HPLC-ESI-Q-TOF-MS/MS were performed on an Agilent 1290 HPLC system (Agilent Technologies, Santa Clara, CA, USA), equipped with an online vacuum degasser, a QuatPump, a manual injection valve with a 20 μL sample loop, a thermostated column compartment, a diode assay detector (DAD), and an Agilent ChemStation, has been selected to analysis of samples. Agilent 6530 Accurate-Mass Q-TOF LC/MS system (Agilent Technologies, Santa Clara, CA) equipped with an electrospray ionization (ESI) interface was coupled in parallel by splitting the mobile phase 1:3 using an adjustable high-pressure stream splitter (Valco Instrument Company, Houston, TX, USA). MS data were acquired across the range m/z 100~1500 in positive and negative ion modes (dry gas: N2, 325°C, 5.0 L/min; sheath gas: N2, 350°C 12 L/min; fragmentor voltage, 135 V; capillary voltage, 3500 V). All operations, acquisition and analysis of data were monitored by Agilent MassHunter Acquisition Software Version A.01.00 and operated under MassHunter Acquisition Software Version B.05.00.
HSCCC was performed on a model TBE-300C HSCCC instrument (Shanghai Tauto Biotechnique Co.Ltd., China). The instrument was equipped with three multilayered preparation coils (diameter of PTFE tube, 1.6 mm, total volume 300 mL) and a 20 mL sample loop. The revolution speed was adjustable from 0 to 900 rpm. The HSCCC system consists of a TBP-5002 pump, a model of TBD-2000UV detector, a DC-0506 constant temperature circulating implement, and an AV2.2.0 B chromatography workstation. During the separation process, the temperature of separation columns was controlled at 25°C, and the effluents were monitored at 214 nm. 1H and 13C NMR spectra were measured on Bruker Avance 600 AV (1H at 600 MHz, 13C at 151 MHz) NMR spectrometers. Using tetramethylsilane (TMS) as internal standard, and all chemical shifts were reported in parts per million (ppm, δ).
Chemicals and plant material
Ethanol, methanol, n-hexane, ethyl acetate, chloroform, hydrochloric acid and Sodium hydroxide, which were used for the preparation of the fractions and the separation of HSCCC are purchased from Hunan Huihong Reagent Co., Ltd. (Changsha, China). Ultrapure water was purified and filtered by a MilliQ water (18.2 MΩ) system (Millipore, Bedford, MA, USA). Formic acid and acetonitrile used for LC-MS were of chromatographic grade (Merk, Germany). Carpesium abrotanoides L. were obtained from Dao County of Hunan Province in China in 2019. A voucher specimen (No.2019-TMJ) was deposited in the Department of Pharmaceutical Chemistry, Research Institute of Chinese Medicine.
Preparation of plant extracts and fractionation
The whole dry crushing plant of Carpesium abrotanoides L. (2.0 kg) were extracted by 95% ethanol (20 L) with third times and soaked for 24 h. After combining and drying, the total ethanol extracts (TEC) (458.1 g) was obtained. TEC fraction (400.0 g) were dissolved in a solution of 2% NaOH to pH=9, and then adjust the pH to 2 with 1% concentrated hydrochloric acid to get precipitate, and finally yielded the total sesquiterpene lactone extracts (SLEC) (42.0 g).
Anti-tumor activity screening by CCK-8 method
Take the cells (Hela, HepG-2, SW620, A549, HL-60, PC-3, SMMC-7721) in the logarithmic growth phase, the cells were inoculated into 96-well plates at a density of 1×105/mL (100 μL/well). Two extracts of TEC and SLEC (50, 100, 150, 200, and 250 μg/mL) or compounds 1-3 (2.5, 5, 7.5, 10, 15, and 20 μM) was added to adherent cells with 96 wells for each group and repeated 3 times. After the adhesion is complete, add the samples, continue to incubate for 48 hours, and add 10 μL CCK-8 solutions to each well. At 37°C, 5% CO2 was incubated for 4 hours, and the absorbance (OD) value at 450 nm was analyzed with a Bio-Tek microplate reader.
Cell proliferation (%) = (mean OD of treated cells / mean OD of untreated cells])×100
HPLC-ESI-Q-TOF-MS/MS was used to analyze the chemical constituents of SLEC. The final conditions of the instrument are as follows: Agilent HPLC column (Eclipse XDB C18, 4.6×250 mm, 5 μm), mobile phase A is 0.1% formic acid water, mobile phase B is acetonitrile, elution procedure (0-8 min, 2% B; 8-10 min, 2%-20% B; 10-50 min, 20%-52% B; 50-60 min, 52% B; 60-65 min, 52%-80% B; 65-68 min, 80-98% B; 68-75 min, 98% B). The column temperature was 25°C, the volume flow rate was 1.0 mL/min, and the injection volume was 5 μL. Spectra were recorded from 190 to 400 nm (peak width 0.2 min and data rate 1.25 s-1) while the chromatogram was acquired at 214 nm.
Measurement of Partition Coefficients
The selection of solvent system is mainly based on the distribution coefficient of the targets. The specific operation of HPLC method is as follows: the upper and lower phase solvents of a certain volume (20 mL) are prepared according to the proportion of the solvent system, and after the phase separation is balanced, the same volume of upper and lower phase solvents are placed in different containers (10 mL). Add a certain amount of sample (200 mg), shake violently to dissolve and rest. The same volume of upper and lower phase solvent was injected into HPLC to determine the peak area of the target components in the upper and lower phase (AS and AM), to calculate the partition coefficient (K) (K = AS/AM).
Five solvent systems consisting of n-hexane-ethyl acetate-methanol-water (1:1:1:1, v/v/v/v), n-hexane-ethyl acetate-methanol-water (1:9:9:1, v/v/v/v), n-hexane-ethyl acetate-methanol-water (3:1:1:1, v/v/v/v), n-hexane-methanol-water (5:4:1, v/v/v), and chloroform-methanol-water (4:3:2, v/v/v) were used for the separation of SLEC. Sample solution was prepared by dissolving 0.1 g of SELC in 20 ml mobile phase of the solvent system for separation.
In each HSCCC separation, the separation column was initiated by filling with the upper phase at 30.0 mL/min, and then the apparatus was rotated 835 rpm. The mobile phase was pumped into the column at 3.0 mL/min. After the equilibration reached, samples were injected into the injection valve. The solvent was continuously monitored at 214 nm and the peak fractions were collected under monitor by with a UV detector.
Quantitative real‐time PCR analysis
Total RNA was extracted from HepG-2 using Trizol reagent (Ambion, Austin, USA). About 500 ng of total RNA for each sample was reverse‐transcribed to prepare cDNA using PrimeScript II RTase reverse transcriptase (Takara, Beijing, China). The quantitative real‐time polymerase chain reaction (qRT‐PCR) was performed using an CFX-Connect 96 (Applied Biosystems, Foster City, CA) with the SYBR FAST qPCR Master Mix (Solarbio, Beijing, China). The primer sequences used for PCR were as follows: JAK2, forward: ACTAAATGCTGTCCCCC, reverse: TTCATGCAGTTGACCGT; STAT3, forward: TGAGGGAGCAGAGATGTG, reverse: TGGGGGCTTGGTAAAA; GAPDH, forward: CCACTCCTCCACCTTTG, reverse: CACCACCCTGTTGCTGT. The relative expression of each target gene compared with GAPDH was calculated using the 2−ΔΔCt method .
The total protein was extracted from HepG-2 cells using RIPA lysis buffer (solarbio, Beijing, China), and the protein concentration was detected by BCA Protein Assay Kit (solarbio, Beijing, China). The loading protein (20 µg) was electrophoresed and separated on a 12 % SDS-polyacrylamide gel (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (0.22µm, Millipore, USA). The PVDF membrane was blocked in 5% skimmed milk at room temperature for 1 hour, the membranes were incubated with primary antibodies (JAK2, p-JAK2, STAT3, p-STAT3, and GAPDH) (Bioswamp) overnight at 4°C.Then, the membranes followed by secondary antibodies goat anti-rabbit IgG (1:10000)) incubation for 1 h at 37°C and detected by ECL.
The results were expressed as means standard deviation (SD). One-way analysis of variance (ANOVA) and t-tests were used for comparisons among and between groups, respectively. All statistical analysis was carried out using SPSS 21.0 with statistical significance set at p < 0.05 or p < 0.01.