3.1. Bergamot Pomace (BP): Antioxidant Characterization
Different Food grade solvents (H
2O and H
2O: EtOH), times, temperatures, and systems to assist the extractability were applied to optimize the extraction yield of bioactive compounds present in the BP. All the obtained extracts were analyzed by spectrophotometric methods, for the determination of total bioactive components, in particular: total polyphenol content (TPC), flavonoid content (TFC), and total antioxidant activity (DPPH and ABTS). Statistical differences were analyzed among all the studied variability (ANOVA). Multivariate analysis carried out on the antioxidant extracts, showed significant differences (
p < 0.01) for extraction methods and used solvents, considering all the total antioxidant assays, and the results are reported in
Table 2. However, significant differences were also highlighted for extraction * solvent, extraction * temperature, and extraction * solvent * temperature (
p < 0.01).
Significative differences in TPC (
p < 0.01) were observed among the different extraction systems (C, UA, MA) and for the different studied variables within the same extraction system (
Table 3).
Conventional and Ultrasound assisted extraction has provided important results in terms of total phenolics recovery. The highest values were measured for the conventional extraction system (C), particularly for C7 and C8 samples (26.30 and 26.06 mg GAE g
−1 dw, respectively), also if the Ultrasound assisted system (UA) led to a good recovery of TPC (about 23.64 mg GAE g
−1 dw in UA5 extract). The obtained values are higher than those reported by Gabriele et al. [
8] (17.44 ± 0.40 mg GAE g
−1 dw) on lyophilized bergamot fruits, and by Multari et al. [
33] (410 ± 36.8 mg kg
−1) on bergamot pomace. Moreover, the content of total phenols was higher compared with other citrus (lemon and orange peel), as reported by Gorinstein et al. [
34]. Among all the evaluated extracts, those obtained through microwave-assisted (MA), showed less extractability (between 10 and 19 mg GAE g
−1 dw).
Regarding the TFC the obtained values were reported in
Table 3. The amount of determined TFC showed great variability in accordance with the applied extraction procedure (
p < 0.01). This spectrophotometric assay showed a different trend compared with the TPC assay; indeed, great extractability was obtained through the application of UA treatment, particularly in the samples prepared using water as an extraction solvent. As demonstrated by some authors, the application of ultrasound to facilitate the extraction of bioactive compounds is very useful, indeed, this technique adopts an acoustic cavitation system that combines ultrasound and traditional solvent extraction. The sound waves induce forces that can break the cell walls intensifying the release of constituents [
25]. In comparison to water, ethanol is characterized by a higher heating efficiency when applied in an aqueous mixture and it is preferred thanks to its better capacity in solving the phenolic compounds. Higher values were estimated in the samples UA1 and UA2 (7.52 and 7.81 mg CE g
−1 dw, respectively). In all the other situations was found a lower content of TFC. Nevertheless, a similar recovery of TFC was evidenced for the sample obtained with a hydroalcoholic mixture (7 and 8) for all the extraction systems (C, UA, and MA).
The antioxidant ability of the different obtained extracts was analyzed in vitro by applying two common spectrophotometric assays: DPPH, and ABTS (
Figure 4a,b). These tests are based on the ability of an antioxidant matrix (represented by the antioxidant extract) to reduce a radical species. The antioxidant molecules present in the extracts react against the radicals and causing oxidation of the reacting molecule, with a related decrease in the solution absorbance (discoloration reaction).
The DPPH assay on the different obtained extracts showed highly significant differences among the samples (
p < 0.01), with values that ranged between 0.71 and 3.21 mM TE g
−1 dw. As clearly shown in
Figure 4a, the antioxidant extracts recovered through the use of a hydroalcoholic solvent (H
2O: EtOH, 50%) highlighted the highest total antioxidant activity (
p < 0.01), particularly, the extracts recovered with ultrasound.
Concerning the ABTS assay, the results are shown in
Figure 4b, where is possible to observe that the obtained values highlighted a higher antioxidant activity compared to the DPPH assay, with values that ranged between 3.22 and 16.93 mM TE g
−1 dw. The best extraction in terms of total antioxidant activity was obtained by applying the hydroalcoholic solution (H
2O-EtOH, 50%) as extraction solvent and assisted by a conventional maceration (samples C6 and C7). The two different antioxidant assays, respond differently to extracts, this trend is probably due to the different interaction of the two free radicals with antioxidant molecules.
For all the analyzed extracts, the two total antioxidant assays showed different results, with higher concentrations of total antioxidants highlighted by the ABTS assay. It could be dependent on the fact that ABTS assay generally is a more sensitive hydrophilic and lipophilic antioxidant, while DPPH assay is more sensitive to lipophilic antioxidants [
35]. This affinity was also described by Zacarías-García [
36] which highlighted the highest value for ABTS compared to DPPH in the hydrophilic fraction.
As already reported in other works, the extractability of bioactive compounds is strongly dependent on different applied extraction variables, such as solvent, time, temperature, etc. [
37] as also evidenced in this work.
In addition, positive correlations were found between TPC/DPPH, particularly for the conventional extraction method (r > 0.7), while the ABTS assay was related to both TPC and TFC (r > 0.95) especially for microwave extraction.
3.2. Quantification of Individual Bioactive Compounds through Liquid Chromatography
The quali-quantitative analysis of the prevalent neohesperidose flavanones (eriocitrin, neoeriocitrin, naringin; neohesperidin, metilidin, and brutieridin) present in bergamot pomace extracts was performed by UHPLC system.
In
Table 4, the validation of chromatographic analysis was reported. The applied methodology was developed by the injection of different concentrations of standard solutions. Particularly, limits of detection (LOD) and quantification (LOQ); the correlation coefficient (R2) and regression equations for each antioxidant standard were studied.
While for Metilidin and Brutieridin compounds the Naringin was used as standard, and the results are expressed as naringin equivalent (mg g−1 dw).
Figure 5 shows the chromatographic profiles of the obtained extracts and the main identified compounds. Particularly, seven phenolic compounds were detected: eriocitrin, neoeriocitrin, narirutin, naringin, neohesperidin, melitidin and brutieridin.
Neoeriocitrin, naringin, neohesperidin, and brutieridin were the main flavonoids detected in BP (
Table 5), as also reported by other authors [
7,
38] and represent the typical compounds that determine the bitterness in the fruit [
39].
Among these, neoeriocitrin was the most abundant in BP extract ranging from 5.03 to 14.14 mg g−1 dw; in particular, it showed its highest amounts (13.95 and 14.14 mg g−1 dw) in the C7 and C8 samples, obtained by conventional extraction at 70 °C for 30 and 60 min. Regarding the other prevailing compounds, the range were 4.69–12.87 mg g−1 dw for naringin; 2.84–7.35 mg g−1 dw for neohesperidin, 2.05–5.67 mg g−1 dw for brutieridin, and 1.12–2.67 mg g−1 dw for melitidin. Lesser quantities of eriocitrin and narirutin were instead determined in all samples.
All the assayed extracts were significantly different for the phenolic content, related to the extraction system (C, UAE, and MA,
p < 0.01) and variables of extraction (times and temperatures,
p < 0.01). The conventional extraction system produced the greatest extractability of this class of antioxidants with similar values in C7 and C8 samples. As observed previously in other food matrices, the hydroalcoholic solvent is the best choice to obtain the maximum yield of antioxidant compounds [
25,
37], when combined with 70 °C temperature [
25].
Additionally, the limonoid content was evaluated on Bergamot pomace extract, as it represents an interesting index to evaluate, indeed, its contribution to the bitter taste of citrus fruits, as also reported by Shi et al. [
40]. In addition, limonoids from Citrus are getting a lot of scientific interest for their bioactive properties in vitro and in vivo such as antitumor, antioxidative, and antibacterial compounds [
41]. The chromatographic analysis showed that also the highest yield of the two mains determined limonoids (limonin and nomilin) was obtained through conventional extraction technique C7 (
Figure 6).
The statistical analysis carried out on the obtained results for limonoid content showed significant differences (
p < 0.01) among all the studied variables (
Table 6).
After identifying the best extraction system, namely conventional maceration for 30 min at 70 °C and using H2O/EtOH 50% as extraction solvent, a correlated study on the antioxidant properties of the different portions of bergamot fruits was carried out. Specifically, this extraction technique was applied to test separately the individual portions of each fruit (albedo/pulp, seeds, and juice) belonging to the three cultivars Fantastico, Femminello, and Castagnaro.
The amounts of total flavonoid content quantified in the albedo/pulp portion showed significant differences (
p < 0.01) among the three cultivars as shown in
Figure 7. The highest content was 46.61 mg CE g
−1 in Fantastico, and the lowest one in Castagnaro (19.11 mg CE g
−1). The same trend was observed for TPC with values that ranged between 61.76 mg GAE g
−1 (Castagnaro) and 129.44 mg GAE g
−1 (Fantastico).
The antioxidant activity of the albedo/pulp portion was studied by DPPH and ABTS radical scavenging assays. Both assays revealed a higher activity than those shown by BP sample. Higher results were expressed by ABTS assay, with statistical differences (p < 0.05) among the samples with ranges from 15.35 mM TE g−1 dw (Castagnaro) and 24.54 mM TE g−1 dw (Fantastico). Lower values were found in the DPPH (2.38 mM TE g−1 dw in Castagnaro and 4.22 mM TE g−1 dw in Fantastico) without statistical differences.
The antioxidant determinations on seeds revealed significant differences for TFC, with higher amounts in Castagnaro and Fantastico (9.16 and 8.64 mg CE g−1, respectively) than in Femminello cultivars (4.59 mg CE g−1).
The distribution of TPC among the cultivars’ seeds was different compared to the albedo/pulp portion. TPC was quantified in the range of 16.57, 21.20, and 37.40 mg GAE g
−1, respectively on Femminello, Fantastico, and Castagnaro seeds. TPC in seeds was comparable with those reported by Al et al. [
42].
The free radical scavenging activity measured with DPPH assay for seed extracts did not show differences with values of about 1 mM TE g−1 dw.
The highest level of scavenging activity based on ABTS assay was determined in Castagnaro seed extract (8.69 mM TE g−1 dw), and the lowest (4.42 mM TE g−1 dw) in Femminello, exhibiting the same trend of TPC, with a high correlation (r = 0.999).
Figure 7 also showed the different amounts of total flavonoid content in tested juices. Although the highest value was found in Castagnaro (160.08 mg CE L
−1), the replications conducted, and the statistical analysis suggested that there are no statistical differences among the analyzed samples.
The phenolic content in the juice was in accordance with the amounts detected by Xu et al. [
43] and Chen et al. [
44], who analyzed respectively the juice of fifteen and twenty-seven different varieties of citrus fruit. Their contents were 78.94 mg GAE 100 mL
−1 in Femminello, 79.33 mg GAE 100 mL
−1 in Fantastico, and 98.73 mg GAE 100 mL
−1 in Castagnaro. The antioxidant activity, evaluated with the DPPH assay was more correlated to the TPC assay (r = 0.768) compared with the other assays. Cautela et al. [
45], in fact, reported relevant differences in the chemical composition and functional properties of juices obtained with different processing methods.
Bergamot portions (albedo/flavedo, seeds, juice) showed high concentrations of flavanones, including eriocitrin, neoeriocitrin, naringin, neohesperidin, melitidin, and brutieridin (
Table 7), as also reported by Walker et al. [
46].
Neoeriocitrin and naringin represent the prevalent flavonoid compounds in the albedo/pulp portion, with values that ranged between 33.19–88.91 and 31.72–58.02 mg g−1 dw, respectively. Lower content of this class of compounds was found in the seeds.
The chromatographic analysis of Bergamot Juice resulted in a high content of neoeriocitrin, naringin, and neohesperidin. They showed values different from those reported by Leporini et al. [
47], but more or less comparable with those reported by Walker et al. [
46], Da Pozzo et al. [
1], and Baron et al. [
48]. The juice of Fantastico cv. showed the highest content in neoriocitrin (196.02 ± 19.13 mg L
−1) while naringin and neohesperidin were higher in Femminello cv. Brutieridin ((hesperetin 7-(200-R-rhamnosyl-600-(30000-hydroxy-30000-methylglutaryl)-β-glucoside)) and melitidin (naringenin7-(200-R-rhamnosyl-600-(30000-hydroxy-30000-methylglutaryl)-β-glucoside)), which have anticholesterolemic activity, exhibiting statin-like properties [
49,
50], were found either in the albedo/pulp, seeds and juice of the three different bergamot cultivars showing higher concentration for brutieridin than melitidin as reported by Di Donna et al. [
51].
The two major limonoid aglycones, limonin, and nomilin, were also detected in the different parts of bergamot fruits. They are responsible for bitterness in citrus fruits. Concerning the limonoids content evaluated on individual bergamot portions, Femminello cv showed a high amount of limonin and nomilin on albedo/pulp (6.77 and 8.79 mg g
−1 dw, respectively). Bergamot seeds possess a higher content of limonin, particularly those of Castagnaro cv (13.29 mg g
−1 dw). The different content of limonin is explained by the variability due to the state of ripeness of the fruit, the variety, and the part of the fruit [
52].