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 4ʹ-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.
The aim of this study was to develop a validated high-performance thin-layer chromatography (HPTLC) procedure for resolution of chemical constituents and identification and quantification of two selected natural anticancer compounds, stigmasterol (PD-1) and cinnamic acid (PD-2), in Pluchea dioscoridis chloroform fraction (PDCF). The chromatographic estimations were conducted on normal HPTLC (20 cm × 10 cm glass-backed silica gel 60 F254) plates with chloroform-methanol-acetic acid (93:5:2, V/V) used as the mobile phase. para-Anisaldehyde was used for the derivatization of the developed plate, and compact spots were scanned at λmax = 513 nm. A well resolved, compact, and intense peaks of PD-1 and PD-2 were recorded at RF = 0.57 and 0.19, respectively. The proposed analytical method for both biomarker compounds was found to be handy, simple, precise, linear (%RSD = 1.03–1.45), accurate (98.91–99.14%), reliable, and sensitive for the analysis of both bio-markers. The LOD/LOQ (ng) for PD-1 and PD-2 were recorded as 38.73/117.37 and 42.58/129.04, respectively, in the linearity range of 200–1400 ng per spot. The obtained result showed maximum quantities of PD-1 and PD-2 (5.36 and 16.98 μg mg−1, respectively). The developed HPTLC was found to be suitable for the routine analysis of these 2 biomarkers in the chloroform fraction of Pluchea dioscoridis and can be further employed in the process quality control of herbal formulations containing the said biomarkers.
In this paper we describe a sensitive and reproducible reversed-phase high-performance liquid chromatography (HPLC) method with photodiode-array detection for isolation and quantification of the bioactive hydrophilic constituent 7-(1-O-β-d-galacturonide-4′-(1-O-β-d-glucopyranosyl)-3′,4′,5,7-tetrahydroxyflavone, 1, from the seeds of Cuminum cyminum. Compound 1 was separated isocratically on a C18 preparative column, in high purity, after removal of solvents. The purity and identity of the compound were established by use of LC-mass spectrometry and by spectroscopic techniques (1H and 13C NMR). The purity of 1 was also confirmed by HPTLC.