*2.5. Antimicrobial Activity*

#### 2.5.1. Growth Conditions of Pathogenic Bacteria

The bacterial strains were supplied by the American Type Culture Collection (ATCC). The antimicrobial activity of PSF extract was studied on three Gram-negative bacteria, specifically *Escherichia coli* (ATCC 25922), *Salmonella enterica* ser. *typhimurium* (ATCC 14028), and *Enterobacter aerogenes* (ATCC 13048), and two Gram-positive bacteria, *Enterococcus faecalis* (ATCC 29212) and *Staphylococcus aureus* (ATCC 25923).

## 2.5.2. Antimicrobial Activity

The growth inhibition of selected bacteria exerted by PSF extract was determined according to Delgado Adámez and colleagues [13], with some modifications.

The tested bacteria were cultured in MHB at 37 ◦C for 16 h and diluted to match the turbidity of 0.5 McFarland standard. Fifty microliters of bacterial suspensions (about 1–5 × 10<sup>5</sup> CFU/mL) was added to 100 μL of MHB and to 100 μL of blackthorn extract (0, 0.25, 0.50, 0.75, and 1.00 mg/mL) in a 96-well plate. A negative control was included on each microplate. A positive control of bacterial growth inhibition consisting of two antibiotics, vancomycin (10 μg/mL) for Gram-positive and gentamicin (10 μg/mL) for Gram-negative bacteria was added to the microplate. The plates were incubated at 37 ◦C for 24 h. Afterwards, the optical density (OD) at 600 nm was determined by a microplate reader (Eti-System fast reader Sorin Biomedica, Modena, Italy). The percentage of growth inhibition was calculated as follows:

$$\% \text{ growth inhibition} = 100 - (\text{OD}\_{\%} \text{OD}\_{\%}) \times 100 \tag{1}$$

where ODS is the optical density of the sample and ODC is the optical density of the negative control (PSF 0 mg/mL).

#### *2.6. In Vitro Antioxidant Activity in Red Blood Cells (CAA-RBC) and Hemolysis Test*

According to the regulations of "Fondazione G. Monasterio CNR-Regione Toscana", human blood samples were obtained from three healthy volunteers in ethylenediaminetetraacetic acid (EDTA)-treated tubes and centrifuged (2300× g at 4 ◦C for 10 min). Plasma and buffy coat were removed, and erythrocytes were washed twice with PBS pH 7.4.

The antioxidant activity of PSF extract (100 mg/mL) was evaluated in an in vitro system with a modified assay in red blood cells as described by Frassinetti and colleagues [14]. Each value was express as CAA units, as follows [12]:

$$\text{CAA unit} = 100 - (\int \text{SA} \, \int \text{CA}) \times 100 \tag{2}$$

where SA is the integrated area of the sample curve and CA is the integrated area of the control curve.

Hemolysis of PSF extract (100 mg/mL) was analyzed according to the protocol described by Frassinetti and colleagues [15] using AAPH, a generator of peroxyl radicals, to cause the red blood cell lysis. The values reported are the percentage of hemolysis compared with the control.
