Antitumor activity on human bladder cancer T-24 cells and composition analysis of the core of Camellia osmantha fruit

In this study, antitumor activity and composition analysis of the core of Camellia osmantha fruit were investigated. The cores were extracted by 80% ethanol to obtain the crude extract ( TF ), which was successively distributed by different concentrations of methanol with MCI resin to obtain three different parts ( Frs 1 to 3 ). Cytotoxicity activities showed that TF , Fr-2 , and Fr-3 exhibited good inhibition against T-24 with IC 50 values of 6.7 ± 1.3, 6.9 ± 0.9, and 6.7 ± 1.74 μg/mL, respectively. Fr-3 has a strong inhibitory activity on T-24 cells, mainly due to effectively increasing the release of intracellular calcium ions and reactive oxygen species. In addition, Fr-3 can inhibit T-24 cells migration and invasion. Further composition analysis on Fr-3 was detected by LC-Q-TOFMS implying the main components to be ellagic acid and its derivatives.

HPLC-grade acetonitrile and methanol were from Fisher Scientific, UK.

Preparation of sample
The cores of C. osmantha fruit (200g) were collected in Nanning, Guangxi. It was dried, smashed, and passed through an 80 mesh sieve. The samples were extracted three times with 80% ethanol and filtered. The solvent was removed under reduced pressure to give 2.02 g of crude product (TF) as a brown solid. The crude extract was dissolved in water and separated into three parts (Frs 1 to 3) by gradient elution with 0%, 50%, and 100% methanol using a column of MCI-gel CHP 20P. All fractions are dried and stored at -

Cell cultures
Five cell strains used for proliferation inhibitory activity in this experiment in vitro, that is, WI-38 human normal liver cell line, MGC-803 human gastric cancer cell line, four human bladder cancer cell lines (T-24, 5637, and EJ), Hela human cervical cancer cell line, and 7402 human liver cancer cell line were obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The above cells were placed in DMEM medium containing 5% fetal bovine serum, 100 U/mL penicillin or RPMI-1640 medium, and cultured in an incubator containing 5% CO 2 at 37 ° C. The cells were observed under a microscope at intervals, and after the cells were grown to 80% to 90%, they were washed twice with a buffer solution (pH=7.43) of 0.05 mol/L PBS. Passage was digested with 0.25% trypsin and logarithmic growth cells were used for the experiment.

Cell viability tests
Cell viability tests were evaluated by MTT assay reported from the literature methods (Elbaz et al. 2012). Seven cells in the logarithmic growth phase were seeded at a density of 1.0 × 10 4 cells/mL in a 96-well culture plate. In the experimental group, 200 μL of sample with HAM / F12 medium were added to each well to a final concentration of 1, 2, 5, 10, and 40 μg/mL, and DMSO with the same concentration as the control group.
After 24 and 48 hours of incubation, 5 g / L of MTT solution was added to each well.
The supernatant was aspirated and shaken by adding 150 μL of DMSO four hours later.
After mixing and dissolving the crystals, the absorbance (OD value) was measured at 490 nm on an enzyme-linked immunosorbent detector, and each dose was repeated to 4 wells in parallel. The inhibition rate was calculated according to the following formula: of each fraction against various cell lines can be calculated by the Bliss method. All experiments were repeated three times and averaged.

Morphology analysis of apoptosis stained by Hoechst 33342
Morphology analysis of T-24 was observed by stained with Hoechst 33342 according to the literature (Tan et al. 2015). T-24 cells in logarithmic growth phase were inoculated into a 24-well plate with a density of 2.5 × 10 5 cells/mL. After adding 24 μg/mL Fr-3 to the plate for 24 h, the culture solution was aspirated and 4% paraformaldehyde was added to fix for 2 h, and then rinse twice with PBS. Finally, 200 μL of Hoechst 33342 staining solution were added with a final concentration of 20 μmol/L, staining for 30 min in the dark and rinse twice with PBS again. Photos were observed with the inverted fluorescence microscope.

Determination of release of intracellular calcium ions
Release of intracellular calcium ions (Driesbaugh et al. 2014). T-24 cell lines were collected at different times and digested after Fr-3 was added with the concentration of 12 and 24 μg/mL. Then T-24 cells were washed twice with RPMI1640 with a mass fraction of 0.1%, stained with trypan blue with a mass fraction of 0.25%, and the viable cells were counted above 95%. The cell suspension was pre-warmed at 37 ° C for 5 min, then washed with PBS and calcium-free Tyrode for 2 to 3 times. 100 μL of Fluo-3/AM dye solution was added and incubated at 37 °C for 45 min. The fluorescence intensity of the cells was recorded using a laser scanning confocal microscope with the excitation wavelength at 488 nm under a 40-fold objective lens. The fluorescence intensity of the cells reflected the concentration of Ca 2+ .

Determination of Reactive Oxygen Production Status in T-24 Cells by FCM
24 μL of T-24 cell suspension (1×10 6 /mL) was treated with different concentrations of

Cell wound-healing assay
T-24 cell migration was measured by a scratch test (Jiang et al. 2010). T-24 cells were seeded in a 6-well plate at a cell concentration of 5×10 5 /mL per well. When the cells were almost full, a 200 μL pipette tip was used to draw a trace in the middle of the cells.
Images were captured using fluorescence microscopic ( were taken with an inverted fluorescence microscope at 0, 36, and 48 h.

Components analysis by HRLC-MS
Chemical composition analysis of the samples was performed using a DIONEX U-3000 HPLC. Gradient elution must be used to separate. The gradient elution conditions were optimized after ensuring that the main components of all samples were able to flow out.

Statistical analysis
Experimental data was performed in triplicate. Data were recorded as the mean and standard deviation and analyzed by Microsoft Excel 2003. The one-sample t-test was run on SPSS 19.0. When P < 0.05 was considered to be statistically significant, P < 0.01 was considered to be statistically significant.

Hoechst 33342 staining
It can be seen from the Figure S1 that the living cells grew adherently and the density was uniform, the nucleus was full and the structure was normal. While treated with Fr-3,

Inhibition of cell migration
As shown in Figure S3, Fr-3 was applied to T-24 cells, the migration ability of T-24 cells was significantly inhibited compared with the control group, and the inhibition effect at high concentration (24 μg/mL) was stronger than that of low concentration (12 μg/mL). The result indicated that the Fr-3 inhibited T-24 cell migration in a dosedependent manner. Figure S3. Effect of Fr-3 on in vitro migration potential of T-24 prostate cancer cells.
From the migration area data of Table S1 (calculated by Image J software), it can be seen that the migration area of the control group gradually decreases as the time increased. The migration area was increased gradually along with the time after Fr-3 treatment at 12 μg/mL, and the velocity also decreased gradually. However, when the concentration of Fr-3 was increased to 24 μg/mL, the migration area was increased with the time, which may be due to the gradual death of the cells and suspended. In summary, Fr-3 can effectively inhibit the migration of T-24 cells.
Table S1 Effect of control and Fr-3 on T-24 cell migration in scratch wound model

HPLC Analysis of the cores of C. osmantha fruit
The chromatograms of TF and Fr-3 under optimized chromatographic conditions are shown in Figure S4. As can be seen from the Figure S4, Fr-3 existed four main components (Peaks 1-4), and each chromatographic peak is basically separated to meet the needs for qualitative analysis. Peaks 1 and 2 have a maximum absorption wavelength of 260 and 362 nm, and it is inferred that may be two compounds with  Figure S4. Chemical constituents' analysis of TF and Fr-3 with wavelength at 254 nm.

Identification of anti-tumor components
The chemical composition of Fr-3 was further analyzed by LC-MS (Table S2, Figures   S5 and S6). In total, four main compounds in Fr-3 were identified by LC Q-TOF MS analysis. The extract ion chromatogram at m/z 300.9974 showed a peak at R t 5.256 min, and was identified as ellagic acid (Chen et al. 2014). At m/z 343.0444, the chromatogram showed a peak at R t 6.521 min, which was identified as 3,3′,4′-tri-Omethylellagic acid (Yasumoto et al. 2006). At m/z 327.2161 the chromatogram showed a peak at R t 6.621 min and m/z 329.2321 a peak at R t 6.821 min, are possibly deduced a type of chain hydroxycarboxylic acids, such as trihydroxyoctadecenoic acid with five oxygen atoms (Falcão et al. 2013).