Fern Leaf Extract Preparation Protocol

Extracts of fern leaves were obtained by solvent extraction, as described by Grzeszczuk et al. [48 (link)]. The solvent was a mixture of methanol (Merck, Darmstadt, Germany) with distilled H₂O (7:3, v/v). Dried leaves were ground in a laboratory mill, then 0.5 g samples were transferred into 50 mL Falcon tubes (Bionovo, Legnica, Poland) and mixed with 40 mL of the mixture. The solutions were then extracted for 30 min in an ultrasonic cleaner (Elmasonic S30H, Elma Schmidbauer GmbH, Singen, Germany) and centrifuged for 5 min at 5000 rpm (Centrifuge 5418 Eppendorf, Warsaw, Poland). The extracts were filtered through 0.22 µm nylon membrane filters (Merck, Darmstadt, Germany). Three repetitions of each extract were prepared and stored in a freezer at −20 °C.