Dextran standard

Samples of Herba Lobellae Chinensis, i.e., dried Lobelia chinensis, were provided by the Kang Qiao Traditional Chinese Medicine Decoction Pieces Co. Ltd. (Shanghai, China), China, and were identified by Prof. Zhili Zhao (Shanghai University of Traditional Chinese Medicine, Shanghai, China 131120). Monosaccharide standards were all from Fluka, USA. Dextran standards and trifluoroacetic acid (TFA) and dimethyl sulfoxide (DMSO) were from Sigma-Aldrich (St. Louis, MO, USA). Other reagents were analytical grade and obtained from China Chemical Reagent Industry (Shanghai, China), unless otherwise specified.

The extraction of polysaccharides was performed according to a previously described method by a previously described procedure [17 (link)]. Briefly, 200 mg of powdered seaweed was incubated in 20 ml of 85% ethanol at 80 °C for 4 h to remove lipids. This extraction operation was repeated three times. Polysaccharides in the residue were extracted with 30 ml of cold water, dialyzed against distilled water, and then freeze-dried (32.0 mg). The total sugar content was determined by phenol–sulfuric acid method using galactose as a standard [18 (link)]. The sulfate content was determined by the BaCl₂-gelatin method [19 (link)] using κ-carrageenan (TCI, Tokyo, Japan) as a positive control. The relative molecular weight of the polysaccharides was determined by high-performance size exclusion chromatography with Shodex OHpak SB-807G (Guard), SB-807 HQ, and SB-806 M HQ (8.0 mm ID × 300 mm length; Showa Denko KK, Tokyo, Japan) at 40 °C and estimated using dextran standards (150, 270, and 670 kDa from Sigma Aldrich Corp., 3,755 kDa from American Polymer Standards Corp., Mentor, OH, USA). Sample (injected volume: 20 μl) was eluted using 0.3 M NaNO₃ at a flow rate of 1 ml/min and was detected using a refractive index (RI) detector RID10 (Shimadzu Corp., Kyoto, Japan).

Pinot noir wines produced at Oregon State University Research Winery were used for wine polysaccharide isolation. All chemicals were of analytical grade unless otherwise specified. Ethanol (200 proof, HPLC-UV grade) was purchased from Pharmco (Brookfield, CT, USA). Acetyl chloride (≥99%), D-glucose (99%), D-galacturonic acid monohydrate (97%), L-arabinose (99%), L-fucose (99%), and L-rhamnose (99%) were purchased from Alfa Aesar (Tewksbury, MA, USA). Dextran standards, myo-inositol (≥99%), lactose (≥99%), and D-galactose (≥99%) were purchased from Sigma-Aldrich (Saint Louis, MO, USA). N-trimethylsilylimidazole (>98%) was bought from TCI Chemicals (Tokyo, Japan), ammonium formate (99%) was obtained from BeanTown Chemical (Hudson, NH, USA), and D-glucuronic acid (≥98%) was purchased from ICN Biomedicals (Irvine, CA, USA). Methanol (extra dry, 99.8%), pyridine (extra dry,99.5%), D-mannose (≥99%), and D-xylose (≥99%) were obtained from Acros Organics (Geel, Belgium).

Samples of DEP-AD PP-U (2 μL at 1 mg/mL in HPLC water, VWR Chemicals) were mixed with 2 μL of the matrix solution (2,5-dihydroxybenzoic acid 10 mg/mL in TFA:CH₃CN:1.75:0.75; v/v). A total of 1 μL of this solution was applied to a stainless steel sample slide and dried [81 (link)]. MALDI-TOF mass spectra of the generated oligomers were recorded on a MALDI-TOF Microflex (Bruker) mass spectrometer. Spectra were acquired in the reflectron mode. Dextran standards of 1 and 5 kDa (Sigma-Aldrich) were used as a calibration mixture for the MALDI-TOF analysis.

The M_w of TSP was measured by HPLC with a TSK gel G5000PWxl column (Tosoh, Tokyo, Japan) using 0.05 mol/L Na₂SO₄ as the mobile phase on an Agilent 1260 HPLC system (Santa Clara, CA, USA) equipped with a refractive index detector (Santa Clara, CA, USA). The column temperature was 35 °C, and the flow rate of the mobile phase was 0.5 mL/min. Dextran standards (1 kDa, 5 kDa, 12 kDa, 50 kDa, 80 kDa, 270 kDa, and 670 kDa; Sigma, Mendota Heights, MN, USA) were used to calibrate the column.
The total sugar content of TSP was analyzed by the phenol-sulfuric acid method [40 (link)] using galactose as the standard. Sulfate content was determined by the barium chloride gelatin method [7 (link)]. The molar ratios of the monosaccharide composition were determined according to the method of Sun et al. [41 (link)]. 1-Phenyl-3-methyl-5-pyrazolone (PMP) precolumn derivation HPLC was used to determine the molar ratio of the monosaccharide composition. Mannose, rhamnose, fucose, galactose, xylose, glucose, and glucuronic acid from Sigma-Aldrich (St. Louis, MO, USA) were used as standards. FTIR spectra of TSP were determined on a Nicolet-360 FTIR spectrometer (Thermo, Waltham, MA, USA) between 400 cm⁻¹ and 4000 cm⁻¹.

DEAE-cellulose (DE-52), Sephadex G-100, dextran standards (1, 12, 50, 80, 270, 670 and 1100 kDa) and monosaccharide standards were purchased from Sigma (St. Louis, MO, USA). CTX was acquired from Shanxi Powerdone Pharmaceutics Co., Ltd. (Datong, China). Lentinan (GuoYaoZhunZi Z20080579) was supplied by Hubei Chuangli Pharmaceutical Co., Ltd. (Zaoyang, China). RPMI-1640 medium was acquired from Gibco (Grand Island, NE, USA). Giemsa stain was supplied by Shanghai Yeasen Biotechnology Co., Ltd. (Shanghai, China). TNF-α, IL-1β, Immunoglobulin A (IgA), immunoglobulin G (IgG) and immunoglobulin M (IgM) kits were acquired from R&D system (Minneapolis, MN, USA). Catalase (CAT), glutathioneperoxidase (GSH-Px), superoxidase dismutase (SOD), malondialdehyde (MDA) and protein carbonyl group (PCG) kits were supplied by Jiancheng Bio-engineering Institute (Nanjing, China).

In order to determine the average molecular mass of the EPS sample, an Agilent1100 series HPLC system (Agilent, Santa Clara, CA, USA) was conducted with an RI detector. EPS samples were eluted with deionized water at a flow rate of 0.8 mL/min. Based on the linear regression equation drawn through dextran standards (Sigma–Aldrich, St. Louis, MO, USA), the molecular mass of EPS was analyzed through GPC data processing software. The monosaccharide composition was determined by gas chromatography coupled with mass spectrograph (GC-MS). Some pretreatments were performed as previously described (Li et al., 2016 (link)). The treated samples were used for GC-MS with the conditions as follows: initial column temperature was set at 140 °C with a rate of 1.5 °C/min to reach 200 °C and with a rate of 10 °C/min to 250 °C, then the highest temperature was held for 5 min, and samples were injected into the column with N₂ as the carrier gas at a flow rate of one mL/min. The monosaccharide composition of EPS was determined by comparison with the retention time of monosaccharide standards.