*2.2. Methods*

#### 2.2.1. Plant Material and Extraction Procedures

SN leaves were collected around Catania (Sicily, Italy) in June 2018 and air-dried at room temperature (24 ± 2 ◦C) with no direct light. A voucher specimen of the plant has been deposited (N. 8234) in the herbarium of the Botany Department of the University of Catania (Italy). The dried and powdered leaves (5 g) were extracted by maceration for 24 h at room temperature with 150 mL each of methanol/water solution 80:20 *v*/*v* (SN1) and water (SN2), respectively. The extraction procedure was repeated three times, and the solvent extracts were combined and separated from the residue by filtration through Whatman N. 1 filter paper in a Buchner funnel under vacuum. The methanol was removed under a reduced pressure below 40 ◦C by using a rotary evaporator, and the aqueous phase remaining after evaporation of the organic phase was freeze-dried. The water extract was freeze-dried. The obtained dried extracts were 1.305 ± 0.24 g for SN1 and 0.975 ± 0.31 g for SN2, respectively. The obtained dried extracts were stored at −20 ◦C until undergoing assays.

#### 2.2.2. Determination of Total Phenolic Content

The amount of total phenolic in the studied extracts was determined using a modified Folin-Ciocalteu method [28]. Extracts were prepared at a concentration of 0.5 mg/mL. An aliquot of a known dilution of the extract was mixed with 5 mL Folin–Ciocalteu reagen<sup>t</sup> (previously diluted 10-fold with deionized water) and was allowed to react for 6 min. Then, 4 mL (70 g/L) of sodium carbonate solution was added to test tubes. The tubes were vortexed for 20 s and allowed to stand for 90 min for color development. Absorbance was measured at 760 nm by using the Perkin Elmer UV–Vis Lambda 25 spectrophotometer (Perkin Elmer Italia spa, Monza, Italy). Extract samples were evaluated at the final concentration of 1 mg/mL. The measurements were compared to a standard curve of prepared gallic acid solutions. The total phenolic content were 92.2 mg/g of extract for SN1 and 40.0 mg/g of extract for SN2, which was expressed as a gallic acid equivalent.
