COMPOSITION FOR PREVENTING OR TREATING NEURODEGENERATIVE DISEASES, CONTAINING DITERPENE-BASED COMPOUND

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating neurodegenerative diseases comprising a diterpene, or a pharmaceutically acceptable salt thereof. Specifically, the diterpene of the present invention can prevent or treat neurodegenerative diseases caused by inhibition of Nurr1 activity by activating Nurr1 and inhibiting the inflammatory response.

Claims

1. A method of treating neurodegenerative diseases comprising administering a pharmaceutical composition comprising a diterpene, or a pharmaceutically acceptable salt thereof as an active ingredient to a subject in need thereof, wherein the diterpene comprises at least one selected from the group comprising Formulas 1 to 16 below. ##STR00021## ##STR00022## ##STR00023## ##STR00024##

2. The method treatment of according to claim 1, wherein the diterpenes is one of the compounds represented by Formulas 2, 8, 13, 14 or 16.

3. The method according to claim 1, wherein the diterpene is the compound represented by Formula 2.

4. The method according to claim 1, wherein the diterpene is the compound represented by Formula 14.

5. The method according to claim 1, wherein the neurodegenerative diseases may be any one selected from the group consisting of Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), Fronto-Temporal Dementia, Cortico Basal Degeneration, and Progressive supranuclear palsy (PSP).

6. The method according to claim 1, wherein the neurodegenerative disease is Parkinson's disease (PD).

7. The method according to claim 1, wherein the diterpene is separated from the extract of flower of Daphne genkwa.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0078] FIGS. 1A-1D show that compounds 11 to 16 of the present invention inhibit the expression of inflammation-related factors in BV-2 cells, which are microglia cells;

[0079] FIG. 1A shows that the inhibition of IL-1b expression was confirmed by Western blotting.

[0080] FIG. 1B is a graph confirming that the inhibition of IL-1b mRNA expression through PCR.

[0081] FIG. 1C is a graph confirming the inhibition of IL-6 mRNA expression through PCR.

[0082] FIG. 1D is a graph confirming through the PCR that inhibit the mRNA expression of TNF-a.

[0083] FIG. 2 is a graph confirming the effect of the compounds 1 to 10 of the present invention on Nurr1 activity

DETAILED DESCRIPTION

[0084] Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1-a: Preparation of the Extract of Daphne Genkwa

[0085] 4.47 kg of dried flowers of Daphne genkwa were immersed in 40 L 80% ethanol for 72 hours, and filtered to obtain a liquid component. The obtained liquid component was concentrated under reduced pressure, and then 435 g of the extract of flower of Daphne genkwa was prepared.

Example 1-b

[0086] 4.47 kg of the stem and root of Daphne genkwa were chopped, and then immersed in 12 L 80% ethanol for 4 hours, filtered to separate the solids and a first liquid component. The separated solid was again immersed in 12 L 80% ethanol for 4 hours, and filtered to obtain a second liquid component. The first liquid component and the second liquid component were mixed, the mixture was concentrated under reduced pressure, and the residue was lyophilized to prepare 255.1 g of extract of Daphne genkwa.

Example 2: Separation of Active Ingredient by Various Solvents from the Extract of Daphne Genkwa

[0087] The extract of the flower of Daphne genkwa obtained in Example 1-a was sequentially fractionated with 2 L of distilled water and 2 L of hexane, chloroform, ethyl acetate, and butanol, respectively. The chloroform layer was concentrated under reduced pressure, and the chloroform fraction (17.6 g) was eluted with a gradient mixed solvent (100:0, 50:1, 20:1, 10:1, 5:1, 2:1, 1:1) of chloroform and methanol with silica gel column chromatography to obtain a total of 3 fractions (Fr. C1, C2, C3). Fr. C2 (4.5 g) was subjected to ODS silica gel chromatography with the gradient mixed solvent (60:40, 80:20, 100:0) of methanol and water to obtain 5 sub-fractions (Fr. C21, C22, C23, C24, C25). Fr. C23 (300 mg) was again subjected to silica gel (40-63 μm; 4 g flash column) MPLC on a gradient mixed solvent of chloroform and acetone (99:1-95:5) to obtain 3 sub-fractions (Fr. C231, C232, C233). Fr. C233 (140 mg) was finally subjected to ODS HPLC with 55% acetonitrile at a flow rate of 5 mL/min to obtain yuanhuafine (25.5 mg, compound of Formula 1) in the form of a white powder. The structure of the compound was identified based on the following NMR, MS, and [α].sup.20.sub.D data.

##STR00005##

[0088] [α].sup.20.sub.D+29.3 (c 0.5, CHCl.sub.3);

[0089] ESI-MS, m/z 573.9 [M+Na].sup.+;

[0090] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 7.68 (dd, J=7.3, 1.8, 2H, H-3′, H-7′), 7.57 (s, 1H, H-1′), 7.41-7.37 (m, 3H, H-4′, H-5′, H-6′), 5.08 (br, 1H, H-12), 5.05 (s, 1H, H-16a), 5.02 (d, J=2.4, 1H, H-16b), 4.98 (br, 1H, H-14), 4.14 (s, 1H, H-5), 4.05 (d, J=12.3, 1H, H-20a), 3.96 (m, 1H, H-10), 3.65 (dd, J=7.3, 4.9, 1H, H-20b), 3.59 (s, 1H, H-8), 2.54 (q, J=7.3, 1H, H-11), 2.00 (s, 3H, H-2″), 1.86 (s, 3H, H-17), 1.76 (d, J=1.1, 3H, H-19), 1.32 (d, J=7.3, 3H, H-18);

[0091] .sup.13C NMR values in ppm (CD.sub.3OD, 101 Hz): δ.sub.C 209.8 (C-3), 171.6 (C-1″), 160.0 (C-1), 145.0 (C-15), 138.3 (C-2), 137.2 (C-2′), 130.5 (C-5′), 128.9 (C-4′, C-6′), 127.2 (C-3′, C-7′), 119.1 (C-1′), 113.9 (C-16), 85.4 (C-13), 82.1 (C-14), 80.3 (C-9), 79.8 (C-12), 74.5 (C-4), 71.3 (C-5), 65.1 (C-20), 64.7 (C-7), 63.2 (C-6), 48.9 (C-10), 45.1 (C-11), 36.6 (C-8), 21.0 (C-2″), 19.1 (C-18), 18.7 (C-17), 10.0 (C-19).

[0092] Fr. C24 (210 mg) was again subjected to Sephadex LH-20 column chromatography with a mixed solvent of chloroform and methanol (1:1), and was finally subjected to ODS HPLC with a 65% acetonitrile at a flow rate of 5 mL/min to obtain genkwadaphnine (25.0 mg, compound of Formula 2) in the form of a white powder. The structure of the compound was identified based on the following NMR, MS, and [ ].sup.20.sub.D data.

##STR00006##

[0093] [α].sup.20.sub.D+56.7 (c 0.1, CHCl.sub.3);

[0094] ESI-MS, m/z 625.5 [M+Na].sup.+;

[0095] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz); δ.sub.H 7.98 (d, J=7.5, 2H, H-3″, H-7″), 7.72 (m, 2H, H-3′, H-7′), 7.61 (m, 1H, H-5″), 7.59 (s, 1H, H-1), 7.48 (t, J=7.7, 2H, H-4″, H-6″), 7.40 (m, 3H, H-4′, H-5′, H-6′), 5.26 (br, 1H, H-12), 5.21 (d, J=2.4, 1H, H-14), 5.13 (s, 1H, H-16a), 5.02 (s, 1H, H-16b), 4.13 (s, 1H, H-5), 4.06 (d, J=12.3, 1H, H-20a), 3.99 (m, 1H, H-10), 3.76 (d, J=2.3, 1H, H-8), 3.68 (s, 1H, H-7), 3.66 (d, J=12.5, 1H, H-20b), 2.69 (q, J=7.3, 1H, H-11), 1.90 (s, 3H, H-17), 1.75 (s, 3H, H-19), 1.42 (d, J=7.3, 3H, H-18);

[0096] .sup.13C NMR values in ppm (CD.sub.3OD, 101 Hz): δ.sub.C 209.8 (C-3), 166.9 (C-1″), 160.0 (C-1), 144.9 (C-15), 138.2 (C-2), 137.2 (C-2′), 134.5 (C-5″), 131.1 (C-2″), 130.7 (C-3″, C-7″), 130.5 (C-5′), 129.7 (C-4″, C-6″), 128.9 (C-4′, C-6′), 127.2 (C-3′, C-7′), 119.1 (C-1′), 114.1 (C-16), 85.7 (C-13), 82.0 (C-14), 80.4 (C-9), 80.2 (C-12), 74.4 (C-4), 71.4 (C-5), 65.2 (C-20), 64.8 (C-7), 63.2 (C-6), 49.9 (C-10), 45.3 (C-11), 37.0 (C-8), 19.1 (C-17), 18.9 (C-18), 10.0 (C-19).

[0097] Fr. C25 (130 mg) was again subjected to Sephadex LH-20 column chromatography with a mixed solvent of chloroform and methanol (1:1), and was finally subjected to ODS HPLC with a 70% acetonitrile at a flow rate of 5 mL/min to obtain genkwanine H (4.0 mg, compound of Formula 3) and genkwanine M (4.0 mg, compound of Formula 4) in the form of a white powder. The structure of the compound was identified based on the following NMR, MS, and [α].sup.20.sub.D data.

##STR00007##

[0098] [α].sup.20.sub.D+46.2 (c 1.5, CHCl.sub.3);

[0099] ESI-MS, m/z 631.6 [M+Na].sup.+;

[0100] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 8.09 (d, J=7.4, 2H, H-3″, H-7″), 7.67 (d, J=9.5, 2H, H-3′, H-7′), 7.60 (t, J=7.4, 1H, H-5″), 7.49 (t, J=7.6, 2H, H-4″, H-6″), 7.36 (m, 3H, H-4′, H-5′, H-6′), 5.07 (s, 1H, H-16a), 4.91 (s, 1H, H-16 b), 4.78 (d, J=11.0, 1H, H-20a), 4.62 (d, J=2.5, 1H, H-14), 4.61 (s, 1H, H-7), 4.51 (d, J=11.1, 1H, H-20 b), 4.16 (d, J=4.1, 1H, H-2), 3.41 (s, 1H, H-5), 2.74 (m, 2H, H-10, H-11), 2.67 (d, J=2.3, 1H, H-8), 2.34 (dd, J=14.1, 8.0, 1H, H-12a), 1.84 (s, 3H, H-17), 1.81-1.65 (m, 4H, H-1, H-2, H-12 b), 1.31 (d, J=6.9, 3H, H-18), 1.06 (d, J=6.0, 3H, H-19);

[0101] .sup.1H NMR values in ppm (CD.sub.3OD, 176 Hz): δ.sub.C 168.4 (C-1″), 148.3 (C-15), 137.9 (C-2′), 134.4 (C-5″), 131.8 (C-2″), 130.8 (C-3″, C-7″), 130.5 (C-5′), 129.7 (C-3′, C-7′), 129.1 (C-4″, C-6″), 127.2 (C-4′, C-6′), 118.6 (C-1′), 111.5 (C-16), 86.9 (C-13), 86.6 (C-14), 85.6 (C-9), 82.7 (C-4), 78.9 (C-3), 77.6 (C-6), 77.2 (C-7), 74.8 (C-5), 68.9 (C-20), 52.9 (C-10), 38.4 (C-8), 37.5 (C-12), 37.4 (C-2), 36.5 (C-11), 36.1 (C-1), 21.5 (C-18), 19.6 (C-17), 13.8 (C-19).

##STR00008##

[0102] [α].sup.20.sub.D−8.4 (c 0.05, MeOH);

[0103] ESI-MS, m/z 613.5 [M+Na].sup.+;

[0104] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 8.07 (d, J=7.3, 2H, H-3″, H-7″), 7.69 (d, J=9.5, 2H, H-3′, H-7′), 7.61 (t, J=7.4, 1H, H-5″), 7.49 (t, J=7.7, 2H, H-4″, H-6″), 7.35 (m, 3H, H-4′, H-5′, H-6′), 5.07 (s, 1H, H-16a), 5.06 (d, J=12.0, 1H, H-20a), 4.91 (s, 1H, H-16 b), 4.63 (d, J=2.6, 1H, H-14), 4.05 (d, J=11.8, 1H, H-20 b), 3.83 (s, 1H, H-3), 3.78 (s, 1H, H-5), 3.53 (s, 1H, H-7), 3.18 (d, J=2.6, 1H, H-8), 2.67 (t, J=9.0, 1H, H-10), 2.48 (m, 1H, H-11), 2.31 (dd, J=14.1, 8.0, 1H, H-12a), 1.84 (s, 3H, H-17), 1.79-1.55 (m, 4H, H-1, H-2, H-12 b), 1.27 (d, J=6.8, 3H, H-18), 1.04 (d, J=5.2, 3H, H-19);

[0105] .sup.13C NMR values in ppm (CD.sub.3OD, 176 Hz): δ.sub.C 168.0 (C-1″), 148.5 (C-15), 138.2 (C-2′), 134.4 (C-5″), 131.6 (C-2″), 130.8 (C-3″, C-7″), 130.3 (C-5′), 129.74 (C-4″, C-6″), 128.9 (C-3′, C-7′), 127.4 (C-4′, C-6′), 118.8 (C-1′), 111.4 (C-16), 86.1 (C-13), 84.0 (C-14), 82.2 (C-4), 81.1 (C-9), 78.8 (C-3), 73.4 (C-5), 69.3 (C-20), 65.8 (C-7), 62.0 (C-6), 50.3 (C-10), 38.4 (C-2), 38.0 (C-8), 37.2 (C-12), 36.6 (C-11), 36.1 (C-1), 21.6 (C-18), 19.6 (C-17), 13.7 (C-19).

[0106] Fr. C3 (4.0 g) was subjected to ODS silica gel chromatography with the gradient mixed solvent (60:40, 80:20, 100:0) of methanol and water to obtain 4 sub-fractions (Fr. C31, C32, C33, C34). Fr. C34 (1.06 g) was subjected to Sephadex LH-20 column chromatography with methanol to obtain 3 sub-fractions (Fr. C341, C342, C343). Fr. C341 (120 mg), C342 (180 mg), and C343 (119 mg) were finally subjected to ODS HPLC with 50% acetonitrile at a flow rate of 4.5 mL/min, respectively, to obtain genkwanin K (9.6 mg, compound of Formula 5) from Fr. C341, yuanhuapine (79.5 mg, compound of Formula 6) from Fr. C342,

[0107] genkwanin A (4.7 mg, compound of Formula 7), orthobenzoate 2 (85.6 mg, compound of Formula 8), 1, 2α-dihydrodaphnetoxin (2.9 mg, compound of Formula 9) and genkwanin I (2.4 mg, Formula 10 Compound) from Fr. C343 in the form of a white powder. The structure of the compound was identified based on the following NMR, MS, and [α].sup.20.sub.D data.

##STR00009##

[0108] [α].sup.20.sub.D+38.5 (c 0.1, CHCl.sub.3);

[0109] ESI-MS, m/z 627.6 [M+Na].sup.+;

[0110] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 7.98 (d, J=7.5, 2H, H-3′, H-7′), 7.90 (d, J=7.5, 2H, H-3″, H-7″), 7.57 (m, 2H, H-5′, H-5″), 7.44 (d, J=7.7, 2H, H-4″, H-6″), 7.42 (d, J=7.6, 2H, H-4′, H-6′), 5.72 (d, J=6.2, 1H, H-14), 5.06 (s, 1H, H-16a), 4.84 (s, 1H, H-16 b), 4.62 (d, J=13.4, 1H, H-20a), 4.42 (d, J=3.0, 1H, H-3), 4.24 (d, J=5.15, 1H, H-20b), 4.19, (d, J=2.4, H-7), 3.33 (s, 1H, H-5), 3.15 (d, J=6.2, 1H, H-8), 2.34 (m, 2H, H-10, H-11), 2.01 (m, 3H, H-2, H-1a, H-12a), 1.81 (s, 3H, H-17), 1.60 (m, 2H, H-1b, H-12b), 1.24 (d, J=7.1, 3H, H-18), 1.08 (d, J=6.5, 3H, H-19);

[0111] .sup.1H NMR values in ppm (CDCl.sub.3, 101 Hz): δ.sub.C 168.3 (C-1″), 166.7 (C-1′), 148.1 (C-15), 133.6 (C-5″), 133.5 (C-5″), 130.2 (C-3″, C-7″), 130.1 (C-3′, C-7′), 129.9 (C-2″), 129.4 (C-2′), 128.7 (C-4″, C-6″), 128.6 (C-4′, C-6′), 111.6 (C-16), 83.8 (C-4), 77.9 (C-9), 77.4 (C-6), 77.3 (C-14), 76.0 (C-7), 75.5 (C-3), 74.5 (C-13), 73.2 (C-5), 67.4 (C-20), 54.6 (C-10), 40.6 (C-8), 37.9 (C-12), 36.6 (C-2), 34.9 (C-1), 34.4 (C-11), 19.5 (C-18), 19.0 (C-17), 13.5 (C-19).

##STR00010##

[0112] [α].sup.20.sub.D+28.5 (c 0.05, CHCl.sub.3);

[0113] ESI-MS, m/z 565.5 [M+Na].sup.+;

[0114] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 7.67 (m, 2H, H-3′, H-7′), 7.37 (m, 3H, H-4′, H-5′, H-6′), 5.07 (s, 1H, H-16a), 5.07 (d, J=2.9, 1H, H-12), 5.04 (s, 1H, H-16 b), 4.95 (d, J=2.7, 1H, H-14), 4.02 (s, 1H, H-5), 4.02 (d, J=12.1, 1H, H-20a), 3.61 (d, J=2.9, 1H, H-8), 3.60 (d, J=10.6, 1H, H-20 b), 3.54 (s, 1H, H-7), 3.18 (dd, J=13.3, 5.8, 1H, H-10), 2.47 (q, J=6.9, 1H, H-11), 2.38 (dt, J=13.3, 6.8, 1H, H-1a), 2.25 (dt, J=13.0, 6.6, 1H, H-1b), 2.01 (s, 3H, H-2″), 1.86 (s, 3H, H-17), 1.46 (d, J=13.1, 1H, H-2), 1.37 (d, J=7.0, 3H, H-18), 1.07 (d, J=6.5, 3H, H-19);

[0115] .sup.13C NMR values in ppm (CD.sub.3OD, 101 Hz): δ.sub.C 218.9 (C-3), 171.6 (C-1″), 145.0 (C-15), 137.4 (C-2′), 130.4 (C-5′), 128.8 (C-4′, C-6′), 127.1 (C-3′, C-5′), 119.3 (C-1′), 113.7 (C-16), 84.8 (C-13), 82.3 (C-14), 80.6 (C-9), 79.2 (C-12), 77.0 (C-4), 70.5 (C-5), 65.3 (C-20), 64.6 (C-7), 63.3 (C-6), 44.9 (C-11), 44.8 (C-10), 43.7 (C-1), 36.8 (C-8), 34.5 (C-2), 21.1 (C-2″), 19.1 (C-18), 19.0 (C-17), 12.8 (C-19).

##STR00011##

[0116] [α].sup.20.sub.D+47.7 (c 0.1, CHCl.sub.3);

[0117] ESI-MS, m/z 505.6 [M+H].sup.+ and m/z 527.4 [M+Na].sup.+:

[0118] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 7.66 (dd, J=6.6, 3.0, 2H, H-3′, H-7′), 7.38 (m, 3H, H-4′, H-5′, H-6′), 5.07 (d, J=6.5, 1H, H-16a), 4.91 (s, 1H, H-16 b), 4.60 (d, J=2.5, 1H, H-14), 4.42 (s, 1H, H-7), 4.13 (d, J=4.6, 1H, H-3), 3.82 (q, J=11.0, 2H, H-20), 3.38 (s, 1H, H-5), 2.71 (m, 2H, H-10, H-11), 2.62 (d, J=2.4, 1H, H-8), 2.32 (dd, J=14.1, 8.0, 1H, H-12a), 1.87 (m, 2H, H-1a, H-2), 1.83 (s, 3H, H-17), 1.69 (m, 1H, H-1b), 1.62 (m, 2H, H-12b), 1.30 (d, J=6.9, 3H, H-18), 1.05 (d, J=6.2, 3H, H-19);

[0119] .sup.13C NMR values in ppm (CD.sub.3OD, 101 Hz): δ.sub.C 148.3 (C-15), 137.9 (C-15), 130.6 (C-5′), 129.1 (C-4′, C-6′), 127.3 (C-3′, C-5′), 118.6 (C-1′), 111.5 (C-16), 86.8 (C-13), 86.5 (C-14), 85.4 (C-9), 82.8 (C-4), 78.5 (C-7), 78.3 (C-6), 77.5 (C-3), 75.0 (C-5), 67.6 (C-20), 52.9 (C-10), 38.5 (C-2), 37.5 (C-8), 37.4 (C-12), 36.5 (C-11), 36.0 (C-1), 21.5 (C-17), 19.8 (C-18), 13.8 (C-19).

##STR00012##

[0120] [α].sup.20.sub.D−16.6 (c 0.05, MeOH);

[0121] ESI-MS, m/z 509.2 [M+Na].sup.+;

[0122] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 7.69 (dd, J=6.6, 2.9, 2H, H-3′, H-7′), 7.35 (m, 3H, H-4′, H-5′, H-6′), 5.06 (s, 1H, H-16a), 4.90 (s, 1H, H-16b), 4.56 (d, J=2.6, 1H, H-14), 4.02 (d, J=12.2, 1H, H-20a), 3.76 (d, J=2.6, 1H, H-3), 3.70 (s, 1H, H-5), 3.59 (d, J=12.3, 1H, H-20 b), 3.42 (m, 1H, H-7), 3.07 (d, J=2.6, 1H, H-8), 2.67 (dd, J=12.6, 5.9, 1H, H-10), 2.47 (p, J=6.9, 1H, H-11), 2.28 (dd, J=13.9, 7.9, 1H, H-12a), 1.82 (s, 3H, H-17), 1.69 (d, J=14.0, 1H, H-1a), 1.64 (dd, J=9.5, 4.8, 1H, H-2), 1.58 (m, 2H, H-1b, H-12 b), 1.27 (d, J=6.8, 3H, H-18), 1.03 (d, J=5.3, 3H, H-19);

[0123] .sup.13C NMR values in ppm (CD.sub.3OD, 101 Hz): SC 148.3 (C-15), 138.1 (C-2′), 130.1 (C-5′), 128.7 (C-4′, C-6′), 127.2 (C-3′, C-7′), 118.5 (C-1′), 111.2 (C-16), 85.8 (C-13), 84.0 (C-14), 82.1 (C-9), 81.0 (C-2), 78.6 (C-4), 73.7 (C-5), 65.7 (C-20), 64.9 (C-7), 63.2 (C-6), 50.0 (C-10), 38.2 (C-2), 37.8 (C-8), 37.0 (C-12), 36.5 (C-11), 35.9 (C-1), 21.4 (C-17), 19.5 (C-18), 13.6 (C-19).

##STR00013##

[0124] [α].sup.20.sub.D+6.2 (c 0.001, MeOH);

[0125] ESI-MS, m/z 507.3 [M+Na].sup.+;

[0126] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 7.72 (m, 2H, H-3′, H-7′), 7.36 (m, 3H, H-4′, H-5′, H-6′), 5.07 (s, 1H, H-16a), 4.91 (s, 1H, H-16b), 4.60 (d, J=2.8, 1H, H-14), 4.01 (s, 1H, H-5), 3.99 (d, J=12.2, 1H, H-20a), 3.57 (d, J=12.2, 1H, H-20 b), 3.44 (s, 1H, H-7), 3.16 (dd, J=13.3, 5.8, 1H, H-10), 3.01 (d, J=2.7, 1H, H-8), 2.55 (m, 1H, H-11), 2.38 (m, 1H, H-12a), 2.29 (m, 2H, H-1), 1.83 (s, 3H, H-17), 1.73 (d, J=14.0, 1H, H-2), 1.53 (m, 1H, H-12 b), 1.30 (d, J=6.8, 3H, H-18), 1.08 (d, J=6.6, 3H, H-19);

[0127] .sup.13C NMR values in ppm (CD.sub.3OD, 101 Hz): δ.sub.C 219.3 (C-3), 148.3 (C-15), 138.1 (C-2′), 130.4 (C-5′), 128.9 (C-4′, C-6′), 127.4 (C-3′, C-5′), 118.8 (C-1′), 111.5 (C-16), 86.0 (C-13), 84.1 (C-14), 82.0 (C-9), 77.2 (C-4), 70.9 (C-5), 65.5 (C-20), 64.9 (C-7), 63.3 (C-6), 45.8 (C-10), 43.9 (C-8), 37.9 (C-1), 37.2 (C-2), 36.7 (C-12), 34.7 (C-11), 21.6 (C-17), 19.6 (C-18), 12.9 (C-19).

##STR00014##

[0128] [α].sup.20.sub.D−22.8 (c 0.005, MeOH);

[0129] ESI-MS, m/z 513.3 [M+H].sup.+ and m/z 535.3 [M+Na].sup.+;

[0130] .sup.1H NMR values in ppm (CD.sub.3OD, 400 MHz): δ.sub.H 7.69 (dd, J=7.5, 1.9, 2H, H-3′, H-7′), 7.36 (m, 3H, H-4′, H-5′, H-6′), 5.09 (s, 1H, H-16a), 4.93 (s, 1H, H-16b), 4.72 (d, J=4.5, 1H, H-3), 4.64 (d, J=2.8, 1H, H-14), 4.10 (s, 1H, H-5), 4.09 (d, J=12.3, 1H, H-20a), 3.53 (d, J=12.4, 1H, H-20 b), 3.49 (s, 1H, H-7), 2.93 (dd, J=13.3, 6.1, 1H, H-10), 2.78 (d, J=2.8, 1H, H-8), 2.35 (dd, J=14.1, 7.8, 1H, H-12a), 2.12 (m, 1H, H-11), 2.04 (dd, J=11.7, 6.0, 1H, H-1a), 1.95 (dd, J=12.0, 6.6, 1H, H-2), 1.84 (s, 3H, H-17), 1.82 (d, J=14.9, 1H, H-12 b), 1.58 (d, J=12.6, 1H, H-1b), 1.31 (d, J=6.7, 3H, H-18), 1.13 (d, J=6.7, 3H, H-19);

[0131] .sup.13C NMR values in ppm (CD.sub.3OD, 101 Hz): δ.sub.C 156.7 (C-21), 148.0 (C-15), 137.8 (C-2′), 130.5 (C-5′), 128.9 (C-4′, C-6′), 127.4 (C-3′, C-7′), 118.8 (C-1′), 111.8 (C-16), 94.1 (C-4), 90.4 (C-3), 85.8 (C-13), 84.2 (C-14), 81.4 (C-9), 71.1 (C-5), 65.0 (C-20), 64.7 (C-7), 62.8 (C-6), 50.7 (C-10), 37.9 (C-2), 37.4 (C-11), 37.3 (C-12), 37.1 (C-8), 36.2 (C-1), 21.6 (C-18), 19.5 (C-17), 12.9 (C-19).

Example 2-b: Separation of Active Ingredient by Various Solvents from Daphne Genkwa

[0132] The extract of Daphne genkwa obtained in Example 1 was dissolved in a 1:1 mixed solvent of 200 mL of distilled water and hexane and fractionated to obtain a hexane layer. The hexane layer obtained by performing the same method twice more was concentrated under reduced pressure to obtain a hexane fraction. The obtained hexane fraction (20 g) is eluted with a gradient mixed solvent (10:1, 5:1, 2:1, 1:1, 1:2) of hexane and ethyl acetate with silica gel column chromatography to obtain a total of 3 fractions (Fr. I, II, III).

[0133] Fr. I (577 mg) was subjected to reverse phase silica gel prep TLC (75% acetonitrile) to obtain an active band. The active band was subjected to ODS HPLC with 83% acetonitrile at a flow rate of 3 mL/min, and acutilonine F (14.0 mg, compound of Formula 11) and wikstroemia factor M1 (7.0 mg, compound of Formula 12) were obtained in the form of a white powder at retention times of 15.2 minutes and 18.5 minutes, respectively. The structure of the compound was identified based on the following NMR, MS, and [α].sup.20.sub.D data.

##STR00015##

[0134] [α].sup.20.sub.D−32.1 (c 1.3, MeOH);

[0135] ESI-MS, m/z 635.6 [M+H].sup.+, 657.7 [M+Na].sup.+;

[0136] .sup.1H NMR (CD.sub.3OD, 500 MHz): δ.sub.H, 7.70 (2H, dd, J=7.35, 2.22 Hz, H-3′, H-7′), 7.37 (1H, m, H-5′), 7.36 (2H, m, H-4′, H-6′), 7.35 (1H, m, H-3″), 6.64 (1H, dd, J=14.82, 10.75 Hz, H-5″), 6.34 (1H, dd, J=14.79, 11.40 Hz, H-4″), 6.22 (1H, dd, J=15.16, 10.69 Hz, H-6″), 6.01 (1H, m, H-7″), 6.00 (1H, m, H-2″), 5.06 (1H, brs, H-16a), 5.03 (1H, d, J=4.52 Hz, H-3), 4.90 (1H, brs, H-16 b), 4.56 (1H, d, J=2.66 Hz, H-14), 3.98 (1H, d, J=12.19 Hz, H-20a), 3.92 (1H, s, H-5), 3.59 (1H, d, J=12.20 Hz, H-20 b), 3.41 (1H, brs, H-7), 3.07 (1H, d, J=2.69 Hz, H-8), 2.83 (1H, dd, J=13.03, 5.32 Hz, H-10), 2.59 (1H, m, H-11), 2.29 (2H, m, H-12), 2.15 (2H, m, H-8″), 1.82 (3H, s, H-17), 1.78 (1H, m, H-2), 1.71 (2H, m, H-1), 1.47 (2H, m, H-9″), 1.31 (3H, d, J=6.89 Hz, H-18), 0.99 (3H, d, J=5.77 Hz, H-19), 0.94 (3H, t, J=7.39 Hz, H-10″);

[0137] .sup.13C NMR values in ppm (CD.sub.3OD, 126 Hz): δ.sub.C 169.4 (C, C-1″), 148.5 (C, C-15), 147.2 (CH, C-3″), 143.3 (CH, C-5″), 141.9 (CH, C-7″), 138.2 (C, C-2′), 131.6 (CH, C-6″), 130.3 (CH, C-5′), 129.2 (CH, C-4″), 128.9 (CH, C-4′, C-6′), 127.4 (CH, C-3′, C-7′), 120.7 (CH, C-2″), 118.7 (C, C-1′), 111.4 (CH.sub.2, C-16), 86.1 (C, C-13), 84.0 (CH, C-14), 82.9 (C, C-4), 82.3 (CH, C-3), 82.1 (C, C-9), 74.1 (CH, C-5), 66.1 (CH.sub.2, C-20), 65.1 (CH, C-7), 63.0 (C, C-6), 37.9 (CH, C-8), 37.7 (CH, C-2), 37.3 (CH.sub.2, C-1), 37.1 (CH.sub.2, C-12), 36.5 (CH, C-11), 36.2 (CH.sub.2, C-8″), 23.4 (CH.sub.2, C-9″), 21.6 (CH.sub.3, C-18), 19.6 (CH.sub.3, C-17), 14.1 (CH.sub.3, C-10″), 13.8 (CH.sub.3, C-19).

##STR00016##

[0138] [α].sup.20.sub.D+18.9 (c 1.0, MeOH);

[0139] ESI-MS, m/z 637.6 [M+H].sup.+, 659.4 [M+Na].sup.+, 635.2 [M−H].sup.−;

[0140] .sup.1H NMR (CDCl.sub.3, 500 MHz): δ.sub.H 7.75 (2H, m, H-3′, H-7′), 7.36 (3H, m, H-4′, H-5′, H-6′), 7.34 (1H, dd, J=15.4 and 10.2, H-3″), 6.21 (1H, dd, J=14.8 and 10.4, H-4″), 5.90 (1H, d, J=15.2, H-2″), 5.05 (1H, brs, H-16a), 4.92 (1H, brs, H-16b), 4.69 (1H, d, J=5.19, H-3), 4.51 (1H, d, J=2.76, H-14), 4.06 (1H, s, H-5), 3.88 (1H, d, J=12.2, H-20a), 3.77 (1H, d, J=12.2, H-20b), 3.44 (1H, s, H-7), 2.96 (1H, d, J=2.8, H-8), 2.82 (1H, dd, J=13.2, 5.5, H-10), 2.48 (1H, m, H-11), 2.20 (2H, overlapped, H-6″), 2.20 (1H, overlapped, H-12a), 1.93 (1H, m, H-1a), 1.83 (3H, s, H-17), 1.78 (1H, m, H-12b), 1.73 (1H, m, H-1b), 1.71 (1H, m, H-2), 1.45 (2H, m, H-7″), 1.33 (3H, d, J=6.9, H-18), 1.32 (2H, overlapped, H-9″), 1.31 (2H, overlapped, H-8″), 1.06 (3H, d, J=6.5, H-19), 0.91 (3H, t, J=6.9, H-10″);

[0141] .sup.13C NMR (CDCl.sub.3, 126 Hz): δ.sub.C 169.6 (C, C-1″), 147.4 (CH, C-3″), 146.8 (CH, C-5″), 146.7 (C, C-15), 136.4 (C, C-2′), 129.4 (CH, C-5′), 128.4 (CH, C-4″), 128.2 (CH, C-4′, C-6′), 126.3 (CH, C-3′, C-7′), 118.0 (CH, C-2″), 117.6 (C, C-1′), 111.4 (CH.sub.2, C-16), 84.5 (C, C-13), 82.7 (CH, C-14), 82.2 (CH, C-3), 81.7 (C, C-4), 80.5 (C, C-9), 75.0 (CH, C-5), 66.3 (CH.sub.2, C-20), 64.2 (CH, C-7), 60.6 (C, C-6), 48.9 (CH, C-10), 36.6 (CH, C-8), 36.4 (CH, C-2), 36.3 (CH.sub.2, C-12), 36.0 (CH.sub.2, C-1), 35.5 (CH, C-11), 33.3 (CH.sub.2, C-6″), 31.6 (CH.sub.2, C-8″), 28.5 (CH.sub.2, C-7″), 22.7 (CH.sub.2, C-9″), 21.1 (CH.sub.3, C-18), 19.4 (CH.sub.3, C-17), 14.2 (CH.sub.3, C-10″), 13.3 (CH.sub.3, C-19).

[0142] Also, Fr. III (400 mg) was subjected to reverse phase silica gel prep TLC (75% acetonitrile) to obtain an active band. The active band was subjected to normal phase silica gel prep TLC (TLC) under CHCl3-MeOH (50:1) conditions to obtain Fr.III-1 and Fr.III-2 as 2 sub-fractions at Rf 0.4 and 0.25, respectively. Fr.III-1 was subjected to ODS HPLC with a 65% acetonitrile at a flow rate of 3 mL/min to obtain prostratin Q (2.1 mg, compound of Formula 13) and yuanhuadine (4.0 mg, compound of Formula 14) in the form of a white powder at retention times of 17.2 min and 23.4 min, respectively. Fr.III-2 was subjected to ODS HPLC in the same manner as Fr.III-1, and to obtain yuanhuatine (4.4 mg, compound of Formula 15) and 12-0n-deca-2,4,6-trienoyl-phorbol-(13)-acetate (1.8 mg, compound of Formula 16) in the form of a white powder at retention times of 19.0 minutes and 21.4 minutes, respectively. The structure of the compound was identified based on the following NMR, MS, and [ ].sup.20.sub.D data.

##STR00017##

[0143] [α].sup.20.sub.D+14.1 (c 0.03, MeOH);

[0144] ESI-MS, m/z 579.5 [M+Na].sup.+;

[0145] .sup.1HNM (CDCl.sub.3, 500 MHz): δ.sub.H 7.60 (1H, s, H-1), 7.24 (1H, m, H-3″), 6.19 (1H, m, H-4″), 6.15 (1H, m, H-5″), 5.79 (1H, d, J=15.41 Hz, H-2″), 5.70 (1H, d, J=4.71 Hz, H-7), 5.47 (1H, d, J=10.27 Hz, H-12), 4.03 (2H, q, J=13.04 Hz, H-20), 3.26 (2H, overlapped, H-8, H-10), 2.53 (2H, m, H-5), 2.17 (2H, overlapped, H-6″), 2.17 (1H, overlapped, H-11), 2.11 (3H, s, H-2′), 1.78 (3H, s, H-19), 1.44 (2H, m, H-7″), 1.32 (2H, m, H-9″), 1.31 (2H, m, H-8″), 1.27 (3H, s, H-16), 1.22 (3H, s, H-17), 1.10 (1H, d, J=5.20 Hz, H-14), 0.91 (3H, d, J=6.93 Hz, H-18), 0.90 (3H, t, J=7.06 Hz, H-10″);

[0146] .sup.13C NMR values in ppm (CDCl.sub.3, 126 Hz); δ.sub.C209.1 (C, C-3), 174.1 (C, C-1′), 167.3 (C, C-1″), 161.0 (CH, C-1), 145.8 (CH, C-3″), 145.5 (CH, C-5″), 140.7 (C, C-6), 133.1 (C, C-2), 129.5 (CH, C-7), 128.5 (CH, C-4″), 119.1 (CH, C-2″), 78.4 (C, C-9), 74.0 (C, C-4), 68.2 (CH.sub.2, C-20), 65.9 (C, C-13), 56.4 (CH, C-10), 43.4 (CH, C-11), 39.3 (CH, C-8), 38.9 (CH.sub.2, C-5), 36.6 (CH, C-14), 33.2 (CH.sub.2, C-6″), 31.6 (CH.sub.2, C-8″), 28.6 (CH.sub.2, C-7″), 25.9 (C, C-15), 24.0 (CH.sub.3, C-17), 22.7 (CH.sub.2, C-9″), 21.3 (CH.sub.3, C-2′), 17.0 (CH.sub.3, C-16), 14.2 (CH.sub.3, C-10″), 10.3 (CH.sub.3, C-19).

##STR00018##

[0147] [α].sup.20.sub.D+7.5 (c 1.3, CH.sub.2Cl.sub.2);

[0148] ESI-MS, m/z 587.6 [M+H].sup.+ and 609.5 [M+Na].sup.+;

[0149] .sup.1H NMR (CDCl.sub.3, 500 MHz): δ.sub.H7.58 (1H, s, H-1), 6.67 (1H, dd, J=15.45, 10.66 Hz, H-3′), 6.05 (1H, dd, J=15.14, 10.71 Hz, H-4′), 5.86 (1H, m, H-5′), 5.65 (1H, d, J=15.46 Hz, H-2′), 5.02 (1H, brs, H-16a), 4.99 (1H, brs, H-12), 4.96 (1H, brs, H-16b), 4.76 (1H, d, J=2.47 Hz, H-14), 4.26 (1H, brs, H-7), 3.94 (1H, dd, J=12.34, 5.86 Hz, H-20a), 3.82 (1H, m, H-10), 3.80 (1H, m, H-20 b), 3.56 (1H, s, H-5), 3.52 (1H, d, J=2.45 Hz, H-8), 2.38 (1H, q, J=7.22 Hz, H-11), 2.10 (2H, q, J=7.20 Hz, H-6′), 2.00 (3H, s, H-2″), 1.84 (3H, s, H-17), 1.80 (3H, d, J=1.31 Hz, H-19), 1.39 (2H, dt, J=14.26, 7.28 Hz, H-7′), 1.30 (2H, m, H-9′), 1.27 (2H, m, H-8′), 0.89 (3H, t, J=6.93 Hz, H-10′);

[0150] .sup.13CNMR values in ppm (CDCl.sub.3, 126 Hz): δ6.sub.C 209.7 (C, C-3), 169.9 (C, C-1″), 160.6 (CH, C-1), 143.3 (C, C-15), 139.6 (CH, C-5′), 137.1 (C, C-2), 135.3 (CH, C-3′), 128.8 (CH, C-4′), 122.5 (CH, C-2′), 117.2 (C, C-1′), 113.5 (CH.sub.2, C-16), 83.9 (C, C-13), 80.7 (CH, C-14), 78.5 (CH, C-12), 78.3 (C, C-9), 72.5 (C, C-4), 72.2 (CH, C-5), 65.3 (CH.sub.2, C-20), 64.5 (C-7), 60.7 (C-6), 47.7 (CH, C-10), 44.3 (CH, C-11), 35.6 (CH, C-8), 32.9 (CH.sub.2, C-6′), 31.5 (CH.sub.2, C-8′), 28.9 (CH.sub.2, C-7′), 22.7 (CH.sub.2, C-9′), 21.4 (CH.sub.3, C-2″), 18.9 (CH.sub.3, C-17), 18.5 (CH.sub.3, C-18), 14.2 (CH.sub.3, C-10′), 10.1 (CH.sub.3, C-19).

##STR00019##

[0151] [α].sup.20.sub.D+52.8 (c 0.5, MeOH);

[0152] ESI-MS, m/z 605.5 [M+H].sup.+, 627.4 [M+Na].sup.+, 603.3 [M−H].sup.−;

[0153] .sup.1HNMR (CDCl.sub.3, 500 MHz): δ.sub.H 7.94 (2H, m, H-3″, H-7″), 7.75 (2H, m, H-3′, H-7′), 7.60 (1H, t, J=7.4, H-5″), 7.48 (2H, m, H-4″, H-6″), 7.40 (3H, m, H-4′, H-5′, H-6′), 5.42 (1H, brs, H-12), 5.07 (1H, brs, H-16a), 5.03 (1H, brs, H-16 b), 4.99 (1H, d, J=2.8, H-14), 4.10 (1H, s, H-5), 3.90 (1H, d, J=12.4, H-20a), 3.85 (1H, d, J=12.3, H-20b), 3.69 (1H, d, J=2.8, H-8), 3.67 (1H, brs, H-7), 3.06 (1H, dd, J=13.3 and 5.9, H-10), 2.59 (1H, q, J=6.9, H-11), 2.40 (1H, m, H-1a), 2.28 (1H, m, H-2), 1.92 (3H, s, H-17), 1.63 (1H, m, H-1b), 1.51 (3H, d, J=6.9, H-18), 1.12 (3H, d, J=6.6, H-19);

[0154] .sup.13CNMR (CDCl.sub.3, 126 Hz): δ.sub.C 220.4 (C, C-3), 165.8 (C, C-1″), 143.2 (C, C-15), 135.7 (C, C-2′), 133.5 (CH, C-5″), 130.0 (CH, C-5′), 129.8 (C, C-2″), 129.7 (CH, C-3″, C-7″), 128.9 (CH, C-4″, C-6″), 128.3 (CH, C-4′, C-6′), 126.2 (CH, C-3′, C-7′), 118.4 (C, C-1′), 113.8 (CH.sub.2, C-16), 83.9 (C, C-13), 81.4 (CH, C-14), 79.3 (C, C-9), 78.7 (CH, C-12), 75.2 (C, C-4), 71.5 (CH, C-5), 65.3 (CH.sub.2, C-20), 64.5 (CH, C-7), 61.0 (C, C-6), 44.3 (CH, C-11), 44.2 (CH, C-10), 43.1 (CH, C-2), 36.3 (CH, C-8), 33.6 (CH.sub.2, C-1), 19.0 (CH.sub.3, C-17, C-18), 12.6 (CH.sub.3, C-19).

##STR00020##

[0155] [α].sup.20.sub.D−15.1 (c 0.2, CHCl.sub.3);

[0156] ESI-MS, m/z 577.5 [M+Na].sup.+, 553.4 [M−H].sup.−;

[0157] .sup.1HNMR (CDCl.sub.3, 500 MHz): δ.sub.H7.61 (1H, s, H-1), 7.28 (1H, dd, J=15.3 and 11.22, H-3″), 6.54 (1H, dd, J=14.9 and 10.7, H-5″), 6.23 (1H, dd, J=14.8 and 11.4, H-4″), 6.15 (1H, dd, J=15.1 and 10.8, H-6″), 5.95 (1H, m, H-7″), 5.84 (1H, d, J=15.3, H-2″), 5.70 (1H, d, J=4.8, H-7), 5.47 (1H, d, J=10.3, H-12), 4.05 (1H, d, J=12.9, H-20a), 4.00 (1H, d, J=12.9, H-20 b), 3.26 (1H, overlapped, H-10), 3.26 (1H, overlapped, H-8), 2.52 (2H, m, H-5), 2.17 (1H, m, H-11), 2.13 (2H, overlapped, H-8″), 2.11 (3H, s, H-2′), 1.78 (3H, d, J=1.5, H-19), 1.45 (2H, dq, J=14.6 and 7.3, H-9″), 1.27 (3H, s, H-16), 1.22 (3H, s, H-17), 1.10 (1H, d, J=5.1, H-14), 0.93 (3H, t, J=7.3, H-10″), 0.91 (3H, d, J=6.4, H-18);

[0158] .sup.13CNMR (CDCl.sub.3, 126 Hz): δ.sub.C 209.2 (C, C-3), 174.1 (C, C-1′), 167.2 (C, C-1″), 161.0 (CH, C-1), 145.6 (CH, C-3″), 141.8 (CH, C-5″), 141.0 (CH, C-7″), 140.7 (C, C-6), 133.1 (C, C-2), 130.2 (CH, C-6″), 129.5 (CH, C-7), 127.9 (CH, C-4″), 119.9 (CH, C-2″), 78.5 (C, C-9), 76.9 (CH, C-12), 74.0 (C, C-4), 68.2 (CH.sub.2, C-20), 65.9 (C, C-13), 56.4 (CH, C-10), 43.4 (CH, C-11), 39.3 (CH, C-8), 38.8 (CH.sub.2, C-5), 36.6 (CH, C-14), 35.5 (CH.sub.2, C-8″), 26.0 (C, C-15), 24.0 (CH.sub.3, C-17), 22.4 (CH.sub.2, C-9″), 21.3 (CH.sub.3, C-2′), 17.0 (CH.sub.3, C-16), 14.6 (CH.sub.3, C-18), 13.9 (CH.sub.3, C-10″), 10.3 (CH.sub.3, C-19).

Example 3: Effect of Compounds on Nurr1 Activity

[0159] As in Example 2, Nurr1 activity according to the concentration of the diterpene isolated from the extract of Daphne genkwa was confirmed through luciferase analysis.

[0160] Specifically, after synthesizing a vector in which the gene having the nucleotide sequence (5′-CTCGGAGGACAGTACTCCG-3 SEQ ID NO:1) to which the GLA4 gene can bind is repeated 8 times to the reporter gene, luciferase, is synthesized, 3 types of plasmid DNA, including DNA containing Nurr1-LBD and DNA with β-galactosidase, were transfected into BE(2)C cells. After 6 hours, the compounds 1 to 10 separated in Example 2 were treated according to the concentrations in Table 1 below. The cells thus treated were cultured in a 5% carbon dioxide incubator at 37° C. for 20 hours, and then luciferase analysis was performed. As a control, 0.1% DMSO was used, and at this time, the activity was increased by multiple, and Amodiaquine was used as a positive control.

TABLE-US-00001 TABLE 1 Final treatment concentration (μM) compound low mid high AQ (Amodiaquine) 5 20 — Formula 1 0.01 0.1 1 Formula 2 0.01 0.1 1 Formula 3 0.1 1 10 Formula 4 0.1 1 10 Formula 5 0.1 1 10 Formula 6 0.01 0.1 1 Formula 7 0.1 1 10 Formula 8 0.1 1 10 Formula 9 0.1 1 10 Formula 10 1 10 100 DG-2 (control) 0.01 0.1 1

[0161] As a result of the analysis, as shown in Table 2 and FIGS. 1A-1D, all compounds of Formulas 1 to 10 activated Nurr1. Specifically, all compounds of Formulas 1 to 10 activated Nurr1 when treated at a concentration of 1 μM, in particular, it was confirmed that the compound of Formula 2 has excellent effect of Nurr1 activity even at a low concentration of 0.01 μM. Thereby, it was found that the compounds isolated from the extract of Daphne genkwa activate Nurr1 and at the same time, Nurr1 activity may be different depending on the structure of the compound.

TABLE-US-00002 TABLE 2 DM- DGII- Conc. SO AQ 1 2 3 4 5 6 7 8 9 10 2 Low 1.0 0.9 1.0 2.1 1.2 1.0 0.9 1.0 1.2 1.7 1.7 1.8 1.1 Midium — 1.8 1.5 2.2 1.4 1.4 1.2 1.8 2.6 2.0 2.4 1.9 2.6 High — — 1.7 1.9 1.4 1.7 1.9 2.4 2.0 1.6 1.5 1.1 2.2

[0162] As a result of the analysis, as shown in Table 1 below, prostratin Q and yuanhuadine of compounds 13 and 14 activated Nurr1 even at a low concentration of 0.003 μM, and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate of compound 16 in addition to compounds 13 and 14 significantly activates Nurr1 at 0.03 μM. Thereby, it was found that the compounds isolated from the extract of Daphne genkwa activated Nurr1 and Nurr1 activity could be different depending on the structure of the compound.

TABLE-US-00003 TABLE 3 density Positive compound compound compound compound compound compound (μM) control 11 12 13 14 15 16 0.003 — — — 1.23 ± 0.09 1.03 ± 0.15 — — 0.01 — — — 1.35 ± 0.28 0.94 ± 0.04 — — 0.03 — 1.28 ± 0.18 1.4 ± 0.18 *1.47 ± 0.17  *1.63 ± 0.18  1.17 ± 0.22 *1.42 ± 0.15  0.1 — 1.16 ± 0.16 **1.29 ± 0.04   *1.42 ± 0.14  **1.68 ± 0.11   1.28 ± 0.35 *1.68 ± 0.43  0.3 — 1.32 ± 0.13 *1.33 ± 0.06  *1.65 ± 0.2   **1.62 ± 0.11   **1.59 ± 0.08   *1.82 ± 0.51  1 0.8 ± 0.03 *1.76 ± 0.08  *1.62 ± 0.21  **1.47 ± 0.02   *1.67 ± 0.32  *2.12 ± 0.37  **1.53 ± 0.07   5 1.1 ± 0.15 — — — — — — 10 **1.6 ± 0.03   — — — — — — 20 **2.7 ± 0.37   — — — — — — (*P < 0.05, **P < 0.01 compared to control treatment)

Example 4: Inhibition Activity on Nitric Oxide Production in a Microglia BV-2 Cell

[0163] The death of cranial nerve cells due to an inflammatory reaction in a microglia cell has been reported as one of the main causes of degenerative brain diseases such as dementia and Parkinson's disease. (Sarkar S et al., Neurotoxicology, 44, 250-262 (2014); Bower J H et al., Neurology, 67, 494-496 (2006)). Accordingly, the inhibition activity on nitric oxide production, a representative inflammatory factor, was investigated for the compounds isolated from Example 2 in a microglia. Specifically, microglia BV-2 cells were put into a 96-well plate at 5×10.sup.4 cells/well, and cultured for 2 days, followed by incubation with LPS (1 mg/mL) for 24 hours with the compound isolated in Example 2 above. The culture supernatant was measured for absorbance at 540 nm using the Griess reagent to quantify nitrite to investigate the amount of nitric oxide production. Minocyline was used as a positive control.

[0164] As a result of the investigation, as shown in Table 3 below, it was confirmed that all compounds inhibit nitric oxide production at a low concentration. In particular, it was confirmed that genkwadaphnine of compound 2 exhibits inhibition activity on nitric oxide production at a very low concentration of 0.06±0.02, and yuanhuadine of compound 14 exhibited inhibition activity of nitric oxide production at a very low concentration of 1.03 μM.

TABLE-US-00004 TABLE 4 Compound IC.sub.50 (μM) Minocycline 21.28 ± 0.48  Formula 1 (yuanhuafine) 0.37 ± 0.15 Formula 2 (genkwadaphnine) 0.06 ± 0.02 Formula 3 (genkwanine H) 1.06 ± 0.12 Formula 4 (genkwanine M) 0.18 ± 0.04 Formula 5 (genkwain K) 4.67 ± 3.10 Formula 6 (yuanhuapine) 0.25 ± 0.06 Formula 7 (genkwanin A) 3.41 ± 0.99 Formula 8 (orthobenzoate 2) 1.22 ± 0.13 Formula 9 (1,2 α-dihydrodaphnetoxin) 1.60 ± 0.37 Formula 10 (genkwanin I) 7.79 ± 0.91

TABLE-US-00005 TABLE 5 Positive compound compound Compound compound compound compound control 11 12 13 14 15 16 IC.sub.50 (μM) 29.9 3.49 2.3 1.8 1.03 3.73 1.78

Example 5: Inhibition Activity of Pro-Inflammatory Cytokines Production in a Microglia BV-2 Cell

[0165] The inhibition activity of compounds production against IL-1b, IL-6 and TNFa, which are representative inflammatory factors in microglia, was investigated. Microglia BV-2 cells were placed in a 96 well plate at 1><10.sup.5 cells/well, and LPS (1 mg/mL) was incubated with the compound for 5 hours. Cells were recovered from each well and subjected to Western blotting and real-time PCR.

[0166] Specifically, the expression level of IL-1b was investigated by Western blotting. Rabbit anti-IL-1b [Cell Signaling (Danvers, Mass., USA); 1:1000]) was used as the primary antibody, mouse anti-actin (Sigma 1:5000) was used as a control. As a secondary antibody, horseradish peroxidase-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) antibody (Amersham, Piscataway, N.Y., USA) was used, and was observed by coloring with an enhanced-chemiluminescent substrate (Amersham).

[0167] In addition, mRNA expression levels of IL-1b, IL-6, and TNF-a were analyzed by real-time quantatitive PCR. Primers of all rat cytokines and GAPDH were purchased from Invitrogen, and the cytokine mRNA expression level was determined by normalizing to the GAPDH mRNA expression level.

[0168] As a result of Western blotting and PCR, as shown in FIGS. 1A and 1B, it was confirmed that the expression level of IL-1b decreased in the administration group of all compounds. In addition, as a result of PCR, as shown in FIGS. 1C and 1D, it was confirmed that the expression level of IL-6 and TNF-a also decreased in the administration group of all compounds.

[0169] In the present invention, the contents that can be sufficiently recognized and inferred by those of ordinary skill in the technical field of the present invention are omitted, and in addition to the specific examples described in the present invention, various modifications are possible within a range that does not change the technical spirit or essential configuration of the present invention. Therefore, the present invention may be implemented in a different manner from those specifically described and exemplified in this specification, which is understood by those skilled in the art of the present invention.

INDUSTRIAL AVAILABILITY

[0170] As described above, the present invention relates to a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases comprising a diterpene, or a pharmaceutically acceptable salt thereof, by showing the effect of suppressing the inflammatory response in neurons, shows the effect of suppressing the inflammatory response in neurons, and can be usefully used for the prevention and treatment of neurodegenerative diseases including Parkinson's disease caused by the inhibition of Nurr1 activity.

TABLE-US-00006 [Sequence list free text] DNA Artificial Sequence GLA4 binding gene SEQ ID NO: 1 ctcggaggac agtactccg