4.1 Chemicals and reagents

The fresh D. rubrovalvata was obtained from Songming, Kunming, China in July 2022.The samples were identified by Zhao Qi, a senior engineer at the Kunming Institute of Botany, Chinese Academy of Sciences.

Standard molecular weight dextrans (1, 5, 12, 25, 470, 610 kDa) were purchased from Sigma-Aldrich Co., Ltd (Shanghai, China). DEAE-52 was purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). Sephacryl S-200 and S-300 were purchased from Cytiva Bio-technology Co., Ltd (Hangzhou, China). Arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, xylose and ribose were purchased from Aladdin Co., Ltd (Shanghai, China). 1-phenyl-3-methyl-5-pyrazolone (PMP) were purchased from Adamas Reagent Co., Ltd. (Shanghai, China). Methyl iodide was purchased from TCI Development Co., Ltd. (Shanghai, China). Deuteroxide (D2O) was purchased from Adamas Reagent Co., Ltd. (Shanghai, China). Congo red was purchased from Sigma-Aldrich Co., Ltd (Shanghai, China). RPMI-1640 medium was purchased from BI Co., Ltd. (Shanghai, China). The mouse IL-10 ELISA kit, mouse IL-6 ELISA kit and mouse TNF-α ELISA kit were purchased from Fine Biotech Co., Ltd. (Wuhan, China).

4.2 Extraction and purification

The fresh fruiting bodies of D. rubrovalvata were divided into volva, thallus and pileus. They were soaked with 95% ethanol to remove pigments and other alcohol-soluble compounds (3 times, each time for 12 h), and ventilated at room temperature to dry, respectively. The dried thallus of D. rubrovalvata were crushed and extracted with hot water (w: v = 1:20, 3 times, each time for 2 h). The water extract was collected by centrifuging, and then concentrated. The protein of water extract was removed by the Sevage method (chloromethane: n-butanol = 4:1, v/v). Subsequently, the water extract was mixed with anhydrous ethanol for the final ethanol concentration to reach 60% (v/v) to precipitate polysaccharides. The precipitates were re-dissolved in hot distilled water and removed the residual ethanol. At last, the crude thallus and volva polysaccharides of D. rubrovalvata (DRTP60 and DRVP60) were obtained by lyophilization.

The crude polysaccharides (DRTP60 and DRVP60, 10 mg/mL) were subjected on DEAE-52, which were eluted with distilled water, 0.1 M NaCl, 0.3 M NaCl and 0.5 M NaCl solution at a flow rate of 2.0 mL/min. And the concentrated eluents were further purified on the Sephacryl S-200 and Sephacryl S-300 gel permeation columns. The columns were eluted with distilled water at a flow rate of 0.5 mL/min. The eluents were tested by by HPLC system (Agilent, USA) equipped with the evaporative light scattering detector (ELSD, Alltech, USA) and Shodex KS-804 column (7.8 mm × 300 mm) and the same fractions were combined. And then the purified and single fractions (DRP-I and DRP-II) were obtained by lyophilization.

4.3 Structure characterization of DRP-I and DRP-II4.3.1 Chemical compositions

The total sugar content was measured with phenol–sulfuric acid method [31]. The protein content was determined by Bicinchoninic-acid method [32]. And the UV spectrophotometer was used to detect the presence of proteins and nucleic acids that have ultraviolet absorption by scanning in the range of 190–600 nm.

4.3.2 Molecular weight and homogeneity determination

The average molecular weight and homogeneity of DRP-I and DRP-II were determined by HPLC-ELSD system equipped with Shodex KS-804 column (7.8 mm × 300 mm). The standard dextrans with different molecular weights (1, 5, 12, 410, 670 kDa) were used to establish a standard molecular weight curve.

4.3.3 Monosaccharide composition analysis

The analytical method of monosaccharide compositions of DRP-I and DRP-II referred to Dai et al. with Simple modification [33]. 2.0 mg of polysaccharides DRP-I and DRP-II were hydrolyzed with 4 mol/L TFA at 90 °C for 8 h, and the residual TFA was removed by adding methanol repeatedly and drying under pressure. The hydrolyzed sample and monosaccharide standard (arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose, xylose and ribose) were derivatized by 1-phenyl-3-methyl-5-pyrazolone (PMP). They were prepared into the solutions of 1 mg/mL and mixed with 50 μL of NaOH (0.6 M) and 100 μL of PMP (0.5 M), respectively. The reaction was performed at 70 ℃ for 100 min. After the reaction and cooling to room temperature, the mixtures were added 100 μL of HCl (0.3 M), 1 mL distilled water and 1 mL chloroform. The supernatant was extracted after chloroform extraction (3 times) and filtered for HPLC analysis.

4.3.4 Infrared (IR) spectral analysis

Approximately 1 mg of dried polysaccharides of DRP-I and DRP-II evenly mixed with KBr powder, tableted. And then they were analyzed with FT-IR spectroscopy (Bruker, Germany) in the range of wave length 4000–500 cm−1.

4.3.5 Methylation analysis

The analytical method of methylation of DRP-I and DRP-II referred to Ciucan, Kerek and Liang et al. with Simple modification [34, 35]. 5.0 mg of polysaccharides DRP-I and DRP-II were dissolved with anhydrous DMSO completely, filled with N2 gas, and then NaOH (20 mg) was added. After ultrasonic mixed for 20 min, the mixture was cooled and solidified. Then dropping CH3I (1.5 mL) slowly and ultrasonic reacted for 30 min. The methylation reactions were terminated by adding 1 mL distilled water. Repeat the above steps until the methylation was complete. The methylated polysaccharides were hydrolyzed with 4 mol/L TFA at 110 °C for 4 h. Subsequently, NaBD4 (10 mg/mL) was added to reduce samples, and acetic anhydride / anhydrous pyridine (v/v = 1:1) were added to acetylate (120 ℃, 120 min). The acetylation products were extracted by CH2Cl2 for GC–MS analysis.

4.3.6 NMR analysis

The freeze-dried polysaccharides of DRP-I and DRP-II (10 mg) were dissolved in D2O (0.5 mL). Repeatedly freeze-dried to replace the H in the polysaccharides with D. The 1D and 2D NMR data were measured by Bruker Advance 800 MHz NMR spectrometer (Bruker, Germany).

4.3.7 SEM analysis

The freeze-dried polysaccharides of DRP-I and DRP-II attached on the conductive adhesive and gold-plated. And then the samples were observed their surface morphology at different magnifications by scanning electron microscope (Carl Zeiss, Germany).

4.3.8 Congo red analysis

Congo red assay was used to analyze whether the polysaccharide had a triple-helix conformation [36], and the steps were as follows: In the experimental group, polysaccharide solution (2 mg/mL) was mixed with Congo red solution (80 μM) in equal volume (50 μL), and then 100 μL NaOH solution with different concentrations was added respectively. The final concentrations of NaOH in the mixed solution were 0.00, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40 and 0.45 M, respectively. After incubating at 25 ℃ for 10 min, the UV absorption of the samples in the range of 400–700 nm was analyzed by microplate reader. The control group was deionized water mixed with Congo red solution in equal volume, and the specific steps were the same as above.

4.4 Immunoactivity on dendritic cells4.4.1 Cells culture

Bone marrow derived dendritic cells (BMDCs) were cultured in RPMI-1640 medium (containing 10% FBS and 1% double antibody) at 37 ℃ and 5% CO2.

4.4.2 Proliferation assay

The proliferation of DRP-I and DRP-II were determined by MTS method on BMDCs [4]. 100 μL of BMDCs (1 × 105 cell/mL) were cultured in 96-well cell plates, after the cells were attached to the wall, the supernatant was discarded. The experimental group was added with DRP-I and DRP-II (100, 200 and 400 μg/mL, 200 μL), and the blank control group was added with RPMI-1640 base medium (200 μL). The culture was continued for different time (24 h and 48 h). Then the supernatant of cells in the 96-well plate was discarded, and MTS solution (20 μL) and RPMI-1640 base medium (100 μL) were added to each well, and cultured under the same conditions for 1 h. The absorbance of each well was measured at 490 nm by microplate reader.

4.4.3 IL-10, IL-6 and TNF-α determination

The effects of DRP-I and DRP-II on cytokines (IL-10, IL-6 and TNF-α) secreted on BMDCs were determined by ELISA kits [4]. The density of BMDCs was adjusted to 1 × 105 cell/mL. 100 μL of BMDCs (1 × 105 cell/mL) were cultured in 96-well cell plates, after the cells were attached to the wall, the supernatant was discarded. The experimental group was added with DRP-I and DRP-II (100, 200 and 400 μg/mL, 200 μL), and the blank control group was added with RPMI-1640 base medium (200 μL). And the supernatant was collected after culture for 24 h. Then the concentrations of IL-6, IL-10 and TNF-α were determined according to the ELISA kit instructions, respectively.

4.5 Statistical analysis

Data were expressed as the mean ± SD (standard deviation) of triplicate determinations. Statistical significance was analyzed by one-way analysis of variance (ANOVA) and GraphPad Prism software (GraphPad, San Diego, CA, USA).  p< 0.05 was considered statistically significant.