Materials and chemicals
Fresh cultivated rhizomes of P. cyrtonema were collected from Jinzhai Senfeng Agricultural Technology Development Co., Ltd., Anhui, China. After removing fibrous root, the rhizomes of P. cyrtonema were cleaned and then cut into thin slices (3 mm ± 1 mm). Species identification was performed by Professor SP Li, one of the corresponding authors. Voucher specimens of these samples were deposited at the Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China.
ABTS, i.e., 2,2'-Azinobis-(3-ethylbenzthiazoline-6-sulphonate), potassium persulfate and ascorbic acid (≥ 99%) were purchased from International Laboratory (San Bruno, CA), Fluka (Selzer, Germany) and Aladdin (Shanghai, China), respectively. Polygalacturonic acid (PGA), galacturonic acid (GA), konjac glucomannan (KG), dextran (DEX), pectinase (EC 220.127.116.11), endo-1,4-β-D-mannanase (EC 18.104.22.168) and endo-1,4-β-D-galactanase (EC 22.214.171.124) were purchased from Megazyme (Wicklow, Ireland). 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Griess reagent, lipopolysaccharides (LPS) and fluorescein isothiocyanate-dextran (FITC-Dextran) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Specific limulus test kit for endotoxin detection was purchased from Bioendo Technology, Co., Ltd. (Xiamen, China). Cell counting kit 8 (CCK8) was purchased from MedChemExpress. Phosphate-buffered saline (PBS), Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin (P/S) were purchased from Gibco-Invitrogen (Paisley, Scotland, UK). Nylon membrane filters (0.22/0.45 μm) were purchased from Millipore (Billerica, MA). Deionized water was prepared using a Millipore MilliQ-Plus system (Millipore, Billerica, MA). All the other reagents were of analytical grade.
Preparation of P. cyrtonema and polysaccharides
Steam of P. cyrtonema
Fresh P. cyrtonema slices (dried as PC0) were steamed, over water, in an autoclave at 115 °C for 2 h at pressure of 0.07 MPa with good repeatability. The steamed material dried under vacuum at 60 °C to constant weight and collected as the sample PC1. Then half of PC1 was moistened with deionized water before further steam and dried under mentioned conditions above to obtain PC2. Similarly, PC3, PC4 and PC5 were prepared, respectively. All samples were grounded, and the particle size was 355 μm ± 13 μm.
Extraction of polysaccharides
Each sample (PC0-PC5) of 50.0 g was soaked at room temperature for 2 h, followed by hot water extraction (95 °C) for further 2 h with solid-to-liquid ratio of 1:15. The water extracts were precipitated with ethanol at a final concentration of 75% to harvest polysaccharides . The supernatant solution (PCS) and precipitate (PCP) were collected. Then small molecules (less than 3 KDa) mixed in polysaccharides were further removed using ultrafiltration. Finally, the polysaccharides were dried with freeze-drier to obtain PCP0, PCP1, PCP2, PCP3, PCP4 and PCP5, respectively.
Sample pretreatment of PCP
Partial and complete acid hydrolysis of PCP
According to a previously reported method with minor modification , PCP solution (2 mg/mL) was treated with trifluoroacetic acid (TFA) at a final concentration of 1.0 mol/L and incubated at 80 °C for 2 h to gather partial acid hydrolysates (PAH). At the same time, the PCP solution (4 mg/mL) of each sample was mixed with an equal volume of 4 mol/L TFA for complete acid hydrolysis at 105 °C for 4 h to gather complete acid hydrolysates (CAH).
Enzymatic hydrolysis of PCP
According to literature , three enzymes of pectinase, β-1,4-galactanase and β-1,4-mannanase were selected to depolymerize PCP (2 mg/mL) at 40 °C for 12 h. After incubation, enzymes were inactivated at 80 °C for 20 min. For PACE analysis, the enzymatic PCP hydrolysates should be firstly freeze-dried and derivatized with ANTS. While no additional treatment is required for HPTLC and monosaccharides analysis. Polysaccharide standards, including PGA, GA and KG, were treated with those enzymes, respectively, under the same conditions. PCP solution without TFA or enzyme treatment was used as blank control.
Physicochemical characterization of polysaccharides
Molecular weights and chain conformation analysis
The molecular weights and their distribution of PCP were determined by HPSEC-MALLS-RID according to our previous report . HPSEC-MALLS-RID method was composed of multi-angle laser light scattering (MALLS) detectors (Wyatt Technology Co., Santa Barbara, CA, USA), Agilent 1260 series LC/DAD system (Agilent Technologies, Palo Alto, CA, USA) and a refractometer (RID, Optilab rEX, Wyatt Technology Co.) in series at 35 °C. The chromatographic columns were TOSOH gel columns TSK-GEL G5000PWXL (300 mm × 7.8 mm) and TSK-GEL G3000PWXL (300 mm × 7.8 mm), and the mobile phase was 0.9% NaCl solution. The flow rate was 0.5 mL/min with 100 μL injection volume. All samples of PCP were dissolved in mobile phase at 2 mg/mL and filtered through 0.45 μm filter membrane before injection. ASTRA 7.3.2 software was used to process the data.
Compositional monosaccharides analysis
The complete acid and pectinase hydrolyzed samples as well as PCS were analyzed by HPAEC-PAD system (Thermo Scientific™ Dionex™ ICS-5000+, Dionex, USA). Ten reference monosaccharides, including Fuc, Ara, Rha, Gal, Glc, Xyl, Man, Fru, GalA and GlcA, were used to calculate the content of each monosaccharide in the samples using one-point calculation of external standard method. All samples were filtered through 0.45 μm membrane before analysis. The mobile phase consisted of 88% deionized water and 12% 10 mM NaOH, running for 22 min at a flow rate of 0.4 mL/min on a CarboPac PA200 (3 mm × 250 mm) analytical column with a system temperature of 25 °C (Additional file 1: Table S1).
PACE analysis of partial acid and enzymatic hydrolysates
PACE was performed according to previous report . Briefly, all lyophilized derivatized hydrolysates of PCP were redissolved in isovolumetric urea (6 mol/L), and separated by Mini-Protean Tetra System, a vertical slab gel electrophoresis apparatus from Bio-Rad. Gels were imaged using an In-Genius LHR CCD camera system (Syngene, Cambridge, UK) under UV 365 nm. Quantity-One software (Ver4.6.2, BioRad) and Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Matlab version, Ver1.315, developed by the Research Center of Modernization of Chinese Herbal Medicine, Central South University and the Hong Kong Polytechnic University) were used for similarity analysis.
HPTLC analysis of complete acid and pectinase hydrolysates
Merck silica gel 60 plates pre-washed with methanol were used for HPTLC analysis. The method was modified according to previous report . In brief, PCP0-PCP5 samples after complete acid or pectinase hydrolysis were prepared into 8 mg/mL, respectively. SP-III electric thin-layer strip sampler (KEZHE SHANGHAI, China) was used for semi-automatic sampling. The bands were 7 mm wide, 5 mm distance, and 10 mm from the bottom edge. Then the plate was firstly developed to 90 mm with 1-butanol/isopropanol/acedic acid/water, 7:5:1:2 (v/v/v/v) as developing reagent at room temperature. Then the plate was dried and placed in the same chamber to develop 95 mm with the same developing reagent as described above. Finally, the developed plates were dried and colorized with aniline-diphenylamine-phosphoric acid solution, 10% sulfuric acid ethanol solution and 0.2% ninhydrin solution, respectively, then heated at 105 °C and photographed under white light.
PCP0-PCP5 (1 mg/mL) were fully dissolved in ultrapure water and diluted to a concentration of 1 × 10−2 μg/mL. Using droplet deposition method, pipette 5μL of solution onto the surface of newly cut mica sheet and dry it at room temperature. After the sample dried, AFM measurement was performed using BioScope Resolve AFM (Bruker Co., Santa Barbara, USA), and NanoscopeAnalysis 1.8 software was used for image analysis.
Antioxidation of PCP against ABTS radicals
In brief, 7 mmol/L ABTS aqueous solution and 2.5 mmol/L potassium persulfate aqueous solution were mixed in a ratio of 1:1, and then stand in the dark for 12 h. This solution was diluted with deionized water to reach a 0.7 ± 0.05 absorbance value at 734 nm and obtained ABTS working solution. After preliminary optimization, series concentrations of PCP0-PCP5 (0.5, 1, 2, 4, 8 mg/mL) and ascorbic acid (0.015, 0.03, 0.06, 0.12, 0.24, 0.5, 1, 2, 4, 8 mg/mL) were used. In a 96-well plate, 200 μL of ABTS working solution and 10 μL of the sample solution were added to each well, and the reaction was kept in the dark for 6 min. Then the absorbance was measured, and the ABTS clearance rate formula was as followed:
C (%) = [1-(A1-A2)/A0] × 100.
C was the clearance rate, A0 is the control absorbance, A1 is the sample absorbance, and A2 was the background absorbance, which was to eliminate the interference of tested solution.
Effects of PCP on macrophage functions
RAW 264.7 cells were purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). Cells were cultured in Dulbecco’s modified eagle medium supplemented with 10% FBS and 1% P/S at 37 °C in a humidified atmosphere of 5% CO2.
RAW 264.7 cells (5 × 103 cells/well) were cultured in 96-well microplates overnight, and then treated with LPS (0.4 μg/mL) and a series of concentrations of PCP for 24 h, respectively. An equal volume of culture medium was used as blank control. Subsequently, the original culture medium was discarded and stained with 100 μL of culture medium containing 10% CCK8 for 4 h in dark. The absorbance values were read at 450 nm and the cell viability was calculated as the ratio of absorbance values between the sample and vehicle control group.
Nitric oxide determination
RAW 264.7 cells (5 × 104 cells/well) were seeded in 96-well microplates overnight, and then cells were treated with a series of concentrations of PCP and LPS (0.4 μg/mL) for 24 h, respectively. An equal volume of culture medium was used as vehicle control. Subsequently, 75 μL of supernatants were collected and mixed with an equal volume of modified Griess reagent at room temperature for 15 min. The absorbance was measured at 540 nm. NO production was expressed as the ratio of absorbance values between sample and LPS treated group.
Phagocytic activity test
FITC-dextran was used for phagocytic assay. RAW 264.7 cells (1 × 105 cells/well) were cultured in 24-well plates overnight and then incubated with culture medium, LPS (0.4 μg/mL) and a series of concentrations of PCP for 18 h, respectively. Then the cells were treated with FITC-dextran (0.1 mg/mL in culture medium) and incubated at 37 °C for an additional 1 h in dark. After incubation, the cells were collected with cold PBS after washed for three times. BD Accuri™ C6 Cytometer (BD Biosciences, San Jose, CA, USA) was used for analysis. The percentage of phagocytosis was expressed as ratio of phagocytic rate between the treatment and control cells.
Determination of endotoxin contamination
Endotoxin detection specific limulus test kit was used for avoiding endotoxin contamination. The results showed that PCP0-PCP5 had no endotoxin contamination in the samples.
GraphPad Prism 8.0.2 was used to analyze and process the data. Data were presented as mean ± SEM from at least three independent experiments for each sample. Statistical significance between the experimental groups was determined by Student’s t-test, and p values less than 0.05 were considered as statistically significant.