Metabolite Profiling of Defatted Methanol Extract

The defatted methanol extract was analyzed using LC–MS–MS. A Shimadzu LC-10 HPLC with a Grace Vydac Everest Narrowbore C-18 column (100 mm × 2.1 mm i.d., 5 µm, 300 Å). LC–MS, connected to an LTQ Linear Ion Trap MS (Thermo Finnigan, San Jose, CA) was utilized with a mass range of 100–2000 m/z. A 2 µL sample was injected using an autosampler. A 35 min method was used as follows: 5 min isocratic run using 5% acetonitrile (Acn) and 0.05% formic acid (FA), then a gradient was run for 25 min until 95% AcN 0.05% FA. Finally, there was 5 min of conditioning the column with 5% AcN and 0.05% FA. The data were processed and analyzed using foundation 3.1_Xcalibur_3.1.6610. Furthermore, the raw data files were converted to mzXML format using MSConvert from the ProteoWizard suite [70 ]. The molecular network was created using the Global Natural Products Social Molecular Networking (GNPS) online workflow [28 (link),29 (link)]. The spectra in the network were then searched against the GNPS spectral libraries and published data.