| Objective"Ban-Zhi-Lian" is the air-dried aerial parts of Scutellaria barbata(Scutellaria genus, Lamiaceae). As a common-used traditional Chinese Medicine, Scutellaria barbata plays an important role in treatment kinds of tumors, including gynecological tumor, leukemia, colon cancer, hepatoma, lung cancer, skin cancer in combination with other traditional Chinese medicines. Scutellaria barbata contains a large number of flavonoids, diterpenoids, alkaloids, volatile oils as well as other organic acids, terides and polysaccharides. The chemical constituent of Scutellaria barbata is very complex. Its rational and efficient utilization depends on the studies of active chemical constituent and its effective mechanism. Recently domestic and foreign scholars carried out many researchs on the anti-tumor effect of Scutellaria barbata. The results indicated that its crude extract had growth inhibitory effects on a number of human cancer cells in vitro. However, the antitumor activity of chemical constituents from Scutellaria barbata has not been fully determined. The underlying mechanism of anti-cancer remains unclear. Numerous studies have also demonstrated some alkaloids of the plant have significant anti-tumor effect. Thus, the present study was carried out to evaluate the anti-cancer activity and to determine the possible mechanisms of proliferation inhibition elicited by AESB in cultured hepatoma HepG-2 and CNE-1 cells. At the same time, the chemical constituents of AESB were isolated. So that we can get some active compounds for anti-tumor and provide a theoretical basis for better development and utilization of Scutellaria barbata. Methods1. Preparation of AESB solutionThe air-dried aerial parts of Scutellaria barbata was finely cut and soaked in acid ethanol (12 mol/L HCl-95% EtOH,2:100, V/V) for 72 h at room temperature, then extracted three times in an ultrasonic bath with acid ethanol for 45 min. After evaporating the solvent under reduced pressure, the extract was dissolved and suspended in 2% HCI solution, stood overnight and filtrated. The acidic solution was basified to pH 10 with NH4OH solution and exhaustively extracted with chloroform. The chloroform phase were combined and dried using a speed vacuum centrifuge to yield the alkaloidal extract. The alkaloidal extract was dissolved in 2% HCl solution again and basified to pH 10 with NH4OH solution, exhaustively extracted with chloroform. The chloroform phases were combined and dried using a speed vacuum centrifuge to yield AESB.0.30 g of AESB was dissolved in 2% HCl solution and basified to pH 7.4 with NaOH solution, then diluted with saline to obtaine AESB solution (30 mg/ml), which was sterilized using a 0.22μm filter and stored at 4℃until use. The final concentrations of AESB which were freshly diluted with culture medium for each experiment were 0.75,1.0,1.5 and 2.0 mg/mL, respectively.2. Cells cultureHepG-2 and CNE-1 cells were maintained with RPMI-1640 medium supplemented with 10% fetal bovine serum, containing 100 U/mL penicillin and 100 U/mL streptomycin in 25cm2 culture flasks at 37℃in a humidified incubator with 5% carbon dioxide. The cells were fed every two days and subcultured once they reached 80% confluence.3. MTT assay measured HepG-2 and CNE-1 cells proliferation inhibition rateHepG2 and CNE-1 cells (1×105 cells/mL) in exponential growth stage were suspended in medium and seeded in 96-well plates with 100 uL/well. After culturing for 24 h to obtain adherent monolayer cells, discarded the medium. Cells were washed with PBS twice and incubated in 100μL of the fresh medium containing various concentrations of AESB solutions (The final concentrations were 0.75 mg/mL,1.0 mg/mL,1.5 mg/mL and 2.0 mg/mL) for 24,48,72 and 96 h, respectively. Positive group was treated with 5-FU solution (The final concentration was 250μg/mL). Wells added the same volume of sterilized saline were set as negative group. At the end of each time point, the drug-containing medium was replaced by 90μL of fresh medium. Then,10μL of MTT was added to each well and the plates were incubated for additional 4 h at 37℃. After removing the supernatant solution,150μL of DMSO were added to each well, and gently vibrated for 10 min. The absorbency at a wavelength of 570 nm of the dissolved solutions was measured with a microplate reader. Results were expressed as the percentage growth inhibition with respect to the untreated cells. The growth inhibition rate was determined using the following formula. Growth inhibition rate(%)=(negative group’s OD-test group’s OD)/negative group’s OD×100%.4. Annexin V-FITC/PI double staining measured HepG-2 and CNE-1 cells apoptosis rateHepG2 and CNE-1 cells (2×105 cells/mL) in exponential growth stage were suspended in medium and seeded in 6-well plates with 2 mL/well. After cultured for 24 h to obtain adherent monolayer cells, the medium was removed. Cells were washed with PBS twice, then 2 ml of the fresh medium containing 0.75 mg/mL AESB was added. Positive group was treated with 5-FU solution (The final concentration was 250μg/mL). Wells added the same volume of sterilized saline were set as negative group. After incubating for 48 h, the detached and attached cells were harvested and washed with PBS twice and centrifuged at 1000 rpm for 5 min to remove PBS. Then the cells were treated according to the Annexin V-FITC/PI apoptosis detection kit steps. The early apoptosis rate of cells was analysis by flow cytometry. Cells positive for Annexin V but negative for PI fluorescence were identified as apoptotic.5. Flow cytometer analyzed HepG-2 and CNE-1 cells cycle phase distributionHepG2 and CNE-1 cells (2×105 cells/mL) in exponential growth stage were suspended in medium and seeded in 6-well plates with 2 mL/well. After cultured for 24 h to obtain adherent monolayer cells, the medium was removed. Cells were washed with PBS twice, then 2mL of the fresh medium containing 0.75 mg/mL AESB was added. Positive group was treated with 5-FU solution (The final concentration was 250μg/mL). Wells added the same volume of sterilized saline were set as negative group. After incubating for 48 h, the detached and attached cells were harvested and washed with PBS twice. The cells were fixed in 70% ice-cold ethanol at-20℃overnight. After fixation, the ethanol was removed. Cells were washed with PBS twice, and resuspended in 500μL of PBS solution. Then 50μL of RNase and 450μL of PI was added and incubated at 37℃for 15 min in the dark. The samples of cells were then analyzed for DNA content by flow cytometer. The cell cycle phase distributions were analyzed by ModiFit LT 2.0 software.6. Isolation of pured compounds from AESBAESB was isolated and purified by modern chromatographic techniques, including silica gel column chromatography, TLC and recrystallization. On the basis of spectroscopic analyses, including MS, IR,1D-NMR (1H-NMR,13C-NMR, DEPT, NOESY-1D and NOE difference spectra),2D-NMR(1H-’H COSY, HSQC and HMBC) and X-ray single crystal diffraction, the structures of these pure compounds were elucidated.7. Statistical analysisExperimental data were expressed with mean±standard deviation (x±s) and analyzed by SPSS 13.0 for windows statistical software. Using analysis of variance of factorial design to analyze the effects of two factors, multiple comparisons adopted Dunnett T3. Groups of Single factor were compared using one-way ANOVA. If variance is homogeneity, LSD will be used, otherwise Dunnett T3. Significant level a = 0.05, P<0.05 showed statistical significance.Results1. The effects of AESB on the proliferation of HepG-2 and CNE-1 cells in vitroAfter the different concentrations of AESB treating HepG-2 and CNE-1 cells for different time, the difference of inhibition rate of HepG-2 and CNE-1 cells at different concentrations of AESB was significant (HepG-2 cells:F=136.216, P=0.000; CNE-1 cells:F=341.470, P=0.000). The difference of inhibition rate of HepG-2 and CNE-1 cells at different times was significant (HepG-2 cells:F=118.114, P=0.000; CNE-1 cells:F=308.147, P=0.000). The interaction of time and AESB was also significantly different (HepG-2:F=3.654, P=0.003; CNE-1:F=7.747, P=0.000). MTT assay showed that AESB could inhibit the proliferation of HepG-2 and CNE-1 cells in dose-and time-dependent manner in vitro.2. The effects of AESB on the apoptosis of HepG-2 and CNE-1 cellsAfter 0.75 mg/mL of AESB treating HepG-2 cells for 48 h, the cells apoptosis rate (25.12±0.91%) had statistical significance as compared with the negative group (2.97±0.49%) (P=0.000). After 0.75 mg/mL of AESB treating CNE-1 cells for 48 h, the cells apoptosis rate (5.13±0.70%) also had statistical significance as compared with the negative group (1.42±0.26%) (P=0.004). These results suggested that 0.75 mg/mL of AESB had positive effects on apoptosis of HepG-2 and CNE-1 cells.3. The effects of AESB on the cell cycle distributions of HepG-2 and CNE-1 cellsSignificant increase in the G2/M phase of the cell cycle (P=0.002) occurred after 0.75 mg/mL of AESB treatment for 48 h by comparison with the corresponding values for cultured HepG-2 cells without AESB. Significant decreases in the G0/G1 (P=0.018) and G2/M (P=0.008) phases of the cell cycle together with significant increase in the S phase (P=0.001) occurred after 0.75 mg/mL of AESB treatment for 48 h by comparison with the corresponding values for cultured CNE-1 cells without AESB. These results suggested that 0.75 mg/mL of AESB could arrest the cell cycle of CNE-1 cells at S phase, the cell cycle of HepG-2 cells at G2/M phase.4. Isolation of pured compounds from AESBThree compounds were isolated from AESB. Their chemical structures were elucidated asβ-sitostero; Loliolide; Ethyl 4-hydroxy-3,5-dimethoxy-benzoate.Conclusion1. AESB could inhibit the proliferation of HepG-2 and CNE-1 cells in vitro, induce HepG-2 cells apoptosis and cell cycle arrest at G2/M phase, induce CNE-1 cells apoptosis and cell cycle arrest at S phase.2.β-sitosterol(1); Loliolide(2); Ethyl 4-hydroxy-3,5-dimethoxy-benzoate(3) were isolated from AESB. Among them, compounds 2 and 3 were isolated from the genus Scutellaria for the first time. |
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