## 3 Results and Discussion
### Effect of Thermal Processing on the Antioxidant Capacity of Broccoli Extracts
All extracts, with the exception of the air-fried broccoli and the water remaining after steaming broccoli, were more efficient in protecting DNA from oxidative damage than the standard Trolox at a concentration of 30 mg/mL. The high level of DNA protection of water remaining after boiling broccoli is consistent with its high antioxidant potential (Table 3) and its high content of polyphenols (Table 1). Polyphenols not only act as antioxidants, but they also have the ability to act as chelating agents and form strong complexes with iron [84]. This lowers the concentration of iron ions in the Fenton's reagent and consequently reduces the concentration of hydroxyl radicals and the extent of oxidative damage. The high level of plasmid DNA protection by water remaining after blanching is probably due to the presence of hydrophilic bioactive components, which were not analyzed in this study. Although the air-fried broccoli exhibited high antioxidant potential, it showed a lower level of preservation of the supercoiled DNA form. This could be due to the high content of endogenous H\({}_{2}\)O\({}_{2}\) in the air-fried broccoli (Figure 1). Namely, H\({}_{2}\)O\({}_{2}\), if not adequately neutralized, can diffuse into assay systems and participate in Fenton-type reactions, producing hydroxyl radicals that damage DNA. Additionally, antioxidant assays are often non-specific and measure overall electron- or hydrogen-donating capacity, not necessarily protection in a biological context. To the authors' knowledge, this is the first study to investigate the effect of thermal processing of vegetables on the potential to preserve plasmid DNA structure. Thermal treatments such as boiling, blanching, steaming, air-frying, and pan-frying, disrupt the plant cell wall matrix to a different level, which reflects on the release, degradation, or transformation of bioactive compounds. The extensive exposure of plant tissues to hot water, combined with prolonged cooking time, contributes to the breakdown of cell walls and the degradation of bioactive compounds, resulting in greater losses of these constituents [85]. Indeed, as shown in Table 1, the share of larger polyphenols and tannins in broccoli was markedly lower in BO, BOW, BL, and BLW samples, suggesting their decomposition to the oligomers and/or monomers. This has also been discussed in the review of [PERSON] et al. (2006) [86]. In addition, enzymes can be released from the cell wall and denatured by heat [87]. Cellulaases are reportedly more stable at high temperatures than other plant cell wall degrading enzymes [88]. The inhibitory activity of plant polyphenols toward gastrointestinal enzymes, such as \(\alpha\)-amylase, \(\alpha\)-glucosidase, and lipase, can also be regulated by cooking conditions [89]. This is due to the fact that different cooking treatments released different types of polyphenols, resulting in different inhibitory enzyme activities. During cooking, the concentration of polyphenics typically decreases because they are polymerized and then oxidized by polyphenol oxidases. These oxidized forms (o-quinones) bind covalently to nitrogen compounds [90]. Due to their stronger molecular  
Figure 4: Effect of thermal processing on the potential of broccoli extracts to protect plasmid supercoiled DNA from damage caused by [PERSON]’s reagent. The results are expressed relative to the concentration of the supercoiled DNA of an intact plasmid. (**A**) The 1.5% agarose gel with three distinct plasmid conformations (supercoiled, linear, and nicked). (**B**) The percentage of preserved supercoiled DNA conformation after exposure to the [PERSON]’s reagent relative to the intact plasmid, alongside the corresponding DNA mass calculated from the standard curve, expressed in nanograms. A band densitometry was performed using the ImageJ (Fiji) software, version 2.2.0. Values represent the average \(\pm\) standard deviation of three biological replicates.