Additional materials and methods
Chemical and reagents
FBS,DMEM, penicillin / streptomycin, Trypsin (purity> 99%) were purchase from Life technology, Gibco; Transwell chamber were purchase from Millipore; MTT powder was purchase from Sangon Biotech(Shanghai, China)；Anexin V/PI kit was purchase from Vazyme Biotech(Nanjing, China);GAPDH, PARP and caspase 3 antibody (purity> 99%) were purchase from Proteintech (Wuhan, China). The control materiel (purity> 99%) were purchased from TAUTO BIOTECH(Shanghai, China).
Sample preparation
Dry powder of the plant samples were sonicated in 95% ethyl alcohol (sample volume : liquor volume = 1:40 ) for 25 min under the ultrasonic power of 50W for twice, followed by centrifugation at 12000 × g for 10mins. The supernatant was transferred to a rotary evaporation bottle to spin-dry, and then dissolved by methanol.
HPLC quantitativedetection
HPLC chromatographic conditions were as followed: The mobile phase consisted of 83% (v/v) methanol and 17% (v/v) water containing glacial acetic acid and triethylamine, with the volume rate of 10:0.03:0.06. The analysis was performed using the flow rate of 1.0mL/min. the column temperature was maintained at 30℃. And the detection wavelength was 203nm.
Preparative HPLC
The experiment was performed on Waters SunFire TM, and the Waters 2489 detector with detection wavelength of 207nm. The separation was carried out on a Waters Prep C18 OBD TM (19×250mm，5µm). The mobile phase consisted of 95% (v/v) methanol and 5% (v/v) water containing glacial acetic acid and triethylamine, with the volume rate of 10:0.03:0.06. The column temperature was maintained at 30 ℃ and the flow rate was 20 mL/min. The sample was filtered by membrane with the pore size of 0.22µm after dissolved by Methanol. The sample size was 2ml. And the fraction of 4.5 min was collected. The separated sample was freeze dried following washing by water just after centrifuge dripping.
UPLC-MS qualitative analysis
The analysis was carried out on Waters UPLC-MS Premier XE with chromatographic column of HSS T3 (2.1×100mm 1.8µm). The analysis was performed using the following a isocratic elution at a flow rate of 0.3 ml/min by mobile phase which consisted of 20% ammonium formate (10mM) and 80% methanol. The column temperature was maintained at 35 ℃. The negative ion electrospray mass spectrometrywas carried out under the capillary voltage of 3.0 KV. For electrospray ( ES )（-）mode , the bore-hole voltage was 25V. For multiplereaction monitoring ( MRM ) mode 488.3→470.4, the fragment voltage was 35V. For MRM mode488.3→424.4, the fragment voltage was 40V. Spray gas flow was 50L/h ,Desolvation Gas Flow was 700L/ h. Desorption temperature was 350℃. theionizationtemperaturewas 110℃.
Cell culture
Human hepatocellular carcinoma HepG2 cell Bel-7405cell and SK-hep-1 cell were purchased from ATCC (Manassas, VA, USA) and cultured with DMEM complemented with 10% FBS and penicillin / streptomycin in a 37°C CO2incubator.
Cell viability
HCC cells were seeded in 96-well plates at 5×104 cells/well for 24 hours, then cells were treated with different concentration TA for another 24 hours. Each well 20 µl of MTT solution (5 mg/ml in PBS) was added and allowed for incubation for 4 hours. The medium was aspirated and replaced with 200 µl/well of DMSO to dissolve the formazan salt formed. Then the plates were shaken for 1 hour to extract the blue products. The optical intensity of the formazan solution, which reflects the cell growth condition, was measured at 490 nm using a microplate spectrophotometer.
Wound healing assay
HCC cells were cultured as confluent monolayers, and wounded by 20µl pipette tip. Cell were treated with different concentration TA for 4 hours and then changed with DMEM (3% FBS complemented) to eliminate influence by proliferation. Wounded monolayers were washed twice with PBS to remove non-adherent cells. Cell migration was recorded under inverted microscope every 24 hours.
Colony formation
HCC cells were seeded in 6-well plates at 1.0×104 cells/well; medium with different TA concentration was changed every three days. Cells were fixed with methanol and stained with violet after 10 days.
Cell migration
HCC cells were starved 12 hours. 1.0×106 cells were seeded per upper chambers in serum-free DMEM whereas the lower chambers were loaded with DMEM containing 10% FBS using Millicell inserts (Millipore). After 24 hours, the non-migrating cells on the upper chambers were removed by a cotton swab, and cells invaded to the underside of the membrane were stained and counted.
Western blot
Cell lysates were lysed in lysis buffer followed by centrifuged at 10,500×g for 15 min at 4ºC to obtain a supernatant. The protein concentrations of the supernatants were determined by the BCA protein assay kit (Pierce). Approximately 30 μg of denatured protein was resolved on 8%-12% SDS-PAGE and electro-blotted onto nitrocellulose membranes (Amersham). After one hour blocking, membranes were incubated with antibodies against GAPDH, PARP, and Caspase 3 at 4ºC overnight followed by further incubation with a secondary antibody. After washing with Tris-buffer saline containing 0.05% Tween 20, the blots were detected by the chemiluminescence (Pierce) followed by exposure to Kodak-X-Omat film.
Flow cytometry
HCC cells were treated with different concentration TA in 6-well dish, After 24 h in the incubator, the cells were digested with EDTA-free trypsin and washed twice with PBS. For each group, 1–5×105cells were collected. The cells were then suspended in binding buffer (500 μl), and then 5 μl Annexin V-FITC and 5 μl PI were added. After 10 min of reaction in a dark environment, the cell apoptotic rate was evaluated by flow cytometry.
The Fig. captions in supplementary resource:
Fig. 1 Chromatorgam map of preparative HPLC
Fig. 2 Compared UPLC-MS chromatorgam maps of standard sample and preparative TA
Top left 488.4>470.4 m/z standard sample，top right 488.4>424.4 m/z standard sample;
Bottom left 488.4>470.4 m/z self made TA, bottom right 488.4>424.4 m/z self made TA.
Fig. 3 Wound healing assay of HCC cell lines
HepG2(A)，Bel-7405(B) and Sk-hep-1 cells(C) were seeded in 6 well plate respectively, a scratch wound was made across each well using a white pipette tip. Cells were treated with different concontration TA for 4 hours, and changed to DMEM medium completed with 2.5% FBS. Take image at the indicated times, compare the wound area between different concentration TA treatment cells at the same time.