*2.1. Animals*

Thirty healthy adult Wistar male rats (8–9 weeks old, 250–300 g) were purchased from ENVIGO Srl (San Pietro al Natisone UD, Italy) and housed randomly in temperature-controlled rooms on a 12 h light cycle at 22–24 ◦C and 50–60% humidity. Animals had free access to standard pellet chow and water ad libitum throughout the experimental protocol. They were acclimatized for one week prior to experimentation. The experimental protocol, followed throughout the study, was conducted in the conformity of the Italian D.Lgs 26/2014", following the criteria outlined by the European Community Council Directive 2010/63/UE, recognized and adopted by the Italian Government, and approved by the Ethical Committee for Animal Experimentation of the University of Palermo and by the Italian Ministry of Health (Authorization n 921/2018–released Rome, Italy).

#### *2.2. Induction of DNBS Colitis and Treatment Protocol*

AphaMax ® extract (Nutrigea Research s.r.l., Borgo Maggiore, Republic of San Marino) was dissolved in 0.5 mL water, and then sonicated (twice for 60 s) immediately prior to the gavage. 20, 50 or 100 mg/kg AphaMax ® solution was administered by oral gavage once a day for 14 days starting 7 days before the induction of colitis (day-7). Rats were randomly assigned into six groups (*n* = 5 animals/each): (1) control group (sham group); (2) group with colitis; (3) sham group + AphaMax ® (100 mg/Kg/day); (4) group with colitis + AphaMax ® (20 mg/Kg/day); (5) group with colitis + AphaMax ® (50 mg/Kg/day); (6) group with colitis + AphaMax ® (100 mg/Kg/day). To induce the colitis intracolonic (i.c.) instillation of 2, 4-dinitrobenzensulfonic acid (DNBS; Sigma-Aldrich Inc., St. Louis, MO, USA), was performed as already described [19–21]. Briefly, rats were fasted overnight and then, under light anesthesia with 1% isoflurane (Merial Italia Spa, Assago, MI, Italy), DNBS (15 mg) dissolved in a solution of 50% ethanol, was deposited in the colon through an 8 cm plastic catheter (PE90).

To avoid reflux actions, the rats were kept for 5 min in a Trendelenburg position, and then allowed to recover with food and water supplied. The control group (sham group) received i.c instillation of vehicle alone (50% ethanol).

None of the rats died during the study or was excluded from the study for any reason. Assessment of colitis-induced damage was performed minimizing the su ffering of animals, in a blinded fashion as previously described [18–21].

#### *2.3. Evaluation of Colitis*

#### 2.3.1. Monitoring of Change in Body Weight, Consistency of Stools and Rectal Bleeding

The animals were monitored daily during the experimental period and scored for body weight loss percentage (initial body = 100%), stool consistency and the presence of rectal blending. All these parameters were ranged and calculated as described by Cooper et al. [22].
