## 3 Results and Discussion
### Anti-Inflammatory Activity of the Extracts  
Different concentrations (10 to 60 \(\upmu\)g/mL) of supercritical extracts were initially evaluated for their cytotoxicity on THP-1/M cells by the MTT method, with the aim of determining the range of concentrations that can be applied for the assay. The results indicated that 40 \(\upmu\)g/mL for heather and heather + marigold combined extracts presented no cytotoxicity, and a value of 20 \(\upmu\)g/mL for marigold extracts. The activation of THP-1/M cells was carried out with the addition of LPS to the medium. These LPS treated cells showed, after an incubation period of 24 h, an important increase in the release of all pro-inflammatory cytokines studied (TNF-\(\upalpha\), IL-1\(\upbeta\) and IL-6), compared to non-activated controls (Figure 1). These activated cells were considered as positive controls for all the cytokines tested. When the activation of THP-1/M cells was carried out in presence of heather (40 \(\upmu\)g/mL) or marigold (20 \(\upmu\)g/mL) extracts, as well as the extracts from the combined heather + marigold (50:50) extraction (40 \(\upmu\)m/mL), no variation was observed in TNF-\(\upalpha\) secretion, compared with levels obtained in the absence of extracts (positive controls). Regarding IL-1\(\upbeta\), only heather extracts containing the highest concentration in triterpen acids (E3-2), especially oleanolic acid (see Table 3), significantly decreased the IL-1\(\upbeta\) secretion. With respect to IL-6, most extracts produced a significant decrease in this interleukin secretion. Thus, heather extracts E1-S2, E2 and E3-2 presented a decrease in 30% of the release of this cytokine. However, it is should be noted that although extracts E2 and E3-2 possess an important quantity of triterpen acids, mainly oleanolic acid, extract E1-S2 only presented a very small concentration in these components. Besides, marigold extract E1-S1 produced the highest inhibition in IL-6 release (45%), meanwhile other extracts with a higher content in triterpen acids (E3-2) did not inhibited this cytokine secretion. Data obtained from both plants (50:50) combined extraction did not present a synergistic effect, regarding anti-inflammatory activity. \begin{table}
\begin{tabular}{c c c c c} \hline \hline  & \multicolumn{2}{c}{**Oleanolic**} & \multicolumn{2}{c}{**Ursolic**} \\ \hline
**Run** & **Experimental** & **Theoretical** & **Experimental** & **Theoretical** \\ \hline E1-S1 & 13.73 \(\pm\) 1.22 & 26.53 & 21.41 \(\pm\) 2.10 & 54.32 \\ E1-S2 & 6.23 \(\pm\) 0.25 & 27.21 & 12.70 \(\pm\) 0.52 & 15.06 \\ E2 & 43.64 \(\pm\) 2.32 & 34.67 & 58.12 \(\pm\) 3.21 & 20.11 \\ E3-1 & 12.87 \(\pm\) 1.14 & 31.29 & 25.19 \(\pm\) 1.22 & 16.79 \\ E3-2 & 36.35 \(\pm\) 0.56 & 43.68 & 41.95 \(\pm\) 1.87 & 29.57 \\ \hline \hline \end{tabular}
\end{table} 
Table 4: Concentration (mg/g extract) of oleanolic and unsolic acid in the extracts obtained from the combined extraction of heather and marigold (50:50) by SFE. Theoretical values are expressed as the arithmetic mean of the experimental concentration in the extracts obtained from each individual plant. In addition to this, two alternative extracts, obtained from mixing the individual heather and marigold extracts (50:50 heather + marigold extract), were assayed (40 u/mL). The extracts selected for the mixture were E2 and E3-2, due to these fractions containing the highest concentration of triterpene acids in the heather + marigold (50:50) combined extraction (see Table 4). Results obtained (data not shown) indicated no differences in cytokine secretion between these new extracts and those obtained from the heather + marigold combined extraction. For both extraction conditions, that is, E3-2 and E2, a similar TNF-\(\alpha\), IL-1\(\beta\) and IL-6 secretion was observed for all samples (extracts from combined extraction or mixed extracts).