# Constituents from _Ageratina pichinchensis_ and Their Inhibitory Effect on Nitric Oxide Production
## 3 Results and Discussion
### Chemical Composition of Anti-Inflammatory Extracts  
The ethyl acetate (leaves) and hydroalcoholic (flowers) extracts obtained from \(A\). _pichinchensis_ were subjected to successive purification processes using the column chro  
Figure 1: Anti-inflammatory effect of in vivo extracts of leaves and stems of \(A\). _pichinchensis_. F-HA: ethanol:H\({}_{2}\)O (95:05 \(v/v\)): hydroalcoholic ethanolic extract of flowers; L-EA: ethyl acetate extract of leaves; L-MeOH: methyl extract of leaves; S-EA: ethyl acetate extract of stems; S-MeOH: methyl extract of stems. Data are expressed as mean \(\pm\) SD values of experiments in three independent assays. Significance was determined using ANOVA followed by [PERSON]’s multiple comparisons test. ***\(p\) < 0.001, ns: not significant vs. indomethacin-treated extracts, and ***\(p\) < 0.001 vs. vehicle acetone. matography technique and normal phase silica gel, which led to the isolation of pure compounds that were identified by analysis of their spectroscopic data and comparison with data from the literature. All the identified compounds are known and have been reported in different species of the _Ageratina_ genus. At the extract level, different biological effects have been demonstrated. From the ethyl acetate extract (leaves), the following were identified: \(O\)-methylenccalinol **(1)**[10; 35], encecalin **(2)**[36; 37], equipatoriochromene **(3)**[38], 6-acetyl-8-hydroxy-2,2-dimethylchrome **(4)**[33; 40], 3, 5-diphenyl-4-hydroxyacetophenone **(5)**[41; 42], (+)-\(\beta\)-eudesmol **(6)**[43; 44], espeletone (7)**[40], dehydrogenase **(8)**[41; 42], encecalinol **(9)**[6; 35; 45], and 5-acetyl-3\(\beta\)-angeloyloxy-2\(\beta\)-(1-hydroxisyopropyl)-2,3-dihydrobenzofuran **(10)**[6; 10]. In the hydroalcoholic extract (flowers), in addition to some compounds **(1, 2, 5, and 6)**, betuletol 3-\(O\)-\(\beta\)-glucoside **(11)** was identified [46; 47]. Compounds **(3, 4, and 11)** were isolated from this plant for the first time, and we investigated the abilities of compounds **1-11** (Figure 2) to inhibit NO production. Figure 2: Compounds isolated from aerial parts of _A. pichinuchensis._  
Compound **3** has been reported in the species _Centaurea solstitialis_ L., _Caccinia macronthera_ Brand var. _macranthera_, _Calea sernata_, _Encelia farinose_, _Helianthus annuus_, and _Pipper mollicommun_ [PERSON] [36; 52; 53; 54; 55; 56]. Regarding compound **4**, it has been reported in the species _Titinia diversifolia_ and _Ageratum houtsonianum_[38; 57], while compound **11** has been reported in the species _Arnica montana_[46; 47]. The compounds identified in our study have been widely described chemically by different authors; however, there is little information on the chemical characterization of compound **11**, and, therefore, we will provide more information on its structural elucidation in this study. Compound **11** was obtained as a yellow-colored amorphous solid. The elucidation of its chemical structure involved a comprehensive analysis using \({}^{1}\)H, \({}^{13}\)C, and DEPT NMR (Figures S1-S3), along with a two-dimensional heteronuclear HMBC experiment (Figure S4) as well as a FAB+ mode mass spectrometry analysis (Figure S5). In the \({}^{1}\)H NMR spectrum, an AA'BB' aromatic spin system was evidence by the two doublets, as there were two doublet signals at \(\delta\) 6.88 and \(\delta\) 8.12 with coupling constants of \(J\) = 8.7 Hz and 8.9 Hz, respectively. Additionally, a singlet signal at \(\delta\) 6.9 was assigned to a penta-substituted benzene. Another doublet signal at \(\delta\) 5.44 was also observed in the spectrum (\(J\) = 7.7 Hz), as well as several signals between \(\delta\) 3.5 and \(\delta\) 4.5, characteristic of a glucopyranose; these signals were evident in the \({}^{13}\)C NMR spectrum, indicating that the aglycone was glycosylated.