Bacterial strains and bacterial culture
Staphylococcus aureus (SA) 25,923 was purchased from Shanghai Tiancheng Bio-information and Technology Co., Ltd., (Shanghai, China). MRSA and ESBLs-SA were provided by Huaihe Hospital (Kaifeng, Henan, China), and identified by Vitek-AMS (Automated Microbic System).
The three S. aureuss trains were activated and inoculated into Broth Agar Medium at 37 °C for 24 h in a thermostat, and then bacterial concentration was diluted with sterile broth agar to 106 CFU/mL.
Preparation of extracts and drugs
The roots of T. asiatica (200801) were collected from Guizhou province, China, in September 2008 and identified by Professor Zhiyou Guo, Qian Nan Normal College for Nationalities, Guizhou, China. The voucher specimen was stored at the Institute of Chinese Materia Medica, Henan University (Kaifeng, Henan, China).
Root powder of T. asiatica (1.3 kg) was extracted three times with methanol for 7 days each time. Then the extracts were evaporated and dried under reduced pressure. The concentrated extract was mixed with silica gel, and eluted successively with petroleum ether, ethyl acetate and methanol to obtain petroleum ether fraction, ethyl acetate fraction and methanol fraction, respectively.
Ethyl acetate fraction was loaded to silica gel column and eluted with CH2Cl2: MeOH (v:v = 100:1~ 8:2). Ten sub-fractions were obtained. After the sixth subfraction was repeatly subjected to silica gel column and Sephadex LH-20, chelerythrine (24.5 mg) was obtained. The purity of chelerythrine was higher than 98%. The NMR data of chelerythrine were published on China Pharmacist [14].
Analysis of chelerythrine by HPLC
The HPLC analysis was carried out in an Agilent 1260. Agilent TC-C18 column (250 mm × 4.6 mm, 5 μm) with acetonitrile and water containing 0.4% phosphoric acid (30:70) as mobile phase were used. The column temperature was set at 30 °C. The detection wavelength was at 258 nm, the flow rate was 1.0 mL/min and the injection volume was 10 μL.
Antibacterial activity
Antibacterial activity of chelerythrine was tested by disc diffusion test. Sample solution was obtained after dissolving chelerythrine (50 μg) with DMSO (1 mL). Filter paper discs of 6 mm diameter were impregnated with 5 μL of sample solution. A disc prepared with corresponding volume of DMSO was used as negative control and that prepared with berberine was used as the positive control. The plates were incubated at 37 °C for 24 h. Antimicrobial activity was evaluated by measuring the diameter of the inhibition zone (IZ) [21].
According to the results of IZ method, bacteriostatic ring was > 8 mm, then its minimum inhibitory concentration (MIC) was determined in triplicate by the tube doubling dilution method, and the test sample was serially obtained a series of concentration. MICs were defined as the lowest sample concentration that exhibited IZ.
Inhibitory effects on the bacterial cell wall
Chelerythrine at MIC and 3 × MIC were mixed with bacterial suspension (5 × 106 CFU). Sterile water was used as negative control group. All the groups were cultured in incubator at 37 °C and time sampling method (0 h, 0.17 h, 0.33 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 7 h, and 9 h) was adopted. Then the testing objects were centrifuged at 3500 rpm for 10 min, the content of AKP in supernatant was measured by the AKP kit assay with time.
Determination of soluble protein
Coomassie brilliant blue method was used to determine the concentration of soluble protein in bacterial suspension treated with chelerythrine (MIC and 3 × MIC) and the control groups, respectively. After resting for 10 min, OD values of all groups were measured with spectrophotometer at 595 nm.
SDS-page
The chelerythrine solution at MIC was added to bacterial suspension and the mixed suspension was cultivated at 37 °C for 24 h. Sterile water was used as negative control. Mixed solution (12 mL) was sampled at 3, 6 and 9 h, respectively. After the samples were diluted and adjusted into the same OD value, the precipitations were obtained by centrifuge, and then 80 μL of sterile water and 20 μL of 5 × SDS sample buffer precipitation were added, mixed thoroughly and boiled in a water bath at 100 °C for 5 min, the solution was centrifuged again and supernatant on standby was collected and used as protein extracts.
The vertical slab gel electrophoresis made from 8% of separating gel and 5% of stacking gel was used to separate proteins. 5 μL of protein maker and 15 μL of each sample were drawn with micro pipette tips. At first, the voltage was set at 60 V and electrophoresis was run, when the bands of samples passed into separating gel, the voltage was adjusted to 120 V and electrophoresis was continuously run until the blue ribbons was one or two centimeters distant from the bottom edge of the gel. The power was turned off and rubber blocks were taken out and stained with Coomassie brilliant blue for 3 h. Thereafter, the stained rubber blocks were de-stained with destainer in a shaking table up to appearing clear bands.
Scanning electron microscope (SEM) and transmission electron microscope (TEM)
Chelerythrine (1 × MIC) was added with bacteria at the concentration of 109 CFU/mL for a shaking Table (150 r/min) for 1.5 h and 6 h at 37 °C, respectively. Distilled water was used as control. The depositions were collected by centrifugation and fixed with 2.5% glutaraldehyde at 4 °C overnight, and washed three times with 0.1 M phosphate buffer solution (PBS, PH 7.2). The samples were dehydrated in a graded series of alcohols (30, 50, 70, 85, 90 and 100%). Tert-butanol was used to replace the ethanol twice before coating onto the metal foil. Eventually, the cells were dried by freeze-drying apparatus (ALPHA1–4, Christ, Germany), and visualized under a scanning electron microscopy (SEM, JSM-5600LV, JEOL, Japan) and a transmission electron microscopy (TEM, JEM-2010, JEOL, Japan), respectively.
