KR20030034420A - Pharmaceutical preparations containing cultured basidiocarps of Phellinus linteus extract for prevention and treatment of neurodegenerative disease - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing, as an active ingredient, an extract of mycelium of Phellinus linteus and a fraction thereof is provided. The composition has nerve cell protecting activity and has no toxicity and is thus effectively used as a therapeutic or treating agent for neurodegenerative diseases such as cerebral apoplexy, dementia, etc. CONSTITUTION: The pharmaceutical composition contains a methanol extract of mycelium of Phellinus linteus, or an EtOAc fraction or a n-hexane fraction thereof having nerve cell protecting activity. It is formulated into a pharmaceutically acceptable formulation with a pharmaceutically acceptable excipient. For an example, 5kg of mycelium of Phellinus linteus is added with 4ml MeOH, followed by sonication, filtration and concentration under reduced pressure to produce 365g of an extract. Thereafter, the extract is suspended in distilled water, followed by fractionation with n-hexane, EtOAc, n-BuOH and concentration under reduced pressure to produce a n-hexane fraction(49g), an EtOAc fraction(34g), a n-BuOH fraction(100g) and a H2O fraction(182g).

Description

Pharmaceutical preparations containing cultured basidiocarps of Phellinus linteus extract for prevention and treatment of neurodegenerative disease

The present invention relates to a situation mushroom mycelium extract having a neuronal protective activity or a fraction thereof and a preventive and therapeutic agent for degenerative cerebral nervous system disease containing the same as an active ingredient.

Recently, as the elderly population increases, the number of patients with degenerative neurological diseases, especially senile dementia, is increasing rapidly. Geriatric dementia is a condition in which memory loss and deterioration in learning ability are the main symptoms and patients and their families must suffer a lot together. As the number of dementia patients due to the increase of the elderly population has become a social problem, the interest in the prevention and treatment of them is increasing day by day, but the severity is getting more serious because there are no treatments yet developed.

Degenerative neurological diseases can occur when the brain is damaged by secondary symptoms caused by adult diseases such as structural degeneration of the neuronal cells due to aging, circulatory disorders, or physical and mechanical factors such as traffic accidents, industrial accidents, and carbon monoxide poisoning. (Rothman, SM (1984) Synaptic release of excitatory amino acid neurotransmitter mediates anoxic neuronal death J. Neurosci 4:... 1884-1891; Weiloch, T. (1985) Hypoglycemia-induced neuronal damage prevented by an NMDA antagonists Science 230: 681-6831-2). In other words, when the blood flow to the brain is reduced or blocked, brain tissue becomes hypoxic and low glucose, so that normal metabolism cannot be performed, and the brain tissue is irreparably damaged. This case is very common in degenerative neurological diseases as well as stroke, trauma and ischemic injury (Benveniste, H., Drejer, J., Schousboe, A. and Diemer, NH (1984) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis.J. Neurochem. 43: 1369-1374; Hagberg, H., Lehmann, A., Saucberg, M., Nystrom , B., Jacobson, I. and Hamberger, A. (1985) Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments.J. Cereb. Blood Flow & Metab. 5: 413-419).

Brain tissue damage is known to be largely due to two mechanisms. First will by increasing the excitatory amino acid glass due to changes in membrane voltage, and second is due to the direct oxidative stress (oxidative stress) (Choi, DW (1988) Glutamate neurotoxicity and disease of the nervous system. Neuron 1: 623-634; Coyle, JT and Purrfarcken, P. (1993) Oxidative stress, glutamateand neurodegenerative disorders Science 262:. 689-695). Therefore, in this study, H ₂ O ₂ or glutamate was used as a toxic substance to induce oxidative stress, which causes brain tissue damage. H ₂ O ₂ is a reactive oxygen species (ROS) that is produced by a variety of causes, both inside and outside cells, and induces the production of other ROS during metabolism (Cross, SAR and Jones, OTG (1991)). ... enzymatic mechanisms of superoxide productions Biochim Biophys Acta 1057:. 281-298). These ROS cause oxidative stress, causing damage to cellular proteins, membranes, DNA, etc., which in turn damage cells and are highly toxic to surrounding cells (Slater, TF (1984) Free-radical mechanisms in tissue injury. Biochem J. 222: 1-15). Cells have various defense mechanisms, such as antioxidants and antioxidant enzymes, against these oxidative stresses. Intracellular antioxidants are represented by GSH, which is mostly present as reduced GSH, and when ROS is increased, it is changed to GSSG. Thus, the increase of GSSG is an indicator of oxidative stress (Huang, J. and Philbert MA (1995) of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells.Brain Res. 680: 16-22).

Recent studies have also shown that in the case of stroke, trauma and ischemic injury, glutamate activates the ionotrophic receptor, causing oxidative stress, leading to glutamatergic abnormalities. Halliwell, B. and Gutteridge, JM (1985a) Oxygen radicals and the nervous system.Trends Neurosci. 5: 22-26; Halliwell, B and Gutteridge JM (1985b) The importance of free radicals and catalytic metal ions in human diseases.Mol. Aspects Med. 8: 89-193). As blood flow in brain tissues decreases, the release of glutamate increases at nerve junctions, and the influx into neurons decreases, rapidly increasing concentrations of glutamate outside the cell, causing toxicity and killing nerve cells (Benveniste, H., Drejer, J., Schousboe, A. and Diemer, NH (1984) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis.J. Neurochem. 43: 1369-1374; Hagberg, H., Lehmann, A., Saucberg, M., Nystrom, B., Jacobson, I. and Hamberger, A. (1985) Ischemia-induced shift of inhibitory and excitatory amino acids from intra- to extracellular compartments. J Cereb.Blood Flow & Metab. 5: 413-419). Neurotoxicity by glutamate is the conversion of glutamate antagonists, Na ⁺ and Ca ²⁺ channel blockers, antioxidants, free radical chain breakers and xanthine dehydrogenase to xanthine oxidases It can be prevented by antiproteases, which are enzymes that inhibit the disease, but antagonists to NMDA receptors are particularly effective (Choi, DW, Koh, J. and Peters, S. (1988) Pharmacology of glutamate neurotoxicity). in cortical cell culture: attenuation by NMDA antagonists J. Neurosci 8:... 185-196; Olney, JW and Sharpe, IG (1969) Brain lesions in an infant rhesus monkey treated with monosodium glutamate Science 166: 368-388).

In the present invention, a natural product that attenuates neurotoxicity induced by H ₂ O ₂ or glutamate in rat cerebral cortical cells primarily cultured with herbal medicines known to be effective for forgetfulness or stroke has been used as a folk medicine or traditional medicine. It was found that the total methanol extract of S. mushroom mycelium culture had significant neuroprotective activity.

Phellinus linteus ( Phellinus linteus ) is a fungus belonging to the genus Mushroom (Hymenochaetceae), which grows on the stems of mulberry and hardwoods and is also called woody mushroom. There are 48 species of mud mushrooms in the world, and 8 species have been found to grow natively in Korea. Similar varieties include Phellinus gilvus , Phellinus ignarius , Black mud mushrooms and Larch mushrooms. Wild mushrooms continue to grow for three to four years, and their shape is maintained in the form of agglomeration of yellow mud in the early stages. The shape of the gat is circular to single or toroidal to horseshoe, and the size is about 10 to 20 cm x 4 to 17 cm, and the surface is initially covered with thin hairs, and is dark brown. The outer surface of the mushroom is a hard piece of wood, with distinct concentric openings and longitudinal cracks in the shape of a cross, and the periphery of the lampshade and the lower part of the tube are initially yellowish or soon yellowish brown, with several layers of opacity.

Situation mushrooms are introduced in sugar liquor pieces in the synonyms of Dongbobo (醫 寶 鑑 木耳). There is a record of the disease being used. Sinnonbonchogyeong (神農 本草 經), herbal herb, phoenix green has also been used for various diseases, especially digestive diseases.

Situation mushrooms are a rare species and no artificial cultivation methods have been developed, making it impossible to study ingredients from the fruiting bodies. The culture method has recently been established (Han, MW, Ko, KS and Chung, KS (1995) Liquid cultivation of Phellinus inteus mycelium and preparation of antitumor and immunostimulating substance.Korea patent Open No. 95-7860) Many studies have been conducted. In this experiment, the mycelium was liquid cultured, the culture filtrate was suction filtered and the remaining mycelium was lyophilized.

Components isolated from S. mushroom mycelium culture include succinic acid, methyl ester of p-hydroxy phenyl acetic acid, p-hydroxybenzaldehyde, and 2,5-dihydroxy. Methylfuran, 2-hydroxymethyl-5-methoxymethylfuran and N-acetyltyramine have been reported (Song, KS, Cho, SM, Ko, KS, Han, MW and Yoo, ID (1994) Secondary metabolites from the myceliumculture broth of Phellinus linteus.Han'guk Nonghwa Hakhoeji 37 (2): 100-104). However, studies on the components and its neuroprotective activity have been insufficient.

Therefore, in the present invention, by extracting the mycelium mushroom mycelium culture and the neuroprotective action of the fraction and its mechanism of action, the present invention was proposed as a candidate of a preparation capable of preventing or treating neurodegenerative diseases such as stroke or senile dementia. As a result, the present inventors have completed the present invention by confirming that the total methanol extract of the situation mushroom mycelium culture, n-hexane fraction thereof, ethyl acetate (EtOAc) fraction has significant neuronal protective activity.

Accordingly, it is an object of the present invention to provide extracts and fractions of the situation mushroom mycelium culture having neuronal protective activity.

Another object of the present invention is to provide an agent for the prevention and treatment of degenerative cerebral nervous system diseases containing the extract of the situation mushroom mycelium culture or a fraction thereof as an active ingredient.

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

Experimental Example

Experimental Animals -Sprague-Dawley rats were supplied from the Seoul National University Animal Breeding Center and were bred in the Laboratory Animal Lab. The environment of the kennel was kept at 22 ± 5 ℃ and the lighting time was fixed from 7 am to 7 pm, and the feed was 23.2% of crude protein, 4.0% of crude fat, 6.0% of crude fiber, 10.0% of crude ash and 0.6% of crude calcium. Solid feed (Seoul, Samyang) containing 0.4% was used.

Isolation and Culture of Cerebral Cortical Cells in Rats-Only the cerebral part of the rat fetus is removed, the meninges are carefully removed under the dissecting microscope, and the cerebral tissues are treated with 0.25% trypsin for 30 minutes to soften the tissues into individual cells. To be liberated. The isolated cerebral cortical cells were transplanted into a culture vessel (Falcon, 15 × 24 mm) coated with poly-L-lysine at 2.0 × 10 ⁵ cells / dish. The culture medium was composed of 90% DMEM, 10% fetal calf serum, 100 IU / ml penicillin and 100 ㎍ / ml of streptomycin. Cultivation of the cells was carried out in a 37 ℃ incubator while continuously supplying a mixture of air (95%) and CO ₂ (5%), and after 4 days of culture to 5 x 10 ^-5 M 5-fluorodeoxyuridine Treatment inhibited the growth of cells other than neurons (Choi, DW (1985) Glutamate neurotoxicity in cortical cell culture is calcium dependent. Neurosci. Lett . 58: 293-297).

Extraction and fractionation of mycelium mushroom mycelium cultures-4 L of MeOH was added to 5 kg of mycelium mushroom mycelium cultures and filtered after ultrasonication 5 times at 40 ° C. This was concentrated under reduced pressure to obtain 365 g of MeOH X. It was suspended in distilled water, and then fractionated with increasing polarity from n -hexane to EtOAc, n- BuOH, and concentrated under reduced pressure, n -hexane fraction (49 g), EtOAc fraction (34 g), n- BuOH fraction (100 g) and H ₂ O fraction (182 g) was obtained (Scheme I).

Scheme 1

Induction of Neurotoxicity by Glutamate- After culturing cerebral cortical cells directly isolated from the fetus of rats for 17 days, the samples to be measured for activity were treated with cultured cells by concentration, and then treated with 50 μM glutamate after 1 hour. Induced toxicity to cells. After 24 hours, the survival rate of the cells was measured by MTT assay to determine the neuronal protective activity of the samples.

Induction of Neurotoxicity by H ₂ O _2- After incubating the cerebral cortex cells directly isolated from the fetus of rats for 10 days, the sample to measure activity was treated to the culture cells by concentration and 50 μM of H ₂ O ₂ was treated to induce cytotoxicity. After 24 hours, the survival rate of the cells was measured by MTT assay to determine the neuronal protective activity of the samples.

Administration of extracts and fractions of S. mushroom mycelium culture-Total extracts and fractions of S. mushroom mycelium culture were dissolved in DMSO (within 0.1% final concentration), diluted with distilled water, and prepared at a concentration of 5 mg / ml, followed by millipore membrane ( 0.22 μm, Millex-GV, USA) to aseptic conditions and administered at different concentrations.

Preparation of Cell Supernatant- After removing the culture medium of the primary cultured cerebral cortical cells, cells were collected by adding 0.1 M potassium phosphate buffer (pH 7.4). The collected cells were sonicated for 20 seconds and then centrifuged at 3,000 g for 20 minutes at 4 ° C. to obtain a cell supernatant.

Determination of Catalase Activity —150 μl of 19 mM H ₂ O ₂ was prepared in vitro. Next, 200 µl of the cell supernatant was added to initiate the reaction. Catalase activity was measured by measuring the depletion of H ₂ O ₂ by measuring the decrease in absorbance at 240 nm for 2 minutes.

Determination of GSH-px Activity —In vitro, 1 ml of reaction solution containing 100 mM potassium phosphate buffer (pH 7.4), 1 mM GSH, 0.2 mM NADPH, 0.5 mM H ₂ O ₂ and 1.5 units / ml GSSG-reductase. Ready. The reaction was started by adding 100 μl of the cell supernatant, and the activity of GSH-px was measured by measuring the decrease in absorbance at 340 nm for 1 minute. GSH-px activity was expressed as μmol NADPH oxidized / mg protein / min using absorption coefficient (6.22μmol ^-1 cm ^-1 ).

Determination of GSSG-R Activity- 1 ml of reaction solution containing 100 mM potassium phosphate buffer (pH 7.4), 1 mM GSSG, 0.1 mM NADPH was prepared in vitro. Next, the reaction was started by adding 100 µl of the cell supernatant, and the activity of GSSG-R was measured by measuring the decrease in absorbance at 340 nm for 2 minutes. The activity of GSSG-R is expressed as μmol NADPH oxidized / mg protein / min.

Determination of Total GSH (GSH + GSSG) Amount- 700 μl of the reaction solution containing 0.3 mM NADPH and 5 mM DTNB was added to the test tube, and 100 μl of the cell supernatant was added thereto. The reaction was then started by adding GSSG-R to a final concentration of 5 units / ml and the increase in absorbance was measured at 412 nm for 1 minute. The amount of GSH was converted to a GSH standard curve to obtain a GSH standard curve.

Determination of GSSG amount -4 μl of 2-vinylpyridine was added to 150 μl of the cell supernatant, mixed well with vortex, and allowed to stand at room temperature for 30 minutes to form a derivative of GSH. The amount of GSSG remaining was determined by the same method as the method of measuring total GSH (20).

Protein Quantitation -The amount of protein was measured by Lowry et al. On the basis of BSA.

Determination of the amount of MDA- 500 µl of 10% TCA was added to 500 µl of the cell supernatant and left for 10 minutes to precipitate the protein. Next, the precipitated protein was removed using microspin. TBA was added to the supernatant at a final concentration of 0.2% to prepare a reaction solution of 1 ml, followed by reaction at 100 ° C. for 1 hour. After absorbance was measured at 535 nm, 1,1,3,3-tetraethoxypropane was converted into MDA using standard.

MTT assay -MTT (5 mg / ml) was added to the culture medium of the cultured cerebral cortical cells to be 10% of the culture solution, and further incubated for 3 hours, and the resulting formazan was dissolved in DMSO and absorbance was measured at 540 nm.

Statistical treatment- Statistical significance test was the number of each group was 3 (n = 3) the variation from the control value was "ANOVA test". When the P value was less than 5%, it was determined to be statistically significant.

Results and Discussion

The effects of total extracts and individual fractions of S. mushroom mycelium cultures on the neurotoxicity of glutamate or H ₂ O ₂ in primary cerebral cortical cells were measured by MTT assay (Table 1). The cerebral cortical cells of the rats were cultured for 17 days, and then the total extract and each fraction of the situation mushroom mycelium cultures were administered by concentration. After 1 hour, 50 μM glutamate was again applied and the culture was continued for 24 hours. At this time, the total extract and each fraction of the situation mushrooms were continued (Throughout treatment). The effects of total extract and each fraction were measured by MTT assay, and the total extract, EtOAc fraction and n -hexane fraction of S. mushrooms significantly blocked neurotoxicity by glutamate, which reduced neuronal cell death. Increased the number of cells. Each extract and fraction showed 20.6%, 71.8% and 52.5% activity at 100 μg / ml concentration, respectively.

Next, we observed the neuroprotective activity of the situational mushroom using H ₂ O ₂ , another toxic substance that can cause oxidative stress and damage. As in the case of inducing toxicity with glutamate, the rat cerebral cortical cells were obtained and cultured for 10 days, and then the total extract and each fraction of the situation mushroom mycelium culture were administered by concentration, and 50 μM H ₂ O ₂ was applied again after 1 hour. Incubate further for 24 hours. At this time, the total extract and each fraction of the situation mushrooms were continued (Throughout treatment). The effects of total extracts and fractions were measured by MTT assay. The total extracts of EtOAc, EtOAc fraction and n -hexane fraction also significantly blocked neurotoxicity by H ₂ O ₂ , respectively. 29.2%, 88.0%, and 58.7%.

In order to determine the mechanism by which the extract and each fraction block neurotoxicity against H ₂ O ₂ , the activity of antioxidant enzymes and the amount of antioxidants in brain neurons were measured.

Representative antioxidant enzymes within cells include catalase, GSH-px and GSSG-R. Catalase plays a role in decomposing H ₂ O ₂ (Freeman, BA and Crapo, JD (1982) Biology of disease.Free radical and tissue injury. Lab Invest . 47: 412-426). GSH-px shows antioxidant activity using GSH in the presence of H ₂ O ₂ and organic peroxide, and GSSG-R plays an important role in the maintenance of GSH by reducing GSSG to GSH (Meister A. and Anderson ME (1983) Glutathione.Annu. Rev. Biochem. 52: 711-760). Induction of H ₂ O ₂ toxicity in cerebral cortical cells of primary cultured rats significantly reduced the activity of these enzymes. However, it was found that the antioxidant enzyme activity significantly increased when the EtOAc fraction of the situation was administered to the concentration of 100 ㎍ / ㎖ in the brain neurons induced by toxicity with H ₂ O ₂ . In the case of GSH-px, the activity of GSH-px reduced by H ₂ O ₂ was maintained to 81.9% at steady state, and the activities of catalase and GSSG-reductase were significantly increased, respectively. 72.8, 64.9% of the time was maintained.

Intracellular antioxidants are typical of GSH. GSH is mostly present as reduced GSH and changes to GSSG as ROS increases, thus increasing GSSG is indicative of oxidative stress (Huang J. and Philbert MA (1995) Distribution of glutathione and glutathione-related enzyme systems in mitochondria and cytosol of cultured cerebellar astrocytes and granule cells.Brain Res. 680: 16-22). GSH is used to remove ROS directly and is also consumed by GSH-px, an enzyme that exhibits antioxidant activity. It is also used by GST for detoxification. Induction of H ₂ O ₂ toxicity in rat cerebral cortex cells significantly reduced the amount of GSH. However, the EtOAc fraction of the situation maintained the total amount of GSH reduced by H ₂ O ₂ at a concentration of 100 μg / ml to 76.4% at steady state and the ratio of GSSG / total GSH, an indicator of GSH redox status. Significantly reduced. It was mentioned earlier that the EtOAc fraction of the situation increased significantly the activity of GSSG-R, so the decrease in the ratio of GSSG / total GSH by the situation EtOAc fraction maintains the activity of GSSG-R and increases the reduction of GSSG to GSH. It was found to be due to.

Depletion of antioxidant enzyme activity by H ₂ O ₂ and consequent depletion of GSH eventually lead to lipid peroxidation. In order to investigate the effect on lipid peroxidation, the production of MDA, an indicator of lipid peroxidation, was measured. The EtOAc fraction of the situation showed a lipid peroxidation inhibitory effect by significantly inhibiting the production of MDA which is an indicator of lipid peroxidation at a concentration of 100 μg / ml.

In conclusion, total methanol extract, EtOAc fraction and n -hexane fraction of S. mushroom mycelium culture showed significant neuroprotective activity against neurotoxicity induced by glutamate or hydrogen peroxide in primary cerebral cortex cells. It was found. It was also found that the protective effect by H ₂ O ₂ is due to the inhibition of lipid peroxidation by maintaining the activity of antioxidant enzymes and increasing the synthesis of GSH.

Table 1. Effects of total extracts and individual fractions of S. mushroom mycelium culture on cell viability in primary cultured rat cerebral cortex cells exposed to H ₂ O ₂ .

Concentration (µg / ml) Viability (%) H ₂ O ₂ Glutamate Control 100 Reference ^a 0 Total extract 100 29.2 20.6 50 14.9 13.8 n -Hexane fr. 100 58.7 52.5 50 20.4 19.4 EtOAc fr. 100 88.0 71.8 50 76.7 34.6 n- BuOH fr. 100 26.6 23.8 50 23.3 15.4 Aqueous fr. 100 10.2 11.3 50 16.7 7.5

Control is the value for primary cultured rat cerebral cortex cells. The MTT value of the control group was 1.343 ± 0.048 optical density (OD).

Reference ^a is for primary cultures of rat cerebral cortical cells exposed to glutamate or H ₂ O ₂ for 24 hours. The baseline for MTT was 0.667 ± 0.059 OD.

Viability was calculated by the following equation.

100x (OD of sample treated-OD of reference) / (OD of control-OD of reference).

The significance level for the reference value of the test results is as follows.

*: p> 0.05, **: p <0.01, ***: p <0.001.

Table 2. Effect of EtOAc fractions of S. mushrooms on antioxidant activity and MDA content in primary cultures of rat cerebral cortical cells exposed to H ₂ O ₂ .

Catalase GSH-px GSSG-R MDA ControlH ₂ O ₂ EtOAc fraction 45.215.832.9 18.26.714.9 5.72.23.7 65.2200.3110.9

The concentration of EtOAc fraction was 100 μg / ml.

Catalase: μmol H ₂ O ₂ consumed / min / mg protein

GSH-px: μmol NADPH consumed / min / mg protein

GSSG-R: μmol NADPH consumed / min / mg protein

MDA: nmol / mg protein

Table 3. Effect of the situation mushroom EtOAc fraction on glutathione content in primary cultures of H ₂ O ₂ exposed rat cerebral cortical cells.

Total GSH Reduced GSH GSSG / total GSH Control 7.2 5.6 0.32 H ₂ O ₂ 2.1 1.0 0.52 EtOAc fraction 5.5 3.4 0.39

The concentration of EtOAc fraction was 100 μg / ml.

GSH: nmol / mg protein

conclusion

1. The total methanol extract, EtOAc fraction and n -hexane fraction of S. mushroom mycelium culture showed significant neuroprotective activity against glutamate or H ₂ O ₂ induced neurotoxicity in primary cultured rat cerebral cortex cells. .

2. The protective effect of H ₂ O ₂ was found to be due to the inhibition of lipid peroxidation by maintaining the activity of antioxidant enzymes and increasing the synthesis of GSH.

3. The above results suggest that the total methanol extract, EtOAc fraction and n -hexane fraction of S. mushroom mycelium culture could be suggested as candidates for the development of preparations for the prevention and treatment of stroke and dementia.

Acute Toxicity Experiment

1. Oral administration

ICR mice (28 ± 6g) and Sprague Dawley (250 ± 12g) rats were divided into 5 groups of 15 rats each. After two weeks of oral administration at doses of 500, 725, 1000 and 5000 mg / kg, no toxicities were observed in any of the five groups and no symptoms were observed.

2. Intraperitoneal administration

ICR mice (25 ± 5g) and spragdoli rats were divided into 5 groups of 10 rats each to obtain 125, 250, 500, 725 and 1000 mg of total mushroom extract, n-hexane fraction and EtOAc fraction, respectively. After 24 hours of intraperitoneal administration at / kg, no toxicities were observed in all 5 groups and no symptoms were apparent in the control group.

In the above results, the extracts and fractions of the present invention was confirmed that there is little acute toxicity.

As described in detail above, the situation mushroom total methanol extract, n-hexane fraction and EtOAc fraction of the present invention can be used as a therapeutic agent for neurodegenerative diseases.

Situation mushroom extract or fractions thereof of the present invention, depending on the sex, age, weight, degree of disease, etc. of the patient, can usually be administered 1 to 3 times 10mg to 5000mg per day.

The situation mushroom extract of the present invention is prepared by pharmaceutical formulations commonly used in pharmaceutical preparations, for example, injections, solutions, syrups, tablets, capsules, etc. together with pharmaceutically acceptable excipients. can do.

Next, the present invention will be described in more detail as formulation examples. The situation mushroom total methanol extract, n-hexane fraction and EtOAc fraction used were prepared in the example according to Scheme 1 (extraction and fractionation of the situation mushroom mycelium culture).

Formulation Example 1

Situation Mushroom Total Methanol Extract 100mg

Appropriate sterile distilled water for injection

pH adjuster

Situation mushroom total methanol extract was dissolved in distilled water for injection, adjusted to pH 7.6 with a pH adjuster, and then the whole was made into 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2

Sichuan mushroom n-hexane fraction 2mg

Appropriate sterile distilled water for injection

pH adjuster

The n-hexane fraction of S. mushrooms was dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, the total amount was 2 ml, and then filled into 2 ml ampoules to prepare an injection.

Formulation Example 3

Situation Mushroom EtOAc Fraction 200mg

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 4

Situation Mushroom Total Methanol Extract 10mg

Lactose 100mg

Starch 50mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 5

Situation Mushroom EtOAc Fraction 100mg

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate appropriate amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 6

Sichuan mushroom n-hexane fraction 5mg

Lactose 100mg

Starch 93mg

Talc 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 7

Situation Mushroom Total Methanol Extract 1000mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

Formulation Example 8

Situation Mushroom EtOAc Fraction 100mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

According to the present invention, a situation mushroom mycelium total methanol extract, n-hexane fraction and EtOAc fraction are provided, and since they have significant neuroprotective activity and no toxicity, they are useful as a preventive and therapeutic agent for degenerative cerebral nervous system diseases such as stroke and dementia. Could be used.

Claims (2)

Total methanol extract, or EtOAc fraction or n -hexane fraction, of the situation mushroom mycelium culture with neuronal cell protective activity. A total methanol extract of S. mushroom mycelium culture, or an EtOAc fraction or an n -hexane fraction thereof as an active ingredient, and is prepared by formulating into a pharmaceutical formulation commonly used in combination with a pharmaceutically acceptable excipient. Prevention and treatment of neurodegenerative diseases.