KR101617775B1 - A composition comprising the extract of black rice (Oryza sativa L.) for treating and preventing neuro-degenerative disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition or a health functional food for the treatment and prevention of forgetfulness and degenerative brain diseases containing an extract of black rice rice as an active ingredient. The extract is excellent in memory in the Morris water maze experiment using the BCCAO- was confirmed the improvement (example 1), glutamate (glutamate) Ca ^{2 +} Signaling experiment the intracellular Ca ^{2 +} signal induced by the glutamate was inhibited from acting on by a (example 2), by glutamate (Experimental Example 1), it was confirmed that glutamate-induced apoptosis was significantly inhibited in the evaluation of efficacy against neuronal cell death induced in brain hippocampus (Experimental Example 1), and a pharmaceutical composition or health functional food for improving memory and treating and preventing degenerative brain diseases to provide.

Description

TECHNICAL FIELD The present invention relates to a composition for treating and preventing degenerative brain diseases containing black rice extract as an active ingredient,

The present invention relates to a composition for improving forgetting and degenerative brain diseases comprising black rice extract.

[Document 1] J.A. Duke, Handbook of phytochemical constituents of GRAS herbs and other economic plants: Herbal Reference Library, CRC Press, Boca Raton, Florida (1992), pp. 143-144.

[2] Jin DQ et al., Anti-oxidant and anti-inflammatory activities of macelignan in murine hippocampal cell line and primary culture of rat microglial cells. Biochem Biophys Res Commun. 2005; 331: 1264-1269.

[3] Lim CS et al., Antioxidant and anti-inflammatory activities of the methanolic extract of Neurhodomela aculeate in hippocampal and microglial cells. Biol Pharm Bull. 2006; 29: 1212-1216.

[4] Bassil and Grossberg, Novel regimens and delivery systems in the pharmacological treatment of Alzheimer's disease. CNS Drugs. 2009; 23 (4): 293-307.

[Literature 5] Choi SW et al. 1996. Food Biotechnol 5: 305-309, Nam SH et al. 1997. Agri Chem. Biotechnol 40: 307-312

[Literature 6] Jo, Min-Kyung et al., 2008. J Korean Soc. Appl Biol Chem 51 (3): 200-206

[Literature 7] Korean Patent Publication No. 10-2005-0024796

[Literature 8] Schiavon et al, Schiavon et al, Neurotoxicity Research, Epub ahead of print, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24532152

Kim et al., 2006. Kim, DH, Hung, TM, Bae, KH, Jung, JW, Lee, S., Yoon, BH, Cheong, JH, Ko, KH, Ryu, JH. Gomisin improves scopolamine-induced memory impairment in mice. Eur. J. Pharmacol. 7 (2006) pp. 129-135

The present invention relates to a composition for improving forgetting and degenerative brain diseases comprising black rice extract.

As the elderly population increases due to the extension of life span due to the development of medical technology, the proportion of patients with degenerative nerve disease, which is a disease of the degenerative nerve, is also increasing rapidly. Dementia is the fourth cause of cancer deaths, heart disease and stroke following death, which greatly reduces the quality of life, both personally and socially, and makes people and people around them unhappy. Dementia is divided into Alzheimer's dementia and vascular dementia. Alzheimer's dementia is a form of dementia commonly observed in western countries. It is a disease in which cholinergic neurons are destroyed by accumulation of beta-amyloid in specific parts of the brain. Vascular dementia is a dementia caused by hypertension, stroke, hyperlipidemia, etc., resulting in ischemic conditions due to abnormal blood vessels to the brain, resulting in death in the hippocampus and peripheral nervous system. These two types of dementia have different mechanisms, but they have the same mechanism in that they reduce the action of acetylcholine, which is known as a memory-transmitting neuron, resulting in impaired memory.

Since dementia is a degenerative disease that is destroying human nature and lasting for a long time, it can not afford socioeconomic burden as a passive method of accommodating patients, so it is necessary to actively try to develop prophylactic and therapeutic agents. However, there have not been developed therapeutic agents capable of treating the underlying cause of Alzheimer's disease so far. Common therapeutic agents include Aricept, an acetylcholinesterase inhibitor of Pfizer, Exelon of Novartis, Reminyl, and recently Ebixa (Memantine) from Lundbeck, an antagonist of NMDA receptors licensed from the US FDA. However, acetylcholinesterase inhibitors improve the declining cognitive ability and do not cure the underlying cause of Alzheimer's disease. In addition, it is difficult to say that it is a fundamental treatment agent because it shows a temporary symptomatic relief effect only in some patients and its effect does not last long. In addition, due to the nature of the disease, a long-term use is required. In the case of the above-mentioned medicines, accompanying toxic effects, vomiting, loss of appetite, Therefore, it is urgent to develop a therapeutic agent that can prevent the progress of the disease. To this end, many multinational pharmaceutical companies have invested heavily in R & D in this field. In particular, beta or gamma secretase inhibitors, which reduce the production of beta amyloid consisting of about 40 amino acids, which is presumed to be a fundamental cause of Alzheimer's disease Is the main type of development. In Korea, basic research on Alzheimer's disease has been done to some extent, but the development of dementia treatment itself is rarely done.

As living standards are improved and human longevity is increasing, interest in various diseases related to aging and aging is growing, and oxidative stress has been found to be an important factor in degenerative brain diseases of the central nervous system such as Alzheimer's syndrome, Parkinson's syndrome, and Huntington's syndrome (Jin DQ et al, Biochemical and Biophysical Research Communications, 331: 1264-1269, 2005; Lim CS et al., Biological and Pharmaceutical Bulletin, 29: 1212-1216, 2006).

Acetylcholine having a quaternary amine structure is a neurotransmitter, and acetylcholinesterase hydrolyzes acetylcholine to choline. In patients with dementia, the concentration of acetylcholine, a neurotransmitter, decreases, and when acetylcholinesterase inhibits, the concentration of acetylcholine in the brain increases and symptoms of dementia are improved. There are four drugs that have been developed and used by the FDA for the treatment of dementia: tacrine, donepezil, galanthamine, and rivastigmine, all of which are acetylcholinesterase inhibitors Bassil and Grossberg, Novel regimens and delivery systems in the pharmacological treatment of Alzheimer's disease. Drugs. 2009; 23 (4): 293-307).

Black rice or black rice is processed into various forms of food due to its unique color and flavor, and consumption is also increasing. Black rice contains many antioxidants such as anthocyanins, polyphenols, flavonoids and vitamins. The ethanol extracts of black rice have been reported to have superior antioxidant activity against rice (Choi SW et al. 1996. Food Biotechnol 5: 305-309, Nam SH et al 1997. Agri Chem. Biotechnol 40: 307-312). On the other hand, the results of a study that administration of rice embryo-derived jelly to rice resulted in improved blood lipid metabolism and increased antioxidant enzyme activity (Cho, Min-Kyung et al., 2008. J Korean Soc. Appl Biol Chem 51 (3): 200 -206). Cyanidin, a kind of black rice extract and anthocyanin, has been reported to lower blood and cholesterol levels in rats given a high cholesterol diet (Korea Publication No. 10-2005-0024796).

However, none of the above documents disclose or teach the amelioration of forgetfulness and the therapeutic effect of degenerative brain disease in black rice extract.

Accordingly, the inventors of the present invention have found that the black rice extract has excellent memory improving effect in the Morris water maze experiment using an animal model in which BCCAO is impaired (Experiment 1) example 1), induced by glutamate (was the glutamate) the intracellular Ca ^{2 +} signal induced by glutamate (glutamate) in effect experiments acting on the Ca ^{2 +} signal by the suppression (example 2), glutamate (glutamate) (Experimental Example 1) was confirmed to be useful for the improvement of memory and for the treatment and prevention of degenerative brain diseases, thereby completing the present invention Respectively.

In order to solve the above-mentioned problems, the present invention provides a pharmaceutical composition for treating amnesia and ameliorating degenerative brain diseases comprising black rice extract as an active ingredient.

Further, the present invention provides a health functional food for amelioration of forgetfulness and prevention and improvement of degenerative brain diseases containing black rice extract as an active ingredient.

The extracts defined herein may be formulated with water, lower alcohols such as methanol, ethanol, butanol or mixtures thereof, preferably water or a mixture of 1-100% water and ethanol, more preferably about 1-70% water And an ethanol-soluble solvent.

Degenerative brain diseases as defined herein include stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease or Huntington's disease, in particular vascular dementia and Alzheimer's disease.

Hereinafter, the method for obtaining the extract of the present invention will be described in more detail.

For example, the extract of the present invention comprises a first step of washing and drying a black rice sample; Water, a C ₁ to C ₄ lower alcohol or a mixed solvent thereof, preferably about 1 to 30 times, preferably about 5 to 15 times (w / v) the weight of the sample, preferably water or ethanol, More preferably 0.01 to 5% (v / w), preferably 0.1 to 5% by volume, of water or 5 to 80% ethanol as the extraction solvent and a weak acid for preventing destruction of the active ingredient, The weak acid such as malic acid, citric acid or formic acid in a weight of 1% (v / w) is added to the extraction solvent and the reaction is carried out at a reaction temperature of about 10 to 120 캜, preferably 20 to 110 캜 for about 1 to 6 hours, A second step of repeating extraction by 1 to 10 times, preferably 2 to 7 times, by a conventional extraction method such as room temperature extraction method, heat extraction method, ultrasonic extraction method and reflux extraction method for 2 to 5 hours, preferably room temperature extraction method; A third step of filtrating and concentrating the extract obtained in the above step under reduced pressure; And the fourth step of lyophilizing the concentrated extract at about -50 ° C to -30 ° C for about 24 to 72 hours to obtain the black rice extract of the present invention.

Accordingly, the present invention provides a method for producing a black rice sample, comprising: a first step of washing and drying a black rice sample; Water, a C ₁ to C ₄ lower alcohol or a mixed solvent thereof, preferably about 1 to 30 times, preferably about 5 to 15 times (w / v) the weight of the sample, preferably water or ethanol, More preferably 0.01 to 5% (v / w), preferably 0.1 to 5% by volume, of water or 5 to 80% ethanol as the extraction solvent and a weak acid for preventing destruction of the active ingredient, The weak acid such as malic acid, citric acid and formic acid in a weight of 1% (v / w) is added to the extraction solvent and the reaction is carried out at a reaction temperature of about 10 to 120 캜, preferably 20 to 110 캜 for about 1 to 6 hours, A second step of repeating extraction by 1 to 10 times, preferably 2 to 7 times, by a conventional extraction method such as room temperature extraction method, heat extraction method, ultrasonic extraction method and reflux extraction method for 2 to 5 hours, preferably room temperature extraction method; A third step of filtrating and concentrating the extract obtained in the above step under reduced pressure; And a fourth step of lyophilizing the concentrated extract at a temperature of about -50 ° C to -30 ° C for about 24 to 72 hours, to prepare a black rice extract of the present invention.

In addition, the present invention provides a pharmaceutical composition and a health functional food for the treatment and prevention of forgetfulness and degenerative brain diseases containing the black rice extract prepared by the above production method and the above production method as an effective ingredient.

The black rice extract prepared above was confirmed that an excellent memory improvement in Morris water maze test with performance degradation in animal models that the BCCAO (Experiment 1), induced by glutamate in effect experiments acting on the Ca ^{2 +} signal by the glutamate the intracellular Ca ^{2 +} was the signal is inhibited (example 2), from the efficacy evaluation tests on the hippocampus nerve cell death caused by glutamate, by checking inhibited significantly the cell death caused by glutamate, red (experiment 1 ) For improving memory and for treating and preventing degenerative brain diseases.

The composition of the present invention contains 0.1 to 50% by weight of the extract, based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

Compositions comprising the extract of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Silicates, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, 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 formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . 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. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the extract of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the administration route and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the extract is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Therefore, the dose is not intended to limit the scope of the present invention in any aspect.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, including, for example, oral and rectal, or intravenous.

Further, the present invention provides a health functional food for amelioration of forgetfulness and prevention and improvement of degenerative brain diseases containing black rice extract as an active ingredient.

The health functional food containing the extract of the present invention can be used variously for medicines, foods and beverages for prevention and improvement of degenerative brain diseases. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamin complexes, health supplements and the like, and they can be used as powders, granules, tablets, capsules or beverages have.

Accordingly, the present invention also provides a food and food additive containing, as an active ingredient, a black rice extract having amelioration of forgetfulness and prevention and improvement of degenerative brain diseases.

The food forms to which the extract of the present invention can be added include various foods of candy, beverages, gums, tea, vitamin complexes, or foods that are health supplement foods.

The extract of the present invention can be added to foods or beverages for the purpose of preventing and improving degenerative brain diseases. At this time, the amount of the extract in the food or beverage is generally 0.01 to 15% by weight of the total food weight of the health food composition of the present invention, and the health beverage composition is 0.02 to 10 g based on 100 ml, Can be added at a ratio of 0.3 to 1 g.

The health beverage composition of the present invention may contain various flavors or natural carbohydrates as an additional ingredient, such as ordinary beverages, in addition to containing a mixture of the above extract as an essential ingredient in the indicated ratios, have. Examples of the above-mentioned natural carbohydrates include monosaccharides such as disaccharides such as glucose and fructose such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like Sugar, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the compositions of the present invention may contain flesh for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

The extract of the present invention was found to have excellent memory improving effect in the Morris water maze experiment using an animal model of BCCAO cognitive impairment (Experimental Example 1), and the effect of glutamate on Ca ²⁺ signal was examined by glutamate induced intracellular Ca ²⁺ was an inhibition signal (example 2), from the efficacy evaluation tests on the hippocampus nerve cell death caused by glutamate, by checking the significantly inhibited the cell death caused by glutamate enemy (example 1) Improvement of memory and degenerative brain diseases were confirmed.

FIG. 1 is a graph showing the inhibitory effect of black rice extracts on morris water from an animal model deprived of cognitive ability (the animals were fed vehicle (vehicle) or black rice extract (black rice) for 1 week before surgery, (BCCAo group) for 17 minutes, followed by vehicle or black rice extract for 1 week, and the time required to find the platform 6 times a day (every 30 minutes) for 4 days was calculated. sham) were not treated with cognitive impairment and did not treat anything, black rice extract, and control (sham) * p <0.05)
FIG. 2 shows an example of the pathway of the mouse in the actual water tank in the experiment with Morris Sumi in the control sham, vehicle, and black rice on the 5th day of the experiment Lt; / RTI &
Figure 3 shows the inhibitory effect of the black rice extract of the intracellular Ca ^{2 +} concentrations increased by glutamate in cultured rat hippocampal neurons (15 μg / ml, 30 μg / ml, 100 μg / ml) (A 1 minutes when 100 μM of intracellular Ca ^{2 +} concentrations of the glutamate-induced every 30 minutes shows that the reproduction of a control. black rice extract, 15 μg / ml on these glutamate by intracellular Ca ²⁺ concentration increase for the ( B), 30 μg / ml (C), and 100 μg / ml (D), respectively, for 30 minutes each. E was the control and each of the black rice extract (15 μg / ml, 30 μg / ml , 100 μg / ml) compared to the control (** p <0.01).
FIG. 4 shows the inhibitory effect of the black rice extract on the hippocampal neuronal cell death by glutamate in pure hippocampal rat hippocampal neurons (control group (control) in which pure hippocampal hippocampal neurons were cultured with HEPES-HBSS solution Glutamate and black rice extracts were incubated with HEPES-HBSS solution containing 15 μg / ml, 30 μg / ml and 100 μg / ml of glutamate and 100 μg / ml of glutamate-containing HEPES- Of the glutamate and black rice extracts were incubated with HEPES-HBSS solution containing 15 μg / ml, 30 μg / ml and 100 μg / ml of glutamate and black rice extract, respectively. Enzo Science). ** p <0.01 compared to vehicle in control and p <0.05 compared to glutamate in control.

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

However, the following Reference Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Reference Examples and Experimental Examples.

Example 1: Preparation of black rice extract

300 kg of black rice (Wando) dried in Kyungdong market (Seoul, Korea) was purchased and completely dried, and 10 times volume of 50% ethanol (with 0.2% v / w malic acid) was added and extracted 3 times at room temperature for 2 hours The resulting extract was concentrated under reduced pressure using a vacuum concentrator and lyophilized in a freeze dryer at about -80 ° C to obtain 15.3 kg of a 50% ethanol extract of black rice (hereinafter referred to as "EtOH 50") (yield: 5.1%). Black rice was extracted from GMP company Silla Biotech (Sasang-gu, Busan).

Reference Example 1. Drugs and reagents

Scopolamine (S1875), tacrine (A3773) were purchased from Sigma-Aldrich Chemistry Co. And other reagents were purchased from the market. Black rice was purchased from Kyungdong Market (Seoul, Korea).

Reference Example 2. Preparation of experimental animals

Six weeks old ICR mice (26 - 28 g) were supplied from Orient (Seoul, Korea) and used for 5 days in a clean cage of Daegu Han University. (23 ± 2 ° C), humidity (55 ± 10%) and light intensity (12 hours) were adjusted automatically.

Reference Example 3. Statistical Processing

All test results were performed significance tests below one-way ANOVA (one way analysis of variance) was a statistical process by using, P <0.05 level using the Student-Newman-Keuls Test, if the significance is admitted.

EXPERIMENTAL EXAMPLE 1. Efficacy for cognitive function using an animal model deprived of cognition

In order to confirm the efficacy of the black rice extracts obtained in the above Examples on the cognitive function using the animal model of reduced cognitive ability, the following procedure was applied by the method described in the literature (Schiavon et al, Neurotoxicity Research, Epub ahead of print, 2014; http://www.ncbi.nlm.nih.gov/pubmed/24532152).

1-1. mouse BCCAO Cognitive ability  Establishment of degraded animal models

The whole brain ischemia model was established by applying Bilateral Common Carotid Artery Occlusion (BCCAO) described in the literature to 9-week-old C57BL / 6 mice (20-22 g, Coatec, male) (Schiavon et al, 2014).

Animals were treated with isoflurane (0.7 N _{2 ­} O, 0.3 O ₂ ; (1% isoflurane, 2% isoflurane, 1% isoflurane) was used to maintain the body temperature at 36.5-37.5 ° C using a heating pad (8315010, CMA Microdialysis AB, Stockholm, Sweden) . After the animals were completely anesthetized, the common carotid artery was exposed using a microaneurysm clip (18055-3, Fine Science Tools, British Columbia, Canada) Was closed for 17 minutes, and blood flow was reperfused.

During the operation, a laser doppler flowmetry probe (PF5010, Perimed, J, Sweden) was attached to the skull of the animal to measure brain local blood flow, and only animals with blood flow less than 12% of the reference blood flow after total occlusion were used for the experiment (Use when the blood flow on one side of the left / right is below the reference value). In the control group (sham), after animal anesthesia, the carotid artery was exposed and then sutured again. Table 1 shows the blood flow measurement results after bilateral total carotid ligation.

Blood flow measurement after bilateral total carotid ligation Group  ( BCCAo ) CBF  (L / R)% Group  ( sham ) CBF  % BCCAo-1 11.4 6.7 sham-1 100 BCCAo-2 20.1 8.4 sham-2 100 BCCAo-3 21.2 10.1 sham-3 100 BCCAo-4 13.8 8.6 sham-4 100 BCCAo-5 11.3 7.5 sham-5 100 sham-6 100

1-2. Observation of memory change by Morris water maze experiment

(Kim et al., 2006. Kim, DH, Hung, TM, et al., Applied Bioscience and Biotechnology, Gomisin A improves scopolamine-induced memory impairment in mice. Eur. J. Pharmacol., 7 (2006), pp. pp. 129-135).

Using a 9 week-old C57BL / 6 mouse (20-22 g), a black rice extract or vehicle was orally administered to mice at a dose of 300 mg / kg once a day for a total of 7 times, followed by a common carotid artery After cerebral ischemia due to occlusion, the black rice extract or vehicle was administered once a day for one week, and then the memory test was carried out with the Morris water maze test described in the literature for 5 days. The control (sham) is a group without treatment. A platform (diameter 10 cm) was installed in a circular water bath of 1.2 m in diameter and then water was filled until the platform was submerged 3 cm. The water temperature was maintained at 22 ± 2 ° C and the edible skim milk was dissolved in water, Respectively. The mouse was placed in a quadrant of the tank and the time to visit the platform was measured.

When the total experiment time exceeded 120 seconds, it was assumed that it was not able to find the platform, it showed the position of the platform, and it sent 10 seconds on the platform. In the present study, the experiment was performed 6 times a day (every 30 minutes) for 5 days starting from 7 days after bilateral total carotid ligation. Animals were photographed with a digital video camera (SCB-4000, Samsung Techone) and the results were calculated using PANLAB's SMART software (PANLAB, Barcelona, Spain).

As a result of the experiment, it took a long time to find the platform in both the vehicle treated with the vehicle on the first day of the test, sham and black rice. However, as time went on, the time to search for the platform was faster than that of the vehicle group in the black rice group (control group = 28.6 (mean) ± 5.3 (Standard error) seconds, black rice extract group 24.9 ± 4.7, vehicle group = 60.1 ± 10.2 sec). This was confirmed on the fifth day through the example of the moving zone in the water tank of the Morris water maze experiment (see FIG. 2). Therefore, the administration of black rice extract inhibited the decline of memory by BCCAo.

Experimental Example 2: Preparation of glutamate-containing Ca ²⁺ Effects on signals

The embodiment a black rice extract have a stroke and application of the rat hippocampal neuron model studies the procedure described in the literature in order to confirm the effect of acting on the Ca ^{2 +} signal by glutamate is known to play an important role in nerve cell death, etc. dementia obtained in Example (Kim et al., 2013).

Sprague Dawley rats (300-350 g, Coatec), 17 days after pregnancy, were anesthetized with 0.8 ml per 100 g body weight with 16% urethane (U2500, Sigma) Blood hippocampal mixed cells are cultured in Dulbecco's modified eagle's medium (DMEM) (16000-044, Invitrogen) with 10% fetal bovine serum-free glutamine. The medium was replaced with DMEM containing 10% horse serum (16050-122, Invitrogen) after 72-90 hours, and 25% of the medium was replaced every week, and the brain hippocampal neurons Respectively. Intracellular Ca ²⁺ concentration was measured by fura-2-AM (F1201, Invitrogen). First, 100 μl of glutamate (G1626, Sigma), which is known to play an important role in neuronal cell death in stroke and dementia, was treated with 1 μM fura-2-AM for 45 min. after the minute processing observed an increase in the intracellular Ca ²⁺ concentration (peak1), it was observed to increase (peak2) of the intracellular Ca ²⁺ concentration in cells by re-glutamate and then processes only 30 minutes vehicle (see Fig. 3A). In the experimental group, the increase of intracellular Ca ²⁺ concentration (peak 1) by the first glutamate was observed, and then 15 μg / ml, 30 μg / ml and 100 μg / ml of black rice extract were treated for 30 minutes, The effect of black rice extract was observed by observing an increase in intracellular Ca ²⁺ concentration (peak ² ) (see FIG. 3B-D). The effect of black rice extract was analyzed with the ratio of the response to the second glutamate treatment (peak 2 / peak 1 = mean ± standard error) for intracellular Ca ²⁺ concentration increase by the first glutamate treatment (see FIG. 3E). In the control group, it was confirmed that the intracellular Ca ²⁺ concentration increase by the first glutamate treatment was well reproduced by the second glutamate treatment after 30 minutes (peak 2 / peak 1 = 102.7 ± 3.1%). The increase in intracellular Ca ²⁺ concentration by glutamate was significantly inhibited by the concentration of black rice extract (15 μg / m = 76.1 ± 3.1%; 30 μg / ml = 55.7 ± 3.5% 100 μg / ml = 17.5 ± 2.1%) (see FIG. 3). Therefore, it is suggested that black rice extract inhibits intracellular Ca ²⁺ concentration by glutamate treatment.

Experimental Example 3. Evaluation of efficacy against glutamate-induced brain hippocampal neuronal apoptosis

In order to confirm the evaluation of the efficacy against glutamate-induced brain hippocampal neuronal cell death of the black rice extracts obtained in the above examples, the following experiment was conducted by applying the rat hippocampal neuron model study method described in literature (Zhao et al., J. Cereb Blood Flow Metab. 20: 550-562, 2000).

By a glutamate observed in the study of intracellular Ca ^{2 +} signal black rice extract suppressed the effect is 17 be a type Sprague Dawley rats after pregnancy the experiment to make sure that affect apoptosis of hippocampus cells by glutamate for the (female , 300-350 g, Coatech). Pregnant rats were anesthetized with 16% urethane, and the hippocampus was separated from the fetus into single cells. To isolate pure neurons, 40,000 cells were plated in 96 wells per well and cultured in Neurobasal medium containing B-27, penicillin / streptomycin (100 U / ml and 100 / ml), 0.5 mM glutamine and 25 glutamate Minus Phenol Red (12348-017, Gibco / Life Technologies) and replaced once every 3-4 days with no glutamate-containing medium (Brewer et al, J Neurosci Res 35: 567576, 1993). After 11 days of cell culture, the cells were suspended in HEPES-buffered Hanks solution (HEPES-HBSS solution, 20 mM HEPES, 137 mM NaCl, 1.3 mM CaCl ₂ , 0.4 mM MgSO ₄ , 0.5 mM MgCl ₂ , 0.4 mM KH ₂ PO ₄ , 0.6 mM The control group, which was cultured for 24 hours in Na ₂ H ₂ PO ₄ , 3.0 mM NaHCO ₃ , 5.6 mM glucose (Sigma), the glutamate group and the black rice extract cultured with 100 glutamate-containing HEPES-HBSS solution, , 30 μg / ml and 100 μg / ml of HEPES-HBSS solution containing 100 μg / ml and 100 μg / ml of glutamate and black rice extract, respectively. (Meng et al, J Neurosci, 33: 13505-13517, 2013). Cell viability was assessed using the Cell Counting Kit-8 method (Enzo Science).

As a result, the survival rate of the control group cultured with HEPES-HBSS solution was 100%, and the survival rate of the glutamate group was significantly reduced to 50.8 ± 1.4%. The survival rate of the black rice extract and glutamate were not significantly different from those of the control group. However, survival rates of the black rice extract and glutamate were 65.4 ± 2.1% at 30 μg / ml and 64.2 ± 3.4% at 30 μg / 5) and 100 μg / ml (63.1 ± 3.0%), respectively (Fig. 4). Therefore, it is suggested that black rice extract inhibits apoptosis in glutamate treatment.

Hereinafter, formulation examples of the composition containing the extract of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation example  One. Sanje  Produce

EtOH 50 ----------------------------------------------- 20 mg

Lactose ------------------------------------------------ 100 mg

Talc ------------------------------------------------- 10 mg

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

Formulation example  2. Preparation of tablets

EtOH 50 ----------------------------------------------- 10 mg

Corn starch ------------------------------------------ 100 mg

Lactose ------------------------------------------------ 100 mg

Magnesium stearate ----------------------------------- 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

EtOH 50 ---------------------------------------------- 10 mg

Crystalline cellulose - 3 mg

Lactose ------------------------------------------ 14.8 mg

Magnesium stearate ------------------------------ 0.2 mg

The above components are mixed in accordance with a conventional method for producing a capsule, and filled in a gelatin capsule to prepare a capsule.

Formulation example  4. Preparation of injections

EtOH 50 ---------------------------------------------- 10 mg

Mannitol --------------------------------------------- 180 mg

Sterile sterile distilled water for injection -------------------------------- 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O ----------------------------------------- 26 mg

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

Formulation example  5. Liquid  Produce

EtOH 50 ----------------------------------------------- 20 mg

Isolation Party ---------------------------------------------- 10 g

Mannitol ------------------------------------------------- 5 g

Purified water ------------------------------------------------

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation example  6. Manufacture of health food

EtOH 50 -------------------------------------------- 1000 mg

Vitamin mixture ----------------------------------------

Vitamin A Acetate --------------------------------- 70 [mu] g

Vitamin E ------------------------------------------- 1.0 mg

Vitamin B1 ----------------------------------------- 0.13 mg

Vitamin B2 ----------------------------------------- 0.15 mg

Vitamin B6 ------------------------------------------ 0.5 mg

Vitamin B12 ----------------------------------------- 0.2 g

Vitamin C -------------------------------------------- 10 mg

Biotin ---------------------------------------------- 10 g

Nicotinic acid amide 1.7 mg

Folic acid ------------------------------------------------ 50 G

Calcium pantothenate -------------------------------------- 0.5 mg

Inorganic mixture ----------------------------------------

Ferrous sulfate - 1.75 mg

Zinc oxide - 0.82 mg

Magnesium carbonate - 25.3 mg

Potassium phosphate monohydrate 15 mg

Secondary calcium phosphate ---------------------------------------- 55 mg

Potassium citrate ----------------------------------------- 90 mg

Calcium carbonate ------------------------------------------ 100 mg

Magnesium Chloride ------------------------------------- 24.8 mg

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.

Formulation example  7. Manufacture of health drinks

EtOH 50 ----------------------------------------- 1000 mg

Citric acid ----------------------------------------- 1000 mg

Oligosaccharides ----------------------------------------- 100 g

Plum concentrate ----------------------------------------- 2 g

Taurine --------------------------------------------- 1 g

Add purified water - 900 ml total

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was heated at 85 DEG C for about 1 hour with stirring, and the solution thus prepared was filtered to obtain a sterilized 2-liter container, which was sealed and sterilized, &Lt; / RTI &gt;

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

Claims (8)

A first step of washing and drying the black rice sample; (V / w) relative to the volume of the extraction solvent in order to remove the water or 5 to 80% ethanol from the sample weight by 5 to 15 times (w / v) A second step of adding malic acid, citric acid, or formic acid in weight to an extraction solvent and repeating extraction 1 to 10 times at room temperature for 1 to 6 hours at a reaction temperature of 10 to 120 캜; A third step of filtrating and concentrating the extract obtained in the above step under reduced pressure; Wherein the concentrated extract is lyophilized at -50 ° C to -30 ° C for 24 to 72 hours to improve dryness and dementia, an Alzheimer's disease, a Parkinson's disease, or Huntington's disease, A pharmaceutical composition for the treatment and prevention of diseases. delete delete A first step of washing and drying the black rice sample; (V / w) relative to the volume of the extraction solvent in order to remove the water or 5 to 80% ethanol from the sample weight by 5 to 15 times (w / v) A second step of adding malic acid, citric acid, or formic acid in weight to an extraction solvent and repeating extraction 1 to 10 times at room temperature for 1 to 6 hours at a reaction temperature of 10 to 120 캜; A third step of filtrating and concentrating the extract obtained in the above step under reduced pressure; Wherein the concentrated extract is lyophilized at -50 ° C to -30 ° C for 24 to 72 hours to improve dryness and dementia, an Alzheimer's disease, a Parkinson's disease, or Huntington's disease, Health functional food for prevention and improvement of disease. The health functional food according to claim 4, wherein the health functional food is powder, granule, tablet, capsule or beverage. delete delete delete

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