Preparation and Characterization of Pp-EE Extract

A 95% ethanol extract of Pp-EE was prepared with the arial part of P. paradoxa Nutt., as reported previously [38 (link)]. The arial part of P. paradoxa Nutt., purchased from Herbmaul (Seoul, Korea), was ground to a powder, which was then used for the extraction process. The extraction was performed with 127 g of the plant material and 890 mL of 95% ethanol for 2 h, three times. The extract was percolated through filter paper (3 mm; Whatman PLC, Kent, UK), condensed using a rotary evaporator (Büchi AG, Flawil, Switzerland), and lyophilized using a freeze dryer (Martin Christ Gefriertrocknungsanlagen, Osterode am Harz, Germany). Pp-EE powder was dissolved and used as a 100 mg/mL stock solution using DMSO as a solvent. The experiment was performed while using DMSO as vehicle control, with the same dilution level as the negative control. For the in vitro experiment, Pp-EE stock solution was dissolved in a culture medium. When making dilutions to treat cells, the dilution coefficient was considered so that the final concentration could be the target concentration we wanted. For the in vivo experiment, the powder was suspended in 0.5% sodium carboxymethylcellulose (0.5% CMC).