KR100588477B1 - Pharmaceutical composition for degenerative central nervous system disease prevention - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of degenerative central nervous system diseases, and more particularly for the prevention and treatment of degenerative central nervous system diseases containing epideusmin isolated from magnolia or magnolia extract as an active ingredient. It relates to a pharmaceutical composition.

Degenerative Central Nervous System Disease, Epipiesmin, Magnolia Extract

Description

Pharmaceutical composition for the prevention and treatment of degenerative central nervous system diseases {PHARMACEUTICAL COMPOSITION FOR DEGENERATIVE CENTRAL NERVOUS SYSTEM DISEASE PREVENTION}

1 is a graph showing the induction of neurite outgrowth in PC12 cells not pretreated as a control,

FIG. 2 is a graph showing the induction of neurite generation factor in PC12 cells by pretreatment of Epiudesmin (20 μg / ml) for 96 hours. FIG.

Figure 3 is a graph showing the induction of new light production factor in PC12 cells by pretreatment of NGF (Nerve growth factor: 2ng / ㎖) for 96 hours,

FIG. 4 is a graph showing the induction of neurite generation factor in PC12 cells by pretreatment of epiyudesmin (20 µg / ml) and NGF (2ng / ml) for 96 hours.

FIG. 5 is a graph showing the effect of PD 98059, a miogen activated protein kinase (MAPK) inhibitor, on the induction of neuronal factor production of epidermamine and NGF,

FIG. 6 is a graph showing the effect of GF 109203X, a calcium dependent protein kinase (PKC) inhibitor, on the induction of neurogenesis factor of epiudesamine and NGF,

7 is a graph showing the effect of H 89, a Cyclic-AMP dependent protein kinase (PKA) inhibitor, on the neutrophil inducer action of epidermamine and NGF.

The present invention relates to a pharmaceutical composition for the prevention and treatment of degenerative central nervous system diseases, and more particularly, to a degenerative center containing Epipiesmin (Epieudesmin) shown below isolated from magnolia extract or magnolia as an active ingredient. It relates to a pharmaceutical composition for the prevention and treatment of diseases of the nervous system.

In general, since neurological diseases are related to the death or damage of nerve cells, the treatment is to suppress the death or damage of nerve cells. More recent methods of treatment include methods of promoting cell regeneration by promoting neuronal production.

To promote the production of neurons, there are neurotrophic factors (Neuroiteic factors) involved in the survival and differentiation of nerves, or neurite outgrowth inducer (Neurite-promoting). Therefore, neurotrophic factor or neurite outgrowth-inducing substance that promotes neurite is a degenerative central nervous system disease such as Parkinson's disease, Alzheimer's disease, and ischemic brain disease. Ischemia), memory disorders, alcoholism, etc. (Gannong WF, 2003, Review of Medical Physiology, 21st ed., 62-63: Seckel BR, 1990, Muscle Nerve, 13, 785-800: Varon S. and Hagg T., 1993, Models to evaluate effects of neurotrophic factors on axonal regeneration, in Neuromethods, Vol 25, 277-289).

NGF (Nerve growth factor) is a representative material that promotes the production of neurons. When NGF pretreatment is performed on PC12 cells, PC12 cells stop dividing, differentiate into sympathetic neuron-like cells, differentiate into neurite outgrowth, and proliferate when NGF is absent Proliferation occurs, but no differentiation occurs, and cell death occurs (Greene, LA, Tischler, AS, Adv. Cell. Neurobiol., 1982, 3, 373-414). Therefore, NGF is associated with neuronal survival and differentiation, and thus, is a neurotrophic factor and promotes neurogenesis, thereby promoting neural cell production as a neurite outgrowth inducer.

In the past, efforts have been made to obtain substances that promote neuron production from natural extracts to treat neurological diseases.

Korean Unexamined Patent Publication No. 2004-14757 discloses that breeding has an effect similar to that of NGF, and Korean Unexamined Patent Publication No. 2003-7111 discloses a composition for protection, growth promotion and regeneration of nerve cells containing golden extract. The present invention discloses a composition related to the protection, growth promotion and regeneration of nerve cells containing rhubarb extract in the Republic of Korea Patent Publication No. 2003-7105, and the bellflower extract in Republic of Korea Patent Publication No. 2002-96925 Disclosed is a composition for preventing and treating degenerative brain disease or for improving memory.

On the other hand, Magnolia kobus is a deciduous broad-leaved arboreous tree of the Magnolia family Magnolia, and epiudesmin is a substance isolated from Magnolia (Latip J., Hartley TG, and Waterman PG, Phytochem., 1999, 51, 107). -110: Miyauchi T. and Ozawa S., 1998, Phytochem., 47 (4), 665-670: Miyazawa M., Kasahara H. and Kameoka H., Phytochem., 1995, 39 (5), 1027-1030 Pan ZX etc, 1987, Phytochem., 26 (5), 1377-1379).

The dried buds of magnolia are called Shin-yi, which is known to cure nasal congestion and to treat chills, fever and sweating and systemic pain due to cold caused by climate change (Lee Young-no, 1998, primary color). Korea Plant Book, etc.). In addition, Korean Patent No. 328478 discloses that Shinyi extract is used as a treatment for asthma, and Japanese Unexamined Patent Publication No. Hei 4-368334 is known as Shinyi thromboembolism treatment agent, but the magnolia extract is known in the prior art. It is not known to be useful for the prevention and treatment of degenerative central nervous system diseases such as the invention.

The present inventors confirmed that epidermine isolated from magnolia is a substance that induces neurite outgrowth in PC12 cells and completed the present invention as a pharmaceutical composition for preventing and treating degenerative central nervous system diseases.

An object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of degenerative central nervous system diseases, comprising Epiyudesmin (Epieudesmin) as an active ingredient.

It is another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of degenerative central nervous system diseases, comprising an extract of magnolia containing epidermine as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of degenerative central nervous system disease, characterized in that it contains Epiudesmin (Epieudesmin) as an active ingredient.

In another aspect, the present invention provides a pharmaceutical composition for the prevention and treatment of degenerative central nervous system diseases, characterized in that it contains a magnolia extract including epidermine as an active ingredient.

Referring to the present invention in more detail as follows.

The present invention may be prepared by containing epidermine as an active ingredient to prepare a pharmaceutical composition for preventing and treating degenerative central nervous system diseases and a pharmaceutical preparation comprising the same.

Epiyudesmin used in the present invention is a Lignan compound, the chemical formula is 3,3 ', 4,4'-tetramethoxy-7,9', 7 ', 9-diepoxylignan, the chemical structure is shown below It is like.

Epiyudesmin is known as Latip J., Hartley TG, and Waterman PG, Phytochem., 1999, 51, 107-110, Miyauchi T. and Ozawa S., 1998, Phytochem., 47 (4) , 665-670), Miyazawa M., Kasahara H. and Kameoka H., Phytochem., 1995, 39 (5), 1027-1030) or Pan ZX etc, 1987, Phytochem., 26 (5) 1377-1379), and are known in magnolia from known methods.

The present invention can be isolated epiyudesmin in the following way.

The present invention obtains a solvent fraction from the magnolia extract extracted using lower alcohol, such as water or ethanol (Petroleum ether), ether (Ether), and then methanol (Methanol) and ethanol to the ether fraction Sepadex LH-20 column chromatography was performed using an Ethanol mixed solvent to separate fractions, and the fractions were subjected to silica gel column chromatography using a mixed solvent of benzene and ethyl acetate. The fractions are separated, and finally, the fractions obtained by performing Sephadex LH-20 column chromatography using methanol solvent can be purified by a conventional purification method to obtain epiyudesmin.

In addition, the present invention may be prepared by containing a magnolia extract including epidermine as an active ingredient to prepare a pharmaceutical composition for preventing and treating degenerative central nervous system diseases and a pharmaceutical preparation comprising the same.

The magnolia extract used in the present invention is dried in the shade by peeling the magnolia, using a magnolia extract extracted with a lower alcohol such as water or ethanol (Ethanol), which is powdered by pulverizing the bark and pulverizing with a grinder. Concentrate the extract which was deposited for 3 days at room temperature using.

The present invention is mixed with diluents or excipients such as fillers, weights, binders, wetting agents, disintegrants and surfactants commonly used, etc., to prevent and treat degenerative central nervous system diseases containing epidermamine isolated from magnolia extract or magnolia. Pharmaceutical compositions can be prepared and prepared by oral administration such as tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, liquids, etc. by conventional methods, or dissolved in distilled water for injection by conventional methods. Can be used.

The present invention compares the pretreatment of epidermis with the pretreatment of NGF to PC12 cells to induce the neurite outgrowth induction experiments. Confirmed to be a better material.

NGF is also activated by Mitogen activated protein kinase (hereinafter referred to as 'MAPK'), Calcium dependent protein kinase (hereinafter referred to as 'PKC') and Cyclic-AMP dependent protein kinase (hereinafter referred to as 'PKA'). It induces neurite outgrowth, so it is PD 98059 (product of SIGMA product code P-215), MGFK inhibitor, GF 109203X (product of SIGMA product code G-2911), inhibitor of PKC, which is an inhibitor of PKA. Experimental effects of epidermine as a control group of H 89 (product of SIGMA product code B-1427) and NGF on PC12 cells at the same time resulted in the effect of epidermine inducing neurite outgrowth. It was confirmed that this was an excellent material.

On the basis of the above results, the present invention is to obtain a pharmaceutical composition for preventing and treating degenerative central nervous system disease containing epiyudesmin or magnolia extract isolated from magnolia and a pharmaceutical preparation comprising the same.

The present invention confirmed that the acute toxicity test results for epidermine or magnolia extract is suitable for use as a pharmaceutical composition for the prevention and treatment of degenerative central nervous system diseases.

In the present invention, an appropriate dosage is selected depending on the degree of absorption, inactivation rate and excretion rate of the active ingredient, the age, sex and condition of the patient, the severity of the disease to be treated, etc. Eusemin 1 ~ 1000mg and magnolia extract 1 ~ 10g can be administered to adults respectively. In addition, the present invention is a formulated unit dosage form may be administered at a predetermined time interval, or using a specialized dosage method according to the judgment of the expert and the needs of the individual to monitor or observe the administration of the drug, if necessary, It may be administered once to three times a day.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited by the examples.

<Example 1>

Preparation of Magnolia Extract

Magnolia was used as a publicly-owned species of magnolia (35 years old) collected at Gajwa Experimental Forest (Gajwa-dong, Jinju-si, Gyeongsangnam-do) in June 2001.

The magnolia was peeled and dried in the shade, and the bark was finely pulverized and pulverized with a grinder to be powdered. The powder was extracted by immersion for 3 days at room temperature using ethanol. Was prepared.

<Example 2>

Isolation of Epiyudesmin from Magnolia Extract

Magnolia extract dissolved in ethanol obtained before drying under reduced pressure in the manufacturing process of the magnolia extract was fractionated by successive successive extraction using petroleum ether, ether, ethyl acetate and butanol. 75 mL using a Sephadex LH-20 column chromatography (60 × 4.5 cm) using methanol-ethanol (methanol: ethanol = 3: 7, v / v) as the eluting solvent for the double ether soluble part (85.0 g). Twelve aliquots were obtained, and each aliquot was subjected to thin layer chromatography (TLC, silica gel 60 F254, developing solvent; toluene: ethyl formate: formic acid = 5: 4: 1, v / v / v). After development, the solution was divided into two fractions by searching with a UV (254 nm) lamp and a color developer (50% H ₂ SO ₄ ).

The second fraction (81.15 g) was eluted by 15 ml using silica gel column chromatography (27.5 × 7.0 cm) using benzene-ethyl acetate (benzene: ethyl acetate = 8: 1, v / v) as the elution solvent. 230 fractions were obtained and 8 fractions were obtained by UV.

The fifth fraction (3.3 g) was fractionated in 5 ml portions using Sephadex LH-20 column chromatography (50 × 4.5 cm) using methanol as the elution solvent to obtain 40 aliquots. The search result was divided into four fractions.

Compound (120 mg) isolated from the second fraction of these was identified as epiyudesmin, a lignan compound, as a result of instrumental analysis and literature search such as NMR and MS.

The MS and NMR results and epichemical structure of epidermine are as shown below.

EI-MS Spectrum m / z ; 386 (M ⁺ ).

¹ H-NMR spectral signal group (ppm); (500 MHz, acetone- d ₆ δ = ppm): δ3.12 (2H, m , H-8, H-8 '), 3.82 (3H, s , OMe), 3.84 (3H, s , OMe), 3.86 (2H, m , H-9a, H-9a '), 4.22 (2H, m , H-9b, H-9b'), 4.73 (2H, m , H-7, H-7 '), 6.92-7.2 (6H, m , arom).

^{Group of 13} C-NMR spectral signals (ppm); (125 MHz, acetone- d ₆ , δ = ppm): δ54. 63 (C-8, 8 '), 55.60 (C-OMe × 4), 71.67 (C-9, 9'), 85.85 (C-7, 7 '), 110.46 (C-6, 6'), 112.11 (C-5, 5 '), 118.46 (C-2, 2'), 134.81 (C-1, 1 '), 149.21 (C-4, 4'), 149.85 (C-3, 3 ').

¹ H- ¹ H COSY correlations: H-7 / H-7'↔H-8 / H-8 ', H-9a / H-9a'↔H-9b / H-9b' / H-8 / H- 8'.

HMBC correlations: H-7 → C-2 / C-6 / C-8 / C-9, H-9 → C-7 / C-8.

Mass (MS) spectra of the isolated compounds were determined by JEOL JMS-600W, nuclear magnetic resonance such as ¹ H-, ¹³ C-NMR, ¹ H- ¹ H Correlation Spectroscopy (COSY), and ¹ H Detected Multiple Bond Connectivity (HMBC). (NMR) spectra were measured using the Varian UI 500 of the Seoul Institute of Basic Science and Technology.

<Example 3>

Neurite outgrowth induction experiment

(1) Culture of PC12 Cells and Pretreatment of Samples

PC12 cells were used as a medium containing serum (5% FBS + 10% HS) RPMI 1640, the culture was incubated in accordance with the conventional culture method in a 37 ℃ incubator. Epiudesmin was added to the serum-containing medium (60 mm culture dish), incubated for 7 days, the medium was changed if necessary, and neurite bearing & Neurite outgrowth was measured at each time period.

(2) Measurement of neurite bearing and neurite outgrowth

After fixation by adding 4% paraformaldehyde to PC12 cells, using a phase-contrast microscope (x100-125 magnification), the cellular process is longer than 1 cell diameter (1x1-mm fields). The number of insides) was counted and measured. The neuritic index was calculated by dividing the number of neurite forming cells by the total number of cells in this field (minimum 200 per count). Comparison of neurite outgrowth by pretreatment of NGF and epidermine was examined by measuring cell number and length of nerite after single or combination pretreatment. The number of neurite forming cells was counted by the substance and measured as a ratio with the total cell number.

After 96 hours of epidermine (20 μg / ml) pretreatment, PC12 cells showed significant neuronal outgrowth induction (100 μm, contrast to scale bar 50 μm). Neurite forming cell numbers increased concentration-dependently with the treatment of epidermine (20 μg / ml).

After 96 hours of epiyudesmin (20 μg / ml) pretreatment, PC12 cells showed significant neuronal outgrowth induction compared to the control (FIG. 1) (FIG. 2).

When co-administration of epidermine and NGF showed a significant neurite outgrowth induction effect compared to the NGF alone group (FIG. 3) (FIG. 4).

In addition, when the neurite length was combined with epidermamine (20 µg / ml) and NGF (2 ng / ml) (250 µm, contrast to scale bar 50 µm), NGF alone administration group (150 µm, contrast to scale bar) As much as 5/3 times longer than that of 50 μm), it showed a remarkable effect of inducing neurite outgrowth.

<Example 4>

Influence of MAPK inhibitors, PKC inhibitors and PKA inhibitors

Neuronal outgrowth induction of NGF is activated by MAPK, PKC, and PKA. Therefore, PD 98059, an inhibitor of MAPK, GF 109203X, an inhibitor of PKC, H 89, an inhibitor of PKA, and the like, were pretreated, and then, whether or not neurogenesis was observed in PC12 cells by epidermine. Neurite outgrowth induction was carried out in accordance with Example 3- (2).

The pretreatment of PD 98059 (3 mM), a MAPK inhibitor, inhibited the neurite outgrowth induction of epidermine (20 µg / ml), and NGF (2 ng / ml) and epidermide (20). Μg / ml) When PD 98059 was administered to the combination treatment group, the number of neurite forming cells was significantly reduced compared to the NGF alone treatment group (FIG. 5).

Pretreatment of PKC inhibitor GF 109203X (100 nM) and PKA inhibitor H 89 (100 nM) inhibited the neurite outgrowth induction of epidermine (20 μg / ml), and NGF (2 ng / ml) The number of neurite forming cells increased in the co-administration of and epiyudesmin (20 μg / ml) was decreased by GF 109203X and H 89 pretreatment (FIGS. 6 and 7).

Taken together, it can be seen that epidermine is a neurite outgrowth inducer.

<Production Example 1>

Manufacture of tablets

2 g of the magnolia extract concentrate of Example 1, 100 mg of lactose and 100 mg of starch were mixed and compressed into tablets according to a conventional tablet manufacturing method.

<Production Example 2>

Manufacture of soft capsule

2 g of magnolia extract concentrate of Example 1, lactose 50 mg, starch 50 mg, talc 2 mg were mixed and filled into a soft capsule type, and then dried first in a drier and finally dried in a ventilated room to obtain a soft capsule. Prepared.

<Production Example 3>

Preparation of Granules

2 g of the magnolia extract concentrate of Example 1 and 100 mg of lactose were mixed and granules were prepared according to a conventional granule preparation method.

Production Example 4

Preparation of liquid

Purified water was added to 2 g of the magnolia extract concentrate of Example 1, 20 g of sugar, and 20 g of isomerized sugar, and 100 ml of the mixture was mixed according to a conventional method for preparing a liquid, and filled and sterilized in a 100 ml brown bottle to prepare a liquid. .

A test on the cytotoxicity of PC12 cells of epiyudesmin isolated from the magnolia of the present invention was performed.

Experimental Example 1

Cytotoxicity Test in Epi12min PC12 Cells

In order to confirm whether the epidermis of the present invention is toxic to humans, it was determined by MTT / LDH activity assay.

As a result, epiudesamine did not show cytotoxic effect in the range of 40 ~ 80㎍ / ㎖ (search by LDH activity measurement), and showed cytotoxic effect only above 80㎍ / ㎖.

Through these safety experiments, it is determined that the epidermine of the present invention does not adversely affect the human body.

The results of all experiments were expressed as mean and standard error, and the significance between means of each group was verified by ANOVA followed by Turkey's test. The statistical significance level between the control group and the experimental group was p <0.05.

As described above, the pharmaceutical composition comprising the epidermis or magnolia extract of the present invention as an active ingredient may be used for the prevention and treatment of degenerative central nervous system diseases.

In particular, the present invention, as can be seen in the above embodiment, it was confirmed that the epidermis is a substance exhibiting the neurite outgrowth-inducing action (epiudesmin and magnolia extract containing the same) to regenerate the central nerve cells It may be effective in preventing and treating degenerative central nervous system diseases such as Parkinson's disease, Alzheimer's disease, ischemic brain disease, memory disorder, and alcoholism.

Claims (6)

Select from Parkinson's disease, Alzheimer's disease, Ischemia, memory disorders, and alcohol addiction, which contain Epipiesmin as an active ingredient with a pharmaceutically acceptable carrier. Pharmaceutical composition for the prevention and treatment of degenerative central nervous system diseases. The method of claim 1, wherein Epideusmin (Epieudesmin) is a pharmaceutical composition for preventing and treating degenerative central nervous system diseases, characterized in that isolated from magnolia. delete Degenerative central nervous system disease selected from Parkinson's disease, Alzheimer's disease, Ischemia, memory disorders and alcoholism, which contain magnolia extract as an active ingredient with a pharmaceutically acceptable carrier Prophylactic and therapeutic pharmaceutical compositions. delete A pharmaceutical composition according to any one of claims 1, 2 or 4 is formulated into a dosage form selected from tablets, capsules, soft capsules, powders, granules, suspensions, syrups, solutions and injections. Pharmaceutical preparations for the prevention and treatment of degenerative central nervous system diseases.

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