KR101554343B1 - Pharmaceutical compositions for the prevention and treatment of the neuro-degenerative Disorders - Google Patents

Abstract

The present invention relates to a composition for ameliorating a neurodegenerative disease caused by oxidative stress, which comprises maize bean-derived meiocin as an active ingredient. More specifically, the present invention relates to a composition for inhibiting neuronal cell death caused by oxidative stress by catalase, glutathione The present invention relates to a method for treating a neurodegenerative disease of the nervous system by inhibiting or inhibiting apoptosis of neurons by increasing mRNA expression of peroxidase-1, superoxide dismutase-1,2, heme oxygenase-1, Lt; / RTI >

Description

[0001] The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases,

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases, and more particularly, to a pharmaceutical composition for preventing or treating neurodegenerative diseases, .

Degenerative brain nervous system diseases are known to be the main cause of neuronal death due to oxidative stress. Recently, the degenerative brain nervous system diseases including Alzheimer's disease and the like have been rapidly increasing the amount of active oxygen species in nerve cells, which is considered to be the main cause of diseases caused by oxidative stress. Therefore, it has been reported that by inhibiting or reducing oxidative stress, such diseases can be prevented or treated.

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

As the aging population increases, the trend of degenerative brain diseases is on the rise. Despite the innovative development of medicine, prevention and treatment methods for degenerative brain neurological diseases are still unclear and no drugs with definite effects have been found .

Although there are currently developed therapeutic agents and therapies for degenerative brain nervous system diseases, the development of materials that can reduce and treat side effects and toxicity is urgent because it often shows side effects and toxicity due to long-term use, to be.

The corn beard has traditionally been ingested as a food or folk remedy for various diseases and is relatively stable and has high potential for development as a new drug derived from natural materials. Although Patent No. 10-1201628 has reported on the pharmacological effect on the antibiotic action and the anticancer effect of the corn beard extracts, the inhibitory activity against neuronal cell death due to oxidative stress and the neuronal cell protective activity have not been reported yet , There is a problem that a composition for ameliorating a degenerative brain neurological disease by inhibiting neuronal apoptosis by reducing oxidative stress caused by H ₂ O ₂ has not been developed.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a pharmaceutical composition for preventing or treating neurodegenerative diseases. A second object of the present invention is to provide a health functional food composition for preventing or ameliorating a degenerative brain disease comprising the composition.

In order to achieve the first object, the present invention provides a pharmaceutical composition for preventing or treating degenerative cerebral neurological diseases containing mexin as an active ingredient.

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 hairs.

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

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

According to another preferred embodiment of the present invention, the composition can inhibit active oxygen species of nerve cells.

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

According to another preferred embodiment of the present invention, the composition is contained at a concentration of 5 to 100 μg / ml.

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

To achieve the second object, there is provided a health functional food composition for preventing or ameliorating a degenerative brain nervous system disease containing the composition.

Hereinafter, terms used in this specification will be briefly described.

In the present invention, the term "prevention" means any action that inhibits the apoptosis of neuronal cells or delays neuronal cell death by administration of the composition, and "treatment" Means any act that improves or benefits.

The present invention effectively inhibits the activity of oxidative stress, thereby reducing the amount of reactive oxygen species, and has an excellent protective effect against damage caused by activated oxidative stress on neurons, thus being useful as a preventive or therapeutic agent for effective degenerative brain diseases have.

FIG. 1A is a graph showing the neuronal cell death inhibition effect using Annexin V / PI fluorescence staining as a result of neuronal cell death by Meishin treatment according to a preferred embodiment of the present invention.
FIG. 1B is a graph showing numerical values obtained by using Annexin V / PI fluorescent staining as a result of experiments on neuronal cell death by Meishin treatment according to a preferred embodiment of the present invention.
FIG. 2 is a graph showing a result of inhibition of DNA fragmentation by Meicin treatment according to a preferred embodiment of the present invention. As shown in FIG. 2, DNA fragmentation induced by H ₂ O ₂ is expressed as ApoDIRECT In Situ DNA fagmentation assay kit.
FIG. 3 is a graph showing experimental results of inhibiting the activity of active oxygen species by Meicin treatment according to a preferred embodiment of the present invention.
FIG. 4A is a graph showing experimental results of observation of increase in expression of catalase (CAT), an antioxidant enzyme in neurons, by Meishin treatment according to a preferred embodiment of the present invention.
FIG. 4B is a graph showing the results of observations of an increase in the expression of glutathione peroxidase-1 (GPx-1), an antioxidative enzyme in neurons, by Meicin treatment according to a preferred embodiment of the present invention.
FIG. 4C is a graph showing the results of experiments in which an increase in the expression of superoxide dismutase-1 (SOD-1), an antioxidative enzyme in neurons, was induced by Meicin treatment according to a preferred embodiment of the present invention.
FIG. 4D is a graph showing the results of observation of an increase in the expression of superoxide dismutase-2 (SOD-2), an antioxidative enzyme in neurons, by Meicin treatment according to a preferred embodiment of the present invention.
FIG. 4E is a graph showing the results of experiments in which the expression of heme oxygenase-1 (HO-1), an antioxidative enzyme in neurons, was observed by treatment with mexin according to a preferred embodiment of the present invention.
FIG. 5A is a graph showing experimental results of observing that cell survival rate of neurons is increased by treatment with mexin according to a preferred embodiment of the present invention. FIG.
FIG. 5B is a graph showing an experiment result of observing that cytotoxicity of neurons is reduced by Meicin treatment according to a preferred embodiment of the present invention. FIG.

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

As mentioned above, corn beard has been reported study the pharmacological effect of the antibiotic and anti-cancer effect, yet not with respect to inhibitory activity and neuroprotective activity against the neuronal death caused by oxidative stress to reported, H ₂ O ₂ has not been developed to reduce the neuronal apoptosis by reducing the oxidative stress induced by the neurodegenerative diseases.

Thus, according to a preferred embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating degenerative cerebral neurological diseases containing mexin as an active ingredient. Thus, it is possible to effectively inhibit the activity of oxidative stress, thereby inhibiting apoptosis of neuronal cells. Thus, it has excellent protective effect against damage of nerve cells due to oxidative stress and thus can be effectively used as a preventive or therapeutic agent for degenerative brain diseases have.

The present invention includes meiocin as an active ingredient.

Maysin is a yellow plant-pigment flavonoid-based substance found in corn, teosinte and centipedegrass, and has been known to have antioxidant and anticancer activities. However, in the present invention, Meishin derived from corn beard confirmed the preventive and therapeutic effect on the degenerative brain nervous system diseases.

Meishin of the present invention is derived from corn hairs. According to a preferred embodiment of the present invention, it is possible to provide a composition for improving the neurodegenerative diseases of the nervous system, which comprises Meishin derived from cornstarch as an active ingredient.

The novel physiological activity of the maize derived from the corn beard was developed. As a result, it was confirmed that Mechin inhibits oxidative stress - induced neuronal cell death, and protects neurons from oxidative stress through increased expression of antioxidant enzymes.

In addition, the active ingredient of the present invention may inhibit apoptosis of neuronal cells 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 mexin. In the graph of Example 1, it was observed that inhibition of cell death by meiocin treatment inhibited cell death by 55 ~ 60% when Meicin 10 μg / ml was treated. Thus, it can be seen that mexin can be used as a therapeutic agent for degenerative brain neurological diseases (see Example 1).

And, when cell death occurs in the cell, the DNA is broken down in the nucleus, and the DNA bound to the histone is fragmented and fragmented little by little. DNA debris can be used to detect apoptosis, and when the apoptosis is detected, the cells can be identified by electrophoresis in an agarose gel. Specifically, as shown in Example 2 of the present invention, the Mechin inhibits H ₂ O ₂ -induced neuronal cell apoptosis and inhibits the activity of DNA fragmentation by apoptosis Can be confirmed.

The term " apoptosis "of the present invention is a programmed cell suicide process at a predetermined time and place. It is a physiological process such as tissue homeostasis and embryonic development, Excessive cell death results in atrophic and degenerative neurological diseases.

Also, according to a preferred embodiment of the present invention, the mechine can inhibit oxidation stress induced by H ₂ O ₂ . Our body regulates the amount of active oxygen in the body itself. If the exercise is excessively increased due to excessive exercise or the function of removing them is decreased, various diseases caused by harmful oxygen are induced. The side effect of harmful oxygen is called oxidative stress. If excess oxygen is produced and oxidative stress accumulates continuously in the body, it affects or damages cellular genes and weakens the immune system.

In addition, according to a preferred embodiment of the present invention, it can be confirmed that the Mechin inhibits oxidative stress and inhibits ROS. When the amount of the active oxygen species is rapidly increased, a degenerative brain nervous system disease including Alzheimer's disease occurs. The active oxygen species produced by H ₂ O ₂ excessively have free radicals, which means that they are not stable and have strong activity. Thus, by oxidizing proteins, lipids, etc. in the cell, it destroys the homeostasis of the cell and kills the cell. Specifically, as shown in Example 3 of the present invention, it was found that the active oxygen species in the neurons induced by H ₂ O ₂ decreased by treating with Meicin, and the control group without H ₂ O ₂ and H ₂ O ₂ - treated neurons, the concentration of reactive oxygen species in the neurons treated with H ₂ O ₂ increased by 1.62 times compared to the control group. In addition, it was confirmed that the concentration of active oxygen species decreased to 40 ~ 45% when the neuron was treated with mexin 10 μg / ml. This indicates that the activity of the active oxygen species is mediated by H ₂ O ₂ .

In addition, as described above, intracellular antioxidant activity is composed of an enzymatic defense system and an antioxidant system. Therefore, antioxidant enzymes are very important in suppressing oxidative stress in cells. According to a preferred embodiment of the present invention, it can be confirmed that the mexin increases the activity of antioxidant enzymes in nerve cells. The antioxidant enzyme may act as a biocatalyst. Biocatalysts such as catalase are contained in many animal and plant systems, and decompose the harmful hydrogen peroxide generated in the metabolic process to make oxygen, and it plays a role of providing oxygen again to oxidation.

Specifically, as shown in Example 4 of the present invention, it was confirmed that enzymes such as catalase, glutathione peroxidase, superoxide dismutase, and heme oxygenase increased from 2.49 to 14.75 times by treating mechicine in neurons. More specifically, when catalase (CAT) was treated with 25 μg / ml of mexin, enzyme activation was increased to 260-265%, and glutathione peroxidase (GPx-1) was treated with 25 μg / ml of mexin , It was confirmed that the enzyme activity was increased to 200 ~ 205%. Also. Superoxide dismutase-1 (SOD-1) increased the enzyme activity to 260 ~ 265% when 25 μg / ml of mexin was treated, and 25 μg / ml of superoxide dismutase-2 The enzyme activity was increased to 230 ~ 235%, and when heme oxygenase (HO-1) was treated with 10 μg / ml of mexin, enzyme activation was increased to 550 ~ 555% . These enzymes transform the active oxygen species produced by oxidative stress in neurons into stable substances and protect them from damage caused by oxidative stress through metabolic processes.

In addition, according to a preferred embodiment of the present invention, it can be confirmed that the Mechin inhibits oxidative stress in nerve cells to protect cells from damage of nerve cells. When Mexicin is used as an active ingredient for preventing or ameliorating a degenerative brain nervous system disease caused by oxidative stress, it is preferable that the concentration of Mexicin is in the range of 0.1 to 1000 占 퐂 / ml, preferably 1.0 to 500 占 퐂 / ml Concentration, more preferably 7.5 to 100 占 퐂 / ml, can achieve the physiological effects described above. On the other hand, in FIG. 3, the inhibitory effect on the production of reactive oxygen species was remarkable at a concentration of meicin between 5.0 / / ㎖ and 10.0 / / ㎖. Therefore, it can be seen that when the concentration of meicin is most preferably 7.5 μg / ml, the effect is remarkably different.

In addition, it can be confirmed that the mexin increases cell viability and cytotoxicity. As shown in Example 5 of the present invention, the cell survival rate was increased according to the concentration of meicin, and the cytotoxicity was evaluated by comparing the control group not treated with H ₂ O ₂ As a result of comparing the toxicity of H ₂ O ₂ treated cells, 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 Meishin was mediated to prevent cytotoxicity of H ₂ O ₂ .

On the other hand, the degenerative brain disease of the present invention may be Alzheimer's, Parkinson's disease, Huntington's disease, multiple sclerosis, or stroke.

The term " degenerative brain disease " refers to a generic term for various diseases related to nerves, particularly, cranial nerves. Preferably, the degenerative brain disease of the present invention refers to a disease caused by H ₂ O ₂ -induced oxidative stress, resulting in neuronal cell damage.

The composition for preventing or treating degenerative brain disease of the present invention may comprise a pharmaceutically acceptable carrier. The composition comprising a pharmaceutically acceptable carrier may be of various oral or parenteral formulations. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

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

Formulations for parenteral administration include sterilized 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, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition may be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze- It can have one formulation.

In addition, these pharmaceutical compositions are useful for administering the TSPO of the present invention to a subject for treating various diseases, including neurodegeneration and / or related symptoms, 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 the effective dose level will depend on the species and severity, age, sex, The time of administration, the route of administration and the rate of excretion, the duration of the treatment, factors including co-administered drugs, and other factors well known in the medical arts.

The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and administer an amount that will achieve the maximum effect in the least amount without side effects. Typical dosages of the pharmaceutical compositions of this invention are in the range of 0.001-100 mg / kg on an adult basis. (Dose, Method of administration)

The route of administration of the pharmaceutical composition may be administered through any conventional route so long as it can reach the target tissue. The composition of the present invention may be administered intraperitoneally, intravenously, intramuscularly, subcutaneously, intradermally, orally, intranasally, intrapulmonarily, or rectally, though it is not intended to be limited thereto. In addition, the composition may be administered by any device capable of transferring the active agent to the target cell.

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

According to another preferred embodiment of the present invention, there is provided a health functional food composition for preventing or ameliorating a degenerative brain disease comprising the composition.

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

The term " health functional food " used in the present invention means a food which is prepared by using a specific ingredient as a raw material for the purpose of health assisting, or by preparing, concentrating, refining, Refers to foods that are designed and processed so that the body control function such as bio-defense, regulation of biorhythm, prevention and recovery of disease, etc. can be sufficiently exhibited to the living body by the above-mentioned ingredients. Recovery of disease, and the like.

There is no limitation on the kind of health food to which the composition of the present invention can be used. In addition, the mulberry extract of the present invention can be prepared by mixing a suitable auxiliary ingredient and a known additive which may be contained in health functional foods according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component.

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

[ Example ]

Example  1: Neuronal death analysis

Example  1-1: H ₂ O ₂ On by Induction of neuronal cell death

Cell death was measured using the Annexin V-FITC apoptosis detection kit (Bio Vision, USA). Cells were cultured in 6-well plates for 24 h and pretreated with 5, 10, 25, and 50 μg / ml of macrophages for 2 h. H ₂ O _{2 was added} for 24 h Respectively. Cells were collected by trypsin-EDTA treatment and centrifuged to remove the supernatant. After the supernatant was suspended in 500 μl of each buffer, 5 μl of Annexin V-FITC and 5 μl of propidium iodide were added The reaction was carried out at room temperature for 5 minutes with blocking of light. And analyzed using flow cytometry.

Example  1-2: inhibition of neuronal cell death by Meicin treatment

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

In order to confirm the suppression effect of H ₂ O ₂ -induced neuronal cell death, SK-N-MC, a human blastoma, was treated with Me ₂ , treated with H ₂ O ₂ , and analyzed with Annexin V / After staining, apoptosis was observed using a flow cytometer. As shown in the results of FIGS. 1A and 1B of Example 1, the effective ingredient of the above Mechin was able to confirm the effect of inhibiting neuronal cell death. Referring to FIG. 1A, control refers to a control group not treated with both macrocyclic and H ₂ O ₂ . to the next H ₂ O ₂ was treated alone, and then H ₂ O ₂ and mexine by concentration. As shown in the graph, it can be seen that the cell death decreases with increasing concentration of meiocin. Fig. 1B, in which the results of Fig. 1A are quantified, shows that there is a significant difference in effect when the concentration of meicin is 5 to 10 μg / ml.

In addition, H ₂ O ₂ is produced in vivo by the disproportionation reaction of superoxide anion and various oxidases, and is also a representative compound of acid disinfection. Since it is removed in vivo by the antioxidant enzyme, the oxidase was removed by the antioxidant enzyme of the above Mechin, and the effect of inhibiting the cell death of the neuron was confirmed.

Example  2: nerve cell DNA fragmentation  Confirm change

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

The DNA fragmentation of neurons induced by H ₂ O ₂ was observed with a light microscope to confirm the inhibitory activity of meicin. Neurons (SK-N-MC) were cultured on a cover glass for 24 hours, pretreated with MeShin at concentrations of 5, 10, 25 and 50 μg / ml for 2 hours and then treated with H ₂ O ₂ for 24 hours . After fluorescent staining with 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: Meishin treatment DNA fragmentation  control

The pretreatment of Mechin at concentrations of 5, 10, 25 and 50 μg / ml, followed by H ₂ O ₂ treatment, showed a decrease in DNA fragmentation as compared to the control group treated with H ₂ O ₂ alone (Fig. 2), which was confirmed by decreasing DNA fragmentation of neurons.

The DNA fragmentation refers to DNA fragmentation, and when the cell death occurs, it is confirmed that the DNA isolated from the nucleus is slightly interrupted.

In other words, the cells are shrunk, gaps are formed between cells, and DNA is regularly cleaved in the cells. At the end, the whole cell is fragmented, and it is consumed by the adjacent cells, and the life is finished. Therefore, it can be seen that inhibition of DNA fragmentation inhibits cell apoptosis.

At this time, comtrol means a control group that did not treat macroin and H ₂ O ₂ . to the next H ₂ O ₂ was treated alone, and then H ₂ O ₂ and Meissin by concentration. As shown in the figure, it can be seen that as the concentration of meiocin increases, DNA fragmentation decreases.

As shown in Figure 2, by treating the meicin, the H ₂ O ₂ -induced DNA fragmentation was suppressed. Thus, the efficacy of meiocin can be effectively used to inhibit apoptosis of neuronal cells by effectively inhibiting DNA fragmentation which causes apoptosis.

Example  3: intracellular activity Oxygen species  ( ROS )

Example  3-1: H ₂ O ₂ in activation Oxygen species  Judo

The amount of reactive oxygen species (ROS) produced in SK-N-MC cells induced by H ₂ O ₂ treatment was measured using a fluorescent probe, 2'7'-dichlorofluorescin diacetate (DCF-DA, Sigma, USA) . SK-N-MC cells are plated in 96-well plates at a density of 1 × 10 5 cells / ml and cultured for 24 hours. 5, 10, 25 and 50 μg / ml of Meicin were pretreated for 2 h and then treated with 200 μM H ₂ O ₂ for 3 h. After completion of the reaction, the cells were washed twice with 1 × PBS, treated with 100 μl of DCF-DA (10 mM in DMSO) to a final concentration of 10 μM, incubated at 37 ° C. for 45 minutes, The 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: Meishin treatment Reactive oxygen species  control

The pretreatment of mexin at 5, 10, 25, and 50 μg / ml for 2 h decreased the amount of intracellular reactive oxygen species by H ₂ O ₂ , And 31.42% of active oxygen species was reduced (Fig. 3).

The active oxygen species is a state in which the active oxygen species has free radicals and can not be stabilized, thereby having strong activity. Excessive production of reactive oxygen species leads to toxicity to the body, that is, oxidative stress, which is sometimes referred to as harmful oxygen. It oxidizes huge molecules (proteins, lipids, etc.) in the cell, destroys the homeostasis of the cells, kills the cells, and causes fatal damage in the cell tissue. Active oxygen species include singlet oxygen, superoxime radicals, hydroxy radicals, and hydrogen peroxide (H ₂ O ₂ ).

That is, the active oxygen species is increased by the H ₂ O ₂ and the oxidative stress is increased accordingly. FIG. 3, which shows the results of the experiment conducted in Example 3 above, confirmed that the active oxygen species was effectively inhibited by treating Meicin. It was confirmed that the active oxygen species decreased to 30 ~ 35% at the macrocein concentration of 5 μg / ml and decreased to 40 ~ 45% at the macrocein concentration of 50 μg / ml.

From the results of the experiment of Example 3, it can be confirmed that meicin is effective in suppressing active oxygen species.

Example  4: Antioxidant enzymes ( CAT , GPx -One, SOD -1,2, HO -1) of mRNA  Change observation

Example  4-1: Total RNA Separation of

To investigate the effect of antioxidant enzymes (CAT, GPx-1, SOD-1,2 and HO-1) on the changes of mecin, mRNA levels of antioxidant enzymes were measured by real-time PCR. Nerve cells (SK-N-MC) to 6-well and then cultured in a plate for 24 hours, and make-receiving process for 2 hours at 5, 10, 25 and a concentration of _{50 μg / ml, H 2 O} 2 for 24 hours Respectively. After the reaction, wash with PBS, dissolve in TRIzol reagent (Invitrogen, USA), add Chloroform (Sigma, USA) and centrifuge at 13,000 rpm for 10 minutes at 4 ℃. Separate the RNA using the Total RNA Extraction kit (intron, Korea). Add 400 μl of binding buffer to the separated RNA, and wash with washing buffer A and B. Finally, 50 μl of RNA is eluted with Elution buffer. The isolated total RNA was quantitated with a spectrophotometer and the concentration was 1 μg.

Example  4-2: 1 st strand cDNA Synthesis of

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

Example  4-3: cDNA Using real - time PCR

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 were added, followed by StepOnePlus ™ system (Applied Biosystems, Foster City, CA) Real-time PCR was performed.

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

The antioxidant enzymes are beneficial substances for eliminating oxidizing enzymes present in cells. SOD-1 and SOD-2 are antioxidant enzymes that inhibit active oxygen by decomposing harmful active oxygen produced in vivo into hydrogen peroxide and oxygen. Hydrogen peroxide decomposed by SOD-1 and SOD-2 acts as an antioxidant enzyme by decomposition into oxygen and water by catalase and glutathione peroxidase. In addition, heme oxygenase is an effective enzyme to maintain body homeostasis by oxidizing heme present in blood. Therefore, it is useful for effectively removing active oxygen by increasing intracellular antioxidant enzymes.

FIG. 4A is a graph illustrating the expression level of catalase through mRNA levels. FIG. It was found that there was a significant difference in the effect between mezin concentration of 5 ~ 10 μg / ml, and when it was treated with 50 μg / ml of mexin, catalase was expressed up to 80 ~ 85% of control. FIG. 4B is a graph illustrating the expression level of glutathione peroxidase-1 through the level of mRNA. When compared with no treatment with meiocin, the expression level increased by 2.42-fold when treated with 50 μg / ml of mexin. FIG. 4C shows the results of the experiment conducted in Example 4. FIG. 4C is a graph showing the expression level of superoxide dismutase-1 expressed in mRNA level. In comparison with no treatment with meiocin, the expression level increased 2.40-fold when treated with 50 μg / ml of mexin. FIG. 4D is a graph showing the expression level of superoxide dismutase-2 expressed in mRNA level. When compared with no treatment with meiocin, the expression level increased 3.15-fold when treated with 50 μg / ml of mexin. 4E, which is an experimental result of Example 4, is a graph that quantifies the expression level of heme oxygenase through the level of mRNA. When compared with no treatment with meiocin, the expression level increased by 14.75-fold when treated with 50 μg / ml of mexin.

Example  5: Measurement of cell viability and cytotoxicity

Example  5-1: MTT assay Cell survival rate

The changes of cell viability by pretreatment of meiocin against H ₂ O _{2 -} treated neuronal cell death were examined by MTT assay. Human SK-N-MC cells were cultured in 96-well plates at 37 ° C and 5% CO2 for 24 hours at a density of 1 × 105 cells / ml. After 5-50 μg / ml mexin was pretreated for 2 hours, 200 μM H2O2 was treated for 24 hours. After the reaction was completed, 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. Of dimethyl sulfoxide (DMSO) was added and the absorbance was measured at 570 nm using a microplate reader (Molecular devices, USA). The relative cell viability of each treatment group was determined by using 100% of the control group to which no sample was added. Cell viability was decreased to 55.8% in H ₂ O _2- treated group, and cell survival rate was significantly increased in pretreatment of mexin in a concentration-dependent manner. Cell survival rate of 80.3% of 50 μg / (Fig. 5A).

Example  5-2: LDH assay Cytotoxicity measurement

Changes in neurotoxicity were measured by LDH assay after pretreatment with mexin and H ₂ O ₂ treatment. SK-N-MC cells were cultured in a 6-well plate for 24 hours at 37 ° C and 5% CO2, pretreated with 5-50 μg / ml mexin for 2 hours, and treated with 200 μM H2O2 for 24 hours . The cell culture was centrifuged at 250 × g, 10 min, and 4 ° C to measure the LDH activity using the 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 calculated as fold increase. The group H ₂ O ₂ only process cytotoxicity was increased about four times, when a pre-make-put of 50 μg / ml concentration was confirmed to be about 33.5% to reduce the cytotoxicity (FIG. 5B).

FIG. 5A showing the results of the experiment conducted in Example 5 shows that the cell survival rate is increased by treating Meicin. At 50 μg / ml concentration, it was confirmed that H ₂ O ₂ was effectively inhibited by increasing to 80-85% of conrtol group. Treatment of meicin increases cell viability of neuronal cells. FIG. 5B is a graph showing that cytotoxicity is reduced by treating meicin. When the macroin was treated with 50 μg / ml, the cytotoxicity was reduced to 60 to 65% as compared with the case without the macroin treatment.

As shown in Example 5 above, it was confirmed that treatment with mexin increased the cell survival rate and decreased cytotoxicity.

[ Manufacturing example ]

Manufacturing example  1: Preparation of pharmaceutical composition

The preparation examples of the pharmaceutical composition containing the above-mentioned Mechin of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described.

Manufacturing example  1-1. Sanje  Produce

Meishin 2 g

Lactose 1 g

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

Manufacturing example  1-2. Manufacture of tablets

Meishin 100 mg

Corn starch 100 mg

Lactose 100 mg

2 mg of magnesium stearate

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

Manufacturing example  1-3. Preparation of capsules

Meishin 100 mg

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.

Manufacturing example  1-4. Manufacture of rings

Meishin 1 g

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.

Manufacturing example  1-5. Manufacture of granules

Meishin 150 mg

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 functional foods

Manufacturing example  2-1. Biscuit  Produce

The grape first grade is 19.59 wt%, the gravity first grade is 22.22 wt%, the white grape is 4.80 wt%, the salt is 0.73 wt%, the glucose is 0.78 wt%, the palm shortening is 11.15 wt%, the ammo is 1.54 wt%, the bezoar is 0.17 wt%, the sodium bisulfate is 0.16 wt% 1.45 wt% of rice flour, 0.0001 wt% of vitamin B1, 0.0001 wt% of vitamin B2, 0.04 wt% of milk fractions, 21.3298 wt% of water, 1.16 wt% of whole milk powder, 0.29 wt% of replacement milk powder, 0.03 wt% 0.27% by weight of water and 7.27% by weight of spray oil and 7% by weight of Meichen were mixed to prepare a biscuit by a conventional method.

Manufacturing example  2-2. Chewing  Manufacture of gum

Chewing gum was prepared in a conventional manner by mixing 20% by weight of a gum base, 70% by weight of sugar, 1% by weight of a flavor and 2% by weight of water and 7% by weight of Meichen.

Manufacturing example  2-3. Manufacture of candy

50% by weight of sugar, 39.8% by weight of starch syrup, 0.2% by weight of fragrance and 10% by weight of Meichen were mixed to prepare a candy by a conventional method.

Manufacturing example  2-4 Manufacture of beverage

0.0001% by weight of honey, 0.0002% by weight of chitosanic acid amide, 0.0004% by weight of nicotinic acid amide, 0.0001% by weight of sodium riboflavin hydrochloride, 0.0001% by weight of pyridoxine hydrochloride, 0.001% by weight of inositol, 0.002% by weight of orthoacids and 89,7362% 10% by weight of deionized water was blended to prepare a beverage in a usual manner.

Claims (10)

Which is selected from the group consisting of Alzheimer's, Parkinson's disease, Huntington's disease, multiple sclerosis and stroke in which the onset is induced by oxidative stress, which comprises Mechin as an active ingredient and exhibits a protective effect of neurons from oxidative stress. A pharmaceutical composition for the prevention or treatment of one or more degenerative brain diseases The method according to claim 1,
Wherein said composition inhibits neuronal cell death. The prevention or treatment of any one or more degenerative brain diseases selected from the group consisting of Alzheimer's, Parkinson's disease, Huntington's disease, multiple sclerosis and stroke in which the onset is induced by oxidative stress A pharmaceutical composition. The method according to claim 1,
The method according to any one of claims 1 to 3, wherein the mexin is derived from a corn beard. The method for preventing or treating one or more degenerative brain diseases selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis and stroke, A pharmaceutical composition. The method according to claim 1,
Wherein said composition enhances the activity of an antioxidant enzyme of a neuron, wherein the composition is selected from the group consisting of Alzheimer's disease caused by oxidative stress, Parkinson's disease, Huntington's disease, multiple sclerosis and stroke, ≪ / RTI > The method of claim 1,
Wherein the composition inhibits oxidative stress induced by H ₂ O ₂ , wherein the composition is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis and stroke, wherein the disease is induced by oxidative stress. A pharmaceutical composition for the prevention or treatment of cerebral neurological diseases.
The method according to claim 1,
Wherein said composition inhibits reactive oxygen species of neuronal cells, characterized in that it inhibits one or more degenerative brain diseases selected from the group consisting of Alzheimer's, Parkinson's disease, Huntington's disease, multiple sclerosis and stroke induced by oxidative stress Or a pharmaceutically acceptable salt thereof. delete The method according to claim 1,
Wherein said mechicine is contained at a concentration of 7.5 to 100 μg / ml. The method of any one of claims 1 to 3, wherein said mechicine is contained at a concentration of 7.5 to 100 μg / ml. A pharmaceutical composition for the prevention or treatment of neurological diseases.
delete Which is selected from the group consisting of Alzheimer's, Parkinson's disease, Huntington's disease, multiple sclerosis and stroke in which the onset is induced by oxidative stress, which comprises Mechin as an active ingredient and exhibits a protective effect of neurons from oxidative stress. A health functional food composition for preventing or improving one or more degenerative brain diseases.

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