KR20160088840A - Composition for prevention or treatment of glutamate toxicity related diseases containing butyrolatone derivatives or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating brain neuronal toxicity-related diseases, which comprises a butyrolactone derivative derived from a strain of Aspergillus terreus (deposit No.: KCTC 0895BP) or a pharmaceutically acceptable salt thereof as an active ingredient. The butyrolactone derivative according to the present invention is highly effective for inhibiting neuronal toxicity caused by glutamate. Thus, the butyrolactone derivative has an excellent effect of preventing brain neuronal death caused by neuronal toxicity, has no cytotoxicity and is safe to the human body. Therefore, the butyrolactone derivative is useful for a pharmaceutical composition for preventing and treating neuron-related diseases, such as brain ischemia, stroke, brain trauma, hypoxia or Huntington disease, caused by neuronal death.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating glutamate toxicity-related diseases, which comprises a butyrolactone derivative or a pharmaceutically acceptable salt thereof as an active ingredient. active ingredient}

The present invention relates to a pharmaceutical composition for preventing or treating a related disease caused by glutamate toxicity comprising a butyrolactone derivative or a pharmaceutically acceptable salt thereof as an active ingredient.

The population distribution of modern society is rapidly changing to the aging trend. As a result, diseases associated with aging, such as stroke or dementia, are increasing in the prevalence of degenerative brain diseases. Cerebral nervous system disease is an annual disease that increases its mortality rate. It is the second leading cause of cancer death in Korea. It is the second most serious cause of death in the world. However, no clear medicines or treatments have yet been proposed to cure this disease, and the social and economic losses are intensifying

Degenerative brain nervous system diseases are caused by various physical and mechanical factors such as secondary symptoms caused by other adult diseases such as structural degeneration of brain nerve cells due to aging, circulatory disorder, or traffic accidents, industrial accidents, carbon monoxide poisoning, , Damaged brain tissue induces the death of brain nerve cells. These mechanisms of neuronal apoptosis are roughly classified into two types, one of which is glutamate, which is an excitatory neurotransmitter present in the brain.

Glutamate acts as a major excitatory neurotransmitter in the central nervous system, where glutamate released into the nerve cell is reabsorbed into the presynaptic terminal or absorbed into glial cells and then converted to glutamine, (Presynaptic terminal) and maintained at a low concentration in the nerve cells. However, if the brain is subject to cerebral ischemia, hypoxia, seizures, hypoglycemia, and brain trauma, the energy supply for extracellular glutamate transport becomes insufficient, leading to a glutamate concentration of about 10,000 times that of the normal state The central nerve cell is killed. Such a high concentration of glutamate (Glutamate) has an increased activity of calcium-dependent enzymes such as the cell increasing the intracellular calcium ion concentration of phospholipase _{A 2 (phospholipase A 2),} NO synthase (nitric oxide synthetase, NOS), Protease (protease) And the increased activity of the enzyme increases the generation of free radicals in the cell, thereby inducing oxidative toxicity and inducing neuronal cell death. Cerebral nerve cell death caused by glutamate toxicity is closely related not only to acute diseases such as cerebral ischemia, stroke and seizure but also to chronic brain diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, etc., Research is urgent.

In recent years, advanced countries such as the US have been attracting attention in the field of next-generation new drug development through research on the separation of useful components of natural products and research into mechanisms, due to low human toxicity of therapeutic agents using microorganisms and natural products in the field of alternative medicine. Accordingly, researches on the treatment of diseases caused by glutamate toxicity using microorganisms and natural products are proceeding from various angles, and on the basis thereof, it is possible to prevent and treat diseases caused by glutamate toxicity Research is underway to develop drugs that can be

First, as a result of observing the increase of cellulase survival rate of the extracts of the extracts of the extracts of the extracts of the extracts of Mt. Paekdusan, the extracts of Mt. Paekdusan showed the protective activities of the brain cells (Non-Patent Document 1).

In addition, 435 medicinal plant extracts which are naturally grown at home and abroad were searched for natural products having neuronal cell protection effect by using a brain nerve cell system hybridoma N18-RE-105 cell line. As a result, Viola mandshurica ) (Patent Document 1), and the round pupa extract and the non-polar solvent soluble extract blocked apoptosis caused by glutamate and hydrogen peroxide neurotoxicity, and thus showed significant protective effect on brain nerve cell protection (Patent Document 2).

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) exhibits excellent inhibitory activity for inhibiting the toxicity of high-concentration glutamate, and can be used for isolation from natural products, Thus completing the present invention.

It is an object of the present invention to provide a pharmaceutical composition for preventing or treating a related disease caused by glutamate toxicity.

It is another object of the present invention to provide a health food composition for preventing or ameliorating a related disease caused by glutamate toxicity.

In order to achieve the above object,

The present invention provides a pharmaceutical composition for preventing or treating a related disease caused by glutamate toxicity, comprising a butyrolactone derivative represented by the formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

The present invention also provides a health food composition for preventing or ameliorating a related disease caused by glutamate toxicity.

The butyrolactone derivative according to the present invention is excellent in the effect of inhibiting glutamate-induced toxicity of nerve cells, and thus has an excellent effect of preventing neuronal cell death induced by nerve cell toxicity, Therefore, it can be effectively used as a pharmaceutical composition for preventing and treating neuron-related diseases such as cerebral ischemia, stroke, brain trauma, hypoxia, Huntington's disease caused by neuronal cell death.

1 is a phase contrast microscope photograph showing the result of N18-RE-105 neuronal cell protection activity through inhibition of glutamate toxicity of a butyrolactone derivative according to Experimental Example 1 (A: glutamate treatment , Neuronal cell line treated with glutamate (20 mM), C: glutamate (20 mM) and bureolactone derivative (20 μg / ml)).
FIG. 2 is a graph showing the survival rate of neurons according to the EZ-Cytox assay of Experimental Example 1 ((-): Cells not treated with glutamate, (+) Cells treated with glutamate (+) B: 5 μg / ml: Cells treated with glutamate and 5 μg / ml of butyrolactone, (+) B: 10 μg / ml: treated with glutamate and 10 μg / ml butyrolactone Cells (+) B: 20 μg / ml: Cells treated with glutamate and 20 μg / ml butyrolactone).

Hereinafter, the present invention will be described in detail.

The present invention provides a pharmaceutical composition for preventing or treating a related disease caused by glutamate toxicity, comprising a butyrolactone derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:

The butyrolactone derivative represented by formula (1) according to the present invention can inhibit cytotoxicity due to accumulation of high concentration of glutamate in neurons.

Glutamate acts as a major excitatory neurotransmitter in the central nervous system, and glutamate released into the nerve cell migrates to the presynaptic terminal, maintaining a low concentration in the nerve cell. However, when the brain lacks the energy supply required for glutamate transport to neuronal cells due to cerebral ischemia, hypoxia, seizures, hypoglycemia, and brain trauma, the glutamate accumulation in neurons increases the concentration. High concentrations of glutamate increase the intracellular calcium ion concentration and induce the activity of calcium ion dependent enzymes, thereby promoting the production of free radicals in the cells, leading to oxidative toxicity and inducing neuronal cell death .

The effect of the butyrolactone derivative represented by the formula (1) according to the present invention for inhibiting high-concentration glutamate toxicity will be described below. As a result of an experiment in which the degree of nerve cell protection against the butyrolactone derivative according to the present invention was compared with the positive control group, vitamin E, the effect of inhibiting the glutamate toxicity of the butyrolactone derivative of the formula (1) (See Experimental Example 1).

In the pharmaceutical composition according to the present invention, the diseases caused by the glutamate toxicity include cerebral ischemia, stroke, brain trauma, hypoxia, Huntington's disease, and the like.

In the pharmaceutical composition according to the present invention, glutamate toxin-induced nerve cells may be nerve cells constituting the central nerve or peripheral nerve, for example, brain nerve cells or spinal nerve cells of the central nerve, A spinal nerve bundle of neurons, a bundle of cranial nerves, or an autonomic nerve bundle.

In the pharmaceutical composition according to the present invention, the butyrolactone derivative represented by the above formula (1) may be used in the form of a pharmaceutically acceptable salt. Such salts are useful as acid addition salts formed by various pharmaceutically or physiologically acceptable organic acids or inorganic acids. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Dioleate, aromatic acid, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

At this time, the acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving a derivative of the formula (1) in an excess amount of an acid aqueous solution, and then mixing the salt with a water-miscible organic solvent such as methanol, ethanol, Followed by precipitation using nitrile. It may also be prepared by evaporating a solvent or excess acid in this mixture and then drying or by suction filtration of the precipitated salt.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (for example, silver nitrate).

Hereinafter, the present invention provides a process for preparing a butyrolactone derivative represented by the above formula (1) according to the present invention, comprising the steps of:

Aspergillus terreus , Accession No .: KCTC 0895BP) Extracting the mycelial culture with ethyl acetate (step 1);

A step (step 2) of obtaining a first fraction by performing silica gel column chromatography using the chloroform-methanol mixed solvent as an elution solvent;

(Sephadex LH-20) column chromatography using methanol as an elution solvent to obtain a second fraction (step 3); And

Performing the reverse phase chromatography using the acetonitrile as the elution solvent (Step 4).

The butyrolactone derivative of the formula 1 according to the present invention can be prepared by extraction, separation and purification from a mycelial culture of Aspergillus terreus (accession number: KCTC 0895BP), but it can be used in the field of organic synthesis Is not excluded from the synthesis by a conventional method.

Hereinafter, the manufacturing method will be described step by step.

First, step 1 is Aspergillus terephthalate mouse (Aspergillus terreus , Accession No .: KCTC 0895BP).

The extract is Aspergillus terephthalate mouse (Aspergillus terreus , Accession No .: KCTC 0895BP) Mycelial cultures can be extracted using an organic solvent commonly used in the field of water, C ₁ -C ₄ lower alcohols, mixed solvents thereof, or mycelium extracts.

At this time, it is preferable to use methanol, ethanol, butanol as the alcohol as the extraction solvent, and chloroform, ethyl acetate or the like as the organic solvent. Most preferably, ethyl acetate can be used.

Next, step 2 is a step of obtaining a fraction (first fraction) of Aspergillus terreus (accession number: KCTC 0895BP) extract obtained from step 1.

As the elution solvent used for fractionating the first fraction, it is preferable to use a mixed solvent of chloroform and methanol. At this time, it is more preferable that the elution solvent is adjusted to have a concentration gradient in a volume ratio of 25: 1 to 1: 1.

Next, step 3 is a step of further fractionating the first fraction obtained from step 2 to obtain a second fraction.

The first fraction used to obtain the second fraction is preferably selected from only those fractions exhibiting glutamate toxicity inhibitory activity among the fractions. As the elution solvent, methanol is preferably used, and it is more preferable to use methanol having a concentration of 100%. As the column used for carrying out the column chromatography, Sephadex LH-20 is preferable.

Next, Step 4 is a step of obtaining the butyrolactone derivative represented by Formula 1 from the second fraction obtained in Step 3.

For this purpose, it is preferable to use acetonitrile as the elution solvent in Step 4, and it is more preferable that the concentration of the solvent is 55%. For column chromatography, reverse phase high performance liquid column chromatography is preferred.

The pharmaceutical composition containing the butyrolactone derivative of the formula 1 or the pharmaceutically acceptable salt thereof of the present invention as an active ingredient can be formulated into various oral or parenteral dosage forms described below, but the present invention is not limited thereto.

Examples of formulations for oral administration include tablets, pills, light / soft capsules, liquids, suspensions, emulsions, syrups, granules, elixirs and the like, , An extender, a wetting agent, a disintegrant, a lubricant, a binder, a surfactant, and the like. As the disintegrant, agar, starch, alginic acid or its sodium salt, anhydrous calcium monohydrogenphosphate, etc. may be used. As the lubricant, silica, talc, stearic acid or its magnesium salt or calcium salt, polyethylene glycol and the like may be used , Magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidine, and low-substituted hydroxypropylcellulose can be used as the binding agent. In addition, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine and the like can be used as a diluent. In general, a commonly known boiling mixture, an absorbent, a colorant, a flavoring agent and a sweetening agent can be used together.

In addition, the pharmaceutical composition containing the derivative of the above formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient may be administered parenterally, and the parenteral administration may be carried out by administering a subcutaneous injection, intravenous injection, intramuscular injection, It depends on the injection method. In this case, in order to formulate the composition for parenteral administration, the derivative of Formula 1 or its pharmaceutically acceptable salt is mixed with water or a stabilizer or a buffer in water to prepare a solution or suspension, which is then administered in unit dosage form of an ampule or vial Can be manufactured.

The composition may be sterilized or may contain other therapeutically useful substances such as preservatives, stabilizers, wettable or emulsifying accelerators, salts for controlling osmotic pressure, buffering agents and the like, and may be mixed, granulated or coated The formulation can be made according to the method.

If necessary, the pharmaceutical composition containing the derivative of formula (I) or a pharmaceutically acceptable salt thereof according to the present invention as an active ingredient may be administered in combination with other medicines, for example, other diabetes medicines.

When the pharmaceutical composition containing the derivative of the formula (I) of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is formulated in unit dose form, the derivative of the formula (I) or a pharmaceutically acceptable salt thereof as the active ingredient It is preferably contained in a unit dose of about 0.1 to 1,500 mg. The dosage depends on the physician's prescription depending on factors such as the patient's weight, age and the particular nature and severity of the disease. However, the dosage required for adult therapy is usually in the range of about 1 to 500 mg per day depending on the frequency and intensity of administration. A total daily dose of about 5 to 300 mg per day may be sufficient for an adult to receive intramuscular or intravenous doses, but in some patients a higher daily dose may be desirable.

Further, the present invention provides a health food composition for preventing or ameliorating a related disease caused by glutamate toxicity, which comprises a butyrolactone derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient do:

[Chemical Formula 1]

.

.

As a health food containing the butyrolactone derivative represented by the formula (1) of the present invention or a pharmaceutically acceptable salt thereof, it is possible to use, as a health food containing a derivative of the butyrolactone, Therapy. The health food of the present invention processed in various forms as described above is excellent in detoxification action without adverse effect on the human body, and is easy to take and can be stored for a long time, so that prevention or improvement of related diseases caused by glutamate toxicity And can be usefully used for a desired health food.

When the butyrolactone derivative represented by the general formula (1) of the present invention or a pharmaceutically acceptable salt thereof is used as a food additive, the derivative compound can be added as it is or can be used together with other food or food ingredients, Can be suitably used. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). Generally, in the production of foods or beverages, the butyrolactone derivative represented by the formula (1) of the present invention is added in an amount of 80 parts by weight or less, preferably 50 parts by weight or less based on 100 parts by weight of the raw material. However, in the case of long-term consumption intended for health and hygiene purposes or health control purposes, the amount may be less than the above range, and there is no problem in terms of safety. Therefore, the active ingredient may be used in an amount of more than the above range.

There is no particular limitation on the kind of the food. Examples of the food to which the butyrolactone derivative represented by the formula 1 of the present invention or a pharmaceutically acceptable salt thereof can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, Gums, dairy products including ice cream, various 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. The above-mentioned natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, 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 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 mL of the butyrolactone derivative represented by the formula (1) of the present invention or a pharmaceutically acceptable salt thereof.

The butyrolactone derivative represented by the general formula (1) of the present invention or a pharmaceutically acceptable salt thereof according to the present invention may be in the form of various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, Colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the butyrolactone derivative represented by the formula (1) of the present invention or a pharmaceutically acceptable salt thereof may contain flesh for the production of natural fruit juice, fruit juice drink and vegetable drink. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the butyrolactone derivative represented by the formula (1) of the present invention or a pharmaceutically acceptable salt thereof.

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

However, the following examples and experimental examples are intended to illustrate the present invention in detail, and the content of the present invention is not limited by the examples and the experimental examples.

< Manufacturing example  1> Aspergillus Tereus  Preparation of mycelial culture

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. Then, the yeast in the fermenter - filling the malt extract (Yeast-malt extract, YM) medium, the Aspergillus terephthalate mouse (Aspegillus terreus, Accession No: KCTC 0895BP) to collect inoculated with strain 3 days 28 ℃, 140 rpm Seeds were cultured. A 5 L fermentor was filled with 3 L of yeast-malt extraction medium, seeded with 3% of the seed culture as described above, and cultured for 7 days to obtain Aspergillus cerevisiae terreus , Accession No .: KCTC 0895BP) mycelial cultures were prepared.

Butyrolactone  Preparation of derivatives

The Aspergillus prepared in Preparative Example 1 mouse's terephthalate (Aspegillus terreus , Accession No .: KCTC 0895BP) The mycelial culture was filtered with a filter paper to obtain filtrated filtrate and unfiltered cells, respectively. The cells were added with acetone, stirred for 4 hours, filtered using a filter paper, and the filtrate was concentrated under reduced pressure to remove acetone. The cell extract of the concentrated aqueous solution and the culture filtrate obtained above were mixed and then extracted with ethyl acetate (Ethylacetate). The extracted organic layer was washed with sodium sulfate (Na ₂ SO ₄ ), and the organic solvent was distilled under reduced pressure to remove Aspgillus terreus , Accession No .: KCTC 0895BP). Next, the obtained extract was started with a mixed solvent of chloroform: methanol = 25: 1, and subjected to column chromatography (silica gel, 100 g Kiesel gel 60 (230- (Sephadex LH-20 (manufactured by Sephadex LH-20), which had been saturated with a 100% methanol solvent, was prepared by collecting only fractions exhibiting toxic inhibitory activity of glutamate among the fractions of the first fraction (ODS) high-performance liquid chromatography (HPLC) using 55% acetonitrile as an elution solvent, and then, the eluted fractions of the second fraction obtained above were subjected to column chromatography (second fraction) To give the desired compound.

[[alpha]] _D &lt; ²⁰ &gt; = + 100 [deg.] (c = 1.0, EtOH);

IR (KBr): 3565, 3480, 3280, 1755, 1740 cm &lt; ^{-1 &} gt ;;

m.p = 95 &lt; 0 &gt;C;

EI-MS (m / z): 424 (M ^&lt; ⁺ &gt;);

UV? _Max (MeOH) = 223 nm (log? = 4.18), 309 nm (log? = 4.31);

_{^{1 H NMR (CDCl 3):}} 7.60 (d, J = 9.0 Hz, 2H), 6.88 (d, J = 9.0 Hz, 2H), 6.54 (dd, J = 2.0 Hz, 9.0 Hz, 1H), 6.50 (d , J = 8.0 Hz, 1H) , 6.42 (d, J = 2.0 Hz, 1H), 5.07 (t, J = 6.5 Hz, 1H), 3.76 (s, 3H), 3.46 (d, J = 14.5 Hz, 1H ), 3.41 (d, J = 14.5 Hz, 1H), 3.09 (dd, J = 6.5 Hz, 1H), 3.07 (dd, J = 6.5 Hz, 1H), 1.66 (s, 3H), 1.57 (s, 3H ) ppm;

_{^{13 C NMR (CDCl 3):}} 171.6, 170.6, 159.1, 154.9, 140.1, 132.9, 132.3, 130.3, 129.7, 128.8, 128.4, 125.1, 123.5, 123.3, 116.6, 115.0, 86.8, 53.8, 39.5, 29.6, 25.9, 17.7 ppm.

<Experimental Example 1> glutamate (Glutamate) the neuroprotective ability of flat with suppressed toxicity

The following experiment was conducted to evaluate the protective ability of the butyrolactone derivative represented by the formula (1) of the present invention by inhibiting glutamate toxicity.

First, N18-RE-105, a neuronal cell line, was used to evaluate the inhibitory activity of glutamate toxicity of butyrolactone derivatives. The N18-RE-105 cell line was cultured in Dulbecco's modified eagles medium (Invitrogen) supplemented with 10% fetal calf serum (FCS) supplemented with HAT supplement (0.1 mM hypoxanthin, 0.14 mM thymidine) Was added to the culture medium. Cell culture conditions were subcultured every 2 days at 37 ° C and 5% CO ₂ (CO ₂ ). N18-RE-105 cells were maintained in Dulbecco's modified eagles medium (Invitrogen) supplemented with 10% dimethyl sulfoxide (DMSO) and 20% fetal calf serum (FCS) For 24 hours and then stored in liquid nitrogen.

Next, the cultured N18-RE-105 cell line was cultured in a T-25 flask for 48 hours and then cultured in an incubator for 2 minutes using trypsin solution (3 ml) to separate the cells from the flask. Then, 1 ml of medium was added to neutralize trypsin, and the supernatant was removed by centrifugation at 1000 rpm for 3 minutes. The supernatant was re-suspended in the removed cells with culture medium, blood converter (Hemocytometer) which counts the number of cells by using the following, the well (well) per cell counts 5 × 10 ⁴ than 100 in 96-well (well) microtiter plates such that respectively. The dispensed cells were incubated at 37 ° C in 5% CO ₂ (CO ₂ ) for 24 hours. The cultured cells were injected with 5 μl of glutamate (Glutamate, 400 mM) solution in FBS buffer, and the butyrolactone compound dissolved in dimethylsulfoxide was diluted 10 times with phosphate buffered saline (PBS) The lactone derivative solution was prepared and the concentration was adjusted so that the final concentration of the prepared derivative solution was 5, 10 and 20 μg / ml, and then the cells were treated. At this time, the untreated group treated with 5 μl of dimethylsulfoxide and the positive control treated with vitamin E (5 μl), which is conventionally known to have a glutamate toxicity inhibitory effect. Cells treated with each compound were cultured in an incubator for 24 hours, and then the protective activity of glutamate toxicity of the sample was observed using a phase contrast microscope. In addition, living cells were quantified by EZ-Cytox assay (dehydrogenase assay). The EZ-Cytox assay was carried out by sample-treating and transferring 70 μl of the reaction culture solution per plate well to the cells cultured for 24 hours into a new 96-well plate, absorbance at 450 nm with a microplate reader. The cell viability was calculated using the following equation (1), and the results were again calculated using the following equation (2). The results are shown in FIG. 1 and FIG.

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 shown in Figs. 1 and 2, the butyrolactone derivative represented by the formula (1) of the present invention protects nerve cells by inhibiting the toxicity of high concentration glutamate in a concentration-dependent manner. Particularly, the nerve cells of the untreated group were swelled, blebbing and lysis due to the toxicity of high-concentration glutamate, and about 60% of the cells were killed. On the other hand, In the neurons treated with the butyrolactone derivative at a concentration of 20 μg / ml, not only the cell shape of the neurons did not change but also the cell survival rate was as high as 95%. The IC ₅₀ values of the butyrolactone derivative according to the present invention and the vitamin E as a positive control were 11.2 μg / ml and 21.5 μg / ml, respectively, The nerve cell protective activity is about two times higher than that of vitamin E.

 Therefore, it can be seen that the butyrolactone fluid represented by the formula (1) of the present invention effectively inhibits the toxicity of glutamate at a high concentration, and thus the effect of protecting nerve cells is remarkably excellent. As a result, the pharmaceutical composition containing the butyrolactone derivative of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is useful as an effective ingredient for preventing or treating cerebral ischemia, stroke, brain trauma, hypoxia and Huntington's disease caused by glutamate toxicity, (Huntington's disease) and the like can be effectively used as a pharmaceutical composition.

< Experimental Example  2> Assessment of cytotoxicity

In order to evaluate the cytotoxicity of the butyrolactone derivative represented by the formula (1) of the present invention, the following experiment was conducted.

Experiments were carried out in the same manner as in Experimental Example 1, except that the concentration of the butyrolactone derivative according to the present invention was adjusted to 5, 10, 20 and 50 μg / ml. As a result, it was confirmed that the butyrolactone derivative was not cytotoxic. In particular, no cytotoxicity was observed at a high concentration of 50 μg / ml, which is 5 times higher than the effective effective concentration of 10 μg / ml, which inhibits the toxicity of high-concentration glutamate.

Accordingly, the butyrolactone fluid represented by the formula (1) of the present invention suppresses the toxicity of glutamate at a high concentration, so that the effect of protecting nerve cells is remarkably excellent, Prevention or treatment of diseases such as cerebral ischemia, stroke, brain trauma, hypoxia and Huntington's disease caused by glutamate toxicity, which comprises a lactone derivative or a pharmaceutically acceptable salt thereof as an active ingredient. And can be usefully used as a pharmaceutical composition.

Meanwhile, the butyrolactone derivative represented by the formula (1) according to the present invention can be formulated into various forms according to the purpose. Hereinafter, some formulation methods in which the compound of Formula 1 according to the present invention is contained as an active ingredient are illustrated, and the present invention is not limited thereto.

< Formulation example  1> Preparation of Tablets (Direct Pressurization)

According to the conventional preparation method of tablets, the following ingredients were added in the prescribed amounts, uniformly mixed and pressed to prepare tablets.

Butyrolactone derivative        5.0 mg

Lactose 14.1 mg

Crospovidone USNF  0.8 mg

Magnesium stearate  0.1 mg

< Formulation example  2> Preparation of tablets (wet assembly)

The butyrolactone derivative was sieved, and lactose and starch were mixed. Thereafter, the polysorbate was dissolved in pure water, and an appropriate amount was added to the active ingredient, lactose and starch mixture, followed by atomization. After drying, the granulate was sieved and mixed with colloidal silicon dioxide and magnesium starate. The granules were pressurized to prepare tablets.

Butyrolactone derivative        5.0 mg

Lactose 16.0 mg

starch  4.0 mg

FreeSorbate 80  0.3 mg

Silicon dioxide  2.7 mg

Magnesium stearate  2.0 mg

< Formulation example  3> Preparation of capsules

The following ingredients were added in the prescribed proportions according to the usual preparation method of the capsules, uniformly mixed, and filled into gelatin capsules of appropriate size to prepare the desired capsules.

Butyrolactone derivative        5.0 mg

Lactose 14.8 mg

Polyvinylpyrrolidone 10.0 mg

Magnesium stearate  0.2 mg

< Formulation example  4> Preparation of injection

In accordance with the conventional method for preparing injections, injections were prepared by incorporating the following ingredients in the stated amounts.

Butyrolactone derivative 100 mg

Mannitol 180 mg

Na ₂ HPO ₄ .2H ₂ O 26 mg

Distilled water      2974 mg

< Formulation example  5> Health food manufacturing

5-1. Manufacturing of beverages

A beverage was prepared using conventional methods with the following composition and contents.

honey 522 mg

Dioctanoic acid amide 5 mg

Nicotinic acid amide 10 mg

Sodium riboflavin hydrochloride 3 mg

Pyridoxine hydrochloride 2 mg

Inositol 30 mg

Ortho acid 50 mg

Butyrolactone derivative    0.48 - 1.28 mg

water 200 ml

5-2. Of chewing gum  Produce

Chewing gum was prepared using a conventional method with the following composition and contents.

Gum base             20%

Sugar       76.36 - 76.76%

Butyrolactone derivative   0.24 - 0.64%

Fruits             One %

water 2 %

5-3. Manufacture of candy

Candies were prepared using the conventional methods with the following composition and contents.

Sugar   50 - 60%

corn syrup   39.26 - 49.66%

Butyrolactone derivative     0.24 - 0.64%

Orange incense 0.1%

5-4. Manufacture of flour food products

0.5 to 5 parts by weight of the butyrolactone derivative according to the present invention was added to 100 parts by weight of wheat flour, and bread, cake, cookies, crackers and noodles were prepared by using this mixture to prepare a food for health promotion.

5-5. Manufacture of dairy products

5 to 10 parts by weight of the butyrolactone derivative according to the present invention was added to 100 parts by weight of milk and various dairy products such as butter and ice cream were prepared using the milk.

5-6. Solar  Produce

Brown rice, barley, glutinous rice, and yulmu were dried by a known method and dried, and then powdered with a grinder to prepare 60 mesh powder. Black beans, black sesame, and perilla were steamed and dried by a known method, and then powdered with a powder of 60 mesh size by a grinder. The grains and seeds prepared above and the butyrolactone derivatives according to the present invention were prepared in the following proportions.

Brown rice 30%

Yulmu 15%

barley 20%

Perilla 7%

Black beans 7%

Black sesame 7%,

Butyrolactone derivative 3%

wisdom 0.5%

Rehab 0.5%

Korea Biotechnology Research Institute KCTC0895BP 20001129

Claims (6)

A pharmaceutical composition for preventing or treating a related disease caused by glutamate toxicity comprising a butyrolactone derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

.
2. The pharmaceutical composition according to claim 1, wherein the butyrolactone derivative represented by the formula (1) inhibits cytotoxicity by accumulation of high concentration of glutamate in neurons.
The pharmaceutical composition according to claim 2, wherein the nerve cell is a brain nerve cell. The pharmaceutical composition for preventing or treating a related disease caused by glutamate toxicity.
The method according to claim 1, wherein the disease caused by glutamate toxicity is cerebral ischemia, stroke, brain trauma, hypoxia, Huntington's disease, or a related disease caused by glutamate toxicity. A pharmaceutical composition for preventing or treating diseases.
The method according to claim 1, wherein the butyrolactone derivative represented by Formula (1)
Aspergillus terreus , Accession No .: KCTC 0895BP) Extracting the mycelial culture with ethyl acetate (step 1);
A step (step 2) of obtaining a first fraction by performing silica gel column chromatography using the chloroform-methanol mixed solvent as an elution solvent;
(Sephadex LH-20) column chromatography using methanol as an elution solvent to obtain a second fraction (step 3); And
(Step 4) of performing a reverse phase chromatography using the acetonitrile as the eluting solvent and the second fraction of the step 3 as an elution solvent. A pharmaceutical composition for preventing or treating a related disease.
A health food composition for preventing or ameliorating a related disease caused by glutamate toxicity comprising an butyrolactone derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient:
[Chemical Formula 1]

.

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