Plant material
Radix Wikstroemiae was purchased from a Chinese medicine pharmacy in Guangzhou, China. The authentication process was carried out by Zhengqiu Mai (Chinese Medicinal Material Company, Guangzhou, China) according to standard protocols [2]. A voucher specimen was deposited in the Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University (Guangzhou, China) with an accession number of 06111205.
General experimental procedures
Optical rotations were determined on a Jasco P-1020 digital polarimeter (JASCO Corporation, Japan). The spectra of electrospray ionization-mass spectrometry (ESI-MS) were recorded on a Finnigan LCQ Advantage Max ion trap mass spectrometer (Thermo Finnigan, USA). The spectra of high resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) were acquired with a Micromass Q-TOF mass spectrometer (Waters Corporation, USA). The spectra of nuclear magnetic resonance spectrometry (NMR) including proton magnetic resonance spectrometry (1H-NMR) and carbon magnetic resonance spectrometry (13C-NMR) were obtained on a Bruker spectrometer (Bruker Corporation, Switzerland) operating at 500 MHz for 1H-MNR and 125 MHz for 13C-NMR respectively. The isolation process was conducted on silica gel (200-300 meshes, Qingdao Marine Chemical, China), Sephadex LH-20 (25-100 μm, Fluka, Switzerland) and semi-preparative HPLC. Semi-preparative HPLC was performed on an Eclipse XDB-C18 column (9.4 mm ID × 25 cm) (Agilent Technologies, USA). Thin layer chromatography (TLC) was carried out on silica gel GF254 plates (0.2 mm thickness, 10 × 20 cm, Qingdao Marine Chemical, China) with FeCl3-EtOH reagent and ultraviolet (UV) illumination as chromogenic methods.
Extraction and isolation
The dried and cut Radix Wikstroemiae (10.0 kg) was soaked in 95% ethanol at room temperature for three times (15 days each time). The ethanol solutions were then combined and concentrated in vacuo to yield a dark brown crude extract (1.0 kg). The ethanol extract was suspended in distilled water and partitioned with petroleum ether, ethyl acetate and n-butanol successively. After evaporation under reduced pressure, the petroleum ether fraction (5.0 g), ethyl acetate fraction (580.0 g) and butanol fraction (400.0 g) were obtained respectively.
The ethyl acetate fraction (550.0 g) was chromatographed on a silica gel column eluted with a solvent system of petroleum ether/ethyl acetate in gradient to obtain 28 subfractions based on the TLC analysis. The subfractions Fr-12 and Fr-23, which yielded positive reaction with FeCl3-EtOH reagent, were further isolated.
Fr-12 (12.6 g) was isolated with repeated silica gel columns eluted with gradient solvent systems of chloroform/acetone and chloroform/methanol, respectively, followed by Sephadex LH-20 column eluted with chloroform/methanol (3/7) to yield compound I (20.0 mg).
Fr-23 (12.6 g) was subjected to repeated silica gel columns eluted with gradient solvent system of chloroform/methanol, followed by semi-preparative HPLC on an Eclipse XDB-C18 column (Agilent Technologies, USA) with a gradient solvent system of 0.1% TFA (A) and methanol (B) to yield compounds II (40.7 mg), III (60.3 mg) and IV (20.7 mg).
Identification
Compound I (neochamaejasmin B), an amorphous brown powder, was positive to FeCl3-EtOH reagent. [α]D = 200° (c 0.1, MeOH). ESI-MS: m/z 541 [M-H] - .1 H-NMR (500 MHz, CD3OD) δ: 7.17 (2H, d, J = 8.4 Hz, H-2''', H-6'''), 6.94 (2H, d, J = 8.4 Hz, H-2', H-6'), 6.81 (2H, d, J = 8.4 Hz, H-3''', H-5'''), 6.67 (2H, d, J = 8.4 Hz, H-3', H-5'), 6.00 (1H, d, J = 1.8 Hz, H-8''), 5.89 (1H, d, J = 1.8 Hz, H-8), 5.80 (1H, d, J = 1.8 Hz, H-6''), 5.78 (1H, d, J = 1.8 Hz, H-6), 5.57 (1H, d, J = 4.6 Hz, H-2), 5.16 (1H, d, J = 8.8 Hz, H-2''), 3.29 (1H, dd, J = 3.2, 8.8 Hz, H-3''), 3.16 (1H, brs, H-3). 13C-NMR (125 MHz, CD3OD) δ: 198.6 (C-4''), 196.3 (C-4), 168.3 (C-7''), 168.1 (C-7), 165.1 (C-8''a), 165.4 (C-5''), 165.2 (C-5), 163.4 (C-8a), 159.0 (C-4'''), 158.6 (C-4'), 130.3 (C-2''', 6'''), 129.1 (C-1'''), 128.8 (C-1'), 128.5 (C-2', 6'), 116.4 (C-3''', 5'''), 116.2 (C- 3', 5'), 105.1 (C-4''a) ,103.9 (C-4a), 97.3 (C-6''), 97.1 (C-6), 96.4 (C-8''), 96.0 (C-8), 83.3 (C-2''), 81.5 (C-2), 50.8 (C-3''), 49.7 (C-3).
Compound II (genkwanol B), a light yellow powder, was positive to FeCl3-EtOH reagent. [α]D = -160° (c 0.1, MeOH). HR-ESI-MS m/z: 557.10805 [M-H] - .1 H-NMR (500 MHz, DMSO-d
6
) δ: 7.09 (2H, d, J = 8.5 Hz, H-2''', H-6'''), 6.73 (2H, d, J = 8.5 Hz, H-3''', H-5'''), 6.58 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.51 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.11 (1H, d, J = 2.0 Hz, H-8''), 6.04 (1H, d, J = 2.0 Hz, H-6''), 5.71 (1H, s, H-6), 4.56 (1H, d, J = 8.2 Hz, H-2), 3.55 (1H, m, H-3), 2.59 (1H, dd, J = 8.9, 16.7 Hz, H-4), 2.05 (1H, dd, J = 5.0, 16.7 Hz, H-4). 13C-NMR (125 MHz, DMSO-d
6
) δ: 190.4 (C-4''), 186.3 (C-5), 168.6 (C-7), 167.7 (C-7''), 163.3 (C-5''), 160.1 (C-8''a), 158.1 (C-4'''), 157.9(C-8a), 156.7 (C-4'), 129.8 (C-2''', 6'''), 127.9 (C-1'), 127.3 (C-2', 6'), 122.0 (C-1'''), 114.6 (C-3''', 5'''), 114.4 (C-3', 5'), 109.2 (C-4a) ,100.4 (C-6), 99.8 (C-4''a), 97.0 (C-6''), 96.1 (C-8''), 89.9 (C-2''), 85.0 (C-8), 82.0 (C-2), 79.8 (C-3''), 66.4 (C-3), 27.0 (C-4).
Compound III (genkwanol C), a light yellow powder, was positive to FeCl3-EtOH reagent. [α]D = +15° (c 0.1, MeOH). HR-ESI-MS m/z: 557.10863 [M-H] -. 1H-NMR (500 MHz, DMSO-d
6
) δ: 7.07 (2H, d, J = 8.5 Hz, H-2''', H-6'''), 6.96 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.74 (2H, d, J = 8.5 Hz, H-3''', H-5'''), 6.51 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.00 (1H, d, J = 2.0 Hz, H-8''), 5.93 (1H, d, J = 2.0 Hz, H-6''), 5.72 (1H, s, H-6), 4.61 (1H, d, J = 6.5 Hz, H-2), 3.77 (1H, m, H-3), 3.16 (1H, d, J = 6.5 Hz, H-4), 2.21 (1H, d, J = 4.0 Hz, H-4). 13C-NMR (125 MHz, DMSO-d
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) δ: 190.3 (C-4''), 186.3 (C-5), 168.5 (C-7), 167.3 (C-7''), 162.6 (C-5''), 160.2 (C-8''a), 158.1 (C-8a), 157.3 (C-4'''), 156.7 (C-4'), 129.6 (C-2''', 6'''), 127.8 (C-1'), 127.4 (C-2', 6'), 121.9 (C-1'''), 114.6 (C-3''', 5'''), 114.5 (C-3', 5'), 109.5 (C-4a) ,100.48 (C-6), 99.2 (C-4''a), 97.8 (C-6''), 95.7 (C-8''), 90.4 (C-2''), 84.6 (C-8), 81.8 (C-2), 79.9(C-3''), 65.0(C-3), 25.5 (C-4).
Compound IV (stelleranol), a light yellow powder, was positive to FeCl3-EtOH reagent. [α]D = -102° (c 0.1, MeOH). HR-ESI-MS m/z: 557.10913 [M-H] -. CD (c 6.25×10-5, MeOH) Δε (nm): 0 (394), -6.8 (342), 0 (327), +31.8 (305), 0 (279), -18.6 (259), -7.5 (242), - 21.2 (219), 0 (206). 1H-NMR (500 MHz, acetone-d
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) δ: 7.22 (2H, d, J = 8.5 Hz, H-2''', H-6'''), 6.83 (2H, d, J = 8.5 Hz, H-3''', H-5'''), 6.71 (2H, d, J = 8.5 Hz, H-2', H-6'), 6.62 (2H, d, J = 8.5 Hz, H-3', H-5'), 6.16 (1H, d, J = 2.0 Hz, H-8''), 6.14 (1H, d, J = 2.0 Hz, H-6''), 6.09 (1H, s, H-2''), 5.68 (1H, s, H-6), 4.96 (1H, s, H-2), 4.18 (1H, brs, H-3), 2.65 (1H, d, J = 17.5 Hz, H-4), 2.49 (1H, dd, J = 3.8, 17.3 Hz, H-4). 13C-NMR (125 MHz, acetone-d
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) δ: 193.1 (C-4''), 188.7 (C-5), 170.4 (C-7), 169.6 (C-7''), 166.1 (C-8''a), 163.1 (C-5''), 160.3 (C-8a), 159.9 (C-4'''), 158.6 (C-4'), 131.7 (C-2''', 6'''), 130.4 (C-1'), 129.2 (C-2', 6'), 124.7 (C-1'''), 116.7 (C-3''', 5'''), 116.5 (C-3', 5'), 111.1 (C-4a) ,102.9 (C-6), 102.0 (C-4''a), 99.0 (C-6''), 98.4 (C-8''), 92.2 (C-2''), 87.5 (C-8), 82.3 (C-2), 82.1 (C-3''), 66.5 (C-3), 28.6 (C-4).
Cell and virus
Human larynx epidermoid carcinoma cell line (HEp-2, CCL-23) and RSV (long strain, VR-26) were purchased from the American Type Culture Collection (ATCC, USA). The cells were grown in Eagle's minimum essential medium (EMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA), 25 μg/ml gentamicin (Sigma, USA) and 200 mM L-glutamine (Sigma, USA) (growth medium). Virus-infected cells were maintained in EMEM with 1% FBS, 25 μg/ml gentamicin and 200 mM L-glutamine (maintenance medium). All the cells were cultured at 37°C in a humidified atmosphere supplied with 5% CO2. Virus titers were determined by the 50% tissue culture infective dose (TCID50) method.
Cytotoxicity assay
Cell viability was tested by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method as described in previous study [10]. Briefly, 100 μl of two-fold diluted samples were added to a 96-well plate containing confluent cell monolayer in triplicates while the dilution medium without the sample was the control. After 72 hours of incubation, 12 μl of the MTT solution (5 mg/ml in phosphate buffered saline) was added to each well. The trays were further incubated for four hours for the formation of blue formazan. After the supernatant was removed, the blue formazan was solubilized in 100 μl DMSO and the optical density (OD) was measured at 570 nm with a microplate reader.
Antiviral assay
CPE reduction assay was adopted for screening the in vitro antiviral activity as described in the previous study [10]. Briefly, 0.1 ml of 100 TCID50 virus suspension and serial two-fold dilutions of the tested samples were added simultaneously to confluent cell monolayers in a 96-well plate. Virus suspension and maintenance medium without samples were added as the virus control and cell control, respectively. The plates were incubated at 37°C in a humidified CO2 atmosphere for 3-5 days. The virus-induced CPE was scored against the virus control under a light microscope. Ribavirin (Sigma, USA) was used as positive control in this experiment.