WO2015072667A1 - Pharmaceutical composition for preventing or treating degenerative cranial nerve diseases - Google Patents

Abstract

The present invention relates to a composition for alleviating oxidative stress-induced cranial nervous system diseases, containing corn silk-derived maysin as an active ingredient and, more particularly, to a pharmacological composition for alleviating degenerative cranial nervous system diseases, wherein maysin increases the expression of mRNAs of antioxidant enzymes (catalase, glutathione peroxidase-1, superoxide dismutases 1 and 2, and heme oxygenase-1), thereby decreasing oxidative stress, and ultimately, inhibiting or blocking nerve cell apoptosis caused by oxidative stress.

Description

Pharmaceutical composition for the prevention or treatment of degenerative neurological disease

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative neurodegenerative diseases, and more particularly, to prevent or treat neurodegenerative neurodegenerative diseases and to prevent neurodegenerative neuroprotective effects. It is about.

Neurodegenerative disease is known to be caused by the death of neurons due to oxidative stress. Recently, degenerative brain neurological diseases including Alzheimer's disease have been cited as the main cause of the disease caused by oxidative stress due to the rapid increase in the amount of free radicals in neurons. Therefore, it is reported that such diseases can be prevented or treated by suppressing or reducing oxidative stress.

Intracellular antioxidant activity consists of an enzymatic defense system and an antioxidant system. Enzymatic defense system protects against oxidative stress by enzymes such as catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and heme oxygenase (HO). It is a mechanism to defend. In particular, HO has been reported to play an important role in maintaining homeostasis through antioxidant activity in the oxidative state.

As the aging population rapidly increases, the incidence of degenerative neurological diseases is on the rise, and despite the innovative advances in medicine, the prevention and treatment of degenerative neurological diseases have not been found and the drugs with decisive effects have not been found. .

Currently, drugs and treatments for degenerative cerebral nervous system disease are being developed, but they often show side effects and toxicity according to long-term use, and there is an urgent need to develop materials that can reduce and treat side effects and toxicity because they only have symptoms that reduce symptoms rather than treatment. to be.

Corn beard has traditionally been ingested as a edible or folk remedy for various diseases, has a relatively high stability, and has a high potential of development as a new drug derived from natural products. In Korean Patent No. 10-1201628, the pharmacological effects of the antimicrobial and anticancer effects of corn beard extracts have been studied, but there has been no report on the inhibitory activity and the neuroprotective activity against neuronal cell death caused by oxidative stress. By reducing the oxidative stress induced by H ₂ O ₂ , there was a problem that a composition for improving neurodegenerative diseases due to inhibition of neuronal cell death has not been developed.

The present invention has been made to solve the above problems, the first problem to be solved of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases of the neurons to inhibit apoptosis. A second object of the present invention is to provide a dietary supplement for the prevention or improvement of neurodegenerative diseases comprising the composition.

The present invention provides a pharmaceutical composition for the prevention or treatment of degenerative cerebral nervous system diseases containing mayin as an active ingredient in order to achieve the first object.

According to a preferred embodiment of the present invention, the composition can inhibit neuronal cell death.

According to another preferred embodiment of the present invention, the composition may be derived from corn beard.

According to another preferred embodiment of the present invention, the composition may increase the activity of antioxidant enzymes of neurons.

According to another preferred embodiment of the present invention, the composition can inhibit the oxidative stress induced by H ₂ O ₂ .

According to another preferred embodiment of the present invention, the composition can inhibit reactive oxygen species of neurons.

According to another preferred embodiment of the present invention, the composition may exhibit a protective effect of neurons.

According to another preferred embodiment of the present invention, the composition may be characterized in that it is contained at a concentration of 5 ~ 100 μg / ml.

According to another preferred embodiment of the present invention, the degenerative neurological disease may include Alzheimer's, Parkinson's disease, Huntington's disease, multiple sclerosis, multiple neurotrophic, epilepsy, brain disease (encephalopathy), or stroke.

In order to achieve the second object, it provides a health functional food composition for the prevention or improvement of neurodegenerative diseases containing the composition.

Hereinafter, the terms used in the present specification will be briefly described.

In the present invention, the term "prevention" refers to any action that inhibits apoptosis of neurons or delays apoptosis of neurons by administration of a composition, and "treatment" refers to symptoms caused by onset by administration of the composition. Means any action that improves or beneficially changes;

The present invention reduces the amount of reactive oxygen species by effectively inhibiting the activity of oxidative stress, and has an excellent protective effect against the damage of activated oxidative stress on nerve cells, which can be usefully used as an effective preventive or therapeutic agent for degenerative cerebral neuropathy. have.

Figure 1a is an experimental result of neuronal cell death by treatment of the mycin according to a preferred embodiment of the present invention, the figure showing the effect of inhibiting apoptosis of neurons using Annexin V / PI fluorescence staining.

FIG. 1B is a graph showing numerical results of using the Annexin V / PI fluorescence stain as an experimental result of neuronal cell death by treatment with Meisin according to a preferred embodiment of the present invention.

Figure 2 is an experimental result of inhibiting DNA fragmentation by the treatment of the mycin according to an embodiment of the present invention, showing the DNA fragmentation induced by H ₂ O ₂ using ApoDIRECT In Situ DNA fagmentation assay kit fluorescent staining Picture.

Figure 3 is a graph showing the experimental results of inhibiting the activity of reactive oxygen species by the treatment of the mycin according to an embodiment of the present invention.

Figure 4a is a graph showing the results of the experiment observed the expression of catalase (CAT) which is an antioxidant enzyme in neurons by treatment of the mycin according to a preferred embodiment of the present invention.

Figure 4b is a graph showing the results of the experiment observed the expression of glutathione peroxidase-1 (GPx-1), an antioxidant enzyme in neurons by the treatment of the mycin according to a preferred embodiment of the present invention.

Figure 4c is a graph showing the results of the experiment observed the expression of superoxide dismutase-1 (SOD-1), an antioxidant enzyme in neurons by treatment of the mycin according to a preferred embodiment of the present invention.

Figure 4d is a graph showing the results of the experiment observed the expression of superoxide dismutase-2 (SOD-2), an antioxidant enzyme in neurons by the treatment of the mycin according to a preferred embodiment of the present invention.

Figure 4e is a graph showing the results of the experimental observation of the increased expression of the antioxidant enzyme heme oxygenase-1 (HO-1) in neurons by the treatment of the mycin according to an embodiment of the present invention.

Figure 5a is a graph showing the experimental results of observing the increase in the cell survival rate of neurons by the treatment of the mycin according to a preferred embodiment of the present invention.

Figure 5b is a graph showing the results of the experiments observed that the cytotoxicity of neurons is reduced by the treatment of the mycin according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

As described above, corn beard has been reported to study the pharmacological effect on the antibiotic and anti-cancer effect, but has not been reported for the inhibitory activity and neuronal protective activity against neuronal cell death caused by oxidative stress, H ₂ O By reducing the oxidative stress induced by ₂ , there is a problem that a composition for improving neurodegenerative diseases due to inhibition of neuronal cell death has not been developed.

Accordingly, according to a preferred embodiment of the present invention, there is provided a pharmaceutical composition for the prevention or treatment of degenerative brain neurological disease containing mayin as an active ingredient. This effectively inhibits the cell death of nerve cells by effectively inhibiting the activity of oxidative stress, thereby excellent protection against neuronal cell damage caused by oxidative stress can be useful as an effective preventive or therapeutic agent of degenerative neurological diseases. have.

The present invention includes the drug as an active ingredient.

Maysin is a yellow plant pigment flavonoid family found in corn, teosinte and centipedes grass, and has been known for its antioxidant and anticancer activity. By the way, the present invention confirmed the prevention and treatment effect of maize mustache-derived degenerative neurological disease.

Meisin of the present invention is derived from corn beard, according to a preferred embodiment of the present invention, it can provide a composition for improving neurodegenerative disorders of the brain, which comprises a mysin derived from corn beard as an active ingredient.

A novel physiological activity of macine derived from the cornbeard was developed. As a result, it was confirmed that the macine inhibited the neuronal cell death by oxidative stress, and the neurons were protected from the oxidative stress by increasing the expression of antioxidant enzymes.

In addition, the active ingredient of the present invention may inhibit the cell death of neurons by increasing the expression of antioxidant enzymes in neurons. Specifically, as shown in Example 1 of the present invention, it was confirmed that apoptosis of human neuroblastoma (SK-N-MC) induced by H ₂ O ₂ was inhibited by treating the mycin. As a result of observing the apoptosis inhibitory effect by the macine treatment in the graph of Example 1, it was observed that when treated with 10 μg / ml of macine, inhibiting cell death by 55 ~ 60%. It can be seen from this that may be used as a therapeutic agent for neurodegenerative diseases of the brain (see Example 1).

When apoptosis occurs in the cell, the DNA is broken down in the nucleus, and the DNA that is bound to the histone is gradually fragmented and fragmented. Cell death could be detected through DNA fragmentation, and when cell death was detected, the cells were placed in an agarose gel and subjected to electrophoresis. Specifically, as shown in Example 2 of the present invention, the macine inhibits apoptosis of neurons induced by H ₂ O ₂ and inhibits DNA fragmentation activity by apoptosis. You can check it.

Meanwhile, the term “apoptosis” of the present invention is a programmed apoptosis process at a certain time and place, and many human diseases as well as physiological processes such as tissue homeostasis and embryonic development. Excessive cell death leads to atrophy and degenerative neuropathy.

In addition, according to a preferred embodiment of the present invention, the may be able to inhibit the oxidative stress induced by H ₂ O ₂ . Our body regulates the amount of free radicals in the body by itself. When excessive exercise is excessively increased due to excessive exercise, or the ability to remove them, various diseases caused by harmful oxygen are caused. The side effects of the harmful oxygen is called oxidative stress. Overproduction of free radicals, which causes oxidative stress to accumulate continuously in the body, affects the genes of cells or damages the immune system.

In addition, according to a preferred embodiment of the present invention, it can be confirmed that the macine inhibits reactive oxygen species (ROS) by inhibiting oxidative stress. When the amount of reactive oxygen species increases rapidly, degenerative neurological diseases including Alzheimer's disease occur. The reactive oxygen species overproduced by H ₂ O ₂ refers to an unstable state having free radicals, and thus has strong activity. Thus, by oxidizing proteins, lipids, and the like in the cells, the homeostasis of the cells is destroyed and the cells are killed. Specifically, as shown in Example 3 of the present invention, the active oxygen species in the neurons induced by H ₂ O ₂ was found to be reduced by the treatment of Meissin, H ₂ O ₂ and the control group not treated with H ₂ O was analyzed by comparing the _second one nerve cell process, the concentration of the active oxygen species of the H ₂ O ₂ treated neurons was confirmed that the increase of 1.62 times compared with the control group. In addition, when treated with 10 μg / ml of macine in neurons, it was confirmed that the concentration of reactive oxygen species decreased by 40 to 45%. It was found that the activity of reactive oxygen species is mediated by H ₂ O ₂ .

In addition, as described above, the intracellular antioxidant action is composed of an enzymatic defense system and an antioxidant system. Therefore, antioxidant enzymes are very important in inhibiting oxidative stress in cells. According to a preferred embodiment of the present invention, the macine may confirm that the activity of antioxidant enzymes in neurons is increased. The antioxidant enzyme may act as a biocatalyst. Biocatalysts, such as catalase, are included in the animal and plant kingdoms, decomposing harmful hydrogen peroxide from metabolic processes into oxygen, and providing oxygen back to oxidation.

Specifically, as shown in Example 4 of the present invention, it was confirmed that the enzymes such as catalase, glutathione peroxidase, superoxide dismutase and heme oxygenase increased by 2.49 to 14.75 times by treating the neurons with neurons. More specifically, catalase (CAT) increased the enzyme activation by 260-265% when 25 μg / ml of macine was treated, and glutathione peroxidase (GPx-1) treated 25 μg / ml of macine In this case, it was confirmed that the activation of the enzyme increased by 200 to 205%. Also. Superoxide dismutase-1 (SOD-1) increased the enzyme activation by 260 ~ 265% when 25 μg / ml of macine was treated, and superoxide dismutase-2 (SOD-2) increased 25 μg / ml of macine In the case of treatment, the activation of enzyme was increased by 230-235%, and heme oxygenase (HO-1) was found to increase the activation of enzyme by 550-555% when 10 μg / ml of mesine was treated. . These enzymes are a mechanism that transforms free radicals produced by oxidative stress in nerve cells into stable substances and protects nerve cells from damage caused by oxidative stress through metabolic processes.

And, according to a preferred embodiment of the present invention, it can be confirmed that the macine protects the cells from damage of neurons by inhibiting oxidative stress in neurons. In addition, when the macine is used as an active ingredient for the prevention or improvement of neurodegenerative diseases caused by oxidative stress, preferably, the concentration of the macine is 0.1 ~ 1000 ㎍ / ㎖, preferably 1.0 ~ 500 ㎍ / ㎖ Concentrations, more preferably at a concentration of 7.5-100 μg / ml, can achieve the physiological effects described above. Meanwhile, in FIG. 3, the inhibitory effect on the generation of reactive oxygen species was remarkable between the concentrations of Mayine between 5.0 μg / ml and 10.0 μg / ml. Therefore, it can be seen that most preferably, when the concentration of macine is 7.5 µg / ml, there is a significant difference in effect.

In addition, it may be confirmed that the macine increases cell viability and decreases cytotoxicity. As shown in Example 5 of the present invention, the cell viability is increased according to the concentration of macine, and the cytotoxicity of the control group is not treated with H ₂ O ₂ . As a result of comparing the toxicity of cells treated with H ₂ O ₂ , The toxicity of the treatment with H ₂ O ₂ cells compared to the control group not treated with H ₂ O ₂ 4 times as it was confirmed that the number of toxicity. It was confirmed that the mycin is mediated in preventing cytotoxicity caused by H ₂ O ₂ .

On the other hand, the neurodegenerative diseases of the present invention may be Alzheimer's, Parkinson's disease, Huntington's disease, multiple sclerosis, multiple neurotrophic, epilepsy, brain disease (encephalopathy), or stroke.

The term "degenerative brain neuropathy" means a term that generically refers to various diseases related to nerves, in particular, cranial nerves. Preferably, the degenerative brain disease according to the present invention means diseases which the nerve cell damage caused by the oxidative stress induced by H ₂ O ₂ induced.

The composition for preventing or treating degenerative neurological disease of the present invention may include a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be in various oral or parenteral formulations. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Solid form preparations for oral administration include tablet pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate, sucrose or lactose in one or more compounds. ) And gelatin. In addition to simple excipients, lubricants such as magnesium stearate, talc and the like are also used. Liquid preparations for oral administration include suspensions, solution solutions, emulsions, and syrups, and various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin, may be included. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition is any one selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations and suppositories. It can have one formulation.

In addition, these pharmaceutical compositions are useful for administering TSPOs of the invention to a subject for the treatment of various diseases, including neurodegeneration and / or symptoms associated therewith, as described above.

The composition of the present invention is administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dose level is determined by the type and severity of the subject, age, sex, activity of the drug, drug Sensitivity, time of administration, route of administration and rate of release, duration of treatment, factors including concurrent use of drugs, and other factors well known in the medical arts.

The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be single or multiple doses. In consideration of all the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects. Typical dosages of the pharmaceutical compositions of the invention are in the range of 0.001-100 mg / kg on an adult basis.

The route of administration of the pharmaceutical composition may be administered via any general route as long as it can reach the target tissue. The composition of the present invention may be administered as desired, but is not limited to intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, pulmonary administration, rectal administration. The composition may also be administered by any device in which the active agent may migrate to the target cell.

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

According to another preferred embodiment of the present invention, it provides a health functional food composition for the prevention or improvement of neurodegenerative diseases comprising the composition.

In the case of using the mycin of the present invention as a food additive, it may be added as it is or may be used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixed amount of the active ingredient can be determined suitably according to the purpose of use (prevention, health or therapeutic treatment).

As used herein, the term "health functional food" refers to a food prepared and processed by extracting, concentrating, refining, and mixing a specific ingredient as a raw material or contained in a food ingredient for the purpose of health supplement. It refers to foods that are designed and processed to sufficiently exert bioregulatory functions on the living body, such as biological defense, regulation of biorhythms, prevention and recovery of diseases, by the above components, and the composition for health foods prevents diseases and It can perform functions related to the recovery of diseases.

In addition, there is no restriction on the kind of health foods in which the composition of the present invention can be used. In addition, tofu extract of the present invention can be prepared by mixing the known additives with other appropriate auxiliary ingredients that may be contained in the health functional food according to the choice of those skilled in the art. Examples of foods that can be added include meat, sausages, breads, chocolates, candy, snacks, confections, pizzas, ramen noodles, dairy products including other noodles, gums, ice creams, various soups, beverages, teas, drinks, alcoholic beverages and Vitamin complexes, and the like, can be prepared by adding the extract according to the present invention as a main ingredient juice, tea, jelly and juice.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

EXAMPLE

Example 1 Neuronal Apoptosis Assay

Example 1-1: H ₂ O ₂ On by Induction of Apoptosis of Neurons

Apoptosis was measured using the Annexin V-FITC apoptosis detection kit (Bio Vision, USA). Neurons (SK-N-MC) were incubated for 24 hours in a 6-well plate and 5, 10, 25, 50 μg / ml of Meisin was pretreated with the cells for 2 hours, followed by H ₂ O ₂ for 24 hours. Treated. Cells were collected by treatment with Trypsin-EDTA, centrifuged to remove supernatant, suspended in 500 μl binding buffer, and 5 μl Annexin V-FITC and 5 μl PI (propidium iodide) were added. The reaction was carried out for 5 minutes with the light blocked at room temperature. Analysis was performed using flow cytometry.

Example 1-2 Inhibition of Apoptosis of Neurons by Meisin Treatment

In the group treated with H ₂ O ₂ only, apoptotic apoptosis was increased by about 8.05 times, and apoptosis was reduced by 85-90% when pretreated with 50 μg / ml of macine (FIGS. 1A and 1B). ).

In order to confirm the inhibitory effect of H ₂ O ₂ induced neuronal cell death, SK-N-MC, a human blastoma, was treated with mayin, followed by H ₂ O ₂ treatment and fluorescence using Annexin V / PI. After staining, cell death was observed using a flow cytometer. As shown in Figure 1A and Figure 1B as a result of Example 1, the active ingredient of the macine was able to confirm the neuronal cell death inhibitory effect. Looking at Figure 1A, control refers to a control group not treated with both Meisin and H ₂ O ₂ . to the next The case where H ₂ O ₂ was treated alone was shown. It is a graph showing that the treatment of the micelle with H ₂ O ₂ and concentration. As shown in the graph, it can be seen that as the concentration of macine increases, cell death decreases. FIG. 1B quantifying the results of FIG. 1A, it can be seen that there is a significant difference in effect when the concentration of the macine is 5 to 10 μg / ml.

In addition, H ₂ O ₂ is produced by disproportionation of superoxide anion and various oxidases in vivo, and is also a representative compound of oxygen poison. Since it is removed by the antioxidant enzyme in vivo, it was confirmed that the oxidase was removed by the antioxidant enzyme of the macine to confirm the effect of inhibiting cell death of neurons.

Example 2: Confirmation of Neuronal DNA Fragmentation Change

Example 2-1: H ₂ O ₂ in Induce DNA fragmentation

The DNA fragmentation of neurons induced by H ₂ O ₂ was observed by light microscopy to confirm the inhibitory activity of macine. Neurons (SK-N-MC) were incubated for 24 hours on a cover glass, and pretreated for 2 hours at 5, 10, 25, 50 μg / ml of mayin, and then treated with H ₂ O ₂ for 24 hours. . After fluorescence staining using an ApoDIRECT In Situ DNA fagmentation assay kit (Biovision Research Products, Mountain View, Calif., USA), morphological changes of neuronal DNA were observed with a fluorescence microscope (× 100).

Example 2-2 Inhibition of DNA Fragmentation by Meisin Treatment

When H ₂ O ₂ was treated after 5%, 10, 25, and 50 μg / ml of mayin, DNA fragmentation was reduced when compared to the control group treated with H ₂ O ₂ only. , It could be confirmed that apoptosis is reduced by reducing DNA fragmentation of neurons (FIG. 2).

The DNA fragmentation means DNA fragmentation, and when apoptosis occurs in a cell, DNA fragmentation separated from the nucleus can be confirmed little by little.

That is, the cells shrink, gaps are created between them, and DNA is regularly cut and fragmented within the cells. At the end, the entire cell is fragmented and eaten by adjacent cells, ending their lifetime. Therefore, it can be seen that inhibiting cell apoptosis by inhibiting DNA fragmentation.

In this case, comtrol refers to a control group not treated with both macine and H ₂ O ₂ . to the next The case where H ₂ O ₂ was treated alone was shown. By H ₂ O ₂ concentration and a diagram showing that the treatment with god mate. As shown in the figure, DNA fragmentation decreases as the concentration of macine increases.

As shown in FIG. 2, H ₂ O ₂ was induced by treating the macine DNA fragmentation was inhibited. Therefore, the active ingredient of mayin can be usefully used to suppress the cell death of neurons by effectively inhibiting DNA fragmentation that causes cell death.

Example 3: Measurement of Intracellular Active Oxygen Species (ROS)

Example 3-1: H ₂ O ₂ in Free radical species induction

The amount of reactive oxygen species (ROS) generated in SK-N-MC cells induced by H ₂ O ₂ treatment was measured using the fluorescent probe 2′7′-dichlorofluorescin diacetate (DCF-DA, Sigma, USA). . SK-N-MC cells are spread on 96-well plates at a density of 1 × 105 cells / ml and incubated for 24 hours. 5, 10, 25, 50 μg / ml of mayin was pretreated for 2 hours followed by 200 μM H ₂ O ₂ for 3 hours. After the reaction was completed, washed twice with 1 × PBS, DCF-DA (10 mM in DMSO) 100μL to each well so that the final concentration is 10 uM and reacted for 45 minutes at 37 ℃, excitation Fluorescence was measured at a wavelength of 485 nm and an emission wavelength of 530 nm. As a result, the treatment with H ₂ O ₂ for three hours compared with the control group not treated with H ₂ O ₂ cells was observed that the active oxygen species by about 1.62 times.

Example 3-2 Inhibition of Reactive Oxygen Species by Meisin Treatment

After 2 hours pretreatment of Meisine at 5, 10, 25, and 50 μg / ml concentrations, the H ₂ O ₂ treated group decreased the amount of intracellular reactive oxygen species by concentration, and at about 50 μg / ml concentration. It was confirmed that 31.42% of reactive oxygen species was reduced (FIG. 3).

Active oxygen species refers to a state in which free radicals are not stable because they have free radicals, and thus have strong activity. Excessive production of reactive oxygen species is called toxic oxygen because it causes toxicity to the living organism, that is, oxidative stress. By oxidizing huge molecules (proteins, lipids, etc.) in the cell, it destroys the homeostasis of the cell, kills the cell, and causes fatal damage in the tissue. Reactive oxygen species include singlet oxygen, superoxite radicals, hydroxy radicals and hydrogen peroxide (H ₂ O ₂ ).

That is, the reactive oxygen species is increased by the H ₂ O ₂ and accordingly the oxidation stress is increased. Figure 3 showing the experimental results carried out in Example 3 was confirmed that the active oxygen species can be effectively inhibited by treating the mycin. At 5 μg / ml, the concentration of reactive oxygen species was reduced to 30 to 35%, and it was confirmed that the concentration of the macine was reduced to 40 to 45% of 50 μg / ml.

Through the experimental results of Example 3, it can be confirmed that the macine is effective in inhibiting reactive oxygen species.

Example 4 Observation of mRNA Changes of Antioxidant Enzymes (CAT, GPx-1, SOD-1,2, HO-1)

Example 4-1: Isolation of Total RNA

In order to investigate the effects on the change of the enzymes of the enzyme (CAT, GPx-1, SOD-1,2, HO-1), the mRNA level of the antioxidant enzymes was examined by real-time PCR. Neurons (SK-N-MC) were incubated for 24 hours in a 6-well plate and treated with mayin at concentrations of 5, 10, 25 and 50 μg / ml for 2 hours, and then H ₂ O ₂ for 24 hours. Treated. After the reaction, the solution was washed with PBS, dissolved with TRIzol reagent (invitrogen, USA), and then added with Chloroform (Sigma, USA) and centrifuged at 4 ° C. and 13,000 rpm for 10 minutes. RNA is isolated using Total RNA Extraction kit (intron, Korea). After the reaction by adding 400 μL of Binding Buffer to the isolated RNA, wash using washing buffer A and B. Finally, 50 μl of RNA is eluted with elution buffer. Total RNA isolated was quantified with a spectrophotometer to a concentration of 1 μg.

Example 4-2: Synthesis of 1st strand cDNA

Total RNA isolated was synthesized first strand complementary DNA (cDNA) using the Power cDNA Synthesis Kit (intron, Korea). 1 μg of Oligo (dT) 15primer was added to 1 μg of total RNA, followed by reaction at 75 ° C. for 5 minutes, followed by reaction at 4 ° C. for 1 minute. Add RNase inhibitor, 5X RT buffer, dNTP, DTT, AMV RT enzyme and react for 1 hour at 42 ℃. CDNA obtained by reacting for 5 minutes at 75 ℃ was used for real-time PCR.

Example 4-3 Real-time PCR with cDNA

Add 1 μl of each synthesized cDNA, 1 μl of sense-antisense primer, 10 μl of 2X Master mix Solution (Applied Biosystems, Foster City, CA), and 8 μl of DEPC Water, and then add StepOnePlus ™ system (Applied Biosystems, Foster City, CA). Real-time PCR was performed.

As a result, mRNA levels of all antioxidant enzymes were decreased in the H ₂ O ₂ treated group compared to the control group without any treatment. CAT, GPx-1, SOD-1,2 were treated in the 50 μg / ml treatment group , HO-1 mRNA level was confirmed to increase 2.49 times, 2.42 times, 2.40 times, 3.15 times, 14.75 times, respectively (Figs. 4A, 4B, 4C, 4D and 5E).

The antioxidant enzymes are beneficial substances for removing oxidase present in cells, and SOD-1 and SOD-2 are antioxidant enzymes that inhibit free radicals by decomposing the over-production of harmful active oxygen into hydrogen peroxide and oxygen. Hydrogen peroxide decomposed by SOD-1 and SOD-2 is decomposed into oxygen and water by catalase and glutathione peroxidase to act as antioxidant enzymes. In addition, heme oxygenase is an effective enzyme that maintains homeostasis by oxidizing heme in the blood. Therefore, the macine is useful for effectively removing free radicals by enhancing intracellular antioxidant enzymes.

Figure 4A showing the experimental results carried out in Example 4 is a graph quantifying the expression amount of catalase through the level of mRNA. It can be seen that there is a significant difference in the effect between the concentration of 5 ~ 10 μg / ml of mesine, the catalase was expressed up to 80 ~ 85% of the control when treated with 50 μg / ml. In addition, Figure 4B showing the experimental results carried out in Example 4 is a graph quantifying the expression amount of glutathione peroxidase-1 through the level of mRNA. It can be seen that the expression amount is increased by 2.42 times when the macine is treated with 50 μg / ml, compared with the case without the treatment with the mycin. In addition, Figure 4C showing the experimental results carried out in Example 4 is a graph quantifying the expression amount of superoxide dismutase-1 through the level of mRNA. When treated with 50 μg / ml of mayin compared to the case without the treatment, it can be seen that the expression amount increases 2.40 times. In addition, Figure 4D showing the experimental results carried out in Example 4 is a graph quantifying the expression amount of superoxide dismutase-2 through the mRNA level. It can be seen that the amount of expression increased by 3.15 times when treated with 50 μg / ml compared to the case without treatment with the mycin. In addition, Figure 4E showing the experimental results carried out in Example 4 is a graph quantifying the expression amount of heme oxygenase through the mRNA level. It can be seen that the expression amount increased by 14.75 times when treated with 50 μg / ml compared to the case without treatment with the mycin.

Example 5: Cell Viability and Cytotoxicity Measurement

Example 5-1 Measurement of Cell Viability by MTT Assay

Changes in cell viability following pre-treatment of macine for neuronal cell death by H ₂ O ₂ treatment were tested by MTT assay. Human neuroblastoma SK-N-MC cells were cultured in 96-well plates at 37 ° C. and 5% CO 2 for 24 hours at a density of 1 × 105 cells / ml. After 5-50 μg / ml Meisin was pretreated for 2 hours, 200 μM H 2 O 2 was treated for 24 hours. After the reaction, 20 μl of 3- [4,5-dimethyl-thiazol] -2,5-diphenyl-tetrazolium bromide (MTT) reagent at a concentration of 5 mg / ml was added thereto and reacted for 4 hours, followed by 200 μl. Dimethyl sulfoxide (DMSO) was added and the absorbance was measured at 570 nm using a Microplate reader (Molecular devices, USA). Relative cell viability of each treatment group was determined using the control group without the sample. In H ₂ O ₂ only group, cell viability decreased by 55.8%, and the concentration of cell-dependent pretreatment significantly increased the cell viability, and 80.3% in case of 50 μg / ml of macine. The figures are shown (FIG. 5A).

Example 5-2: Cytotoxicity Measurement by LDH Assay

After the pre-treatment of the mycin, the change in neuronal toxicity when H ₂ O ₂ treatment was measured by LDH assay. After SK-N-MC cells were incubated in a 6-well plate for 24 hours at 37 ° C. and 5% CO 2 conditions, 5-50 μg / ml mayin was pretreated for 2 hours, and 200 μM H 2 O 2 was treated for 24 hours. . After taking the cell culture, the supernatant was taken by centrifugation at 250 × g, 10 min, 4 ℃ condition and LDH activity was measured using LDH cytotoxicity detection kit (Takara bio inc, Tokyo, Japan). Finally, the absorbance was measured at 490 nm using a microplate reader, and the relative cytotoxicity of each treatment group was determined as fold increase using 1 as the control without the sample. In the group treated with H ₂ O ₂ only, cytotoxicity was increased by about 4 times, and pretreatment with 50 μg / ml concentration of mesine was found to reduce about 33.5% cytotoxicity (FIG. 5B).

5A showing the results of the experiment conducted in Example 5, it was found that the cell viability was increased by treating the mycin. When the concentration of 50 μg / ml, it was confirmed that to increase the 80 to 85% of the conrtol group effectively inhibit the H ₂ O ₂ . Treatment of macine increases the cell viability of neurons. In addition, Figure 5B is a graph showing a decrease in cytotoxicity by treatment with macine. When treated with 50 μg / ml of mayin, it was confirmed that the cytotoxicity is reduced by 60 ~ 65% compared to the case without the treatment with the mycin.

As shown in Example 5, it was confirmed that the cell viability was increased and cytotoxicity was reduced through the treatment of Meicin.

[Production example]

Preparation Example 1 Preparation of Pharmaceutical Composition

Examples of the formulation of the pharmaceutical composition comprising the above-mentioned mesine of the present invention will be described, but the present invention is not intended to be limited thereto but only to be described in detail.

Preparation Example 1-1. Manufacture of powder

Meishin 2 g

1 g lactose

The above ingredients were mixed and filled in airtight cloth to prepare a powder.

Preparation Example 1-2. Manufacture of tablets

Meisin 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Preparation Example 1-3. Preparation of Capsule

Meisin 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.

Preparation Example 1-4. Manufacture of rings

Meishin 1 g

Lactose 1.5 g

1 g of glycerin

Xylitol 0.5 g

After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.

Preparation Example 1-5. Preparation of Granules

Meisin 150 mg

Soy extract 50 mg

Glucose 200 mg

Starch 600 mg

After mixing the above components, 100 mg of 30% ethanol was added, dried at 60 ° C. to form granules, and then filled into fabrics.

Preparation Example 2 Preparation of Health Functional Food

Preparation Example 2-1. Preparation of Biscuits

Force Grade 1 19.59 wt%, Gravity Grade 1 22.22 wt%, White sugar 4.80 wt%, Salt 0.73 wt%, Glucose 0.78 wt%, Palm shortening 11.15 wt%, Ammo 1.54 wt%, Neutral 0.17 wt%, Sodium bisulfite 0.16 wt% , Rice flour 1.45%, Vitamin B1 0.0001%, Vitamin B2 0.0001%, Milk flavor 0.04%, Water 21.3298%, Whole milk powder 1.16%, Substitute powder 0.29%, calcium phosphate 0.03%, Spray salt Biscuits were prepared in a conventional manner by combining 0.29% by weight, 7.27% by weight of spray oil, and 7% by weight of the macine.

Preparation Example 2-2. Preparation of Chewing Gum

Chewing gum was prepared in a conventional manner by combining 20% by weight of gum base, 70% by weight of sugar, 1% by weight of perfume, 2% by weight of water, and 7% by weight of the above-mentioned macine.

Preparation Example 2-3. Candy

Candy was prepared in a conventional manner by combining 50% by weight of sugar, 39.8% by weight of starch syrup, 0.2% by weight of fragrance, and 10% by weight of the mesine.

Preparation Example 2-4 Preparation of Beverages

0.26% by weight of honey, 0.0002% by weight of thioctoamide, 0.0004% by weight of nicotinic acid, 0.0001% by weight of sodium riboflavinate, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoic acid and 89.7362% by weight of water and the may A beverage was prepared by the conventional method by combining 10% by weight of thinner.

Claims (10)

1. Pharmaceutical composition for the prevention or treatment of degenerative cerebral nervous system disease containing mayin as an active ingredient.
2. The method of claim 1,

   The composition is a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, characterized in that to inhibit neuronal cell death.
3. The method of claim 1,

   The composition is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that derived from corn beard.
4. The method of claim 1,

   The composition is a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, characterized in that to increase the activity of the antioxidant enzymes of nerve cells.
5. The method of claim 1,

   The composition is a pharmaceutical composition for the prevention or treatment of degenerative cerebral nervous system disease, characterized in that to inhibit the oxidative stress induced by H ₂ O ₂ .
6. The method of claim 1,

   The composition is a pharmaceutical composition for the prevention or treatment of degenerative cerebral nervous system disease, characterized in that to inhibit the reactive oxygen species of neurons.
7. The method of claim 1,

   The composition is a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, characterized in that it shows a protective effect of nerve cells.
8. The method of claim 1,

   The Meisin is a pharmaceutical composition for the prevention or treatment of degenerative neurological diseases, characterized in that it is contained in a concentration of 7.5 ~ 100 μg / ml.
9. The method of claim 1,

   The neurodegenerative disease is any one selected from the group consisting of Alzheimer's, Parkinson's disease, Huntington's disease, multiple sclerosis, multiple nerve atrophy, epilepsy, brain disease (encephalopathy), or stroke is characterized in that the prevention or treatment of neurodegenerative diseases. Pharmaceutical composition for.
10. Health functional food composition for the prevention or improvement of degenerative cerebral nervous system disease containing mayin as an active ingredient.

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