**2. Results and Discussion**

### *2.1. Red Onion Chemical Properties*

The treatments with fertilizer pads, SB, SBOR and SBOP, influenced positively, but to different extents, the properties of red onions compared to the control (CTR). Pads containing orange positively affected the red onion quality, followed by SBOP and SB. These were due to the presence of organic components in the pads, as reported in previous

publications [21,22] that evidenced a great level of flavonoids in organically grown Welsh onions and red onion. Ren et al. [21] also found high amounts of phenolics, total flavonoids, and anthocyanins, as well antioxidant activities, in two different onion varieties grown under organic production. Muscolo et al. [17] evidenced a positive effect of sulphur bentonite-organic-based fertilizers on secondary metabolite (SMs) production in red onions, suggesting that sulphur bentonite organic fertilization was able to stimulate the plant's secondary metabolism, inducing the production of phytochemicals that can be useful in preserving human health. Human natural antioxidant systems, if perfectly working, are able to mitigate damage to important biomolecules, such as DNA, proteins, lipids, and carbohydrates, avoiding the insurgence of diseases [23]. The additional intake of antioxidants with the diet represents a very important way to prevent the diseases caused by oxidative stresses. There is, nowadays, a growing interest to enrich the human diet with functional foods naturally rich in antioxidant compounds. Polyphenols represent the most important natural antioxidant compounds with beneficial effects on human health [24].

Our results evidenced in red onion bulb the greatest increase in polyphenols (Table 1) in the presence of SBOR at both concentrations (low and high); SBOP also increased the quantity of polyphenols with respect to the control but less than SBOR. In contrast, an inverse trend was observed for the total flavonoids (Table 1) that increased more in the presence of SBOP than SBOR LP and HP. Anthocyanins were the highest in all fertilized red onion bulbs. Phenolic acids (Figure 1) found in the CTR and fertilized onions were caffeic and chlorogenic. Gallic acid was present only in the fertilized onions (Figure 1), while *p*-coumaric acid in the CTR and, in the greatest amount, in SBORLP. Caffeic and chlorogenic acids did not show significant differences with respect to the CTR, except for the onion fertilized with SBORHP. *p*-coumaric and gallic acids are antioxidants with diverse physiological functions that are beneficial for human health with ascertained anticancer, anti-inflammatory, and antimicrobial properties [25–27]. The mechanisms of action of polyphenols are various and complex and depend on their chemical structures. The antioxidant property of *p*-coumaric acid is ascribed to its phenyl hydroxyl group (-OH) that enables it to donate hydrogen or electrons. In vivo studies on the *p*-coumaric mechanism of action evidenced, on a rat model, that it was able to reduce basal oxidative DNA damage, inducing glutathione (GSH) and glutathione S-transferase Mu 2 (GST-M2) in colonic mucosa. Additionally, it was demonstrated that *p*-coumaric acid was capable of decreasing the expression of the inflammatory mediators, such as TNF-α and IL-6, regulating the production of cytokines [28]. Nasr Bouzaiene et al. [29] showed how the proliferation of human lung (A549) and colon (HT29-D4) cancer cells was significantly inhibited by ferulic, caffeic, and *p*-coumaric acids. These inhibitory effects were likely to be mediated by the suppression of DNA synthesis induced by the phenolic acids in MCF-7. Caffeic acid, among the phenolic acids, was found more able to block the many modulators involved in tumor progression, including NF-kB, COX-2, TNF-a, IL-6, Nrf2, iNOS, NFAT and HIF-1α, repressing cancer angiogenesis and therefore recognized as an inducer of tumor cell death and performer of cancer growth blockage [30].

**Table 1.** Total phenols (mg·GAE·g−<sup>1</sup> DW), flavonoids (mg·rutin·g−<sup>1</sup> DW), and anthocyanins (mg·cyanidin-3-glucosideg−<sup>1</sup> DW) found in red onion bulbs differently fertilized: control (CTR), sulphur bentonite (SB), sulphur bentonite-low percentage orange residue (SBOR LP), sulphur bentonitehigh percentage orange residue (SBOR HP), and sulphur bentonite-olive pomace (SBOP). Data are the mean of three replicates ± the standard error.


Means followed by different letters in the same column are significantly different (Tukey's test at *p* < 0.05).

**Figure 1.** Phenolic acids and flavonols (mg·100·g−<sup>1</sup> FW) found in red onion bulbs differently fertilized: control (CTR), sulphur bentonite (SB), sulphur bentonite-low percentage orange residue (SBOR LP), sulphur bentonite-high percentage orange residue (SBOR HP), and sulphur bentonite—olive pomace (SBOP). Data are the mean of three replicates ± the standard error. Different letters indicate significant differences at *p* < 0.05.

Anthocyanidins (Figure 2), increased in treated onions compared to the CTR. Equally, S methyl-cysteine sulfoxide and the majority of organosulphides (Table 2) increased with respect to the CTR, mostly in red onions treated with SBOR pads and particularly with SBORLP. Anthocyanidins have health-promoting effects linked with antioxidant, antiinflammatory, and anticarcinogenic properties. Their antioxidant nature was observed in all neurological diseases through MMP2, MMP3 and MMP9 metalloproteinase inhibition; reactive oxygen species generation inhibition; endogenous antioxidants modulation as superoxide dismutase and glutathione; the formation and aggregation of beta-amyloid (β-A) protein inhibition; and brain protective action through the modulation of brain-derived neurotrophic factor (BDNF), important for neural plasticity [31]. Additionally, organosulphur compounds have a well-recognized antiproliferative activity in several tumor cell lines that is mediated by the induction of apoptosis and alterations of the cell cycle. Organosulphur compounds generally act by modulating the activity of several metabolizing enzymes that activate (cytochrome P450s) or detoxify (glutathione S-transferases) carcinogens and inhibit the formation of DNA adducts in several target tissues [32]. Their low amounts found in SBORHP and SBOP treated onions can be related to the contemporary increase in other SMs with antioxidant properties. This suggests that the fertilizers used were able to influence the biosynthesis and accumulation of other SMs, evidencing that these fertilizers are capable of redirecting the metabolism to consequently regulate the production of specific bioactive constituents, as already reported by [33].

**Figure 2.** Anthocyanidins (mg·100·g−<sup>1</sup> FW) found in red onion bulbs differently fertilized: control (CTR), sulphur bentonite (SB), sulphur bentonite-low percentage orange residue (SBOR LP), sulphur bentonite-high percentage orange residue (SBOR HP), and sulphur bentonite-olive pomace (SBOP). Data are the mean of three replicates ± the standard error. Different letters indicate significant differences at *p* < 0.05.

**Table 2.** S-Methyl-L-cysteine sulfoxide (μg·g−<sup>1</sup> FW) and the relative concentration <sup>μ</sup>g·g−<sup>1</sup> FW of volatile organic compounds in the onion bulbs differently fertilized: control (CTR), sulphur bentonite (SB), sulphur bentonite-low percentage orange residue (SBOR LP), sulphur bentonite-high percentage orange residue (SBOR HP), and sulphur bentonite-olive pomace (SBOP). Data are the mean of three replicates ± the standard error.


Means followed by different letters in the same column are significantly different (Tukey's test at *p* < 0.05).

The in vitro antioxidant capacity, determined with DPPH, ABTS and ORAC (Figure 3), increased in red onion grown mainly with SBOR and SBOP than the CTR (Figure 3). Specifically, ORAC was the highest in bulbs of red onion grown with SBOR LP, while DPPH and ABTS were the highest in bulbs of red onion grown with SBOR, both LP and HP. Cavalheiro et al. [34] demonstrated an increase in the antioxidant activities in bulbs treated with organic fertilizers. The antioxidant activities are generally related to the chemical composition of the plants in terms of the typology of antioxidant compounds. Each single compound has its own biological activity with different effects on human health [35]. Flavonoids can scavenge free radicals and can form complexes with catalytic metal ions rendering them inactive. There is also evidence of an additional mechanism by which total phenols protect against oxidative stress by producing hydrogen peroxide (H2O2), which can then help to regulate immune response actions, such as cellular growth [36].

**Figure 3.** Antioxidant activities (ORAC, DPPH and ABTS) detected in red onion bulbs differently fertilized: control (CTR), sulphur bentonite (SB), sulphur bentonite-low percentage orange residue (SBOR LP), sulphur bentonite-high percentage orange residue (SBOR HP), and sulphur bentoniteolive pomace (SBOP). Data are the mean of three replicates.

Pearson's correlation (Figure 4) evidenced that the total phenols were positively and significantly correlated with ABTS (r = 0.96), DPPH (r = 0.57), and ORAC (r = 0.62); the flavonoids correlated only with ABTS (r = 0.63), while the anthocyanins correlated with DPPH (r = 0.57) and ORAC (r = 0.87). Among the single phenolic acids, caffeic acid correlated with all the antioxidant activities, gallic acid correlated with DPPH and ABTS and chlorogenic acid with DPPH and ORAC, while *p*-coumaric acid correlated only with ORAC (r = 0.85). Among the flavonoids, the flavanol quercetin correlated with DPPH and ABTS. S-methyl cysteine sulfoxide was not involved in the antioxidative system; conversely the organic volatile compounds correlated with the antioxidant activities, mostly with DPPH and ORAC and less with ABTS.


**Figure 4.** Pearson's correlations (r) between phytochemicals and antioxidant activities. The boxed dots show the significant correlations between values; the color shows the level of correlation (yellow boxed dots *p* < 0.05 and green boxed dots *p* < 0.01). The red dots indicate a negative correlation.

#### *2.2. Effects Red Onion Phytochemicals in Terms of Cell Proliferation or Cytotoxicity*

To evaluate the possible effects of the phytochemical contents of onion samples in terms of cell proliferation or cytotoxicity, in this work, the H9c2 cells, found to be closer to normal primary cardiomyocytes for their energy metabolism features, were successfully used as an in vitro cellular model [18]. The H9c2 cells were incubated with red onion samples, fertilized, and not with the different pads in a range of concentrations between 0.5 and 10 mg/mL, and the cell viability was determined 24, 48 and 72 h after treatment following the chemical reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) by mitochondrial reductases in live cells [37]. As reported in Figure 5, the fertilization of red onion with recycled sulphur bentonite pads modified the red onion samples' ability to affect the proliferation rate and/or the oxidative metabolism of H9c2 cells with respect to the 'CTR' one. No significant toxic effects on cell viability were detected in the different conditions for all the onion samples up to a concentration of 10 mg/mL, except for the 'CTR' and 'SBORLP' at the highest concentration. In the latter, the toxic effect was very strong, and it was already observed at 24 h of treatment. Furthermore, at 72 h of incubation time, a toxic effect was also observed with 'SBOP' at a low concentration.

Noteworthy is the significant increase in cell viability of H9c2 cells treated with 'SB', 'SBOR LP', and 'SBOR HP' samples as compared with the 'CTR' one, even at low concentrations and already after 24 h of treatment, which could be caused by an increase in the cells' number and/or by an improvement in the oxidative metabolism. The effect was noticeable as early as after 24 h of treatment at very low concentrations (0.5 and 1 mg/mL) and up to 72 h for the 'SB' sample. 'SBOP' pads reduced these effects, as indicated by the overall similar results obtained with the 'CTR' and 'SBOP' treatments. Red onion samples' capabilities to alter the proliferation rate and/or the oxidative metabolism were more evident after 24 h of treatment with 'SBOR HP' and after longer exposure times, 48 and 72 h, in the presence of 'SBOR LP'. Overall, these data show a positive effect on the cell viability of H9c2, possibly related to an increase in energy metabolism, in the presence of 'SB' alone or with the addition of orange residue both at low and high percentages as

compared to the 'CRT', likely due to the greatest level in bioactive compounds. To evaluate if the different red onion samples were able to protect H9c2 in oxidative stress conditions, the cells were pretreated with them for 24, 48 and 72 h before the exposure for 45 min to tert-butyl hydroperoxide (TBHP), an exogenous oxidative stress inducer. According to the treatment conditions of the previous screening, the cells were treated with two concentrations of onion samples, 0.5 and 5 mg/mL, except for 'SBOR LP', for which the concentrations of 1 and 5 mg/mL were used.

**Figure 5.** MTT assay performed on the H9c2 cell line. Cells have been treated for 24 h, 48 h and 72 h with different samples of red onions at different concentrations ranging from 0.5 mg/mL to 10 mg/mL. Data were the means ± SEM from at least 3 independent experiments under each condition and were expressed as the percentage of vehicle-treated cells. Statistical analyses were performed using Brown-Forsythe and Welch one-way analysis of variance, and mean comparisons were made using the unpaired *t*-test with Welch's correction. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, and \*\*\*\* *p* < 0.0001.

Firstly, the basal ROS levels after treatment with the onion samples for 24, 48 and 72 h were measured (Figure 6). No changes were observed in the ROS levels for all onion samples, except for an increase observed in the presence of 5 mg/mL 'SBOP' at 24 h of incubation, which returned to the basal level already at 48 h of incubation. A significant decrease of the basal ROS levels was, however, observed at the longest incubation time, with all the onion samples, albeit at different concentrations and, in particular, in the presence of 'SBOR HP' and 'SBOP' samples, at 0.5 mg/mL. Intriguingly, these same samples showed no effects at the highest concentration. On the contrary, the 'SB' and 'SBOR LP' samples showed the same effects elicited by the 'CTR' red onions, inducing a decrease of the basal ROS level at a higher concentration (5 mg/mL).

**Figure 6.** ROS detection performed on the H9c2 cell line. Cells were treated for 24 h, 48 h and 72 h with different samples of red onions at two different concentrations, and the ROS levels were measured by the DCF assay under the basal conditions and after exposure to the exogenous inducer of oxidative stress, T-BHP. Data were the, means ± SEM from at least 3 independent experiments under each condition and were expressed as the percentage of vehicle-treated cells (dotted line) or tert-Butyl hydroperoxide-treated-cells (T-BHP) (red line). Statistical analyses were performed using Brown–Forsythe and Welch one-way analysis of variance, and mean comparisons were made using the unpaired *t*-test with Welch's correction. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, \*\*\*\* *p* < 0.0001.

Next, the effects of red onion samples were measured on the ROS levels under TBHPinduced oxidative stress conditions (Figure 6). Even in this stress condition, only the longest pretreatment with the red onion samples showed an evident effect restoring the ROS basal levels or, in the case of the highest concentration of the 'SB' sample, further reducing them. Notably, 'SBORLP' showed the same capability of reducing the ROS levels to the basal ones at both concentrations used in the pretreatment. An early significant effect, at 24 h, was observed after incubation with the lower concentration of the 'CTR' extract and in the presence of 5 mg/mL of 'SB' at 48 h. The results that the different onion samples were able to decrease TBHP-induced oxidative stress could be ascribed either to a direct scavenger activity or to an enhancement of the activity of the antioxidant defenses that neutralize the ROS levels [38].

Finally, the 'SB', 'SBOR' and 'SBOP' treatments influenced, even if to different extents, the H9c2 viability and oxygen radical homeostasis with respect to the 'CTR' sample. In particular, the 'SBOR' treatments showed an interesting influence on the viability of H9c2 cells, dependent on the concentration of orange residue and time of exposure, requiring

longer exposure times, in some cases, when the percentage was lower. Notably, 'SBORLP' fertilized onions reduced the ROS levels in the basal and in oxidative stress conditions, confirming the results related to the in vitro antioxidant capacity determined by the ORAC, DPPH and ABTS assays. In particular, the results related to the oxygen radical homeostasis for the 'SBOR' treatments were not due only to the greatest content of the phenolic component present in these samples but also to the higher level of the organosulphides that have been shown to scavenge ROS and prevent damage caused by oxidative stress [39].

To assess the potential benefits of the differentially fertilized red onion samples in a pathological scenario, highly characterized primary human skin fibroblasts isolated from a healthy subject (control fibroblasts) and from a patient affected by early-onset Parkinson's disease (*parkin*-mutant) fibroblasts [40–48] were treated as previously described for H9c2 treatment. Indeed, *parkin*-mutant fibroblasts are representative of oxidative stress-correlated chronic diseases, as they display mitochondrial defects associated with deregulated reactive oxygen species (ROS) production, along with impaired energy metabolism and lipid oxidation [42]. As described in Figure 7, the incubation of control fibroblasts at low concentrations of the 'CTR' sample (0.5 and 1 mg/mL) showed an increase in the cell viability after 24, 48 and 72 h and a gradual decrease at the highest concentrations (5 and 10 mg/mL), resulting in a significant inhibition of cell proliferation. Furthermore, an increase in cell proliferation was also observed after 24 h of treatment in the presence of low concentrations of 'SBOR HP' and after 48 h and 72 h in the presence of 'SBOP'. Noteworthy, treatments with all the different onion samples at high concentrations and at long incubation times induced an inhibition of cell vitality of control fibroblasts, except for 'SBOP', which showed a protective action. In *parkin*-mutant fibroblasts, whereas the treatment with the 'CTR' sample induced a reduction in cellular viability, even at low concentrations and short incubation times, in the presence of all the other onion treatments, except for 'SBOR LP' at the highest concentrations, no change in the cellular vitality was observed. The lack of increase in the cellular vitality in the *parkin*-mutant fibroblasts, which was instead observed in control fibroblasts, could be due to the specific impairment of these cells. *Parkin*-mutant fibroblasts adapted to live in an environment characterized by a condition of oxidative stress showed a deficit in the mitochondrial biogenesis process, which could not lead to an increase in the cellular proliferation induced by the red onion samples. Finally, in the control fibroblasts, 'CTR', 'SBOR HP' and 'SBOP' onion extracts were able to increase the cellular vitality not observed in *parkin*-mutant fibroblasts. In these latter fibroblasts, the treatments with fertilized red onion samples could avoid the decrease in cellular vitality that was instead observed in the presence of the 'CTR' sample.

As described previously for the H9c2 cell line, it was evaluated if the different red onion samples were able to protect human fibroblasts in TBHP-induced oxidative stress conditions. According to the findings of the viability screening, cells were treated with 0.5 and 5 mg/mL for 24, 48 and 72 h (Figure 8A). In control fibroblasts, a decrease in the basal ROS levels was observed already at 24 h of incubation in the presence of high concentrations of 'CTR' but low concentrations of 'SB' samples. Furthermore, a decrease in the ROS basal levels was induced by 'SB' at 48 h of incubation, as well as by 'SBOR HP', 'SBOR LP', and at a higher extent, by 'SBOP'. It is possible to assume that the increase in the ROS basal level observed at 24 h of incubation in the presence of 0.5 mg/mL and 5 mg/mL of 'SBOR HP' and 'SBOP', respectively, might have induced an antioxidant enzymatic response, which, in turn, resulted in a ROS scavenger effect at 48 h of incubation. In *parkin*-mutant fibroblasts at 24 h of incubation, the 'CTR' induced a decrease at the basal ROS levels at low and high concentrations, and this effect persisted also at the highest concentrations and longest incubation times, similar to what was observed in the control cells. In addition, the decrease in the basal ROS levels was observed at 24 and 48 h of incubation in the presence of 5 mg/mL of 'SB' and at low and high concentrations of 'SBORHP' after 48 h of treatment. The scavenger effect of the 'CTR' sample, observed in *parkin*-mutant fibroblasts, especially in oxidative stress conditions, demonstrated the effectiveness of red onion already rich in bioactive compounds.

**Figure 7.** The MTT assay performed on the control and *parkin*-mutant fibroblasts. Cells were treated for 24 h, 48 h and 72 h with different samples of red onions at different concentrations ranging from 0.5 mg/mL to 10 mg/mL. Data were the means ± SEM from at least 3 independent experiments under each condition and were expressed as the percentage of vehicle-treated cells. Statistical analyses were performed using Brown-Forsythe and Welch one-way analysis of variance, and mean comparisons were made using the unpaired *t*-test with Welch's correction. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, and \*\*\*\* *p* < 0.0001.

**Figure 8.** ROS detection performed on the control and *parkin*-mutant fibroblasts. Cells were treated for 24 h, 48 h, and 72 h with different samples of red onions at two different concentrations, and the ROS levels were measured by the DCF assay in the basal condition (**A**) and after exposure to the exogenous inducer of oxidative stress, T-BHP (**B**). Data were the means ± SEM from at least 3 independent experiments under each condition and were expressed as a percentage of vehicle-treated cells (dotted line) (**A**) or tert-Butyl hydroperoxide-treated cells (T-BHP) (red line) (**B**). Statistical analyses were performed using one-way analysis of variance, and mean comparisons were made using Fisher's LSD test. \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001, and \*\*\*\* *p* < 0.0001.

Next, the effects of red onion samples on the ROS levels under TBHP-induced oxidative stress conditions were assessed (Figure 8B). In this condition, the behavior observed in the control and *parkin*-mutant fibroblasts was substantially different, mainly the long incubation time. A significant decrease was observed in the control cells at 24 h of incubation with all the treated onion samples, albeit at different concentrations, as compared with the 'CTR'. In *parkin*-mutant fibroblasts in the same conditions, a reduction of TBHP-induced oxidative stress was observed at higher concentrations of the 'CTR' and in the presence of 'SB', 'SBOR HP' and 'SBOR LP' samples. At 48 and 72 h of incubation, no effect of red onion treatment on TBHP-induced oxidative stress was observed in the control cells, except for the reduction of the ROS level in the presence of a high concentration of 'CTR' over long time of incubation, whereas a reduction at 48 h of incubation was observed in *parkin*mutant fibroblasts in the presence of a different concentration of 'CTR', 'SB' and 'SBOR HP' samples, as well as at 72 h with low and high concentrations of 'SBOR LP'. Furthermore, in the control cells, an increase in the ROS level with respect to TBHP-induced oxidative stress was observed with 0.5 mg/mL of 'SB' and with low and high concentrations of 'SBOR HP', highlighting a possible toxic effect, as already shown by the decrease in cellular viability in this condition. The scavenger effect observed in *parkin*-mutant fibroblasts and in basal and TBHP-induced oxidative stress conditions pointed out an effective protective role of red onion samples fertilized with sulphur bentonite containing orange, particularly at low concentrations with respect to the 'CTR' sample.
