PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING NEURODEGENERATIVE DISEASES, CONTAINING ANGELICA GIGAS NAKAI EXTRACT OR MIXED EXTRACT OF ANGELICA GIGAS NAKAI AND BROCCOLI

Abstract

Provided is a use of an Angelica gigas Nakai extract or a mixed extract of Angelica gigas Nakai and broccoli for the prevention and/or treatment of neurodegenerative diseases and/or amelioration, the protection of nerve cells and/or the generation of nerve cells.

Claims

1. A pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising as an active ingredient an Angelica gigas Nakai extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol at 40 to 80° C.

2. The pharmaceutical composition for preventing or treating neurodegenerative diseases according to claim 1, wherein the neurodegenerative disease is dementia, Parkinson's disease (PD), Huntington's disease, or amyotrophic lateral sclerosis.

3. (canceled)

4. A pharmaceutical composition for protecting nerve cells or regenerating nerve cells, comprising as an active ingredient an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol aqueous solution at 40 to 80° C.

5. A pharmaceutical composition for preventing or treating neurodegenerative diseases, comprising a mixed extract of Angelica gigas Nakai and Brassica oleracea var. italic as an active ingredient, wherein the mixed extract of Angelica gigas Nakai and Brassica oleracea var. italic is: one comprising an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol at 40 to 80° C., and a Brassica oleracea var. italic ethanol extract obtained by extracting Brassica oleracea var. italica with 20 to 80% (v/v) ethanol at 10 to 80° C., or an extract of a mixture of Angelica gigas Nakai and Brassica oleracea var. italica obtained by extracting a mixture of Angelica gigas Nakai and Brassica oleracea var. italica with 40 to 100% (v/v) ethanol at 30 to 80° C.

6. (canceled)

7. The pharmaceutical composition for preventing or treating neurodegenerative diseases according to claim 5, wherein a mixing ratio between Angelica gigas Nakai extract or Angelica gigas Nakai and Brassica oleracea var. italica extract or Brassica oleracea var. italica in the mixed extract is 1:0.1 to 10 based on the weight (the weight of Angelica gigas Nakai extract or Angelica gigas Nakai: the weight of Brassica oleracea var. italica extract or Brassica oleracea var. italica).

8. The pharmaceutical composition for preventing or treating neurodegenerative diseases according to claim 7, wherein a mixing ratio between Angelica gigas Nakai extract or Angelica gigas Nakai and Brassica oleracea var. italica extract or Brassica oleracea var. italica in the mixed extract is 1:0.2 to 5 based on the weight (the weight of Angelica gigas Nakai extract or Angelica gigas Nakai: the weight of Brassica oleracea var. italica extract or Brassica oleracea var. italica).

9. The pharmaceutical composition for preventing or treating neurodegenerative diseases according to claim 5, wherein the neurodegenerative disease is dementia, Parkinson's disease (PD), Huntington's disease, or amyotrophic lateral sclerosis.

10. (canceled)

11. A pharmaceutical composition for protecting nerve cells or regenerating nerve cells, comprising a mixed extract of Angelica gigas Nakai and Brassica oleracea var. italica as an active ingredient, wherein the mixed extract of Angelica gigas Nakai and Brassica oleracea var. italic is: one comprising an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol at 40 to 80° C., and a Brassica oleracea var. italic ethanol extract obtained by extracting Brassica oleracea var. italica with 20 to 80% (v/v) ethanol at 10 to 80° C., or an extract of a mixture of Angelica gigas Nakai and Brassica oleracea var. italica obtained by extracting a mixture of Angelica gigas Nakai and Brassica oleracea var. italica with 40 to 100% (v/v) ethanol at 30 to 80° C.

12. A health functional food for preventing or improving neurodegenerative diseases or protecting nerve cells or regenerating nerve cells, comprising, an Angelica gigas Nakai extract, or a mixed extract of Angelica gigas Nakai and Brassica oleracea var. italica as an active ingredient, wherein the mixed extract of Angelica gigas Nakai and Brassica oleracea var. italic is: one comprising an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol at 40 to 80° C., and a Brassica oleracea var. italic ethanol extract obtained by extracting Brassica oleracea var. italica with 20 to 80% (v/v) ethanol at 10 to 80° C., or an extract of a mixture of Angelica gigas Nakai and Brassica oleracea var. italica obtained by extracting a mixture of Angelica gigas Nakai and Brassica oleracea var. italica with 40 to 100% (v/v) ethanol at 30 to 80° C.

13. A method for preventing or treating neurodegenerative diseases, comprising administrating an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol at 40 to 80° C. to a subject in need of the prevention or treatment of neurological disorders.

14. The method for preventing or treating neurodegenerative diseases according to claim 13, wherein the neurodegenerative disease is dementia, Parkinson's disease (PD), Huntington's disease, or amyotrophic lateral sclerosis.

15. The method for preventing or treating neurodegenerative diseases according to claim 14, wherein the dementia is Alzheimer's disease.

16. A method for protecting nerve cells or regenerating nerve cells, comprising administrating an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol at 40 to 80° C. to a subject in need of the protection of nerve cells or the regeneration of nerve cells.

17. A method for preventing or treating neurodegenerative diseases, comprising administrating a mixed extract of Angelica gigas Nakai and Brassica oleracea var. italica to a subject in need of the prevention or treatment of neurological disorders, wherein the mixed extract of Angelica gigas Nakai and Brassica oleracea var. italic is: one comprising an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol at 40 to 80° C., and a Brassica oleracea var. italic ethanol extract obtained by extracting Brassica oleracea var. italica with 20 to 80% (v/v) ethanol at 10 to 80° C., or an extract of a mixture of Angelica gigas Nakai and Brassica oleracea var. italica obtained by extracting a mixture of Angelica gigas Nakai and Brassica oleracea var. italica with 40 to 100% (v/v) ethanol at 30 to 80° C.

18. The method for preventing or treating neurodegenerative diseases according to claim 17, wherein the Brassica oleracea var. italica ethanol extract is extracted under conditions of pH 7 to 9.

19. The method for preventing or treating neurodegenerative diseases according to claim 17, wherein a mixing ratio between Angelica gigas Nakai extract or Angelica gigas Nakai and Brassica oleracea var. italica extract or Brassica oleracea var. italica in the mixed extract is 1:0.1 to 10 based on the weight (the weight of Angelica gigas Nakai extract or Angelica gigas Nakai: the weight of Brassica oleracea var. italica extract or Brassica oleracea var. italica).

20. The method for preventing or treating neurodegenerative diseases according to claim 19, wherein a mixing ratio between Angelica gigas Nakai extract or Angelica gigas Nakai and Brassica oleracea var. italica extract or Brassica oleracea var. italica in the mixed extract is 1:0.2 to 5 based on the weight (the weight of Angelica gigas Nakai extract or Angelica gigas Nakai: the weight of Brassica oleracea var. italica extract or Brassica oleracea var. italica).

21. The method for preventing or treating neurodegenerative diseases according to claim 17, wherein the neurodegenerative disease is dementia, Parkinson's disease (PD), Huntington's disease, or amyotrophic lateral sclerosis.

22. The method for preventing or treating neurodegenerative diseases according to claim 21, wherein the dementia is Alzheimer's disease.

23. A method for protecting nerve cells or regenerating nerve cells, comprising administrating a mixed extract of Angelica gigas Nakai and Brassica oleracea var. italica to a subject in need of the protection of nerve cells or the regeneration of nerve cells, wherein the mixed extract of Angelica gigas Nakai and Brassica oleracea var. italic is: one comprising an Angelica gigas Nakai ethanol extract obtained by extracting Angelica gigas Nakai with 90 to 100% (v/v) ethanol at 40 to 80° C., and a Brassica oleracea var. italic ethanol extract obtained by extracting Brassica oleracea var. italica with 20 to 80% (v/v) ethanol at 10 to 80° C., or an extract of a mixture of Angelica gigas Nakai and Brassica oleracea var. italica obtained by extracting a mixture of Angelica gigas Nakai and Brassica oleracea var. italica with 40 to 100% (v/v) ethanol at 30 to 80° C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 is a schematic diagram showing the administration schedule of beta-amyloid and the mixed extract for performing a test for confirming the neuroprotective effect of the extract on the induction of nerve damage by beta-amyloid in a mouse model.

[0055] FIG. 2 is a photograph showing the results of CV (cresyl violet acetate) staining of the hippocampal tissue of mice to which beta-amyloid was administered after administration of the extract according to one embodiment.

[0056] FIG. 3 is a graph showing the results of quantifying the stained hippocampal tissue of FIG. 2.

[0057] FIG. 4 shows the result of confirming the expression level of BDNF (brain-derived neurotrophic factor) through Western blotting when the extract according to one embodiment was administered to an Alzheimer's disease mouse model (3×-TG).

[0058] FIG. 5 is a graph showing the result of quantifying the expression level of BDNF (BDNF expression level/beta-actin expression level) confirmed by the Western blotting of FIG. 4 using Western blot densitometry analysis.

[0059] FIG. 6 shows the result of confirming the expression level of MAP2 (microtubule-associated protein 2), Synap (synaptophysin), and PSD-95 (postsynaptic density protein-95) through Western blotting when the extract according to one embodiment was administered to an Alzheimer's disease mouse model (3×-TG).

[0060] FIG. 7 is a graph showing the results of quantifying the expression level of MAP2 (MAP2 expression level/beta-actin expression level) confirmed by the Western blotting of FIG. 6 using Western blot densitometry analysis.

[0061] FIG. 8 is a graph showing the results of quantifying the expression level of Synap (Synap expression level/beta-actin expression level) confirmed by Western blotting of FIG. 6 using Western blot densitometry analysis.

[0062] FIG. 9 is a graph showing the result of quantifying the expression level of PSD-95 (PSD-95 expression level/beta-actin expression level) by Western blotting of FIG. 6 using Western blot densitometry analysis.

[0063] FIG. 10 shows the result of confirming the expression level of TrkB (tyrosine kinase receptor B) through Western blotting when the extract according to one embodiment was administered to an Alzheimer's disease mouse model (3×-TG).

[0064] FIG. 11 is a graph showing the results of quantifying the expression level of TrkB (TrkB expression level/beta-actin expression level) confirmed by the Western blotting of FIG. 10 using Western blot densitometry analysis.

[0065] FIG. 12 shows the results of confirming the expression level of CAMKII (Ca2.sup.+/calmodulin-dependent protein kinase II) through Western blotting when the extract according to one embodiment was administered to an Alzheimer's disease mouse model (3×-TG).

[0066] FIG. 13 is a graph showing the result of quantifying the expression level of phosphorylated CAMKII (phosphorylated CAMKII expression level/non-phosphorylated CAMKII expression level) confirmed by Western blotting of FIG. 12 using Western blot densitometry analysis.

[0067] FIG. 14 shows the result of confirming the expression level of ERK (extracellular signal-regulated kinase) through Western blotting when the extract according to one embodiment was administered to an Alzheimer's disease mouse model (3×-TG).

[0068] FIG. 15 is a graph showing the results of quantifying the expression level of phosphorylated ERK (phosphorylated ERK expression level/non-phosphorylated ERK expression level) confirmed by Western blotting of FIG. 14 using Western blot densitometry analysis.

[0069] FIG. 16 shows the results of confirming the expression level of Akt (RAC-alpha serine/threonine-protein kinase) through Western blotting when the extract according to an embodiment was administered to an Alzheimer's disease mouse model (3×-TG).

[0070] FIG. 17 is a graph showing the results of quantifying the expression level of phosphorylated Akt (phosphorylated Akt expression level/non-phosphorylated Akt expression level) confirmed by the Western blotting of FIG. 16 using Western blot densitometry analysis.

[0071] FIG. 18 shows the result of confirming the expression level of CREB (cAMP-responsive element-binding protein) through Western blotting when the extract according to one embodiment was administered to an Alzheimer's disease mouse model (3×-TG).

[0072] FIG. 19 is a graph showing the results of quantifying the expression level of phosphorylated CREB (phosphorylated CREB expression level/non-phosphorylated CREB expression level) confirmed by Western blotting of FIG. 18 using Western blot densitometry analysis.

[0073] FIG. 20 is a photograph showing the results of CV (cresyl violet acetate) staining of the hippocampal tissue of a mouse to which beta-amyloid was administered after administration of an extract extracted with various concentrations of the extraction solvent.

[0074] FIG. 21 is a graph showing the results of quantifying the hippocampal tissue stained in FIG. 20.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0075] Hereinafter, the present invention will be described with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited thereto. It will be obvious to those skilled in the art that the examples described below may be modified without departing from the scope of the essential gist of the present invention.

Example 1: Preparation of Extract

[0076] 1.1: Preparation of Angelica gigas Nakai Extract

[0077] 1.1.1. Preparation of 98% Ethanol Extract

[0078] Angelica gigas Nakai roots were washed with clean water and dried sufficiently. The dried Angelica gigas Nakai roots were crushed, 5 volume times (500 ml) of ethanol (98% (v/v) ethanol (alcohol)) was added to 100 g of the obtained powder, extracted at 40 to 60° C. for 4 hours or more, and then filtered through a 1 um (micrometer) filter, and the filtrate was heated and concentrated until it became 10 wt. % of the original weight. Crystalline cellulose was gradually added to the obtained concentrate, continuously concentrated, dried completely, and then powdered to prepare an Angelica gigas Nakai ethanol extract powder (hereinafter, referred to as AGE).

[0079] 1.1.2. Preparation of 90% Ethanol Extract

[0080] Angelica gigas Nakai roots were washed with clean water and dried sufficiently. The dried Angelica gigas Nakai roots were crushed, 5 volume times (500 ml) of ethanol (90% (v/v)) was added to 100 g of the obtained powder, extracted at about 4060° C. for 4 hours or more, and then filtered through a 1 um (micrometer) filter, and the filtrate was heated and concentrated until it became 10 wt. % of the original weight. Crystalline cellulose was gradually added to the obtained concentrate, continuously concentrated, dried completely, and then powdered to prepare an Angelica gigas Nakai ethanol extract powder.

[0081] 1.2: Preparation of Broccoli Extract

[0082] The outpost of Brassica oleracea var. italica was washed with clean water and dried sufficiently. The dried Brassica oleracea var. italica was crushed, 5 volume times (500 ml) of ethanol (50% (v/v)) was added to 100 g of the obtained powder, and NaOH was added until the pH reached 8. Thereafter, the mixture was extracted three times at 40° C. for 3 hours or more, and then filtered through a 1 um (micrometer) filter, and the filtrate was heated and concentrated until it became 10 wt. % of the original weight. The obtained concentrate was continuously concentrated, dried completely, and then powdered to prepare a Brassica oleracea var. italica ethanol extract powder (hereinafter, referred to as BKE).

[0083] 1.3. Preparation of Test Animals

[0084] All animals used in the test (ICR mice and 3×-TG mice) were reared under the conditions of a constant light and dark cycle adding light by each 12 hours from 7 am to 7 pm, a temperature of 22° C. to 25° C., and a humidity of 60%. Water and food were freely ingested, and a general pellet-dried feed was used.

Example 2: Neuroprotective Activity Test

[0085] Beta-amyloid 1-42 (American Peptide, USA) and beta-amyloid 42-1 (Bachem, Switzerland) were dissolved at a concentration of 37 μl/μl in sterilized 0.1 M phosphate-buffered saline (pH 7.4), and stored until before using the preparative solution. On the other hand, 200 mg/kg of Angelica gigas Nakai ethanol extract (AGE) prepared in Example 1.1.1, 400 mg/kg of Brassica oleracea var. italica ethanol extract (BKE) prepared in Example 1.2, or a mixture of 200 mg/kg of the Angelica gigas Nakai ethanol extract (AGE) and 400 mg/kg of the Brassica oleracea var. italica ethanol extract were dissolved in 10 ml of distilled water, and then administered to the normal mice prepared in Example 1.3 (ICR mice; body weight 18-26 g, 5 mice per group) once daily for 4 weeks. For comparison, a group in which distilled water was administered in an amount of 10 ml instead of the above extract was prepared as a control group. In the bregma of mice to which the extract was administered for 4 weeks, injection was performed at a depth of 2.4 mm using a 50 μl Hamilton microsyringe equipped with a 26-gauge needle, and the above prepared beta-amyloid 1-42 and beta-amyloid 42-1 were each administered in an amount of 5 For the bregma of mice to which the above extract was administered for 4 weeks, injection was injected at a depth of 2.4 mm using a 50 μl Hamilton microsyringe equipped with a 26-gauge needle, and the prepared beta-amyloid .sub.1-42 and beta-amyloid .sub.42-1 were respectively administered in an amount of 5 The process of the test is schematically shown in FIG. 1.

[0086] For the mouse to which the extract was administered for 4 weeks (28 days) and beta-amyloid was administered as described above, on the third day after administration of beta-amyloid, the experimental animals were subjected to general anesthesia using a gas in which 3% (v/v) isoflurane (Baxtor, USA) was mixed with a mixed gas of nitrogen and oxygen in a ratio of 7:3. While maintaining the anesthetized condition of the experimental animal by using a gas in which 2.5% (v/v) isoflurane was mixed with a mixed gas of nitrogen and oxygen, surgery was performed on the experimental animals Thiophental sodium (Yanhan Corporation, Korea) was injected intraperitoneally at a dose of 30 mg each per 1 kg of body weight, anesthetized, and then a physiological saline (4° C.) containing 1,000 IU of heparin per 1,000 ml was injected into the left ventricle, and washed by perfusion. The perfused and washed animal was subjected to a perfusion fixation using 4% (w/v) paraformaldehyde (4° C.) in 0.1 M phosphate buffer; PB), pH 7.4).

[0087] The bone space of the head of the experimental animal for which perfusion fixation was completed was opened using a bone cutter, and the brain was removed. The removed brain of the experimental animals were then post fixed in a 4% paraformaldehyde (0.1 M phosphate buffer (PB), pH 7.4) solution (4° C.) for 4 hours using a stirrer at room temperature. The post fixed brain was placed in a 30% (w/v) sucrose solution (in 0.1 M phosphate buffer) and allowed to settle until it sinks on the bottom. Then, the brain tissue was cut to a thickness of 30 μm with a sliding microtome (Reichert-Jung, Germany) to make a tissue section. The tissue section was placed in a 6 well plate containing a storage solution and stored at 4° C. until staining was performed.

[0088] The tissue in which the hippocampal formation was well made in the prepared tissue section was selected. In order to eliminate the preservative solution adhering to the tissue section, the tissue was washed 3 times with 0.01M PBS (pH 7.4) by each 10 minutes. The washed tissue section was smeared on a gelatin-coated slide glass and sufficiently dried at 37° C. The tissue sections were immersed in distilled water for a while, and then immersed in a 2% (w/v) cresyl violet acetate (Sigma, USA) solution for 1 minute, and the tissue section was stained. Subsequently, the stained tissue section was sufficiently washed with running water to remove excess dye adhering to the slide, immersed in distilled water for a while, and then treated sequentially with 50% (v/v), 70% (v/v), 80% (v/v), 90% (v/v), 95% (v/v), and 100% (v/v) ethanol solutions, and dehydration and excess crease violet washing were performed. After confirming that the Nissl body was visible in the tissue section, it was immersed in xylene (Junsey, Japan) to make it transparent, and then encapsulated in Canada balsam (Kanto, Japan).

[0089] The respective tissues of the normal group (ICR mice; beta-amyloid-untreated), the control group (extract-untreated and beta-amyloid-treated) and the experimental group (extract-treated and beta-amyloid-treated) were enlarged 40 times the entire hippocampal region and 20 times the CA1 region with an Axio M1 microscope equipped with a digital camera (Axiocam, Cal Zeiss, Germany), and the respective tissue sections were photographed. The photographs of the entire hippocampal region are shown in FIG. 2. Further, after staining the hippocampal region measured in FIG. 2, the result of quantification using a microscope is shown in FIG. 3 as a relative value to the control group.

[0090] As shown in FIGS. 2 and 3, it was confirmed that the protective effect of the hippocampus region in the group treated with beta-amyloid after treatment with the extract was excellent as compared with the control group (extract-untreated and beta-amyloid-treated). In particular, in the group treated with Angelica gigas Nakai extract (AGE) and the group treated with Angelica gigas Nakai extract (AGE) and Brassica oleracea var. italica extract (BKE) together, an excellent hippocampal area protective effect similar to that of the normal group was observed. These results show that the Angelica gigas Nakai extract and the mixed extract of Angelica gigas Nakai and Brassica oleracea var. italica have a neuroprotective effect against nerve damage caused by neurotoxic substances. Furthermore, it shows that it has a preventive effect on neurodegenerative diseases caused by the neurotoxic substances.

Example 3: Nerve Regeneration Activity Test

[0091] Mice in which Alzheimer's disease was induced through genetic manipulation (3×Tg-AD, triple-transgenic mouse model of AD, 12 months old, hereinafter “3×-TG mice”; THE JACKSON LABORATORY (USA); female, 4 mice per group, weight about 40 g) was purchased and used. These mice gradually expressed Plaques and tangles, and showed deficiencies in learning ability and memory.

[0092] The prepared 12-month-old 3×-TG mice were orally administered for 6 weeks, wherein 200 mg/kg of Angelica gigas Nakai ethanol extract (AGE) prepared in Example 1.1.1 and/or 400 mg/kg of Brassica oleracea var. italica ethanol extract (BKE) prepared in Example 1.2 were dissolved in 10 ml of distilled water and administered alone or in combination 6 times a week for 6 weeks. For comparison, a group to which 10 ml of distilled water was administered instead of the extract was prepared as a control group. The same test was performed using B6129SF2/J mice (female; 4 mice, weight 40 g; THE JACKSON LABORATORY (USA)) as a normal group.

[0093] For immunohistochemical analysis, the mouse to which the extract was administered for 6 weeks was anesthetized, and subjected to intracardiac perfusion with 0.01M PBS and 4% (w/v) paraformaldehyde (PFA). Brain was removed and post fixed in 4% (w/v) PFA for 2 days, and cryopreserved in PBS containing 30% (w/v) sucrose for 2 days. Then, it was frozen on powdered dry ice and stored at −80° C. until use. Thereafter, changes in biomarkers related to nerve regeneration were confirmed by Western blot analysis as follows.

[0094] Specifically, the Western blot analysis was performed as follows:

[0095] The frozen and prepared brain tissues and cells were homogenized in a radioimmunoprecipitation analysis buffer (cell Signaling Technology, Beverly, MIA, USA), and the protein analysis reagents (Bio-Rad, Hercules, Calif., USA) were used to measure the concentration of biomarker proteins that could confirm the production of brain (nerve) cells. Protein was separated by electrophoresis on 10% (w/v) sodium dodecyl sulfate polyacrylamide gel, and electrophoretically transferred to a polyvinylidene fluoride membrane (Millipore, Billerica, Mass., USA). The membrane was blocked with 5% (v/v) non-fat dry milk or 5% (v/v) PBS, and cultured with a primary antibody against biomarkers such as beta-actin. After incubation with Horseradish-peroxidase-conjugated secondary antibody, the protein band was measured using an enhanced chemiluminescence detection kit (GE Healthcare, St. Giles, UK). The obtained results were quantified using ImageJ software (National Institutes of Health, Bethesda, Md., USA).

[0096] The biomarker proteins used in this Example and antibodies thereto are summarized in Table 1 below:

TABLE-US-00001 TABLE 1 Source of primary Secondary Biomarker antibody antibody BDNF (brain-derived Santa Cruz Horseradish- neurotrophic factor) peroxidase- MAP2 (microtubule- Millipore conjugated associated protein 2) antibody Synaptophysin Millipore (Sigma Aldrich) PSD-95 (postsynaptic Cell density protein-95) signaling TrkB (tyrosine kinase Cell receptor B) signaling CAMKII (Ca.sup.2+/calmodulin- Cell dependent protein kinase II)* signaling Phosphorylated-CAMKII Cell signaling ERK (extracellular Santa Cruz signal-regulated kinase)* Phosphorylated-ERK Santa Cruz Akt (RAC-alpha serine/ Cell threonine-protein kinase)* signaling Phosphorylated-Akt Cell signaling CREB (cAMP-responsive Cell element-binding protein)* signaling Phosphorylated-CREB Cell signaling beta-actin*

[0097] (in Table above, ‘*’ is reference marker) The results of electrophoresis results obtained from the biomarker proteins and the results of quantification thereof are shown in FIGS. 4 to 19.

[0098] As in FIGS. 4 to 19, in most cases of the extract-treated 3×-TG mice (‘3×-TG mice+AGE’, ‘3×-TG mice+BKE’, and ‘3×-TG mice+AGE+BKE’) ‘or’ Non-3×-TG mice’), the effect of generating nerve cells similar to that of the control group (extract-untreated 3×-TG mice; indicated as ‘3×-TG mice’) was confirmed. In particular, the group (‘3×-TG mice+BKE’) treated with Brassica oleracea var. italica extract (BKE) and the group (‘3×-TG mice+AGE+BKE’) treated with Angelica gigas Nakai extract (AGE) and Brassica oleracea var. italica extract (BKE) together were observed to have an excellent effect of generating nerve cells. Among them, the effect of generating nerve cells in ‘3×-TG mice+AGE+BKE’ was particularly excellent. These results show that the Angelica gigas Nakai extract, the Brassica oleracea var. italica extract, and the mixed extract of Angelica gigas Nakai and Brassica oleracea var. italica have a neurogenic (regeneration) effect on a neurological injury mouse model (e.g., Alzheimer's disease mouse model). Furthermore, it shows that it has a therapeutic effect on neurodegenerative diseases (e.g., Alzheimer's disease) having nerve damage.

Example 4: Analysis of Active Ingredients According to Extraction Conditions (HPLC)

[0099] 4.1. Preparation of Test Solution

[0100] The Angelica gigas Nakai 98% ethanol extract prepared in Example 1.1.1 (extract (1) of Table 2) and the Angelica gigas Nakai 90% ethanol extract prepared in Example 1.1.2 (extract (2) in Table 2) were prepared.

[0101] For comparison, referring to Example 1.1.1 above, six types of Angelica gigas Nakai extracts (extracts (2) to (8) in Table 2) were prepared by varying the types of extraction solvent (ethanol or water) and concentration (30%, 50%, 75%, 80%; w/v) under various temperature conditions.

[0102] 1 g of the prepared Angelica gigas Nakai extract was exactly taken, placed in a 50 ml volumetric flask, and then dissolved by adding about 30 ml methanol (100%), filled with methanol to a marked line, filtered, and used as a sample solution.

[0103] 4.2. Preparation of Standard Solution

[0104] 10 mg of decursinol standard product (purity 98% or more), 5 mg of decursinol standard product (purity 98% or more), and 5 mg of decursinol angelate standard product (purity 98% or more) were taken and put in a 25 ml flask, and dissolved by adding 100% methanol, filled with methanol to a marked line, filtered, and used as a sample solution. A standard solution having a concentration of 12.5-25-50-100-200 μg/ml was prepared from this standard solution and used for measuring the calibration curve.

[0105] 4.3. HPLC Operating Conditions

[0106] Testing was performed with the prepared sample solution and standard solution according to the operating conditions of the liquid chromatography below to calculate the content of decursinol, and decursinol angelate:

[0107] Column: Cadenza CW C18, (150*4.6 mm, 3 μm) or equivalent column;

[0108] Detector: Ultraviolet spectrophotometer (detection wavelength: 330 nm);

[0109] Flow rate: 0.7 ml/min;

[0110] Mobile phase: Water (A %), Acetonitrile (B %),

[0111] 0-5 min (20, B), 5-6 min (20.fwdarw.40, B), 6-22 min (40.fwdarw.55, B), [0112] 22-23 min (55.fwdarw.80, B), 23-25 min (80, B), 25-27 min (20, B);

[0113] Sample injection amount: 10 μl.

[0114] 4.4. Result

[0115] The ingredient analysis results of each of the obtained extracts are shown in Table 2 below.

TABLE-US-00002 TABLE 2 (1) (2) (3) (4) (5) (6) (7) (8) Extraction solvent 98% 90% 98% 80% 75% 50% 30% Water Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol Ethanol (hot water) Extraction temperature 40~60° C. 90° C. 40~60° C. — Total Decursin 3243 2911 1640 2351 2089 1560 1101 19 extracted Decursinol 1995 1724 790 1477 1327 1116 865 10 compounds angelate from 100 g Nodakenin 2982 2562 1332 2318 1913 1571 543 559 dried β-Sitosterol 324 269 85 212 145 88 35 13 Angelica gigas Nakai (mg)

[0116] As shown in Table 1 above, it can be confirmed that the ethanol extract of Angelica gigas Nakai of Examples 1.1.1 and 1.1.2 is remarkably higher in the content of useful compounds in the extract as compared with other extracts having different extraction conditions (extraction solvent concentration, type, extraction temperature, etc.).

Example 5: Neuroprotective Activity Test According to the Concentration of Extraction Solvent (Ethanol)

[0117] Among the Angelica gigas Nakai extracts obtained under various extraction conditions prepared in Example 4, the neuroprotective activity test was performed on 98% (w/v) ethanol extract (extract (1); Example 1.1.1 extract) and 90% (w/v) ethanol extract (extract (2); Example 1.1.2 extract), and 80% (w/v) ethanol extract (extract (4); equivalent to a conventional extract INM-176), 75% (w/v) ethanol extract (extract (5)), and 50% (w/v) ethanol extract (extract (6)). (Extraction temperature of the extract: 40-60° C.). Neuroprotective activity test was performed with reference to the test procedure of Example 2.

[0118] The result of CV (cresyl violet acetate) staining of the obtained hippocampal tissue is shown in FIG. 20 below, and the quantitative results of the stained hippocampal tissue is shown in FIG. 21.

[0119] As shown in FIGS. 20 and 21, it can be confirmed that 98% ethanol extract (extract (1)) and 90% ethanol extract (extract (2)) has excellent neuroprotective activity as compared with the extracts under conditions outside the concentration range of the extraction solvent,