## 4 Discussion
Anthocyanin (delphinidin 3-O-sambubioside and cyanidin 3-O-sambubioside) are one of the flavonoids with the greatest presence and activity [44], giving several interesting properties in _H. sabdariffa_: An increase in anthocyanin content causes an increase in activity against free radicals; this metabolite contributes 51% of the Roselle antioxidant capacity [45]; they may potentially prevent diseases; give the color of the calves, dark calyces have more anthocyanin than light calyces, but whites or greens lack these flavonoids [46], reason why this compound have been studied extensively [47]. Although white Roselle calves had no anthocyanin and tannin content, the antioxidant activity of its extract could be due to the presence of phenolic acids and other polyphenols [34]. Also, tannins are valuable compounds with antioxidant properties, of which they have been identified in aqueous extracts of _H. sabdariffa_, chlorogenic acid [48], gallic acid [49], and the Hibiscus protocatechuic acid [40]. Moreover, it was reported that the extract of Roselle was found to be very high in ascorbic acid content or ascorbate, which is a well-known natural antioxidant [50], and caffe acid [43]. The prevention of genotoxic damage by _H. sabdariffa_ extracts was evaluated using the comet assay. This is a sensitive and rapid method for detecting DNA strand breakdown in individual cells, and have different applications, including human biomonitoring [51]. In this method DNA lysis and electrophoresis are performed using acridine orange for staining the DNA, resulting in an image that looks like a \"comet\" with a distinct head consisting of intact DNA, and tail, which contains damaged or broken pieces of DNA [51]. Our results showed that _H. sabdariffa_ extracts, tested at polyphenol concentration of 13.019 mg GAE/g dw for the red, and 12.953 mg GAE/g dw for white varieties, reduced DNA damage and reversed damage at the cellular level, as evidenced by the tail lengths of 9.98 um and 10.15 um, respectively. These lengths were shorter than the 15.25 um of the sample with mutagen EMS (positive control), but close to the 9.3 um of the sample without mutagen (negative control). Also, Hibiscus anthocyanins quenched DPPH free radicals, reduced the cytotoxicity induced by tertbutyl hydroperoxide [52] and to attenuate hepatotoxicity in rats as well as to protect DNA damage [53], and cytotoxicity [54]. Finally, the effects of acoustic ultrasound have been studied for many years. It is well accepted that acoustic cavitation result in the creation, expansion, and sudden implosive collapse of bubbles, sometimes referred to as hot spots [55; 56], with shock waves of several hundred atmospheres and around 5000 \({}^{\circ}\)K [57]. Near of the surface of the plant particles the collapse generates a high-speed jet of liquid [55]. Many of the bioactive compounds are located within the cell wall of vegetables, so that the efficiency of the UAE methods in their extraction would be due to the physical impacts over cell wall. In Figure 3 we present a scheme on the effect of ultrasonic cavitation on the cell wall and the extraction of metabolites. The highly recalcitrant structure (Figure 3a) of lignocellulosic (cellulose, lignin and hemicellulose) creates limitations for penetration of solvents [58]. The acoustic cavitation causes disruption and damage of the cell walls (Figure 3b) and provides continuous circulation of solvent into the plant particles, continuous solvent mixing and sometimes particle size reduction, and in addition many hollows are opening [59; 60]. The disruption of the cell wall and the penetration of the solvent enhances the extraction and solubilization of bioactive compounds (non-structural components). UAE is an advanced method of compound extraction, contemplate as a green and sustainable process. It is a clean method that allows high yields with low solvent consumption (generally safe (GRAS) solvents), and short extraction times, therefore lower energy consumption than conventional methods.