# Antioxidant Activity of Sulfate Metabolites of Chlorogenic Acid
## 4 Discussion
As was mentioned before, the FC assay is based on the SET mechanism in a basic medium (adjusted with a sodium carbonate solution to pH\(\sim\)10), while the CUPRAC assay is based on the SET mechanism in neutral pH (acetate buffer, pH = 7). The pH values have an important effect on the reducing capacity of antioxidants. In particular, at basic pH, proton dissociation of phenolic compounds may enhance their vulnerability to reduction [39]. The above studies highlighted the poor correlation of CUPRAC and FC assays due to the different reduction potentials, which are well known for CUPRAC, while the exact potential of the Folin reagent with a presumably higher potential is not definitely known. Consequently, the authors noted that the FC assay was originally thought to be specific for phenolic compounds and should oxidize samples more efficiently than the CUPRAC assay [40]. Interestingly, the DPPH\(\bullet\) assay results show the opposite: hydrogenated analogues and dihydroexcaffeic and dihydroferulic acids exhibited higher antioxidant activities than their precursor compounds. However, it has already been highlighted that due to the different mechanisms involved, analytes can react differently in different assays [28]. The results obtained during the radical neutralization experiment show that the reduction of the carbon-carbon double bond in hydroxcinnamic acid derivatives considerably enhanced the antioxidant activity with respect to the precursor compounds. Since changes in antioxidant structure result in changes in electron delocalization possibilities, the antioxidant activity might vary. Furthermore, the structure of cinnamic acid derivatives is characterized by a conjugated double-bond system, which is a more stable structure than that of their hydrogenated analogues. In parallel, changes in hydrophobicity could significantly influence the reducing capacity of compounds. Therefore, for a more precise estimation of the antioxidant activity of a given sample, one should use at least two distinct methods based on different reaction mechanisms. With regard to sulfate esters of caffeic and ferulic acids, our results are in line with the results reported by [PERSON] et al. [30]. We provide evidence that the sulfation of hydroxyl groups results in inhibition of antioxidant properties with respect to precursor compounds [30]. Similar results were obtained for dihydroexcaffeic and dihydroferulic acids and a significant decrease in the antioxidant properties of the sulfate esters was observed. It is generally believed that \(O\)-sulfation reduces or even inhibits reducing efficiency due to the modification of the hydroxyl groups which are responsible for the antioxidant activity of the phenolic compounds. Firstly, the \(O\)-glucuronidation and \(O\)-sulfation processes modify the hydrophobicity and the possibilities of electron delocalization, so the antioxidant activity of conjugates might be different from that of the precursor compounds. Secondly, considering the model of the aromatic sulfates (Figure 6A), the main reason for the reduced antioxidant activity of caffeic acid and dihydroexcaffeic acid sulfates can be noted. The ortho-position of the sulfate moiety favors the intramolecular H bond and, in consequence, stabilizes the phenol (Figure 6B). This effect has been widely discussed in the literature [41]. Considering ferulic acid and dihydroferulic acid sulfates, the inhibition of antioxidant activity is caused by the lack of a free -OH group. This means that the reducing capacity of phenolic acids, after the metabolic process, decreases due the conjugation of hydroxyl groups. Consequently, our results highlight the importance of the presence of unsubstituted hydroxyl groups in the antioxidant activity of phenolic acids and their metabolites.