p <0.05), respectively. On the other hand, when treated with 40μM H2O2 was 183.1% (33.5 ± 4.3) (p <0.01) significantly increased compared to the control group not treated with H2O2. MCV value of LDH activity was shown in 20μM H2O2 treatment. The protective effect of REUU against H2O2 damaged by H2O2 on LDH activity surface was 10-100μg / ml 2 hours before exposure for 5 hours at 20μM H2O2 concentration, which is MCV value of H2O2. After the treatment with REUU containing each, the protective effect was investigated. As a result, when only 20μM H2O2 was treated, it was 77.9% (11.3 ± 1.3) compared to the control 100% (14.5 ± 1.8). However, 72.5% (10.3 ± 1.1) of 10Ug / ml REUU treatment was found to be 62.5% (9.0 ± 0.5) and 51.1% (6.9) of 25g / ml and 50μg / ml H2O2 treatment, respectively. ± 0.8) (p <0.05). In addition, the treatment of 100 μg / ml REUU showed a very significant decrease of 40.5% (5.1 ± 0.6) (p <0.01) compared to the control group (Table 9, 10, FIG. 7).

Table 9.Dose-response relationship of hydrogen peroxide (H2O2) on lactate dehydrogenase (LDH) release in cultured mouse DRG neurons

H2O2 (μM) 0 One 10 20 40 Amount of LDH Release 18.3 ± 1.6 21.3 ± 2.4 22.8 ± 2.1 28.6 ± 3.4 * 33.5 ± 4.3 **

Cultured mouse spinal DRG neurons were exposed to various concentrations of hydrogen peroxide (H2O2) for 5 hours. LDH release was measured at wavelength of 570 nm. The values represent the mean ± SE for 6 experiments. *

: significantly different from the value of control group. * p <0.05; ** p <0.01

Table 10.Dose-response relationship of Ramulus et Uncus Uncariae (REUU) for its neuroprotective effect on hydrogen peroxide (H2O2) in lactate dehydrogenase (LDH) release on cultured mouse DRG neurons

H2O2 (μM) LDH Release (% of control) concentration of REUU (µg / ml) 0 10 25 50 100 0 14.5 ± 1.8 14.2 ± 1.6 13.7 ± 1.5 13.5 ± 1.2 12.6 ± 1.4 20 11.3 ± 1.3 10.3 ± 1.1 9.0 ± 0.5 6.9 ± 0.8 * 5.1 ± 0.6 **

Cultured mouse spinal DRG neurons were preincubated with various concentrations of Ramulus et Uncus Uncariae (REUU) for 2 hours before treatment with 20μM H2O2 for 5 hours. The values are the mean ± SE for 6 experiments. Significant differences between groups are marked with asterisks. * p <0.05; ** p <0.01

Experimental Example 7

Quantitative determination of Lipid peroxidation was based on the determination of TBARS (thiobarbituric acid reactive substances) in supernatant and cell lysate of 經 節 細胞 根 神經 節 細胞, which had been used for several hours. 12N H2SO4 and 10% phosphotungstic acid were added to each of 2.0ml and 0.3ml for 10 minutes. After the reaction was completed, 1.0 ml of TBA was added, heated at 90 ° C. for 1 hour, and cooled to n-butanol. After completion of n-butanol treatment, it was centrifuged to remove it, and then determined by fluorometry at 553 nm. Lipid peroxidation is a fluorescence assay that measures the amount of lipid peroxidation of cells by light absorption at 553 nm. In order to measure the quantitative change in lipid peroxidation according to the concentration of H2O2 in H2O2-damaged 經 節 細胞 神經 節 細胞, H2O2 was treated with 脊髓 後 根 神經 節 細胞 for 5 hours in the soil containing H2O2 at a concentration of 1-50μM. After that, thibarbituric acid reactive substances (TBARS) were identified and cell viability was compared with the control group. As a result, in 1μM H2O2 treatment, TBARS was 41.7 ± 4.8 compared to the control group (36.8 ± 4.2) and the cell survival rate was 113.3% compared to the control group (100%). In addition, TBARS was 46.4 ± 5.3 and 59.7 ± 6.5 (p <0.01), respectively, when treated with 10μM and 30μM H2O2, and cell viability was decreased by 126.1% and 162.2% (p <0.01), respectively. In the treatment of 50μM H2O2, TBARS was 75.9 ± 8.7 (p <0.01) and cell viability was decreased by 206.3% (p <0.01). To investigate the effect of REUU on H2O2-induced 經 節 細胞 根 神 脊髓 後 on lipid peroxidation, 15-120㎍ 2 hours before exposure for 2 hours at 30μM H2O2 concentration, midcytotoxicity value of H2O2. / ml REUU was treated in each containing 후, and their effects were investigated. In case of H2O2 treatment, TBARS was 22.7 ± 3.8 compared with control group (32.7 ± 5.3), and cell survival rate was 69.4% compared with control group (100%). However, TBARS was 20.2 ± 2.6 and 17.4 ± 1.6 (p <0.05) in 15 µg / ml and 30 µg / ml REUU treatments, respectively, and the rate of decrease in cell viability was lower than that of the control group (100%). 62.3% and 53.0% (p <0.05), respectively. In addition, TBARS was 13.4 ± 2.3 (p <0.01) and 9.5 ± 0.7 (p <0.01) in 60μg / ml and 120μg / ml REUU treatment, respectively. 100%), 41.7% (p <0.01) and 30.1% (p <0.01), respectively (Table 11, Figure 8).

Table 11.Dose-response relationship of hydrogen peroxide (H2O2) on total protein synthesis

in cultured mouse spinal DRG neurons

H2O2 (μM) Total Protein (% of control) 0 100 ± 8.3 10 75.6 ± 6.4 25 68.2 ± 7.6 50 47.2 ± 4.8 ** 100 32.3 ± 5.2 **

Cultured mouse spinal DRG neurons were exposed to 10, 25, 50 and 100 μM H2O2 for 5 hours. Amount of total protein was measured by SRB assay (540nm), and shown as% of control. The values are the mean ± SE for 6 experiments. *: significantly different from the value of control group. ** p <0.01

Experimental Example 8

In order to investigate the effect of H2O2 on the total protein level, the changes of protein synthesis by H2O2 after 5 hours of immersion in H2O2 containing from 10μM to 100μM, respectively As a result, the protein synthesis was 75.6% in 10μM H2O2 treatment and 68.2% in 25μM H2O2 treatment. In the case of 50μM and 100μM H2O2 treatment, protein synthesis was 47.2% (p <0.01) and 32.3% (p <0.01)%, respectively. To investigate the effect of REUU on H2O2-induced cytotoxicity in the surface of quantitative changes in total protein, 10 to 80 hours before exposure for 5 hours at 50 μM H2O2 concentration, the MCV value of H2O2 After the pretreatment in each containing ㎍ / ml REUU was investigated the protective effect. As a result, when only H2O2 was treated, the quantitative change in total protein was 42.6% compared to the control (100%). On the other hand, the treatment of 10µg / ml REUU was 63.8% compared to the control, and the treatment of 20µg / ml and 40µg / ml was 71.5% and 76.7%, respectively. In addition, the treatment of 80 ㎍ / ml REUU was 81.2% (p <0.05) significantly increased compared to the control (Table 12, Figure 9).

Table 12.Dose-response relationship of Ramulus et Uncus Uncariae (REUU) for its neuroprotective effect on hydrogen peroxide (H2O2) by SRB assay in cultured mouse DRG neurons

H2O2 (μM) Total protein (% of control) concentration of REUU (µg / ml) 0 10 20 40 80 0 100 ± 6.3 100 ± 5.8 100 ± 7.1 100 ± 8.6 100 ± 7.5 50 42.6 ± 5.2 63.8 ± 7.4 71.5 ± 8.3 76.7 ± 6.5 81.2 ± 9.8 *

Cultured mouse spinal DRG neurons were preincubated with various concentrations of Ramulus et Uncus Uncariae (REUU) for 2 hours before treatment with 50μM H2O2 for 5 hours. The values are the mean ± SE for 6 experiments. Significant differences between groups are marked with asterisks. * p <0.05

As confirmed from the above experimental results, the novel composition of the present invention has an excellent antioxidative function while eliminating excellent reactive oxygen species. Therefore, the composition of the present invention can be usefully used as a pharmaceutical composition. The pharmaceutical composition of the present invention may vary depending on the sex, age, degree of disease, etc. of the patient, but if the composition of the present invention is used without extracting the composition of the present invention with water or an organic solvent, 1.0 g to 50 g per day is used. If used in the form of X, 10mg to 1000mg per day can be administered once to several times per day.

Compounds of the present invention are associated with stroke, stroke, cerebral infarction, hypertension, Alzheimer's disease, vascular disease (multi-infarct dementia), mixed Alzheimer's disease and multi-infarct dementia, Parkinson's disease, hypothyrodism, alcoholicity Since Alzheimer's dementia has a therapeutic effect, water extract is already used as a drug in the composition of the present invention, which is already used as a drug CGS 19755, MK-801, Dextrophan, Remacemide, Eliprodil, Magnesium sulfate, CNS 1102, GV 150526 , Nimodipine, Lifarizine, SNX111, Isradipine, GM1 ganglioside, Lubeluzole, Riluzole, Fosphenytoin, rofermin, further containing one or more of the following pharmaceutically acceptable excipients, adjuvants, diluents, isotonic agents, preservatives, It may also be used in combination with drugs such as lubricants, dissolution aids, and the like.

By using the compound of the present invention and the above drug, it is possible to reduce the normal dose of the existing drug, thereby reducing the problems with the existing drugs.

The compounds of the present invention may be mixed with excipients, adjuvants, analgesics, isotonic agents, preservatives, and other pharmaceutically acceptable adjuvants, which are commonly used pharmaceutically, and formulated into pharmaceutically acceptable formulations for pharmaceutical purposes. Suitable formulations may be prepared. Such pharmaceutical preparations may be formulated into injections, solutions, tablets, capsules, powders, syrups and the like.

Next, the present invention will be described in more detail as formulation examples.

Formulation Example 1

Water extract 500 mg of Example 2

Appropriate sterile distilled water for injection

pH adjuster

500 mg of Example 1 was dissolved in distilled water for injection, adjusted to pH about 7.6 with a pH adjuster, and then the total amount was 2 ml, and then filled in a 2 ml ampoule and sterilized to prepare a medicinal needle injection.

Formulation Example 2

500 mg of water extract powder of Example 2

Appropriate sterile distilled water for injection

pH adjuster

500 mg of Example 1 was dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, the whole was made to 2 ml, and then filled in an ampoule of 2 ml to prepare a medicinal needle injection.

Formulation Example 3

500 mg of water extract powder of Example 1

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 4

500 mg of water extract powder of Example 1

Lactose 100mg

Starch 50mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 5

500 mg of water extract powder of Example 1

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the inside is filled with a polyethylene coated coated chloride and sealed to prepare a powder.

Formulation Example 6

X 500mg of Example 1

Lactose 50mg

Starch 50mg

Talc 2mg0

Magnesium stearate appropriate amount

Capsules are prepared by mixing the above ingredients and filling gelatin capsules according to a conventional method of preparing capsules.

Formulation Example 6

X powder 500 mg of Example 1

Lactose 100mg

Starch 93mg

Tackle 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 7

X 5g of Example 1

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Purified water is added, and 100 ml of the above components are mixed according to a conventional method for preparing a liquid, and filled into a 100 ml brown bottle and sterilized to prepare a liquid.

Formulation Example 8

Example 5 x 5 g

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water and mix the above components according to a conventional method for preparing a liquid, and fill a 100 ml brown bottle and sterilize to prepare a liquid.

Formulation Example 9

X 500mg of Example 1

MK-801 10mg

Appropriate sterile distilled water for injection

pH adjuster

X 500 mg of Example 1 was dissolved in distilled water for injection, adjusted to pH about 7.6 with a pH adjuster, and then the total amount was 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 10

X 500mg of Example 1

CGS 19755 10mg

Appropriate sterile distilled water for injection

pH adjuster

X 500 mg of Example 1 was dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, and the whole was made to 2 ml, and then filled in a 2 ml ampoule to prepare an injection.

Formulation Example 11

X 500mg of Example 1

SNX111 20mg

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 12

X 500mg of Example 1

GM1 ganglioside 50mg

Lactose 100mg

Starch 50mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 13

X 500mg of Example 1

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate appropriate amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 14

X 1000mg of Example 1

Riluzole 10mg

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate appropriate amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 14

X 100mg of Example 1

Fosphenytoin 20mg

Lactose 100mg

Starch 93mg

Tackle 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 16

X 1000mg of Example 1

rofermin 1000mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water and mix the above components according to a conventional method for preparing a liquid, and fill a 100 ml brown bottle and sterilize to prepare a liquid.

As described in detail above, REUU contains, as a main component, an extract derived from Uncariae Ramulus et Uncus, powder, or a solvent selected from water, lower alcohol, ethyl acetate, aromatic hydrocarbon, and chlorinated hydrocarbon, and if necessary, dermis (Aurantii Nobilis). Pericarpium, Hoelen, Ligusticum acutilobum, Uncariae Ramulus et Uncus, Acorus gramineus, Atractyodis Rhizoma, Angelica dahurica, Sanji (Zizyphi Spinosi Semen) The composition containing X extracted with one or more auxiliary herbs selected from Preparata, Cornus officinalis, and Polygala tenuifolia, or a solvent selected from water, lower alcohol, ethyl acetate, aromatic hydrocarbons, and chlorinated hydrocarbons, is stroke, cerebral infarction. , Hypertension, Alzheimer's disease, vascular disease (Multi-infarct dementia), a combination of Alzheimer's disease and multi-infarct dementia, Parkinson's disease, low As a pharmaceutical composition which has an excellent effect on a merchant ship increases (hypothyrodism), alcoholic dementia, Alzheimer treated as a pharmaceutical composition has an excellent effect in neuroprotection.

Claims (4)

REUU is a stroke, cerebral infarction, hypertension, Alzheimer's disease, blood vessels containing X extracted with one or more auxiliary herbs selected from Uncariae Ramulus et Uncus or solvents selected from water, lower alcohols, ethyl acetate, aromatic hydrocarbons, and chlorinated hydrocarbons. A pharmaceutical composition having excellent neuroprotective effect as a pharmaceutical composition for treating diseases (Multi-infarct dementia), Alzheimer's disease and multi-infarct dementia, Parkinson's disease, hypothyrodism, and alcoholic dementia. REUU contains 50 to 500 parts by weight of Uncariae Ramulus et Uncus or its extract as a main ingredient, if necessary, 50 to 500 parts by weight of dermis (Aurantii Nobilis Pericarpium), 50 to 500 parts by weight of Hoelen, Ligusticum acutilobum) 50 to 500 parts by weight, 50 to 500 parts by weight of Uncariae Ramulus et Uncus, 50 to 500 parts by weight of Acorus gramineus, 50 to 500 parts by weight of Atractyodis Rhizoma, 50 to 500 parts by weight of Angelica dahurica 500 parts by weight, 50 to 500 parts by weight of Zizyphi Spinosi Semen, 50 to 500 parts by weight of Rehmanniae Radix Preparata, 50 to 500 parts by weight of Cornus officinalis, 50 to 500 parts by weight of Polygala tenuifolia Stroke, cerebral infarction, hypertension, Alzheimer's disease, vascular disease (multi-infarct dementia) containing extracts extracted with selected solvents from one or more selected supplements or water, lower alcohols, ethyl acetate, aromatic hydrocarbons, chlorinated hydrocarbons : Multi-infarctdementia), a combination of Alzheimer's disease and multi-infarct dementia, Parkinson's disease, hypothyrodism, and alcoholic dementia. A paralytic formulated with a pharmaceutically acceptable excipient, adjuvant, diluent, isotonicity agent, preservative, ointment agent and solubilizing agent in the form of a pharmaceutically acceptable pharmaceutical formulation. Excellent neuroprotective effect in the treatment of cerebral infarction, hypertension, Alzheimer's disease, vascular disease (Multi-infarct dementia), Alzheimer's disease and multi-infarct dementia, Parkinson's disease, hypothyrodism, alcoholic dementia Alzheimer's Pharmaceutical formulations having a. According to claim 3, CGS 19755, MK-801, Dextrophan, Remacemide, Eliprodil, Magnesium sulfate, CNS 1102, GV 150526, Nimodipine, Lifarizine, SNX111, Isradipine, GM1 ganglioside, Lubeluzole, Riluzole, It further contains at least one drug selected from Fosphenytoin, rofermin, and is a pharmaceutically acceptable pharmaceutical agent together with excipients, adjuvants, diluents, tonicity agents, preservatives, suspending agents, and dissolution aids. For the treatment of stroke, cerebral infarction, hypertension, Alzheimer's disease, vascular disease (multi-infarct dementia), mixed Alzheimer's disease and multi-infarct dementia, Parkinson's disease, hypothyrodism, alcoholic dementia Alzheimer's treatment A pharmaceutical formulation having a pharmaceutical composition having an excellent neuroprotective effect as a pharmaceutical composition.