Simultaneous quantification of two phenolic biomarkers by a validated high-performance thin-layer chromatographic method in antimicrobial and antioxidant active ethyl acetate fraction of Allium cepa L. (Peel)

Abstract

In this study, we have developed a validated high-performance thin-layer chromatographic (HPTLC) method for the simultaneous quantification of two phenolic biomarkers, protocatechuic acid (compound 1) and quercetin 4'-O-β-d-glucopyranoside (compound 2) in antimicrobial and antioxidant active A. cepa ethyl acetate extract (ACEAE). The quantitative analysis was carried out on normal- phase HPTLC (glass-backed silica gel 60 F254) plates with solvents toluene, ethyl acetate, and formic acid in the ratio of 3:6:1, v/v (as the mobile phase). Well-resolved, compact, and intense peaks of compound 1 (Rf = 0.56+ 0.001) and compound 2 (Rf = 0.05+0.001) were found at λmax = 275 nm. The linear regression equation / r2 for compound 1 (Y = 8.89X + 250.71 / 0.994) and compound 2 (Y = 6.64X + 209.34 / 0.998) in the concentration range of 100-700 ng spot-1 indicated good linear relationship. The low values of percent relative standard deviation (%RSD) for intra-day / inter-day precision of compound 1 (1.14-1.26 / 1.08-1.23) and compound 2 (0.97-1.18 / 0.93-1.16) suggested that the method is precise. The (%) recoveries for compound 1 / compound 2 were found as 98.07-99.55 / 98.20- 99.89 which confirms the good accuracy of the proposed method. The quantities (%w/w) of compound 1 / compound 2 in ACEAE were found as 18.84% / 13.64% of the dried weight of the extract. In vitro 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay showed the promising free radical scavenging activity of ACEAE (69.00+ 2.99%) and compound 2 (63.86+ 2.02%) which were comparable to ascorbic acid tested at 400 μg mL-1. ACEAE was found to be highly active against all tested bacterial strains, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa; however, Candida albicans was found to be susceptible to both compound 2 and ACEAE. The presence of compound 2 in high quantity in the ethyl acetate fractions of A. cepa peel (ACEAE) validated its antimicrobial and antioxidant property. The above developed HPTLC method can be further employed in the analysis of these markers in marketed formulations and in the quality control of herbal drugs.