New Dibenzocyclooctadiene Lignan from Kadsura induta and Their Cytotoxic Activities

Experimental

General. Optical rotations were determined on a Jasco DIP-370 automatic polarimeter. The NMR spectra were recorded using a Bruker DRX 500 spectrometer (1H, 500 MHz; 13C, 125 MHz), and the FAB-MS using a JEOL JMS-HX/HX110A tandem mass spectrometer. The HR-ESI-mass spectra were obtained using an AGILENT 6550 iFunnel Q-TOF LC/MS system. Column chromatography was perform-ed using a silica-gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) or YMC RP-18 resins (30-50 μm, Fujisilisa Chemical Ltd.), and thin layer chromatography (TLC) using a pre-coated silica-gel 60 F254 (0.25 mm, Merck) and RP-18 F254S plates (0.25 mm, Merck)

Figure 3.Important NOESY correlations of compound 1.

Plant Material. The leaves of K. induta were collected in Tamdao National Botanical Park, Vietnam during July 2012, and identified by Dr Bui Van Thanh, Institute of Ecology and Biological Resources, VAST. A voucher specimen (KI1207) was deposited at the Herbarium of the Institute of Chemistry, VAST.

Extraction and Isolation. The leaves of K. induta (1.5 kg) were extracted with MeOH by the sonication for 3 times at room temperature to yield 56.0 g of a dark solid extract, which was then suspended in water and successively parti-tioned with n-hexane and ethyl acetate (EtOAc) to obtain the n-hexane (KI1), EtOAc (KI2), and water (KI3) layers. KI1 was chromatographed on a silica gel column and eluted with gradient elution of n-hexane–acetone (40:1, 20:1, 10:1, 5:1, and 1:1) to obtain fractions, KI1A–KI1E. KI1B was chromato-graphed on a silica gel column eluting with n-hexane–EtOAc (15:1) to yield 2 (0.5 g). KI2 was chromatographed on a silica gel column eluting with CHCl3–MeOH (100:1, 50:1, 25:1, 10:1, and 5:1) to obtain fractions KI2A–KI2E. KH2B was chromatographed on an YMC column eluting with acetone–water (2:1) to yield 1 (8.8 mg). KH2C was chromatographed on an YMC column eluting with MeOH–water (5:1) to yield 3 (8.0 mg) and 4 (6.0 mg). KH2D was chromatographed on an YMC column eluting with acetone–water (2:1) to yield 5 (120.0 mg) and 6 (100.0 mg). KH2E was chromatographed on a silica gel column eluting with CHCl3–MeOH (6:1) to yield 7 (117.0 mg).

Schizanrin O (1): A white amorphous powder, [α]D25 −56 (c 0.1, CHCl3), CD (c 1 × 10−3 M, MeOH) ∆ε245 = –3.4, ∆ε212 = +1.8, HR-ESI-MS found at m/z 631.2172 (Calcd C33H37O11 for 631.2150), 1H- and 13C-NMR: see Table 1.

Cytotoxicity Assay. The effect of compounds 1-7 on the growth of human cancer cells was determined by measuring the cytotoxic activity using a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay.11 The A-549 (human lung cancer), HT-29 (human colon cancer) and OVCAR (human ovarian cancer) cell lines were obtained from the Korea Cell Line Bank (KCLB) and were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL and 100 g/ mL, respectively) at 37 °C in a humidified 5% CO2 atmosphere. The MTT assays were performed as follows: human cancer cells (1.5-2.5 × 105 cells/mL) were treated for 3 days with 1, 10, 50 and 100 µM of the isolated compounds. Mitoxantrone was used to final concentrations of 1, 3, 10 and 20 µM as a reference. After incubation, 0.1 mg (50 µL of a 2 mg/mL solution) MTT (Sigma, Saint Louis, MO, USA) was added to each well and the cells were then incubated at 37 °C for 4 h. The plates were centrifuged at 1000 rpm for 5 min at room temperature and the media was then carefully aspirated. Dimethylsulfoxide (150 µL) was then added to each well to dissolve the formazan crystals. The plates were read im-mediately at 540 nm on a microplate reader (Amersham Pharmacia Biotech., USA). All the experiments were performed three times and the mean absorbance values were calculated. The results are expressed as the percentage of inhibition that produced a reduction in the absorbance by the treatment of crude extract or solvent fractions compared to the untreated controls. A dose-response curve was generated and the inhibitory concentration of 50% (IC50) was deter-mined for each compound as well as each cell line.