*3.6. LC*−*MS Analysis and Molecular Networking Generation*

The crude extract and fractions A−H were dissolved in MeOH at 0.5 mg/mL. A 50 μL aliquot of each sample was injected via LC−MS/MS on a Thermo Dionex Ultimate 3000 LC-PDA system coupled to a Bruker Maxis impact QTOF system in an ESI positive mode and eluted with a gradient of H2O with 0.1% formic acid and CH3CN with a gradient method as follows: 10% CH3CN/H2O for 2 min, 10% CH3CN/H2O to 45% in 8 min, held at 45% CH3CN/H2O for 2 min, 45% CH3CN/H2O to 99% in 4 min, held at 99% CH3CN/H2O for 1 min, then 99% CH3CN/H2O to 10% CH3CN/H2O in 1 min, and finally held at 10% CH3CN/H2O for 2 min with the flow rate of 0.6 mL/min at room temperature. The UV chromatogram was measured at 210, 230, 280, 360 nm by photodiode array detection. Data-dependent (automated) MS/MS spectra were collected during the same run. The raw data of MS/MS spectra from the all fractions were converted to mzXML format using the ProteoWizard tool MSConvertGUI, and the processed files were uploaded to the GNPS website (http://gnps.ucsd.edu) to generate a molecular network that was visualized using Cytoscape 3.8 software (Weblinks S1 and S2). A molecular network was created using the online workflow on the GNPS website (https://ccms-ucsd.github.io/ GNPSDocumentation). The precursor ion mass tolerance was set to 1 Da and a MS/MS fragment ion tolerance of 0.5 Da. The spectra in the network were then searched against available GNPS spectrometric libraries. The library spectra were filtered in the same manner as the input data. All matches kept between network spectra and library spectra were required to have a score above 0.7 and at least 4 matched peaks [33].

#### *3.7. Cell Culture*

The human colorectal cancer cell line, HCT116, was purchased from American Type Culture Collection (ATCC; Bethesda, MD, USA). Cells were maintained in DMEM medium, supplemented with 1% L-glutamine, 10% fetal bovine serum (FBS), 1% sodium pyruvate and 1% PenStrep (penicillin + streptomycin) (Biological Industries, Beit Haemek, Israel). Cells were grown in a humidified incubator at 37 ◦C with 5% CO2 in air, and served twice a week with fresh medium.

### *3.8. XTT Cell Proliferation Assay*

Evaluation of the effect of each crude organic extract and fractions A-H, as well as subfractions C1-C6 and C3–1–C3–12 on cell viability was performed using the standard XTT assay and an established protocol [44]. In brief, HCT116 cells were seeded in 96-well plates (104 cell/well) and 24 h later were treated for a period of 24 h with two doses from the crude extract and fractions A-H; 200 and 400 μg/mL, and with 4 doses for each subfraction; 15, 25, 50, and 100 μg/mL. Medium and DMSO were added to control wells. For sub-fraction C3–5, the XTT assay was additionally conducted using 30 μg/mL for 24 h. Following treatment, cell viability was determined by the XTT assay (Biological Industries, Beit Haemek, Israel) according to the manufacturer's instructions using a plate reader (version, BioTek, Winooski, VT, USA). Experiments were repeated 3 times. Data were presented as the average proliferation percentage of the respective control.
