KR20210148802A - Pharmaceutical Composition Comprising Woohuangchengsimhwan for Preventing or Treating Parkinson's Disease - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating Parkinson's disease, comprising Uhwang-cheongsimhwan (a pill formulated with thirty medicinal ingredients including ox bezoar), wherein the pharmaceutical composition has a neuroprotective effect on nerve cells as well as exhibits an excellent effect in improving the expression of tyrosine hydroxylase involved in motor performance and dopamine precursor production for an animal model of Parkinson's disease, thereby being able to be usefully used for preventing or treating Parkinson's disease.

Description

Pharmaceutical composition for preventing or treating Parkinson's disease comprising Woohwangcheongsimhwan {Pharmaceutical Composition Comprising Woohuangchengsimhwan for Preventing or Treating Parkinson's Disease}

The present invention relates to a pharmaceutical composition for preventing or treating Parkinson's disease, comprising Woohwangcheongsimhwan.

Parkinson's disease (Parkinson's disease) is a degenerative neurological disease in which the muscles of the arms, legs, or the whole body are cramped or stiff and cannot focus. These symptoms are caused by the gradual destruction of certain nerve cells in the brain as an abnormality occurs in the secretion of dopamine, a neurotransmitter, due to a problem in the brain nerve cells. Typical symptoms of Parkinson's disease include movement disorders including convulsions, muscle stiffness and slow motion, and non-motor disorders such as olfactory disorders, sleep disorders, depression, anxiety disorders, cognitive disorders, and schizophrenia.

Dopamine precursor levodopa (L-dopa), the most widely used therapeutic agent in the early stages of Parkinson's disease, is converted to dopamine by DOPA decarboxylase to break the blood-brain barrier. It is the amino acid precursor of dopamine that passes through. However, it is known that most L-dopa is metabolized before reaching the blood-brain barrier and causes various side effects such as anorexia, nausea, vomiting, etc.

Accordingly, in order to reduce the occurrence of side effects due to the existing therapeutic agents for Parkinson's disease, the development of new therapeutic agents for Parkinson's disease using natural materials is being actively conducted in recent years. According to Korean Patent Nos. 10-1909814, 10-1867238, and 10-1643737, juniper extract, coriander extract, New Green extract, etc. were confirmed to exhibit protective activity on brain nerve cells, but excellent enough to be applied to clinical practice. It wasn't an effect. Therefore, for the prevention and treatment of Parkinson's disease, the development of a new material for improving the symptoms of Parkinson's disease while preventing the destruction of brain cells is required.

Korean Patent Registration No. 10-1909814 Korean Patent No. 10-1867238 Korean Patent Registration No. 10-1643737

According to one embodiment of the present invention, it is an object of the present invention to provide a pharmaceutical composition for preventing or treating Parkinson's disease containing woohwang cheongsimhwan as an active ingredient.

One aspect of the present invention provides a pharmaceutical composition for preventing or treating Parkinson's disease containing woohwang cheongsimhwan as an active ingredient.

Woohwangcheongsimhwan in the present invention is licorice, health, cinnamon, gilgyeong, angelica, soybean hwanggwon, jujube, maekmundong, windbreak, baekryeom, baekchul, bokryeong, musk, sanyak, swoogak, shiho, shingok, glue, nutritious ginseng, uranium, ginseng, Contains peony, cheongung, pohwang, dragon brain, passerby, and gold, from ancient times stroke (stroke, general paralysis, incapacity of hands, limbs, speech disorder, coma, mental confusion, facial nerve palsy, cerebral hemorrhage), hypertension, heart palpitations, mental anxiety, It is known to be effective against acute and chronic epilepsy, autonomic ataxia, and incapacity. However, there was no known correlation between Woohwangcheongsimhwan and Parkinson's disease.

In the present invention, it may be described as woohwang cheongsimhwan or cheongsimhwan.

The woohwang cheongsimhwan may be one in which each component is mixed according to a specific mixing ratio so that the medicinal effect can be sufficiently exhibited.

According to one embodiment of the present invention, the woohwang cheongsimhwan is 100 to 300 parts by weight of licorice, 10 to 100 parts by weight of health, 10 to 150 parts by weight of cinnamon, 10 to 100 parts by weight of gilgyeong, 10 to 100 parts by weight of angelica, 10 parts by weight of soybean hwangwon to 150 parts by weight, jujube 0.1 to 10 parts by weight, maekmundong 10 to 100 parts by weight, windbreak 10 to 100 parts by weight, baekchul 10 to 100 parts by weight, bokryeong 10 to 100 parts by weight, musk 1 to 30 parts by weight, acid about 200 to 400 parts by weight, 10 to 100 parts by weight of Shiho, 50 to 200 parts by weight of Shingok, 10 to 150 parts by weight of glue, 5 to 50 parts by weight of nutmeg, 5 to 50 parts by weight of oxen, 50 to 200 parts by weight of ginseng (KP), peony 10 to 100 parts by weight, 10 to 100 parts by weight of cheongung, 50 to 200 parts by weight of pohwang, 10 to 100 parts by weight of dragon brain, 10 to 100 parts by weight of passerby, and 10 to 100 parts by weight of gold, but limited thereto no.

The woohwang cheongsimhwan may be extracted using a conventional solvent under conventional temperature and pressure conditions according to a conventional method known in the art to which the present invention pertains.

According to one embodiment of the present invention, the woohwang cheongsim ring may be extracted using water, an alcohol having 1 to 4 carbon atoms, or a mixture thereof as a solvent. For example, water is used as the solvent, or one or two or more solvents selected from the group consisting of methanol, ethanol, propanol, butanol, ethylene glycol, propylene glycol, 1,3-butylene glycol, ethyl acetate and acetone. A mixed solvent may be used. In this case, the amount of the solvent used may be 1 to 60 times, 10 to 50 times, 20 to 40 times, or 30 times compared to the raw material of woohwang cheongsimhwan.

The extraction of the woohwang cheongsimhwan can be extracted using various methods such as hot water extraction, hot water extraction, cold-chim extraction, reflux extraction, and ultrasonic extraction. Extraction conditions may be adjusted according to the extraction method, the temperature may be 60 to 120 °C, 70 to 110 °C, 80 to 100 °C, or 90 °C, and the time may be 1 to 5 hours, 2 to 4 hours, or 3 It may be time, but is not particularly limited. Thereafter, the extracted woohwang cheongsimhwan extract may be subjected to one or more of filtration, concentration, and drying processes to remove the solvent contained in the extract, and filtration, concentration, and drying processes may all be performed as necessary. For example, filtration may be performed by filtration using a filter paper, filtration under reduced pressure using a reduced pressure filter, concentration may be performed by a reduced pressure concentration method, and drying may be performed by a freeze drying method, but is not particularly limited.

According to one embodiment of the present invention, the pharmaceutical composition comprising Uhwangcheongsimhwan as an active ingredient is mixed with a pharmaceutically acceptable carrier, diluent, and other pharmaceutically acceptable adjuvants, and a pharmaceutically acceptable formulation. It can be formulated in the form to prepare a pharmaceutical preparation. When formulating the composition, it is usually prepared using a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, and a surfactant. The carrier, diluent and adjuvant include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The formulation may be in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, or external preparations, suppositories, and sterile injection solutions. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, and the like, and liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.

The method of administering the pharmaceutical composition of the present invention is to administer a pharmaceutically effective amount, and the effective dose level is the patient's disease type, severity, drug activity, drug sensitivity, administration time, administration route and excretion rate, The duration of treatment may depend on factors including concomitant medications and other factors well known in the medical field. In order to enhance the therapeutic effect of the composition, the dosage of the pharmaceutical composition of the present invention may be administered at 1 to 1,000 mg/kg, 5 to 500 mg/kg, or 10 to 250 mg/kg per day. Administration may be administered once a day, or divided into several administrations. Any route of administration is possible, for example, oral, subcutaneous, arterial, intravenous, intramuscular, intrarectal, intrauterine, intraperitoneal, intrasternal, intrathecal, or intracerebroventricular injection may be administered.

According to one embodiment of the present invention, the pharmaceutical composition may have a neuroprotective action.

According to an embodiment of the present invention, in an in vitro experiment using nerve cells, Woohwangcheongsimhwan reduced neuronal cell death caused by 6-OHDA, thereby confirming excellent neuroprotective efficacy.

According to one embodiment of the present invention, the pharmaceutical composition may improve exercise ability. In addition, according to one embodiment of the present invention, the pharmaceutical composition may be to improve the expression of tyrosine hydroxylase in the substantia nigra.

According to an embodiment of the present invention, in an animal model experiment for MPTP-induced Parkinson's disease (in vivo), Woohwang Cheongsimhwan exercised through behavioral experiments at a level similar to or superior to L-dopa, which is an existing Parkinson's disease treatment. It was confirmed that not only the ability but also the expression of tyrosine hydroxylase involved in the production of dopamine precursors in the substantia nigra was improved.

The pharmaceutical composition containing Woohwangcheongsimhwan as an active ingredient according to the present invention has an excellent effect in improving the expression of tyrosine hydroxylase involved in motor ability and dopamine precursor production for an animal model of Parkinson's disease as well as having a neuroprotective effect on nerve cells. Therefore, it can be usefully used for the prevention or treatment of Parkinson's disease.

1 is a result of Experimental Example 1 according to the present invention, a result of confirming the neuronal protection effect by Woohwangcheongsimhwan through MTT assay. (a) is a graph showing the cell viability for 10, 20, 50, 100, 200, 400 μg/ml of Woohwang Cheongsimhwan, (b) is a graph showing the neuroprotective effect of Woohwang Cheongsimhwan on nerve cells damaged by 6-OHDA It is a graph.
2 is a result of Experimental Example 2 according to the present invention, a result of confirming the effect of improving exercise ability by Woohwang Cheongsimhwan through a rotarod experiment among behavioral experiments.
3 is a result of Experimental Example 2 according to the present invention, a result of confirming the effect of improving exercise ability by Woohwangcheongsimhwan through a rod experiment during a behavioral experiment.
4 is a result of Experimental Example 3 of the present invention, a result of confirming the effect of increasing dopamine precursor-related enzymes by Woohwangcheongsimhwan through immunochemical staining.
5 is a result of Experimental Example 3 of the present invention, a result of confirming the dopamine precursor-related enzyme increase effect by Woohwangcheongsimhwan through Western blot analysis.

Hereinafter, with reference to the accompanying drawings, the present invention will be described in more detail through examples. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

Example 1. Preparation of Uhwang Cheongsimhwan

100 to 300 parts by weight of licorice, 10 to 100 parts by weight of health, 10 to 150 parts by weight of cinnamon, 10 to 100 parts by weight of ginseng, 10 to 100 parts by weight of angelica, 10 to 150 parts by weight of soybean yellow bean, 0.1 to 10 parts by weight of jujube, Maekmundong 10 to 100 parts by weight, windbreak 10 to 100 parts by weight, Baekchul 10 to 100 parts by weight, Bokryeong 10 to 100 parts by weight, Musk 1 to 30 parts by weight, San about 200 to 400 parts by weight, Shiho 10 to 100 parts by weight, Shingok 50 to 200 parts by weight, 10 to 150 parts by weight of glue, 5 to 50 parts by weight of nutmeg, 5 to 50 parts by weight of oxen, 50 to 200 parts by weight of ginseng (KP), 10 to 100 parts by weight of peony, 10 to 100 parts by weight of cheongung A cheongshim ring was prepared according to a conventional method using 50 to 200 parts by weight of pohwang, 10 to 100 parts by weight of yongnoe, 10 to 100 parts by weight of haengin, and 10 to 100 parts by weight of gold.

Preparation Example 1. Neuronal cell culture

In order to confirm the neuroprotective effect of Cheongshimhwan, the human neuroblastoma-derived cell line SH-SY5Y was used. SH-SY5Y was cultured in DMEM (Invitrogen, USA) medium containing 10% FBS and 100 unit/ml penicillin and 100 ug/ml streptomycin as antibiotics to prevent contamination. Trypsin-EDTA was treated and subcultured. The medium was exchanged every 2-3 days.

Experimental Example 1. Confirmation of neuronal protection effect by Woohwang Cheongsimhwan

SH-SY5Y cells were subcultured ^{at 5x10 4} cells in a 24 well plate. The next day, after replacing with DMEM medium without FBS for at least 16 hours, cells were treated with Uhwangcheongsimhwan and 6-OHDA at different concentrations, and reacted for 24 hours. In order to observe the cell viability by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method, MTT 2 mg/ml was added to the medium, and incubated for 4 hours in an incubator. After that, the medium was removed, dissolved in DMSO (dimethylsulfoxide), and absorbance was measured with a microplate reader at a wavelength of 580 nm to calculate cell viability.

6-OHDA (6-hydroxydopamine) is a neurotoxin, and it is known that it induces immediate dysfunction and movement disorders in the axon terminal of dopaminergic cells through oxidative stress, and induces retrograde dopaminergic neuronal cell death.

MTT assay is a test method using the ability of cells to reduce the yellow water-soluble substrate MTT tetrazolum to the blue-violet water-insoluble MTT formazan by dehydrogenase action. The absorbance of MTT formazan is maximized at a wavelength of 580 nm, and the absorbance measured at this wavelength reflects the redox power of living and metabolically active cells.

Cell viability (%) was defined as follows. Using the untreated cells as the value of the control, the OD (optical density) value at this time was defined as 100% cell viability, and the cells treated with Woohwangcheongsimhwan or 6-OHDA were used as the value of the experimental group. Measured OD Values were converted to relative values as follows: Cell Viability = (experimental group / control group).

All measured values were expressed as mean ± standard error (mean ± SE), and statistical analysis between each experimental group was tested with one-way ANOVA of SPSS program ver. was considered

As a result, as shown in Figure 1, Woohwang Cheongsimhwan did not show cytotoxicity even at a concentration of 400 μg/ml, and in particular, when Uhwang Cheongsimhwan 50, 100, and 200 μg/ml were treated, nerve cells damaged by 6-OHDA were appeared to recover. Through these results, it was found that Woohwangcheongsimhwan has a neuroprotective effect against damaged nerve cells.

Preparation Example 2. Parkinson's disease induction model

Experimental animals were apparently healthy ICR mice purchased from Orient Bio (Co., Ltd.), male 6 weeks old (25 ~ 30g), temperature 22±1℃, humidity 55±1%, 12 hours light-dark cycle. Food and drinking water were allowed to be freely consumed in the controlled breeding room of

The experimental group was equally divided into 4 groups of 7 animals by weight, and each group was treated as follows.

MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is a Complex I inhibitor of the mitochondrial electron transport chain, and induces the production of the neurotoxin MPP+, thereby nigra) induces apoptosis of dopaminergic neurons and Parkinson's disease-related lesions, thereby inducing motility disorders similar to those of Parkinson's disease.

L-dopa is a precursor of dopamine, which is converted to dopamine by L-dopa decarboxylase in the brain, and is clinically used to treat Parkinson's disease.

1) Normal: Non-Parkinson's disease group administered with saline intraperitoneally (individual: 7 animals)

2) Control group (MPTP): Parkinson's disease group in which MPTP-HCL (30 mg/kg of free base) diluted in saline was administered intraperitoneally every 24 hours for 5 days (individual: 7 animals)

3) Woohwang Cheongsimhwan administration group (Chungsimhwan): After oral administration of Woohwang Cheongsimhwan 100 mg/kg/day for 1 week, MPTP-HCL (30 mg/kg of free base) diluted in saline was administered intraperitoneally at 24 hour intervals for 5 days. Parkinson's disease group (individuals: 7 animals)

4) Dopamine administration group (L-dopa): After oral administration of L-dopa 10 mg/kg for 1 week, MPTP-HCL (30 mg/kg of free base) diluted in saline was administered intraperitoneally every 24 hours for 5 days. One Parkinson's disease group (7 animals)

The following experiments were performed using each treatment group. All measured values were expressed as mean ± standard error (mean ± SE), and statistical analysis between each experimental group was tested with one-way ANOVA of SPSS program ver. was considered

Experimental Example 2. Confirmation of exercise ability improvement effect by Woohwang Cheongsimhwan - Behavioral experiment

2-1. Rota-rod test

After placing the mouse on the rotarod equipment and acclimating it to the speed of 4 rpm at first, the time for the mouse to fall from the cylinder was measured while gradually increasing it to 4 ~ 35 rpm as an acceleration test (acceleration speed test).

As a result, as shown in FIG. 2 , the experimental group administered with Woohwang Cheongsimhwan showed a significantly increased exercise time compared to the control group administered with MPTP only.

2-2. Pole test

After turning the mouse's head upward, it was placed at the end of a rough vertical pole (diameter: 8 mm, height: 55 cm) and the time for the mouse to come down to the floor was measured (locomotor activity time, maximum: 60). candle). If the mouse could not turn its head downward or fell off the rod, the locomotor activity time was recognized as 60 seconds (default value), and the average value was measured by measuring a total of three times.

As a result, as shown in FIG. 3 , the experimental group administered with Woohwang Cheongsimhwan had a head turning time (orient down time) and head turning time compared to the control group administered only with MPTP, and then the time to descend the bar (traverse down time) was appeared to be significantly reduced.

These behavioral experimental results suggest that administration of Woohwang Cheongsimhwan can restore exercise ability to a level similar to that of the normal group without Parkinson's disease.

Experimental Example 3. Confirmation of dopamine precursor-related enzyme increase effect by Woohwang Cheongsimhwan - Immunochemical staining

Parkinson's disease induction model was anesthetized with chloral hydrate overdose (300 mg/kg) 7 days after the last administration of MPTP or saline. Thereafter, cardiac puncture was performed and fixed with 4% paraformaldehyde (paraformaldehyde in 0.05 M phosphate buffer), and the brain was removed and post-fixed. After 24 hours, the tissue was sectioned by putting it in 30% sucrose for 24 hours. Immunohistochemistry was a free-floating method, and the tissue was cut to a thickness of 40 μm using a cryomicrotome, and a region containing substantia nigra (SN) was used. After suspension in 0.05 M PBS solution containing 3% H ₂ O ₂ for 10 minutes, anti-TH (tyrosin hydroxylase) antibody (1:1000, manufactured by SantaCruz Biotechnology) was bound for at least 16 hours at room temperature, and then Vectastain Elite ABC kit ( The color was developed using Vector Laboratories).

TH is an enzyme that attaches a hydroxyl group (-OH) to the phenol group of tyrosine and produces L-dopa, a precursor of dopamine.

As a result, as shown in Fig. 4, the experimental group administered with woohwang cheongsimhwan showed a significant increase in TH expression in the substantia nigra, which was reduced by MPTP, and the level was the same as in the experimental group administered with the Parkinson's disease treatment L-dopa. appeared to be at a similar level.

Experimental Example 4. Confirmation of dopamine precursor-related enzyme increase effect by Woohwang Cheongsimhwan - Western blot analysis

After extracting the brain in the same manner as in Experimental Example 3, the SN was separated and the buffer solution (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 30 mM sodium pyrophosphate, 10 mM NaF, 2 mM Na ₃ VO ₄ and protease inhibitor cocktail) and homogenized using a homogenizer. The homogenate was centrifuged at 12,000 rpm for 15 minutes at 4°C to obtain only the supernatant. For protein quantification, BCA (bicinchoninic acid, manufactured by Pierce) was used. The supernatant was added with 4X Lammlie's buffer (62.5 mmol/1 Tris-HCl, pH 6.8, 2% SDS, 20% glycerol and 10% 2-mercaptoethanol) and boiled at 95°C. 50 ug of the quantified protein sample was separated by electrophoresis using 4-12% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gradient gel, manufactured by Invitrogen), and transferred to an NC membrane (nitrocellulose paper, manufactured by Amersham). The membrane to which the protein has been transferred is stained with Ponceau-S to confirm that the protein has been completely transferred, washed with Tris-buffered saline (TBS-T) containing 0.1% Tween 20 and blocked with 5% skim milk powder for more than 30 minutes. ) was done. Anti-TH (manufactured by antaCruz Biotechnology) and anti-β-actin (manufactured by Cell signaling tech) were reacted at 4°C for 16 hours, respectively, and then the membrane was washed 3 times in TBS-T for 10 minutes each, followed by blot with secondary antibody. The reaction was carried out for 1 hour. After the secondary antibody reaction, the membrane was washed and the desired protein was visualized with ECL (enhanced chemiluminiscence system, manufactured by Pierce). Protein visualization and quantitative analysis were performed using image equipment (LAS-3000, Fuji).

As a result, as shown in FIG. 5, the experimental group administered with woohwang cheongsimhwan showed a significant increase in TH expression in the substantia nigra, which was reduced by MPTP, and the level was the same as the experimental group administered with the Parkinson's disease treatment L-dopa. appeared to be at a similar level. This Western blot analysis result was confirmed to be the same as the immunochemical staining result of Experimental Example 3.

Therefore, it was found that Woohwangcheongsimhwan significantly improved the expression of dopaminergic precursor production-related enzymes reduced by Parkinson's disease.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

Claims (6)

A pharmaceutical composition for preventing or treating Parkinson's disease, comprising Woohwang Cheongsimhwan as an active ingredient.
The method according to claim 1,
The woohwang cheongsimhwan is licorice, health, cinnamon, gilgyeong, angelica, soybean hwanggwon, jujube, maekmundong, windbreak, baekryeom, baekchul, bokryeong, musk, sanyak, swoogak, shiho, shingok, glue, yanggak, woohwang, ginseng, peony, cheongung , Pohwang, Yongnoe, a pharmaceutical composition comprising gold.
The method according to claim 1,
The pharmaceutical composition further comprises a pharmaceutically acceptable carrier, adjuvant or diluent.
The method according to claim 1,
The pharmaceutical composition is a pharmaceutical composition that acts to protect nerve cells.
The method according to claim 1,
The pharmaceutical composition is a pharmaceutical composition to improve athletic performance.
The method according to claim 1,
The pharmaceutical composition is a pharmaceutical composition to improve the expression of tyrosine hydroxylase in the substantia nigra.

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