Monosaccharide

Monosaccharide content was determined via HPLC [Chromatographic conditions: chromatographic column (Biorad Aminex HPX-87P), Deashing packed column, Detector (evaporative light scattering detector), Injection volume (35 μL), mobile phase (Ultrapure water), flow velocity (0.6 mL/min), Nitrogen pressure (30 psi); drift tube (heating mode, 80 ± 25°C), Sprayer (60%); running time (20min)]. Exactly 4 mL of the filtrate was obtained, and the pH was adjusted to 5–6 with CaCO₃. The supernatant was collected by centrifugation and filtered through a 0.22 μm filter membrane. Then, HPLC was used to determine the content of monosaccharides. Both monosaccharides and calcium carbonate are pure reagents (Sigma) for chromatographic analysis.
The cellulose and hemicellulose contents were calculated from the monosaccharide content as follows:
where Ac is the dehydration correction coefficient. The Ac values of pentose and hexose were 0.88 and 0.90, respectively. %Glu, %Xyl, %Ara, %Gal, and %Man represent the contents of the corresponding monosaccharides obtained by the regression curve method.

RAW 264.7 murine macrophages were purchased from National Centre for Cell Science, Pune, India. 2-Deoxy-D-ribose, NaOH, ferric chloride, hydrogen peroxide, thiobarbituric acid, ferrozine, trichloroacetic acid, potassium ferricyanide, trifluoroacetic acid, DPPH, ABTS, sodium persulfate, ammonium molybdate, sodium borohydride, pyridine, ascorbic acid, ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (BSA), LPS and monosaccharides were procured from Sigma chemicals Co. (St. Louis, MO, USA). A mushroom β glucan kit was used from Megazyme Institute Wicklow, Ireland. Dulbecco’s Modified Eagle Medium (DMEM), neutral red, sulfanilamide, naphthylethylenediamine dihydrochloride, phosphoric acid, Congo red, 2′,7′-dichlorofluorescin diacetate (DCFDA), 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Himedia, Mumbai, India. WST was obtained from Takara Bio Inc, Japan. Fetal bovine serum (FBS) and TRIzol were purchased from Invitrogen, New Delhi, India. PenStrep, amphotericin B and cDNA preparation kit were used from MP Biomedicals, Santa Ana, CA, USA. PowerUp^TM SYBR^® Green Master Mix was procured from Applied Biosystems, USA.

Iron (II) sulfate, potassium ferricianyde, sulfuric acid and acetonitrile were obtained from Merck (Darmstadt, Germany). Nitro Blue Tetrazolium (NBT), monosaccharides, methionine and ammonium molybdate were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Cell culture medium components (Dulbecco’s Modified Eagle Medium-DMEM), trypsin and newborn calf serum (FCS) were obtained from Cultilab (Campinas, Brazil). l-glutamine, sodium bicarbonate, sodium pyruvate and phosphate buffered saline (PBS) were purchased from Invitrogen Corporation (Burlington, ON, USA). All other solvents and chemicals were of analytical grade.
Eukaryotic cells—Mouse embryonic fibroblast cells (NIH/3T3 ATCC^® CRL-1658™-3T3, Manassas, VA, USA) were grown in Dulbecco’s modified Eagle’s medium (DMEM) with 10% of fetal bovine serum (FBS), 10 mg/mL streptomycin, and 10,000 IU penicillin.

Acetic acid, Folin-Ciocalteu phenol reagent, ethanol, and sulfuric acid were obtained from Merck (Darmstadt, Germany). Monosaccharides, 3-isobutyl-1-methylxanthine (IBMX), insulin, dexamethasone, and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Sodium bicarbonate, culture medium components (minimum essential Dulbecco's modified Eagle's medium (DMEM)), nonessential amino acids, fetal bovine serum, sodium pyruvate, and phosphate-buffered saline (PBS) were purchased from Invitrogen Canada Inc. (Burlington, ON, Canada). Water was purified with a Milli-Q system (Millipore®, Bedford, MA, USA). All other solvents and chemicals were of analytical grade.

The levels of total carbohydrates were determined using the phenol-sulfuric acid method and d-glucose as standard [58 (link)]. The levels of sulfate were determined using the barium chloride-gelatin method [59 (link)].
The molar ratio of Monosaccharides was determined by the 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization assay, as described previously, with slight modifications [60 (link)]. Briefly, 10 mg of polysaccharides was hydrolyzed in the presence of 4 mL of trifluoroacetic acid (TFA, 4 M) in an ampoule at 110 °C for 6 h. The hydrolyzed polysaccharides were dried and dissolved in 0.1 mL of distilled water. Then, the hydrolysate (100 μL) was derivatized by adding 100 μL of NaOH (0.3 M) and 120 μL of PMP methanol solution (0.5 M) and incubating at 70 °C for 1 h. The mixture was neutralized with 100 μL of HCl (0.3 M) and extracted with chloroform. Finally, the aqueous phase passed through a 0.45 μm membrane and the resulting solution (100 μL) was injected into a high-performance liquid chromatography (Shimadzu-20A, Shimadzu, Kyoto, Japan) equipped with a YMC-Pack ODS-AQ column (250 mm × 4.6 mm, 5 μm) (Agilent, Santa Clara, CA, USA). The mobile phase was composed of water and acetonitrile, and the flow rate was 1.0 mL/min. Monosaccharides (fucose, galacturonic acid, galactose, glucose, mannose, rhamnose, and xylose) (Sigma, St. Louis, MO, USA) were used as controls.