High performance liquid chromatography (HPLC)-grade methanol and formic acid were obtained from Merck KGaA. Deionized water was prepared via Milli-Q water purification (EMD Millipore). Baicalein reference standards (cat. no. 18031608) were purchased from the Beijing Aoke Biological Technology Corporation, and stock solutions were prepared in dimethyl sulfoxide (DMSO) and stored at 4°C. Methyl p-hydroxybenzoate (internal standard; purity >98% by HPLC-UV) was obtained from Sigma-Aldrich (Merck KGaA).

In order to detect and analyze baicalein in rat plasma after oral administration of S. baicalensis extract, UPLC-MS/MS was conducted as previously described (27). An Acquity UPLC system (Waters Corporation) and an API 4000 Triple Quadrupole mass spectrometer (Shanghai AB SCIEX Analytical Instrument Trading Co.) equipped with an electrospray ionization (ESI) source were used as a part of this system, using Analyst 1.5 software (Applied Biosystems; Thermo Fisher Scientific, Inc.) for assay control. An Acquity UPLC HSS BEH C18 column (100 × 2.1 mm, 1.7 µm; Waters Corporation) maintained at 40°C was used for chromatographic separation, with a mobile phase of (A) 0.1% formic acid, and (B) methanol, with a gradient elution of 60–90% (A) for 0–6 min, 90% (A) for 6–7 min, 90–35% (A) for 7–10 min, and 35% (A) for 10–12 min. The flow rate of the mobile phase was 0.20 ml/min, with a 1-µl injection volume. The electrospray ionization source was performed in negative ionization mode with the following parameters: −4.5 kV ion spray voltage, 500°C turbo spray temperature. For gas pressures, nebulizer, heater and curtain gas were set to 55, 50 and 25 psi, respectively, with a dwell time of 50 msec. Detection analysis was conducted in multiple reaction monitoring mode at the transition m/z [M-H]⁻ 269.2à195.0 for baicalein and 150.9à136.0 for IS, with collision energies of −35 eV and −19 eV, and cone voltages of −70 V and −56 V, respectively.

To obtain an aqueous extraction of S. baicalensis, 100 g S. baicalensis roots were immersed in distilled water for 30 min with occasional stirring, and then boiled three times for 30 min each; plant:water ratios were maintained at 1:10. The three separate decoctions were combined and concentrated into a final volume of 100 ml to yield S. baicalensis extract, which was used for UPLC-MS/MS analysis. The raw materials were identified by Professor Minghua Qiu of Kunming Institute of Botany, Chinese Academy of Sciences (Kunming, China), where voucher specimens are retained.

A total of six male Sprague-Dawley rats (weight, 250±20 g; age, 8 weeks) were provided by Kunming Medical University (Yunnan, China). The rats were housed under standard conditions (20±2°C with 60±5% humidity and 12-h light/dark cycles) with free access to food and water, and acclimated for 1 week. Animals were observed daily throughout the study. All rats were fasted, with free access to water, for 12 h prior to the experiment. The rats were then randomized into 2 groups (n=3 per group); the experimental group received S. baicalensis extract (4.5 g/kg) by intragastric administration once (28), and the control rats received distilled water only (10 ml/kg of body weight). At 45 min after oral administration, rats were then anesthetized using pentobarbital sodium at a dose of 50 mg/kg (i.p.), and blood samples (0.5 ml) were collected via retro-orbital bleeding. The anesthetic agent doses selected were based on existing literature (29). The sampling time-points were selected based on previous pharmacokinetic studies (27). Subsequently, the rats were sacrificed by cervical dislocation. Plasma was then separated from blood after centrifugation at 5,000 × g for 10 min at 4°C. The humane endpoint of this experiment was as follows: A marked reduction in food or water intake, labored breathing, inability to stand, and no response to external stimuli. No abnormal signs that signified the humane endpoints of the experiment were observed from any of the rats during the experiment. All animal procedures were approved and performed in compliance with the guidelines set by the Animal Care Committee of the First People's Hospital of Yunnan Province (30).

A total of 10 µl IS (methyl p-hydroxybenzoate, 1 µg/ml in methanol) and 50 µl 0.2 M HCl were spiked into a 100-µl sample of rat plasma, mixed and allowed to rest for 10 min. Next, 800 µl ethyl acetate was added and mixed for 3 min, followed by centrifugation for 5 min at 5,000 × g (4°C). The supernatants were transferred to fresh tubes and evaporated using a nitrogen gas stream at room temperature. Any remaining residue was dissolved in 100 µl mobile phase, mixed for 1 min by vortexing and centrifuged at 15,000 × g for 5 min at 4°C . Finally, 1 µl supernatant was injected into the UPLC-MS/MS system for baicalein detection.

HT29 and DLD1 human colorectal cancer cell lines were obtained from Shanghai Cell Biological Institute of the Chinese Academy of Sciences, and cultured in RPMI-1640 media (Hyclone; GE Healthcare Life Sciences) containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin (Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂.

The CCK-8 assay (Beyotime Institute of Biotechnology) was used to assess the effects of baicalein on cancer cell viability. HT29 and DLD1 cells were seeded into a 96-well plate at a density of 2×10³ cells/well, and cultured until complete adherence. The cells were treated with baicalein at concentrations of 0, 20, 40, 60, 80, 100 and 120 µmol/l for 24, 48 and 72 h, using DMSO as a negative control. The media was then replaced with fresh media containing 10% CCK-8 solution. After a further 3 h of incubation, the optical density (OD) of each well was assessed using a spectrophotometer at a wavelength of 450 nm. The inhibition rates were calculated as follows: Inhibition rate = [1- (OD drug treated - OD blank)/(OD control-OD blank)] ×100%. The half inhibitory concentration (IC₅₀) for baicalein was determined using the Logit method (31), indicating the concentration of baicalein necessary to inhibit 50% cell proliferation at a given time-point.

HT29 cells were plated in a 6-well plate at 1×10⁶ cells/well, and cultured to 90% confluency. A 10-µl sterile micropipette tip was used to create a wound across the monolayer, and the cells were washed twice with sterile phosphate-buffered saline (PBS) to remove debris. The cells were treated with 10, 20, and 30 µmol/l baicalein in RPMI-1640 media without FBS, and the control group was treated with 0.05% DMSO only. Cell migration was assessed using an inverted microscope (Zeiss Axio Vert.A1), original magnification, ×10.

To assess the effects of baicalein on cancer cell invasiveness, a Transwell invasion assay was performed using 24-well Transwell chambers (pore size, 8 µm), pre-coated with Matrigel^® for 1 h at 37°C. (BD Biosciences). Following treatment with 10, 20, and 30 µmol/l baicalein at 37°C for 24 h, HT29 cells were digested with 0.25% trypsin and resuspended in serum-free RPMI-1640 medium at a density of 1×10⁶/ml, and 100 µl cell suspension was added into the upper chambers. The lower chambers were filled with 500 µl medium supplemented with 10% FBS. Following incubation at 37°C for 24 h, the inserts were detached and non-invasive cells were gently removed with a cotton wool swab. Invaded cells were fixed with 4% paraformaldehyde for 20 min at room temperature and stained with 0.1% crystal violet for 15 min at room temperature. Stained cells were visualized using an inverted microscope (Zeiss Axio Scope.A1) and counted in 5 randomly selected fields (magnification, ×10).

pcDNA3.1-vector and pcDNA3.1-Snail plasmids were obtained from Shanghai GeneChem Co., Ltd., and verified by DNA sequencing. The pcDNA3.1-vector plasmids were used as the controls. HT29 and DLD1 cells (4×10⁵ cells/well) were seeded into 6-well plates with complete medium and incubated at 37°C for 24 h prior to transfection. For transient transfections, cells were transfected with 2.5 µg plasmid using Lipofectamine^® 3000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The cells were harvested 1–2 days after transfection for further investigation.

For western blotting, HT29 and DLD1 cells were plated at 3×10⁵ cells/well in 6-well plates, and treated with 10, 20, and 30 µmol/l baicalein or DMSO only for 24 or 48 h at 37°C. Cells were washed with PBS and lysed using ice-cold RIPA buffer (Beyotime Institute of Biotechnology) for 30 min to extract the total protein, which was quantified using the bicinchoninic acid assay method. The lysates were then denatured in loading buffer containing 4% SDS, and incubated at 95°C for 10 min. In total, 50 µg total protein per sample was separated by 10% SDS-PAGE gel and transferred to PVDF membranes, which were subsequently blocked for 2 h using 5% non-fat dry milk in TBS and 0.05% Tween 20. The membranes were incubated overnight at 4°C with primary antibodies targeted against: E-cadherin (cat. no. 14472; 1:1,000; Cell Signaling Technology, Inc.), vimentin (cat. no. 5741; 1:1,000; Cell Signaling Technology, Inc.), Snail (cat. no. 3879; 1:1,000; Cell Signaling Technology, Inc.), Twist1 (cat. no. 46702; 1:1,000; Cell Signaling Technology, Inc.), p53 (cat. no. 60283-2-Ig; 1:800; ProteinTech Group, Inc.), p21 (cat. no. 10355-1-AP; 1:800; ProteinTech Group, Inc.) and β-actin (cat. no. 4970; 1:10,000; Cell Signaling Technology, Inc.). Following primary incubation, the membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse (cat. no. 6946; 1:8,000; Abcam) and anti-rabbit (cat. no. 6721; 1:6,000; Abcam) IgG (H+L) secondary antibodies for 2 h at room temperature. Protein bands were visualized in a darkroom using enhanced chemiluminescence reagents (New Cell & Molecular Biotech Co., Ltd.). Protein expression was quantified using ImageJ software (v.1.48; National Institutes of Health) with β-actin as the loading control.

All experiments were performed in triplicate. Data are presented as the mean ± standard deviation. Statistical analyses were performed using SPSS software (v.22.0; IBM Corp.). One-way ANOVA followed by Dunnett's post hoc test was used to compare the treatment and control groups. P<0.05 was considered to indicate a statistically significant difference. All graphs were generated using GraphPad Prism software (v.5.0; GraphPad Software, Inc.).

The S. baicalensis herb comprises a complex mixture of different phytochemicals, and contains >60 chemical components. Thus, the purpose of using the UPLC-MS/MS technique was to detect and identify baicalein in rat plasma following the oral administration of S. baicalensis extract. A representative chromatogram of baicalein is presented in Fig. 1.

The anti-proliferative activity of baicalein was assessed with a CCK-8 assay, using HT29 and DLD1 cells treated with 0–120 µmol/l baicalein to identify the minimal non-lethal dose. Baicalein was revealed to inhibit the viability of HT29 and DLD1 cells in a dose- and time-dependent manner; in HT29 cells, the IC₅₀ values at 24, 48 and 72 h were 49.77, 34.35 and 16.91 µmol/l, respectively; and in DLD1 cells, the IC₅₀ values were 60.49, 34.70, and 18.75 µmol/l at the same time-points, respectively (Fig. 2A). To avoid growth suppression, all subsequent experiments were conducted using baicalein concentrations <34 µmol/l.

p53 and p21 are reportedly involved in the baicalein-associated inhibition of CRC HCT116 cell proliferation (24). Therefore, the expression levels of p53 and p21 in HT29 and DLD1 cells were investigated, with or without baicalein treatment. As revealed in Fig. 2B and D, both p53 and p21 expression were significantly increased in baicalein-treated cells compared with the control cells, which was consistent with the aforementioned study (24).

p53 is also important for the regulation of metastasis and E-cadherin expression (32). Chang et al (33), revealed that p53 inhibits the invasiveness of CRC cells by regulating EMT. To further explore the effects of baicalein on CRC cells, the migration and invasion abilities of baicalein-treated HT29 cells were investigated. The wound-healing assay results demonstrated that baicalein inhibited the migratory ability of CRC cells in a dose-dependent manner (Fig. 3A). Following treatment with 10, 20 or 30 µmol/l baicalein for 48 h, HT29 cell motility was inhibited by 49.65, 67.41 and 83.17%, respectively (Fig. 3B). In the Transwell assays, cells from the control group exhibited a higher invasive capacity than those that had been treated with baicalein, indicating that baicalein significantly inhibited the invasiveness of CRC cells in a dose-dependent manner (Fig. 3C and D). These findings indicated that baicalein may act as a suppressor of CRC cell migration and invasion.

EMT is an important process which is characterized by the decreased expression of epithelial markers such as E-cadherin, and the concomitant increased expression of mesenchymal markers such as vimentin, matrix metalloproteinase 9 and various transcription factors (34). In the present study, the effects of baicalein on the expression of EMT markers was assessed in HT29 and DLD1 cells, following a 24- or 48-h treatment with 0–30 µmol/l baicalein. Compared with the control group, Snail, Twist1 and vimentin expression was decreased in baicalein-treated HT29 cells, while E-cadherin expression was increased at 24 and 48 h, respectively (Fig. 4A and B). Similar effects were observed in DLD1 cells (Fig. 4C and D). These results indicated that baicalein was able to impede EMT in CRC cells.

Since Snail plays a key role in metastasis and baicalein suppresses the expression of Snail and Snail-associated target genes (35), it was hypothesized that the antitumor activity of baicalein was influenced by Snail. In order to confirm this hypothesis, CRC cells were transfected with a pcDNA3.1-Snail plasmid, and Snail overexpression was confirmed by western blotting (Fig. 5A). The overexpression of Snail was revealed to increase the migratory and invasive capacities of CRC cells, which were reversed by baicalein treatment (Fig. 5B and C). Snail is reported to be one of the most important EMT-associated transcription factors; therefore, the effects of baicalein on Snail-induced EMT were also evaluated. As revealed in Fig. 5D, transfection with Snail-pcDNA3.1 markedly increased the expression levels of Snail, vimentin and Twist1, while the level of E-cadherin expression was significantly decreased, compared with the control-transfected cells; these results were consistent with the EMT expression profile. Notably, following baicalein treatment, Snail-induced vimentin and Twist1 upregulation, as well as E-cadherin downregulation were decreased both in HT29 and DLD1 cells, indicating that baicalein exhibits its suppressive effect partly through the inhibition of Snail-induced EMT.