Daphnane and Phorbol Diterpenes, Anti-neuroinflammatory Compounds with Nurr1 Activation from the Roots and Stems of Daphne genkwa

Abstract

The methanol extract of the roots and stems of Daphne genkwa and its constituents yuanhuacin (1) and genkwanine N were previously reported to have Nurr1 activating effects and neuroprotective effects in an animal model of Parkinson’s disease (PD). In this study, four more daphnane-type diterpenes (acutilonine F (2), wikstroemia factor M₁ (3), yuanhuadine (5), and yuanhuatine (6)) and two phorbol-type diterpenes (prostratin Q (4) and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate (7)) were isolated as Nurr1 activating compounds from the D. genkwa extract. Consistent with their higher Nurr1 activating activity, compounds 1, 4, 5, and 7 exhibited higher inhibitory activity on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine microglial BV-2 cells with an IC₅₀ (µM) of 1–2, which was 15–30 times more potent than that of minocycline (29.9 µM), a well-known anti-neuroinflammatory agent. Additionally, these diterpenes reduced expression and transcription of LPS-induced pro-inflammatory cytokines in BV-2 cells. Thus, the daphnane-type and phorbol-type diterpenes had anti-neuroinflammatory activity with Nurr1 activation and could be responsible for the anti-PD effect of the roots and stems of D. genkwa.

Parkinson’s disease (PD) is one of the most common degenerative neurological disorders, with a prevalence that increases with age. One of the key features of PD pathology is neuroinflammation.^1–3) Several studies have shown that the microglia in the substantia nigra (SN) and striatum of PD affected humans and PD animal models are activated.⁴⁾ In addition, pro-inflammatory cytokines such as interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α activate microglia in the brain.¹⁾ Anti-neuroinflammatory agents were shown to prevent the degeneration of dopaminergic neurons and significantly mitigate the risk of having PD.^5,6) One such agent is minocycline, which has anti-inflammatory potential and is being investigated in humans.^7,8)

Nurr1, an orphan nuclear receptor, is well-known as a key regulator of dopamine (DA) neuronal differentiation^9–11) and is essential for the maintenance of DA neurons.¹²⁾ Recently, other roles of Nurr1 have received attention. In microglia and astrocytes, Nurr1 inhibits the expression of pro-inflammatory genes. In addition, the repression of inflammation by Nurr1 activation decreases PD symptoms and DA neuronal loss from in vivo and in vitro PD models.¹³⁾ Thus, activation of Nurr1 could improve the pathogenesis of PD. Indeed, small-molecule Nurr1 agonists such as amodiaquine have been reported to improve behavioral deficits in animal models of PD.¹⁴⁾

During screening for Nurr1 activators from Korean medicinal plants, we previously reported that a methanol extract of the stem and root portions of Daphne genkwa SIEBOLD et ZUCC. (Thymelaeaceae) and daphnane-type diterpenes, yuanhuacin (1) and genkwanine N, had Nurr1-activating activity as well as neuroprotective effects in an animal model of PD.¹⁵⁾ Nurr1-activating components of D. genkwa, however, was not studied in depth. In this study, we continued to isolate Nurr1 activating compounds from the methanol extract of the stem and root portions of D. genkwa. This resulted in the isolation of four more daphnane-type diterpenes (acutilonine F (2),¹⁶⁾ wikstroemia factor M₁ (3),¹⁷⁾ yuanhuadine (5),¹⁸⁾ and yuanhuatine (6)¹⁹⁾) and two phorbol-type diterpenes (prostratin Q (4)²⁰⁾ and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate (7)²¹⁾) (Fig. 1). We report the isolation, Nurr1 enhancing and anti-neuroinflammatory activities of these diterpenes.

Fig. 1. Structures of Yuanhuacin (1), Acutilonine F (2), Wikstroemia Factor M1 (3)_, Prostratin Q (4), Yuanhuadine (5), Yuanhuatine (6), and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate (7) Isolated from Root and Stem Parts of D. genkwa

MATERIALS AND METHODS

General Experimental Methods

NMR spectra were recorded on a Bruker Biospin Avance 500 spectrometer (Korea Basic Science Institute). The electrospray ionization (ESI)-MS data were recorded with a Jeol JMS-HX110/110 A mass spectrometer. Column chromatography on silica gel (Kieselgel 60, 70–230 mesh, Merck, Darmstadt, Germany) and TLC on pre-coated 60 F₂₅₄ silica gels (0.25 mm, Merck) were conducted. Mouse microglia BV-2 cells were a kind gift from Dr. Jau-Shyong Hong (National Institute of Environmental Health Sciences, NC, U.S.A.).²²⁾ For cell culture, fetal bovine serum (FBS) was purchased from HyClone (Piscataway, NJ, U.S.A.). Horse serum, penicillin, streptomycin, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Invitrogen (Carlsbad, CA, U.S.A.). Dulbecco’s modified Eagle’s medium (DMEM), L-glutamine, and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich Co. (St. Louis, MO, U.S.A.).

Materials

A sample of the stems and roots of D. genkwa was purchased from Hantaek Botanical Garden, Yongin, Gyeonggi Province, South Korea. The sample was identified by the staff at the Plant Extract Bank, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. A voucher specimen (DG-2010-8) was deposited in the Superbacteria Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea. Plants were immediately transferred to the laboratory, washed with tap water and finally rinsed with distilled water. The stem and root portions were dried for one week in the shade and then cut into smaller pieces.

Extraction and Isolation

Dried material (4.47 kg) of the stem and root portions of D. genkwa was extracted twice with 12 L 80% aqueous ethanol. After the ethanol solution was concentrated in vacuo, the dried residue (255.1 g) was suspended in water and successively partitioned three times using 200 mL of hexane. After the n-hexane layer was concentrated in vacuo, the resultant dried residue (20 g) was subjected to silica gel (Merck art no. 7734.9025) column chromatography, followed by stepwise elution with n-hexane–EtOAc (10 : 1, 5 : 1, 2 : 1, 1 : 1, and 1 : 2) to give three fractions, Fractions I–III. Fraction I (577 mg) was purified by preparative ODS TLC (Merck No. 1.15389.0001; Merck) and developed with acetonitrile–water (75 : 25) to give an active band, which was finally purified by preparative ODS HPLC with acetonitrile–water (83 : 17) at a flow rate of 3 mL/min to give 2 (14.0 mg), and 3 (7.0 mg) with retention times of 15.2, and 18.5 min, respectively. Fraction II (320 mg) was purified by preparative ODS TLC developed with acetonitrile–water (75 : 25) to give an active band. The active band (130 mg) was further purified by a second TLC (silica gel F254, Merck No. 1.05715.0001) developed with CHCl₃–MeOH (50 : 1) and finally purified by preparative ODS HPLC with acetonitrile–water (79 : 21) at a flow rate of 3 mL/min to give 1 (29.2 mg) with a retention time of 16 min. Fraction III (400 mg) was further purified in the same way as Fraction II to give two sub-fractions, Fr. III-1 and III-2, with Rf values of 0.40 and 0.25, respectively. Compounds 4 (2.1 mg) and 5 (4.0 mg) were obtained from Fr. III-1 (22.0 mg) by preparative ODS HPLC with acetonitrile–water (65 : 35) as eluent at a flow rate of 3 mL/min and retention times of 17.2 and 23.4 min, respectively. A work up of Fr. III-2 with acetonitrile–water (58 : 42) as eluent was performed to yield 6 (4.4 mg) and 7 (1.8 mg) with retention times of 19.0 and 21.4 min, respectively.

Acutilonine F (2)

White powder; [α]_D²⁰ −32.1 (c 1.3, MeOH) [lit.¹⁶⁾ [α]_D −37.6 (MeOH)]; [α]_D²⁰, ¹H, ¹³C-NMR, and MS data in accordance with those of acutilonine F.¹⁶⁾

Wikstroemia Factor M₁ (3)

White powder; [α]_D²⁰ +18.9 (c 1.0, MeOH); ESI-MS, m/z 637.6 [M+H]⁺, 659.4 [M+Na]⁺, 635.2 [M−H]⁻; ¹H-NMR (CDCl₃, 500 MHz): δ_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, 10.2 Hz, H-3″), 6.21 (1H, dd, J=14.8, 10.4 Hz, H-4″), 5.90 (1H, d, J=15.2 Hz, H-2″), 5.05 (1H, br s, H-16a), 4.92 (1H, br s, H-16b), 4.69 (1H, d, J=5.19 Hz, H-3), 4.51 (1H, d, J=2.76 Hz, H-14), 4.06 (1H, s, H-5), 3.88 (1H, d, J=12.2 Hz, H-20a), 3.77 (1H, d, J=12.2 Hz, H-20b), 3.44 (1H, s, H-7), 2.96 (1H, d, J=2.8 Hz, H-8), 2.82 (1H, dd, J=13.2, 5.5 Hz, 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 Hz, H-18), 1.32, (2H, overlapped, H-9″), 1.31 (2H, overlapped, H-8″), 1.06 (3H, d, J=6.5 Hz, H-19), 0.91 (3H, t, J=6.9 Hz, H-10″); ¹³C-NMR (CDCl₃, 126 Hz): δ_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 (CH₂, 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 (CH₂, 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 (CH₂, C-12), 36.0 (CH₂, C-1), 35.5 (CH, C-11), 33.3 (CH₂, C-6″), 31.6 (CH₂, C-8″), 28.5 (CH₂, C-7″), 22.7 (CH₂, C-9″), 21.1 (CH₃, C-18), 19.4 (CH₃, C-17), 14.2 (CH₃, C-10″), 13.3 (CH₃, C-19).

Prostratin Q (4)

White powder; [α]_D²⁰ +14.1 (c 0.03, MeOH) [lit.²⁰⁾ [α]_D +16.3 (MeOH)]; [α]_D²⁰, ¹H, ¹³C-NMR, and MS data in accordance with those of prostratin Q.²⁰⁾

Yuanhuadine (5)

White powder; [α]_D²⁰ +7.5 (c 1.3, CH₂Cl₂) [lit.²³⁾ [α]_D +9.4 (CHCl₃)]; [α]_D²⁰, ¹H, ¹³C-NMR, and MS data in accordance with those of yuanhuadine.²⁴⁾

Yuanhuatine (6)

White powder; [α]_D²⁰ +52.8 (c 0.5, MeOH); ESI-MS, m/z 605.5 [M+H]⁺, 627.4 [M+Na]⁺, 603.3 [M−H]⁻; ¹H-NMR (CDCl₃, 500 MHz): δ_H 7.94 (2H, m, H-3″, H-7″), 7.75 (2H, m, H-3′, H-7′), 7.60 (1H, t, J=7.4 Hz, H-5″), 7.48 (2H, m, H-4″, H-6″), 7.40 (3H, m, H-4′, H-5′, H-6′), 5.42 (1H, br s, H-12), 5.07 (1H, br s, H-16a), 5.03 (1H, br s, H-16b), 4.99 (1H, d, J=2.8 Hz, H-14), 4.10 (1H, s, H-5), 3.90 (1H, d, J=12.4 Hz, H-20a), 3.85 (1H, d, J=12.3 Hz, H-20b), 3.69 (1H, d, J=2.8 Hz, H-8), 3.67 (1H, br s, H-7), 3.06 (1H, dd, J=13.3, 5.9 Hz, H-10), 2.59 (1H, q, J=6.9 Hz, 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 Hz, H-18), 1.12 (3H, d, J=6.6 Hz, H-19); ¹³C-NMR (CDCl₃, 126 Hz): δ_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 (CH₂, 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 (CH₂, 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 (CH₂, C-1), 19.0 (CH₃, C-17, C-18), 12.6 (CH₃, C-19).

12-O-n-Deca-2,4,6-trienoyl-phorbol-(13)-acetate (7)

White powder; [α]_D²⁰ −15.1 (c 0.2, CHCl₃) [lit.²¹⁾ [α]_D −15.3 (CHCl₃)]; ESI-MS, m/z 577.5 [M+Na]⁺, 553.4 [M−H]⁻; ¹H-NMR (CDCl₃, 500 MHz): δ_H 7.61 (1H, s, H-1), 7.28 (1H, dd, J=15.3, 11.22 Hz, H-3″), 6.54 (1H, dd, J=14.9, 10.7 Hz, H-5″), 6.23 (1H, dd, J=14.8, 11.4 Hz, H-4″), 6.15 (1H, dd, J=15.1, 10.8 Hz, H-6″), 5.95 (1H, m, H-7″), 5.84 (1H, d, J=15.3 Hz, H-2″), 5.70 (1H, d, J=4.8 Hz, H-7), 5.47 (1H, d, J=10.3 Hz, H-12), 4.05 (1H, d, J=12.9 Hz, H-20a), 4.00 (1H, d, J=12.9 Hz, H-20b), 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 Hz, H-19), 1.45 (2H, dq, J=14.6, 7.3 Hz, H-9″), 1.27 (3H, s, H-16), 1.22 (3H, s, H-17), 1.10 (1H, d, J=5.1 Hz, H-14), 0.93 (3H, t, J=7.3 Hz, H-10″), 0.91 (3H, d, J=6.4 Hz, H-18); ¹³C-NMR (CDCl₃, 126 Hz): δ_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 (CH₂, C-20), 65.9 (C, C-13), 56.4 (CH, C-10), 43.4 (CH, C-11), 39.3 (CH, C-8), 38.8 (CH₂, C-5), 36.6 (CH, C-14), 35.5 (CH₂, C-8″), 26.0 (C, C-15), 24.0 (CH₃, C-17), 22.4 (CH₂, C-9″), 21.3 (CH₃, C-2′), 17.0 (CH₃, C-16), 14.6 (CH₃, C-18), 13.9 (CH₃, C-10″), 10.3 (CH₃, C-19).

Nurr1 Assay

Nurr1 activity was measured as previously reported.¹⁴⁾ In brief, SK-N-BE(2)-C human neuroblastoma cells were maintained in DMEM supplemented with 10% FBS. All culture media contained 100 units/mL penicillin and 100 µg/mL streptomycin. For transfection of the reporter construct p4xNL3-Luc and Nurr1-expression vector (pCMV-Nurr1 construct), the day before transfection, cells were plated on 24-well plates at 1.5×10⁵ cells/well in DMEM without antibiotics. Transfections were performed using Lipofectamine^® LTX with Plus™ Reagent (Invitrogen) according to the manufacturer’s protocol. The total amount of DNA was 0.5 µg/well, with 0.1 µg pRSV-β-gal as an internal control. Six hours after transfection, extracts diluted at 0.5% concentration in DMEM with 1% charcoal-stripped fetal calf serum were added and incubated overnight. Cells from each well were lysed with 100 µL lysis buffer, which contained 25 mM Tris-phosphate (pH 7.8), 2 mM dithiothreitol (DTT), 2 mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 10% glycerol, and 1% Triton X-100. An equal volume of firefly luciferase substrate was added, and luciferase activity was measured using a luminometer plate reader, and normalized for β-galactosidase activity.

Anti-inflammation Assay

The effect on the expression of the proinflammatory cytokine gene was measured as previously reported.¹⁴⁾ In brief, BV-2 cells, a microglial-like mouse cell line, were maintained in six-well culture plates (Corning Costar, Corning, NY, U.S.A.) at a density of 1.0×10⁵ cells/well in DMEM supplemented with 10% FBS heat-inactivated at 56°C for 30 min, 2 mM L-glutamine, and 1X Pen-Strep (Invitrogen). To begin the experiment, BV-2 cells were washed with DMEM and treated with LPS (1 µg/mL) in the presence or absence of test compounds. The plates were incubated in a tissue culture incubator at 37°C in an atmosphere of 5% CO₂/95% air for 5 h. Cells from each well were withdrawn for Western blotting and real-time PCR analysis. For Western blotting, cells were washed twice with phosphate-buffered saline (PBS) and harvested in a solution containing 1% Triton X-100, 20 mM Tris (pH 7.6), 150 mM sodium chloride, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The cell suspensions were sonicated and boiled in an equal volume of sodium dodecyl sulfate (SDS) sample buffer. Samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, U.S.A.). After blocking, the membranes were incubated with primary antibodies diluted in PBS containing 0.1% bovine serum albumin (BSA). The following primary antibodies were used: rabbit anti-IL-1β [Cell Signaling (Danvers, MA, U.S.A.); 1 : 1000], and mouse anti-actin (Sigma 1 : 5000). The membranes were incubated with a 1 : 3000 dilution of horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin G (IgG) antibody (Amersham, Piscataway, NY, U.S.A.). Detection was achieved using an enhanced-chemiluminescent substrate (Amersham).

Real-Time Quantatitive PCR Analysis

Real-time quantatitive PCR analysis was performed using the ABI-Prism7700 sequence detection system (Applied Biosystems, Foster City, CA, U.S.A.) as described previously.^25,26) Briefly, it was performed in 0.5 mL tubes (Applied Biosystems) on cDNA equivalent to 50 ng deoxyribonuclease (DNase)-digested RNA in a volume of one twenty-fifth, containing two twenty-fifths of SYBRGreen universal master mix, and forward and reverse primers, following the manufacturer’s protocol. All primers for mouse cytokines and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Invitrogen. The mRNA expression of cytokines was normalized to the level of GAPDH mRNA.

Nitric Oxide (NO) Quantification

The accumulation of nitrite, extensively used as an indicator of NO production, was assayed using the Griess reagent. BV-2 microglial cells were seeded in 96-well tissue culture plates at 5×10⁴ cells/well containing medium. After incubation for 2 d and media change, the cells were treated with LPS (1 µg/mL) in the presence or absence of test compounds for 24 h. The supernatants were mixed with equal amounts of Griess reagent, and then the absorbance was read at 540 nm using a microplate reader.

Statistical Methods

Two-sample comparisons were performed using the Student’s t-test. All data are presented as the means±standard deviation (S.D.), and significant differences were accepted at the 5% level unless otherwise indicated.

RESULTS AND DISCUSSION

Our continued isolation of Nurr1-activating compounds from the stems and roots of D. genkwa resulted in the identification of four more daphnane-type diterpenes (2, 3, 5, and 6) and two phorbol-type diterpenes (4 and 7) from the n-hexane layer. Compounds 2–7 were isolated via activity-guided fractionation using n-hexane extraction, SiO₂ column chromatography, and finally ODS HPLC. Although a biscoumarin, daphnorectin,²⁷⁾ was isolated as a major compound from the ethyl acetate layer, daphnorectin showed no Nurr1 activating activity (data not shown).

The ¹H- and ¹³C-NMR data of 5 were similar to those of 1, a daphnane-type diterpene containing an orthoester acyl chain, except for the absence of signals corresponding to an aromatic ring. Thus, 5 was identified as yuanhuadine¹⁸⁾ based on ¹H, ¹³C-NMR, MS and [α]_D data in the literature. The ¹H- and ¹³C-NMR data of 2, 3, and 6 were similar to those of genkanine N,²⁸⁾ a daphnane-type diterpene containing an orthoester aromatic ring. Compound 2 was identified as acutilonine F¹⁶⁾ based on ¹H, ¹³C-NMR, MS and [α]_D data in the literature. In the heteronuclear multiple bond connectivity (HMBC) spectrum of 3, the aromatic protons at δ 7.75 (2H, m, H-3′ and H-7′) were long-range coupled with the orthoester carbon (C-1′) at δ 117.6. The HMBC correlations of H-2″ at δ 5.90 and H-3″ at δ 7.33 in the acyl chain to the ester carbonyl C-1″ at δ 169.6 indicated the linkage of the acyl chain to C-1″. The geometry of the double bonds at C-2″ and 4″ in the acyl chain was determined as Z based on the larger coupling constants of H-2″, 4″, and 6″. Thus, 3 was identified as wikstroemia factor M₁.¹⁷⁾ The ¹³C-NMR data of 3 and the geometry of the acyl chain were reported for the first time in this study. The ¹H- and ¹³C-NMR data of 6 indicated two aromatic rings similar to genkanine N. The HMBC spectrum of 6 assigned the two aromatic rings to C-1′ and C-1″. Thus, 6 was identified as yuanhuatine.¹⁹⁾ The ¹³C-NMR data of 6 were reported for the first time in this study. The NMR spectra of 4 and 7 showed signals characteristic of phorbol-type diterpene²⁹⁾ with a long chain aliphatic ester. The difference between 4 and 7 was that 7 had an acyl chain with one more double bond. In the HMBC spectrum of 7, the long-range correlations of H-2″ at 5.84 and H-3″ at 7.28 in the acyl chain to the ester carbonyl C-1″ at 167.2 indicated the linkage of the acyl chain to C-1.” The geometry of the double bonds at C-2″, 4″, and 6″ in the acyl chain was determined as Z based on the larger coupling constants of H-2″, 4″, and 6″. The larger coupling constant J=10.3 Hz of H-12 suggested the β-configuration of the acyl chain. Thus, 4 and 7 were identified as prostratin Q²⁰⁾ and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate,²¹⁾ respectively. The ¹³C-NMR data of 7 were reported for the first time in this study.

To determine the effects of 1–7 on Nurr1, a cell-based luciferase reporter assay was used. Compounds 1–7 significantly activated Nurr1 at 0.03, 1, 0.1, 0.03, 0.03, 0.3, and 0.03 µM, respectively, whereas amodiaquine, as a positive control, activated Nurr1 at 10 µM (Table 1). Daphnorectin, a major compound in the ethylacetate layer, showed no Nurr1 activating activity even at 100 µM (data not shown).

Table 1. Activation of Nurr1 with Compounds 1–7 (Mean±S.D.)^a)

Conc. (µM)	AQb)	1	2	3	4	5	6	7
0.003	—	0.95±0.04	—	—	1.23±0.09	1.03±0.15	—	—
0.01	—	1.13±0.19	—	—	1.35±0.28	0.94±0.04	—	—
0.03	—	**1.87±0.09	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	—	*1.77±0.35	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	—	*1.65±0.14	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	*1.63±0.18	*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	—	—	—	—	—	—	—

a) Ratio of luciferase activity vs. DMSO treatment. b) Amodiaquine as a positive control. * p<0.05, ** p<0.01 compared to DMSO treatment. Luciferase assay to measure nurr1 activity was performed (n=6).

Because Nurr1 is known to reduce the expression of inducible nitric oxide synthase (iNOS), IL-1β, and TNF-α mRNA by recruiting the CoREST corepressor complex and subsequent clearing of nuclear factor-kappaB (NF-κB)-p65,¹³⁾ it was investigated whether compounds 1–7 inhibit NO production and expression of the proinflammatory cytokines. Compounds 1–7 dose-dependently inhibited NO production, measured as nitrite accumulation, by LPS-activated microglial BV-2 cells with IC₅₀ values (µM) of 1.03–3.73, which were 10–30 times higher in activity than minocycline,⁸⁾ a well-known anti-neuroinflammatory agent, with an IC₅₀ of 29.9 µM (Fig. 2 and Table 2). Among them, consistent with higher Nurr1 activating activity, 1, 4, 5, and 7 exhibited higher inhibitory activity against NO production. Amodiaquine showed cytotoxicity at the concentration inhibiting NO production.

Fig. 2. Effect of Compounds 1–7 on NO Production by LPS-Activated Microglial BV2 Cells (A) and Their Effect on Cell Viability (B)

n=6, * p<0.01, ** p<0.001 compared to LPS-treated.

Table 2. Effect of 1–7 on NO Production by LPS-Activated Microglial BV2 Cells

	1	2	3	4	5	6	7	AQa)	MCb)
IC50 (µM)	1.24	3.49	2.3	1.8	1.03	3.73	1.78	>10	29.9

a) Amodiaquine showed cytotoxicity at 10 µM. b) Minocyline as a positive anti-neuroinflammatory agent.

The effect of 1–7 on the expression of IL-1β was examined by Western blotting analysis (Fig. 3A). Compounds 1–7 prominently reduced the expression of LPS-induced IL-1β in BV-2 cells at 3 µM. Additionally, expression of IL-1β, IL-6, and TNF-α mRNA was investigated to see whether 1–7 inhibit transcription of the proinflammatory cytokines. The LPS-induced expression of IL-1β, IL-6, and TNF-α mRNA in BV 2 cells was determined by real-time quantitative PCR (Fig. 3B–D). IL-1β, IL-6, and TNFα mRNA were dramatically induced in the LPS treatment, but 1–7 significantly inhibited the transcription of IL-1β, IL-6, and TNF-α at 3 µM. The phorbol-type diterpenes, 4 and 7, comprising C12-unsaturated fatty ester group increased the levels of IL-1β than daphnane-type diterpenes. These results suggested that the orthoester group in daphnane-type diterpenes could be involved in inhibition of the transcription of IL-1β.

Fig. 3. Effect of Compounds 1–7 on the LPS-Induced Expression of Pro-inflammatory Cytokines in Microglial BV-2 Cells

Western blotting analysis (A) of IL-1β and real time-PCR analysis (B, C, and D) of IL-1β, IL-6, and TNF-α mRNA in LPS-induced BV-2 cells with or without compounds 1–7 at 3 µM and AQ (amodiaquine) at 20 µM.

Known biological activities of the daphnane-type diterpene (acutilonine F,¹⁶⁾ wikstroemia factor M₁,^20,30) yuanhuadine,³¹⁾ and yuanhuatine³²⁾) and prostratin Q²⁰⁾ are mainly cytotoxic activity against human cancer cell lines. No biological activity of 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate is known. To our knowledge, anti-neuroinflammatory activity of the daphnane-type diterpenes and the phorbol-type diterpenes is nearly unreported.

Acutilonine F has been isolated from D. acutiloba REHD.¹⁶⁾ Wikstroemia factor M₁ has been isolated from Wikstroemia mekongenia¹⁷⁾ and D. genkwa.³⁰⁾ Prostratin Q has been isolated from the buds of Wikstroemia chamaedaphne.²⁰⁾ 12-O-n-Deca-2,4,6-trienoyl-phorbol-(13)-acetate has been isolated from Sapium japonicum and Euphorbia tirucalli.²¹⁾ To our knowledge, this study is the first report of the isolation from D. genkwa of acutilonine F and the phorbol-type diterpenes (prostratin Q and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate).

In conclusion, four daphnane-type diterpenes (acutilonine F, wikstroemia factor M₁, yuanhuadine and yuanhuatine) and two phorbol-type diterpenes (prostratin Q and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate) were isolated from the stems and roots of D. genkwa. The six diterpenes activated Nurr1 at 0.03–1 µM and inhibited LPS-induced NO production in BV-2 microglial cells with approximately 10–30 times higher potency than minocycline, a well-known anti-neuroinflammatory agent. Additionally, the diterpenes reduced expression and transcription of proinflammatory cytokines such as IL-1β, IL-6, and TNF-α. This result indicated that the daphnane-type and phorbol-type diterpenes could be responsible for the anti-PD effect of the stems and roots of D. genkwa. To our knowledge, acutilonine F and the phorbol-type diterpenes (prostratin Q and 12-O-n-deca-2,4,6-trienoyl-phorbol-(13)-acetate) were isolated for the first time from D. genkwa in this study. Nurr1 activating and anti-neuroinflammatory activity of the daphnane-type diterpenes and the phorbol-type diterpenes was reported for the first time in this study.

Acknowledgments

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET) through the Agri-Bio Industry Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (114145-3). Support was also obtained from the KRIBB Research Initiative Program, Republic of Korea.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) McGeer EG, McGeer PL. The role of anti-inflammatory agents in Parkinson’s disease. CNS Drugs, 21, 789–797 (2007).
• 2) Tansey MG, McCoy MK, Frank-Cannon TC. Neuroinflammatory mechanisms in Parkinson’s disease: potential environmental triggers, pathways, and targets for early therapeutic intervention. Exp. Neurol., 208, 1–25 (2007).
• 3) Sarkar S, Raymick J, Imam S. Neuroprotective and therapeutic strategies against Parkinson’s disease: recent perspectives. Int. J. Mol. Sci., 17, 904 (2016).
• 4) Sarkar S, Chigurupati S, Raymick J, Mann D, Bowyer JF, Schmitt T, Beger RD, Hanig JP, Schmued LC, Paule MG. Neuroprotective effect of the chemical chaperone, trehalose in a chronic MPTP-induced Parkinson’s disease mouse model. Neurotoxicology, 44, 250–262 (2014).
• 5) Bower JH, Maraganore DM, Peterson BJ, Ahlskog JE, Rocca WA. Immunologic diseases, anti-inflammatory drugs, and Parkinson’s disease: a case-control study. Neurology, 67, 494–496 (2006).
• 6) Wolozin B, Wang SW, Li NC, Lee A, Lee TA, Kazis LE. Simvastatin is associated with a reduced incidence of dementia and Parkinson’s disease. BMC Med., 5, 20 (2007).
• 7) NINDS NET-PD Investigators. A randomized, double-blind, futility clinical trial of creatine and minocycline in early Parkinson’s disease. Neurology, 66, 664–671 (2006).
• 8) Tikka T, Fiebich BL, Goldsteins G, Keinanen R, Koistinaho J. Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia. J. Neurosci., 21, 2580–2588 (2001).
• 9) Saucedo-Cardenas O, Quintana-Hau JD, Le WD, Smidt MP, Cox JJ, De Mayo F, Burbach JP, Conneely OM. Nurr1 is essential for the induction of the dopaminergic phenotype and the survival of ventral mesencephalic late dopaminergic precursor neurons. Proc. Natl. Acad. Sci. U.S.A., 95, 4013–4018 (1998).
• 10) Zetterström RH, Solomin L, Jansson L, Hoffer BJ, Olson L, Perlmann T. Dopamine neuron agenesis in Nurr1-deficient mice. Science, 276, 248–250 (1997).
• 11) Chung S, Leung A, Han BS, Chang MY, Moon JI, Kim CH, Hong S, Pruszak J, Isacson O, Kim KS. Wnt1-lmx1a forms a novel autoregulatory loop and controls midbrain dopaminergic differentiation synergistically with the SHH-FoxA2 pathway. Cell Stem Cell, 5, 646–658 (2009).
• 12) Kadkhodaei B, Ito T, Joodmardi E, Mattsson B, Rouillard C, Carta M, Muramatsu S, Sumi-Ichinose C, Nomura T, Metzger D, Chambon P, Lindqvist E, Larsson NG, Olson L, Bjorklund A, Ichinose H, Perlmann T. Nurr1 is required for maintenance of maturing and adult midbrain dopamine neurons. J. Neurosci., 29, 15923–15932 (2009).
• 13) Saijo K, Winner B, Carson CT, Collier JG, Boyer L, Rosenfeld MG, Gage FH, Glass CK. A Nurr1/CoREST pathway in microglia and astrocytes protects dopaminergic neurons from inflammation-induced death. Cell, 137, 47–59 (2009).
• 14) Kim CH, Han BS, Moon J, Kim DJ, Shin J, Rajan S, Nguyen QT, Sohn M, Kim WG, Han M, Jeong I, Kim KS, Lee EH, Tu Y, Naffin-Olivos JL, Park CH, Ringe D, Yoon HS, Petsko GA, Kim KS. Nuclear receptor Nurr1 agonists enhance its dual functions and improve behavioral deficits in an animal model of Parkinson’s disease. Proc. Natl. Acad. Sci. U.S.A., 112, 8756–8761 (2015).
• 15) Han BS, Kim KS, Kim YJ, Jung HY, Kang YM, Lee KS, Sohn MJ, Kim CH, Kim KS, Kim WG. Daphnane diterpenes from Daphne genkwa activate Nurr1 and have a neuroprotective effect in an animal model of Parkinson’s disease. J. Nat. Prod., 79, 1604–1609 (2016).
• 16) Huang SZ, Zhang XJ, Li XY, Kong LM, Jiang HZ, Ma QY, Liu YQ, Hu JM, Zheng YT, Li Y, Zhou J, Zhao YX. Daphnane-type diterpene esters with cytotoxic and anti-HIV-1 activities from Daphne acutiloba Rehd. Phytochemistry, 75, 99–107 (2012).
• 17) Dagang W, Sorg B, Adolf W, Seip EH, Hecker E. Oligo- and macrocyclic diterpenes in thymelaeaceae and euphorbiaceae occurring and utilized in yunnan (Southwest China) 3. Two new daphnane type 9,13,14-orthoesters from Wikstroemia mekongenia. Phytother. Res., 7, 72–75 (1993).
• 18) Zhang S, Li X, Zhang F, Yang P, Gao X, Song Q. Preparation of yuanhuacine and relative daphne diterpene esters from Daphne genkwa and structure–activity relationship of potent inhibitory activity against DNA topoisomerase I. Bioorg. Med. Chem., 14, 3888–3895 (2006).
• 19) Hu BH, Sha H, Wang CR, Yu DF, Wu XC, Yu XG. Studies on the anti-fertility constituent of the flower yuan-hua isolation and structure of yuanhuatine. Acta Chimi. Sinica, 43, 460–462 (1985).
• 20) Guo J, Zhang J, Shu P, Kong L, Hao X, Xue Y, Luo Z, Li Y, Li G, Yao G, Zhang Y. Two new diterpenoids from the buds of Wikstroemia chamaedaphne. Molecules, 17, 6424–6433 (2012).
• 21) Gunasekera SP, Kinghorn AD, Cordell GA, Farnsworth NR. Plant anticancer agents. XIX. Constituents of Aquilaria malaccensis. J. Nat. Prod., 44, 569–572 (1981).
• 22) Sohn MJ, Hur GM, Byun HS, Kim WG. Cyclo(dehydrohistidyl-l-tryptophyl) inhibits nitric oxide production by preventing the dimerization of inducible nitric oxide synthase. Biochem. Pharmacol., 75, 923–930 (2008).
• 23) Hong JY, Nam JW, Seo EK, Lee SK. Daphnane diterpene esters with anti-proliferative activities against human lung cancer cells from Daphne genkwa. Chem. Pharm. Bull., 58, 234–237 (2010).
• 24) Zhang S, Li X, Zhang F, Yang P, Gao X, Song Q. Preparation of yuanhuacine and relative daphne diterpene esters from Daphne genkwa and structure–activity relationship of potent inhibitory activity against DNA topoisomerase I. Bioorg. Med. Chem., 14, 3888–3895 (2006).
• 25) Hering R, Petrovic S, Mietz EM, Holzmann C, Berg D, Bauer P, Woitalla D, Muller T, Berger K, Kruger R, Riess O. Extended mutation analysis and association studies of Nurr1 (NR4A2) in Parkinson’s disease. Neurology, 62, 1231–1232 (2004).
• 26) Kim SY, Han YM, Oh M, Kim WK, Oh KJ, Lee SC, Bae KH, Han BS. DUSP4 regulates neuronal differentiation and calcium homeostasis by modulating ERK1/2 phosphorylation. Stem Cells Dev., 24, 686–700 (2015).
• 27) Cordell GA. Studies in the Thymelaeaceae I. NMR spectral assignments of daphnoretin. J. Nat. Prod., 47, 84–88 (1984).
• 28) Li L-Z, Gao P-Y, Peng Y, Wang L-H, Yang J-Y, Wu C-F, Zhang Y, Song S-J. Daphnane-type diterpenoids from the flower buds of Daphne genkwa. Helv. Chim. Acta, 93, 1172–1179 (2010).
• 29) Gustafson KR, Cardellina JH 2nd, McMahon JB, Gulakowski RJ, Ishitoya J, Szallasi Z, Lewin NE, Blumberg PM, Weislow OS, Beutler JA, Buckheit RW Jr, Cragg GM, Cox PA, Bader JP, Boyd MR. A nonpromoting phorbol from the samoan medicinal plant Homalanthus nutans inhibits cell killing by HIV-1. J. Med. Chem., 35, 1978–1986 (1992).
• 30) Chen YY, Duan JA, Tang YP, Guo S. Cytotoxic daphnane-type diterpenoids from Daphne genkwa. Chem. Nat. Compd., 50, 163–164 (2014).
• 31) Hong J-Y, Chung H-J, Lee H-J, Park H-J, Lee S-K. Growth inhibition of human lung cancer cells via down-regulation of epidermal growth factor receptor signaling by yuanhuadine, a daphnane diterpene from Daphne genkwa. J. Nat. Prod., 74, 2102–2108 (2011).
• 32) Shao ZY, Shang Q, Zhao NX, Zhang SJ, Xia GP, Bai XX, Dong HL, Han YM. Daphnane diterpene esters from flower buds of Daphne genkwa and their cytotoxic effects on cancer cells. Chin. Tradit. Herb. Drugs, 44, 128–132 (2013).