Ika rv 10

The whole epigeal part (flowers, stems and leaves) from C. libanotis, C. ferulacea and C. pungens were collected in Calabria, Southern Italy (leg. det. Carmine Lupia). Voucher specimens were deposited at Mediterranean Etnobotanical Conservatory, Sersale (Catanzaro), in Apiaceae section (numbers 18, 31 and 52, respectively). Dry plant material was cut into small pieces and extracted by Naviglio extractor^® (Atlas Filtri, s.r.l., Limena, PD, Italy) in methanol (MeOH) with the following conditions: plant:solvent ratio 1:10 g/mL, 2 cycles. Extracts were filtered using qualitative filter paper (particle retention 10–20 μm, VWR International, Leuven, Belgium) and dried at 37 °C under reduced pressure using a rotary evaporator (IKA^® RV 10, VWR International, Milan, Italy). Each extract was quantified and standardized in furanocoumarin content (xanthotoxin, bergapten and isopimpinellin) and two formulations were prepared for each extract: “a” and “b”, in which the sum of the three chosen standards was equal to the concentration of bergapten 10 µM (2.16 µg/mL) and 5 µM (1.08 µg/mL), two concentrations able to exert a biological activity without affecting cell viability. The concentration of bergapten 10 µM used as positive control was chosen based on previous literature studies [8 (link)] and tested in our conditions in order to confirm concentration and activity.

According to the results obtained from the response surface plots and PCA analysis in a previous study [11 (link)], the combination of 180 °C and 16.7 MPa applied during 30 min were the optimal PLE conditions for Tuber aestivum and Terfezia claveryi truffles, although extractions from Terfezia truffles were more effective than from the Tuber genus. Thus, the optimized PLE method was applied to seven other truffles species.
Truffle powders (0.5 g) were submitted to pressurized liquid extraction using an Accelerated Solvent Extractor (ASE) (Dionex Corporation, ASE 350, USA) [6 (link)]. Briefly, samples were submitted to 16.7 MPa at 180 °C for 30 min. Fractions obtained with water were immediately frozen and freeze-dried in a LyoBeta 15 lyophilizer (Telstar, Madrid, Spain), and those obtained with ethanol were dried using a rotary vacuum evaporator at 40 °C (IKA^® RV 10, VWR International, Barcelona, Spain). The water:ethanol extracts were dried and then lyophilized. Afterwards, samples were stored in darkness at −20 °C.

The aerial parts from wild Centranthus ruber and Tropaeolum majus were collected in Calabria (southern Italy) in June and May, respectively (leg. F. Conforti, det. F. Conforti). Voucher specimens are deposited in the Herbarium of the University of Calabria under the registration numbers CLU- 26241 (for C. ruber) and CLU- 26244 (for T. majus). Dried plant material was extracted with methanol (analytical grade, VWR International, Milan, Italy) at room temperature through maceration (plant to solvent ratio 1:10 g/mL, 48 h × 3 extractions). The obtained solutions were then filtered using qualitative filter paper with particle retention 10–20 μm (VWR International, Leuven, Belgium) and dried using a rotary evaporator IKA^® RV 10 (VWR International, Milan, Italy).
A fraction of each raw extract was then suspended in methanol:water, 9:1 and extracted with n-hexane. The residue was then suspended in distilled water and extracted successively with dichloromethane and ethyl acetate (analytical grade, VWR International, Milan, Italy).