2.3. Substrate, Inhibitor/Flavonoid and Sample Preparation

Stock solutions (250 mM) of sucrose, maltose and isomaltose (substrates), plus glucose and fructose (standards only), were prepared in sodium phosphate buffer (SPB, 10 mM, pH 7.0). Stock solutions of acarbose (1 mM) and all flavonoids (10 mM) were prepared by dissolving in their respective solvents, stored at −20°C and used within 2 weeks. Acarbose and EGCG were prepared in H₂O, quercetin and quercetagetin were prepared in DMSO, while kaempferol and galangin were dissolved in absolute ethanol. The maximum working concentrations for each compound was pre-determined before enzymatic reaction assay to ensure no precipitation occurred in the system. Compared to acarbose and EGCG, lower solubility of tested flavonols was expected due to their structural differences, where compounds with less than 100 µg/mL solubility were considered poorly soluble, as reported previously [28,29]. Inhibitors were tested at various concentrations: acarbose (0.1–100 µM), EGCG (5–1500 µM), quercetin (20–200 µM), quercetagetin (1–50 µM), kaempferol (5–40 µM) and galangin (1–25 µM). The flavonols were tested up to their maximum soluble concentrations. Working solutions were prepared fresh at various concentrations in SPB buffer immediately before assaying. The maximum concentrations (v/v) of DMSO were ≤2% and ≤0.5% for quercetin and quercetagetin, respectively, and ethanol was ≤0.5% for kaempferol and galangin. The solvents did not affect enzyme activity, as demonstrated by vehicle controls. Cell-free extracts (CFE) were prepared as described in Section 2.6 and used as the enzyme source.

All prepared standards and assay samples, for both method validation and post-assay quantification, underwent the same treatment prior to injection on the HPAE-PAD system. All were deproteinated by mixing with an equal volume of acetonitrile, vortexed for 30 s and centrifuged at 17,000× g, 15 min at 4 °C. The resulting supernatants were then diluted at least 10× in H₂O (maximum final acetonitrile concentration of 5% (v/v)). Additionally, all standards and samples containing enzyme and substrates were filtered through 0.2 µM polyether sulfone (PES) filters (Sartorius, Göttingen, Germany). All standards, samples and blanks were kept at 4–8 °C until analysis by HPAE-PAD, as described in Section 2.2, was complete.