# 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
Eneccalin (**2**)  
Yellow oil; \({}^{1}\)H-NMR (400 MHz, CDCl\({}_{3}\)), \(\delta_{\rm H}\):7.23 (1H, s, H-5), 6.27 (1H, s, H-8), 6.12 (1H, d, \(J\) = 9.8 Hz, H-4), 5.44 (1H, d, \(J\) = 9.7 Hz, H-3), 3.55 (3H, s, -OMe), 2.10 (3H, s, CH\({}_{3}\)-12), 1.20 (6H, s, CH\({}_{3}\)-13 and CH\({}_{3}\)-14). \({}^{13}\)C-NMR (100 MHz, CDCl\({}_{3}\)). \(\delta_{\rm C}\): 197.68 (C-11), 161.37 (C-7),158.28 (C-10), 128.94 (C-5), 128.36 (C-3), 121.36 (C-4), 120.42 (C-6), 114.22 (C-9), 99.76 (C-8), 77.21 (C-2), 55.72 (MeO-), 32.26 (C-12), 26.68 (C-13), 28.32 (C-14); these data match those in the literature [36; 37]. The spectra of \({}^{1}\)H and \({}^{13}\)C-NMR are shown in Figures S3 and S4. Euparoriochromene (**3**)  
Yellow needles; mp: 78-80 \({}^{\circ}\)C; \({}^{1}\)H-NMR (200 MHz, CDCl\({}_{3}\)), \(\delta_{\rm H}\):7.21 (1H, s, H-5), 6.24 (1H, s, H-8), 6.21 (1H, d, \(J\) = 10 Hz, H-4), 5.41 (1H, d, \(J\) = 10 Hz, H-3), 2.49 (3H, s, CH\({}_{3}\)-12), 1.39 (6H, s, CH\({}_{3}\)-13 and CH\({}_{3}\)-14). \({}^{13}\)C-NMR (50 MHz, CDCl\({}_{3}\)). \(\delta_{\rm C}\): 198.13 (C-11), 162.18 (C-7), 159.18 (C-10), 128.68 (C-5), 128.53 (C-3), 126.14 (C-6), 122.08 (C-4), 114.38 (C-9), 104.26 (C-8), 77.43 (C-2), 32.12 (C-12), 28.86 (C-13), and 28.78 (C-14); these data match those in the literature [38]. The spectra of \({}^{1}\)H and \({}^{13}\)C-NMR are shown in Figures S2 and S5. 6-Acethyl-8-Hydroxy-2,2-Dimethylchromene (**4**)  
White powder; mp: 98 \({}^{\circ}\)C; \({}^{1}\)H-NMR (200 MHz, CDCl\({}_{3}\)), \(\delta_{\rm H}\):7.62 (1H, d, \(J\) = 1.2 Hz, H-7), 7.43 (1H, d, \(J\) = 1.2 Hz, H-5), 6.34 (1H, d, \(J\) = 10 Hz, H-4), 5.71 (1H, d, \(J\) = 10 Hz, H-3), 2.56 (3H, s, CH\({}_{3}\)-12) and 1.43 (6H, s, CH\({}_{3}\)-13 and CH\({}_{3}\)-14). \({}^{13}\)C-NMR (50 MHz, CDCl\({}_{3}\)), \(\delta_{\rm C}\): 202.98 (C-11), 165.23 (C-8), 160.68 (C-10), 128.61 (C-6), 128.32 (C-3), 125.34 (C-9), 122.24 (C-4), 118.38 (C-5), 114.31 (C-7), 78.26 (C-2), 28.58 (C-13 and C-14), and 26.38 (C-12); these data match those in the literature [39; 40]. The spectra of \({}^{1}\)H and \({}^{13}\)C-NMR are shown in Figures S7 and S8. 3.5-Diprenyl-4-Hydroxyacetophenone (**5**)  
Crystalline solid; mp: 93-95 \({}^{\circ}\)C; \({}^{1}\)H-NMR (400 MHz, CDCl\({}_{3}\)), \(\delta_{\rm H}\):7.44 (2H, s, H-2 y H-6), 5.98 (1H, s, OH), 5.31 (2H, m, H-2\({}^{\prime}\) y H-2\({}^{\prime\prime}\)), 3.37 (2H, d, \(J\) = 7.1 Hz, H-1\({}^{\prime}\)y H-1\({}^{\prime\prime}\)), 2.48 (3H, d, \(J\) = 19.8 Hz, CH\({}_{3}\)-8), 1.74 (12H, d, \(J\) = 11.1 Hz, CH\({}_{3}\)-4\({}^{\prime}\), CH\({}_{3}\)-4\({}^{\prime\prime}\)y CH\({}_{3}\)-5\({}^{\prime}\), CH\({}_{3}\)-5\({}^{\prime\prime}\)), \({}^{13}\)C-NMR (100 MHz, CDCl\({}_{3}\)), \(\delta_{\rm C}\): 197.52 (C-7), 157.59 (C-4), 135.28 (C-1), 130.15 (C-31, C-3\({}^{\prime\prime}\)), 129.02 (C-2, C-6), 127.30 (C-3, C-5), 121.58 (C-2\({}^{\prime}\), C-2\({}^{\prime\prime}\)), 29.82 (C-1\({}^{\prime}\), C-1\({}^{\prime\prime}\)), 26.51 (C-8), 25.99 (C-5\({}^{\prime\prime}\), C-4\({}^{\prime}\)), and 18.10 (C-4\({}^{\prime\prime}\), C-5\({}^{\prime}\)); these data match those in the literature [41; 42]. The spectra of \({}^{1}\)H and \({}^{13}\)C-NMR are spectra of \({}^{1}\)H and \({}^{13}\)C-NMR are shown in Figures S9 and S10. \(\beta\)-Eudesmol (**6**)  
White amorphous solid; mp = 78-79 \({}^{\circ}\)C; \({}^{1}\)H-NMR (200 MHz, CDCl\({}_{3}\)), \(\delta_{\rm H}\):4.70 (1H, d, \(J\) = 2 Hz, H-15b), 4.43 (1H, d, \(J\) = 1.6 Hz, H-15a), 2.38 (2H, m, Hs-3), 1.98 (1H, m, H-10), 1.25-1.62 (6H, m, CH\({}_{2}\)-5, CH\({}_{2}\)-7 and CH\({}_{2}\)-8), 1.00-1.36 (5H, m, CH\({}_{2}\)-1, CH\({}_{2}\)-2 and CH-6), 1.39 (3H, s, CH\({}_{3}\)-11), 1.40 (3H, s, CH\({}_{3}\)-12), 0.69 (3H, s, CH\({}_{3}\)-14). \({}^{13}\)C-NMR (50 MHz, CDCl\({}_{3}\)), \(\delta_{\rm C}\): 152.41 (C-4), 105.14 (C-15), 72.23 (C-11), 49.58 (C-10), 49.42 (C-6),42.12 (C-3), 41.86 (C-1), 41.12 (C-8), 36.89 (C-9), 26.76 (C-12 and C-13), 24.73 (C-5), 23.45 (C-2), 22.76 (C-7), and 16.24 (C-14); these data match those in the literature [43; 44].