Anthrone

Soluble sugar content was spectrophotometrically determined according to Calatayud et al. (2008 (link)) with several modifications. The sample extract (0.3 g) was mixed with 15 mL of 80% ethanol (v/v), which was previously heated. The mixture was incubated in a water bath for 10 min at 85°C and then vortexed. Samples were centrifuged at 10,000 g at 23°C for 10 min. The resulting supernatant was reserved in a flask. This same process was repeated 2 more times by adding hot ethanol to the mixing tube. The ethanol present in the reserved supernatant was then evaporated by a rotary evaporator (R-210, Buchi) at 50°C. The sugar concentrate was diluted in 100 mL of distilled water and filtered to be reserved in a volumetric flask for 24 h at 4°C. Next 0.5 mL of this solution was mixed with 2 mL of distilled water and placed on ice. Once cooled, 5 mL of 4 μM anthrone (Acros Organics B.V.B.A.) solution, diluted in 96% (v/v) sulphuric acid, were added to each tube. Samples were incubated in a water bath for 7.5 min at 85°C and then placed on ice for 30 min. The absorption of solution was measured at 630 nm in a spectrophotometer (Uvikon XS, Bio-Tek). Total sugar concentration was compared to a standard curve using a diluted (1:25) stock solution of 55.6 μM glucose and 70 μM sodium benzoate as the standard. Total sugar content was expressed as g glucose equivalent 100 g⁻¹ FW.