## 4 Discussion  
It has been suggested in previous studies that the antioxidant ability of a series of compounds is strongly affected by pH, which influences the relative abundance of the corresponding acid-base forms [36]. Acid-base equilibria have been reported to strongly affect the UV-vis spectra of several phenolics, including flavonoids. This is observed as significant changes in both the shapes and the intensities of the bands owing to variation in the pH values [37]. The absorption and fluorescence spectra of luteolin have been reported to strongly depend on pH [38]. Therefore, the corresponding acidity constant values are an essential parameter in predicting the functional mechanism of proteins in chemistry and biochemistry. We calculated here the acidic constants of all the possible sites of quercetin, luteolin and L-ascorbic acid. According to our results, the increase in the pH of the medium at values higher than 3 prompted the proton removal from the protonated carbonyl group (i.e., from H\({}_{n+1}\)L\({}^{+}\) in Equation (3)), leading to the formation of an electroneutral compound (generically H\({}_{n}\)L). In contrast, in the narrow pH range of 8-10, in an aqueous solution of quercetin and luteolin, six or five species can simultaneously exist, respectively. These ion molecular forms are in dynamic equilibrium in the aqueous solution, depending on the acidity of the medium. The experimental assignment for the constants of quercetin and luteolin could not be assessed. Although some efforts have been carried out in this way, a systematic and detailed study factoring in all the possible acid forms has not been carried out, and the computational evaluation is still debated [31; 35; 36; 37; 38; 39]. On the basis only of their structures and, in particular, on the number of phenolic -OH groups, the existence of five and four pK\({}_{\text{a}}\) values for quercetin and luteolin, respectively, can be hypothesized. However, for quercetin, in previous works dealing with its acidity constants, sets of only two [40; 41], three [41; 42; 43; 44] and four [43] pK\({}_{\text{a}}\) values have been reported. Interestingly, in the work of [PERSON] and colleagues [36], through a combined experimental and computational approach, a complete set of constants has been reported for quercetin. In particular, the deprotonation order was supposed to be 4', 7, 3, 3' and 5. Luteolin is structurally analogous to quercetin--except that it has four phenolic -OH groups, as there is no -OH group on ring C. Indeed, the \(K_{\text{a1}}\) and \(K_{\text{a2}}\) of quercetin and those of luteolin are the same. Hence, we can suppose that the first two protolysis equilibria involve -OH groups that are less influenced by the deprotonation on the C ring. Only the first acidity constant has been experimentally measured in an aqueous medium [14], and the -OH group in position 4' has been theoretically calculated as the most acidic site [38]. In the case of L-ascorbic acid, the monoanionic form, derived from the deprotonation of the hydroxy group linked to carbon 3 [O(3)-H] (see Figure 1c), is particularly stable thanks to the delocalization of the negative charge on the oxygen atoms at positions 1 and 3. The second deprotonation occurs at higher pH values, and the corresponding constant is difficult to measure in aqueous media [45; 46; 47]. An analysis of the acidity constants in the literature shows major variation among the published values. Furthermore, most of the reported pK\({}_{\text{a}}\) values were acquired using alcohol and water combinations; thus, there is limited knowledge on pK\({}_{\text{a}}\) values found in aqueous solutions. Therefore, the acidic constant values determined in this work are not directly comparable with the others in literature, owing to differences in the experimental conditions (Table A1)--except in [11] and [14], which have reported values that are in excellent agreement with our data. Only a qualitative comparison can be carried out with the values obtained in other works, which reveals acceptable agreement.