# Antioxidant Activity of Sulfate Metabolites of Chlorogenic Acid
## 4 Discussion  
Phenolic acids can be found in all plant-originating food, such as grains, coffee, tea, etc. They are one of the most common ingredients in the food we eat every day. Although the human diet was not originally heavily dependent on plant-originating food like nowadays, over centuries of evolution, our bodies have 'learned' how to efficiently metabolize phenolic acids. They circulate in plasma as metabolites, mainly as \(O\)-sulfates and \(O\)-glucuronates, rather than their native forms. However, surprisingly, in the literature on this subject, very little attention has been given to the antioxidant activity of polyphenol metabolites. The papers which do discuss it often report inconclusive results; for example, quercetin 3-O-sulfate was found to retain antioxidant properties, but less efficiently than unconjugated quercetin [34]. The \(O\)-methylation of catechol B-ring hydroxyls caused a decrease in the antioxidant activity with regard to the precursor compounds. However, at pH 7.4, they maintained significant radical neutralization activity [35; 36]. Moreover, [PERSON] et al. [37] reported that the phenolic metabolites of coffee provide an effective defense against DEP (diesel exhaust particle)-induced oxidative stress by supporting its antioxidant activities. On the other hand, [PERSON] et al. [30] synthesized the metabolites of caffeic acid-4-\(O\)-sulfate, caffeic acid-4-\(O\)-sulfate and caffeic acid-3-\(O\)-sulfate and showed that monosulfate derivatives of caffeic and ferulic acids exhibited very low antioxidant activity. Nevertheless, it is worth noting that the main metabolites circulating in plasma after CGA administration are dihydrocaffeic and dihydrocrule acids, as well as their \(O\)-sulfate conjugates [12; 13; 22]. To our knowledge, there are no studies that compare the antioxidant activity of their precursors, caffeic and ferulic acids, to their hydrogenated analogues, dihydrocaffeic and dihydrocrule acid, as well as their \(O\)-sulfate derivatives. Herein, we followed [PERSON] et al. 's [32] method and synthesized sulfate monoesters of caffeic, ferulic and their hydrogenated analogues according to their pathway. The LC-MS/MS analyses allowed for the full characterization of these derivatives, which makes them reliable standards for further research. The reaction of ferulic and dihydrocrule acids with ClSO\({}_{3}\)H resulted in the formation of two monoesters, ferulic acid-4-\(O\)-sulfate and dihydroferulic acid-4-\(O\)-sulfate, since they possess only one hydroxyl group at which sulfation may occur. Synthesis of sulfate conjugates of caffeic acid resulted in a mixture of two monoesters, caffeic acid-3-\(O\)-sulfate and caffeic acid-4-\(O\)-sulfate, at a ratio of 1:1, which were separated under chromatographic conditions. The results obtained for monoesters of dihydrocaffeic acid indicated co-elution of two different monoesters under these conditions: dihydrocaffeic acid-3-\(O\)-sulfate and dihydrocaffeic acid-4-\(O\)-sulfate. Although the \(O\)-sulfation of caffeic and dihydrocaffeic acids could occur at both hydroxyl groups simultaneously, disulfates were not observed. Our results shed new light on the antioxidant capacity of the main metabolites of CGAs. Firstly, all assays used in the experiment showed that caffeic acid exhibited higher antioxidant efficiency than ferulic acid. This phenomenon has already been presented in the literature and correlated with the substitution of the hydroxyl group in the third position of the aromatic ring (caffeic acid) with a less polar methoxyl group (ferulic acid) [26; 38]. In CUPRAC and FC assays, dihydroexcaffe acid exhibited lower antioxidant activity than its precursor compound. Furthermore, the same results were observed for dihydroferulic acid in the FC assay, while in the CUPRAC assay, the antioxidant abilities of dihydroferulic acid and its precursor fertile acid were comparable. However, these two assays use different chromogenic redox reagents as well as different conditions of measurement.