Antioxidant Metabolites from Justicia gendarussa Fractions based on UHPLC-Q-Orbitrap-HRMS Metabolomics and Density Functional Theory Approach

The herb Justicia gendarussa, known as gendarussa, has the potential as an antioxidant. A previous study has identified that ethanol extract from J. gendarussa has the highest antioxidant activity. The study profiled the metabolites of n-hexane, ethyl acetate, n-butanol, and water fractions of its ethanol extract using ultrahigh performance liquid chromatography-high resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS). The antioxidant capacities of metabolites were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylenebenzothiazoline)-6-sulfonic acid (ABTS), ferric ion reducing antioxidant power (FRAP), and cupric reducing antioxidant capacity (CUPRAC) method combined with chemometric technique. Finally, a computational density functional theory (DFT) study was carried out to ensure the ability of the active compounds as an antioxidant. The results showed that the highest antioxidant capacity of the DPPH method was on the n-butanol fraction of 40.5 µmol (trolox equivalent) TE/g dry fraction, while the ABTS, FRAP, and CUPRAC methods were on the ethyl acetate fraction with antioxidant capacity were 69.0, 189, and 624 µmol TE/g dry fraction. Principal component analysis (PCA) and heatmap succeeded in classifying the metabolites in J. gendarussa fraction based on polarity. There were 37 and 21 identified metabolites in positive and negative ionization, respectively. Using orthogonal partial least square-discriminant analysis (OPLS-DA), the predictive active antioxidant metabolites were gendarusin A, justridusamide A, sesamol, 6-{[(Benzyloxy)carbonyl]amino}-6-deoxy-1,2-O-isopropylidene-α-D-glucofuranose, methyl 2-[cyclohex-2-en-1-yl(hydroxy)methyl]-3-hydroxy-4-(2-hydroxyethyl)-3-methyl-5-oxoprolinate, and 3,4-Dihydroxy-6-(N-ethylamino) benzamide. In addition, 14 unknown metabolites in the ethyl acetate fraction were predicted to be active antioxidants, which can be isolated for further research to obtain new metabolites. Density functional theory (DFT) proves that 6-{[(Benzyloxy)carbonyl]amino}-6-deoxy-1,2-O-isopropylidene-α-D-glucofuranose and gendarusin A have a smaller energy gap than trolox as a standard, indicating it has a better antioxidant ability. Therefore, those two compounds are the best antioxidant compounds. Additional tests in vitro or in vivo research on the isolation compounds results need to be carried out to prove the research results further.