# Constituents from _Ageratina pichinchensis_ and Their Inhibitory Effect on Nitric Oxide Production
## 2 Materials and Methods
### Plant Material
#### 2.2.2 Purification and Identification of Compounds from \(A\). pichichenensis Flowers  
Selected flowers from the collected plants were dried in the shade at room temperature. The plant material (256.7 g) was successively extracted with ethanol/H\({}_{2}\)O (95:05 \(v/v\)) three times (each time with 4 L of the solvent). The hydroalcoholic extract obtained was concentrated to dryness by distillation under reduced pressure using a rotary evaporator, obtaining 6.7 g of residue.  
Fractionation of the hydroalcoholic extract by open CC (silica gel, 70-230 mesh; 10 cm i.d. \(\times\) 60 cm) was performed with a gradient system of \(n\)-hexane-CH\({}_{2}\)Cl\({}_{2}\)/MeOH 90:10:00 to 100% MeOH. Fractions of 100 mL were obtained (53 fractions). Based on TLC analysis, these fractions were grouped according to their chemical profile into two main groups: AP-M-1A (1-32, 2.02 g) and AP-M-1B (33-53, 3.98 g). The groups were subjected to column chromatography using silica gel (70-230 mesh).  
The AP-M-1A fraction was adsorbed on 3 g of silica gel and placed in a glass column (80 cm high and 3.5 cm in diameter) packed with 70 g of silica gel. Elution was carried out using a gradient system, \(n\)-hexane/CH\({}_{2}\)Cl\({}_{2}\) (100:00\(\rightarrow\)80:20), and 37 fractions, each comprising 100 mL, were obtained. These were concentrated and monitored by TLC and grouped into three groups of fractions: AP-M-1A-1 (fractions 1-17, 0.87 g), AP-M-1A-2 (fractions 18-26, 0.606 g), and AP-M-1A-3 (fractions 27-37, 0.87 g).  
The three groups of fractions were subjected to successive purification processes using a gradient system (CH\({}_{2}\)Cl\({}_{2}\)/MeOH 95:05 to 80:20), obtaining \(O\)-methylencecalinol (**1**, 32.4 mg) as the main product from the AP-M-1A-1 fraction; eneccalin (**2**, 24 mg) and 3, 5-diphenyl-4-hydroxyacetophenone (**5**, 16 mg) were from the AP-M-1A-2 group; and speletone (**7**, 14 mg), dehydrogenase (**8**, 11 mg), eneccalinol (**9**, 17 mg), and betuletol 3-\(O\)-\(\beta\)-glucoside (**11**, 64 mg) were purified from the AP-M-1A-3 group.  
Betuletol 3-\(O\)-\(\beta\)-Glucoside (**11**)  
Yellow amorphous solid; mp: 152-154 \({}^{\circ}\)C; \({}^{1}\)H-NMR (500 MHz, DMSO-d6); \(\delta_{\rm H}\):12.58 (1H, s, OH), 8.00 (2H, d, \(J\) = 8.9 Hz, H-2' H-6'), 6.90 (1H, s, H-8), 6.82 (2H, d, \(J\) = 8.9 Hz, H-3' H-5'), 5.25 (1H, d, \(J\) = 7.7 Hz, H-1''), 3.85 (3H, s, OMe-H-4'), 3.70 (3H, s, OMe-H-6), 3.58-3.56 (1H, ddd, \(J\) = 9.7, 7.7, 4.7 Hz, H-4''), 3.32-3.31 (1H, m, H-5''), 3.37-3.36 (1H, m, H-3''), 3.47-3.45 (1H, m, H-2''), 3.33-3.29 (1H, m, H-6''). \({}^{13}\)C-NMR (125 MHz, DMSO-d\({}_{\rm c}\)), \(\delta_{\rm C}\): 177.86, (C-4), 160.13, (C-4'), 158.80 (C-7), 156.78, (C-2), 151.81 (C-5), 151.68 (C-9), 133.29 (C-3), 131.76 (C-6), 128.57 (C-2', C-6'), 120.65 (C-1'), 115.56 (C-3', C-5'), 105.36 (C-10), 101.99 (C-1''), 91.82 (C-8), 73.95 (C-5''), 73.54 (C-3''), 74.13 (C-2''), 70.59 (C-4''), 60.63 (C-6''), 60.64 (C-OMe-6), 56.97 (C-OMe-4'); these data match those in the literature [46; 47]. The spectra of \({}^{1}\)H and \({}^{13}\)C, DEPT, and HMBC NMR are shown in Figures S19-S22. C\({}_{23}\)H\({}_{25}\)O\({}_{12}\) (MSFAB\({}^{+}\)\(m/z\) = 493) MS is shown in Figure S23.