# Phytochemical Analysis of _Symphytum officinal_ Root Culture Extract
## 3 Results
\begin{table}
\begin{tabular}{c c c} \hline \hline
**Samples** & **Concentration, \(\mu\)g/mL** & **Anti-Inflammatory Activity, \%** \\ \hline \multirow{3}{*}{I} & 10.0 \(\pm\) 0.5 \({}^{\mathrm{a}}\) & 60 \(\pm\) 2 \({}^{\mathrm{a}}\) \\  & 20.0 \(\pm\) 0.7 \({}^{\mathrm{b}}\) & 75 \(\pm\) 3 \({}^{\mathrm{b}}\) \\  & 30.0 \(\pm\) 0.8 \({}^{\mathrm{c}}\) & 75 \(\pm\) 3 \({}^{\mathrm{b}}\) \\ \hline \multirow{3}{*}{II} & 10.0 \(\pm\) 0.6 \({}^{\mathrm{a}}\) & 63 \(\pm\) 2 \({}^{\mathrm{a}}\) \\  & 20.0 \(\pm\) 0.8 \({}^{\mathrm{b}}\) & 75 \(\pm\) 3 \({}^{\mathrm{b}}\) \\ \cline{1-1}  & 30.0 \(\pm\) 0.8 \({}^{\mathrm{c}}\) & 75 \(\pm\) 2 \({}^{\mathrm{b}}\) \\ \hline \hline \end{tabular} I-extract samples of _Symphytum officinal_, containing type I polymer. Data presented as a mean \(\pm\) SD (\(\mu\) = 3). Values in columns followed by the same letter do not differ significantly (\(p\) > 0.05).  
\end{table} 
Table 4: The results of determining anti-inflammatory activity of _Symphytum officinal_ extract samples. Figure 4: Mass spectrum at the top of the chromatographic peak at 5.547 min, corresponding to 7-Angeloylheliotridine extracted from _Symphytum officinal_ (reference mass ions [M + H]\({}^{+}\)\(m\)/ \(z\) 136 and \(m\)/\(z\) 124.03). Figure 5: Mass spectrum at the top of the chromatographic peak 5.58 min, corresponding to 9-angeloylretronectin extracted from _Symphytum officinal_ (reference mass ions [M + H]\({}^{+}\)\(m\)/ \(z\) 138 and \(m\)/\(z\) 126). Considering the importance of cyclooxygenaseases in the inflammatory process, the possibility of inhibiting COX-1 and/or COX-2 with comfrey extract samples was studied. First, HUVEC was stimulated with combinations of IL-1 and comfrey, and COX enzymatic activity was determined using a fluorescence-based assay that measures COX-1 and COX-2. Comfrey extract samples significantly reduced IL-1-induced but not constitutive COX activity; since it is COX-2 that is induced by IL-1, while COX-1 is expressed constitutively, it can be concluded that comfrey selectively affects the activity of COX-2. Western blot of the same samples using an antibody specific for COX-2 confirmed that COX-2 protein levels were induced by IL-1 and decreased by comfrey extract samples. In addition, when reanalyzing samples, COX-2 mRNA was lower in cells treated with comfrey extract samples compared to control. The potential COX-2 selectivity of comfrey was further tested using recombinant COX-1. While the nonspecific inhibitor diclofenac readily suppressed the enzymatic activity of COX-1, comfrey extract samples did not. Finally, unstimulated HUVECs (which express only constitutively present COX-1) were treated with either the nonspecific COX inhibitor diclofenac, the specific COX-1 inhibitor SC-560, or comfrey extract samples. This COX activity, which is attributed exclusively to COX-1, was inhibited by diclofenac and SC-560, but not by comfrey. Thus, we concluded that comfrey extract samples do not suppress the enzymatic activity of both COX isoforms but act on COX-2, preventing its mRNA and protein synthesis, and therefore, unlike diclofenac, act indirectly through inhibition of NF-kB but specifically inhibit COX-2. It was found that comfrey extract samples were characterized by a high antioxidant status; the antioxidant activity of the samples was 235.00 mg AA/g. Both methods (amperometric and DPPH) showed similar results. The results of determining the antioxidant activity of the _Symphytum officinal_ extract samples are presented in Table 5.