# Antioxidant Activity of Sulfate Metabolites of Chlorogenic Acid
## 3 Results
### Characterization of Antioxidant Activity of Synthesized Metabolites  
We were not able to fully examine the antioxidant activity of metabolites of phenolic acids because of the lack of commercially available standards. To evaluate the antioxidant properties of the synthesized compounds, we used FC and CUPRAC (summarized in Table 2) and DPPH\(\bullet\) neutralization assays (summarized in Table 3). FC and CUPRAC assays are based on single-electron transfer (SET) and reflect the reducing capacity of the sample. As a reference compound, we used gallic acid and Trolox, while the antioxidant  
Figure 4: (**A**) TIC of mixture after synthesis of caffeic acid sulfate; (**B**) chromatogram of caffeic acid sulfate obtained after purification; (**C**) fragmentation spectra of caffeic acid sulfate. Figure 5: (**A**) TIC of mixture after synthesis of dihydrocaffeic acid sulfate; (**B**) chromatogram of dihydrocaffeic acid sulfate obtained after purification; (**C**) fragmentation spectra of dihydrocaffeic acid sulfate. activity of the analyzed compound was expressed as a GAE and TE. The higher the value of TE and GAE, the better the reducing capacity of the analyzed compound. The DPPH\(\bullet\) test is based on the neutralization of a synthetic radical. This mechanism in a polar and hydrogen bonding solvent such as methanol is a two-step process involving the sequential loss of an acidic proton followed by an electron transfer (SPLET) [33]. The results are expressed as a scavenging effect (%) corresponding to the percentage of neutralized DPPH\(\bullet\) as well as TE. Table 2 shows the comparison of the reducing capacity of GCA metabolites according to CUPRAC and FC assays. As is shown in both assays, caffeic acid possesses a higher reducing capacity in comparison to ferulic acid. In the CUPRAC assay, caffeic acid and ferulic acid corresponded to 0.77 and 0.33 mM TE, respectively, and in the FC assay, 86 and 80 ppm GAE, respectively. Similar results were obtained with the DPPH\(\bullet\) assay; caffeic and ferulic acids neutralized about 21% and 14% of DPPH\(\bullet\), respectively, corresponding to approximately 0.3 and 0.2 mM Trolox (Table 3). The hydrogenated analogue of caffeic acid--dihydrocaffeic acid--demonstrated lower reducing abilities in both the FC and CUPRAC assays than its precursor compound. Surprisingly, in the FC test, ferulic acid and its hydrogenated analogue showed a similar reducing capacity, unlike in the CUPRAC assay, where ferulic acid showed a higher reducing power than dihydroferulic acid. Interestingly, the results obtained using the DPPH\(\bullet\) assay are contradictory; the hydrogenated analogues of caffeic and ferulic acids were shown to have higher ability to neutralize DPPH\(\bullet\) via their antioxidant activity. The antioxidant activity of dihydrocaffeic acid was 1.8 times higher than that of the precursor caffeic acid, while the antioxidant activity of dihydroferulic acid was 1.7 times higher compared to precursor ferulic acid. This shows that the reduction of carbon-carbon \begin{table}
\begin{tabular}{c c c} \hline
**Compound** & **CUPRAC** & **Folin-Ciocalteu** \\
**TE (mM)** & **GAC (ppm)** \\ \hline Caffeic acid & 0.77 \(\pm\) 0.02 & 86.87 \(\pm\) 7.05 \\ Caffeic acid monosulfate 1 & 0 & 4.37 \(\pm\) 0.22 \\ Dihydrocaffeic acid & 0.49 \(\pm\) 0.02 & 72.07 \(\pm\) 2.00 \\ Dihydrocaffeic acid monosulfate 2 & 0 & 2.19 \(\pm\) 0.11 \\ Ferulic acid & 0.33 \(\pm\) 0.04 & 80.01 \(\pm\) 4.77 \\ Ferulic acid-4-O-sulfate & 0 & 14.29 \(\pm\) 0.71 \\ Dihydroferulic acid & 0.39 \(\pm\) 0.05 & 66.58 \(\pm\) 2.48 \\ Dihydroferulic acid-4-O-sulfate & 0 & 15.20 \(\pm\) 0.76 \\ \hline \end{tabular} 1 Mixture of caffeic acid-3-O-sulfate and caffeic acid-4-O-sulfate at a ratio of 1:1. 2 Mixture of dihydrocaffeic acid-3-O-sulfate and dihydrocaffeic acid-4-O-sulfate at an unknown ratio.  
\end{table}