Starch

Chemicals such as ascorbic acid, sodium nitrite, glucose, citric acid, potassium iodide, potassium iodate were obtained from VWR Life Science (Randor, PA, USA). The pH indicators such as phenol red, bromophenol blue, chlorophenol red, and bromocrescol green were purchased from BDH-VWR Analytical (Randor, PA, USA). Enzymes such as glucose oxidase and horseradish peroxidase were procured from Sigma Aldrich (St. Louis, MO, USA). Indicators such as Griess reagent, starch, phenolphthalein, and guaiacol were obtained from VWR Life Science (Randor, PA, USA). Lactaid capsules were purchased from Amazon, USA. Whatman filter paper No.1 (11 µm pore diameter) was provided from Bio rad Laboratories, USA. Distilled water used for experimentation was obtained from a Milli-Q water purifier.

The α-amylase disc assay was conducted according to (Correia et al., 2004 (link)) with minor modifications. Root extracts were diluted 1:4, 1:16 and 1:64 with ultrapure water (Purelab Flex, ELGA Veolia, United Kingdom). 80 µl of these dilutions or, as control, ultrapure water was mixed with 20 µl porcine pancreatic α-amylase (Sigma-Aldrich, Taufkirchen, Germany) (20 mg/ml 20 mM sodium phosphate buffer, pH 6.9). Acarbose added at concentrations of 0.1, 1, and 10 mM was used as a reference inhibitor. Four filter discs (diameter of 0.5 cm) were placed on Petri plates (92 × 16 mm, Sarstedt, Nürnbrecht, Germany) filled with medium containing 1% agar-agar (Carl Roth, Karlsruhe, Germany) and 1% starch (VWR, Darmstadt, Germany). 20 µl of the mixtures were given onto a filter disc and the plates were incubated overnight at 37°C. After removing the filter discs, 5 mL of iodine stain solution (5 mM iodine in 3% potassium iodide solution, both chemicals from Merck, Darmstadt, Germany) were added to each plate. Following 15 min incubation excess iodine stain was drained, before the diameter of the cleared zones was measured. The percentage inhibition of α-amylase at each extract concentration was calculated by using the following equation: $Inhibition(\%)=\left(1-\frac{Diameter of sample}{Diameter of control}\right)\times 100$

All of the reagents were of an analytical grade or better. The de-ionized water was used in all the experiments. The raw-Re nanostructures were obtained from the precursor solution of ammonium perrhenate(vii) (NH₄ReO₄), which contains 2000 mg L⁻¹ of Re(vii) ions. The reagents for the determination of the selected RNS and ROS were purchased from Hanna Instruments (Salaj, Romania) and Sigma-Aldrich (Steinheim, Germany), respectively. The reagents used for ROS determination were: (i) potassium iodide, (ii) starch, and (iii) agar, and they were purchased from Avantor Performance Materials (Gliwice, Poland), Sigma-Aldrich (Steinheim, Germany), and BTL (Lodz, Poland), respectively. The 4-NP and NaBH₄ (MERCK, Branch Poland) used for the catalytic hydrogenation were applied as received.

Chitosan (low molecular weight 50,000–190,000 Da; deacetylation degree 75–85%), diacrylate poly(ethylene glycol) (crosslinker, Mn = 700 g/mol), 2-hydroxy-2-methylpropiophenone (photoinitiator, d = 1.077 g/mol, 97%, Mw = 164.2 g/mol) and L-aspartic acid (≥98%) were purchased in Merck (Darmstadt, Germany). Starch (natural potato-Starch) was bought in Avantor Performance Materials Poland S.A. (Gliwice, Poland). All reagents were applied without further purification.