Hieracium calophyllum, R. Uechtr.

Milutinović, Violeta, Niketić, Marjan, Krunić, Aleksej, Nikolić, Dejan, Petković, Miloš, Ušjak, Ljuboš & Petrović, Silvana, 2018, Sesquiterpene lactones from the methanol extracts of twenty-eight Hieracium species from the Balkan Peninsula and their chemosystematic significance, Phytochemistry 154, pp. 19-30 : 28-29

4.3. Preparation of MeOH extract of H. calophyllum flowering heads and isolation of SLs

Dried and powdered flowering heads of H. calophyllum (13 g) were macerated with CH 2 Cl 2 (1:15 w/v) at room temperature for 48 h. The extract was filtered, and the residual material dried at room temperature and re-extracted with MeOH (1:15 w/v) by bimaceration procedure (2 × 48 h). Combined MeOH extracts were evaporated under reduced pressure using a rotary evaporator (Büchi Rotavapor R- II, Flawil, Switzerland). The thus obtained dried MeOH extract (3 g) was then suspended in H 2 O (300 ml), extracted successively with CH 2 Cl 2 (300 ml), EtOAc (600 ml) and n -BuOH (600 ml), and then lyophilised. The thus obtained dry residue (0.5 g) was redissolved in H 2 O (8 mg /ml) and subjected to preparative HPLC with MS detection, on an Agilent LC/MS System, equipped with Zorbax SB-C 18 column (250 × 9.4 mm; 5 μm particle size). SLs were eluted using 0.1% (v/v) HCO 2 H/H 2 O (mobile phase A) and MeOH (mobile phase B), and selected gradient program: 10–20% B (5 min), 20–30% B (5 min), isocratic 30% B (3 min), 30-20% B (7 min), 20–50% B (4 min), 50–90% B (6 min), with the total run time of 30 min, post run time of 5 min, flow rate of 2 ml/min, and the injection volume (Vinj) of 100 μl. DAD was operating at 210 nm, 254 nm, 320 nm and 350 nm wavelengths. MSD was recording in the positive ion and full-scan mode in the range of m/ z 80–700 to acquire Total Ion Chromatogram (TIC). Optimized ion source parameters were as follows: fragmentor voltage of 200 V, nebulising drying gas flow of 9 L/min at 350 ̊C, nebulizer pressure of 40 psi and capillary voltage of 3000 V. Splitter was set at 1000:1. Time-based fractionation afforded eleven fractions, F 1–11, which were collected during 0.5 min (F 1–3), 1 min (F 4–6, 9), or 3 min (F 7, 8, 10, 11). Fraction F 4 (tR = 14.32 min) afforded compound 1 (1 mg), F 6 (tR = 15.55 min) compound 2 (1.5 mg), F 7 (tR = 16.72 min) compound 3 (3 mg) and F 9 (tR = 31.25 min) compound 4 (5 mg). Compounds 2 and 3 gradually decomposed upon one-week standing in D 2 O solution at temperature above 303 K. The mixture of 2 and its degradation products was further separated on Zorbax SB-C 18 column (50 × 9.4 mm; 5 μm particle size) affording 2a (0.5 mg). For this purpose, mobile phases: 0.1% (v/v) HCO 2 H/H 2 O (A) and MeOH (B), and fast gradient program: 20–30% B (5 min), 50–90% B (6 min), 90- 20% B (3 min), with the total run time of 15 min, flow rate of 2 ml/ min, and Vinj of 60 μl were used. The mixture of 3 and its degradation products was fractioned using the same column, mobile phases, flow rate and Vinj, with slightly different gradient program, i.e. 20–30% B (5 min), 30-20% B (5 min), 50–90% B (2 min), 90-20% B (1 min), and the total run time of 14 min, affording 4.

4.3.1. Calophyllamine B (1, 3 β -(β -glucopyranosyl)-oxy-8 α -hydroxy-13 α - (N-prolyl)-eudesma-1,4(15)-dien-5 α, 6 β, 7 α, 11 β H-12,6-olide)

White solid [α] D +106 (c 0.5, H 2 O). 1 H, 13 C, DEPT, HMBC, and NOESY spectroscopic data are presented in Table 2 . HRMS m/z: 540.2456 [M + H] + (calcd 540.2439 for [M + H] + C 26 H 38 NO 11), qTOF-MS/MS (15 eV) m/z (rel. int.): 540.2456 (100), 494.2445 (22), 316.2084 (3), 128.0718 (4), 100.0759 (4).

4.3.2. Calophyllamine A (2, 3 β, 8 α -dihydroxy-13 α -(N-prolyl)-guaia- 4(15),10(14)-dien-5 α, 6 β, 7 α, 11 β H-12,6-olide)

White solid. 1 H, 13 C, HMBC, ROESY spectroscopic data are presented in Table 2 . HRMS m/z: 378.1916 [M + H] + (calcd 378.1911 for [M+H] + C 20 H 28 NO 6), qTOF-MS/MS (15 eV) m/z (rel. int.): 378.1892 (100), 332.1865 (22), 128.0710 (10), 100.0729 (4).

4.3.3. 8-Epiixerisamine A (3, 3 β -(β -glucopyranosyl)-oxy-8 α -hydroxy-13 α - (N-prolyl)-guaia-4(15),10(14)-dien-5 α, 6 β, 7 α, 11 β H-12,6-olide)

White solid [α] D −3.6 (c 1.2, H 2 O). 1 H, 13 C, DEPT, ROESY and HMBC spectroscopic data are presented in Table 2 . HRMS m/z: 540.2456 [M + H] + (calcd 540.2439 for [M + H] + C 26 H 38 NO 11), qTOF-MS/MS (15 eV) m/z (rel. int.): 540.2456 (100), 494.2414 (9), 378.1885 (22), 360.1806 (5), 332.1909 (5), 316.1909 (3), 128.0734 (10), 100.0886 (6).

4.3.4. Crepiside E (4, 3 β -(β -D-glucopyranosyl)-oxy-8 α -hydroxy-guaia- 4(15),10(14),11(13)-trien-5 α, 6 β, 7 α -12,6-olide)

Brownish gum. [α] D +24.3 (c 1.2, MeOH). 1 H, 13 C, COSY, NOESY,

TOCSY, HMBC spectroscopic data are presented in Table S1 . HRMS m/ z: 447.1645 [M + Na] + (calcd 447.1625 for [M + Na] + C 21 H 28 O 9 Na), qTOF-MS/MS (25 eV) m/z (rel. int.): 263.1304 (29); 245.1204 (16); 227.1058 (100); 217.1268 (57); 209.1308 (14); 201.1261 (14); 199.1140 (62); 183.1078 (11); 181.104 (72); 175.0810 (5); 171.1170 (55); 143.0926 (12); 131.0869 (19).

4.3.5. Desacylcynaropicrin (2a, 3 β, 8 α -dihydroxy-guaia-4(15),10(14), 11(13)-trien-5 α, 6 β, 7 α -12,6-olide)

White solid, [α] D = +34 (c 0.5, H 2 O). 1 H NMR (500 MHz, D 2 O, TMS): δ H 6.25 (d, J = 3.4 Hz, H-13b); 6.16 (d, J = 3.0 Hz, H-13a); 5.45 (s, H-15b); 5.34 (s, H-15a); 5.16 (s, H-14b); 5.04 (s, H-14a); 4.64 (t, J = 6.2 Hz, H-3); 4.36 (t, J = 9.8 Hz, H-6); 4.06 (dd, J = 7.6, 4.7 Hz, H- 8); 3.06 (m, H-1); 2.99 (q, J = 9.9 Hz, H-5); 3.00 (t, J = 9.8 Hz, H-7); 2.72 (dd, J = 13.6, 4.9 Hz, H-9β); 2.33 (dd, J = 14.6, 3.8 Hz, H-9α); 2.22 (td, J = 13.5, 7.0 Hz, H-2β); 1.76 (td, J = 11.9, 9.2 Hz, H-2α). 13 C NMR (125 MHz, D 2 O): δ C 173.0 (C-12); 151.6 (C-4); 141.6 (C-10); 123.7 (C-13); 116.8 (C-14); 112.4 (C-15); 79.6 (C-6); 72.6 (C-3); 71.2 (C-8); 50.1 (C-7); 49.3 (C-5); 44.7 (C-1); 40.2 (C-9); 37.6 (C-2).