Composition for preventing, alleviating or treating neurodegenerative diseases

Abstract

Disclosed herein is a pharmaceutical composition for preventing, alleviating or treating neurodegenerative diseases. The pharmaceutical composition contains, as active ingredients, Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome, and Pulvis aconiti tuberis purificatum. The pharmaceutical composition is prepared by a method including the steps of: mixing Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome, and Pulvis aconiti tuberis purificatum to obtain a mixture, and extracting the mixture to obtain a mixture extract; filtering the mixture extract; concentrating the mixture extract; and drying the mixture extract.

Claims

1. A composition for preventing, alleviating or treating a neurodegenerative disease, the composition containing, as an active ingredient, an extract of a mixture of the herbal materials consisting essentially of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome, and Pulvis aconiti tuberis purificatum, wherein the extract of the mixture is contained in the composition at a concentration of 10 to 200 g/ml and wherein the composition reduces LPS-induced nitrite production in cultured BV2 microglial cells by at least 30% when provided at a concentration of 50 ug/ml, at least 60% when provided at a concentration of 100 ug/ml, and at least 80% when provided at a concentration of 200 ug/ml.

2. A composition as in claim 1 wherein said neurodegenerative disease is selected from Lou Gehrig's Disease, motor neuron disease, dementia, Parkinson's Disease, cerebral atrophy, Creutzfeldt-Jakob Disease, multiple system atrophy, and Huntington's Disease.

3. A composition as in claim 2 wherein said composition alleviates neuroinflammation and protects neural cells.

4. A composition as in claim 2 wherein said neurodegenerative disease is Lou Gehrig's Disease.

5. A composition as in claim 1 wherein said composition contains 3 to 4 parts by weight of Paeoniae radix, 3 to 4 parts by weight of Glycyrrhizae radix, 1 to 2 parts by weight of Pulvis aconiti tuberis purification, and 0.5 to 1 part by weight of each of Curcumae longae rhizome, Gastrodia rhizome, Polygalae radix, Salviae radix, Chaenomeles fructus, and Atractylodis rhizome.

6. A composition as in claim 1 wherein said composition inhibits LPS-induced inflammation and glutamate neurotoxicity in microglial BV-2 cells.

7. A composition as in claim 1 wherein the extract is produced by the steps of: (a) extracting the mixture of herbal materials to form an extract; (b) concentrating the extract; and (c) drying the extract.

8. A composition as in claim 7 wherein the extracting step (a) is performed by solvent extraction, microwave extraction, percolation extraction, steam distillation, or carbon dioxide supercritical extraction.

9. A composition as in claim 8 wherein the extracting step (a) is by solvent extraction in a solvent selected from water, ethanol, methanol, fatty oil, glycerin, horse oil, propylene glycol ether, chloroform, petroleum ether, hexane, benzene, methylene chloride, ethyl acetate, acetone, butanol, and isopropanol.

10. A composition as in claim 9 wherein the solvent is ethanol at 30 wt %.

11. A composition as in claim 10 wherein the extraction process is performed 1 to 4 times at a temperature of 80-95 C. for 1 to 5 hours.

Description

DESCRIPTION OF DRAWINGS

(1) FIGS. 1 and 2 are graphs showing the anti-neuroinflammatory effect of a composition (indicated as Mecacin) obtained according to Embodiment 1 of the present invention;

(2) FIG. 3 is a graph showing the results of measuring the cytotoxicity of the composition (indicated as Mecacin) of Embodiment 1 of a present invention and the protective effect of the composition against glutamate neurotoxicity; and

(3) FIG. 4 is a graph showing the results of the Rotarod test performed after the animal models with Lou Gehrig's disease were treated with the composition (indicated as MSC) of Embodiment 1 of the present invention.

MODE FOR INVENTION

(4) The terms and words used in the specification and claims should not be interpreted as being limited to typical meanings or dictionary definitions, but should be interpreted as having meanings and concepts relevant to the technical scope of the present invention, based on the principle that an inventor can appropriately define the concept of the terms to describe their invention in the best way.

(5) Accordingly, it should be understood that the experimental examples and reference examples described in the specification are merely examples and do not represent all the technical spirits of the present invention, and thus there may be various equivalents and modifications that can replace the above examples at the time when the present application is filed.

Embodiment 1: Composition (1) for Preventing, Alleviating or Treating Neurodegenerative Diseases

(6) (1) Step of Preparing Mixture Extract

(7) Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome and Pulvis aconiti tuberis purificatum are mixed, and then extracted to obtain a mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome and Pulvis aconiti tuberis purificatum.

(8) The herbal materials that are used to prepare the mixture extract may be used at varying weight ratios. For example, based on 3 to 4 parts by weight of Paeoniae radix or Glycyrrhizae radix, Pulvis aconiti tuberis purificatum may be used in an amount of 1 to 2 parts by weight, and each of Curcumae longae rhizome, Salviae radix, Chaenomeles fructus, Polygalae radix, Glycyrrhizae radix and Atractylodis rhizome may be used in an amount of 0.5-1 parts by weight, but the present invention is not limited thereto.

(9) To extract a mixture of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizoma and Pulvis aconiti tuberis purificatum, various methods may be used, including solvent extraction, steam distillation, carbon dioxide supercritical extraction, microwave extraction, percolation extraction, and the like. Preferably, solvent extraction may be used, but is not limited thereto.

(10) Two or more extraction methods may be used to extract the mixture.

(11) Depending on the kind of extraction solvent, the extraction temperature, the extraction time, the amount of solvent used and a method of treating residual components may vary. The extraction solvent may also be selected from various solvents. Extraction solvents that may be used in the present invention include water, ethanol, methanol, fatty oil, glycerin, horse oil, propylene glycol, ether, chloroform, petroleum ether, hexane, benzene, methylene chloride, ethyl acetate, acetone, butanol, isopropanol and the like. Preferably, ethanol may be used, but is not limited thereto.

(12) In addition, two or more solvents having different distribution coefficients may also used together for extraction, and two or more solvents may be used for second-step extraction.

(13) The extraction solvent may be used at varying concentrations. Preferably, the solvent may be used at a concentration of 30 wt %, but is not limited thereto.

(14) The extraction time, the extraction temperature and the number of extractions may also vary over wide range. Preferably, the extraction process may be performed 1 to 4 times at a temperature of 80 to 95 C. for 1 to 5 hours, but is not limited thereto.

(15) (2) Step of Filtering Mixture Extract

(16) The mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizoma and Pulvis aconiti tuberis purificatum is filtered.

(17) For filtration of the mixture extract, various methods may be used, including atmospheric pressure filtration, suction filtration, vacuum filtration, ultrafiltration, microfiltration, and reverse osmosis filtration.

(18) The filtration time, the filtration temperature and the number of filtrations may vary over a wide range.

(19) The filtration process may be performed using methods other than the above-listed methods, and two or more methods may also be used for filtration.

(20) When microfiltration or ultrafiltration is performed, the filter membrane may preferably have a pore size of 50 m, but is not limited thereto.

(21) (3) Step of Concentrating Mixture Extract

(22) The mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizoma and Pulvis aconiti tuberis purificatum is concentrated.

(23) For concentration of the mixture extract, various methods may be used, including freeze concentration, reduced-pressure concentration, membrane concentration, vacuum concentration, heating concentration, and evaporation concentration. Preferably, reduced-pressure concentration may be used, but is not limited. In addition, two or more concentration methods may also be used.

(24) The concentration process may be performed at varying temperatures. Preferably, the concentration process may be performed at a temperature of 5 to 60 C., but is not limited thereto. Concentration duration and the number of concentrations may also vary over a wide range.

(25) (4) Step of Drying Mixture Extract

(26) The mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizoma and Pulvis aconiti tuberis purificatum is dried.

(27) For drying of the mixture extract, various methods may be used, including shade-drying, sun-drying, hot-air drying, freeze drying, electric drying, and cold-air drying. Two or more drying methods may be used.

(28) The drying temperature, the drying time and the number of drying cycles may vary over a wide range.

Embodiment 2: Composition (2) for Preventing, Alleviating or Treating Neurodegenerative Diseases

(29) (1) Step of Preparing Extract 1

(30) Paeoniae radix, Glycyrrhizae radix and Pulvis aconiti tuberis purificatum are mixed, and then extracted to obtain extract 1.

(31) Preparation of extract 1 is performed in the same manner as described in the step of preparing the mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome and Pulvis aconiti tuberis purificatum in Embodiment 1.

(32) The mixing ratio between Paeoniae radix, Glycyrrhizae radix and Pulvis aconiti tuberis purificatum may vary over a wide range. Preferably, based on 3 to 4 parts by weight of Paeoniae radix, Glycyrrhizae radix may be used in an amount of 3 to 4 parts by weight, and Pulvis aconiti tuberis purificatum may be used in an amount of 1 to 2 parts by weight, but the present invention is not limited thereto.

(33) (2) Step of Preparing Extract 2

(34) Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus and Atractylodis rhizome are mixed, and then extracted to obtain extract 2.

(35) Preparation of extract 2 is also performed in the same manner as described in the step of preparing the mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome and Pulvis aconiti tuberis purificatum in Embodiment 1.

(36) The mixing ratio between Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus and Atractylodis rhizome may vary over a wide range. Preferably, each of Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus and Atractylodis rhizome may be used in an amount of 0.5 to 1 part by weight, but is not limited thereto.

(37) (3) Step of Preparing Extract Mixture

(38) Extracts 1 and 2 prepared as described above are mixed to obtain an extract mixture.

(39) (4) Step of Filtering Extract Mixture

(40) The extract mixture is filtered.

(41) Filtration of the extract mixture is performed in the same manner as described in the step of filtering the mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome and Pulvis aconiti tuberis purificatum in Embodiment 1.

(42) (5) Step of Concentrating Extract Mixture

(43) The extract mixture is concentrated.

(44) Concentration of the extract mixture is performed in the same manner as described in the step of concentrating the mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome and Pulvis aconiti tuberis purificatum in Embodiment 1.

(45) (6) Step of Drying the Mixture of Extract 1 and Extract 2

(46) The mixture of extracts 1 and 2 are dried.

(47) Drying the mixture of extracts 1 and 2 is performed in the same manner as described in the step of drying the mixture extract of Curcumae longae rhizome, Gastrodiae rhizoma, Polygalae radix, Salviae radix, Chaenomeles fructus, Paeoniae radix, Glycyrrhizae radix, Atractylodis rhizome and Pulvis aconiti tuberis purificatum in Embodiment 1.

Experimental Example 1: Effect Against Neuroinflammation

(48) 1-1. Experimental Method

(49) In order to examine the effect of the composition of the present invention against neurodegenerative diseases, the inflammation inhibitory effect of the composition of the present invention against LPS-induced neuroinflammation in microglia BV-2 cells was evaluated.

(50) In this experiment, the composition according to embodiment 1 was used at varying concentrations (10, 50, 100 and 200 g/ml).

(51) In order to examine the inflammation inhibitory effect of the composition in neural cells, the expression levels of COX-2 and iNOS, which are pro-inflammatory genes, were measured, and the expression levels of PGE2, nitrite, p65, p50, p-IB- and NF-B, which are pro-inflammatory genes, were also analyzed.

(52) 1-2. Experimental Results

(53) FIGS. 1 and 2 are graphs showing the anti-neuroinflammatory effect of a composition (indicated as Mecacin) obtained according to Embodiment 1 of the present invention.

(54) As can be seen therein, the experimental results indicated that when the BV-2 cells with LPS-induced neuroinflammation were treated with the composition of the present invention, the secretion of pro-inflammatory proteins such as COX-2 and iNOS was inhibited in a concentration-dependent manner.

(55) In addition, it was shown that the secretion of PGE2, nitrite, p65, p50, p-IB- and NF-B, which are pro-inflammatory genes, was also inhibited in a concentration-dependent manner, when the BV-2 cells with LPS-induced neuroinflammation were treated with the composition of the present invention.

(56) This suggests that the composition according to the present invention has the effect of preventing, alleviating or treating neurodegenerative diseases by inhibiting neuroinflammation.

Experimental Example 2: Effect Against Glutamate Neurotoxicity

(57) 2-1. Experimental Method

(58) Glutamate is an excitatory neurotransmitter that increases excitation of neural cells and plays an important role in memory and learning.

(59) However, it is known that when glutamate is excessively released, it changes into an excitotoxic compound which kills neural cells, thereby causing neurodegenerative diseases such dementia, Lou Gehrig's disease or the like.

(60) Therefore, in this Experimental Example, the protective effect of the composition of the present invention against glutamate-induced neurotoxicity was evaluated in order to confirm the effect of the composition of the present invention against neurodegenerative diseases.

(61) In the experiment, BV-2 cells were treated with 5 mM of glutamate to induce neurotoxicity and treated with varying concentrations (10, 20, 50 and 100 g/ml) of the composition of the present invention, and the viability of the cells was measured. As a positive control, Trolox was used.

(62) Meanwhile, in order to whether or not the composition of the present invention is cytotoxic, BV-2 cells were treated with varying concentrations (1, 2, 5, 10, 50, 100 and 200 g/ml) of the composition of the present invention, and the viability of the cells was measured.

(63) 2-2. Experimental Results

(64) FIG. 3 is a graph showing the results of measuring the cytotoxicity of the composition (indicated as Mecacin) of Embodiment 1 of the present invention and the protective effect of the composition against glutamate neurotoxicity.

(65) Specifically, FIG. 3A shows the results of measuring the cytotoxicity of the composition (indicated as Mecacin) of the present invention. As can be seen therein, the cell viability reached 100% even at a concentration of up to 100 g/ml, indicating that the composition of the present invention is not cytotoxic at a concentration of up to 100 g/ml. In addition, at a concentration of 200 g/ml, a cell viability of about 90% was observed.

(66) FIG. 3B shows the results of measuring cell viability after neural cells with glutamate-induced neurotoxicity were treated with the composition of the present invention. As can be seen therein, the cell viability increased with concentration.

(67) Particularly, it could be seen that the group treated with 100 g/ml of the composition exhibited a better protective effect compared to the positive control Trolox.

(68) Therefore, it can be seen that the composition according to the present invention has the effect of preventing, alleviating or treating neurodegenerative diseases by protecting neural cells from glutamate neurotoxicity.

Experimental Example 3: Effect in Animal Models with Lou Gehrig's Disease

(69) 3-1. Experimental Method

(70) Lou Gehrig's disease (also known as amyotrophic lateral sclerosis (ALS)) is a neurodegenerative disease that shows symptoms, such as movement disorders, muscle weakness, respiratory failure and the like, due to motor neuron death.

(71) In this Experimental Example, animal models with Lou Gehrig's disease were treated with the composition of Embodiment 1 of the present invention, and then the motor activities of the mice were measured by a Rotarod test.

(72) The Rotarod is a device that evaluates the motor function of a test animal by pacing the test animal on a beam that rotates at a fixed speed or a gradually accelerating speed and measuring the time taken for the test animal to fall. Using the Rotarod, the effect of the composition of the present invention on motor ability improvement was evaluated.

(73) 3-2. Experimental Results

(74) FIG. 4 is a graph showing the results of the Rotarod test performed after the animal models with Lou Gehrig's disease were treated with the composition (indicated as MSC) of Embodiment 1 of the present invention.

(75) As can be seen therein, the experimental results indicated that the time taken for the test animal to fall from the rotating beam increased in the test group treated with the composition of the present invention, compared to the untreated control group.

(76) This suggests that the composition according to the present invention improves the motor function of Lou Gehrig's disease animal models.