Fruits of Silybum marianum (L.) Gaernt are the main source of taxifolin derived flavonolignans. Together, these molecules constitute a mixture called silymarin with many useful applications for cosmetic and pharmaceutic industries. Here, a validated method for the separation of the silymarin constituents has been developed to ensure precision and accuracy in their quantification. Each compound was separated with a high reproducibility. Precision and repeatability of the quantification method were validated according to the AOAC recommendations. The method was then applied to study the natural variability of wild accessions of S. marianum. Analysis of the variation in the fruits composition of these 12 accessions from Pakistan evidenced a huge natural diversity. Correlation analysis suggested a synergistic action of the different flavonolignans to reach the maximal antioxidant activity, as determined by cupric ion reducing antioxidant capacity (CUPRAC) and ferric reducing antioxidant power (FRAP) assays. Principal component analysis (PCA) separated the 12 accessions into three distinct groups that were differing from their silymarin contents, whereas hierarchical clustering analysis (HCA) evidenced strong variations in their silymarin composition, leading to the identification of new silybin-rich chemotypes. These results proved that the present method allows for an efficient separation and quantification of the main flavonolignans with potent antioxidant activities.

Melatonin as plant growth regulator induces differential effects on metabolites that are responsible for reduction, capping and stabilization of zinc oxide nanoparticles. Phytochemical analysis of callus cultures was performed and results were compared with callus cultures supplemented with other plant growth regulators (α-napthalene acetic acid, 2,4-dichlorophenoxy acetic acid and thidiazuron). Highest total phenolic and flavonoid content [42.23 mg of gallic acid equivalent (GAE) g−1 DW and 36.4 mg of (quercetin equivalent) g−1 DW, respectively] were recorded at melatonin (1.0 µM) + NAA (13.5 µM). ZnONPs were synthesized from NAA (13.5 µM) and melatonin (1.0 µM) + NAA (13.5 µM)-induced calli extracts separately and characterized via X-ray diffraction, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). FTIR analysis confirmed the presence of phenolics and flavonoids that were mainly found responsible for reduction and capping of ZnONPs. SEM analysis showed triangular shaped ZnONPs synthesized from melatonin + NAA callus extract and these NPs were more dispersed as compared to the spherical-agglomerates of ZnONPs synthesized from NAA-mediated callus extract. Melatonin + NAA callus extract-mediated ZnONPs (having smaller size) were more potent against multiple drug resistant bacterial strains, e.g. Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa by producing zone of inhibitions 17 ± 0.76 mm,10 ± 0.57 mm and 13 ± 0.54 mm, respectively.