Plant materials
P. japonicum roots (harvested from Geumo-do, Nam-myeon, Yeosu-si, Jeollanam-do, Korea) were purchased from the Dongbu Herbal Farming Cooperative. (Suncheon, Korea) in January 2015.
Chemicals and reagents
RPMI 1640 was purchased from Thermo Scientific (Waltham, MA, USA); Dulbecco's modified Eagle medium (DMEM) was obtained from Gendepot (Baker, TX, USA); cell counting kit-8 (CCK-8) was procured from Dojindo Laboratories (Kumamto, Japan); dimethyl sulfoxide (DMSO) and propidium iodide (PI) were acquired from Sigma Aldrich (St. Louis, MO, USA); Annexin V-FITC was purchased from BD Biosciences (Franklin Lakes, NJ, USA), and the JC-1 Assay Kit was obtained from Invitrogen (Carlsbad, CA, USA). Methanol, dichloromethane, hexane (Duksan Co., Ansan, Korea), HPLC grade acetonitrile, water, methanol (J. T Baker, Phillipsburg, NJ, USA), chloroform-d (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA), silica gel 60 (70-230 mesh, Merck Co., Kenilworth, NJ, USA), Luna 5u (C18 100A 250×10 mm, Phenomenex Inc., Torrance, CA, USA), and YMC-actus (Triart C18 250×20 mm, YMC, Kyoto, Japan) were also used.
Extraction and isolation of compounds from P. japonicum
Roots of P. japonicum (dry wt. 1.0 kg) were extracted three times with MeOH in CH2Cl2 (1:1, v/v) at room temperature. These extracts were combined and partitioned three times between MeOH and n-hexane. The MeOH layer was further partitioned between H2O and ethyl acetate to establish an H2O fraction and an ethyl acetate fraction (8.41 g). The ethyl acetate-soluble fraction was subjected to silica gel flash column chromatography and eluted with a step gradient solvent system of 100% to 0% CH2Cl2 in MeOH, which resulted in 11 fractions (1-11). Compounds 1 (1.0 mg, tR 23 min) and 2 (1.2 mg, tR 37 min) were obtained by reverse-phase HPLC (Shiseido CAPCELL C18 5 μm, 250×10 mm, 2.0 mL/min, UV detection at 210 nm) using 70% CH3CN in H2O as the eluant. Compounds 1 and 2 were identified as (-)-isosamidin and 3ʹS,4ʹS disenecioylkhellactone, respectively, by comparing their spectroscopic data and optical rotations with previously reported data [27, 28]. For (-)-isosamidin(1); 1H NMR (CDCl3, 700 MHz) δ 7.61 (1H, d, J = 9.5), 7.36 (1H, d, J = 8.7), 6.81 (1H, d, J = 8.5), 6.57 (1H, d, J = 4.8), 6.23 (1H, d, J = 9.5), 5.69 (1H, s), 5.35 (1H, d, J = 4.8), 2.17 (3H, d, J = 1.2), 2.11 (3H, s), 1.90 (3H, d, J = 1.2), 1.45 (3H, s), 1.41 (3H, s); 13C NMR (CDCl3) δ169.5 (C), 164.9 (C), 159.5 (C), 158.4 (C), 156.4 (C), 153.6 (C), 128.7 (CH), 114.8 (CH), 114.1 (C), 112.8 (CH), 112.3 (C), 106.7 (C), 77.7 (C), 68.7 (CH), 27.4 (CH3), 24.5 (CH3), 23.1 (CH3), 20.6 (CH3), 20.3 (CH3); LRESIMS m/z 286.4 (M – 99 + H)+, 793.3 (2M + Na)+ (13C NMR chemical shifts were determined using 2D NMR spectroscopy data and confirmed by comparing them with previously reported data) (Fig. 1). For 3ʹS, 4ʹS disenecioylkhellactone(2); 1H NMR (CDCl3, 700 MHz) δ7.60 (1H, d, J = 9.2), 7.37 (1H, d, J = 8.7), 6.82 (1H, d, J = 8.7), 6.65 (1H, d, J = 4.8), 6.24 (1H, d, J = 9.5), 5.69 (1H, s), 5.64 (1H, s), 5.37 (1H, d, J = 4.8) 2.22 (3H, s), 2.17 (3H, s), 1.91 (3H, s), 1.90 (3H, s); 13C NMR (CDCl3) δ 167.5 (C), 167.3 (C), 162.7 (C), 160.8 (C), 160 (C), 158.8 (C), 155.9 (C), 146.6 (CH), 131.7 (CH), 117 (CH), 117 (CH), 114.8 (CH), 114.4 (CH), 113.6 (C), 109.4 (C), 79.4 (C), 72 (CH), 61.8 (CH), 28.3 (CH3), 28.2 (CH3), 26.5 (CH3), 23.2 (CH3), 21.3 (CH3), 21.2 (CH3); LRESIMS m/z 327.1 (M – 100 + H)+, 876.3 (2M + Na)+ (13C NMR chemical shifts were determined using 2D NMR spectroscopic data and confirmed by comparing them with previously reported data) (Fig. 2).
Cells and cell culture
Human acute promyelocytic leukemia cells (HL-60; KCLB 10240), human lung adenocarcinoma cells (A549; KCLB 10185) and human breast adenocarcinoma cells (MCF-7; KCLB 30022) were obtained from the Korean Cell Line Bank (Seoul, Korea) and cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 μg/mL). Madin-Darby canine kidney (MDCK; KCLB 10034) cells were also obtained from the Korean Cell Line Bank and cultured in DMEM containing 10% FBS, 100 U/mL penicillin G, and 100 μg/mL streptomycin sulfate. The purities of all compounds tested by HPLC were confirmed to be >95%. Samples of test compounds were dissolved in DMSO and added to the medium to a final concentration of 0.03%. Cultures were maintained at 37°C under 5% CO2/95% air, and media were changed every two days.
Cytotoxicity assay
Cell viabilities were determined using the cell counting kit-8 (CCK-8) assay. First, MCF-7, A549 and HL-60 cells were resuspended in RPMI 1640 medium at densities of 3×104 cells/mL, 1×104 cells/mL, 2×104 cells/mL, and 1×105 cells/mL, respectively. Next, 100 μL of the cell suspensions was added per well to 96-well flat-bottomed microtiter plates, followed by 100 μL of compounds 1 and 2 to achieve final concentrations of 1, 3, 10, and 30 μM. The plates were then incubated for 24 h at 37°C in 5% CO2/95% air. Second, HL-60 cells were resuspended in RPMI 1640 medium at 1×105 cells/mL whereas MDCK cells were suspended in DMEM at 1×104 cells/mL. Next, 100 μL of cell suspension was added to the wells of 96-well flat-bottomed microtiter plates followed by 100 μL of compounds 1 and 2 (to final concentrations of 1, 10, and 30 μM), and the plates were incubated for 24 h at 37°C in 5% CO2/95% air. Three replicates per condition were used in the experiments. After 24 h, 100 μL of medium was replaced with an equal volume of fresh medium containing 10 μL of CCK8, after which cell viability was measured.
Cell cycle assay
To determine the cell cycle distribution, HL-60 cells (5×105/well) were seeded and treated with compound 1 or 2 at 30 μM for 12 h, 18 h, and 24 h. After incubation, the cells were harvested, fixed in 70% ethanol, and treated with ribonuclease A (to increase cell permeability). Cell pellets were then incubated with PI for 2 h at 4°C in the dark before they were analyzed by flow cytometry. The percentages of cells in the G0/G1, S and G2/M phases of the cell cycle and the sub-G1 peak were determined after excluding cell debris and aggregates.
Annexin V/PI assay of apoptotic cells
Apoptosis induced by compounds 1 and 2 was quantified by flow cytometry using an Annexin V-FITC and propidium iodide (PI) solution according to the manufacturer's instructions. Briefly, HL-60 cells (5×105/well) were seeded into 24-well plates and treated to compounds 1 or 2 (30 µM for both) for 12 h, 18 h, and 24 h. Apoptosis was analyzed by staining with Annexin V-FITC/PI and performing flow cytometry, and the apoptosis percentages were calculated by counting the number of Annexin V- and PI-positive cells.
Detection of changes in mitochondrial membrane potentials (MMPs)
HL-60 cells were seeded in 24-well plates at a density of 5×105 cells/mL and treated with compounds 1 or 2 at 30 μM for different periods (12 h, 18 h, and 24 h). Cells were then harvested and stained with the lipophilic dye JC-1 (5,5ʹ,6,6ʹ-tetrachloro-1,1ʹ,3,3ʹ-tetraethylbenzimidazole-carbocyanine iodide), which was used to measure the MMP by flow cytometry. When JC-1 enters the mitochondria, it aggregates and fluoresces red. Upon loss of the MMP, the dye diffuses throughout the cytoplasm and fluoresces green. The histogram shows the population of cells showing green (JC-1 monomers) and red (JC-1 aggregates) fluorescence. Frequency plots were prepared with FITC and PE gating to determine the percentages of mitochondria stained green (loss of the MMP) and red (normal MMP) [29].
Activation of caspase-3, -8, and -9
The activities of caspase-3, -8, and -9 were determined using the caspase-3, -8, -9 activity kit according the manufacturer’s protocol. HL-60 cells were seeded into 24-well plates and treated with compounds 1 and 2 at 25 μM for 12 h, 18 h, and 24 h. Then, the cells were collected, washed, fixed, and stained with FITC before they were analyzed by flow cytometry.
Statistical analysis
Differences between groups are presented as the means±S.D. of 3 replicates. Statistical differences were analyzed using Student’s t-test. Probability values less than 0.05 were considered significant (P values *<0.05, **<0.01, ***<0.001 vs control, #<0.05, ##<0.01, ###<0.001 vs 24 h sample).