KR101537579B1 - Neuroprotective composition for comprising extracts or fractions of Aspergillus terreus as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for neuroprotection containing an Aspergillus terreus extract or a fraction thereof as an active ingredient, wherein the Aspergillus stearus extract or a fraction thereof contains a glutamate- And thus it has an excellent effect of preventing neuronal cell death induced by neuronal cell toxicity, and is safe for human body because of its lack of cytotoxicity. Therefore, it is possible to prevent cerebral ischemia, stroke, brain trauma, Hypoxia, Huntington's disease, and the like. The present invention also provides a pharmaceutical composition for preventing or treating neuron-related diseases such as hypoxia and Huntington's disease.

Description

[0001] The present invention relates to a neuroprotective composition comprising aspergillus stearus extract or fractions thereof as an active ingredient,

The present invention relates to a nerve-protecting composition containing fungal Aspergillus stearothial extract or a fraction thereof as an active ingredient, or a composition for preventing or treating brain diseases.

Glutamate acts as a major excitatory neurotransmitter in the central nervous system (CNS) and acts as a metabotropic receptor and an ionotropic receptor (NMDA-, quisqualate-, kainate-type ) And exhibits various neurophysiological effects. Glutamate released outside the nerve cell is absorbed directly into the presynaptic terminal or absorbed into the astrocyte (glial cell) by a Na ⁺ -dependent high affinity transporter, It is converted to glutamine by the glutamine synthase and moves to the pre-synaptic terminal (see FIG. 3).

The glutamate concentration outside the cells is maintained at a low concentration of 0.3 μM by the action of these pre-synaptic terminals and astrocytes. However, when the brain is subjected to ischemia, hypoxia, seizure, hypoglycemia, trauma, etc., the energy supply required for glutamate transportation becomes insufficient, and the extracellular glutamate concentration is reduced to a normal state 3,000 mM, which is 10,000 times, which causes the central nerve cell to die. These glutamate-induced neuronal cell deaths are referred to as glutamate-induced neurotoxicity and are closely related to oxidative stress due to the generation of free radicals in the cells . Therefore, it has been clarified that the death of neurons by glutamate toxicity is caused by the destruction of cell components such as lipids, sugars, nucleic acids and proteins caused by free radicals generated in cells excessively. Thus, a free radical scavenger lipid peroxidation inhibitors, such as free radical scavengers, are very important as new therapeutic agents for protecting brain cells.

Increased extracellular concentrations of glutamate are associated with acute diseases such as cerebral ischemia, stroke, brain trauma, hypoxia, hypoglycemia and seizures as well as Huntington's disease (HD), Parkinson's disease (PD), amyotrophic sclerosis lateral sclerosis, ALS), and Alzheimer's disease (AD). NIH research shows that stroke, one of the acute illnesses, is the third leading cause of death in the United States and is the leading cause of adult disability. About half a million patients die each year, of which 30% die, 20-30% become permanent cramps, and the rest suffer from physical disabilities, body paralysis, vision loss, and memory loss. Also, in Korea, cancer is the second leading cause of death following cancer, and a treatment for such diseases is strongly demanded.

Glutamate binds to two receptors, an ionotropic receptor and a metabotropic receptor. Ionic receptors are associated with ion channels and act independently on nerve cells, resulting in excitotoxic damage. On the other hand, in metabolic receptors, glutamate does not cause direct neuronal death, unlike ionic receptors.

Neuronal cell death caused by binding of glutamate to ionic receptors can be divided into Na ⁺ , Cl ^- dependent and Ca ^{2 +} dependent. Na ⁺ and Cl ^- dependent neuronal cells develop within a few minutes after exposure to glutamate and show neuronal cell death similar to the depolarizing agent treatment. Glutamate query Squelch acid receptor (quisqualate receptor) to stimulate the Na ⁺ ion is is excess flow into the cell, with the influx of Na ⁺ ion Cl ^-, finally dissolved in H ₂ O is entered into the cells, cell expansion (swelling) lt; / RTI >

Ca ^{+ 2-dependent} neuronal cell death delay DE nerve injury (delayed neuronal damage) to occur after a few hours after glutamate treatment, similar to when the processing of A23187 Ca ^{2 +} ion permeable carrier (ionophore). Influx of Na ⁺ ion is open the NMDA receptor Ca ^{2 +} channel which is closed by causing depolarization (depolarization) of the cell membrane, which causes the voltage-sensitive Ca ^{2 +} channels (voltage sensitive Ca ^{2 +} channel, VSCC) and Mg ^{2 +} with do. This causes a large amount of Ca ^{+ 2} are to be introduced into the cell. The thus introduced Ca ^{2 +} ion is Phospholipase _{(phospholipase A 2, PLA 2)} , nitric oxide synthase (nitric oxide synthetase, NOS), Protease (protease), endonuclease (endonuclease) Ca ^{2 +} dependence of such Energize enzymes. In particular, PLA ₂ degrades phospholipids to produce arachidonic acid, and free radicals such as superoxide are produced during the metabolism of arachidonic acid.

In addition, arachidonic acid and peroxides promote glutamate release in presynaptic neurons, further increasing the extracellular glutamate concentration. Peroxides are formed by the action of xanthine oxidase. Nitric oxide produced by the action of NOS reacts with peroxides to pass through peroxynitrite and reacts with hydroxyl radicals (hydroxyl radical is generated. In other words, enzymatic action of PLA ₂ , NOS, xanthine oxidase, etc., results in excess radicals in the cells, resulting in oxidative stress resulting in DNA, protein, lipid, And nonselective destruction of the nerve cells, and also endonuclease (endonuclease) by the action of apoptosis may occur.

In order to investigate the mechanism of toxicity of glutamate, Malouf et al. Examined various nerve cell lines and selected N18-RE-105 cell line sensitive to glutamate toxicity. The N18-RE-105 cell line was generated by cell fusion of mouse neuroblastoma clone N18TG-2 and Fisher rat 18-day embryonic neural retina. The N18-RE-105 cell line has a receptor similar to the glutamate receptor present in the hippocampus and exhibits delayed calcium dependent cell death. These characteristics of the N18-RE-105 cell line allow the N18-RE-105 cell line to be used as an in vitro ischemic model. Glutamate toxicity in the N18-RE-105 cell line is known to result from the inhibition of cysteine uptake by glutamate. Cysteine is an important amino acid that constitutes glutathione in cells. It is antiported with glutamate in N18-RE-105 cells. If the concentration of extracellular glutamate increases and cysteine does not enter the cell, the concentration of glutathione decreases and free radicals accumulate in the cell, eventually causing the neuron to die. This fact is also confirmed in the primary culture of the embryonic cortical neurons of the embryo, and the quisqualate-type glutamate toxicity is mainly due to oxidative stress. .

As described above, glutamate causes acute cranial nerve diseases such as stroke and trauma, and oxidative stress due to glutamate toxicity is one of the causes in chronic brain diseases such as PD, AD and HD It is known. Accordingly, antioxidant active substances such as lipid peroxidation inhibiting substances capable of inhibiting or alleviating oxidative stress due to glutamate toxicity are expected to be therapeutic agents for acute and chronic neurodegenerative diseases. Accordingly, in It is anticipated that searching for glutamate toxicity inhibitors using tissues or cultured cells close to vivo will be able to create novel substances with new neuronal cell protection mechanisms as well as substances acting on known targets.

BACKGROUND ART As stroke caused by glutamate is a serious disease worldwide, development of a therapeutic agent for it is actively under way. Particularly, active oxygen has been actively studied for antioxidants such as novel free radical scavengers or lipid peroxidation inhibitors since it is the main ultimate source of neuronal death due to glutamate toxicity. Upjohn developed by 21-amino steroid of the amine unit and vitamin E (vitamin E) of tetramethyl-chroman ring (tetramethyl-chroman ring) lipid peroxidation inhibitor consisting of a compound in U78517F vivo It has been reported that IRFI-016, a basic skeleton of benzofuran, significantly inhibits the damage of brain cells in cerebral ischemia after reperfusion. In addition, quinonone idebenone, known as the active oxygen scavenging agent, strongly inhibits glutamate cytotoxicity in N18-RE-105 cells, while in Vivo experiments also show therapeutic effects on cerebral artery sclerosis and cerebral vascular dementia. It is now known as a substance that is being tested in Japan as a cerebral metabolism activator after cardiac surgery or organ transplantation. The TAK-218 compound, a peroxidase inhibitor developed by Takeda in Japan in June 1996, showed neuroprotective and anti-ischemic effects by eliminating free oxygen and abnormal free dopamine .

In addition, in relation to glutamate toxicity, a study evaluating brain cell protective activity by observing an increase in cell survival rate of a plant extract of Pseudomonas aeruginosa from HT22 cell line, a hippocampus-derived cell line of mice induced by glutamate toxicity, was reported (Journal of the Pharmaceutical Sciences, Kor. J. Pharmacogn. 39 (3): 213-217 (2008)). In addition, 435 medicinal plant extracts naturally grown in Korea and abroad were searched for natural products having neuronal cell protection effect using a hybridoma cell line Hybridoma N18-RE-105 cell line. As a result, Viola mandshurica ), and the contents thereof have been registered (Korean Patent No. 10-0982022). In addition, it has been confirmed that the round crude extract and the non-polar solvent soluble extract inhibit cell death caused by glutamate and hydrogen peroxide neurotoxicity, and thus have a significant effect on the protective activity of brain cells (Korean Patent No. 10-0776347). However, there has been no report on the neuronal cell protection activity of Aspergillus stearus extract or its fractions.

Accordingly, the present inventors have conducted studies to inhibit the toxicity of high-concentration glutamate (Glutamate), and found that Aspergillus terreus , Accession No .: KCTC 0895BP) The culture or mycelial extract or its organic solvent fraction of the strain inhibits the toxicity of glutamate at high concentration and is excellent in neuroprotective activity, The present invention has been completed.

It is an object of the present invention to provide a pharmaceutical composition for neuroprotection or a health food containing an Aspergillus terreus extract or a fraction thereof as an active ingredient.

It is still another object of the present invention to provide a pharmaceutical composition or health food for preventing and treating brain diseases containing Aspergillus stearus extract or a fraction thereof as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for neuroprotection containing Aspergillus terreus extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a pharmaceutical composition for preventing and treating brain diseases comprising Aspergillus stearus extract or a fraction thereof as an active ingredient.

The present invention also provides a health food for nerve protection comprising Aspergillus oryzae extract or a fraction thereof as an active ingredient.

In addition, the present invention provides a health food for preventing or ameliorating cerebrospinal fluid comprising Aspergillus cereus extract or a fraction thereof as an active ingredient.

The Aspergillus oryzae extract of the present invention or its fractions are excellent in the effect of inhibiting glutamate-induced neuronal cell toxicity, and thus are excellent in preventing neuronal cell death induced by neuronal cell toxicity And is safe for the human body since it has no cytotoxicity. Therefore, it is useful as a pharmaceutical composition for preventing and treating neuron-related diseases such as cerebral ischemia, stroke, brain trauma, hypoxia and Huntington's disease caused by neuronal cell death Can be used.

FIG. 1 is a phase contrast microscope photograph showing the result of N18-RE-105 neuronal cell protection activity through the glutamate toxicity inhibitory activity of Aspergillus stearothrix.
A: Neuronal cell line not treated with glutamate
B: Neuronal cell line treated with glutamate (20 mM); And
C: Neuronal cell line treated with glutamate (20 mM) and Aspergillus stearus extract (45 ug / ml).
Figure 2 is a graph showing the inhibitory effect of glutamate toxicity on the N18-RE-105 neuronal cell line of Aspergillus stearothial extract and its fractions:
(-) Glut: N18-RE-105 cell line not treated with glutamate (20 mM); And
(+) Glut: N18-RE-105 cell line treated with glutamate (20 mM).
Figure 3 is a simplified illustration of the process involved in the excitotoxic effect of glutamate.
VSCC; Voltage-sensitive calcium channel,
NOS; Nitric oxide synthase,
PLA ₂ ; Phospholipase A ₂ (phospholipase A _2),
AA; Arachidonic acid,
DG; Diacyl glycerol,
Glut; Glutamate; And
IP ₃ ; Inositol triphosphate.
4 is a schematic diagram showing a method for producing an Aspergillus stearus fraction.

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for neuroprotection containing Aspergillus terreus extract or a fraction thereof as an active ingredient.

Aspergillus stearus is an Aspergillus sp. Fungus strain deposited with accession number KCTC 0895BP.

The Aspergillus stearus extract is preferably prepared by inoculating an Aspergillus stearus strain into an Erlenmeyer flask or a fermenter filled with a yeast-malt extract medium, but not limited thereto.

Preferably, the extract is obtained by extracting the mycelium or culture medium of the strain, but not limited thereto.

The extract is preferably extracted with water, C ₁ to C ₂ lower alcohol or a mixture thereof as a solvent. As the lower alcohol, ethanol or methanol is preferably used, and methanol is more preferably used, but not limited thereto Do not.

The fraction is preferably prepared by further extracting Aspergillus stearus extract with any one selected from the group consisting of chloroform, ethyl acetate and butanol, but not always limited thereto.

In addition, the present invention provides a pharmaceutical composition for preventing and treating brain diseases comprising Aspergillus stearus extract or a fraction thereof as an active ingredient.

 Aspergillus stearus is an Aspergillus sp. Fungus strain deposited with accession number KCTC 0895BP.

The Aspergillus stearus extract is preferably prepared by inoculating an Aspergillus stearus strain into an Erlenmeyer flask or a fermenter filled with a yeast-malt extract medium, but not limited thereto.

Preferably, the extract is obtained by extracting the mycelium or culture medium of the strain, but not limited thereto.

The extract is preferably extracted with water, C ₁ to C ₂ lower alcohol, or a mixture thereof. As the lower alcohol, ethanol or methanol is preferably used, and methanol is more preferably used, but not limited thereto .

The fraction is preferably prepared by further extracting Aspergillus stearus extract with any one selected from the group consisting of chloroform, ethyl acetate and butanol, but not always limited thereto.

The brain diseases include stroke, paralysis, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em-bolism, transient ischemic but is not limited to, any one selected from the group consisting of stroke, attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disturbance and brain function coma.

Examples of the method for extracting the Aspergillus stearus extract include filtration, hot water extraction, immersion extraction, reflux cooling extraction, ultrasonic extraction, and the like, and extraction using the hot water extraction method one to five times And may be, but is not limited to, three more iterations. The extraction solvent may be added to Aspergillus stearus 0.1 to 10 times, preferably 0.3 to 5 times. The extraction temperature may be 20 to 40, but is not limited thereto. In addition, the extraction time may be 12 to 48 hours, but is not limited thereto.

In the above method, the vacuum concentration may be performed using a vacuum decompression concentrator or a vacuum rotary evaporator, but is not limited thereto. The drying may be, but not limited to, vacuum drying, vacuum drying, boiling, spray drying or lyophilization.

In a specific example of the present invention, the N18-RE-105 cell line, which is a neuronal hybridoma cell (mouse neuroblastoma clone N18TG-2 × Fisherrat 18-day embryonic neuronalretina) sensitive to glutamate toxicity, was cultured in Aspergillus oryzae extract or its extract fraction It was confirmed that the activity to protect neurons was extremely excellent by inhibiting the death of N18-RE-105 cell line treated with this high concentration of glutamate.

Specifically, the extract of Aspergillus stearus was prepared by extracting with an alcohol and water mixture solution and then concentrating under reduced pressure. The extract of Aspergillus stearus was suspended in water and extracted sequentially with chloroform (CHCl ₃ ), ethyl acetate (EtOAc) and butanol (C ₄ H ₉ OH).

The inventors of the present invention treated the Aspergillus stearus extract and its fractions with the N18-RE-105 cell line to analyze the glutamate toxicity of the Aspergillus stearus extract and its fractions by treating the Aspergillus stearus extract and its fractions, The inhibitory activity of glutamate toxicity on the N18-RE-105 cell line according to the treatment concentration of the Aspergillus stearus extract and its fractions was measured (Fig. 1) (Fig. 2), and the extract of Aspergillus stearus and its ethylacetate extract (Fig. 2) were obtained. As a result, The neuroprotective activity of the fractions was measured using glutamate for the N18-RE-105 cell line As a result of cell viability measurement of the degree of inhibition of glutamate toxicity, it was confirmed that the Aspergillus stearus culture extract and its ethyl acetate extract fraction did not show cytotoxicity even at a high concentration of 80 ug / ml, (See Table 1).

Therefore, it has been confirmed that the Aspergillus stearus extract or its fractions have no cytotoxicity and excellent glutamate toxicity inhibitory activity on neurons. Therefore, the Aspergillus oryzae extract or its fractions can be used as a neuroprotective agent And can be used as an active ingredient of a pharmaceutical composition for preventing and treating brain diseases.

The pharmaceutical composition for neuroprotection and the pharmaceutical composition for the prevention and treatment of brain diseases of the present invention may contain Aspergillus cereus extract and active fractions thereof and may further contain an active ingredient exhibiting the same or similar function And may contain one or more species.

The Aspergillus stearothial extract of the present invention, its active fractions or a mixture thereof 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 stearate 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, freeze-dried preparations and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao paper, laurin, glycerol and gelatin can be used. The composition of the present invention may 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 0.01 to 10 g / kg, preferably 0.1 g to 5 g / kg, and can be administered 1 to 6 times a day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

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

The present invention also provides a health food for nerve protection comprising Aspergillus oryzae extract or a fraction thereof as an active ingredient.

The Aspergillus stearus has the amino acid sequence of SEQ ID NO: It is a fungal strain deposited with 6178.

The Aspergillus stearus extract is preferably prepared by inoculating an Aspergillus stearus strain into an Erlenmeyer flask or a fermenter filled with a yeast-malt extract medium, but not limited thereto.

Preferably, the extract is obtained by extracting the mycelium or culture medium of the strain, but not limited thereto.

The extract is preferably extracted with water, C ₁ to C ₂ lower alcohol, or a mixture thereof. As the lower alcohol, ethanol or methanol is preferably used, and methanol is more preferably used, but not limited thereto .

The fraction is preferably prepared by further extracting Aspergillus stearus extract with any one selected from the group consisting of chloroform, ethyl acetate and butanol, but not always limited thereto.

In addition, the present invention provides a health food for preventing or ameliorating cerebrospinal fluid comprising Aspergillus cereus extract or a fraction thereof as an active ingredient.

 Aspergillus stearus is Aspergillus sp. F80161 deposited with accession number KCTC 0895BP.

The Aspergillus stearus extract is preferably prepared by inoculating an Aspergillus stearus strain into an Erlenmeyer flask or a fermenter filled with a yeast-malt extract medium, but not limited thereto.

Preferably, the extract is obtained by extracting the mycelium or culture medium of the strain, but not limited thereto.

The extract is preferably extracted with water, C ₁ to C ₂ lower alcohol, or a mixture thereof. As the lower alcohol, ethanol or methanol is preferably used, and methanol is more preferably used, but not limited thereto .

The fraction is preferably prepared by further extracting Aspergillus stearus extract with any one selected from the group consisting of chloroform, ethyl acetate and butanol, but not always limited thereto.

The brain diseases include stroke, paralysis, dementia, Alzheimer's disease, Huntington's disease, Pick's disease, Creutzfeld-Jakob disease, thrombosis, em-bolism, transient ischemic but is not limited to, any one selected from the group consisting of stroke, attack, lacune, stroke, cerebral hemorrhage, cerebral infarction, head trauma, cerebral circulatory metabolic disturbance and brain function coma.

When the Aspergillus stearothial extract of the present invention, its active fractions or a mixture thereof is used as a food additive, the extract, fraction or mixture may be added as it is or may be used together with other food or food ingredients, Can be suitably used. The extract of Aspergillus stearus is preferably extracted with hot water and ethanol, and the concentration of ethanol is preferably 50% to 100%. 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 Aspergillus stearothial extract of the present invention, its active fractions or a mixture thereof can be added include meat, sausage, bread, chocolate, candy, snack, confectionery, pizza, ramen, other noodles, Dairy products including beverages, soups, beverages, tea, drinks, 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. Natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as 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, the Aspergillus stearothial extract of the present invention, the active fraction thereof, or the mixture thereof may be used as various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the Aspergillus stearothial extract of the present invention, its active fractions or a mixture thereof may contain flesh for the production of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination.

Therefore, it has been confirmed that the Aspergillus cerevisiae extract or its fractions have no cytotoxicity and excellent glutamate toxicity inhibitory activity on nerve cells. Therefore, the Aspergillus cerevisiae extract or its fractions can be used for neuroprotective Functional foods, and health functional foods for prevention and improvement of brain diseases.

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> Aspergillus  Preparation of Tereus extract

The strains isolated from the soil were cultured in an Erlenmeyer flask in an amount of 100 ml each. Then, the prepared culture broth and the compounds isolated therefrom were examined for their ability to inhibit glutamate toxicity. Aspergillus terreus (accession number: KCTC 0895BP) were selected. The cells were then inoculated into an Erlenmeyer flask or a fermenter filled with yeast-malt (YM) medium (10 g of glucose, 3 g of yeast extract, 5 g of Tryptone, 3 g of Malt extract and 1 L of water) and cultured for 6 days (28 ° C. and 140 rpm) Respectively. The cultured mycelium of Aspergillus terreus (KCTC 0895BP) obtained after culturing was filtered with a filter paper to obtain filtrated filtrates and unfiltered cells, respectively. The cells were added with acetone and stirred for 4 hours. Then, the cells were filtered using a filter paper, and the filtrate was concentrated under reduced pressure to remove acetone. The mixture of the cell extract of the concentrated aqueous solution and the culture filtrate obtained above was extracted with ethylacetate and the extracted organic layer was washed with sodium sulfate (Na ₂ SO ₄ ) and the organic solvent was distilled off under reduced pressure, Aspergillus terreus , Accession No .: KCTC 0895BP).

< Example  2> Aspergillus  Tereus Fraction  Produce

The aspergillus theraustic strain prepared in Example 1 The extract was suspended in water and, using chloroform (CHCl _3), ethyl acetate (EtOAc), and butanol (C ₄ H ₉ OH) to extract it served successively. Through the above examples, the process for obtaining chloroform fraction, ethyl acetate fraction and butanol fraction was prepared as shown in [Fig. 4].

< Example  3> N18 - RE -105 cell culture

The N18-RE-105 cell line is a neuronal hybridoma cell sensitive to glutamate toxicity. The N18-RE-105 cell line has a receptor similar to that of the glutamate receptor present in the hippocampus of the brain and has a receptor that causes cell death due to oxidative damage through the accumulation of free radicals in the cell delayed calcium-dependent cell death, which is very similar to actual brain cell death due to glutamate toxicity. Therefore, in the present invention, the N18-RE-105 cells were used to measure the activity of inhibiting glutamate-induced toxicity. N18-RE-105 cell culture was as follows.

The N18-RE-105 cell line was cultured in Dulbesco's modified Eagle's medium (DMEM) supplemented with HAT (0.1 mM hypoxanthin, 0.04 mM aminopterin, 0.14 mM thymidine) and heat-treated fetal bovine serum fetal calf serum, FCS). Cell culture conditions were 5% CO 2 and 37 ° C, and subcultured every 2 days. N18-RE-105 cells were maintained in DMEM medium containing 10% DMSO and 20% fetal bovine serum for 24 hours at -75 ° C and stored in liquid nitrogen.

< Experimental Example  1> Aspergillus  Tereus extract and its object The fraction  By processing Neuronal cell  Protective activity assay

<1-1> N18-RE-105 cell culture and treatment of Aspergillus stearus extract and its fractions

The N18-RE-105 cell line was cultured in a T-25 flask for 48 hours and then cultured for 2 minutes in an incubator using trypsin solution (3 ml) to separate the cells from the flask. The medium was neutralized by adding 1 ml of the medium, centrifuged at 1000 rpm for 3 minutes, the supernatant was removed, and the cells were suspended in the medium.

The number of cells was calculated using a hemocytometer and 100 쨉 l was dispensed into a 96-well microplate such that the number of cells per well was 5 × 10 4 cells / well. Cells were cultured in 5% CO 2 at 37 ° C for 24 hours, and then 5 μl of glutamate (400 mM) solution dissolved in phosphate buffer (PBS) and 10 μl of diluted samples in DMSO (DMSO) was dissolved in DMSO to lower the concentration of DMSO in the cells so that the DMSO was not toxic because the DMSO was toxic to the cells. The sample is diluted 10 times with PBS in a safe manner and the concentration of DMSO added to the final cells is 0.005%.) The specific treatment concentration is as follows. The throughput of each Aspergillus tereus extract obtained in Example 1 and the fraction of each Aspergillus tereus extract prepared in Example 2 was 1.0 mg / ml (the amount of Aspergillus stearus extract added to cells (Final concentration of 45 / / ml), 0.5 mg / ml (final concentration 22.7 / / ml), and 0.3 mg / ml (final concentration 13.6 / / ml).

<1-2> Aspergillus  Tereus extract or its Fraction N18 - RE -105 cells Glutamate  Toxicity-inhibiting activity measurement

<1-2-1> Phase difference  Using a microscope Aspergillus  Tereus Extract N18 - RE -105 cells Glutamate  Toxicity-inhibiting activity measurement

The Aspergillus stearus extract prepared in Example <1-1> was treated with the cells as in Experimental Example <1-1>, and the cells treated with the sample were cultured in an incubator for 24 hours. After incubation, the protective activity of the sample against glutamate toxicity was observed using a phase contrast microscope.

As a result of the observation by phase contrast microscope, the Aspergillus cereus culture extract of the present invention and its fractions were evaluated for their ability to inhibit glutamate toxicity on N18-RE-105 cell line, a neuronal hybridoma cell, Activity. Compared with the control without Glutamate treatment, glutamate-treated cells in N18-RE-105 cells were swelled, cell membrane bubbled and lysed to about 60% The cells were killed, but the cells of the specimens to which 45 ug / ml of the fungal extract had been added did not change the shape of the cells, and the survival rate of the cells was remarkably reduced due to the extinction of more than 80% of the cells (FIG. 1).

<1-2-2> Cell Survival  Used Aspergillus  Tereus extract and Fraction  For N18-RE-105 cells Glutamate  Toxicity-inhibiting activity measurement

Aspergillus stearus extract prepared in Example <1-1> and Aspergillus stearus prepared in Example <2-1> The fraction of the extract was treated with the cells as in Experimental Example < 1-1 > and the cells treated with the sample were cultured in an incubator for 24 hours. After culturing, the cytotoxic activity of the sample through the glutamate toxicity-inhibiting activity was evaluated by EZ-Cytox analysis (dehydrogenase assay) in each experimental group.

In addition, living cells were quantified by EZ-Cytox analysis. For EZ-Cytox analysis, 10 μl of EZ-Cytox analytical sample per plate well was cultured for 24 hours after the sample treatment, and then cultured for 3 hours in an incubator. Then, 70 μl of the reaction culture was transferred to a new 96-well plate The cell viability was measured by measuring the absorbance at 450 nm using a microplate reader (Molecular Devices), and the measured absorbance was calculated using the following formula (1) to calculate cell viability , And the resultant value was again calculated using the following equation (2).

A is the absorbance of each well; And

Ac is the absorbance of a well not treated with glutamate.

Vc is the cell survival rate of a well not treated with glutamate (Glutamate);

Vg is the cell survival rate of a well treated with Glutamate; And

Vs is the cell survival rate of glutamate and well treated with the sample.

As a result, the activity of inhibiting glutamate toxicity on the N18-RE-105 cell line according to the treatment concentration of Aspergillus stearus extract and its fractions was measured by measuring the viability of the cells. As a result, (Fig. 2).

As a result of measuring the nerve cell protection activity of the Aspergillus stearus extract of the present invention and the organic solvent extract fraction thereof which inhibit the glutamate toxicity of the N18-RE-105 cell line, which is a neuronal hybridoma cell, by 50% The IC ₅₀ of Tereus extract was very good at 35.5 ㎍ / ml. Among the fractions, the ethyl acetate fraction was the highest at 23.5 ㎍ / ml, followed by the vitamin E (IC ₅₀ 21.5 ug / ml) was high (Table 1).

sample IC ₅₀ (ug / ml) Aspergillus stearus extract 35.5 n-hexane extract fraction > 90.0 Chloroform-extracted fraction > 70.0 Ethyl acetate extract fraction 23.5 Butanol extract fraction > 70.0 Vitamin E 21.5

Aspergillus stearus extract and fractions did not show cytotoxicity even at a high concentration of 100 ㎍ / ml, which was 3 and 4 times higher than the active concentration, respectively.

< Manufacturing example  1> Preparation of pharmaceutical preparations

<1-1> Sanje  Produce

2 g of the Aspergillus stearus extract of the present invention

Lactose 1 g

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

<1-2> Preparation of tablets

100 mg of the Aspergillus stearothial extract of the present invention

Corn starch 100 mg

100 mg of milk

Magnesium stearate 2 mg

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

&Lt; 1-3 > Preparation of capsules

100 mg of the Aspergillus stearothial extract of the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

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

&Lt; 1-4 >

1 g of the Aspergillus oryzae extract of the present invention

Lactose 1.5 g

Glycerin 1 g

0.5 g of xylitol

After mixing the above components, they were prepared so as to be 4 g per one ring according to a conventional method.

<1-5> Preparation of granules

150 mg of the Aspergillus stearothial extract of the present invention

Soybean extract 50 mg

200 mg of glucose

600 mg of starch

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

< Manufacturing example  2> Manufacture of health food

Aspergillus stearus extract 1000 mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg of vitamin

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> Manufacturing of food

<3-1> Production of flour food

0.5-5.0 parts by weight of the Aspergillus stearothial extract of the present invention was added to wheat flour and the mixture was used to prepare bread, cake, cookies, crackers and noodles.

<3-2> soup  And juicy ( gravies )

0.1 to 5.0 parts by weight of the Aspergillus theraustic extract of the present invention was added to the soup and the juice to prepare health promotion meat product, noodle soup and juice.

<3-3> Ground Beef  Produce

10 parts by weight of the Aspergillus stearus extract of the present invention was added to ground beef to prepare ground beef for health promotion.

<3-4> Dairy products ( dairy products )

5-10 parts by weight of the Aspergillus stearus extract of the present invention was added to milk and various dairy products such as butter and ice cream were prepared using the milk.

<3-5> Solar  Produce

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

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

The Aspergillus tereus extract of the present invention was concentrated under reduced pressure in a vacuum concentrator, dried by spraying, and dried with a hot-air drier, and the resulting dried product was pulverized to a size of 60 mesh with a pulverizer to obtain a dry powder.

The grains, seeds and the aspergillus cereus of the present invention were prepared in the following proportions.

(30 parts by weight of brown rice, 15 parts by weight of yulmu, 20 parts by weight of barley), seeds (7 parts by weight of perilla seeds, 8 parts by weight of black beans, 7 parts by weight of black sesame seed), Aspergillus stearus extract (0.5 part by weight), guinea pig (0.5 part by weight).

<3-6> Of chewing gum  Produce

Gum base 20%

Sugar 76.36-76.76%

Aspergillus stearus extract 0.24-0.64%

Fruit flavor 1%

Water 2%

Chewing gum was prepared using the above-mentioned composition and content by a conventional method.

<3-7> Manufacture of candy

Sugar 50-60%

Starch syrup 39.26-49.66%

Aspergillus stearus extract 0.24-0.64%

Orange fragrance 0.1%

The composition and the content of the candy were prepared using a conventional method.

< Manufacturing example  4> Manufacturing of beverages

<4-1> Health drink  Produce

5 g of the Aspergillus stearus extract of the present invention was uniformly blended with a material such as liquid fructose (0.5%), oligosaccharide (2%), sugar (2%), salt (0.5% After sterilization, they were packaged in small containers such as glass bottles and plastic bottles.

<4-2> Production of vegetable juice

5 g of the Aspergillus theraustic extract of the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice.

<4-3> Preparation of fruit juice

1 g of the Aspergillus stearus extract of the present invention was added to 1,000 ml of apple or grape juice to prepare fruit juice.

Korea Biotechnology Research Institute KCTC0895BP 20001129

Claims (15)

delete delete delete delete delete delete A method for preventing and treating brain diseases, which is selected from the group consisting of stroke, Alzheimer's disease, Huntington's disease, thrombosis, embolism, head injury, brain circulatory metabolic disorder, and brain function coma containing Aspergillus stearothial extract or its fraction as an active ingredient A pharmaceutical composition for therapeutic use.
delete [Claim 7] The method according to claim 7, wherein the extract is selected from the group consisting of stroke, Alzheimer's disease, Huntington's disease, thrombosis, embolism, head injury, brain circulatory metabolic disorder, A pharmaceutical composition for the prevention and treatment of brain diseases.
The method according to claim 7, wherein the extract is extracted with water, a C ₁ to C ₂ lower alcohol, or a mixture thereof as a solvent. The method of claim 7, wherein the extract is selected from the group consisting of stroke, Alzheimer's disease, Huntington's disease, thrombosis, embolism, head injury, And cerebral function coma. The pharmaceutical composition for preventing and treating brain diseases according to any one of claims 1 to 3,
The use according to claim 7, wherein the Aspergillus stearus is Aspergillus sterae deposited with the deposit number KCTC 0895BP. The method according to claim 7, wherein the cerebrospinal fluid is selected from the group consisting of stroke, Alzheimer's disease, Huntington's disease, thrombosis, embolism, head injury, Functional coma. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
[Claim 7] The method according to claim 7, wherein the fraction is prepared by extracting Aspergillus stearus extract with methanol or ethanol and then extracting the extract with one selected from the group consisting of n-nucleic acid, chloroform, ethyl acetate and butanol. Alzheimer's disease, Huntington's disease, thrombosis, embolism, head injury, brain circulatory metabolic disorder, and brain functional coma.
delete delete A method for preventing and treating brain diseases, which is selected from the group consisting of stroke, Alzheimer's disease, Huntington's disease, thrombosis, embolism, head injury, brain circulatory metabolic disorder, and brain function coma containing Aspergillus stearothial extract or its fraction as an active ingredient Health food for improvement.

Images