*3.1. Materials*

All the solvents used for extraction and fractionation purposes, acetonitrile (LC–MS grade), formic acid (98%, for mass spectrometry) were purchased from Sigma-Aldrich (Buchs, Switzerland). Cell culture media and reagents for cytotoxicity testing were purchased from Invitrogen (Paisley, Scotland, UK), except MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] which was from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany).

### *3.2. Plant Extraction and Fractionation*

One hundred g of commercial hemp seeds (*Hanf* & *Natur* product, by Bioland, a certified company for the cultivation of ecological and sustainable hemp located in Lindlar, Germany), after mechanical reduction, underwent ultrasound assisted maceration (UAM) using sequentially *n*-hexane and ethanol as extracting solvents. The drug/solvent ratio used was 1:5. Ultrasound (mechanic waves able to propagate through an elastic medium) maceration cycles were in total 4 and performed using a BransonicTM M3800-E device (Branson UltrasonicsTM, Danbury, CT, USA) operating in sweep-frequency mode at 40 kHz. The duration of each ultrasound cycle was 30 min. The alcoholic fraction (4.4 g) was then chromatographed through SiO<sup>2</sup> column chromatography (h 16 cm, Ø 2.0 cm), eluting with chloroform, ethyl acetate and methanol providing three subfractions. The methanol extract, named LnHS, underwent chemical investigation by means of UHPLC-HR-MS/MS and HPLC-UV-DAD analyses and cytotoxicity assessment towards U-87 human glioblastoma cells. The extraction/fractionation scheme is depicted in Figure S1.
