HPLC grade methanol, ethanol and acetonitrile were obtained from Tedia Co. (Fairfield, OH, USA). Milli-Q water was supplied by a Milli-Q Water Purification System (Millipore, MA, USA). Four flavonoid standards, i.e., baicalin, wogonoside, baicalein and wogonin, were obtained from the National Institute for Control of Pharmaceuticals and Biological Products (Beijing, China). All standards were of biochemical-reagent grade and at least 95% pure confirmed by HPLC. Cellulase (XW-G-F Cellulase, 140,000 U/g) was obtained from Novozymes Biologicals Co. (Shenyang, China). Trypsin, McCoy’s 5A and DMEM medium, fetal bovine serum (FBS), and penicillin-streptomycin solution (200X) were obtained from Mediatech (Herndon, VA, USA). A CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit was obtained from Promega (Madison, WI, USA). Propidium iodide (PI) and RNase were obtained from Sigma (St. Louis, MO, USA). FITC Annexin V Apoptosis Detection kit was obtained from BD Biosciences (San Diego, CA, USA).

The S. baicalensis root, collected from Gansu, China, was obtained from Tianyi Drugstore Co. (Huaian, China). The voucher samples were deposited at the Group of BioTCM and Biocatalysis, Faculty of Life Science and Chemical Engineering, Huaiyin Institute of Technology. Dried S. baicalensis roots were ground with a blade-mill (FW135 Medicine Mill, Nanjing, China) to obtain a relatively homogeneous drug powder, and then sieved through a 100-mesh (0.15 mm) screen. The powder was dried in a desiccator with silica gel at ambient temperature until constant weight before extraction.

Ultraviolet (UV) spectra of flavonoids were recorded on a Shimadzu UV-2401PC UV-Vis spectrophotometer (Shimadzu, Japan). Quantitative determination was performed on an Agilent 1100 liquid chromatographic system (Agilent Technologies, CA, USA) consisted of a G1313A-ALS autosampler, a G1316A-Colume thermostated compartment, a G1311A-QuatPump and a G1379A degasser, a G1315B-DAD detector, and ChemStation software for peak identification and integration. The separation was carried out on an Agilent Zorbax Extend-C18 reversed-phase column (4.6×250 mm, 5.0 μm). A ternary isocratic solvent system of methanol-acetonitrile-water-acetic acid (18:25:57:0.2, v/v) was used. The flow rate was 0.8 ml/min and the detection wavelength was set to 275 nm. Temperature of column compartment was maintained at 30ºC. The injection volume was 10 μl. All tested solutions were filtered through a 0.2-μm membrane filter. The content of the constituents were calculated using calibration curves of four flavonoid standards.

Cellulase was quantified accurately and dispersed in phosphoric buffer solution to obtain 20 ml of enzyme solution with a certain activity unit. One gram of herbal powder was added into the enzyme solution. After adjustment of the pH value, the flask containing the reaction mixture was fixed on a thermostatic water bath shaker (130 rpm) for a period of treatment time at a controlled temperature. After the treatment was finished, absolute ethanol was added to the reaction solution and the concentration of ethanol in the solution was 75% (v/v). The solution was sonicated in an ultrasonic bath (Kunshan Ultrasonic Instrument Co., China) for 30 min at 25ºC before the extract was filtered. The residues were ultrasonically extracted twice with 20 ml of 75% ethanol. All the filtrates were transferred into a 250 ml volumetric flask and adjusted to the volume with 75% ethanol. A 5.0 ml of extract solution was used for HPLC analysis. The solvent of the extract solution was evaporated under vacuum, and then the residue was completely dissolved in methanol. The following reaction conditions were tested: cellulase concentration (5–30 U/g), temperature (30–60ºC), pH value (4.0–5.0), and reaction time (0–10 h).

After optimal conditions were obtained, a series SbE samples were prepared for biological evaluation. Briefly, 10 g of S. baicalensis root powder were added to 200 ml of reaction solution containing cellulase 20 U/g. The pH value was adjusted to 4.8. The temperature was set at 50ºC. The flask containing the reaction mixture was fixed on the shaker (130 rpm), and the reaction was conducted for 2, 4, 6 and 8 h. Subsequent treatment steps were the same as the above protocol. The filtrate collected was concentrated under vacuum (<60ºC) in a rotary evaporator to remove solvent. Then the extracts were lyophilized to obtain 2, 4, 6 and 8 h catalyzed SbE extracts: SbE2, SbE4, SbE6 and SbE8, respectively. Another control extract SbE0, which was without cellulase transformation, was also prepared. Before biological evaluation, the contents of the four flavonoids in SbEs were determined by HPLC.

Two cell lines, HCT-116 human colorectal carcinoma cells and MCF-7 human breast cancer cells (ATCC, Manassas, VA, USA) were cultured in McCoy’s 5A and DMEM Media, respectively, supplemented with 10% fetal bovine serum and penicillin-streptomycin (50 U/ml). Cells were maintained at 37ºC in a humidified atmosphere, with 5% carbon dioxide and 95% air.

Cells were seeded in 96-well plates. After one day, various concentrations of SbEs (dissolved in DMSO) were added to the wells. The final concentration of DMSO was 0.5%. Controls were exposed to culture medium containing 0.5% DMSO without drugs. All experiments were performed in triplicate. Cell proliferation was evaluated using an MTS assay according to the manufacturer’s instructions. Briefly, at the end of the drug exposure period, the medium was replaced with 100 μl of fresh medium and 20 μl of MTS reagent (CellTiter 96 Aqueous Solution) in each well, and the plate was returned the incubator for 1–2 h. A 60-μl aliquot of medium from each well was transferred to an ELSA 96-well plate and its absorbance at 490 nm was recorded. Results were expressed as a percentage of the control (DMSO vehicles set at 100%).

To further evaluate the effects of SbEs on anticancer potential, we performed cell cycle analysis of HCT-116 treated with different concentrations of SbE0 and SbE8. HCT-116 cells were seeded in 24-well tissue culture plates. On the second day, the medium was changed, and cells were treated with different concentrations of SbE0 or SbE8 for 48 h. After harvesting, the cells were fixed gently by adding 80% ethanol and placing them in a freezer for 2 h. They were then treated with 0.25% Triton X-100 for 5 min in an ice bath. The cells were resuspended in 300 μl of PBS containing 40 μg/ml propidium iodide (PI) and 0.1 mg/ml RNase. Cells were incubated in a dark room for 20 min at room temperature, and then subjected to cell cycle analysis using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA, USA) and FlowJo 10.0.4 software (Tree Star, Ashland, OR, USA). For each measurement, at least 20,000 cells were counted.

Cells were seeded in 24-well tissue culture plates. After one day, the medium was changed, and SbE0, SbE4 and SbE8 were added. After treatment for 48 h, cells floating in the medium were collected. The adherent cells were detached with 0.25% trypsin. Then, culture medium containing FBS (and floating cells) was added to inactivate trypsin. After pipetting gently, the cells were centrifuged for 5 min at 1500 × g. The supernatant was removed and cells were stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer’s instructions. Untreated cells were used as a control for double staining. The cells were analyzed immediately after staining using a FACScan flow cytometer. For each measurement, at least 20,000 cells were counted.

Cell proliferation, and flow cytometry experiments were performed in triplicate. The data are presented as mean ± standard error (SE). A one-way ANOVA determined whether the results had statistical significance. In some cases, a Student’s t-test was used for comparing two groups. The level of statistical significance was set at P<0.05.

Fig. 1A shows the chemical structures of four flavonoids (baicalin, wogonoside, baicalein and wogonin) identified from S. baicalensis. Ultraviolet spectroscopy of these four flavonoids is shown in Fig. 1B. The maximum absorption wavelength (λ_max) of the four flavonoids was 277.5, 275.5, 275.0 and 275.5 nm, respectively. Thus, for the HPLC analysis, 275 nm was a suitable wavelength for the determination of the four flavonoids. Fig. 1C is the HPLC chromatogram recorded at 275 nm. The four flavonoids were well separated under the established HPLC condition, and this HPLC method can be utilized to accurately measure these compound contents in the test samples.

To determine the optimal conditions of cellulase in catalyzing flavonoids from S. baicalensis, variable conditions were tested. Fig. 2A indicates the effects of cellulase concentrations from 5–30 U/g. Along with an increase of the enzyme concentration, an increase of the two aglycon contents and a decrease of the two glycoside contents were observed. Using the selected cellulase concentration of 20 U/g, the effect of temperature (30–60ºC) on transformation was tested. Fig. 2B indicates that 50ºC was the optimal reaction temperature. Using the selected cellulase concentration and temperature, Fig. 2C indicates the effects of pH value from 4.0–5.0, and a pH of 4.8 was selected for further evaluation. Fig. 2D shows the effects of treatment time from 0 to 10 h. The content curves of the two aglycons and two glycosides were obtained and they clearly indicate that treatment time significantly affected the flavonoid conversion. Overall, the optimized catalyzing conditions are: 20 U/g of enzyme, pH 4.8, treatment at 50ºC for 8 h.

Based on Fig. 2D observation, at fixed conditions (cellulase 20 U/g, pH 4.8 and 50ºC), different lengths of time were used to treat S. baicalensis. Fig. 3A shows HPLC chromatograms of five SbE sample treatments for 0, 2, 4, 6 and 8 h (SbE0, SbE2, SbE4, SbE6 and SbE8). Significantly different proportions of the four flavonoids in the five SbEs are shown in Fig. 3B. For SbE0, the content of the two glycosides was highest, and the content of the two aglycons was the lowest. However, for SbE8, the content of the two glycosides was the lowest, and the content of the two aglycons was the highest. The proportion of baicalin, wogonoside, baicalein and wogonin, was 43.3, 23.6, 8.5 and 1.6% in SbE0; 34.9, 16.8, 21.5 and 2.3% in SbE2; 21.5, 14.9, 35.6 and 4.1% in SbE4; 5.5, 9.4, 57.7 and 6.7% in SbE6; and 1.0, 1.7, 68.6 and 9.1% in SbE8, respectively. The results indicate that the proportion of the four flavonoids in the SbEs was significantly changed after cellulase treatment.

Effects of cellulase-catalyzed extracts on cancer cell growth inhibition were assayed. As shown in Fig. 4, after treatment with 20 μg/ml for 48 h, SbE0 inhibited HCT-116 cell growth by 15.9%, while SbE8 inhibited cell growth by 92.6%. Antiproliferative potential of other SbEs were between SbE0 and SbE8 (Fig. 4A). Sensitivity of MCF-7 cells by SbEs was lower than that of HCT-116. At 100 μg/ml, SbE0 inhibited MCF-7 cell growth by 20.5%, while SbE2, SbE4, SbE6 and SbE8 inhibited cell growth by 26.1, 55.4, 64.8 and 86.4%, respectively (Fig. 4B). Of note, at some lower concentrations, SbE0 and SbE2 actually improved MCF-7 cell growth. The IC₅₀ (50% inhibitory concentration) of SbE8 for HCT-116 and MCF-7 cells was ~5 and 30 μg/ml, respectively. Incubation time also influenced antiproliferative effects of SbEs on cancer cells. In treatment with 10 μg/ml of SbE8 for 24, 48 and 72 h, HCT-116 cell growth was inhibited by 57, 68 and 84%, respectively (Fig. 4C). Similar results were also observed on MCF-7 cells (Fig. 4D), suggesting that SbEs inhibit cancer cell proliferation in a time-dependent manner. In addition, SbE8 had the strongest antiproliferative effects on both cancer cell lines. These results demonstrated that 8 h of cellulase catalyzing significantly increased the antiproliferative potential of S. baicalensis.

To explore the potential mechanism by which cellulase-catalyzed extract inhibits HCT-116 cell growth, the cell cycle profile was assayed by flow cytometry. As shown in Fig. 5, compared to the control, SbE0 did not change the cell cycle profile obviously, whereas significant changes were observed with SbE8. Although treatment with SbE8 at 1 and 2.5 μg/ml did not change the cell cycle profile, when the treatment concentration was increased, the cell cycle profile was changed. Compared to the control (57.1% of G-phase, 25.3% of S-phase, and 19.9% of G2/M-phase), the percentage of G1-, S- and G2/M-phase cells after treatment with SbE8 for 48 h were 33.1, 46.0 and 15.6% at 10 μg/ml; and 25.4, 52.1 and 21.7% at 20 μg/ml, respectively. These results indicate that SbE8 significantly induced cell cycle arrest in the S-phase.

The cell proliferation assay results suggested that the cancer cell growth inhibitory capacity of SbEs is related to the cellulase pretreatment time (Fig. 4). To further explore the mechanisms of actions of SbEs, we carried out an apoptotic assay. As shown in Fig. 6, compared to the control (early apoptosis 4.7–5.6%, late apoptosis 2.2–3.6%), after 48 h of treatment with 5 and 10 μg/ml of extracts, SbE0 did not increase early and late apoptosis while SbE4 moderately increased the early apoptosis to 17.6%, whereas SbE8 quickly increased the early apoptosis to 48.1%. At the same time, we observed SbE4 and SbE8 significantly increased late apoptosis. This result suggests that the antiproliferative effect of SbE4 and SbE8 was mediated by the induction of apoptosis, and induced apoptosis in a dose-dependent manner. Furthermore, it was shown that apoptotic induction potential was positively correlated with the cellulase treatment time.