2.3. Phytochemical analysis

The methanolic extract and its fractions of E. arvense were dissolved in liquid chromatography mass spectrometry grade methanol (1 mg/mL) and filtered through 0.2 µm membrane filter into HPLC autosampler glass vials.

Metabolomics profiling of the extract and fractions was carried out using a Shimadzu (Kyoto, Japan) Nexera UHPLC system coupled with Impact II quadrupole/time-of-flight mass spectrometer (Bruker, MA, USA). The electrospray ionization technique was used to acquire the spectra in positive ion mode. For chromatographic separation, a CORTECS UHPLC C-18 column was used with particle size of 1.6 μm, internal diameter of 2.1 mm and 100 mm column length (Waters, Milford, MA, USA) with a pre-column (2.1 mm × 5 mm). The gradient program was set using 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). Gradient elution profile was as follows: 5% B (2.50 min), 5–100% B (6 min), 100% B (2.40 min) and 5% B (3 min) and total run time 13.90 min. The oven temperature was set at 40 °C. The sample injection volume was 5 µL and the flow rate was kept at of 0.4 mL/min. Spectra were recorded with m/z range of 80 to 1000.

The LC-MS/MS data of the E. arvense crude extract and fractions were subjected to Global Natural Product Social molecular networking analysis available online at (https://gnps.ucsd.edu/ProteoSAFe/stat 6 ic/gnps-splash.jsp). The data were converted to GNPS compatible (“.mzML” format) files via MSConvert package (Version 3.0.19330, Proteowizard Software Foundation, USA). The converted “.mzML” files were uploaded on the GNPS platform using WinSCP version 5.17.6. The spectral networks were visualized with the help of Cytoscape 3.7.2 (Wang et al., 2016).