Bacteria strain and cultural condition
S. suis (ATCC 700794) strain was purchased from American Type Culture Collection (ATCC) and maintained in 50% glycerin at -40 °C. The bacteria were cultured at 37 °C in Todd Hewitt Broth (THB) medium (Summus Ltd, Harbin, Heilongjiang, China) when performing the experiments
Sample preparation and reagents
Samples of dried rhubarb (Rheum officinale Baill) plants were purchased from Three-trees herbal medicine market (Harbin, China). All the samples were smashed into powdery form using a disintegrator (xuman-2000y, Boou, Zhejiang, China), then filtered using a 50-mesh sieve (Beijing, China). Subsequently, the R. officinale powder was dried in an oven (YHG-9055A, Yaoshi instrument, Shanghai, China) at a temperature of 80 °C for 24 h and stored in a vacuum dish. Standard emodin of 98% purity was purchased from Solarbio (Beijing, China). Besides, all of other reagents used were of analytical grade. The methanol, crystal violet and glacial acetic acid were purchased from Aladdin (Shanghai, Chian).
Establishment of standard curve for emodin
First, 6.8 mg of standard emodin was added to a volumetric flask containing 50.0 mL of absolute ethanol. Aliquot of 1.0, 2.0, 3.0, 4.0 and 5.0 mL standard emodin solutions were mixed thoroughly with 1% magnesium acetate-ethanol solution in the volumetric flasks, and then these flasks were kept at room temperature for 15 min. Before measuring the absorbance of the standard emodin solutions, full wavelength scanning was carried out to determine the maximum absorption wavelength (λmax). Finally, the absorbance was measured at λmax of 507 nm against a blank sample without emodin using a UV spectrophotometer (UV-6000PC, Shanghai Yuan Analysis Instrument, Shanghai, China). A relationship between absorbance and concentration of emodin was obtained and expressed by the following regression equation:
An ultrasonic cell breaker (JY92-2D, Ningbo Scientz Technology Co. Ltd., Jiangsu, China) has been used for UAE of emodin from R. officinale. 0.5 g of R. officinale powder was poured into a beaker and a certain volume of ethanol with different concentrations was added at different liquid to solid ratios. The ultrasonic tip was submerged in the solution with a fixed depth of 2.0 cm and then UAE was conducted under ultrasonic power of 500 W for various periods of time at room temperature. The temperature of the system was maintained by adding ice on beaker surrounding. The suspension obtained was centrifuged at 8000 rpm (GL-20G-Ⅱ, Shimadzu, Japan) for 15 min at room temperature. Finally, the samples were measured at 507 nm according to the method mentioned above and the contents of emodin in solutions were computed according to the standard curve obtained in 2.3.1.
Single-factor experiments of UAE
In order to explore the best conditions of UAE for emodin, three variables including ultrasonic time, ethanol concentration and liquid to solid ratio were investigated. The ultrasonic time varied from 10 to 30 min; the variation of ethanol concentration was from 50% to 90%; and the liquid to solid ratio was from 5 to 15 (mL/g), respectively. When one of the variables was investigated, the remaining variables were fixed at a certain level.
Based on the results obtained from the single-factor experiments, ethanol concentration (X1), ultrasonic time (X2) and liquid to solid ratio (X3) were the three variables selected and the effect of their interactions on the yield of emodin (Y) was investigated by using RSM. The three-factor-three-levels Box-Behnken design (BBD) of RSM was carried out by using Design-expert software (version 8.0), and all the independent variables that to be encoded were varied over three levels (-1, 0, 1). As a result, X1 was varied over a range of 70%–90%. X2 was varied from 15 to 25 min. Likewise, X3 was changed from 10 to 15 mL/g. Besides, the codes and its representative values were shown in Table 1. The process of coding was carried out based on equation below :
BBD-matrix consisted of 17 groups of experimental runs including 12 factorial points and 5 central points. Additionally, the details of the experimental conditions and the relevant experimental and predicted values of emodin yields were given in Table 2. The relationship between dependent and independent variables was expressed as a second-order polynomial equation:
All experiments were tested in triplicate and data were analyzed by Design-expert software (version 8.0). The adequacy of the mathematical model was evaluated by analysis of variance (ANOVA), the regression coefficients, coefficient of determination, and the line of best fit. On the basis of the optimum conditions predicted by RSM, additional experiments were done to compare the obtained data with the predicted value, which in turn verify the validity of the model.
Under the optimum UAE conditions, the extraction solution was collected, concentrated, lyophilized, and the crude extract was obtained. Hereafter, the crude extract was purified by using a macroporous resin and then stored at -20 °C prior to further analysis. The purified sample was termed “RUEP” and its purity (P) was calculated according to the formula: P (%) = CRVR*100/mR, where in CR and VR were the concentration of emodin (mg/mL) and the volume (mL) of sample solution, respectively, and mR was the mass of frozen sample (mg).
Put Table 2 here
Identification of emodin in RUEP by HPLC-MS/MS
HPLC-MS/MS was applied to analyze the active ingredient in RUEP. During the process of separation of sample, an ACQUITY UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 mm) was connected to a HPLC system (SCIEX ExionLC™ AD) and kept at a temperature of 40 °C. Importantly, gradient elution was carried out in this experiment. Details were as follows: 0 min, 5% solvent B; 5 min, 95% solvent B; 11 min, 95% solvent B; 12 min, 5% solvent B and 15 min, 5% solvent B. Besides, the mobile phase A and B were 0.1% formic acid and acetonitrile, respectively. The injection volume and flow rate of mobile phase were set at 5 μL and 0.4 mL/min, respectively. The detection system was mass spectrometer (TripleTOF® 5600+, SCIEX, USA) equipped with an electrospray ionization (ESI) source. Identification process was performed under positive mode and the electrospray ionization voltage was set at 5500 V. Besides, the temperature of the source was kept at 550 °C. The pressure of nebulizer gas and heater gas was 50 psi, while the curtain gas was maintained at 35 psi. In positive ESI mode, the scan range of MS was 100–1200 m/z and MS2 were 100–1000 m/z. Nitrogen was used in each gas path with pressure of 50 psi, and the flow rate was of 10.0 L/min. ESI-MS and ESI-MS2 were 90 V in the declustering potential. The collision energy for ESI-MS and ESI-MS2 were 10 V and 35 V, respectively. The collision energy spread of ESI-MS2 was 15 V. The multiple reaction monitoring mode was applied on detection. Data collection and analysis were performed by using Analyst 1.5 software.
Determination of minimal inhibitory concentration (MIC)
MIC of RUEP was determined by using the Clinical and Laboratory Standards Institute (CLSI) guidelines as referenced . Briefly, overnight culture of S.suis was dissolved in 5.0 mL of THB medium at 37 °C and then was diluted to a final concentration of 1×105 CFU/mL. Subsequently, serial dilutions of the antibiotics (20 μL) were added into a 96-well microplate and then 180 μL of bacterial suspension was added. 200 μL of bacterial suspension without any antibiotics was selected as a control. After incubation at 37 °C for 24 h, the sample was taken from 96-well plate for observation. MIC was determined as the lowest concentration of antibiotics showing no visible bacterial growth and the control group was viewed as a reference
Effect of RUEP on biofilm formation
Biofilm assay was performed in line with the procedure described previously . Briefly, based on the MIC procedures, the culture time was prolonged to 72 h. The medium was discarded, and then phosphate buffer saline (PBS, pH 7.2) was added into each well to wash away the floating bacteria. After pouring out PBS, 200 μL of 99% methanol was added and the mixture was incubated for 30 min to fix the biofilm. Subsequently, the methanol was poured out, and every well was dyed with 200 μL of crystal violet solution for 30 min. In the end, crystal violet dye solution was discarded and wells were washed with distilled water, and then 200 μL of glacial acetic acid (33%) was added. The optical density value of this mixed solution was measured by using a microtiter plate reader (DG5033A, Huadong, Ltd., Jiangsu, China)
Scanning electron microscopy (SEM) observation
Two pieces of sterilized frosted glass were firstly put into a 6-well microplate, and then the biofilm culture was conducted according to the method mentioned above. One well without adding RUEP was used as a control group, the other well adding RUEP was used as an experimental group. After the biofilm formation of S.suis on frosted glass, the culture medium was discarded and 2.5% of glutaraldehyde was added, then, the mixture was placed in a refrigerator (4 °C) for 1.5 h. At the rinsing stage, 0.1 mol/L of PBS (pH 6.8) was applied three times at an interval of 10 min each. In order to achieve the purpose of dehydration, 70%, 80%, and 90% ethanol was respectively added in experimental units for one time, and then 100% ethanol was added three times at an interval of 10 min each. Next, a mixture of tert-butyl alcohol solution and ethanol (1:1) and a pure tert-butyl alcohol solution were added respectively at an interval of 15 min. The glass was stored in a refrigerator at -20 °C, and then it was pre-frozen for 30 min and dried in a Freeze dryer (ES-2030, HITACHI, Japan) for 4 h. The glass was fixed on the sample table with conductive tape and coated the gold film with a thickness of 100–150 Å using an ion sputtering coater (E1010, HITACHI, Japan). Finally, the observation of S.suis biofilm was carried out under an electron microscopy (S-3400N, HITACHI, Japan).
Molecular docking was one of the main methods in Computer-Aided Drug Design. The method was widely applied on discovery of drug target and study of potential mechanism of drug action . The three-dimension structure of LuxS of S.suis was obtained from the Protein Data Bank (https://www.rcsb.org) (PDB ID: 4XCH) . The emodin structure was obtained from Pubchem (https://pubchem.ncbi.nlm.nih.gov) . CDOCKER was selected for docking, which was a docking method performed based on CHARMm. Besides, semi flexible docking is adopted in the docking process, namely, all the residues of protein was fixed and the chemical compound was movable. The best conformation of compound with the lowest energy was selected for further analyzed. The binding model was analyzed and visualized by PyMoL v2.0.6. software .
All experiments were repeated in triplicate. Statistical analysis was carried out using SPSS 19.0. And p < 0.05 was considered as statistically significant.