Phytol

Phytol (Fig. 1) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and praziquantel tablets were purchased from Merck (São Paulo, SP, Brazil). For in vitro studies, drugs were dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) to obtain stock solutions of 4 mg/mL. For in vivo studies, Phytol was suspended in 3.7 mL of phosphate buffered saline (PBS) and orally administered at final concentration of 40 mg/kg.

Thylakoid membranes were prepared from 50 g of fresh leaves from 18-day-old pea (Pisum sativum) plants, which were grown under control conditions with minor modifications (Fitzpatrick and Keegstra, 2001 (link); Smith et al., 2003 ; Kummerová et al., 2006 (link), 2008 (link)). The Hill reaction was adapted and carried out to measure the direct effect of Phytol on the photosynthetic primary reaction at the level of PSII. The total reaction mixture (4 ml) contained 0.03 mM sodium salt solution of 2,6-dichlorophenolindophenol (DCPIP; redox system), 20% dimethyl sulfoxide (DMSO, Sigma-Aldrich) and 0.5 and 1 mg thylakoid membranes in suspension buffer. Phytol (97%, mixture of isomers) was purchased from Sigma-Aldrich and suspended in DMSO at a final concentration of 10 mM. Phytol was used to obtain final concentrations of 500 µM and 50 µM. The reactions were incubated for 5 minutes and then exposed for 6 minutes to an irradiance of 200–250 µmol m⁻² s⁻¹ at room temperature. A 1 ml aliquot was removed at 3 and 6 minutes, centrifuged for 1 min at 16000 g, and measured spectrophotometrically at 600 nm. Photosynthetic activity was determined by measuring DCPIP reduction using an UltraViolet-Visible Spectroscopy (UV/VIS) Ultrospec 3100 pro (Amersham Biosciences) in 1 cm UV-visible cuvettes. Measurements were repeated at least three times at each time point.

Hexadecanal (palmitic aldehyde, 16:0al) was obtained from Cayman Chemical. Phytol (cis- and trans-isomers) was obtained from Sigma Aldrich. Phytenal was synthesized by oxidation of Phytol with pyridinium chlorochromate (22 , 23 (link)). Briefly, a solution of Phytol and pyridinium chlorochromate (in a nanomolar ratio of 0.01–0.09) in dichloromethane was stirred for 90 min. The reaction mixture was passed through a silica column. The reaction product (phytenal) was collected in the flow-through and eluted with 5-ml dichloromethane. Pristanol was synthesized from pristanic acid (Larodan) as follows. Pristanic acid methyl ester was produced by methylation with 1 N methanolic HCl at 80 °C for 30 min. The methyl esters were extracted after addition of 1 ml 0.9% NaCl and 1-ml hexane. The hexane was evaporated and the methyl ester reduced to pristanol with LiAlH₄ (24 ). Pristanal (pristyl aldehyde) was obtained from pristanol by oxidation with pyridinium chlorochromate as described for phytenal. Nonadecanal (19:0al), eicosanal (icosanal, 20:0al), and phytanal were produced from the corresponding fatty acids (nonadecanoic acid, eicosanoic acid, from Sigma Aldrich; phytanic acid from Larodan). The fatty acids were converted into their methyl esters and then reduced to the alcohols with LiAlH₄, and the alcohols oxidized with pyridinium chlorochromate as described above.