KR101394650B1 - Pharmaseutical compositions for prevention or treatment of cerebrovascular disease, or for improving impairments, containing the extracts of Curcuma Longae Radix, Phellinus Linteus and Scutellariae Radix as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for the prevention or treatment of cerebrovascular diseases or a composition for improving memory damage, which contains Curcuma Longae Radix, Phellinus Linteus and Scutellariae Radix extract as active ingredients, Alcohol, or a mixture thereof as a solvent inhibits autosomal death, Ca ²⁺ production and active oxygen production of mouse neurons and effectively inhibits memory damage in mice that have induced neurotoxicity, A pharmaceutical composition for the prevention or treatment of vascular disease, or for ameliorating memory damage, or an effective ingredient of a health functional food.

Description

FIELD OF THE INVENTION The present invention relates to a pharmaceutical composition for preventing or treating cerebrovascular diseases or for improving memory impairment, which comprises, as an active ingredient, an extract of Curcuma Longae Radix , Phellinus Linteus and Scutellariae Radix as an active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating cerebrovascular diseases or for improving memory damage, which comprises a plant extract as an active ingredient.

Cerebral apoplexy, or cerebral infarction, is the main cause of cardiogenic embolism due to thrombosis of the cerebral arteries, embolism and heart disease caused by vascular atherosclerosis. Cerebral infarction caused by blockage of cerebral blood vessels is divided into cerebral hemorrhage and cerebral embolism. Cerebral hemorrhage is caused by arterial sclerosis due to hypertension, diabetes, hyperlipidemia, and the wall of the artery becomes thick or hard. As a result, blood vessels become narrow, blood vessel walls are easily damaged, and blood clots become clogged due to the lack of smoothness, resulting in marked reduction or interruption of blood supply, resulting in insufficient oxygen and nutritional supply to brain cells, resulting in impaired brain function. Cerebral embolism is caused by abnormalities of the blood flow in the heart due to diseases such as heart valve disease or atrial fibrillation, so that a part of the blood is partially stopped in the heart, and thus the blood coagulates and the blood remains. Cerebral infarction occurs.

Vascular dementia refers to a decrease in acquired irreversible cognitive function due to brain damage due to cerebrovascular disease. Dementia caused by stroke and cerebral infarction accounts for about one-third of all dementia, followed by Alzheimer's dementia. It is known that vascular dementia occurs as a result of large blood vessels or small blood vessel occlusion of brain in patients with vascular risk factors such as general stroke and cerebral infarction, have.

Vascular dementia may be caused by multiple infarct dementia, subcortical ischemic vascular dementia, and dementia due to a single lesion, depending on the location of the lesion causing the vascular pathology or dementia. dementia) and the like. Dementia caused by a single lesion due to occlusion means that a single lesion occurs in the caudate nucleus, mesial frontal lobe, and genu of internal capsule, resulting in dementia. Leukoaraiosis (white matter hyperintensity) caused by laconic cerebral infarction or incomplete infarction due to small blood vessel disease usually damages the prefrontal-subcortical circuit. This may result in subcortical dysfunctions, such as bradyphrenia and apraxia, rather than symptoms of traditional dementia, such as memory impairment.

Alzheimer's disease (AD) is characterized by a loss of neurons and an extracellular senescent half-life with amyloid-beta (Aβ), a 39-43 amino acid peptide derived from amyloid precursor protein senile plaque. In vitro and in vivo studies have shown that A [beta] or A [beta] peptide fragments have toxic effects, suggesting that A [beta] plays an important role in the pathogenesis of AD (Butterfieldmeat get .,Free Radical Biology and Medicine ,2002, 32: 1050-1060; ButterfIeldmeat get.,Free Radical Biology and Medicine ,2007, 43: 658-677). At the time of culture, Aβ directly induces neuronal cell death and makes neuronal cells vulnerable to excitotoxic and oxidative damage. NMDA (N-methyl-D-aspartate receptor) receptors act as mediators of Aβ-binding selective substrate or Aβ-induced glutamate excitotoxicity. NMDA receptors are especially Ca^{2 +}Is a highly permeable ligand-gate / voltage-sensitive cation channel. [Ca^{2 +}]_i, Leads directly to cell dysfunction, excessive excitement, or death. Thus, the neurotoxic effect of A? Is a noncompetitive NMDA receptor antagonist (5R.10S) - (+) - 5-methyl-10,11-dihydro-5H- dibenzo [a, d] cycloheptene- -Imine maleate (MK-801) [5R.10S) - (+) - 5-methyl-10,11-dihydro-5H-dibenzo (a, b) cyclohepten-5,10-imine maleate] As evidenced by the report, the expression of Ca via NMDA receptors by A [beta] exposure^{2 +}Inflow plays a crucial role in A [beta] -induced neurotoxicity. The formation of reactive oxygen species (ROS) is also believed to be involved in the onset of degenerative brain diseases. Some evidence supports the involvement of oxidative stress as an active factor in A [beta] -mediated neuropathy by triggering or facilitating neurodegeneration through a broad molecular shape that interferes with neuronal homeostasis. However, the clinical benefit of direct blockers of NMDA receptor antagonists and neuronal channels is controversial, as they lack the verifiable efficacy or have serious adverse effects.

Most degenerative brain diseases involve dementia, especially cognitive and memory impairment (memory loss). Therefore, a desirable therapeutic agent for degenerative brain diseases including dementia should delay brain cell destruction and aging to protect brain cells and restore cognitive function. The drugs that have been developed so far include antioxidants such as vitamin E and selegiline that inhibit the breakdown of brain cells by active oxygen, acetylcholine such as Tacrine, Aricept, and Exelon And acetylcholinesterase-inhibiting drugs. However, these drugs have been reported to cause considerable side effects, and their efficacy is not excellent.

Curcuma Longae is a perennial plant classified as Curcuma of the genus Zingiberaceae. It is grown in India, China, Southeast Asia, and is used as edible, medicinal and natural dyes. Recently, cucuminoids and curcumin have been reported to have various physiological effects such as antioxidation, anticancer, anti-mutation, anti-inflammation, prevention of dementia, protection of liver function and reduction of cholesterol (Guo LY et al ., 2008, Archives Pharmacal Research , 31: 490-496; Adaramoye OA et al ., 2002, Biochemistry 12: 127-135).

The situation was mud mushroom ( Phellinus sp. ). Mud mushrooms are known to 220 species worldwide, including P. igniarius , P. robustus , and P. pine . The situation has been shown to be excellent in anti-tumor immunity, gastrointestinal function, and detoxification, and 96.7% in animal studies (Ajith TA et al ., 2002 Journal of Ethnopharmacology , 2002, 81: 387-391; Sliva et al . , 2008, British Journal of Cancer , 98: 1348-1356). Its efficacy is superior to other mud mushrooms. In addition, the stability of clinical trials has been proven in clinical trials, and industrial application studies have been actively conducted. Due to the availability of pharmaceuticals, large quantities have been distributed from China and India to high prices.

Golden ( Scutellariae Radix) is a medicinal herb that has been stripped of the spruce grass (Scutellaria baicalensis Gergi) and dried its roots. In general, in Oriental medicine, inflammatory diseases are described as heat (heat) or fire (fire), and medicines that have an action to treat them are said to have an efficacy of chelating heat for reducing heat. As the medicinal herbs that have been used in the past, they have been used with gold, chrysanthemum, yellow whites, rhubarb and gypsum. Among them, the most representative gold is used in the fever of the intestines and lungs come. Thus, it is mainly applied to diseases such as fever, seawater, fever, diarrhea, jaundice, and hematemesis, which are caused by external fraud involving the human body. Currently pharmacologically, baicalin, baicalein, and wogonin have been isolated and have been reported to have antidiarrheal, antipyretic, anti-inflammatory and anti-cancer effects (Bensky D et al ., 1992, Chinese Herbal Medicine Materia Medica . Eastland Press , pp . 107-109; Huang KC, 1999, The Pharmacology of Chinese Herbs , pp. 385).

Thus, the present inventors have self-cell death by beta amyloid in the brain cell culture are of, turmeric, conditions and Gold extract was efforts to develop natural material with a cerebrovascular disease preventing and treating effect from rat fetuses, Ca ^{2 +} generation and activity The present invention relates to a pharmaceutical composition for preventing or treating cerebrovascular disease or for improving memory damage, and a method for effectively and effectively inhibiting memory damage by inhibiting oxygen production and effectively inhibiting memory damage in a mouse caused by beta amyloid-induced neurotoxicity The present invention has been completed.

The object of the present invention is to provide curcuma Longae Radix), the situation ( Phellinus Linteus ) and golden ( Scutellariae Radix extract as an active ingredient for the prevention or treatment of cerebrovascular diseases, or for improving memory damage, and a health functional food.

In order to achieve the above object, the present invention is turmeric (Curcuma Longae Radix), the situation ( Phellinus Linteus ) and golden ( Scutellariae Radix) as an active ingredient for the prevention or treatment of cerebrovascular diseases, or a pharmaceutical composition for improving memory damage.

Further, the present invention provides a health functional food for preventing or ameliorating cerebrovascular disease or improving memory damage, which contains turmeric, a condition and a golden extract as an active ingredient.

The curcuma of the present invention Longae Radix), the situation ( Phellinus Linteus ) and golden ( Scutellariae Radix extract inhibited beta-amyloid-induced auto-apoptosis, Ca ²⁺ production, and reactive oxygen production of brain cells cultured from rat fetuses and effectively inhibited memory damage in mice that induced beta-amyloid-induced neurotoxicity A composition for preventing or treating cerebrovascular diseases, a pharmaceutical composition for improving memory damage, and a health functional food.

Fig. 1 is a graphical representation of the curcuma Longae Radix), the situation ( Phellinus Linteus ) and the golden ( Scutellariae (25-35) -induced neuronal apoptosis inhibitory effect of Aβ (Radix) extract.
Fig. 2 is a graph showing the effect of Aβ (25-35) -induced cell death on cell death in the turmeric, the situation and the golden extract according to the present invention.
3 is Aβ (25-35) of turmeric, conditions and Gold extract according to the invention - a graph showing the induced Ca ^{2 +} increase inhibitory effect.
FIG. 4 is a graph showing the inhibitory effect of Aβ (25-35) -induced reactive oxygen species on turmeric, a situation, and a gold extract according to the present invention.
FIG. 5 is a graph showing the inhibitory effect of A? (25-35) -induced memory damage of turmeric, a situation and gold extract according to the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to a process for preparing curcuma Longae Radix), the situation ( Phellinus Linteus ) and golden ( Scutellariae Radix extract (hereinafter abbreviated as HS0608) as an active ingredient for the prevention or treatment of cerebrovascular diseases or for the amelioration of memory damage.

It is preferable but not limited to use in a weight ratio of 1: 1: 1, and it can be used without limitation such as turmeric, circumstance and gold.

The HS0608 is preferably but not limited to be produced by a manufacturing method comprising the following steps.

1) mixing the turbulence, the situation and the gold;

2) extracting the mixture of step 1) with an extraction solvent;

3) cooling the extract of step 2) and filtering; And

4) reflux condensation of the filtered extract of step 3) followed by drying.

The extraction solvent is preferably water, alcohol or a mixture thereof. As the alcohol, C ₁ to C ₄ lower alcohol is preferably used, and as the lower alcohol, ethanol or methanol is preferably used. As the extraction method, it is preferable to use shaking extraction, Soxhlet extraction or reflux extraction, but it is not limited thereto. It is preferable that the extraction solvent is added by 5 to 15 times the amount of the dried condition, the mixture of turmeric and gold, and it is more preferable to perform extraction by adding 10 times. The extraction temperature is preferably 40 to 100 ° C, more preferably 60 to 80 ° C, but is not limited thereto. Further, the extraction time is preferably 4 to 24 hours, more preferably 8 to 15 hours, but is not limited thereto. The number of times of extraction is preferably 1 to 5 times, more preferably 3 times, but is not limited thereto.

In the above method, drying in step 4) is preferably, but not exclusively, reduced-pressure drying, vacuum drying, boiling drying, spray drying or lyophilization.

The cerebrovascular disease is preferably any one selected from the group consisting of cerebral infarction, cerebral infarction, stroke, dementia, Alzheimer's disease, Huntington's disease, pick and Creutzfeld-Jakob disease Do not.

In a specific embodiment of the present invention, neuronal cells were isolated from the fetus of the rat and cultured, and the effect of the neuron was examined. As a result, HS0608 suppresses the generation of my self apoptosis cells by Aβ (apoptotic cell death) (see Fig. 2), Ca ^{2 +} concentrations increase (see Fig. 3), the intracellular reactive oxygen species (ROS) (see Fig. 4) Respectively. Therefore, it was confirmed that HS0608 of the present invention inhibits apoptosis caused by Aβ, which is known to be a causative agent of dementia.

In order to confirm the memory damage inhibitory effect of HS0608, the present inventors conducted a Passive Avoidance Test. As a result, the group to which HS0608 was administered showed a dose-dependent inhibition of memory impairment (see FIG. 5).

Therefore, HS0608 of the present invention can be used as an active ingredient of a pharmaceutical composition for the prevention or treatment of cerebrovascular diseases or for ameliorating memory damage.

The composition containing HS0608 of the present invention preferably includes 0.1 to 50% by weight based on the total weight, but is not limited thereto.

The composition of the present invention can be administered orally or parenterally at the time of clinical administration and can be used in the form of a general pharmaceutical preparation. In other words, the composition of the present invention can be administered in various formulations of oral and parenteral administration at the time of actual clinical administration. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant and a surfactant, . ≪ / RTI > Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. Such solid preparations can be prepared by mixing the pharmaceutical composition of the present invention with at least one excipient such as starch, calcium carbonate, sucrose, lactose And gelatin. In addition to simple excipients, lubricants such as magnesium, styrene, and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. Various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included in addition to water and liquid paraffin, which are commonly used simple diluents. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilisers and suppositories. Examples of non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol and gelatin can be used. The pharmaceutical composition of the present invention can be administered parenterally, subcutaneously, intravenously or intramuscularly.

The dosage unit may contain, for example, 1, 2, 3 or 4 times the individual dose or may contain 1/2, 1/3 or 1/4 times the dose. The individual dosages preferably contain amounts in which the active drug is administered in one go, which usually corresponds to the full, half, one-third or one-fourth of the daily dose. The effective dose of the composition of the present invention is 0.0001 to 10 g / kg, preferably 0.0001 g to 5 g / kg, and can be administered 1 to 6 times a day.

The composition of the present invention can be used alone or in combination with methods for the prevention and treatment of cerebrovascular diseases or using surgery, hormone therapy, chemotherapy and biological response modifiers.

Further, the present invention provides a health functional food for preventing or ameliorating cerebrovascular disease or improving memory damage, which contains turmeric, a condition and a golden extract as an active ingredient.

In a specific example of the present invention, the inventors of the present invention examined HS0608 for the prevention and treatment of cerebrovascular diseases and found that HS0608 induced apoptotic cell death by Aβ It inhibited the production of (apoptotic cell death), Ca ^{+ 2} concentration increases, the intracellular reactive oxygen species (ROS). The group receiving HS0608 in mice that induced neurotoxicity showed dose-dependent inhibition of memory impairment.

Accordingly, the present invention can be used for preventing or ameliorating cerebrovascular diseases or improving health-promoting foods for memory impairment, which contains turmeric, a condition and a golden extract as an active ingredient.

The health functional food of the present invention can be used as it is, or can be used with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, the composition of the present invention is added in an amount of not more than 15 parts by weight, preferably not more than 10 parts by weight, based on the raw material, when the food or beverage is produced. 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.

There is no particular limitation on the kind of the food. Examples of the food to which the above substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen and other noodles, gums, ice cream, various soups, drinks, tea, Alcoholic beverages, and vitamin complexes, all of which include health foods in a conventional sense.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. The above-mentioned natural carbohydrates are sugar alcohols such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the composition of the present invention.

In addition to the above, HS0608 of the present invention can be used for various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, A carbonating agent used in beverages, and the like. In addition, HS0608 of the present invention may contain pulp for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.02 to 1 part by weight per 100 parts by weight of the composition of the present invention.

Since HS0608 of the present invention has little toxicity and side effects, it can be safely used for prolonged use for preventive purposes.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Production Examples. However, the following Examples, Experimental Examples and Preparation Examples illustrate the present invention concretely, and the contents of the present invention are not limited by Examples, Experimental Examples and Production Examples.

< Example  1> curcuma ( Curcuma Longae Radix ), situation( Phellinus Linteus ) And gold Scutellariae Radix ) Preparation of extract

<1-1> Preparation of ethanol extract

I used the fragrance, the situation and the gold in the Seoul Kyungdong Chinese medicine market. 100 g each of virulence, circumstance and gold were prepared and combined, and then extracted with 3 L of 80% ethanol for 3 hours in a reflux condenser at room temperature three times. The solution was filtered through Watman No.1 paper and dried to finally yield 64 g of ethanol extract (HS0608). The final obtained turmeric, the situation and the golden mixed extract (HS0608) were dissolved in dimethylsulfoxide (DMSO) at a concentration of 50 mg / ml and stored at -20 ° C. In the experiment, the dilution was used so that the final concentration of DMSO was 0.1% or less in the experimental buffer.

<1-2> Preparation of water extract

(35.5% yield) was obtained in the same manner as in the extraction method of Example <1-1>, except that water was used in place of 80% ethanol as an extraction solvent.

<1-3> Preparation of 100% ethanol extract

(Yield: 26.4%) was obtained in the same manner as in the extraction method of Example <1-1>, except that 100% ethanol was used instead of 80% ethanol as an extraction solvent.

<1-4> Preparation of 100 Methanol Extract

(33.1% yield) was obtained in the same manner as in the extraction method of Example <1-1>, except that 100 methanol was used instead of 80% ethanol as an extraction solvent.

< Experimental Example  One > Cultivation of cerebral cortical neurons

Primary cerebral cortical neurons were prepared from Sprague-Dawley rat fetuses 15-16 days old by known methods (Ban JY et al ., Life Science , 2006, 79: 2251-2259).

< Experimental Example  2 > HS0608 Of nerve cell death inhibition

The present inventors conducted the following experiments to confirm the effect of the mixed sulfur extract (HS0608) obtained from the above Example 1 on neuronal cell death.

The cerebral cortical neurons prepared in Experimental Example 1 were used for the experiment at 3-4 days after culturing. The nerve cells were either not treated or HS0608 obtained in Example 1 was treated with 0.1, 1, 10 and 50 mg / ml. 15 minutes after the treatment, 10 [mu] M A [beta] (25-35) (Bachem, Switzerland) dissolved in serum-free Dulbecco's modified Eagle's medium (DMEM) ) And cultured at 37 캜 for 36 hours (Aβ single treatment group and Aβ + HS0608 treatment group). Thereafter, a known method was used to prepare 3- (4,5-dimethylthiazol-2-yl-2,5-diphenyltetolol bromide) [3- bromide (MTT)] analysis was performed (Ban JY et al ., 2006). At this time, when the cells without Aβ treatment were used as a control group, the absorbance of the Aβ single treatment group and the HS0608 treatment group in% was shown in a graph in FIG. 1 and Table 1.

As shown in Fig. 1 and Table 1, in the group treated with 10 μM Aβ (25-35) alone, the absorbance was reduced to 70.6% as compared with the control (100%). This means that about 39% of neurons were killed by Aβ. In contrast, in the HS0608 treated group, the absorbance was 73.5% at 0.1 ㎍ / ㎖, 81.4% at 1 ㎍ / ㎖ treatment, 83.7% at 10 ㎍ / ㎖ treatment and 95.1% at 50 ㎍ / ㎖ treatment, Was decreased in proportion to the concentration of HS0608. In the subsequent experiments, only the ethanol aqueous solution extract having the best cell death inhibitory effect was tested by MTT analysis.

Aqueous ethanol extract Water extract Ethanol extract Methanol extract  Control group 100 100 100 100  Aβ 71 72 72 72  Aβ + HS0608 0.1 74 73 72 73  Aβ + HS0608 1 81 74 75 78  Aβ + HS0608 10 84 77 76 79  Aβ + HS0608 50 95 79 81 82

< Experimental Example  3> HS0608 Neuron Autosomal death  Measurement of inhibitory effect

The present inventors performed Hoechst 33342 staining in a neuron treated in the same manner as in Experimental Example 2 to prove that apoptosis by ap is apoptosis (Ban JY et al. al ., 2006). The results are shown in Fig.

As shown in FIG. 2, in the control group, autologous cell death was observed at 14.8%, whereas in the Aβ treatment group, 39.2% of autologous cell death was observed. In HS0608 treatment group, 22.9% at 1 μg / (20.3%) and 50 ㎍ / ㎖ (13.4%), respectively, and autologous cell death was reduced by HS0608.

< Experimental Example  4> HS0608 Measurement of inhibitory effect of intracellular calcium increase

In order to confirm whether HS0608 of the present invention causes biochemical changes in the cells, a fluorescent dye 4-AM (fluo-4 AM) (Molecular Probes inc., USA) was added to the cells for 40 minutes, washed, treated with nothing, or HS0608 obtained in Example 1 was treated with 1, 10 and 50 占 퐂 / ml. 15 minutes after the treatment, changes in the intracellular calcium concentration of Aβ (25-35) immediately after treatment of Aβ (25-35) dissolved in 10 μM in serum-free DMEM to the neurons were analyzed by calcium-sensitive fluorescence Increasing fluorescence intensities were measured by a fluorescent dye Fluo-4 AM (Molecular Probes, USA) using a Laser Scanning Convocal microscope (LSM 510, Carl Zeiss Germany) [488 nm excitation Argon laser and 515 nm long Pass radiation filter] by the intracellular calcium change (Ban JY et al ., 2006; Lee SB et al ., Korean Journal of Medicinal Crop Science , 2007, 15: 444-450). The measurement results are shown in Fig.

As shown in Fig. 3, fluorescence intensity increased when intracellular calcium concentration ([Ca ^&lt; ^{2 + &} gt ^; ] _i ) increased and fluorescence intensity increased when applied to nerve cells treated with fluoro-4 AM, After increasing to the maximum value of 153, the maximum value gradually decreased. On the other hand, in the case of pretreatment of 50 μg / ml of HS0608, it was confirmed that the increase of [Ca ^{2 +} ] _i induced by Aβ was completely inhibited throughout the measurement period.

< Experimental Example  5> HS0608 Of active oxygen ( ROS ) Production inhibition effect measurement

In order to confirm the inhibitory effect of HS0608 on ROS generation in neurons, neurons prepared in Example 1 were treated for 36 hours in the same manner as Experimental Example 1, and then treated with H ₂ DCF-DA (Molecular Probes (USA) was used to absorb and use H ₂ DCF-DA (Sigma, USA) for 10 min in a well known manner using a laser scanning con- cocal microscope MRC1021ES Bio-red, UK [488 nm excitation and 515 nm emission filter] ROS was measured as a change in fluorescence intensity by Kim JY meat al ., Korean Journal of Medicinal Crop Science 2008, 16: 409-415, Lee, SB et al al ., 2007). The results are shown in Fig.

As shown in FIG. 4, the fluorescence intensity was 242.8 due to the production of active oxygen by Aβ, which was about 2.5 times higher than that of control cells (47.6). In comparison, HS0608 showed fluorescence intensities of 138.7, 114.2 and 47.5, respectively, at 1, 10 and 50 ㎍ / ㎖ treatment.

< Experimental Example  6> Passive avoidance test ( Step through ) &Lt; / RTI &gt;

Animals were sacrificed by oral administration of HS0608 (25, 50 and 100 ㎎ / ㎏ po) on the first day of the experiment (1 day) Min, 15 μl of 15 nmol of Aβ (25-35) was slowly injected into the ventricle using a microsylinge equipped with a 27-gauge needle (Hamilton, USA) (Maurice T et al ., Brain Reserch , 1996,706: 181-193). Thereafter, HS0608 at 25, 50 and 100 mg / kg po, respectively, were orally administered once a day for one week. On day 7, HS0608 was orally administered and after 30 minutes the mice were mastered with a single-pass step-passive-task. The manual avoidance box is divided into two sections, a grid floor and an impact generator. In darkness, the mouse is placed at the starting point. After 50 seconds, the door is opened. When the mouse enters the dark box, the door is closed so that the electric stimulus (0.3 mA, 2 seconds) I remembered the experience of electric shocks within. After 24 hours, when animals were placed in light boxes and the same experiment was carried out, the animals that remember the shock of the previous day did not move to dark boxes. The time of setting the animals in a light box was measured and used as an index of memory formation, and it was 5 minutes to have a stay time of 5 minutes or more. The results are shown in Fig.

As shown in FIG. 5, the group administered with 15 nmol Aβ (25-35) migrated to the dark room in 274 seconds, whereas the group administered with Aβ (25-35) moved into the dark room at 62 seconds. The results suggest that memory is not formed. Compared with the control group, the time to enter the dark room increased dose - dependently in the groups treated with HS0608 25, 50 and 100 ㎎ / ㎏ for 1 week, 165.4 seconds in the 25 ㎎ / ㎏ group and 222 Sec, and 100 ㎎ / ㎏, respectively, in all HS0608 - treated group, and it was found that the inhibition of memory impairment by Aβ (25-35) was significantly inhibited.

< Manufacturing example  1>: Preparation of pharmaceutical preparations

<1-1> Preparation of powder

HS0608 300 mg

Lactose 100 mg

10 mg of talc

The above components are mixed and filled in airtight bags to prepare powders.

<1-2> Preparation of tablets

HS0608 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

After the above components are mixed, they are tableted according to a conventional preparation method of tablets,

.

&Lt; 1-3 > Preparation of capsules

HS0608 50 mg

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

&Lt; 1-4 > Preparation of injection

HS0608 50 mg

Sterile sterilized water for injection

pH adjuster

(2 ml) per ampoule in accordance with the usual injection method.

<1-5> Preparation of liquid agent

HS0608 1,000 mg

20 g of sugar

20 g per isomer

Lemon incense quantity

Purified water was added to adjust the total volume to 1000 ml. The above components were mixed according to a conventional method for producing a liquid agent, and then filled in a brown bottle and sterilized to prepare a liquid agent.

<Manufacturing Requirement 2>: Manufacture of health food

HS0608 1,000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

< Manufacturing example  3>: Manufacture of health drinks

HS0608 1000 mg

Citric acid 1000 mg

100 g of oligosaccharide

Plum concentrate 2 g

Taurine 1 g

Purified water was added to a total of 900 ml

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 ° C for about 1 hour. The resulting solution was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.

Claims (10)

For the prevention or treatment of cerebrovascular diseases containing curcuma longae radix, Phellinus linteus and gold ( Scutellariae radix) in an amount of 1: 1: 1 by weight as an active ingredient, A pharmaceutical composition.
delete The pharmaceutical composition for the prevention or treatment of cerebrovascular diseases according to claim 1, wherein the extract is extracted with water, alcohol or a mixture thereof as a solvent.
The pharmaceutical composition according to claim 3, wherein the alcohol is a C ₁ to C ₄ lower alcohol.
[Claim 5] The pharmaceutical composition according to claim 4, wherein the lower alcohol is ethanol or methanol.
6. The method of claim 1 or 5, wherein said cerebrovascular disease is selected from the group consisting of cerebral infarction, brain weakness, stroke, dementia, Alzheimer's disease, Huntington's disease, pick, and Creutz feld-Jakob disease Wherein the compound is any one selected from the group consisting of:
A health functional food for preventing or ameliorating cerebrovascular disease or improving memory impairment containing an extract of a mixture of turmeric, the situation and gold in a weight ratio of 1: 1: 1 as an active ingredient.
delete The health functional food for preventing or ameliorating cerebrovascular diseases according to claim 7, wherein the extract is extracted with water, ethanol or a mixture thereof as a solvent.
10. The method according to claim 7 or 9, wherein said cerebrovascular disease is selected from the group consisting of cerebral infarction, cerebral infarction, stroke, dementia, Alzheimer's disease, Huntington's disease, pick and Creutzfeld- Or a pharmaceutically acceptable salt thereof, for the prevention or amelioration of cerebrovascular diseases or for the amelioration of memory damage.

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