*3.5. Physicochemical Analysis*

Total sugar content determination was performed by the phenol-sulphuric acid method using galactose as the reference sugar [61]. Sulfate content was determined by the barium chloride-gelatin method [62]. Protein content was measured as described by Bradford [63]. Monosaccharide composition was determined by nuclear magnetic resonance spectroscopy as described by Sassaki and coworkers (2014) [64]. Briefly, samples (5 mg) were hydrolyzed with 2 M HCl for 4 h at 100 ◦C. Then, the solution was neutralized, evaporated, and the residue dissolved in D2O for NMR analysis (~600 μL). A diluted D2O–H2SO4 solution was used for adjusting pH value to 3.5. Finally, quantitative-HSQC (Q-HSQC) spectra were obtained at 27 ◦C using a hsqcetgpsisp2.2 pulse sequence on Bruker spectrometers (Bruker, Billerica, MA, USA).

#### *3.6. Molecular Weight and Homogeneity Determination*

Molecular weight and homogeneity SPs from *C. cupressoides* were determined by gel permeation chromatography (GPC). Each fraction was dissolved to a final concentration of 10 mg/mL and applied to a column containing Sephadex ® G-100 (135 × 1 cm i.d.; Sigma Chemical Company, St. Louis, MO, USA). GPC was performed using an isocratic elution mode. The molecular weight was estimated by reference to a calibration curve made by dextran standards (10, 40, 70, 147, and 500 kDa). Homogeneity of SPs was evaluated by chromatographic profile. The SPs elution was monitored by sugar determination according to Dubois et al. [61].
