2.17. ABTS Antioxidant Assay

The antioxidant capacity of the various extracts was investigated according to the method described by Messaili. S. et al. [36]. Briefly, 7 mM of ABTS and 2.45 mM potassium persulfate were mixed and agitated in the dark for 16 h at RT to form the blue-green ABTS radical solution. The ABTS radical solution was then diluted with ethanol/water (25/75) at a 1:12.5 volume ratio. To carry out the antioxidant assays, 190 µL of the ABTS diluted solution was mixed with 10 µL of the extract or positive control solution in a 96 well-microtiter plate. Plant extracts were prepared in deionized H₂O and their antioxidant capacities determined at 1, 0.1, and 0.01 mg mL⁻¹. Similarly, Trolox was prepared at 1, 0.1, and 0.01 mg mL⁻¹ in pure ethanol and used as a positive control. The absorbance of the plate was recorded at 734 nm after 30 min incubation in the dark at RT. The antioxidant assays were performed in triplicate (n = 3) and plate readings in duplicates (n = 2). The antioxidant activities of the various extracts and the positive control were assessed based on their ability to induce decolorization of the ABTS radical solution by electron transfer. This was manifested by a reduction of the absorbance compared to the absorbance of the ABTS radical solution. The percentage reduction in absorbance is calculated according to Equation (4):

where A_sample is the absorption of the mixture of the ABTS radical and extract samples or trolox and A_ABTS is the absorption of the ABTS radical solution only.

Table 1 summarizes all the experiments realized on the three plants with the corresponding lot numbers, the extraction mode, and the granulometry.

List of samples (with lot numbers) studied in this work with the corresponding experimental investigation. For each lot number, three plant extract samples coming from independent extractions were tested. ^a: on raw and ground 1 mm granulometry; ^b: on raw, ground 1 mm and ground ‘fine’ granulometry; ^c: on ground ‘fine’ granulometry; ^d: all extraction modes were tested; ^e: optimized infusion protocol only; ^f: ground 1 mm granulometry. For the granulometry, see Section 2.2. All extraction modes are described in Section 2.3, Section 2.4, Section 2.5, Section 2.6, Section 2.7, Section 2.8