*3.3. Antioxidant Activity*

Table 3 reports the mulberry AA measured by three different tests. Data indicate clearly that *M. nigra* showed the highest antioxidant activity corresponding to about 33, 26 and 21 μmol Trolox/g FW, respectively by the DPPH, ABTS and FRAP tests.

**Table 3.** Antioxidant activity of different mulberry genotypes evaluated by DPPH, ABTS and FRAP test. Results are expressed as μmol Trolox Equivalents/g FW. Values 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.


In the DPPH scavenging activity test, all the mulberry genotypes showed an AA significantly higher than ABTS and FRAP test. In fact, *M. alba Legittimo nero* exhibited an antioxidant capacity that was about two times greater than that measured with the other methods and *M. alba Nello* showed a capacity that was about 10 times lower when assessed by the FRAP test.

These data could be the result of a different qualitative and quantitative composition of phenolic compounds of the three mulberries. Further studies are needed to prove this. Moreover, we cannot exclude the possibility that the quenching mechanisms of the different compounds are more efficient against DPPH• than ABTS•+.

However, the observed antioxidant activity is probably due to the set of phenolic compounds present in the juice; in addition to the anthocyanin effect, it is known that there are significant quantities of other phenolic compounds such as gallic and cinnamic acid, procyanidin B1, catechin and quercetin [32].

Moreover, these results are in agreemen<sup>t</sup> with the literature data, in particular with the work of Ercisli et al. [33] that reports for *M. nigra* and *M. rubra* an AA ranging between 16 and 21.2 μmol Trolox/g FW and between 9.2 and 12.1 μmol Trolox/g FW when it was estimated by DPPH; instead, the range was between 12.3 to 14.1 μmol Trolox/g FW and from 4.9 to 8.1 μmol Trolox/g FW, respectively for *M. nigra* and *M. rubra* when measured by the FRAP test [33]. Also, the radical scavenging activity measured using ABTS system was in agreemen<sup>t</sup> with the values reported for different mulberry genotypes (black and/or red mulberries) grown in various Turkish regions, ranging from 6.8 to 14.4 μmol Trolox/g FW and from 5.1 to 7.3 μmol Trolox/g FW, respectively, in *M. nigra* and *M. rubra* [34].

#### *3.4. Anti-Inflammatory Activity (AI)*

The anti-inflammatory activity is related to the ability of some compounds to inhibit the two isoforms of the cyclooxygenase enzyme, COX-1 and COX-2; this activity was demonstrated for the anthocyanins isolated from raspberries and sweet cherries (cyanidin-3-glucosyl-rutinoside and cyanidin-3-rutinoside [42] and also for the anthocyanidins as cyanidin and malvidin [43].

The results of the AI in vitro assay are reported in Table 4; data indicate for all the extracts a relevant biological activity. The IC50 values relative to COX-1 ranged between 125 and 185 μg/mL TA and from 64 and 97 μg/mL TA relative to COX-2 for *M. nigra* and *M. alba Nello* genotypes, respectively. Employing identical analysis conditions, the AI of ibuprofen and nimesulide (positive controls, two synthetic anti-inflammatories compounds), were equal to 6 and 5 μg/mL, respectively, for COX-1 assay, and to 5 and 2 μg/mL for COX-2 assay. To our knowledge, it is the first time that mulberry extracts AI was determined in vitro using the ELISA methodology. Several authors have evaluated pure anthocyanins or di fferent fruit extracts (such as cherry, blueberry, blackberry, etc) employing di fferent assay methods showing that among aglycones, cyanidin was particularly active in the COX-2 assay [39]. Comparing IC50 values for di fferent fruits from literature data, for pomegranate values were reported relative to COX-1 between 249 and 145 μg/mL and from 175 and 75 relative to COX-2, respectively for di fferent genotypes grown in Salento area (Southern Apulia) [27]; the cherries showed values up to 130 μg/mL whereas blueberries, which are known to have high AI, had a range of 400–800 μg/mL depending to the ecotypes [42].

**Table 4.** Anti-inflammatory activity (AI) of mulberry genotypes measured as COX-1 and COX-2 inhibitory activity expressed as IC50, μg/mL extract. Values represent the results of three determinations ± SD; means with di fferent letters in the same column are significantly di fferent from each other (*p* < 0.05) according to the multicomponent Duncan's test.

