# Constituents from _Ageratina pichinchensis_ and Their Inhibitory Effect on Nitric Oxide Production
## 3 Results and Discussion
### Inhibition of LPS-Induced NO Production by Compounds 1-11  
The compounds were solubilized in DMSO and evaluated at concentrations of 9.375, 18.75, 37.50, 75.00, and 150.00 uM (Table S1). Table 1 shows the effect found for the compounds at a concentration of 75 uM, highlighting that only compound **6** exhibited a cytotoxic effect at this concentration. The rest of the compounds did not reveal an important reduction in cell viability compared to the positive control (etoposide) at the concentrations evaluated (Table S2). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Compounds** & **Cell Viability (\%) a** & **NO Inhibition 75 uM (\%)** & **NO Inhibition (IC\({}_{\text{50}}\), uM)** \\ \hline
**1** & \(109.9\pm 7.16\) & \(0.95\pm 1.35\) & \(>\)75 \\
**2** & \(112.4\pm 24.08\) & \(16.75\pm 5.36\) & \(>\)75 \\
**3** & \(115.6\pm 1.58\) & \(11.98\pm 7.85\) & \(>\)75 \\
**4** & \(99.33\pm 12.39\) & \(22.63\pm 10.38\) & \(>\)75 \\
**5** & \(100.20\pm 2.95\) & \(29.77\pm 18.27\) & \(>\)75 \\
**6** & \(61.14\pm 6.31\) & \(\longrightarrow\) & \(\longrightarrow\) \\
**7** & \(104.7\pm 1.82\) & \(5.90\pm 8.35\) & \(>\)75 \\
**8** & \(103.9\pm 3.83\) & \(36.73\pm 16.93\) & \(>\)75 \\
**9** & \(110.9\pm 8.3\) & \(29.77\pm 9.37\) & \(>\)75 \\
**10** & \(121.2\pm 10.20\) & \(5.98\pm 5.22\) & \(>\)75 \\
**11** & \(101.3\pm 1.62\) & \(75.08\pm 3.07\) & \(20.55\pm 0.27\) \\ DMSO b & \(\longrightarrow\) & \(\longrightarrow\) & \(\longrightarrow\) \\ Indomethacin c (84 uM) & \(\longrightarrow\) & \(65.93\pm 6.03\) & \(54.69\pm 10.34\) \\ Etoposide d (68 uM) & \(42.02\pm 4.23\) & \(\longrightarrow\) & \(\longrightarrow\) \\ \hline \hline \end{tabular}  
* Cell viability at 75 uM, b blank control; c positive control for the NO production assay; d positive control for cytotoxicity against RAW 264.7 cells.  
\end{table} 
Table 1: Percentage of inhibition of NO production and cell viability in RAW 264.7 macrophages at a concentration of 75 uM for compounds 1–11. Compound **6** was not evaluated to determine its effect on NO production, because it only promoted a survival rate of 61.14% at a concentration of 75 uM. \(\beta\)-eudesmol is a sesquiterpene that has already been isolated from several species; to mention a few, there are _Atracylodes lancea_[58], _Zinggher Zerumbet_[59], _Gutteria friesiana_[60], and _Muraya tetr genera_[61]. Likewise, several biological effects have been demonstrated for this secondary metabolite, for which we can highlight its anti-inflammatory effect, tumor suppressor effect, and anticancer effect [62; 63; 64]. The above agrees with the inhibitory effect that compound **6** had on cell viability. Therefore, we proceeded to evaluate the inhibitory effect of compounds **1-5** and **7-11** on the production of NO in RAW 264.7 macrophages previously stimulated with LPS for 24 h after 2 h of incubation. The results revealed that the NO level was increased in RAW 264.7 cells with LPS stimulation, and this effect was significantly decreased by treatment with the compounds. The inhibition percentages and IC\({}_{50}\) values are shown in Table 1. It was observed that compound **11** was the best inhibitor of NO secretion, with an IC\({}_{50}\) of 20.55 \(\pm\) 0.27, and it competed with indomethacin, which IC\({}_{50}\) value was 54.69 \(\pm\) 10.34. Regarding specifically the activity displayed by compound **11**, it was observed that it presents a dose-dependent effect (Figure 3a), even improving the inhibition exhibited by the positive control indomethacin. Consequently, its relationship with the proinflammatory cytokine IL-6 and the nuclear factor NF-k\(\beta\) was determined. The results indicate a statistically significant inhibition of interleukin IL-6; likewise, NF-k\(\beta\) activation is also inhibited (Figure 3b,c). This result suggests that compound 11 exhibits a proinflammatory effect which application could represent alternatives for the treatment of diseases such as rheumatoid arthritis, chronic hepatitis, pulmonary fibrosis, sepsis, and hypertension, among others [65; 66; 67; 68].