We used the ethyl acetate and acetone extracts as samples for qualitative phytochemical screening via HPLC via the Agilent-1260 infinity system, according to the reported method of Zeb79. Briefly, we mixed one-gram sample extract in methanol and water (1:1; 20 mL; v/v) and heated at 70˚C for 1 h in a water bath. We centrifuged this mixture at 4000 rpm for 10 min and filtered 2 ml of the supernatant into HPLC vials through Whatman filter paper. We performed the separation via the Agilent-Zorbax-Eclipse column (XDB-C18). Column gradients system comprised solvent B and C. Solvent B consisted of deionized water: methanol: acetic acid having a ratio of 180: 100: 20; v/v while solvent C had deionized water: methanol: acetic acid in the ratio of 80: 900: 20; v/v. We started the gradient system by solvent B for 100%, 85%, 50% and 30% at 0, 5, 20 and 25 min followed by solvent C (100%) from 30 to 40 min. Elution occurred after 25 min. We set the ultraviolet array detector (UVAD) at 280 nm for the antioxidants analysis and documented the chromatogram using retention times. We carried out the UV spectra of compounds and accessible standards along with quantification by taking the per cent peak area. We measured the quantity of the antioxidants by the formula:

Cx = Sample concentration; As = Standard peak area; Ax = Sample peak area; Cs = Standard concentration (0.09 µg/ml).

We analyzed a mixture of standards and new metabolites found in the ethyl acetate and acetone fractions via LC–MS, exactly as per the method reported by Yap et al.80. To eradicate systematic errors, we used a reference solution with the two ions, with m/z of 121.0508 and 92,266.0097, being selected for mass calibration. Finally, we ran the mass spectra for the compounds present in ethyl acetate (EA) and acetone (AC) fractions against the database of NIST (National Institutes of Standard and Technology, Gaithersburg, MD, USA) Mass Spectral Search Program-2009 version 2 for the documentation of homologous compounds over Agilent Mass-Hunter Qualitative Analysis B.05.00 software.

We subjected the ethyl acetate and acetone fractions to gas chromatography-mass spectrometry (GC–MS) analysis, using Agilent technologies model 7890B GC System coupled with Pegasus HT High Throughput TOFMS (Leco Corp., MI, USA). We injected an aliquot of an extract of 1 ml into the GC–MS apparatus. Next, we used Agilent J&W HP-5MS (phenylmethyl siloxane, length 30 m, Dia. 0.32 mm, Film, 0.25 µm) analytic column to separate components under an inert atmosphere of helium (1.5 mL/min). Other standardized parameters utilized during the process: oven temperature of 80 °C (2 min) was increased to a temperature of 300 °C at the rate of 3 °C/min, solvent delay time was 5 min, inlet line temperature was 225 °C, and ion source temperature was 250 °C. Mass spectra were taken at 70 eV and the acquisition mode-scan was 20–1000 amu while sixty-four (64) minutes was the GC run time. We achieved the interpretation of mass spectrum and the documentation of phytochemicals present in the fractions via the database of NIST libraries.

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