# Constituents from _Ageratina pichinchensis_ and Their Inhibitory Effect on Nitric Oxide Production
## 2 Materials and Methods
### Plant Material
#### 2.2.1 Purification and Identification of Compounds from _A. pichinchenensis_ Leaves  
The dry stems and leaves of _A. pichinchenensis_ were separately extracted three times with ethyl acetate (EtOAc) by sonication; each extraction lasted 30 min. The solvent was concentrated in a rotatory evaporator under reduced pressure. The plant material was subjected to a second extraction with the solvent methanol following the same procedure as that used for ethyl acetate. An ethanol/H\({}_{2}\)O (95:05) extract of the flowers was obtained under the same conditions. Leaves were selected from the collected plants and dried at room temperature. This material (1.085 kg) was extracted with EtOAc three times (each time with 4 L of the solvent). The EtOAc extracts obtained were concentrated to dryness by distillation under reduced pressure using a rotary evaporator, obtaining 20.3 g of residue. Fractionation of the EtOAc 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/EtOAc 80:20 to 0:100, collecting 35 fractions of 300 mL. The fractions were grouped according to their chemical profile into four groups: AP-1A (fractions 1-5, _n_-hexane: EtOAc 80:20, 4.12 g), AP-1B (fractions 6-19, _n_-hexane: EtOAc 60:40 and 40:60, 5.23 g), AP-1C (fractions 20-32, _n_-hexane: EtOAc 20:80, 5.94 g), and AP-1D (fractions 33-35, EtOAc 100%, 3.52 g). AP-1A contains aliphatic esters, fatty acids, and _O_-methylencalinol (1, 48 mg) as the main product; in a similar way, AP-1D contains the same compounds but also sugars. The groups AP-1B and AP-1C were subjected to column chromatography using silica gel (70-230 mesh). The AP-1B fraction (5.23 g) was adsorbed on 4.9 g of silica gel and placed in a glass column (70 cm high and 3.5 cm in diameter) packed with 157 g of silica gel. Elution was performed using a gradient system, _n_-hexane/EtOAc (100:00\(\rightarrow\)50:50), and 164 fractions were obtained in 50 mL. The fractions were concentrated by reducing pressure using a rotatory evaporator; monitored by TLC; and grouped into 5 groups (AP-1B-1, AP-1B-2, AP-1B-3, AP-1B-4, and AP-1B-5). The AP-1B-1 group (fractions 1-39, 0.306 g, _n_-hexane: EtOAc 95:05 to 85:15) was purified and identified as _O_-methylencecalinol (**1**, 32 mg) as the main product. From AP-1B-2 (fractions 40-69, 3.32 g, _n_-hexane: EtOAc 80:20 to 70:30), _7_-hydroicenecalin (**3**, 7 mg) and 8-hydroicenecalin (**4**, 6 mg) were identified. The compounds enececalin (**2**, 16 mg), 3,5-dipropyl-4-hydroxyacetophenone (**5**, 32 mg), and (+)-\(\beta\)-eudesmol (**6**, 8 mg) were isolated from the AP-1B-3 group (fractions 70-99, 0.391 g, _n_-hexane: EtOAc 65:35 to 55:45). The AP-1B-4 group (fractions 100-129, 0.821 g, _n_-hexane: EtOAc 50:50 to 40:60) led to the identification of the compounds (+)-\(\beta\)-eudesmol (**6**, 7 mg), dehydrogenase (**8**, 12 mg), and speletone (7, 14 mg). Through successive chromatography of the AP-1B-5 group (fractions 130-149, 0.427 g, _n_-hexane: EtOAc 35:75 to 25:75), enececalinol (**9**, 28 mg) and 5-acetyl-3\(\beta\)-angeloxy-2\(\beta\)-(1-hydroxyisopropyl)-2,3-dihydrobenzofuran (**10**, 36 mg) were isolated. _O_-Methylencecalinol (**1**)  
Colorless oil; \({}^{1}\)H-NMR (200 MHz, CDCl\({}_{3}\)), \(\delta_{\rm H}\):6.95 (1H, s, H-5), 6.32 (1H, s, H-8), 6.26 (1H, d, \(J\) = 9.6 Hz, H-4), 5.43 (1H, d, \(J\) = 10 Hz, H-3), 4.61 (1H, q, \(J\) = 12.8, 6.8 Hz, H-11), 3.75 (3H, s, -OMe), 3.21 (3H, OMe), 1.40 (3H, s, CH\({}_{3}\)-13), 1.39 (3H, s, CH\({}_{3}\)-14), 1.34 (3H, d, \(J\) = 6.8 Hz, CH\({}_{3}\)-12). \({}^{13}\)C-NMR (50 MHz, CDCl\({}_{3}\)). \(\delta_{\rm C}\): 157.70 (C-7), 153.25 (C-10), 127.76 (C-3), 124.06 (C-5), 122.30 (C-4), 120.02 (C-6), 114.19 (C-9), 99.43 (C-8), 76.55 (C-2), 72.97 (C-11), 56.59 (-OMe-C11), 56.03 (OMe-C-7), 28.32 (C-13), 28.26 (C-14), 22.65 (C-12); these data match those in the literature [10;35]. The spectra of \({}^{1}\)H and \({}^{13}\)C-NMR are shown in Figures S1 and S2.