Materials
Dry Z. bungeanum was purchased from Xufang Traditional Chinese Medicine Co., Ltd. (Hebei, China), which was identified by Dr. Zhao (College of Pharmacy, Dalian Medical University). DEAE-52 cellulose and Sephadex G-75 were purchased from Beijing Solarbio Technology (Beijing, China). MN9D cells were purchased from the Institute of Biochemistry and Cell Biology (Shanghai, China). N-methyl-4- phenylpyridinium (MPP+) was supplied from MedChemExpress (New Jersey, USA). Dextran Standard 1.152 kDa was obtained from Yuanye Biotechnology Inc. (Shanghai, China). Dextran standards 5, 11.6, 23.8, 48.6, 273, 409.8 and 667.8 kDa were purchased from Sigma-Aldrich (St. Louis, MO, USA). Standard monosaccharides and methylation kit were obtained from Bo Rui Saccharide Biotech Co., Ltd (Jiangsu, China). All antibodies were obtained from ZEN-BIOSCIENCE Co., Ltd (Chengdu, China). ROS detection kit was supplied by Beyotime (Shanghai, China).
Acquisition of the crude polysaccharides
Crude polysaccharides were extracted according to the conventional method [26]. Total of 250 g crude Z. bungeanum powders (20–40 mesh) were weighed, and 2 L solvent (95% ethanol–petroleum ether = 1: 1, v/v) was added for extraction under reflux for 4 h at 70°C. The extract solution was filtered and the residues were collected and dried at 45°C in oven. Then, the herbs were processed with 2500 mL boiling water for 2.5 h. The extracts were concentrated to 250 mL under reduced pressure, and the supernatant was collected and added with 95% ethanol until the final concentration of ethanol reached to 80%, and the solutions were left to alcoholic sedimentation at 4°C for 24 h. The precipitate was obtained by vacuum filtration and washed with 95% ethanol, and dried in a vacuum drying oven. Then, the crude polysaccharide from Z. bungeanum (WZBP) was obtained.
In the study, the crude polysaccharides from Z. bungeanum were primary purified by AB-8 macroporous resin column chromatography. The crude polysaccharide solution (5 mg/mL) was prepared and loaded into the column, which was eluted with ultrapure water. The eluent was collected and concentrated.
Polysaccharide purification
The crude polysaccharide was further purified by DEAE-52 cellulose column chromatography. The crude polysaccharide aqueous solution (10 mg/mL) at 65°C was added to the DEAE-52 cellulose column (φ 45 mm × 30 cm) and equilibrated for 10 min. Then, the column was eluted with distilled water, 0.1 M NaCl solutions, 0.2 M NaCl solutions and 0.3 M NaCl solutions at the flow rate of 1 mL/min, successively. Each of the elution fraction were dialyzed (3500 Da) and freeze-dried. The content of polysaccharides were determined by phenol-sulfuric acid method [27]. The fractions eluted by 0.2 and 0.3 M NaCl solutions named WZBP-1 and WZBP-2 were obtained.
WZBP-1 and WZBP-2 were prepared into 10 mg/mL solutions, and loaded into Sephadex G-75 column (φ 20 mm × 50 cm) which were eluted with ultrapure water at the flow rate of 0.8 mL/min. In each tube, 10 mL of eluent was collected, and the absorbance values (490 nm) of the eluent were measured. Further, the elution fractions were dialyzed (3500 Da) and freeze-dried. The polysaccharide samples were obtained by weighing 1.0 g of sample precisely, adding it to 1 M TFA solution (100 mL), shaking vigorously at 25°C for 8 h, and freeze-drying after dialysis.
2.4. Homogeneity and molecular weight of WZBP-1-2 and WZBP-2
The homogeneity and molecular weights of WZBP-1-2 and WZBP-2 were analyzed by high-performance gel-permeation chromatography (HPGPC) [28]. High performance liquid chromatography (HPLC) equipped with BRT105-104-102 gel column (8 × 300 mm) and RI-10A refractive index detector were used. A series of standard substances and the polysaccharide samples were accurately weighed to prepare 5 mg/mL solution. 0.05 M NaCl solution was used to the mobile phase with the flow rate of 0.6 mL/min. The injection volume was 20 mL, the column temperature was 40°C. The solution of standard substance was injected into for the analysis, and the molecular weights of WZBP-1-2 and WZBP-2 were determined.
Monosaccharide composition
The monosaccharide composition were determined by Ion chromatography (ThermoFisher ICS5000, USA) with a DionexCarbopacTMPA20 column and an electrochemical detector [29, 30]. Polysaccharide (10 mg) was dissolved in 10 mL 3 M trifluoroacetic acid (TFA), and hydrolyzed at 120°C for 3 h. The acid hydrolysis solution was blow-dried with nitrogen to remove TFA. Then, 10 mL water was added, mixed, and then take 0.1 mL of the solution, diluted 10 times and centrifuged (12000 rpm, 5 min). The supernatant was taken for the analysis.
Methylation analysis
Methylated sugar alcohol acetate was evaluated by gas chromatography -mass spectrometry (GC-MS) [31, 32]. Reduction and complete acid hydrolysis of the glyoxylate were performed prior to methylation, and the compositions of the monosaccharide were determined using ion chromatography to confirm that all of the glyoxylate was reduced.
Methylation reaction was carried out according to the kit. The samples were determined by gas chromatography-mass spectrometry (Shimadzu, Japan). GC-MS analytical conditions were as follows: RXI-5-SIL-MS column (30 m × 0.25 mm × 0.25 µm), programmed temperature (starting temperature was 120°C, heating to 250°C/min at a rate of 3°C/min, maintain 5 min), injector temperature and detector temperature were 250°C, flow rate was 1 mL/min.
Nuclear magnetic resonance (NMR)
The polysaccharide sample (30 mg) was dissolved in 0.5 mL D2O and freeze-dried. The 1D NMR (1H, 1C and Dept135) and 2D NMR (HSQC, COSY, HMBC and NOESY) were measured using 600 MHz Bruker NMR spectrometer at 25°C (Bruker, Canada). MestReNova v12.0.3 software was used to process the data.
Cytotoxicity assay
MN9D cells were cultured in DMEM medium supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) at 37°C in 5%CO2 in a humidified incubator. The cells (1 × 105 cells/mL, 200 µL) were inoculated into 96-well plates and then treated with the polysaccharide solutions (0, 5, 10, 20, 40, 80, 160 µg/mL) in DMEM medium for 3, 6, 9 h, respectively. Total of 10 µL CCK-8 solution was added, and cell viability was detected by microplate reader at 450 nm (TECAN, Switzerland).
Cell viability assessment
MN9D cells were inoculated into 96-well plates (1 × 105 cells/mL, 200 µL). After growth for 18 h, polysaccharide solutions (10, 20, 40 µg/mL) were added into the cells for 6 h. Next, MPP+ (1200 µM) was added for 24 h, and CCK-8 method was used to measure the cell viability.
ROS assay
MN9D cells were inoculated into 24-well plates for 18 h (1 × 105 cells/mL, 500 µL). Then, the cells were processed through the same way mentioned above. In the end, 300 µL of DCFH-DA solution (10 µM) was added to each well for 30 min, and the sample was observed by inverted fluorescence microscopy and photographed (ZEISS, Germany).
Real-time PCR assay
MN9D cells were inoculated into 6-well plates (1 × 105 cells/mL, 2mL). Then, the cells were treated. TransZol UP kit was used to extract the total RNA, which was operated to produce cDNA according to the TransScript® All-in-One First-Strand cDNA Synthesis SuperMix for qPCR kit instruction. Finally, the mRNA levels were measured according to the instruction of TransStart® Top Green qPCR SuperMix kit, and the results were calculated by the 2−△△Ct method. All primers were designed by Real-time PCR primer design principles.
Western blot assay
MN9D cells were inoculated into 6-well plates (1 × 105 cells/mL, 2mL). Then, the cells were treated, and cellular protein was extracted. After determination of protein concentration by BCA protein quantitation kit (Seven-biotech, China), the samples were mixed with the solution (5 × loading buffer), boiled in water for 5 min and store at -20°C. The protein samples were transferred to a polyvinylidene fluoride membrane (Millipore, USA) by SDS-PAGE. After 5% skim milk was used to block at 25°C for 2 h, primary antibody was added to membranes overnight at 4°C. Then, the membranes were incubated with secondary antibody for at 25°C 2 h. Protein levels were visualized by enhanced chemiluminescence reagents (ECL) and imaged using Bio-Spectrum Gel Imaging System (UVP, USA). The bands were normalized and GAPDH was used as internal control.
Statistical analysis
The data of all experiments recorded as means ± standard deviations (SD) were analyzed with GraphPad Prism 7.0 analysis software. SPSS 20.0 software was used to analyze anti-inflammatory activity data by one-way ANOVA. Differences at p < 0.05 or p < 0.01 were generally considered as significant.