*2.1. Chemicals*

2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), Gallic acid, Folin-Ciocalteu reagent, Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), acetonitrile, acetone, methanol and water HPLC grade were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and they are of analytical grade. Cyanidin-3-O-Glucoside analytical grade was purchased from Extrasyntese (Genay, France), and anti-inflammatory Kit was purchased from Cayman Chemicals cat. Number 560131 (Ann Abor, MI, USA). Sugar kit and organic acids kit were purchased from R-Biopharm Italia Srl, cat. Numbers 10716260035, 10139076035, 10139068035 (Cerro al Lambro, Milano, Italy).

#### *2.2. Extraction and Purification Juice*

The analytical determinations were carried out on one local variety of *Morus nigra* and on two varieties of *Morus alba* known as *Morus alba cv Legittimo nero* and *Morus alba cv Nello*, which were harvested at full maturity in the Corigliano town fields (40◦938",16 N, 18◦1536",72 E) (Province of Lecce) during June–July 2015 and stored at −20◦C prior to the analysis.

Juice was obtained by centrifugation and used to quantify the sugars (sucrose, fructose and glucose) and citric and malic acid amounts by an enzymatic spectrophotometric kit provided by R-Biopharm Italia (see "Chemicals" paragraph 2.1) and expressing the results as g/100 g of fresh weight (FW) and, after filtration with Watmann n. 1, for determination of antioxidant activity. The moisture was determined in accordance to the AOAC method [24].

For the determination of phenolic compounds, a raw extract of each mulberry sample was obtained homogenizing 25 g of fresh plant material (3 min. at 10,000 rpm with Ultraturrax) using 100 mL of cold acetone 70% (*v*/*v*) acidified with 0.1% HCl. The homogenate was stirred for two hours in the dark at 4 ◦C, then centrifuged at 5000× *g* for 10 min. On the pellet, two further extractions were carried out in the same way. The supernatants were dried at reduced pressure and re-suspended with ultrapure water (HPLC grade) acidified with 0.1% HCl, thus obtaining a final volume of 100 mL (1:4 *w*/*v*).

#### *2.3. Total Phenolics and Anthocyanins Determination*

Total phenolics compounds (TPC) were measured by the Folin Ciocalteau spectrophotometric method using gallic acid as a standard and expressing the results as mg Gallic Acid Equivalent (GAE)/100 g FW [25]. Moreover, *o*-diphenolics compounds (ODC) were determined by the Arnow spectrophotometric method and expressing the results as mg chlorogenic acid/100 g FW [26].

In order to evaluate the anthocyanin amount using high performance liquid chromatography/diode array detector/mass spectrometry (HPLC/DAD/MS), the mulberry raw extracts were purified by solid phase extraction (SPE) using cartridge Strata X (Phenomenex Italia, Castel Maggiore, Bologna, Italy). After activation of the SPE cartridge with 2 mL pure methanol and 5 mL bi-distilled water, 20 mL raw extract were loaded and washed with 10 mL acidified bi-distilled water and 5 mL ethyl acetate to remove sugars and less polar flavons respectively; finally, 20 mL acid methanol were used to recover the anthocyanins. The purified extract was dried under vacuum and re-suspended with HPLC water acidified with 0.1 % HCl.

The identification was completed using an HPLC Agilent 1100 system (Agilent Tecnnologies, palo Alto, CA, USA) coupled with an Agilent DAD sensor (detection wavelength 520 nm) and Agilent ESI/MS spectrometer 6100 in positive ionization mode as reported by Negro et al. [27]. The separation was carried out at 30 ◦C with a gradient elution program at a flow rate of 0.8 mL/min using a Phenomenex Gemini C18 250 × 4.6 mm, 5 μm separation column. The mobile phases consisted of water plus 2% formic acid (A) and water:formic acid:acetonitrile 48:2:50 (B). The following multistep linear gradient was applied: 0 min, 6% B; 15 min, 30% B; 25 min, 50% B; 30 min, 60% B. The injection volume in the HPLC system was 20 μL. The quantification of anthocyanin was achieved using calibration curves of the authentic chemical standards cyanidin 3-glucoside (C3G) and moreover, total anthocyanins (TA) was determined as being the sum of the area of the single compounds.
