4.2. Sesquiterpene Lactones Isolation

Fifty grams of dried aerial parts of each plant species were extracted twice at room temperature with dichloromethane (DCM) (500 mL, 3 min.). Filtrates were concentrated on a rotatory evaporator at 40 °C under reduced pressure. S. maimarensis and S. gilliesii crude extracts were dissolved in warm ethanol (140 mL), the solution was placed in a dropping funnel, water (60 mL) was added (ratio ethanol–water 70:30 v/v) and the mixture was extracted three times with hexane (3 × 50 mL) and then with DCM (3 × 50 mL). DCM sub-extracts were reunited and evaporated to yield dewaxed extract, rich in STLs.

S. alpina and M. minima crude extracts and S. maimarensis and S. gilliesii dewaxed extracts were fractionated by silica gel column chromatography (50 × 4.5 cm, 180 g, 230–400 mesh) and eluted isocratically with dichloromethane-ethyl acetate mixtures: 1:2 for S. maimarensis and S. gilliesii, 9.5:0.5 for S. alpina and 2:1 for M. minima. Column chromatography eluates were monitored by thin layer chromatography (TLC) using STL standards. Fractions showing a single spot corresponding to the desired compounds were pooled and dried in a rotary evaporator. Fractions enriched in the desired compound were also pooled and rechromatographed as above to recover additional amounts of lactone.

The STL eupatoriopicrin precipitated from S. maimarensis fractions as white crystals. The crystals were washed twice with a 7:3 mixture of ethyl ether-ethyl acetate. Recrystallization from n-heptane-ethyl acetate yielded needles mp 158–160 °C [9]. Eupahakonenin B was obtained as a greenish gum when S. gilliesi fractions showing a single spot on TLC with R_f identical to a reference standard [10] were reunited and evaporated in rotavapor. Less pure fractions were reunited and rechromatographed under the same conditions described above in order to increase the amount of the isolated compound. The rechromatographed lactone was isolated as a colorless gum. Estafietin and minimolide were purified by column chromatography from crude extracts of S. alpina and Mikania minima, respectively (See supplementary materials). In both cases, STLs-rich fractions were pooled, the solvent was evaporated in a rotary evaporator and the residue was suspended in a minimum volume of warm heptane-ethyl acetate (1:2) to near complete dissolution and then kept in the refrigerator overnight. Both STLs precipitated as white needles and were recrystallized from heptane-ethyl acetate; estafietin, mp 104–105 °C [11]; minimolide, mp 112–113 °C [18].

The purity of the compounds was determined by HPLC using a RP-C18 column (250 × 4.6 mm - 5 µm mesh) and a water: acetonitrile gradient (35% to 95% acetonitrile in 30 min. for eupatoriopicrin, minimolide and eupahakonenin B and 45% to 95% acetonitrile in 30 min. for estafietin. A flow rate of 1 mL/min was used. The diode array detector was set at 210 nm. The purity of the sesquiterpene lactones determined by HPLC was: eupatoriopicrin: 94.6%, minimolide: 99.8%, estafietin: 93.3% and eupahakonenin B: 93.9%. Further recrystallization of eupatoriopicrin, estafietin and eupahakonenin B yielded analytical samples with a purity >98.2% in all three cases. The identity of the compounds was confirmed by UV, IR, NMR and MS spectroscopy.