# Constituents from _Ageratina pichinchensis_ and Their Inhibitory Effect on Nitric Oxide Production
## 4 Conclusions  
For the first time, we report the anti-inflammatory effect of _A. pichinchensis_, specifically the ethyl acetate extracts of leaves and ethanolic extracts of flowers, which exhibited in vivo anti-inflammatory effects, significantly inhibiting TPA-induced edema at a dose of 0.01 per ear. Additionally, the inhibitory effect on NO production in an in vitro inflammatory model of compounds **1**-**5 and **7**-**11**, isolated from the leaves and flowers of _A. pichinchensis_, was evaluated. Among them, betuletol 3-O-\(\beta\)-glucosidase (**11**) inhibited NO production in a dose-dependent manner; in particular, treatment with 75 uM significantly decreased NO production by 75.08 \(\pm\) 3.07% (IC\({}_{50}\) = 20.55 \(\pm\) 0.27) compared to indomethacin, which inhibition was 65.93 \(\pm\) 6.03% (IC\({}_{50}\) = 54.69 \(\pm\) 10.34). The nitric oxide inhibition effect associated with the upregulation of proinflammatory cytokine (IL-6) expression and inhibition of NF-k\(\beta\) activation indicates that compound **11** may be useful as a therapeutic agent in the treatment of inflammation-related diseases caused by macrophage overactivation. Therefore, the effect attributed to _A. pichinchensis_ species is due to compound **11**, and these findings corroborate the effect attributed to the species in traditional medicine.  
Figure 3: Effect of compound **11** on (**a**) NO, (**b**) IL-6, and (**c**) NF-k\(\beta\) of RAW 264.7 macrophages activated with LPS. Data were expressed as the mean \(\pm\) SD values of experiments in three independent assays. *** \(p\) < 0.01, *** \(p\) < 0.001 vs. LPS-treated cells, and *** \(p\) < 0.001 vs. vehicle control.  
**Supplementary Materials:** The following supporting information can be downloaded at [[https://www.mdpi.com/article/10.3390/app14093942/s1](https://www.mdpi.com/article/10.3390/app14093942/s1)]([https://www.mdpi.com/article/10.3390/app14093942/s1](https://www.mdpi.com/article/10.3390/app14093942/s1)): Tables S1 and S2. Effect of compounds **1-11** at different concentrations on cell viability and NO production in RAW 264.7 cells. Figures S1-S12 and S15-S18. \({}^{1}\)H and \({}^{13}\)C NMR spectra of compounds **1-6** and **9** and **10**. Figures S13 and S14. \({}^{1}\)H NMR spectra of compounds **7** and **8**. Figures S19-S22. One-dimensional and two-dimensional NMR spectra of compound **11**. Figure S23. Mass-mass spectrum (FAB+).  
**Author Contributions:** Conceptualization, S.M.-B. and F.C.-S; formal analysis, A.R.-E., A.R.-G. and J.G.-C.; funding acquisition, F.C.-S., J.J.A.-F. and L.A.; investigation, M.S.-R. and S.M.-B.; methodology, J.J.A.-F., A.R.-E., A.G.-A., J.G.-C. and M.S.-R.; resources, F.C.-S. and L.A.; supervision, L.A., A.R.-G., F.C.-S. and M.S.-R.; writing, S.M.-B., J.J.A.-F. and M.S.-R.; original draft, A.R.-G., L.A. and J.J.A.-F.; writing--review and editing, S.M.-B., [PERSON]-R., [PERSON] and F.C.-S. All authors have read and agreed to the published version of the manuscript.  
**Funding:** This research was supported in part by Conahcyt (Grant CB 240801).  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy.  
**Acknowledgments:** The authors thank the Laboratorio Nacional de Estructura de Macromolecules (Conahcyt 279905) for the spectroscopic and mass analyses. The authors thank [PERSON], curator of the HUMO Herbarium, for his support with the taxonomic identification.  
**Conflicts of Interest:** The authors declare no conflicts of interest.