*3.3. Antioxidant Properties*

Using different in vitro screening assays for evaluating the antioxidant properties of prepared aqueous extracts of field-grown leaves, in vitro leaves and callus cultures of blackberry and blueberry gives better insight into their potential opportunities for health improvement and further applications. In order to obtain more detailed information on the antioxidant properties of the analyzed samples, it is very important to apply several different tests based on different mechanisms of action. Three mechanisms of action of bioactive compounds are generally known: hydrogen atom transfer (HAT), single electron transfer (SET), and the ability to chelate transition metals [41–43]. These mechanisms include several tests most commonly used to assess the antioxidant properties of phenolics extracts of leaves and callus cultures of berry plants [11,12,20,31,44]. In this study, three in vitro screening antioxidant assays, FRP, ABTS•<sup>+</sup> and DPPH• scavenging activity, were used with the aim of evaluating the antioxidant potential of the leaves and calluses of blueberry and blackberry cultivars (Figure 3).

According to our knowledge, this is the first time the antioxidant properties of in vitro leaves and calluses of these berry plant cultivars has been evaluated by performing these three assays.

**Figure 3.** (**a**) Ferric reducing power (FRP), (**b**) ABTS•<sup>+</sup> radical scavenging activity, and (**c**) DPPH• radical scavenging activity of field-grown leaves, in vitro leaves and callus cultures of blackberry 'Caˇ ˇ canska Bestrna' and blueberry 'Toro'. The bars with (±) standard deviation represent mean values. The different lowercase letters indicate a significant difference (*p* < 0.05) between field-grown leaves, in vitro leaves and callus culture of the same berry. Different uppercase letters indicate a significant difference (*p* < 0.05) between the same types of samples of two different cultivars.

### *3.4. Ferric Reducing Power (FRP)*

The Ferric reducing antioxidant power (FRAP) assay was most commonly used in the literature to assess the reducing properties of extracts of blueberry [11,12] and blackberry [9,40] leaves. However, this assay has some limitations, such as the low pH value applied and the reactivity rate of different types of molecules, giving variable results [42]. On the other hand, the ferric reducing power assay is based on the ability of bioactive compounds to reduce Fe3+-ferricyanide complexes to their ferrous (Fe2+) form in more relevant physiological conditions (pH = 6.6) [42], and can serve as a significant indicator of antioxidant activity of prepared leaves and callus culture extracts of blueberry and blackberry.

The aqueous extracts of field-grown and in vitro leaves of blueberry 'Toro' and blackberry 'Caˇ ˇ canska Bestrna' analyzed in this study showed good ferric reducing ability (Figure 3a). More precisely, the FRP values for field-grown leaves were 7722.75 ± 1125.62 ('Toro') and 9815.27 ± 155.52 ('Caˇ ˇ canska Bestrna') mg GAE/g DW, while for in vitro leaves the values were 5153.76 ± 88.37 and 6731.56 ± 50.96 mg GAE/g DW, respectively. As can be seen in Figure 3a, the FRP values of field-grown leaves for both berry plants leaves were significantly higher (*p* < 0.05) in comparison with the FRP values of in vitro leaves, which

is in agreemen<sup>t</sup> with TPC and TFC results. Good ferric reducing ability of field-grown blackberry and blueberry leaves has also been reported by other authors [9,11,12]. However, a direct comparison is not possible, primarily due to the previously mentioned FRAP method used by the authors to measure the reducing ability of leaf extracts. Interestingly, field-grown and in vitro leaves of blackberry 'Caˇ ˇ canska Bestrna' had better FRP activity than field-grown and in vitro leaves of blueberry 'Toro' (Figure 3a). This contrasts with the results obtained by chromatographic, TPC and TFC analysis, which showed a more diverse and higher content of phenolic compounds in the leaves of 'Toro'. This can be explained by the fact that field-grown and in vitro leaf extracts of blackberry contain other highly effective antioxidants in addition to the quantified phenolic compounds, such as ellagic acid derivatives, ellagitannins [5,9], vitamin C [45] and some terpenes [46] in significant quantities, which have not been analyzed in this study. On the other hand, aqueous extracts of callus cultures in both berry plants showed very low ferric reducing ability, which is consistent with the results obtained for their individual and total phenolic content. Correlation analysis revealed a significant positive correlation (*p* < 0.05) between FRP and TPC (*r* = 0.87).

### *3.5. ABTS*•*<sup>+</sup> and DPPH*• *Scavenging Activity*

The ABTS•<sup>+</sup> scavenging activity of aqueous extracts of field-grown leaves of blackberry 'Caˇ ˇ canska Bestrna' was 3574.10 ± 426.78 μgAAC/mL, which was significantly higher than ABTS•<sup>+</sup> values for aqueous extracts of in vitro leaves of the same cultivar (2933.51 ± 147.84 μgAAC/mL) or field-grown and in vitro leaves of blueberry 'Toro' (Figure 3b). ABTS•<sup>+</sup> scavenging activity of aqueous extracts of field-grown and in vitro leaves of blueberry were 2644.22 ± 98.43 and 2442.74 ± 155.24 μgAAC/mL, without significant differences. As with the FRP results, aqueous extracts of field-grown and in vitro leaves of blackberry showed ABTS•<sup>+</sup> values significantly higher than ABTS•<sup>+</sup> values for aqueous extracts of field-grown and in vitro leaves of blueberry. This is not in accordance with the chromatographic and spectrophotometrically obtained results for individual and total phenolics, which are significantly higher for blueberry 'Toro'. A previous study by Pavlovi´c et al. [5] using UHPLC-LTQ OrbiTrap MS/MS analysis showed that the leaves of 'Caˇ ˇ canska Bestrna' blackberry predominantly contain ellagic acid and its derivatives, as well as ellagitannins, which probably contribute to the high ABTS•<sup>+</sup> values. Due to the large number of hydroxyl groups, ellagic acid and its derivatives, as well as ellagitannins, are potentially good hydrogen ion donors and scavengers of ABTS•+. Other authors have also noticed good ABTS•<sup>+</sup> scavenging activity by different leaves of wild blackberry [9] and blueberry cultivars, including 'Toro' [11,12], but a direct comparison with our results is not possible due to differences in measurement units and the applied methods. The ABTS•<sup>+</sup> scavenging activity of callus cultures were 701.48 ± 18.27 ('Toro') and 703.14 ± 46.50 ('Caˇ ˇ canska Bestrna') μgAAC/mL, without significant difference (*p* < 0.05) (Figure 3b). The obtained ABTS•<sup>+</sup> values for callus cultures are about three-fold (blueberry), that is, about four-fold (blackberry) less than the ABTS•<sup>+</sup> values obtained for field-grown and in vitro leaves of the same berry plants. To the best of our knowledge, this is the first time that the ABTS•<sup>+</sup> scavenging activity of in vitro leaves and callus cultures of blueberry and blackberry has been determined.

The DPPH• scavenging assay is most commonly used to evaluate the antioxidant properties of field-grown leaves [5,6,38,39,44] and callus cultures [30,31] of different blueberry and blackberry cultivars. In this study, similar to the FRP results, aqueous extracts of field-grown and in vitro leaves of blackberry 'Caˇ ˇ canska Bestrna' showed ABTS•<sup>+</sup> values significantly higher than ABTS•<sup>+</sup> values for aqueous extracts of field-grown and in vitro leaves of blueberry 'Toro'. This is not in compliance with the chromatographic and spectrophotometrically obtained results for individual and total phenolics, which are significantly higher for blueberry. A previous study by Pavlovi´c et al. [5], using UHPLC-LTQ OrbiTrap MS/MS analysis showed that the leaves of 'Caˇ ˇ canska Bestrna' blackberry predominantly contain ellagic acid and its derivatives, as well as ellagitannins, which probably contribute to high

ABTS•<sup>+</sup> values. Due to a large number of hydroxyl groups, ellagic acid, its derivatives, and ellagitannins are potentially good hydrogen ion donors and scavengers of ABTS•+. Other authors have also noticed good ABTS•<sup>+</sup> scavenging activity of different leaves of wild blackberry [9] and blueberry cultivars, including 'Toro' [11,12], but a direct comparison with our results is not possible due to differences in measurement units and the applied methods. The ABTS•<sup>+</sup> scavenging activity of callus cultures were 701.48 ± 18.27 ('Toro') and 703.14 ± 46.50 ('Caˇ ˇ canska Bestrna') μg AAC/mL, respectively, without significant differences (*p* < 0.05) (Figure 3b). The obtained ABTS•<sup>+</sup> values for callus cultures are about three-fold (blueberry), that is, about five-fold (blackberry) less than the ABTS•<sup>+</sup> values obtained for field-grown and in vitro leaves of the same berry plant. As far as we know, this is the first time that the ABTS•<sup>+</sup> scavenging activity of in vitro leaves and callus cultures of blueberry and blackberry cultivars has been determined. Aqueous extracts of field-grown and in vitro leaves of blackberry 'Caˇ ˇ canska Bestrna' showed significantly higher percentage of inhibition of DPPH radicals (75.77% and 59.44%, respectively), than field grown (39.15%) and in vitro (39.65%) leaves of blueberry 'Toro' (Figure 3c). These DPPH• results show the same trend as the results obtained for the ABTS•<sup>+</sup> scavenging activity and FRP assays. Correlation analysis revealed a significant positive correlation (*p* < 0.05) between the FRP and ABTS•<sup>+</sup> (*r* = 0.98) and DPPH• (*r* = 0.95) scavenging activities, indicating that aqueous extracts of investigated samples that showed good ferric reducing power also possessed a good ability to scavenge free radicals.

However, the radical scavenging assays are also not consistent with the results obtained for individual and total phenolics analyzed in this study. The activity of complex extracts according to DPPH• is different and closely dependent on the nature of the phenolic compounds present in the extract, because DPPH• is known as a stable and lipophilic radical [47]. Good DPPH• scavenging activity has been previously reported for methanolic and aqueous extracts of field-grown leaves in various blackberry cultivars [38] and methanolic extracts of 'Caˇ ˇ canska Bestrna' leaves also collected in Serbia (83.77%) [5]. Furthermore, several studies have shown good DPPH• scavenging activity for extracts of field-grown leaves of various blueberry cultivars, including leaves of 'Toro' [6,11,12]. Callus cultures of both berry cultivars had significantly lower DPPH• scavenging activity, that is, 3.89% ('Toro') and 4.01% ('Caˇ ˇ canska Bestrna').

Based on the obtained results of the three antioxidant assays, aqueous extracts of fieldgrown leaves of blackberry 'Caˇ ˇ canska Bestrna' showed the best antioxidant properties, while aqueous callus culture extracts of both berry cultivars had the lowest phenolic content and significantly lower antioxidant properties than the corresponding leaves.
