Dpph reagent

Chicken eggs were obtained at a local market (Harbin, China). Gallic acid and ABTS were purchased from Solarbio (Beijing, China). The DPPH reagent was obtained from Sigma (St. Louis, MO, USA). Other reagents were obtained from Aladdin (Shanghai, China).

Antioxidant activity of the açaí extracts was measured using the conversion of the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) or ABTS reagent to the radical cation ABTS+• by potassium persulfate that reacts with the antioxidant compounds present in the extracts, such as polyphenols as described before [29 (link)]. In this assay adapted to a micro plate, 100 μL of açaí extract was added to ABTS+• solution and the absorption was measured spectrophotometrically at 734 nm at 30 °C 10 min after initial mixing and is presented as the Trolox equivalent used as standard reference.
The other radical scavenging assay involves the free radical 2,2-diphenyl- 1-picrylhydrazyl or DPPH•, which reacts with an antioxidant molecule resulting in a discoloration of the solution, measured at 515 nm [30 (link)], and adapted to a micro-plate UV-Vis reader. The radical 150 μM DPPH• solution was prepared by mixing the DPPH reagent (Sigma-Aldrich, St. Louis, MO, USA) in 80% methanol (HPLC grade, Sigma-Aldrich, St. Louis, MO, USA). In a 96 well-plate, 180 μL of the radical solution was added to 20 μL of the açaí extract, standards or 70% methanol, which was used as a blank. The plate was stored in the dark for 40 min and read at 515 nm. The results were also quantified using a calibration curve of Trolox (Sigma-Aldrich, St. Louis, MO, USA) over the range of 100 to 500 μM.

Morphological analysis has been performed with transmission electronic microscopy (TEM) by a TEM microscope JEOL JEM1400Plus (Peabody, MA, USA), hydrodynamic diameter, and ζ-potential measurements were performed through the instrument Nano ZS90 (Malvern Instruments, Malvern, UK), as previous described [62 (link)].
Determination of antioxidant activity has been performed by neutralization of DPPH-free radicals (DPPH reagent from Sigma Aldrich, Italy). Briefly, 0.5 ml of samples (3 mg/ml of PAE and certain amounts of nanocrystals containing 3 mg/ml of PAE) was added to 2.5 ml of 0.1 mM DPPH-methanolic solution and vigorously shaken in the dark at room temperature. The absorbance of samples at 515 nm was measured after 30 min. Ascorbic acid and methanol were used as positive (standard) and negative controls, respectively. The antioxidant capacity (AC) of PAE, nanoCaCO₃@PAE, and nanoCaCO₃@PAE@CH solutions has been calculated using the following equation: $\begin{array}{c}\text{AC}\left(\%\right)=\left[\frac{\left(\text{Abs\hspace{0.17em}control}-\text{Abs\hspace{0.17em}sample}\right)}{\text{Abs\hspace{0.17em}control}}\right]\times 100,\end{array}$ where control is the DPPH-methanolic solution.