KR100951185B1 - A compound extracted from Phellinus linteus for protection of neuronal cells, method for preparing the compound and a composition comprising the compound as an effective component - Google Patents

Abstract

The present invention relates to a compound for protecting nerve cells extracted from a situation mushroom, a method for producing the same, and a composition for protecting a nerve cell containing the compound as an active ingredient, and more specifically, a compound for protecting a nerve cell extracted from a situation mushroom represented by Formula 1 below. In addition, the present invention relates to a method for preparing the same and a neuronal cell protective composition containing the compound as an active ingredient:

(1)

(One)

According to the present invention, experimentally verified the applicability of the DHP derivative extracted from the situation mushroom as a neuroprotective compound, and effectively inhibits the death of neurons mediated by hydrogen peroxide, thereby protecting the neuronal protective compound having an excellent neuronal protective effect. Can provide.

Description

A compound extracted from Phellinus linteus for protection of neuronal cells, method for preparing the compound and a composition comprising the compound as an effective component}

The present invention relates to a compound for protecting nerve cells extracted from a situation mushroom, a method for producing the same, and a composition for protecting a nerve cell containing the compound as an active ingredient, and more specifically, the applicability of the DHP derivative extracted from a situation mushroom as a compound for nerve cell protection. By experimentally verifying, the present invention relates to a nerve cell protective compound having an excellent neuronal cell protective effect, a method for preparing the same, and a nerve cell protective composition containing the compound as an active ingredient.

Various neuropathological changes, including edema, ischemia, etc., increase the production of intracellular calcium and free radicals and affect secondary damage, which leads to regeneration of central nervous system neurons. It is caused by an improper central nervous system environment rather than a lack of ability. Therefore, various studies are being actively conducted to promote the regeneration of the central nervous system by neutralizing or replacing an inappropriate environment in the central nervous system and stimulating a violent cellular response.

In general, Spinal Cord Injury (SCI) has three stages: acute phase, secondary tissue damage followed by significant ischemic necrosis, and chronic phase. Increased production of radicals, excessive release of excitatory neurotransmitters, and inflammatory reactions occur.

On the other hand, oxidative stress is assumed to play an important role in the pathophysiology of SCI, and this oxidative stress is mediated by Reactive Oxygen Species (ROS). ROS generate oxidative ruptures or free radicals as part of cell lysis and immune reactions, which can occur only when there are sufficient free transition metals that can act as catalysts, mainly as a product of cellular respiration associated with mitochondrial electron transport. Can be. Oxidative damage mediated by ROS is caused by an imbalance between ROS production and prophylactic mechanism activity, whereby excessively produced ROS can cause excessive exposure of cells, leading to free radical mediated damage Can be. The damaging effects mediated by ROS can be inhibited by antioxidant systems including enzymes that act to remove ROS such as glutathione, ascorbic acid and superoxide dismutase, glutathione peroxidase and catalase. However, functional defects of the antioxidant system by ROS cause various oxidative damages and consequently cell death.

In particular, neurons such as cultured spinal cord-derived neural progenitor cells (NPCs) and neural stem cells (NSCs) are sensitive to an increase in ROS, resulting in cell death. ROS-mediated apoptosis in neurons is often observed during neural stem cell therapy and therapeutic radiotherapy of the brain. Therefore, various cellular defense systems have been reported, such as converting ROS into inactive species by enzymatic methods, chelating transition metal catalysts, or decoding ROS using antioxidants.

On the other hand, 4- (3,4-dihydroxyphenyl) (DHP) derivative is a fat-soluble organic compound, phellinus linteus ) can be extracted and purified. In particular, DHP derivatives are known to effectively inhibit cell death mediated by ROS in a variety of cells, exert a variety of biological activities through reduced ROS production at low concentrations, including anti-cancer, anti-inflammatory and anti-infective activity. It has various pharmacological effects. In this regard, Korean Unexamined Patent Publication No. 2005-74070 discloses a DHP derivative, a preparation method thereof, and a use thereof as a novel compound having antioxidant activity isolated from a situation mushroom, and the antioxidant activity of the compound is determined by free radical removal test. It is identified through, and discloses a method for preparing various types of antioxidant formulations including the same.

However, there is no research on how the antioxidant activity of the DHP derivative affects the protection of nerve cells such as neural progenitor cells and neural stem cells, and the application of the DHP derivative to a compound or composition for protecting nerve cells There is no experimental support for the possibility.

Therefore, the first problem to be solved by the present invention, by experimentally verifying the applicability of the DHP derivative extracted from the situation mushroom as a neuronal protective compound, to provide a neuronal protective compound having an excellent neuronal protective effect.

The second problem to be solved by the present invention is to provide a method for producing the compound for nerve cell protection from the situation mushroom.

In addition, the third problem to be solved by the present invention is to provide a neuronal protective composition containing the neuronal protective compound as an active ingredient.

In order to achieve the first object of the present invention,

It provides a compound for protecting nerve cells extracted from the situation mushroom represented by the following formula (1):

In order to achieve the second object of the present invention,

Collecting and drying the situation mushrooms, extracting with ethanol, filtering, and concentrating the filtrate to obtain an ethanol extract;

Dissolving the ethanol extract in a mixture of hexane and H ₂ O to obtain a hexane extract;

Obtaining an active fraction by performing column chromatography on the hexane extract using a hexane-ethyl acetate concentration gradient; And

Drying the active fraction

It provides a method for producing a compound for protecting nerve cells extracted from the situation mushroom represented by the formula (1) comprising:

In addition, the present invention to achieve the third object,

It provides a neuronal protective composition containing a neuronal protective compound extracted from the situation mushroom represented by the formula (1) as an active ingredient:

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

In the present invention, by using the antioxidant activity of the DHP derivative represented by the following formula 1 it was intended to use it as a neuronal protective compound:

(1)

(One)

In the present invention, it is understood that the compounds of formula 1 affect neurotoxic substances in neurons such as neural progenitor cells and neural stem cells, and this effect is assessed by reduced lipid peroxidation. It may be related to the regulation of reduction reaction. The regulation of the redox reaction is directly related to the fact that the compound of formula 1 has various antioxidant activities including expression of thioredoxin reductase, removal of hydrogen peroxide, activation of caspase-3 and caspase-9, and the like. It is presumed to be related, and the present invention has been specifically proved through various experimental methods.

The compound according to Formula 1, known as a DHP derivative, has various antioxidant activities and functions as a useful defense mechanism against active oxygen species. In addition, antioxidants generally perform cell growth, survival, cytotoxicity, transcriptional regulation of various genes, as well as transformation regulation related to redox response and chemical toxicity, but DHP derivatives still have some function in the organism. It is not fully understood whether this is done. Therefore, the present invention was to find that the compound of formula 1 has an excellent neuronal protective effect, and to apply this property as a compound for neuronal protection.

In addition, in the present invention, as a manufacturing method for the compound for protecting the neuron,

Collecting and drying the situation mushrooms, extracting with ethanol, filtering, and concentrating the filtrate to obtain an ethanol extract; Dissolving the ethanol extract in a mixture of hexane and H ₂ O to obtain a hexane extract; Obtaining an active fraction by performing column chromatography on the hexane extract using a hexane-ethyl acetate concentration gradient; And it provides a method for producing a compound for protecting nerve cells extracted from the situation mushroom, characterized in that it comprises the step of drying the active fraction,

Furthermore, it provides a neuronal protective composition containing the neuronal protective compound as an active ingredient.

The nerve cell protective composition according to the present invention may further comprise a pharmaceutically acceptable carrier, in addition to the compound of formula 1 as an active ingredient, such carriers, excipients, binders, lubricants, disintegrating agents, lubricants, coatings And one or more carriers selected from the group consisting of emulsifiers, suspensions, solvents, stabilizers, absorption aids, water for injection and isotonic agents. Specifically, lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin as a binder, dicalcium phosphate as an excipient, corn starch or sweet potato starch as a disintegrating agent, magnesium stearate as a lubricant, Calcium stearate, sodium stearyl fumarate, polyethylene glycol wax, etc. are mentioned.

The composition according to the present invention may be formulated as an oral or parenteral preparation. Examples of oral preparations include granules, tablets, capsules, solutions, and the like, and when administered parenterally, intravenous injection, subcutaneous injection, muscle It may be applied in the form of internal injection, intraperitoneal injection, or chest injection, and the dosage may vary depending on the age, sex, and weight of the patient. To formulate into a parenteral formulation, the composition may be mixed in water with stabilizers or buffers to prepare a solution and formulated in unit dosage forms of ampoules or vials.

The composition may be prepared in the form of health food, in which case the compound represented by the formula (1) is added to food materials such as beverages, teas, spices, gum, confectionery, or the like prepared by encapsulation, powdered, suspension, etc. As a food, it means that it has a certain health effect.

According to the present invention, experimentally verified the applicability of the DHP derivative extracted from the situation mushroom as a neuroprotective compound, and effectively inhibits the killing of neurons mediated by hydrogen peroxide to provide a neuroprotective compound having an excellent neuronal protective effect. Can provide.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the following Examples are only to aid the understanding of the present invention and should not be construed as limiting the scope of the present invention.

Example  1. Preparation of Compounds According to the Invention

300 g of the mushrooms were taken, dried at 60 ° C. for 3 days, and then extracted with 500 ml of ethanol for 1 week. Subsequently, the extract was dissolved in 100 ml of a mixture of hexane and H ₂ O (volume ratio of hexane: H ₂ O = 4: 1) to obtain 5 ml of hexane extract. Next, 3 ml of active fractions were purified by separation using silica gel (0.063-0.200 mm, Merck) column chromatography using a hexane-ethyl acetate concentration gradient as an eluent. 0.3 g of compound was prepared.

Example  2. Culture of Adult Spinal Cord-derived Neural Progenitor Cells and Neural Stem Cells

Mice were anesthetized for culturing adult spinal cord derived neural progenitor cells (NPCs) and neural stem cells (NSCs). To obtain adult spinal cord-derived neuronal progenitor cells, a portion of the complete cervical enlargement (spinal cord level C3 through T1) was excised and removed. In addition, in order to obtain neural stem cells derived from the adult brain, mouse fetal brain was extracted.

After removing the brain membrane, the tissue was cut finely, washed with PBS and treated with 0.125% trypsin and 0.01% DNAase (DNase) and incubated for 30 minutes at 37 ℃. Cells were cultured in NB medium containing B27 supplement, bFGF (20 ng / ml), EGF (20 ng / ml), and cultured in Petri dishes with neurospheres or in PDL-treated culture dishes.

Example  3. NPCs Wow NSCs For Example  Treatment of the compounds according to H ₂ O ₂ Exposure to

Cells were seeded in NB medium and then cultured in a CO ₂ incubator at 5% O ₂ concentration. Cells at 80% concentration were pretreated with the compound according to Example 1 (1 ng / ml) for 8 hours and then treated with H ₂ O ₂ (0.3 mM) for 6 hours. The optimal concentration of the compound according to Example 1 and H ₂ O ₂ was determined by preliminary studies related to cytotoxicity and cell survival effects at various concentrations of reactants. H ₂ O ₂ dilutions were prepared by diluting a 30% stock solution with DMEM immediately prior to the experiment. Cells were measured over time as cultures and toxic or biochemical measurements were taken. The H ₂ O ₂ treatment was confirmed to reduce the toxicity of H ₂ O ₂ if not done within 5 minutes after dilution, which may be due to the high reaction force of H ₂ O ₂ and the short half-life in the dilute solution.

Example  4. Cell viability measurement

Cell viability was measured at least three times under the same conditions via Tryphan Blue staining.

Example  5. Spinal Cord Injury Animal Model Induction

Rats (adult female Wistar, 260g, 6 weeks-old) were used as a method for causing spinal cord injury. The following method was used to induce trauma to experimental animals.

Rats were anesthetized with ketamine (75 mg / kg) and xylazine (10 mg / kg), followed by spinal cord destruction by inducing physical damage to the T9 and T10 sites. To assess the effect of the compound according to Example 1 on acute stage spinal cord injury (SCI) pathophysiology, rats that induced spinal cord injury were observed in three groups (n = 40). The experimental group was mixed with matrigel 10 ng / ml of the compound according to Example 1 directly into the damaged area (n = 30), the control group was mixed with DMSO and the matrigel into the damaged area (n = 10).

Example  6. About Spinal Cord Injury Models Example  Treatment of the compounds according to 1

Purified compound according to Example 1 (10 ng / ml) was dissolved in DMSO, mixed with Matrigel and injected directly into the site of injury immediately after spinal cord injury. As a control, the same amount of DMSO was mixed with Matrigel and added to the damaged area. 40 experimental animals were used, divided into two groups of 30 spinal cord injury groups treated with the compound according to Example 1 and 10 spinal cord injury groups as a control group. After spinal cord injury, experimental treatment (compound according to Example 1 mixed with Matrigel) or control treatment (DMSO mixed with Matrigel) was immediately injected into the intra-thecal space of the injured spinal cord, and the skin The incision was closed.

Example  7. ATP  Measure

Protein quantitation was measured using a protein quantification kit (Bio-Rad, Hercules, CA, USA). Cells were (150 mmol / L KCl, 25 mmol / L Tris-HCl; pH 7.6, 2 mmol / L EDTA pH 7.4, 10 mmol / L KPO4 pH 7.4, 0.1 mmol / L MgCl ₂ and 0.1% (w / v) BSA (1 mg protein concentration in 1 ml of buffer) buffer was suspended. ATP synthesis was performed with 250 ml of cell suspension in 750 μl of substrate buffer (10 mmol / L malate, 10 mmol / L pyruvate, 1 mmol / L ADP, 40 μg / ml digittonin and 0.15 mmol / L adenosine pentaphosphate). It was started by putting. ATP quantification was measured by a luminometer (Berthold, Detection Systems, Pforzheim, Germany) using the ATP Bioluminescence Assay Kit (Roche Diagnostics, Basel, Switzerland). Experimental data were analyzed using change analysis on Fisher or t- test (see FIG. 6).

Example  8. Caspase  Analysis and Quantification

For measuring caspase-3 and caspase-9 activity, proteins were mixed with caspase-Glo 3 or 9 reagent (Promega). After reacting for 2 hours at room temperature, luminescence was measured using a TD 20/20 luminometer (Turner Designs, Sunnyvale, Calif.).

Example  9. Intracellular Reactive oxygen species  Measure

Intracellular ROS was measured using the fluorescent labeling index DCFDA. For observation by fluorescence microscopy, cells were plated and treated with 0.3 mM H ₂ O ₂ only or with the compound according to Example 1 followed by 0.3 mM H ₂ O ₂ . Next, cells were washed and then treated with 10 μM of DCFDA at 37 ° C. for 30 minutes and observed with a fluorescence microscope (ex / em = 495/525 nm). To quantify ROS development, neural progenitor cells derived from spinal cord were washed with HBSS and then treated with 10 μM of DCFDA at 37 ° C. for 30 minutes. Cells were washed three times with HBSS and exposed with 100 μM of t BHP or SS-31. Oxidation of DCF was measured in real time by microplate spectrofluorometer (ex / em = 485/530 nm) (Molecular Devices, Sunnyvale, Calif.).

Example  10. Flow Cytometry  ( Flow cytometry )

Flow cytometry was performed on the cells with BD Biosciences FACScan (San Jose, Calif.). Cell ratios of G0 / G1, S, G2 / M phases of the cell cycle were measured by DNA histogram fitting program (MODFIT; Verity Software, Topsham, ME).

Example  11. Histological Analysis

Experimental animals were anesthetized with sodium pentobarbital and the spinal cord was fixed with 4% paraformaldehyde. The fixed spinal cord was maintained at 30% sucrose for 48 hours and cut to 10 μm thickness with a frozen microtome. To analyze the upregulation of proteins in the injured spinal cord, tissues at the center of the injury were labeled with immunochemical methods using specific antibodies such as ED-1 and GFAP, and then analyzed using confocal microscopy.

Example  12. Statistical Analysis

All data were presented using ± SEM from five or more independent experiments. The statistical significance of the differences between groups was calculated using Student's two tailed t- test.

Evaluation of Experiment Results

Figure 1 shows the growth effect on the cultured RAW 264.7 macrophages (5 × 10 ⁵ / ml) of the compound according to formula (1). Referring to FIG. 1, the growth rate of macrophages is slowed or reduced in proportion to the concentration of the compound. This result suggests that the compound according to Chemical Formula 1 indirectly suggests the possibility of acute treatment of central nervous system diseases by inhibiting the growth of immune cells involved in inflammatory reactions including macrophages.

On the other hand, it was possible to determine the following items from the experimental results according to Examples 1 to 12.

1) The compound according to Example 1 protects NPCs and NSCs from apoptosis mediated by hydrogen peroxide and affects cell growth.

As can be seen from the results of FIG. 2, hydrogen peroxide-mediated apoptosis was clearly observed on NPCs and NSCs on the second day, but apoptosis was observed in the culture pretreated with the compound (1 ng / ml) according to Example 1 Not observed. Therefore, it was found that pretreatment of 1 ng / ml of the compound according to Example 1 protects NPCs and NSCs from apoptosis induced by hydrogen peroxide treatment at a concentration of 0.3 mm or 0.05 mm. In addition, when NPCs and NSCs were pretreated with 1 ng / ml of the compound according to Example 1, the growth of NPCs and NSCs by the pretreatment of the compound according to Example 1 was achieved. It was found that this increase, in this case it was found that the optimum concentration of the compound according to Example 1 is 1 ng / ml. On the other hand, when the concentration of the compound according to Example 1 in Figure 2 10 ng / ㎖ over time it can be seen that the effect of cell growth is lower compared to the much smaller concentration of 1 ng / ml. After all, the optimum concentration of the compound according to Example 1 is 1 ng / ml, 10 ng / ㎖ when the concentration of the compound according to Example 1 is 0.5 ~ 5 ng / ㎖ when considering that the effect is rather deteriorated It can be expected that the effect is excellent.

2) NPCs, NSCs, Spinal Cord Injury In the animal model, the compound according to Example 1 inhibits apoptosis characteristics mediated by hydrogen peroxide.

Hydrogen peroxide increases cellular oxidative stress and induces cell death. Thus, the effect of the compound according to Example 1 on ROS generation after hydrogen peroxide treatment was evaluated in NPCs. Hydrogen peroxide increased the oxidation of DCF in a concentration dependent manner and the increased DCF fluorescence intensity was inhibited by the compound according to Example 1 at 1 ng / ml (see FIG. 3). It is believed that the compound itself according to Example 1 did not affect ROS generation, but reduced ROS generation mediated by hydrogen peroxide and inhibited apoptosis mediated by hydrogen peroxide by maintaining mitochondrial integrity.

In addition, since hydrogen peroxide stimulates caspase activity, it was observed whether the compound according to Example 1 inhibited caspase-3 and caspase-9 activity in hydrogen peroxide-treated NPCs and NSCs and spinal cord injury animal models (FIG. 4, 5, 7a).

Experimental results showed that after 24 hours and 48 hours of hydrogen peroxide treatment in NPCs and NSCs, the activity of caspase-3 and caspase-9 was significantly increased, but the compound pretreatment according to Example 1 was caspase mediated by hydrogen peroxide. It was found to significantly inhibit activation (see FIGS. 4 and 5). After hydrogen peroxide exposure for 1 and 7 days in spinal cord injury animal model, the activity of caspase-3 and caspase-9 was significantly increased, but pretreatment of the compounds according to Example 1 significantly increased the caspase activation mediated by hydrogen peroxide. Inhibiting (FIG. 7A),

Furthermore, the effect of the compound according to Example 1 on ATP production after hydrogen peroxide treatment in NPCs and NSCs was evaluated (see FIG. 6). As a result of the evaluation, after treatment with hydrogen peroxide for 24 and 48 hours, respectively, ATP production was significantly reduced, but the compound pretreatment according to Example 1 significantly restored ATP production. These results indicate that the compound according to Example 1 inhibits hydrogen peroxide mediated apoptosis characteristics in NPCs and NSCs, spinal cord injury animal models as well as brain injury models.

3) The compounds according to Example 1 protect cells from hydrogen peroxide mediated apoptosis in NPCs, NSCs, and spinal cord injury animal models.

In order for the cells to survive, inhibition of apoptosis is required, and spinal cord injury shows extensive proliferation of many microglia and macrophages around the epicenter (see FIG. 7B), but is performed. Animal groups treated with the compound according to Example 1 were found to reduce the infiltration and activation of ED-1 positive macrophages and microglyal cells.

4) The compound according to Example 1 reduces apoptosis at the site of spinal cord injury.

Spinal cord injury is a result of cell death, and cell death caused by injury before and after treatment with the compound according to Example 1 mixed with Matrigel as the injury site was confirmed by TUNEL staining. Compound pretreatment according to Example 1 effectively inhibited TUNEL positive cells mediated by injury.

As can be seen from the results of Examples 1 to 12, in the present invention, the compound according to Example 1 has an immunomodulatory function and an antioxidant reaction, in particular, the pathology of inflammatory diseases such as macrophage and microglyal cells activation and acute spinal cord injury. It has been experimentally demonstrated that it plays a key role in suppressing the development of ROS, a critical mediator in physiology.

In addition, in the present invention, the pathway regulated by the compound according to Example 1 is in vitro NPCs and NSCs, in vivo spinal cord injury animal model p-P38, p-SAPK / JNK, PARP, p-ERK, Bax, caspase 3, 9 activation and cytochrome c was found (see Figures 4, 5 and 7). The short-term effect of the compound according to Example 1 was mainly inhibition of apoptosis, and cell damage mediated by ROS was significantly reduced by the compound pretreatment according to Example 1. Pretreatment of the compounds according to Example 1 in vitro culture (NPCs and NSCs) not only lowered intracellular ROS levels (see FIG. 3), but also effectively inhibited the activation of caspase-3 and caspase-9 (FIG. 3). 4 and 5), the cell's ability to produce ATP was significantly recovered (see FIG. 6).

Activation of caspase occurs after spinal cord injury, and also increases in PI3K / p-AKT, p-ERK1 / 2 and Bcl-2 proteins play an important role in the execution of apoptosis in the injured spinal cord, in particular caspase-3 And caspase-9 contribute to apoptosis through PARP cleavage (see FIGS. 5 and 7A). In addition, cleavage of DNA repair enzymes by PARP during apoptosis preserves the intracellular pool of ATP essential for the inhibition of DNA repair as well as for the progression of apoptosis.

Thus, it is clear that the main protective effect of the compounds according to Example 1 in spinal cord injury is the inhibition of secondary apoptosis with immunomodulatory function (see FIG. 7B). The results of Examples 1-12 above demonstrate that the compound according to Example 1 significantly reduces apoptosis and functional defects immediately after SCI. The animal group treated with the compound according to Example 1 recovered hind limb reflexes even faster, which showed a higher response rate compared to the control.

Application of the compound according to the present invention as a compound for protecting nerve cells is very useful for improving nerve function and preventing secondary pathological damage after spinal cord injury, and as a result, to achieve new drug development goals for the treatment of human spinal cord injury. It will be a starting point.

1 is a diagram showing the growth effect on the cultured RAW 264.7 macrophages (5x10 ⁵ / ml) of the compound according to formula (1).

Figures 2a and 2b shows the effect of reducing the apoptosis mediated by hydrogen peroxide of the compound according to formula 1, Figure 2a is a pre-treatment of the cultured NPCs with a compound according to formula (1 ng / ml) for 8 hours FIG. 2B shows the results of treatment of 0.3 mm H ₂ O ₂ for 5 days, and FIG. 2B shows pretreatment of cultured NSCs with a compound according to Formula 1 (1 ng / ml) for 8 hours and H of 0.05 mm. the ₂ O ₂ is a diagram showing results of processing for 5 days.

FIG. 3 shows the effect of pretreatment of a compound according to Formula 1 on hydrogen peroxide mediated ROS generation in cultured NPCs.

Figure 4 is a diagram showing the effect of the pretreatment of the compound according to formula (1) on the inhibition of apoptosis mediated by hydrogen peroxide in cultured NPCs.

5 is a diagram showing the effect of the pretreatment of the compound according to formula (1) on the inhibition of apoptosis mediated by hydrogen peroxide in cultured NSCs.

FIG. 6 shows the effect of a compound according to Formula 1 on ATP production after hydrogen peroxide treatment in NPCs and NSCs .

7 is a diagram showing the inhibitory effect on cell death characteristics in a spinal cord injury experimental animal model of the compound according to formula 1, Figure 7a is a caspa mediated by hydrogen peroxide of the compound according to formula 1 in a spinal cord injury experimental animal model FIG. 7B is a diagram showing the results of immunostaining of the damaged part of the spinal cord with ED-1 and GFAP antibodies. FIG.

Claims (3)

delete delete Neuronal cell protective composition containing 0.5 ~ 5 ng / ㎖ as an active ingredient neuron protective compound extracted from the situation mushroom represented by Formula 1:

(One)

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