*3.2. Anthocyanins Analysis*

Figure 1 reports a typical HPLC/DAD/MS separation of the anthocyanins present in mulberry fruits. Five components were identified on the basis of the UV and MS spectra and retention time, corresponding to cyanidin-3-O-sophoroside (C3S, m/z 611, peak 1), cyanidin-3-O-glucoside (C3G, m/z 449, peak 2), cyanidin-3-O-rutinoside (C3R, m/z 595, peak 3), pelargonidin-3-O-glucoside (P3G, m/z 433, peak 4) and pelargonidin-3-O-rutinoside (P3R, m/z 579, peak 5). Our results are in agreemen<sup>t</sup> with the data reported some years ago by Dugo et al. [39] that identified a mixture of five different anthocyanidin glycosides in Italian mulberries cultivars but are discordant with Pawloska et al. [13], which revealed four anthocyanins in *M. nigra* fruits harvested in Benevento (South Italy).

**Figure 1.** Typical HPLC/DAD/MS analysis (recorded at 520 nm) of the anthocyanins present in mulberry fruit extracts (*M. nigra*). (1) cyanidin-3-*O*-sophoroside, (2) cyanidin-3-*O*-glucoside, (3) cyanidin-3-*O*-rutinoside, (4) pelargonidin -3-*O*-glucoside and (5) pelargonidin -3-*O*-rutinoside.

The anthocyanins identified and the total anthocyanin content (TA), calculated as the sum of the single compounds, were shown in Table 2. The TA in *M. alba Nello* was 24.7 mg/100 g FW, was about 8 and 12 times lower than that determined in *M. nigra* and *M. alba Legittimo nero*, corresponding to 206 and 289 mg/100 g FW, respectively. According to earlier reports, TA content was 2–30, 0.3–83, 1.5–615 mg/100g FW respectively for genotypes grown in Turkey, Korea and India [34,40,41]. C3G and C3R are the anthocyanins most represented with amounts ranging from 18.2 to 212 and from 5.2 to 71.8 mg/100 g FW, respectively. P3G ranged from 1.6% to 5% of the TA content while C3S was sligtly higher in *M. nigra*, representing about 1.5% of the TA in comparison to 0.17% and 0.56% for the other genotypes.

**Table 2.** Amounts of cyanidin-3-*O*-sophoroside (C3S), cyanidin-3-*O*-glucoside (C3G), cyanidin-3-*O*-rutinoside (C3R), pelargonidin-3-*O*-glucoside (P3G) and pelargonidin-3-*O*-rutinoside (P3R) and Total anthocyanins (TA) in mulberry fruits. Values are expressed as C3G mg/100 g FW and represent the results of three determinations ± SD. Means with different letters in the same column are significantly different from each other (*p* < 0.05) according to the multicomponent Duncan's test.

