Additional file 1 of Structural properties and immunomodulatory activity of an α-glucan purified from the fruiting body of Stropharia rugosoannulata
Creators: name: Zuofa, Zhang
name: Tingting, Song
name: Guoying, Lv
name: Jie, Liu
name: Qunli, Jin
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Molecular Biology
subject: Chemical Sciences not elsewhere classified
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified

description: Additional file 1: Methods S1. Monosaccharide composition analysis of PSRa-2. Approximately 5 mg of sample was hydrolyzed with trifluoroacetic acid (2 M) at 121 °C for 2 h in a sealed tube. Dry the sample with nitrogen. Add methanol to wash, then blow dry, repeat methanol, and wash 2–3 times. The residue was re-dissolved in deionized water and filtered through 0.22 μm microporous filtering film for measurement. The sample extracts were analyzed by high-performance anion-exchange chromatography (HPAEC) on a CarboPac PA-20 anion-exchange column (3-by-150 mm; Dionex) using a pulsed amperometric detector (PAD; Dionex ICS 5000 system) by Sanshu Biotech. Co., LTD (Shanghai, China). Flow rate, 0.5 mL/min; injection volume, 5 μL; solvent system A: (ddH2O), solvent system B: (0.1 M NaOH), solvent system C: (0.1 M NaOH, 0.2 M NaAc); gradient program, volume ratio of solution A, B, C was 95:5:0 at 0 min, 85:5:10 at 26 min, 85:5:10 at 42 min, 60:0:40 at 42.1 min, 60:40:0 at 52 min, 95:5:0 at 52.1 min, and 95:5:0 at 60 min. Data were acquired on the ICS5000 (Thermo Scientific), and processed using chromeleon 7.2 CDS (Thermo Scientific). Methods S2. Homogeneity and molecular weight determination. The samples were dissolved in 0.1 M NaNO3 aqueous solution containing 0.02% NaN3 at the concentration of 1 mg/mL and filtered through a filter of 0.45 μm pore size. The samples were dissolved in DMSO solution containing lithium bromide (0.5% w/w) (DMSO/LiBr) at the concentration of 1 mg/mL and filtered through a filter of 0.45 μm pore size. The homogeneity and molecular weight of various fractions were measured using SEC-MALLS-RI. The weight and number-average molecular weight (Mw and Mn) and polydispersity index (Mw/Mn) of various fractions in 0.1 M NaNO3 aqueous solution containing 0.02% NaN3(or DMSO solution containing 0.5% LiBr) were measured on a DAWN HELEOS-II laser photometer Wyatt Technology Co., USA) equipped with Three tandem columns (300 × 8 mm, Shodex OH-pak SB-805, 804 and 803; Showa Denko K.K., Tokyo, Japan) which was held at 45 ℃ (or 60 ℃) using a model column heater by Sanshu Biotech. Co., LTD (Shanghai, China). The flow rate is 0.5 mL/min (or 0.3 mL/min). A differential refractive index detector (Optilab T-rEX, Wyatt Technology Co., USA) was simultaneously connected to give the concentration of fractions and the dn/dc value. The dn/dc value of the fractions in 0.1 M NaNO3 aqueous solution containing 0.02% NaN3 was determined to be 0.141 mL/g, and in DMSO solution was determined to be 0.07 mL/g. Data were acquired and processed using ASTRA6.1 (Wyatt Technology). Methods S3. Congo red test. The conformation of PSRa-2 was determined by the Congo red test. Various concentrations of NaOH were added to a mixture of PSRa-2 (2 mg/mL) and Congo red (80 μM) and the mixtures were left to stand for 10 min. The maximum absorption wavelength of the samples was recorded using a spectrophotometer (Shanghai Precision and Scientific Instrument Co., Ltd, Shanghai, China). Methods S4. The methylation analysis of PSRa-2. The polysaccharide sample was dissolved in DMSO. The solutions were methylated in DMSO/NaOH with CH3I. After complete methylation, the permethylated products were hydrolyzed with 2 mol/L TFA at 121 ℃ for 1.5 h, reduced by NaBD4 and acetylated with acetic anhydride for 2.5 h (100 ℃). The acetates were dissolved in chloroform and analyzed with GC–MS on an Agilent 6890A-5975C equipped with Agilent BPX70 chromatographic column (30 m × 0.25 mm × 0.25 µm, SGE, Australia), and high purity helium (split ratio 10:1) was used as the carrier gas with an injection volume of 1 μL by Sanshu Biotech. Co., LTD (Shanghai, China). Mass spectrometry analysis was performed at the initial temperature of 140 ℃ for 2.0 min, and the temperature is increased to 230 ℃ by 3 ℃/min for 3 min. Methods S5. Immunomodulatory effect of PSRa-2 on the splenocytes of mice. The potential immunomodulatory effect of PSRa-2 was evaluated by determining its potential activities on proliferation of splenocytes and protection on spleocytes against 5-Fu induced immunosuppression. Briefly, the aqueous treatment solution of PSRa-2 was first prepared in serial concentrations. Splenocytes were cultured in 96-well plates for 24 h, respectively. After incubated with treatment solution of PSRa-2 for another 48 h, the cell viability was measured based on MTT method, and the absorbance at 570 nm was obtained to calculate the cell viability of splenocytes. The cytokines (TNF-α and IL-2) exist in the culture supernatants of splenocytes were measured by ELISA kits according to the manufactures’ manner. All experiments were performed in triplicate.