*2.7. Statistical Analysis*

All of the results were performed in triplicate and presented as the mean values ± standard deviation (SD). Data for TPC, TFC and antioxidant properties were analyzed using Two-way ANOVA considering the origin of the samples (field-grown leaves, in vitro leaves and callus cultures) and berry plant cultivars as fixed effects and the replicates as a random effect. Significant differences (*p* < 0.05) between means were determined by Tukey's test, using GraphPad Prism6 software (USA). Significant differences (*p* < 0.05) between the means for individual phenolics were determined by Student's *t*-test. The correlation analyses were performed by calculating Pearson's correlation coefficient (*r*), (*p* < 0.05), using the Statistica software version 12.0 (StatSoft Co., Tulsa, OK, USA). Figures were drawn using GraphPad Prism6 software (USA).

### **3. Results and Discussion**

### *3.1. Total Phenolic and Flavonoid Content*

The TPC of aqueous extracts of blueberry and blackberry leaves and calluses are presented in Figure 2a.

**Figure 2.** (**a**) Total phenolic content and (**b**) Total flavonoid content of blackberry 'Caˇ ˇ canska Bestrna' and blueberry 'Toro' leaves and callus samples. 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 type of samples of two different berry cultivars.

The TPC of aqueous extracts of field-grown and in vitro leaves of 'Toro' blueberry were 14.06 ± 0.51 and 13.47 ± 0.42 mg GAE/g DW, without a statistically significant difference (*p* < 0.05). These values are significantly lower (almost 10-fold lower) than the TPC values for field-grown leaves of six cultivated blueberry cultivars, including 'Toro' (132.92 mg GAE/g leaves material), reported by S, tefănescu et al. [6]. In addition, Wu et al. [11] also reported significantly higher TPC values for blueberry field-grown leaves from 73 different cultivars collected in southern China, which ranged from 32.18 ± 0.01 ('O'Neal') to 224.1 ± 3.4 ('Blackpearl') mg GAE/g DW. Moreover, the same study determined TPC values in the leaves of field-grown 'Toro' plants of 75.07 ± 1.48 mg GAE/g

DW, which is 5.3- and 5.6-fold higher than the TPC values obtained in this study for both field-grown and in vitro leaves for the same cultivar, respectively. Goyali et al. [39] also obtained higher TPC values for the leaves of wild lowbush blueberry clone QB9C (*Vaccinium angustifolium* Ait.) originating from both ex vitro (propagation by stem cuttings) and in vitro propagated plants. These differences are mainly due to the differences in extract solvents. Namely, in this study the methanol extracts of leaves and calluses were evaporated, resuspended in milliQ water and, after filtration through 0.45 μm syringe filters, subjected to spectrophotometric analysis, whereas the aforementioned authors used 40% ethanol, 85% methanol or 80% acetone. Furthermore, differences in method of extraction (solid-liquid against ultrasound-assisted extraction), applied in vitro propagation methods as well as the different geographical areas where the berry plant is grown, climatic factors and soil composition for field-grown plants can also affect the TPC value of analyzed samples [4,5,36]. Furthermore, total phenolics for both aqueous extracts, field-grown and in vitro leaves of 'Toro' blueberry were significantly higher (*p* < 0.05), in comparison to those of field-grown and in vitro leaves of 'Caˇ ˇ canska Bestrna', whose TPC values were 11.46 ± 0.27 and 8.97 ± 0.35 mg GAE/g DW, respectively. These values are significantly lower than previously reported TPC value for field-grown leaves of the same blackberry cultivar, also collected in Serbia [5]. However, the TPC value of field-grown blackberry leaves obtained in this study was significantly higher than the TPC values obtained for water extracts from the leaves of wild-grown and cultivated blackberry (*Rubus fruticosus* L. 'Thornfree') collected during different seasons of the year [40]. In the study conducted by Fathy et al. [20], TPC values for methanolic extracts obtained from the leaves of in vitro blackberry (*Rubus fruticosus* L.) shoots grown on media with different concentrations of PGRs (benzyladenine (BA) at different concentrations, applied alone or in combination with α-naphthaleneacetic acid (NAA)), were in the range from 1.17 to 2.39 mg GAE/g, which is significantly less than the TPC value obtained for in vitro blackberry leaves in this study. Thus, PGR combinations applied to in vitro cultivation of blackberry 'Caˇ ˇ canska Bestrna' should be considered as promising, because in vitro leaves can be a good source of phenolic compounds for further applications in the food industry and pharmacy. As can be seen in Figure 2a, callus culture induced from both berry plants had significantly lower TPC in comparison with their leaves; the values were 1.52 ± 0.03 and 0.78 ± 0.03 mg GAE/g DW in blueberry 'Toro' and blackberry 'Caˇ ˇ canska Bestrna', respectively. The TPC value of blueberry callus culture was significantly lower than those reported by Ramata-Stunda et al. [31], and significantly higher than TPC values reported by Ghosh et al. [30] in callus culture of different blueberry cultivars. The TPC value of blackberry callus culture in this study was in agreemen<sup>t</sup> with the TPC values obtained for callus culture induced from blackberry (*Rubus fruticosus* L.) leaves treated with different concentrations of NAA and/or 2,4-dichlorophenoxyacetic acid (2,4-D) [20].

The total flavonoid content of aqueous extracts of 'Toro' and 'Caˇ canska Bestrna' leaves and their callus cultures are illustrated in Figure 2b. TFC values of aqueous extracts of field-grown and in vitro blueberry leaves were 18.56 ± 0.98 and 10.88 ± 0.66 mg CE/g DW, respectively. On the other hand, the TFC values of aqueous extracts of field-grown and in vitro leaves in blackberry were significantly lower (almost five-fold) in comparison with corresponding blueberry samples, i.e., 4.42 ± 0.11 and 2.44 ± 0.34 mg CE/g DW, respectively. Interestingly, the TFC value in the extract of field-grown blueberry leaves was significantly higher in comparison to the TPC value of the same extract, which was also observed in the study reported by Wu et al. [11]. TFC values in field-grown leaves of blueberry 'Toro' previously reported by S, tefănescu et al. [6] and Wu et al. [11], showed significantly higher flavonoid content in comparison to the value obtained in this study. As shown in Figure 2b, callus culture for both berry plants had significantly lower TFC values in comparison to the results obtained for their leaves, which is in accordance with the obtained results for TPC. The TFC values of blueberry and blackberry callus cultures (1.31 ± 0.03 and 0.57 ± 0.01 mg CE/g DW, respectively) were not significantly different (*p* < 0.05). The literature survey led us to the conclusion that the data on the TFC

ˇ

of blueberry and blackberry callus culture are rather scarce. Ghosh et al. [30] obtained considerably higher TFC values for the callus culture of different blueberry cultivars, in comparison with TFC value obtained for 'Toro' blueberry callus culture in this study. On the other hand, Fathy et al. [20] obtained very low TFC values for methanolic extract of the callus culture induced from blackberry leaves which were treated with different concentrations and combinations of PGRs, such as 2,4-D and NAA.

#### *3.2. UHPLC-DAD MS/MS Analysis of Blueberry and Blackberry Leaves and Callus Cultures*

Characterization and quantification of phenolic compounds from aqueous extracts of blueberry and blackberry leaves, as well as their callus cultures, were performed using UHPLC-DAD MS/MS analyzer (Table 3).

Depending on the berry plant genotype and specific characteristics of samples tested, a total of 20 phenolic compounds was confirmed and quantified. As can be observed from Table 3, field-grown and in vitro leaves of blueberry 'Toro' are better source of phenolic compounds in comparison to field-grown and in vitro leaves of blackberry 'Caˇ ˇ canska Bestrna'. The most abundant PCs of 'Toro' leaves belong to phenolic acids, flavonols and flavan-3-ols, with respective shares of 41.3, 54.3, and 4.3% for field-grown, and 29.7, 54.6, and 14.9% for in vitro leaves. The dominant presence of phenolic acids and flavonols has been shown in some previous PC characterizations of leaves of different blueberry cultivars using the chromatographic technique [6,10–12]. Quercetin derivatives, chlorogenic acid and gallocatechin were dominant the PCs for both field-grown and in vitro blueberry leaves. Other studies have also reported quercetin derivatives [6,11] and chlorogenic acid [4,12,31] as the most abundant PCs in the leaves of various blueberry cultivars, including 'Toro'. Among quercetin derivatives, quercetin-3- *O*-glucoside and quercetin-3- *O*-rutinoside were the most predominant, with respective contents of 160.113 ± 3.059 and 49.639 ± 1.526 mg/kg DW for extracts of field-grown leaves and 115.598 ± 5.041 and 62.293 ± 4.403 mg/kg DW for extracts of in vitro leaves. This is in agreemen<sup>t</sup> with the result reported by S, tefănescu et al. [6] for field-grown leaf extract of various blueberry cultivars, including 'Toro'. Interestingly, significant amounts of flavonols, such as quercetin-3- *O*-rhamnoside, aesculetin, and the aglycones of quercetin and kaempferol, have been identified only in extracts of in vitro blueberry leaves.

On the other hand, the quercetin derivatives syringic and chlorogenic acid were the most abundant phenolic compounds detected in field-grown and in vitro leaves of blackberry 'Caˇ ˇ canska Bestrna', making more than 95% of all quantified phenolics. Some previous studies have reported significantly higher contents of different phenolic acids and flavonoids in methanolic extracts of field-grown leaves of three blackberry wild genotypes and three cultivars [7], or 26 different wild blackberry genotypes collected from various localities throughout Poland [9]. Flavan-3-ols were not detected in the leaves of 'Caˇ ˇ canska Bestrna', which is not in accordance with the results reported by Pavlovi´c et al. [5], which found significant amounts of catechin derivatives in methanolic extract of field-grown leaves of the same blackberry cultivar, also collected in Serbia. The absence of flavan-3-ols can be attributed to their ability to rapidly polymerize in aqueous extract into complex forms, which were removed by filtration through a 0.45 μm filter before UHPLC-DAD MS/MS analysis. Differences in the phenolic profiles of leaves originating from in vitro cultivated plants may be due to applied PGRs [17] such as zeatin and indol-3-acetic acid for blueberry 'Toro'; that is, gibberellic acid (GA3), BA and indole-3-butyric acid (IBA) for blackberry 'Caˇ ˇ canska Bestrna' (Table 1).

*Horticulturae* **2021**, *7*, 420


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field-grown and in vitro leaves for both berry plant cultivars.

Generally, the field-grown and in vitro blueberry/blackberry leaves have shown to be a much better source of phenolic compounds (PCs) in comparison to in vitro induced callus culture obtained from in vitro leaves of the same berry cultivars. The low yield of phenolic compounds in the callus culture of blueberry 'Toro' and blackberry 'Caˇ ˇ canska Bestrna' can be explained by the lack of cell differentiation [15]. The callus culture of 'Toro' produced higher levels of specific phenolic acids, flavan-3-ols and flavonols, in comparison with the callus culture of 'Caˇ ˇ canska Bestrna'. However, both aqueous extract of blueberry and blackberry callus cultures contained small amounts of individually detected PCs. Interestingly, except for quercetin-3- *O*-glucoside and quercetin-3- *O*-rutinoside, other identified phenolics are specific for blueberry callus culture and have not been detected in field-grown and in vitro blueberry leaf extracts. For example, small amounts of phenolic acid such as vanillic and ferulic acid (0.330 ± 0.021 mg/kg DW), or flavan-3-ols such as catechin (0.567 ± 0.045 mg/kg DW) and catechingallate, were found in the callus extract of 'Toro', while chlorogenic acid and gallocatechin were not detected. However, in the study of Ramata-Stunda et al. [31], chlorogenic acid was the most abundant PC in the callus culture of 'Duke' and 'Bluecrop' blueberries, while other phenolic acids were present in traces. This may be due to the presence of phytohormones and elicitors in nutrient medium, which are able to initiate the synthesis of specific PCs [16,17]. On the other hand, although in small amounts, quercetin-3- *O*-glucoside, gallic and ferulic acid were found as dominant PCs in the callus culture of blackberry 'Caˇ ˇ canska Bestrna', while other phenolics were detected in traces. Interestingly, the predominantly confirmed syringic and chlorogenic acids in field-grown and in vitro blackberry leaf extracts were not found in the callus culture of this berry plant, but gallic and ferulic acid were. However, the effect of different PGRs on the synthesis of specific PCs in the callus cultures of 'Toro' and 'Caˇ ˇ canska Bestrna' requires a more complex experiment and additional research.
