Anti-inflammatory dimeric furanocoumarins from the roots of Angelica dahurica

    Anti-inflammatory dimeric furanocoumarins from the roots of Angelica dahurica Wan-Qing Yang, Yue-Lin Song, Zhi-Xiang Zhu, Cong Su, Xu Zhang, Juan Wang, She-Po Shi, Peng-Fei Tu PII: DOI: Reference:

S0367-326X(15)30045-9 doi: 10.1016/j.fitote.2015.07.006 FITOTE 3223

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

25 May 2015 3 July 2015 7 July 2015

Please cite this article as: Wan-Qing Yang, Yue-Lin Song, Zhi-Xiang Zhu, Cong Su, Xu Zhang, Juan Wang, She-Po Shi, Peng-Fei Tu, Anti-inflammatory dimeric furanocoumarins from the roots of Angelica dahurica, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.07.006

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ACCEPTED MANUSCRIPT Anti-inflammatory dimeric furanocoumarins from the roots of Angelica

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dahurica

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Wan-Qing Yang a,b,1, Yue-Lin Song a,1, Zhi-Xiang Zhu a, Cong Su a,b, Xu Zhang a,b, Juan

Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine,

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a

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Wanga,b, She-Po Shi a,*, Peng-Fei Tu a

Beijing 100029, People’s Republic of China;

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, People’s

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b

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Republic of China;

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*To whom correspondence should be addressed: Tel/Fax: 86-10-64286350, E-mail: [email protected] (S.-P. Shi).

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These two authors contributed equally to this work.

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ACCEPTED MANUSCRIPT ABSTRACT Seven new dimeric furanocoumarins, dahuribiethrins A–G (1–7), were isolated from the roots

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of Angelica dahurica. Their structures were determined by chemical derivatization and

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extensive spectroscopic techniques, including 1H NMR, 13C NMR, HSQC, 1H-1H COSY, HMBC, and NOESY experiments. Compounds 2, 3, 4, and 5 exhibited significant inhibition of nitric oxide production in the lipopolysaccharide (LPS)-stimulated RAW264.7

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macrophage cells with IC50 values in the range of 8.8–9.8 μM.

Keywords:

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Angelica dahurica; Dimeric furanocoumarin; Coumarin; Anti-inflammatory; Nitric oxide

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production.

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ACCEPTED MANUSCRIPT 1. Introduction Angelica dahurica, belonging to the family Apiaceae, is a perennial plant widely

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distributed in China, Korea, Japan, and Russia. As a well-known herbal medicine, the roots of

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A. dahurica are commonly used for the treatment of headache, toothache, abscess, furunculosis, and acne [1]. Up to date, more than 100 coumarins [2–16] have been obtained from A. dahurica, exhibiting notable and diverse pharmaceutical properties, such as

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anti-tumor [17–18], anti-inflammatory [19–23], anti-oxidative [24], and acetylcholinesterase

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inhibitory activities [25]. Our previous investigations on the coumarins of A. dahurica by LCMS revealed that dimeric furanocoumarins were most probably occurred in this plant with

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trace amount [26]. However, only a handful of dimeric furanocoumarins [16,27] were

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obtained from A. dahurica. On the other hand, condensed furanocoumarins, including dimers,

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trimers, and even tetramers of furanocoumarins, have been frequently reported to be isolated from other Apiaceae plants, such as Heracleum candicans [28–31], Pleurospermum

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rivulorum [32–35], and Ferula sumbul [36]. Attracted by the structural complexity and remarkable bioactivities of this kind of condensed furanocoumarins, an HPLC-MS-guided separation and purification procedure was performed to targetedly isolate the condensed furanocoumarins in A. dahurica. As a result, seven new dimeric furanocoumarins, Dahuribiethrins A–G (1–7), were isolated from the roots of A. dahurica. Herein, the isolation and structural elucidation of the new compounds are described as well as their inhibitory effects on nitric oxide (NO) production in LPS-stimulated RAW264.7 macrophage cells. 2. Experimental 2.1. General Experimental Procedures 3

ACCEPTED MANUSCRIPT Optical rotations were obtained on a Rudolph Autopol IV automatic polarimeter (NJ, USA). IR spectra were recorded on a Thermo Nicolet Nexus 470 FT-IR spectrophotometer

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(MA, USA) with KBr pellets. UV spectra were obtained using a Shimadzu UV-2450

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spectrophotometer (Tokyo, Japan). NMR spectra were recorded on a Varian INOVA-500 spectrometer (CA, USA) operating at 500 MHz for 1H NMR and 125 MHz for 13C NMR. HRESIMS was recorded on an LCMS-IT-TOF system, fitted with a Prominence UFLC

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system and an ESI interface (Shimadzu, Kyoto, Japan). Silica gel (200-300 mesh, Qingdao

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Marine Chemical Inc., Qingdao, People′s Republic of China), LiChroprep RP-C18 gel (40–63 μm, Merck, Germany), and Sephadex LH-20 (Pharmacia) were used for open column

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chromatography (CC). HPLC was performed on a Shimadzu LC-20AT pump system

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(Shimadzu Corporation,Tokyo, Japan), equipped with a SPD-M20A photodiode array

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detector monitoring at 365 nm. A semi-preparative HPLC column (YMC-Pack C18, 250 × 10 mm, 5 μm) was employed for the isolation. TLC was performed using GF254 plates.

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2.2 Plant material

The roots of Angelica dahurica (Fisch. ex Hoffm.) Benth. et Hook. were collected in Bozhou, Anhui province, People’s Republic of China, in December 2013, and plant authentication was performed by one of the authors (P.-F.T.). A voucher specimen (SPSHI-ADB-201312) is deposited in the Modern Research Center for Traditional Chinese Medicine, Beijing University of Chinese Medicine. 2.3. Extraction and isolation The air-dried roots of A. dahurica (30 kg) were refluxed with 80% EtOH (3 × 180 L, 2 h each). After removal of the solvent under reduced pressure, the residue (1.5 kg) was 4

ACCEPTED MANUSCRIPT suspended in H2O (4 L) and partitioned with petroleum ether (4 × 4 L), EtOAc (4 × 4 L), and n-BuOH (4 × 4 L), successively. The EtOAc extract (400 g) was subjected to silica gel CC,

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eluting with a stepwise gradient of petroleum ether–EtOAc (10:1 → 0:1) and then

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CH2Cl2-MeOH (20:1 → 0:1), to afford six fractions (Fr. A–F). Consequently, an HPLC-MS-guided separation and purification procedure [0.1% formic acid aqueous solution (v/v, solvent A) and acetonitrile (solvent B): 0–40 min, 20–45% B; 40–55 min, 45–60% B;

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55–65 min, 60–80% B; 65–70 min, 80–100% B, v/v)] was performed to screen out the

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targeted fractions containing dimeric furanocoumarins. Fr. A (10.0 g) proposed containing dimeric furanocoumarins was subjected to silica gel CC eluting with petroleum ether–EtOAc

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(5:1 → 0:1) and then CH2Cl2–MeOH (15:1 → 0:1) to give nine subfractions (Fr. A1–A9). Fr.

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A8 (2.0 g) containing the targeted compounds was chromatographed over Sephadex LH-20

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CC eluting with CH2Cl2–MeOH (1:1) to yield three portions (Fr. A8a–A8c). Fr. A8b (1.5 g) was resolved by ODS C18 CC eluting with 60% aqueous MeOH to afford SubFr. A8b1–A8b3.

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SubFr. A8b2 (1.0 g) was chromatographed over semi-preparative HPLC using a mobile phase of 75% aqueous MeOH to afford three fractions Sub-Fr. A8b2a–A8b2c. Sub-Fr. A8b2a was separated and purified by semi-preparative HPLC (51% aqueous MeCN) to give compounds 1 (8.0 mg, tR 37.0 min), 3 (1.0 mg, tR 55.0 min), and 4 (5.0 mg, tR 50.5 min). Sub-Fr. A8b2b was separated and purified by semi-preparative HPLC (53% aqueous MeCN) to afford compounds 2 (8.0 mg, tR 53.0 min) and 5 (6.0 mg, tR 47.0 min). Sub-Fr. A8b2c was separated by semi-preparative HPLC (54% aqueous MeCN) to give compounds 6 (1.2 mg, tR 67.0 min) and 7 (2.8 mg, tR 73.0 min). 2.3.1. Dahuribiethrins A (1) 5

ACCEPTED MANUSCRIPT Pale yellow, amorphous powder; [α]25D : -59.1° (c 0.05, MeOH); UV λMeOH max (log ε): 221 (4.67), 249 (4.46), 267 (4.48), 310 (4.34); IR (KBr) νmax: 3433, 2920, 2850, 1735, 1623, 1591,

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603.1840 [M + H]+ (calcd for C33H31O11, 603.1861).

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1478, 1150, 1073 cm−1; 1H and 13C NMR data, see Table 1; positive-ion HRESIMS: m/z

2.3.2. Dahuribiethrins B (2)

Pale yellow, amorphous powder; [α]25D : -20.3° (c 0.06, MeOH); UV λMeOH max (log ε): 222

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(4.60), 249 (4.41), 267 (4.41), 310 (4.31); IR (KBr) νmax: 3439, 2920, 2851, 1732, 1624, 1480,

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1350, 1147, 1070 cm−1; 1H and 13C NMR data, see Table 1; positive-ion HRESIMS m/z 603.1860 [M + H]+ (calcd for C33H31O11, 603.1861).

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2.3.3. Dahuribiethrins C (3)

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Pale yellow, amorphous powder; [α]25D : -29.1° (c 0.10, MeOH); UV λMeOH max (log ε): 219

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(4.41), 248 (4.33), 306 (4.10); IR (KBr) νmax: 3444, 2920, 2851, 1724, 1625, 1587, 1455, 1156, 1132, 1099, 1074 cm−1; 1H and 13C NMR data, see Table 1; positive-ion HRESIMS:

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m/z 573.1763 [M + H]+ (calcd for C32H29O10, 573.1755). 2.3.4. Dahuribiethrins D (4) Yellow, amorphous powder; [α]25D : +19.1° (c 0.06, MeOH); UV λMeOH max (log ε): 220 (4.54), 249 (4.36), 267 (4.33), 308 (4.22); IR (KBr) νmax: 3445, 2919, 2850, 1732, 1627, 1579, 1456, 1153, 1134, 1070 cm−1; 1H and

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603.1868 [M + H]+ (calcd for C33H31O11, 603.1861). 2.3.5. Dahuribiethrins E (5) Pale yellow, amorphous powder; [α]25D : -15.0° (c 0.06, MeOH); UV λMeOH max (log ε): 221 (4.54), 249 (4.34), 267 (4.36), 310 (4.23); IR (KBr) νmax: 3439, 2976, 2851, 1727, 1624, 1480, 6

ACCEPTED MANUSCRIPT 1350, 1147, 1133, 1091, 1070 cm−1; 1H and 13C NMR data, see Table 2; positive-ion HRESIMS: m/z 603.1853 [M + H]+ (calcd for C33H31O11, 603.1861).

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2.3.6. Dahuribiethrins F (6)

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Yellow, amorphous powder; [α]25D : +22.2° (c 0.05, MeOH); UV λMeOH max (log ε): 221 (4.11), 266 (3.95), 309 (3.83); IR (KBr) νmax: 3444, 2921, 2850, 1726, 1626, 1480, 1349, 1138, 1071 cm−1; 1H and 13C NMR data, see Table 3; positive-ion HRESIMS: m/z 603.1853 [M + H]+

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(calcd for C33H31O11, 603.1861).

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2.3.7. Dahuribiethrins G (7)

Cl Pale yellow, amorphous powder; [α]25D : +76.0 (c 0.11, CH2Cl2); UV λCH (log ε): 221 max 2

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(4.01), 266 (3.82), 307 (3.70); IR (KBr) νmax :3442, 2919, 2851, 1724, 1625, 1480, 1350,

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1150, 1135, 1072 cm−1; 1H and 13C NMR data, see Table 3; positive-ion HRESIMS: m/z

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603.1850 [M + H]+ (calcd for C33H31O11, 603.1861). 2.4. Preparation of the (R)- and (S)-MTPA Esters of Compounds 1 and 2

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The (R)- and (S)-MTPA ester derivatives of compounds 1 and 2 were prepared as previously described [37-38]. In brief, 5 μL (R)-(-)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride were added into 400 μL deuterated pyridine solutions of each sample (1 and 2, 2.0 mg). The reaction mixture was homogenized and kept at room temperature for 12 h to get (S)-MTPA esters of Compounds 1 and 2. Similarly, the (R)-MTPA esters of Compounds 1 and 2 were prepared. (R)-MTPA Ester of dahuribiethrins A (8) 1H-NMR (Pyridine-d5) δH 1.76 (3H, s, H-14), 4.98 (1H, br s, H-15a), 5.24 (1H, br s, H-15b), 1.46 (3H, s, H-14′), 1.39(3H, s, H-15′), 5.67 (1H, d, J = 10.0 Hz, H-11′a), 5.26 (1H, t, J = 10.0 Hz, H-11′b). 7

ACCEPTED MANUSCRIPT (S)-MTPA Ester of dahuribiethrins A (9) 1H-NMR (Pyridine-d5) δH 1.79 (3H, s, H-14),

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(1H, d, J = 10.0 Hz, H-11′a), 5.12 (1H, t, J = 10.0 Hz, H-11′b).

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5.00 (1H, br s, H-15a), 5.28 (1H, br s, H-15b), 1.59 (3H, s, H-14′), 1.49(3H, s, H-15′), 5.58

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(R)-MTPA Ester of dahuribiethrins B (10) 1H-NMR (Pyridine-d5) δH 1.89 (3H, s, H-14), 5.05 (1H, br s, H-15a), 5.33 (1H, br s, H-15b), 1.66 (3H, s, H-14′), 1.50(3H, s, H-15′), 5.36 (1H, d, J = 10.0 Hz, H-11′a), 5.12 (1H, t, J = 10.0 Hz, H-11′b).

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(S)-MTPA Ester of dahuribiethrins B (11) 1H-NMR (Pyridine-d5) δH 1.85 (3H, s, H-14),

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5.01 (1H, br s, H-15a), 5.27 (1H, br s, H-15b), 1.53 (3H, s, H-14′), 1.39(3H, s, H-15’), 5.52 (1H, d, J = 10.0 Hz, H-11’a), 5.28 (1H, t, J = 10.0 Hz, H-11’b).

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2.5. Biological assays

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The murine macrophage RAW264.7 cell line was purchased from Peking Union Medical

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College (PUMC) Cell bank (Beijing, People's Republic of China), and was cultured in DMEM supplemented with 10% Fetal Bovine Serum, 100 U/mL penicillin G and 100 μg/mL

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streptomycin, in a humidified 5% CO2 at 37 °C. Cell viability was evaluated using MTT assay. The NO concentration was detected by the Griess method [39–40]. Briefly, RAW264.7 macrophage cells were seeded into 96-well plates at a density of 5 × 104 cells/well and stimulated with 0.5 μg/mL LPS (Sigma, USA) in the presence or absence of test compounds. After incubation for 24 h at 37 °C, treated RAW264.7 macrophage cells were incubated with 100 μL MTT solution (0.5 mg/mL in medium) for another 4 h at 37 °C , subsequently, the supernatants were removed and residues were dissolved using 150 μL DMSO for each well; 50 μL of cell-free supernatant was mixed with 100 μL of Griess reagent containing equal volumes of 2% (w/v) sulfanilamide in 5% (w/v) phosphoric acid and 0.2% (w/v) 8

ACCEPTED MANUSCRIPT N-(1-naphthyl) ethylenediamine solution to measure nitrite production. The absorbance was detected at 540 nm using a microplate reader (Thermo, USA). Compared with a calibration

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curve prepared using NaNO2 standards. The experiments were performed in triplicate.

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Indomethacin was conducted as a positive control. All the compounds were prepared as stock solutions in DMSO (final solvent concentration less than 0.3% in all assays). 3. Results and discussion

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The 80% EtOH extract of the roots of A. dahurica was suspended in H2O and extracted

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successively with petroleum ether, EtOAc, and n-BuOH. The EtOAc-soluble fraction was repeatedly subjected to silica gel, Sephadex LH-20, Lichroprep RP-C18 gel CC, and

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semi-preparative HPLC, to afford seven new dimeric furanocoumarins, dahuribiethrins A–G

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(1–7) (Fig. 1).

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Compound 1 was obtained as a pale yellow, amorphous powder, [α]25D -59.1° (c 0.05, MeOH). Its molecular formula was assigned as C33H30O11 by the [M + H]+ ion peak at m/z

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603.1840 in the HRESIMS spectrum, which was supported by the 13C NMR data (Table 1). The 1H NMR spectrum of 1 showed the presence of a C-5 and C-8 disubstituted linear-type furanocoumarin moiety signals at δH 8.04 (1H, d, J = 10.0 Hz, H-4), 6.19 (1H, d, J = 10.0 Hz, H-3), 7.48 (1H, d, J = 2.5 Hz, H-9), 6.94 (1H, d, J = 2.5 Hz, H-10), a C-5 monosubstituted linear-type furanocoumarin moiety signals at δH 8.14 (1H, d, J = 10.0 Hz, H-4′), 6.08 (1H, d, J = 10.0 Hz, H-3′), 7.46 (1H, d, J = 2.0 Hz, H-9′), 6.97 (1H, d, J = 2.0 Hz, H-10′), 6.94 (1H, s, H-8′), a 3-methyl-3-butenyl-1,2-dioxy group signals at δH 4.13 (1H, m, H-11a), 4.15 (1H, m, H-11b), 4.65 (1H, dd, J = 7.5, 3.0 Hz, H-12), 4.98 (1H, br s, H-15a), 5.18 (1H, br s, H-15b), 1.80 (3H, s), a 2-hydroxy-3-methylbutyl-1,3-dioxy group signals at δH 4.52 (1H, m, H-11′a), 9

ACCEPTED MANUSCRIPT 4.85 (1H, m, H-11′b), 4.17 (1H, m, H-12′), 1.44, 1.35 (each 3H, s), and a methoxy group signal at δH 4.18 (3H, s). The 1H NMR data mentioned above suggested that compound 1

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might be a dimeric furanocoumarin, which was also supported by the 13C NMR data of 1

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(Table 1). All the protons and carbons were unambiguously assigned by 2D NMR experiments, including HSQC, 1H-1H COSY, and HMBC. In the HMBC spectrum of 1 (Fig. 2), the long range correlation between the protons at δH 4.18 (3H, s) and C-5 (δC 144.3)

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revealed that the methoxy group was attached at C-5. Additionally, the long range

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correlations of H-11/C-8, and H-11′/C-5′ established that the groups of 3-methyl-3-butenyl-1,2-dioxy and 2-hydroxy-3-methylbutyl-1,3-dioxy were attached at C-8

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and C-5′, respectively, suggesting that compound 1 was a condensed furanocoumarin formed

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by neobyakangelicol and oxypeucedaninhydrate. Comparison of the NMR data with those of

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the known compounds neobyakangelicol and oxypeucedaninhydrate, allowed the establishment of the planar structure of 1 was as shown in Fig. 1.

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The absolute configuration of C-12′ was resolved by modified Mosher’s method. The treatment of 1 with (R)-(-)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride, and (S)-(+)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride gave the (S)-MTPA ester (9) and (R)-MTPA ester (8) of 1, respectively. The 1H NMR of two MTPA esters indicated that the absolute configuration of C-12′ was to be S form (Fig. 3). However, determination of the absolute configuration of C-12 was frustrated by this method. Accordingly, the structure of 1 was elucidated as shown in Fig. 1, named as dahuribiethrins A. Compound 2 was obtained as a pale yellow, amorphous powder, [α]25D -20.3° (c 0.06, MeOH). Its molecular formula was determined as C33H30O11 by the presence of a [M + H]+ 10

ACCEPTED MANUSCRIPT ion peak at m/z 603.1860 in the HRESIMS spectrum. Comparison of the NMR data with those of 1 revealed that compound 2 shared a very similar skeleton with that of 1.

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Unambiguous assignment of the protons and carbons of 2 by 2D NMR experiments allowed

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to establish the planar structure of 2, which was completely same to that of 1. However, when compounds 1 and 2 were analyzed by HPLC using the same analytic method, the absolutely different retention time of compounds 1 and 2 suggested that these two compounds are a pair

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of isomers. Thus, a modified Mosher’s method was applied, which led to determine the

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absolute configuration of C-12′ was to be R form (Fig. 3). Accordingly, the structure of 2 was assigned as shown in Fig. 1, named as dahuribiethrins B.

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Compound 3 was isolated as a pale yellow amorphous powder, with a molecular formular

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of C32H28O10 deduced from the [M + H]+ ion peak at m/z 573.1763 in the HRESIMS

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spectrum. The 1H NMR and 13C NMR spectra of 3 were closely resemble to those of 2, except that the signal due to a methoxy group in 2 was replaced by an aromatic proton

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presented at δH 7.36 (1H, s, H-5). Unambiguous assignment of the protons and carbons by 1H NMR, 13C NMR, and 2D NMR, including HSQC, 1H-1H COSY, and HMBC experiments led to establish the planar structure of 3 as shown in Fig. 1. Restricted by the small amount of 3, the absoluted configuration were not determined. However, the similar optical activities of compounds 2 and 3 allowed to tentively determine the structure of 3 as shown in Fig. 1, named as dahuribiethrins C. Compound 4 was isolated as a pale yellow, amorphous powder, [α]25D +19.1° (c 0.06, MeOH). The HRESIMS spectrum exhibited the presence of a quasimolecular ion peak [M + H]+ at m/z 603.1868, corresponding to the molecular formula C33H30O11. Analysis of the 1H 11

ACCEPTED MANUSCRIPT NMR and 13C NMR data (Table 2) of 4 revealed that 4 was formed by the condensation of byakangelicin and pabulenol, which was further confirmed by 2D NMR experiments,

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including HSQC, 1H-1H COSY, and HMBC (Fig. 2). In the HMBC spectrum, the long-range

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correlation between H-12′ and C-13 led to establish the structure of 4 as shown in Fig. 1, named as dahuribiethrins D.

Compound 5 was obtained as a pale yellow, amorphous powder, [α]25D -15.0° (c 0.10,

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MeOH). Its molecular formula was assigned as C33H30O11 by the [M + H]+ ion peak at m/z

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603.1853 in the HRESIMS spectrum. Comparison of the NMR data of 5 with those of 4 suggested that the planar structure of 5 was completely identical with that of 4. However,

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compounds 4 and 5 showed opposite optical activities with the value of +19.1° and -15.0°,

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respectively, which suggested that compounds 4 and 5 are a pair of isomers. Further analyzed

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by HPLC revealed that 4 and 5 were separable on an Agilent Eclipse XDB C18 column (250 × 4.6 mm, 5 µm). In consequence, the structure of 5 was established as shown in Fig. 1, named

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as dahuribiethrins E.

Compound 6 was obtained as a pale yellow, amorphous powder, [α]25D +22.2° (c 0.05, MeOH). The 1H NMR and 13C NMR spectra indicated the presence of a C-5 monosbustitued linear-type furanocoumarin moiety, a C-5 and C-8 disubstituted linear-type furanocoumarin moiety, and two 2-hydroxy-3-methylbutyl-1,3-dioxy groups. The HRESIMS spectrum of 6 indicated the presence of a [M + H]+ ion peak at m/z 603.1853, in accordance with an empirical molecular formula of C33H30O11. Although a terminal double bond due to the 3-methyl-3-butenyl-1,2-dioxy group in 1 was replaced by a saturated hydrocarbon bond in 6, the molecular formula and the calculated indices of hydrogen deficiency of compounds 6 and 12

ACCEPTED MANUSCRIPT 1 are exactly identical, suggesting the formation of a dioxane between the two 2-hydroxy-3-methylbutyl-1,3-dioxy groups in 6. Analysis of the 2D NMR spectra, including

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HSQC, 1H-1H COSY, and HMBC led to the establishment of the planar structure of 6. The

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relative configuration of H-12 and H-12′ was achieved by NOESY experiment. In the NOESY spectrum of 6 (Fig. 4), both of H-12 and H-12′ indicated correlations with H-15 and H-15′, which suggested that H-12 and H-12′ were in cis-orientation. Therefore, the structure

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of 6 was elucidated as shown in Fig. 1, named as dahuribiethrins F.

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Compound 7 was isolated as a yellow, amorphous powder , [α]25D +76.0° (c 0.11, CH2Cl2). The molecular formula C33H30O11 of 7 was determined same as that of 6 from the 13C NMR

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and HRESIMS data. Unambiguous assignment of the protons and carbons by 1H NMR, 13C

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NMR and 2D NMR experiments achieved the establishment of the planar structure of 7,

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which was exactly identical to that of 6. Additionally, the NOESY correlations between H-14/14′and H-12′, H-15/15′ and H-12 suggested that H-12 and H-12′ were in

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trans-orientation (Fig. 4). Additionally, when compounds 6 and 7 were analyzed by HPLC using same analytic method, the absolutely different retention time of compounds 6 and 7 confirmed that these two compounds are a pair of isomers. Thus, the structure of 7 was elucidated as shown in Fig.1, named as dahuribiethrins G. Compounds 1-7 were evaluated for their inhibitory effects on the NO production in LPS-stimulated RAW264.7 macrophage cells. Compounds 2, 3, 4, and 5 exhibited potent inhibition of NO production with IC50 values of 8.8 μM, 9.2 μM, 9.6μM, and 9.8μM, respectively (Table 4). Although compounds 1 and 2 share a completely identical planar structure, compound 1 did not indicated inhibitory effects on NO production. On the other 13

ACCEPTED MANUSCRIPT hand, both 4 and 5, a pair of isomers sharing identical planar structures, indicated notable inhibitory activities. It seems that stereochemical configurations curiously related to

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inhibitory activities. Unfortunately, restricted by the low yield, unambiguous determination

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of the absolute configurations of these dimeric furanocoumarins by preparation of crystals for X-ray diffraction analysis were frustrated. Therefore, a scale-up separation should be needed,

these structural interesting furanocoumarins.

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Acknowledgment

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which would be undoubtedly advantage to investigate the structure-activity relationship of

We are thankful to the financial supported by Beijing Natural Science Foundation (No.

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5132022) and the Program for New Century Excellent Talents in University (No.

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NCET-11-0604) and graduate students independent subject of Beijing University of Chinese

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Medicine (No. 2015-JYBZZ-XS-080)

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ACCEPTED MANUSCRIPT Reference [1] Chinese Pharmacopoeia Commission. Pharmacopoeia of the People's Republic of China,

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volume I. Beijing: Chemical Industry Press; 2010 97.

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[2] Kwon YS, Shin SJ, Kim MJ, Kim CM. A new coumarin from the stem of Angelica dahurica. Arch Pharm Res 2002;25:53-6.

[3] Kwon YS, Kobayashi A, Kajiyama SI, Kawazu K, Kanzaki H, Kim CM. Antimicrobial

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constituents of Angelica dahurica roots. Phytochemistry 1997;44:887-9.

MA

[4] Thuong PT, Hung TM, Ngoc TM, Ha DT, Min BS, Kwack SJ, et al. Antioxidant activities of coumarins from Korean medicinal plants and their structure–activity

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relationships. Phytother Res 2010;24:101-6.

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[5] Thanh PN, Jin WY, Song GY, Bae K, Kang SS. Cytotoxic coumarins from the root of

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Angelica dahurica. Arch Pharm Res 2004;27:1211-5. [6] Baek NI, Ahn EM, Kim HY, Park YD. Furanocoumarins from the root of Angelica

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dahurica. Arch Pharm Res 2000;23:467-70. [7] Liu DP, Luo Q, Wang GH, Xu Y, Zhang XK, Chen QC, et al. Furocoumarin derivatives from radix Angelicae dahuricae and their effects on RXRα transcriptional regulation. Molecules 2011;16:6339-48. [8] Seo WD, Kim JY, Ryu HW, Kim JH, Han SI, Ra JE, et al. Identification and characterisation of coumarins from the roots of Angelica dahurica and their inhibitory effects against cholinesterase. J Funct Foods 2013;5:1421-31. [9] Lee SH, Li G, Kim HJ, Kim JY, Chang HW, Jahng Y, et al. Two new furanocoumarins from the roots of Angelica dahurica. B Kor Chem Soc 2003;24:1699-701. 15

ACCEPTED MANUSCRIPT [10] Kim SH, Kang SS, Kim CM. Coumarin glycosides from the roots of Angelica dahurica. Arch Pharm Res 1992;15:73-7.

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[11] Zhao XZ, Feng X, Jia XD, Dong YF, Wang M. Neolignan glycoside from Angelica

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dahurica. Chinese Chem Lett 2007;18:168-70.

[12] Zhao XZ, Feng X, Jia XD, Wang M, Shan Y, Dong YF. New coumarin glucoside from Angelica dahurica. Chem Nat Compd 2007;43:399-401.

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[13] Lu J, Jin L, Jin YS, Chen HS. Chemical constituents in roots of Angelica dahurica var.

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formosana. Acad J Sec Mil Med Univ 2007;28:294-8. [14] Jia XD, Zhao XZ, Fen X, Wang M, Sun H, Dong YF. Coumarins of Angelica dahurica

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var. formosana (II). Chin Trad Herb Drugs 2008;39:1768-71.

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[15] Zhao AH, Yang XW, Yang XB, Liu JX, Wang QL, Wang WQ. A new natural product

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from root of Angelica dahurica cv. Qibaizhi. Chin J Chin Mat Med 2012;37:2400-7. [16] Deng GG, Wei W, Yang XW, Zhang YB, Xu W, Gong NB, et al. New coumarins from the

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roots of Angelica dahurica var. formosana cv. Chuanbaizhi and their inhibition on NO production in LPS-activated RAW264.7 cells. Fitoterapia 2015;101:194-200. [17] Luo KW, Sun JG, Chan JY, Yang L, Wu SH, Fung KP, et al. Anticancer effects of imperatorin isolated from Angelica dahurica: induction of apoptosis in HepG2 cells through both death-receptor-and mitochondria-mediated pathways. Chemotherapy 2010;57:449-59. [18] Choochuay K, Chunhacha P, Pongrakhananon V, Luechapudiporn R, Chanvorachote P. Imperatorin sensitizes anoikis and inhibits anchorage-independent growth of lung cancer cells. J Nat Med 2013;67:599-606. 16

ACCEPTED MANUSCRIPT [19] Hua JM, Moon TC, Hong TG, Park KM, Son JK, Chang HW. 5-Methoxy-8-(2-hydroxy-3-buthoxy-3-methylbutyloxy)-psoralen isolated from Angelica

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dahurica inhibits cyclooxygenase-2 and 5-lipoxygenase in mouse bone marrow-derived

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mast cells. Arch Pharm Res 2008;31:617-21.

[20] Lee MY, Seo CS, Lee JA, Lee NH, Kim JH, Ha H, et al. Anti-asthmatic effects of Angelica dahurica against ovalbumin-induced airway inflammation via upregulation of

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heme oxygenase-1. Food Chem Toxicol 2011;49:829-37.

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[21] Kang OH, Chae HS, Oh YC, Choi JG, Lee YS, Jang JH, et al. Anti-nociceptive and anti-inflammatory effects of Angelicae dahuricae radix through inhibition of the

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2008;36:913-28.

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expression of inducible nitric oxide synthase and NO production. Am J Chin Med

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[22] Ban HS, Lim SS, Suzuki K, Jung SH, Lee YS, Shin KH, et al. Inhibitory effects of furanocoumarins isolated from the roots of Angelica dahurica on prostaglandin E2

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production. Planta Med 2003;69:408-12. [23] Lee MY, Lee JA, Seo CS, Ha H, Lee H, Son JK, et al. Anti-inflammatory activity of Angelica dahurica ethanolic extract on RAW264.7 cells via upregulation of heme oxygenase-1. Food Chem Toxicol 2011;49:1047-55. [24] Piao XL, Park IH, Baek SH, Kim HY, Park MK, Park JH. Antioxidative activity of furanocoumarins isolated from Angelicae dahuricae. J Ethnopharmacol 2004;93:243-6. [25] Kim DK, Lim JP, Yang JH, et al. Acetylcholinesterase inhibitors from the roots of Angelica dahurica. Arch Pharm Res 2002;25:856-9.

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ACCEPTED MANUSCRIPT [26] Li B, Zhang X, Wang J, Zhang L, Gao BW, Shi SP, et al. Simultaneous characterisation of fifty coumarins from the roots of Angelica dahurica by off-line two-dimensional

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mass spectrometry. Phytochem Analysis 2014;25:229-40.

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high-performance liquid chromatography coupled with electrospray ionisation tandem

[27] Wang NH, Yoshizaki K, Baba K. Seven new bifuranocoumarins dahuribirin A-G from Japanese Bai Zhi. Chem Pharm Bull 2001;49:1085-8.

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[28] Taniguchi M, Inoue A, Shibano M, Wang NH, Baba K. Five condensed furanocoumarins

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from the root of Heracleum candicans Wall. J Nat Med 2011;65:268-74. [29] Nakamori T, Taniguchi M, Shibano M, Wang NH, Baba K. Chemical studies on the root

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of Heracleum candicans WALL. J Nat Med 2008;62:403-12.

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[30] Inoue A, Taniguchi M, Shibano M, Wang NH, Baba K. Chemical studies on the root of

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Heracleum candicans Wall.(Part 3). J Nat Med 2010;64:175-81. [31] Doi M, Nakamori T, Shibano M, Taniguchi M, Wang NH, Baba K. Candibirin A, a

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furanocoumarin dimer isolated from Heracleum candicans WALL. Acta Crystallogr C 2004;60:833-5.

[32] Xiao YQ, Liu XH, Taniguchi M, Baba K. Bicoumarins from Pleurospermum rivulorum. Phytochemistry 1997;45:1275-7. [33] Taniguchi M, Xiao YQ, Liu XH, Yabu A, Hada Y, Guo LQ, et al.. Rivulobirin E and Rivulotririn C from Pleurospermum rivulorum. Chem Pharm Bull 1999;47:713-5. [34] Taniguchi M, Xiao YQ, Baba K. Three novel cyclospirobifuranocoumarins, cyclorivulobirins A-C, from Pleurospermum rivulorum. Chem Pharm Bull 2000;48:1246-7. 18

ACCEPTED MANUSCRIPT [35] Taniguchi M, Xiao Y Q, Liu XH, Yabu A, Hada Y, Baba K. Rivulobirins C and D, two novel new spirobicoumarins, from the underground part of Pleurospermum rivulorum.

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Chem Pharm Bull 1998;46:1065-7.

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[36] Zhou P, Takaishi Y, Duan H, Chen B, Honda G, Itoh M, et al. Coumarins and bicoumarin from Ferula sumbul: anti-HIV activity and inhibition of cytokine release. Phytochemistry 2000;53:689-97.

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[37] Su BN, Park EJ, Mbwambo ZH, Santarsiero BD, Mesecar AD, Fong HHS, et al. New

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chemical constituents of Euphorbia quinquecostata and absolute configuration assignment by a convenient mosher ester procedure carried out in NMR tubes. J Nat Prod

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2002;65:1278-82.

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[38] Li J, Du L, Kelly M, Zhou YD, Nagle DG. Structures and potential antitumor activity of

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sesterterpenes from the marine sponge Hyrtios communis. J Nat Prod 2013;76:1492-7. [39] Su XQ, Song YL, Zhang J, Huo HX, Huang Z, Zheng J, et al. Dihydrochalcones and

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homoisoflavanes from the red resin of Dracaena cochinchinensis (Chinese dragon's blood). Fitoterapia 2014;99:64-71. [40] Li MM, Su XQ, Sun J, Gu YF, Huang Z, Zeng KW, et al. Anti-inflammatory ursane-and oleanane-type triterpenoids from Vitex negundo var. cannabifolia. J Nat Prod 2014;77: 2248-54.

19

ACCEPTED MANUSCRIPT Table 1 H (500 MHz) and 13C (125 MHz) NMR data of compounds 1–3 a (δ in ppm, in CDCl3) 2a

δH, multi. (J in Hz)

2

δC, type

3a

δH, multi. (J in Hz)

160.2, C

δC, type 160.0, C

δH, multi. (J in Hz)

T

1a position

IP

1

δC, type 160.0, C

6.19, d (10.0)

112.7, CH

6.24, d (9.5)

112.8, CH

6.15, d (9.5)

112.5, CH

4

8.04, d (10.0)

139.2, CH

8.09, d (9.5)

139.3, CH

7.73, d (9.5)

144.1, CH

107.4, C

5

144.3, C

6

114.3, C

7

149.9, C

8

126.7, C

8a

143.6, C

107.4, C 144.8, C

NU

4a

SC R

3

116.5, C 7.36, s

113.8, CH

114.3, C

126.0, C

150.0, C

147.8, C

126.5, C

131.4, C

143.9, C

143.2, C

7.48, d (2.5)

144.8, CH

7.52, d (2.0)

144.8, CH

7.62, d (2.5)

146.7, CH

10

6.94, d (2.5)

105.2, CH

6.98, d (2.0)

105.4, CH

6.81, d (2.5)

106.9, CH

11

4.13, m

77.0, CH2

4.18, dd (9.5, 2.5)

77.1, CH2

4.36, m

76.7, CH2

4.15, m

4.22, dd (9.5, 8.5)

4.65, dd (7.5, 3.0)

13

75.7, CH 144.5, C

4.70, dd (8.5, 2.5)

D

12

MA

9

4.37, m 75.5, CH

4.70, dd (8.0, 3.0)

144.1, C

75.4, CH 144.1, C

1.80, s

18.9, CH3

1.80, s

18.9, CH3

1.83, s

18.9, CH3

15

4.98, br s

113.5, CH2

4.98, br s

113.5, CH2

5.01, br s

113.7, CH2

TE

14

5-OCH3

4.18, s

2′

60.7, CH3

CE P

5.18, br s

5.18, br s 4.19, s

5.20, br s 60.7, CH3

161.1, C

161.2, C

161.3, C

6.08, d (10.0)

112.1, CH

6.08, d (9.5)

112.4, CH

6.33, d (10.0)

114.8, CH

4′

8.14, d (10.0)

139.4, CH

8.25, d (9.5)

139.8, CH

8.25, d (10.0)

139.7, CH

4′a 5′ 6′ 7′ 8′

AC

3′

6.94, s

8′a

106.9, C

107.5, C

107.3, C

149.1, C

149.1, C

149.1, C

113.8, C

114.2, C

114.1, C

157.9, C

158.0, C

158.0, C

93.8, CH

7.06, s

94.2, CH

152.3, C

7.08, s

152.3, C

94.2, CH 152.5,C

9′

7.46, d (2.0)

144.6, CH

7.51, d (2.0)

145.0, CH

7.50, d (2.0)

144.8,CH

10′

6.97, d (2.0)

105.2, CH

7.00, d (2.0)

105.0, CH

6.99, d (2.0)

105.0, CH

11′

4.52, dd (9.5, 8.0)

74.6, CH2

4.54, dd (10.0, 8.5)

74.4, CH2

4.53, dd (10.0, 8.0)

74.4, CH2

4.85, dd (9.5, 2.0) 12′

4.17, m

13′

4.65, dd (10.0, 2.5) 76.8, CH

4.12, dd (8.5, 2.5)

77.7, C

4.65, dd (10.0, 3.0) 75.6, CH

4.11, dd (8.0, 3.0)

77.8, C

75.8, CH 78.0, C

14′

1.44, s

21.4, CH3

1.41, s

22.0, CH3

1.40, s

22.0, CH3

15′

1.35, s

23.9, CH3

1.34, s

23.8, CH3

1.35, s

23.8, CH3

a

Assignments were carried out based on HSQC and HMBC experiments.

20

ACCEPTED MANUSCRIPT Table 2 H (500 MHz) and 13C (125 MHz) NMR data of compounds 4–5 a (δ in ppm, in CDCl3)

position

5a

δH, multi. (J in Hz)

δH, multi. (J in Hz)

δC, type

160.1, C

160.1, C

3

6.20, d (10.0)

112.6, CH

6.20, d (10.0)

112.6, CH

4

8.03, d (10.0)

139.3, CH

8.05, d (10.0)

SC R

2

δC, type

5

144.5, C

6

114.3, C

7

140.2, C

8

126.9, C

8a

144.3, C

144.6, C 114.0, C

9

7.53, d (2.0)

145.0, CH

10

6.95, d (2.0)

105.3, CH

11

4.28, dd(10.0, 8.5)

75.6, CH2

4.00, dd (8.5, 3.0)

127.1, C 143.9, C 145.0, CH

6.96, d (2.0)

105.3, CH

4.28, dd (10.0, 8.5)

75.7, CH2

4.22, dd (10.0, 3.5)

76.2, CH 78.2, C

TE

13

150.2, C

7.51, d (2.0)

D

4.62, dd(10.0, 3.0) 12

139.4, CH 107.2, C

NU

107.3, C

MA

4a

4.08, dd (8.5, 3.5)

76.1, CH 78.2, C

1.32, s

22.4, CH3

1.32, s

21.9, CH3

15

1.39, s

22.8, CH3

1.38, s

24.0, CH3

5-OCH3

4.18, s

60.6, CH3

4.18, s

60.6, CH3

CE P

14

2′

161.1, C

161.0, C

6.15, d (10.0)

112.6, CH

6.14, d (10.0)k

112.7, CH

4′

8.05, d (10.0)

139.1, CH

8.10, d (10.0)

139.1, CH

5′ 6′ 7′ 8′

AC

3′

4′a

7.08, s

8′a

107.0, C

107.2, C

148.6, C

148.5, C

113.8, C

114.0, C

158.0, C

158.0, C

94.3, CH

7.09, s

94.5, CH

152.5,C

152.5,C

9′

7.55, d (2.0)

144.9,CH

7.55, d (2.0)

145.0,CH

10′

6.96, d (2.0)

104.9, CH

6.98, d (2.0)

104.9, CH

11′

4.22, dd (9.5, 4.0)

75.3, CH2

4.36, dd (10.0, 8.0)

75.6, CH2

4.36, dd (9.5, 7.0) 12′

4.53, dd (7.0, 4.0)

13′

T

4a

IP

1

4.60, dd (10.0, 3.0) 74.9, CH

4.63,dd (8.0, 3.0)

143.8, C

75.0, CH 144.4, C

14′

1.82, s

18.5, CH3

1.80, s

18.6, CH3

15′

5.04, br s

114.1, CH2

5.01, br s

114.3, CH2

5.19, br s a

5.17, br s

Assignments were carried out based on HSQC and HMBC experiments.

21

ACCEPTED MANUSCRIPT Table 3 H (500 MHz) and 13C (125 MHz) NMR data of compounds 6–7 a (δ in ppm, in CDCl3)

position

7a

δH, multi. (J in Hz)

δH, multi. (J in Hz)

δC, type

160.3, C

160.3, C

3

6.30, d (10.0)

113.0, CH

6.29, d (10.0)

112.8, CH

4

8.14, d (10.0)

139.4, CH

8.13, d (10.0)

SC R

2

δC, type

5

144.5, C

6

114.7, C

7

150.1, C

8

127.1, C

8a

143.9, C

144.6, C 114.4, C

9

7.64, d (2.0)

145.1, CH

10

7.02, d (2.0)

105.2, CH

11

4.34, m

73.5, CH2

150.4, C 127.3, C 144.0, C

7.63, d (2.5)

145.1, CH

7.01, d (2.5)

105.2, CH

4.22, dd (10.5, 7.0)

73.9, CH2

4.26, dd (10.5, 4.0 )

4.09, dd (7.0, 4.0)

74.8, C

TE

13

73.3, CH

D

4.39, m 12

139.4, CH 107.6, C

NU

107.6, C

MA

4a

4.09, dd (7.0, 4.0)

75.5, CH 72.6, C

1.26, s

22.5, CH3

1.28, s

17.9, CH3

15

1.35, s

23.7, CH3

1.33, s

26.4, CH3

5-OCH3

4.19, s

60.8, CH3

4.19, s

60.8, CH3

CE P

14

2′

161.2, C

161.1, C

6.32, d (10.0)

112.9, CH

6.31, d (10.0)

112.1, CH

4′

8.22, d (10.0)

139.4, CH

8.22, d (10.0)

139.4, CH

5′ 6′ 7′ 8′

AC

3′

4′a

7.19, s

8′a

107.3, C

106.9, C

148.7, C

149.1, C

113.8, C

113.8, C

158.1, C

157.9, C

94.5, CH

7.19, s

93.8, CH

152.6,C

152.3,C

9′

7.61, d (2.5)

145.1,CH

7.60, d (2.0)

144.6,CH

10′

6.96, d (2.5)

104.8, CH

6.96, d (2.0)

105.2, CH

11′

4.30, m

72.9, CH2

4.17, dd (10.0, 7.5)

74.6, CH2

4.36, m 12′

4.02, dd (7.5, 3.5)

13′

4.33, dd (10.0, 3.0) 73.2, CH

3.98, dd (7.5, 3.0)

74.0 C

76.8, CH 71.7, C

14′

1.16, s

22.4, CH3

1.17, s

17.7, CH3

15′

1.22, s

23.4, CH3

1.21, s

26.3, CH3

a

Assignments were carried out based on HSQC and HMBC experiments.

22

T

6a

IP

1

ACCEPTED MANUSCRIPT Table 4 Inhibitory Effects of Compounds 1–7 on LPS-Stimulated NO Production in RAW264.7

IC50 (μM)b

2

9.6 ± 0.3

3

9.8 ± 0.2

4

8.8 ± 0.4

5

9.2 ± 0.2

Indomethacinc

23.6 ± 0.4

NU

MA

a

SC R

compound a

IP

T

Macrophage cells

Compunds 1, 6 and 7 were inactive (< 50% inhibition b

at 10 μM). Values are represented as means ± SD based

AC

CE P

TE

D

on three independent experiments. cPositive control.

23

ACCEPTED MANUSCRIPT Figures and Legends Fig. 1 Structure of compounds 1-7

T

Fig. 2 Key HMBC (H→C) correlations of compounds 1–7.

IP

Fig. 3 Δδ (δS − δR) values (in ppm, data obtained in pyridine-d5) for the MTPA esters of

SC R

compounds 1 and 2.

AC

CE P

TE

D

MA

NU

Fig. 4 Key NOE (H–H) correlations of compounds 6 and 7.

24

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 1

25

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 2

26

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 3

27

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

SC R

IP

T

Fig. 4

28

ACCEPTED MANUSCRIPT Graphical Abstract

T

Anti-inflammatory dimeric furanocoumarins from the roots of Angelica

SC R

IP

dahurica

Wan-Qing Yang a,b,1, Yue-Lin Song a,1, Zhi-Xiang Zhu a, Cong Su a,b, Xu Zhang a,b, Juan

CE P

AC

Angelica dahurica

TE

D

MA

NU

Wanga,b, She-Po Shi a,*, Peng-Fei Tu a

29