KR20160011731A - Composition for prevention, improvement or treatment of neurodegenerative disease containing grateloupia filicina extract - Google Patents

Abstract

The present invention relates to a composition comprising a Grateloupia filicina extract as an active ingredient for preventing, alleviating, or treating brain diseases. More specifically, the present invention relates to a composition for preventing or treating brain diseases, which comprises a Grateloupia filicina extract as an active ingredient and has no cytotoxicity with respect to nerve cells. In addition, the composition is capable of preventing, alleviating, or treating the nerve cells damaged by oxidative stress, and improving the resistance against oxidative stress, and antioxidant effects.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing, ameliorating or treating brain diseases,

The present invention relates to a composition for preventing, ameliorating or treating brain diseases, which comprises as an active ingredient a genus ali extract. More particularly, the present invention relates to a composition for preventing, ameliorating or treating a damaged neuron due to oxidative stress, Or an antioxidative effect and an oxidative stress tolerance to a composition for preventing or treating brain diseases.

Oxygen (O ₂ ), an indispensable element in the life of an organism, is a necessary substance to sustain life. However, oxygen, which is an essential element, does not produce free radicals in the body's reactive metabolites and becomes unbalanced, causing oxidative stress, and this oxidative stress induces a change in intracellular redox status . This change induces the formation of a superoxide anion (O ₂ .sup.-), which is a progenitor of another ROS, by binding one electron to oxygen (O ₂ ) and inducing the production of xanthine oxidase ), And an enzyme such as glucose oxidase binds electrons to oxygen to generate hydrogen peroxide (H ₂ O ₂ ).

Reactive Oxygen Species (ROS), such as H ₂ O ₂ produced by oxidative stress, are known to cause oxidative damage to cells or tissues. In addition, reactive oxygen species are known to be involved in various diseases such as aging and inflammation, ischemia, stroke, Parkinson ' s disease, Alzheimer ' s disease through enzyme catalysis in vivo, electron transfer in mitochondria, cell signal transduction system and gene expression, It is causing diseases such as illness.

Astrocytes are the most important neurons in the central nervous system (CNS), and play a role in maintaining normal brain physiology by simultaneously functioning with supportive function and metabolism. In addition, astrocytes have been reported to play an important role in regulating the response of the brain to neuronal damage and also in the mechanism of expression of various neurological diseases.

These stellate cells are activated upon damage of the central nervous system and function to regenerate the central nervous system by cell and molecular interaction. However, in the case of activated astrocytes, glial scars composed of chondroitin sulphate proteoglycan (CSPG) are formed to inhibit the regeneration of nerve cells (Michale V. Sofroniew, Acta Neuropathol, 2010 , Astrocytes: biology and pathology). In recent years, some researchers have attempted to use neural tissue-derived cells as therapeutic agents. Astrocytes have been studied for the treatment of astrocytic cells, which protect against reactive oxygen species and which cure by implanting astrocytes into damaged brain tissue, which is a supporting cell that occupies a significant portion of the brain.

Recently, attention has been paid to natural products as an antioxidant and anti-inflammatory agent for oxidative damage caused by reactive oxygen species such as hydrogen peroxide (H ₂ O ₂ ). Natural products are becoming increasingly popular because they have no side effects than conventional chemotherapeutic agents and do not need to be synthesized, which saves time and money, and is more effective and safe. Thus, there is an increasing number of studies on algae such as green algae, brown algae, and red algae that grow in the ocean, one of the environmentally friendly sources.

Among these seaweeds, Grateloupia filicina , which belongs to the red algae, is a reddish red algae that grows on the east coast of Korea. It is also used for edible purposes and is known to have an effect as a natural antioxidant. It is also known to be effective for lipid peroxidation and DNA oxidation, which is an antioxidant that mediates cell damage, and is effective for preventing aging and obesity. Currently, there are various researches on environmentally friendly seedling methods of Jinuari plants, and it has been reported that mass production of Jinuari biomass is possible by preserving the marine coasts without pollution.

Korean Patent Publication No. 10-2011-0117898 Korean Patent Publication No. 10-2007-0089310

In order to solve the problems of the prior art as described above, the present invention provides a method for preventing or treating neuronal cells damaged by oxidative stress, which is free from cytotoxicity to nerve cells, Prevention or amelioration or treatment of diseases caused by cancer.

The present invention also relates to a method for preventing, ameliorating or treating brain diseases, which comprises enhancing the molecular activity of cells and enhancing antioxidative and tolerance to oxidative stress by enhanced neurons as an active ingredient And to provide a composition.

It is another object of the present invention to provide a composition for preventing, ameliorating or treating brain diseases, which comprises as an active ingredient an extract of Genus alba, which is suitable for use in medicine such as cell therapy or drug delivery by oxidative damage.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating brain diseases, which comprises as an active ingredient an extract of cinnamurus.

The extract can be extracted with water or an organic solvent. In particular, the extract is preferably a water extract or a 70% ethanol extract.

Preferably, the extract contains 50% by weight or more of phycoerythrin in the extract.

It is preferable that the genus ali extract is contained in an amount of 0.01 to 95% by weight based on the total weight of the pharmaceutical composition.

The brain disease may be selected from the group consisting of stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em- Transient ischemic attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, brain circulatory metabolic disorder, brain function coma, and the like.

The present invention also provides a food composition for preventing or ameliorating brain diseases, which comprises as an active ingredient an extract of Guinigi.

According to the present invention, it is possible to prevent, ameliorate, or treat damaged neurons due to oxidative stress without cytotoxicity to nerve cells, including genus extract as an active ingredient, to strengthen the molecular activities of cells, The nerve cells can improve the antioxidative and tolerance to oxidative stress. In addition, the compositions of the present invention are suitable for use in medical applications such as cell therapy or drug delivery due to oxidative damage.

1 is a graph showing the results of reaction of mouse astrocytic cells with oxidative stress according to an embodiment of the present invention.
FIG. 2 is a graph showing the results of measurement of the content of phytoerythrin contained in a 70% ethanol extract of Guinoura and a water extract of Guinouraia according to an embodiment of the present invention.
FIG. 3 is a graph showing the results of exposure to oxidative stress (B, D) and unexposed (A, C. standard) mice exposed to oxidative stress by hydrogen peroxide in mouse astrocytes pretreated with a 70% Lt; / RTI > cell proliferation and cell proliferation.
FIG. 4 is a graph showing the results of measurement of cell viability (A) and cell proliferation (B) when exposed to oxidative stress by hydrogen peroxide in mouse astrocyte cells pretreated with a water extract of Juniperia according to an embodiment of the present invention to be.
FIG. 5 is a graph showing the cell survival rate of oxidatively damaged mouse astrocyte cells and the cytotoxicity through cell proliferation using a water extract of Jinuari according to an embodiment of the present invention. FIG.
FIG. 6 is a graph showing the results of confirming the ability to remove reactive oxygen species in astrocytes exposed to oxidative stress by hydrogen peroxide in mouse astrocyte cells pretreated with water extract of Juniperia according to an embodiment of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for treating asthma cells isolated from a mouse cerebral cortex by treating the extract of Jinnuri with an effect on cell viability and proliferation, The present invention has been completed based on the finding that the extract of Jinuari, which is an active ingredient of the present invention, has a novel potential as a therapeutic agent for neuronal cells.

The present invention provides a pharmaceutical composition for preventing or treating brain diseases, which comprises as an active ingredient an extract of cinnamurus.

The extract can be obtained by extracting and isolating from nature using an extraction and separation method known in the art. The 'extract' defined in the present invention is extracted from guinea using an appropriate solvent, For example, crude extracts of guinea, polar solvent-soluble extracts, or non-polar solvent-soluble extracts.

As an appropriate solvent for extracting the extract from the genus Arina, any pharmaceutically acceptable organic solvent may be used, and water or an organic solvent may be used, but the present invention is not limited thereto. Examples of the solvent include water, alcohols having 1 to 4 carbon atoms such as methanol, ethanol, propanol, isopropanol, and butanol, acetone, ether various solvents such as ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane and cyclohexane may be used alone or as a mixture of two or more thereof. Can be used. Particularly, it is preferable to use water or 70% ethanol as the solvent.

Examples of the extraction method include hot water extraction, cold extraction, reflux cooling, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression. The desired extract may further be subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation on the method for producing the extract of Guinoura extract of the present invention, and any known method can be used.

For example, the genus allium extract contained as an active ingredient in the composition of the present invention may be prepared in powder form by an additional process such as vacuum distillation, freeze drying, spray drying, or the like, by extracting the primary extract extracted by the hot water extraction or solvent extraction method can do. In addition, the primary extract can be further purified into a further purified fraction using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography and the like Can be obtained.

Therefore, the Jinuari extract used in the present invention is a concept including all the extracts, fractions and tablets obtained in each step of extraction, fractionation or purification, their diluted solutions, concentrates or dried products.

Hereinafter, a method for preparing the extract of Guinoura extract according to an embodiment of the present invention will be described in detail.

In the present invention, the Jinuori is washed and ground with a blender, and is then washed with water or a lower alcohol having 1 to 4 carbon atoms, preferably water or 70% ethanol, corresponding to about 5 to 15 times the weight of the Jinuari sample, For 3 to 15 hours. The obtained guanyllium or 70% ethanol extract may be subjected to filtration and concentration under reduced pressure according to a conventional method to obtain a water extract of Guinoura or a 70% ethanol extract of Guinari.

Preferably, the extract is a water extract of Jinuari or a 70% ethanol extract of Jinuari. In the case of the water extract of Jinuari or the extract of 70% ethanol of Jinuari, the activity of the cell and the viability of the astrocytes due to oxidative stress And phycoerythrin which acts to increase the proliferation in high purity.

Therefore, it is preferable that the genino extract of the present invention contains a large amount of phycoerythrin isolated from cinnamon. Particularly, it is preferable that 50% or more by weight of phycoerythrin is contained in the above extract, more preferably 100% of phycoerythrin. When the content of the picoerythrin is less than 50% by weight, it is preferable to increase the viability, proliferation and molecular activity of damaged astrocytes due to oxidative stress, and to reduce active oxygen species in cells or tissues.

The above-mentioned extract is preferably contained in the pharmaceutical composition of the present invention in an amount of 0.01 to 95% by weight, more preferably 1 to 80% by weight. If the content is less than 0.01% by weight, the efficiency of taking may be poor. If the content is more than 95% by weight, formulation may be difficult.

The brain disease may be selected from the group consisting of stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em- But are not limited to, transient ischemic attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, brain circulatory metabolic disorders, brain functional coma, and the like.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of pharmaceutical compositions. In addition, it can be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to a conventional method. Suitable formulations known in the art are preferably those as disclosed in Remington ' s Pharmaceutical Science, recently, Mack Publishing Company, Easton PA. Examples of carriers, excipients and diluents which may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When the composition is formulated, it is prepared using a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, or an excipient 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 and 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 sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. 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 term "administering" as used herein is meant to provide any desired composition of the invention to a subject in any suitable manner.

The present invention relates to pharmaceutical compositions comprising an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as contemplated by a researcher, veterinarian, physician or other clinician, RTI ID = 0.0 > effective < / RTI > amount. It will be apparent to those skilled in the art that the therapeutically effective dose and frequency of administration for the pharmaceutical compositions of the present invention will vary with the desired effect. Thus, the optimal dosage to be administered can be readily determined by those skilled in the art and will vary with the nature of the disease, the severity of the disease, the amount of active and other ingredients contained in the composition, the type of formulation, and the age, The age, body weight, sex, diet, time of administration, route of administration and fraction of the composition, duration of treatment, concurrent medication, and the like. For a desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg / kg / day, preferably 1 to 200 mg / kg / day, or may be administered once a day, . ≪ / RTI >

The pharmaceutical compositions of the present invention can be administered to a subject in a variety of routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

In addition, the composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy or biological response modifiers for the prevention or treatment of brain diseases.

The present invention also provides a food composition for preventing or ameliorating brain diseases, which comprises as an active ingredient an extract of Guinigi. When the extract of the present invention is used as a food additive, it can be suitably used according to a conventional method such as adding the extract as it is or mixing it with other foods or food ingredients.

It is of course possible to appropriately change the extract according to the intended use (preventive, health or therapeutic treatment), and it is particularly preferable that the extract is contained in the food composition of the present invention in an amount of 0.01 to 95% by weight, Preferably 1 to 80% by weight. If the content is less than 0.01% by weight, the efficiency of taking may be poor. If the content is more than 95% by weight, formulation may be difficult.

As a specific example, the juice extract of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material. However, when it is intended for health and hygiene purposes or for the purpose of controlling health, it can be added in an amount below the above range, and there is no problem in terms of safety. Therefore, the active ingredient can be used in an amount exceeding the above range have.

There is no particular limitation on the kind of the food. Examples of the food to which the extract of the present invention can be added include food products such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, Tea, a drink, an alcoholic beverage, a vitamin complex, and the like.

When the food composition of the present invention is prepared as a beverage, it may contain additional ingredients such as various flavors or natural carbohydrates such as ordinary beverages. Examples of the natural carbohydrate include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame, and the like. The natural carbohydrate is contained in an amount of 0.01 to 10% by weight, preferably 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, , Carbonating agents used in carbonated beverages, and the like. In addition, the compositions of the present invention may include flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of the above additives is not limited to a great extent, but may be in the range of 0.01 to 0.1% by weight based on the total weight of the food composition of the present invention.

The composition of the present invention comprising the extract of Guinneia as an active ingredient protects nerve cells, particularly astrocytes, damaged by oxidative stress caused by active oxygen species such as hydrogen peroxide (H ₂ O ₂ ) Increase proliferation and cell activity, and reduce tissue and intracellular reactive oxygen species. In addition, the extract of Jinroi, which is an active ingredient of the present invention, strengthens the molecular activity of the cells, and the strengthened astrocytes can improve the antioxidative and tolerance by oxidative stress, It is also suitable for use in applications such as therapy and drug delivery.

Hereinafter, the present invention will be described in more detail with reference to examples. These embodiments are for purposes of illustration only and are not intended to limit the scope of protection of the present invention.

EXAMPLES Example 1 Preparation of Genuari 70% Ethanol Extract

The Jinuari used in this example was taken directly from the coast of Samcheok Executive Offices in Gangwon Province. To remove contaminants such as salt, epiphytes, and sand in Jinuni harvested, they were washed twice with clean water and then thoroughly washed with distilled water. The washed guinea pigs were quickly replaced with a blender. 20 g of Blender Jinuari was placed in a beaker, and 70% ethanol was added as an extraction solvent, followed by stirring at 60 ° C for 6 hours. The extract was dialyzed with distilled water for 3 days and then lyophilized to obtain a guinari 70% ethanol extract.

Example 2 Preparation of water extract of cinnamon

A water extract of Guinapialy was obtained in the same manner as in Example 1, except that 20 g of guanilla obtained in Example 1 was placed in a beaker and water was added as an extraction solvent and stirred at 60 ° C for 6 hours.

Experimental Example

Isolation and culture of astrocytes

Primary astrocytes were isolated from the brain cortex of Day 1 Sprague-Dawley mice (SAMTACO, Korea). The density of the water film (meninges) is removed, a Sprague Dawley (SD) 4 × 10 6 cells / well in a mixed cell 75T- flask containing the medium of 16㎖ extracts of mouse cerebral finely pulverized with a Pasteur pipette haejugo enzyme treatment , And cultured in an incubator of 5% CO ₂ at 37 ° C. One week after the culture, the medium was removed, and DMEM medium was added, followed by further incubation for 24 hours. After 24 hours, all of the medium is removed and only the astrocytes remain on the bottom of the culture flask. The isolated stellate cells were used in the experiment with fresh DMEM medium once every 3 days in a 5% CO ₂ incubator at 37 ° C.

Experimental roadmap for external factor therapy and analysis of astrocytes

To investigate the survival and proliferation of astrocytic cells, 6 x 10 ³ cells / well of passage 2 astrocytic cells were inoculated into a 96-well plate containing 200 μl of medium. For analysis of reactive oxygen species (ROS), 2.5 x 10 ⁴ cells / well of passage 2 astrocytes were inoculated into a 24-well plate containing 1 ml of medium.

Experimental Example 1. Response of astrocytes to oxidative stress

To determine the response of mouse astrocytic cells to oxidative stress, cell viability of Passage 3 astrocytes exposed to oxidative stress by hydrogen peroxide was investigated using MTT assay.

First, the mouse Passage 3 astrocytes were inoculated on a plate for each assay. When oxidative stress caused by hydrogen peroxide was applied, the astrocytic culture medium was further cultured for 24 hours at 37 ° C with 5% CO _{2 in} a medium containing 500 μM hydrogen peroxide. 200 [mu] l (0.5 mg / ml) of the MTT solution was added to each of the cell culture plates and further cultured for 1 hour. Then, the MTT solution was removed, 200 ml of DMSO was added to each well, and the mixture was dissolved by shaking at 150 rpm for 1 hour. The absorbance of the solution was then measured at 540 nm using a Sun rise ELISA reader (Tecan, Austria). The results are shown in Fig.

As shown in Fig. 1, similar cell survival rate was observed on day 1 when exposed to oxidative stress by hydrogen peroxide (Fig. 1A) and when not exposed to oxidative stress by hydrogen peroxide (Fig. 1B). However, in the case of not being exposed to oxidative stress by hydrogen peroxide, the cell survival rate of astrocytes was gradually increased (FIG. 1B), but when the oxidative stress was exposed by hydrogen peroxide, the cell survival rate was gradually decreased (Fig. 1A).

These results show that the cell survival rate of astrocytes is reduced by oxidative stress caused by hydrogen peroxide and that oxidative stress due to hydrogen peroxide can be prevented by pretreatment of the extract of the present invention.

Experimental Example 2: Absorption and fluorescent spectrum of guinari

In order to measure the content of phytoerythrin, which is known to enhance the activity of the cells contained in the extracts of P. cinerea and antioxidant effect, the absorption spectrum of the Cinnamoi extract of Examples 1 and 2 was measured using an automatic spectrophotometer, a fluorescence analyzer (Thermo fisher sctentific , Finland). Absorption spectra were recorded at 300-700 nm with increasing 10 nm at room temperature. Purity was measured according to the OD560 / OD280 absorbance ratio. Fluorescence emission and excitation wavelength were measured at 580 nm, 498 nm. The results are shown in Fig.

As shown in FIG. 2, the phytoerythrin component can be confirmed in both the 70% ethanol extract of Jinuari and the water extract of Jinuari in 70% ethanol and water in Examples 1 and 2 above. In particular, the water extract of Example 2 using water as the extraction solvent showed a slightly higher absorption peak than that of the extract of Shinuari 70% ethanol, and the purity was also higher than that of the water extract of Shinuari (OD560 / OD280 = 3.75) It was confirmed that the purity was higher than the 70% ethanol extract of Jinju (OD560 / OD280 = 1.93).

Experimental Example 3 Effect of Genuari 70% Ethanol Extract on Stellate Cell Survival and Proliferation Damaged by Oxidative Stress

In order to observe whether or not the extract of Jinuari protects damaged mouse astrocytes due to oxidative stress, the cells pretreated with the Jinuari 70% ethanol extract of Example 1 for 1, 2, and 3 days were exposed to hydrogen peroxide, The proliferation was observed, and the results are shown in Fig. At this time, cell viability was confirmed by the same MTT assay as in Experimental Example 1, and cell proliferation was confirmed by BrdU assay.

The BrdU assay was performed as follows. First, 100 μl of each medium was removed from the prepared samples, 10 μl / well of BrdU labeling solution was added to a final volume of 100 μl / well, and the cells were cultured in a 5% CO ₂ incubator at 37 ° C. for 2 hours. After the labeling medium was removed by tapping off and suction, 200 μl of FixDenate solution was added to the medium and incubated at room temperature for 30 minutes. Subsequently, FixDenat solution was removed by tapping and flicking, and 100 μl / well of anti-BrdU-POD solution was added to the sample, followed by incubation at room temperature for 90 minutes. After removing the antibody complex by tapping, each sample was washed three times with 200 μl / well of PBS solution and reacted with 100 μl / well of substrate solution for 30 minutes at room temperature. 25 [mu] l / well 0.1 M sulfuric acid was added to the sample to stop the reaction. Then, the fluorescence was measured by 450 nm ELISA after shaking for 1 minute to confirm the reaction.

Experimental Results As shown in FIG. 3, the cell survival rate of astrocytes exposed to oxidative stress by hydrogen peroxide after the pre-treatment with the 70% ethanol extract of Jinuari in Example 1 was found to be time-dependent , And it was higher than that of the control without the extract of guinea pig. In particular, it was confirmed that the cell survival rate was higher at a concentration of 1,000 to 2,000 ng / ml (FIG. 3D). On the other hand, in the standard state, when the 70% ethanol extract of Guinoura was added, the cell survival rate was time-dependent as compared with the control not added (Fig. C). Also, in the case of cell proliferation, astrocytes exposed to oxidative stress caused by hydrogen peroxide showed a significant increase in cell proliferation at a concentration of 70 ng / ml ethanol at 1,000 ng / ml (Fig. 3B).

From these results, it can be seen that the 70% ethanol extract of Jinroi, which is an active ingredient of the present invention, positively influences the cell survival rate and cell proliferation against oxidative stress.

EXPERIMENTAL EXAMPLE 4 Effect of Water Extract of Jinuari on Stellate Cell Survival and Proliferation Damaged by Oxidative Stress

In order to observe whether or not the extract of Jinuari protects damaged mouse astrocytes due to oxidative stress, the cells pretreated with the water extract of Example 2 of Example 2 were exposed to hydrogen peroxide for 1, 2, and 3 days, and cell viability and cell proliferation The results are shown in Fig. At this time, cell viability and cell proliferation were confirmed in the same manner as in Experimental Example 3.

As shown in FIG. 4, the astrocytes exposed to oxidative stress by hydrogen peroxide after pretreatment with the water extract of Example 2 had a significantly increased cell proliferation compared to the control without the pre-treatment of Jinuari extract , And it was confirmed that the cell proliferation was maximally increased at a concentration of 1,000 to 2,000 ng / ml (Fig. 4A). In addition, the most prominent increase in cell proliferation was observed at the concentration of 1000 ng / ㎖ of water extract of Jinuari, compared with that of untreated astrocytes in the case of astrocytes pretreated with water extract of Jinuari It was confirmed that the cell proliferation was very high.

As a result, the water extract of Cinnamus japonica, an active ingredient of the present invention, improved the survival rate and cell proliferation of astrocytes damaged by oxidative stress and decreased the oxidative stress caused by oxidative damage caused by active oxygen species such as hydrogen peroxide And the survival rate can be efficiently recovered.

Experimental Example 5: Cytotoxicity test

The cytotoxicity of the water extract of Example 2 was tested to confirm that it was stable without toxicity in oxidatively damaged mouse astrocytic cells, and the results are shown in FIG.

In the standard state shown in Fig. 5A, the cell survival rate on day 1 and day 2 was small, while the increase in survival rate between mouse astrocytes treated with water extract of Example 2 and control was insignificant, but on day 3, The results are shown in Fig. In addition, the cell proliferation shown in FIG. 5B showed that the cell proliferation was increased depending on the concentration and treatment time of the water extract. Especially, the cell survival rate and proliferation were significantly increased at a concentration of 1,000 ~ 2,000 ng / ㎖ of water extract. From the above results, it can be seen that the water extract of Jinroari as an active ingredient of the present invention is stable without toxicity to mouse astrocytic cells.

Experimental Example 6. Determination of the ability to remove active oxygen species from the extract of Cinnamoides

2 ', 7'-dichlorodihydrofluorescein diacetate (H2DCF-DA, Sigma) was used to examine the ability of the water extract of Example 2, which is an active ingredient of the present invention, to remove ROS generated by hydrogen peroxide (DCF) and CM-H2DCFDA (Invitrogen, Carlsbad, Calif., USA) were used to measure the fluorescence intensity of the active oxygen species (ROS).

For the fluorescent reagent, DCF stock solution was added to DMSO and diluted in PBS before measurement. CM-H2DCFDA (Invitrogen, Carlsbad, CA, USA) with a final concentration of 10 μM was incubated in a CO ₂ incubator for 15 min. The reactive oxygen species were observed by fluorescence microscope after replacing the reaction solution with PBS. To quantify fluorescence, some cells were treated with a fluorescent reagent and analyzed by BCA protein assay (Sigma-Aldrich, St. Louis, Mo., USA). RIPA was treated with cell lysis buffer at 4 DEG C for 30 minutes. Samples were observed using a fluorescence reader at excitation 492 nm and emission 527 nm. The results are shown in Fig.

As shown in FIG. 6, the astrocytes exposed to oxidative stress by hydrogen peroxide pretreated with the addition of water extract of Jinuari showed that the amount of reactive oxygen species produced by oxidative stress due to hydrogen peroxide was less than that of the control It can be confirmed that it represents the phenomenon. Also, similar to the cell viability and cell proliferation results, it was confirmed that when treated with a water extract of Jinnuri water at a concentration of 1,000 ~ 2,000 ng / ml, the active oxygen species was significantly mitigated. In oxidative stress caused by hydrogen peroxide, mouse astrocytes treated with 1,000 and 2,000 ng / ㎖ water extracts showed 21.64% and 18.36% reduction of reactive oxygen species, respectively.

As a result of the above results, it can be seen that the extract of Jinuari, which is an active ingredient of the present invention, increases cell survival rate, cell proliferation and molecular activity in damaged mouse astrocytes by oxidative stress caused by hydrogen peroxide, And it was found that the Jinuari extract having this function can enhance the tolerance by the oxidative stress of the astrocytes and also give the antioxidative effect. In addition, the extract of Genus alba, which is an active ingredient of the present invention, can be used as a drug or an adjuvant for the treatment of neural tissue-derived cells and can be expected to be suitable for use in prevention, improvement or treatment of brain diseases caused by oxidative stress there was.

Hereinafter, specific diseases that can be prevented or treated using the genus extract of the present invention will be described.

Application example 1: Alzheimer's disease (AD)

Alzheimer's disease is accompanied by necrosis of glutamatergic neurons in the cerebral cortex and hippocampus and cholinergic neurons in the basal forebrain of the brain, and is involved in the formation of amyloid plaques and nerve fibers in nerve cells Neurofibrillary tangles are found to be common phenomena. In addition, excessive amounts of active oxygen cause lipid peroxidation, 8-hydroxy deoxyguanosine, protein carbonyls, nitration, oxidative cross-linking of proteins oxidative crosslinking of proteins are known to be increased in Alzheimer's disease (Vitek et al., 1994, Proc. Natl Acad Sci USA, 91, 4766-4770, Smith et al., 1995, Trends. Neurosci Smith, et al., 1996, Mol. Chem. Neuropathol., 28, 41-48, Smith et al., 1997, Proc. Natl Acad Sci USA, 94, 9866-9868 ; Montine et al., 1996, J. Neuropathol. Exp. Neurol., 55, 202-201). Therefore, the Jinuari extract of the present invention, which is excellent in lipid peroxidation inhibitory activity and antioxidative enzyme activity, may be useful for preventing and treating Alzheimer's disease.

Application Example 2: Parkinson ' s disease

Parkinson's disease is a degenerative nervous system disease that is accompanied by necrosis of dopaminergic neurons present in the black matter and exhibits various symptoms such as progression, muscle stiffness, exercise completion, abnormal posture, and inability to exercise. Oxidative toxicity is presented as a major cause of nerve cell necrosis, with increased levels of lipid peroxidation, DNA oxidation, protein carbonyl, and nitrotyrosine found in blacks (Bowling, AC and Beal, MF , Life Sci., 1995, 56 (14), 1151-1171). Therefore, the extract of Guinoura extract of the present invention having excellent lipid peroxidation inhibitory activity and antioxidative enzyme activity can be effectively used for preventing and treating Parkinson's disease.

Application Example 3: Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis mainly starts from the lower motor neurons of the spinal cord and progresses to the upper motor neurons and causes damage to the cerebral cortex, Accompanied by muscle atrophy, weakened respiratory muscle strength, and fibrous spasm, and they die within 2-5 years of the first appearance of symptoms. In particular, genetic amyotrophic lateral sclerosis patients showed mutations in the SOD-1 gene, which removes the superoxide radical located at chromosome 21 (chromosome 21), resulting in oxidative toxicity, (Bowling et al., 1993, J. Neurochem., 61, 2322-2325) have been reported to increase the protein carbonyl group as an indicator of toxicity. Therefore, the extract of Jinuari of the present invention which is excellent in lipid peroxidation inhibitory activity and antioxidation-related enzyme activity can be effectively used for preventing and treating Louuker's disease.

Application Example 4: Hypoxic-ischemic injury

Stroke is a disease caused by a deficiency of glucose and oxygen supplied by a certain amount of blood circulation disorder (thrombosis, embolism, stenosis) of the brain. It is caused by loss of body function regulation by a damaged brain, Cognitive dysfunction occurs. After stroke, membrane depolarization of the cell membrane causes calcium to enter the cell, which then enters the mitochondria, damaging the repiratory chain and increasing the production of reactive oxygen species do. The resultant active oxygen causes neuronal cell necrosis by inducing destruction of cell membrane lipids, genetic damage, protein denaturation, etc., whereas antioxidant has the effect of inhibiting necrosis of ischemic neurons (Holtzman et al., 1996 Et al., 2001, Acta neuropathol. (Berl.), 101 (3), 229-38; Hall et al., 1990, Stroke, 21, 11183-11187). Therefore, the extract of Guinoura extract of the present invention having excellent lipid peroxidation inhibitory activity and antioxidative enzyme activity can be effectively used for preventing and treating ischemic stroke.

Formulation Example 1. Preparation of pharmaceutical preparations

Acid production

20 mg of guinolite extract, 100 mg of lactose, and 10 mg of talt were mixed and filled in airtight bags to prepare powders.

Tablet manufacture

10 mg of cinnamon extract, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate were mixed, and tablets were prepared by tableting according to a conventional preparation method of tablets.

Capsule preparation

10 mg of guineyi extract, 3 mg of crystalline cellulose, 14.8 mg of lactose and 0.2 mg of magnesium stearate were mixed and filled in gelatin capsules according to a conventional capsule preparation method to prepare capsules.

Injection manufacturing

(2 ml) guinea extract extract (10 mg), mannitol (180 mg), sterile distilled water for injection (2,974 mg) and Na ₂ HPO ₄ .2H ₂ O (26 mg) according to the usual injection preparation method.

Liquid preparation

In accordance with a conventional method for preparing a liquid preparation, 20 mg of Jinuari extract, 10 g of isomerized sugar and 5 g of mannitol were added to purified water and dissolved, and the lemon flavor was added in an appropriate amount. Then, purified water was further added thereto, adjusted to a total volume of 100 ml, filled in a brown bottle, and sterilized to prepare a liquid preparation.

Formulation Example 2. Preparation of food preparation

Health food manufacturing

A vitamin A acetate, 70 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 g of vitamin B12, 10 mg of vitamin C, 10 g of biotin, 10 g of nicotinamide 1.7 mg, folate 50 g, calcium pantothenate 0.5 mg, an appropriate amount of inorganic mixture, ferrous sulfate 1.75 mg, zinc oxide 0.82 mg, magnesium carbonate 25.3 mg, potassium phosphate monohydrate 15 mg, dibasic calcium phosphate 55 mg, potassium citrate 90 mg , Calcium carbonate (100 mg) and magnesium chloride (24.8 mg), granules were prepared, and a health food was prepared according to a conventional method. At this time, although the composition ratio of the vitamin and mineral mixture is relatively mixed with the ingredient suitable for health food, it may be arbitrarily modified.

Health drink manufacturing

100 g of vitamin C, 100 g of vitamin E (powder), 19.75 g of iron lactate, 3.5 g of zinc oxide, 3.5 g of nicotinic acid amide, 0.2 g of vitamin A, 0.25 g of vitamin B1, 0.3 g of vitamin B2 and a predetermined amount of water were mixed and heated at 85 DEG C for about 1 hour with stirring. The resulting solution was filtered and sterilized in a 2 L container to be sterilized by sealing, and then stored in a refrigerator to prepare a health drink. At this time, although the composition ratio of the ingredients suitable for the beverage is comparatively mixed, the mixture ratio may be arbitrarily varied according to the demand, the demanded country, the intended use, and the regional or national preference.

Although the present invention has been described in terms of the preferred embodiments mentioned above, it is possible to make various modifications and variations without departing from the spirit and scope of the invention. It is also to be understood that the appended claims are intended to cover such modifications and changes as fall within the scope of the invention.

Claims (9)

A pharmaceutical composition for prevention or treatment of brain diseases, which comprises as an active ingredient an extract of Cinnamoides. The method according to claim 1,
Wherein the extract is extracted with water or an organic solvent as an extraction solvent. The method according to claim 1,
Wherein the genus allium extract is a water extract or a 70% ethanol extract. The method according to claim 1,
Wherein the extract comprises 50% or more by weight of phycoerythrin in the extract. The method according to claim 1,
The pharmaceutical composition for preventing or treating cerebral diseases according to claim 1, wherein the extract is in an amount of 0.01 to 95% by weight based on the total weight of the pharmaceutical composition. The method according to claim 1,
The brain disease may be selected from the group consisting of stroke, stroke, dementia, Alzheimer's disease, Parkinson's disease, Lou Gehrig's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em- Wherein the disease is any one selected from transient ischemic attack, lacune, stroke, cerebrospinal fluid, cerebral infarction, head trauma, cerebral circulatory metabolic disorder, and brain function coma. ≪ / RTI > A food composition for preventing or ameliorating brain diseases, which comprises as an active ingredient an extract of Cinnamoides. 8. The method of claim 7,
Wherein the extract comprises 50% or more by weight of phycoerythrin in the extract. 8. The method of claim 7,
The food composition for preventing or improving brain diseases according to claim 1, wherein the extract is 0.01 to 95% by weight based on the total weight of the food composition.

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