Sources and authentication of herbal materials
Radix Astragali, dried root of Astragalus membranaceus (Fisch.) Bge., Radix Codonopsis, dried root of Codonopsis pilosula (Franch.) Nannf. and Cortex Lycii, dried root bark of Lycium chinense Mill., were purchased from a herbal pharmaceutical company in Hong Kong. All herbs were authenticated by morphological observation and thin layer chromatography (TLC) according to the method described in Pharmacopoeia of the People's Republic of China 2000 . The reference compounds and reference herbs used in authentication were purchased from National Institute for the Control of Pharmaceutical and Biological Products in China. The authenticated voucher specimen were deposited in the Institute of Chinese Medicine, The Chinese University of Hong Kong with voucher numbers (Radix Astragali, 2005-2580; Radix Codonopsis, 2005-2597; Cortex Lycii, 2005-2601).
Preparation of herbal extracts
SR10 was prepared as described previously by boiling 214.3 g of Radix Astragali, 214.3 g of Radix Codonopsis and 71.4 g of Cortex Lycii in 5 L of distilled water for 2 hours under reflux  and collecting the extract. Another 5 L of distilled water was added and the boiling process was repeated. Two batches of water extract were mixed together and centrifuged to remove the herbal debris. The extract was vacuum dried and the resulting herbal powder was stored at -20°C until use. The powder contained 25.5 g/100 g of the starting raw material.
A7r5, a rat aorta smooth muscle cell line, was purchased from American Type Culture Collection (ATCC number CRL-1444) and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified atmosphere of 5% CO2 at 37°C.
Measurement of free radical-induced erythrocyte hemolysis
Blood was collected from adult Sprague-Dawley (SD) rat from thoracic aorta by heparinised tube. Red blood cells were obtained by centrifugation at 1500 × g for 10 minutes and washed twice with 0.15 M NaCl solution. After centrifugation, 20% RBC suspension was obtained by resuspending RBC in four times the volume of 0.15 M NaCl solution. RBC lysis reaction was set up in microcentrifuge tubes, each containing 10% RBC suspension, 100 mM of 2,2'-azo-bis-(2-amidinopropane) dihydrochloride (AAPH) and ascorbic acid (positive control) or various concentrations of SR10 in a total volume of 1 ml. Control was set up using PBS instead of SR10. RBC with ascorbic acid or SR10 was added first, and the reaction initiated by adding 100 mM of AAPH. The mixtures were then incubated in an oscillator at 37°C for 200 minutes. After incubation, the mixtures were diluted with PBS or distilled water by 20-fold respectively. The diluted mixtures were centrifuged at 1500 × g for 10 minutes. The supernatant (200 μl) of each mixture was transferred to a 96-well microtiter plate for measurement at 540 nm by microplate reader. Percentage inhibition of RBC hemolysis was calculated by the equation: Inhibition % = (A - B) × 100%, whereas A = (DWsample - PBSsample)/DWsample; B = (DWcontrol - PBScontrol)/DWcontrol
Measurement of LDL peroxidation
The reaction was set up in a quartz cuvette, each containing 75 μg of LDL, 5 μM of copper (II) chloride and various concentrations of SR10 in a total volume of 1 ml. LDL and the testing drug were added to the cuvette before adding copper (II) chloride to initiate the reaction. Conjugated dienes formation was continually monitored at 37°C by measuring UV absorption at 234 nm in 5-minute intervals for a total of 24 hours. The lag time (L.T.) for the formation of conjugated dienes was determined to be the intercept of the slopes for the lag and propagation phases, and was compared to the control (using PBS instead of SR10).
Cell proliferation assay
A7r5 cells (0.5 × 104/well) were seeded in each well of 96-well culture plate. After overnight incubation, PDGF-BB (25 ng/ml) was added to the cells in the presence or absence of various concentrations of SR10. Cells without the addition of PDGF-BB and SR10 was used as negative control. After further incubation for 24 hours, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay was performed to measure cell viability . Briefly, medium was removed and 40 μl of MTT solution (5 mg/ml in PBS) was added to each well. After incubation for 4 hours at 37°C, MTT solution was removed and 100 μl of dimethyl sulfoxide was added to dissolve the crystals formed. Then, absorbance at 540 nm was read using a microplate reader. The percentage cell viability was calculated as [Absorbance(treatment)/Absorbance(negative control)] × 100%.
Determination of DNA synthesis
DNA synthesis in A7r5 cells was determined by 3H-thymidine uptake assay. Cells (2 × 103/well) were seeded in a 96-well plate and incubated overnight. The cells were synchronized by starving in 1% fetal bovine serum for another 24 hours. PDGF-BB was added in the presence or absence of various concentrations of SR10 and further incubated at 37°C with 5% CO2 for 24 hours. Subsequently, tritiated thymidine (0.5 μCi per well) was added into each well and incubated for 6 hours. After that, the cells were harvested on glass fiber filters by a cell harvester. Radioactivity in the filters was measured by Microplate Scintillation and Luminescence Counter (Topcount NXT™).
Cell cycle analysis by PI staining using flow cytometer
Cells (2 × 105/well) were seeded in a 6-well plate and incubated overnight. The cells were synchronized by starving in 1% fetal bovine serum for another 24 hours. PDGF-BB was then added in the presence or absence of various concentrations of SR10 and further incubated at 37°C with 5% CO2 for 24 hours. Cell cycle analysis was performed by PI staining using flow cytometry as described previously . In brief, the cells were harvested, washed twice with PBS and fixed overnight with 70% ethanol. After fixation, the cells were washed with PBS and resuspended in 400 μl of PBS, 50 μl of RNase A (10 mg/ml) and 10 μl of propidium iodide (PI, 2 mg/ml). The cells were further incubated at 37°C for 30 minutes before analysis by FACSort flow cytometry (Becton Dickinson) using 'Cell Quest' software. The cell population was chosen by forward scatter (FSC) light and side scatter (SSC) light. The signal was detected by FL3 channel for PI with log scale.
Cell migration assay
Cell migration assay was performed in modified Boyden chambers using Transwell (Costar) culture chambers with membrane pore size of 8 μm. A7r5 cells (1.5 × 104/well) in serum-free DMEM were loaded in the upper compartment (100 μl). PDGF-BB dissolved in plain DMEM was placed in the lower compartment (600 μl) in the presence or absence of various concentrations of SR10. The chamber was incubated for 3 hours at 37°C in a humidified atmosphere containing 5% CO2/95% air. Cells on the membrane were fixed in 1% paraformaldehyde and stained in hematoxylin. Non-migrated cells on the upper surface were scraped away gently. The number of migrated cells at the lower surface was determined under microscope. Five regions were counted per filter. Three chambers were used for each treatment and control group. The experiment was performed in triplicate.
Western blot analysis of extracellular regulated kinases 1 and 2 (ERK1/2) and cyclin D1
Western blot was performed as described previously . After appropriate treatment, the cells were lysed in buffer containing 0.02% Aprotinin, 2% SDS, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8 and the protein concentration was determined using the bicinchonic acid protein assay. Samples with equal amount of protein (25 μg) were analysed by polyacrylamide gel electrophoresis. The proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 10% non-fat milk in PBS-T (PBS with 0.1% Tween-20), the membranes were incubated overnight with antibodies against ERK1/2 or cyclin D1 or β-actin at 4°C. The membranes were further incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour and the signal detected by enhanced chemiluminescence (ECL) detection reagents (GE Healthcare).
All experimental results were presented as mean ± standard deviation (S.D.). The Mann-Whitney test was used for comparison between PDGF-BB-treated group and each SR10 treatment group. The test was two-sided with a significance level of 0.05.