Differentiating calli of Catharanthus roseus according to their relative locations by 1H NMR and principal-components analysis

Abstract

A bioassay-guided approach was employed to identify the glycolate oxidase (GOX, EC 1.1.3.15) inhibitory agent in a Tribulus terrestris L. extract using a microplate enzyme assay. This permitted the isolation of two flavonols quercetin (1) and kaempherol (2). Inhibition kinetics revealed quercetin (1) as non-competitive inhibitor of GOX with K m 2.5 mM and K i 1.8 mM, whereas kaempherol (2) showed competitive inhibition with K m 4-20 mM and K i 0.5 mM. Molecular docking studies were performed on spinach GOX with PDB entry 1al7 using BioMed CAChe software (v 6.1.1). The docking studies revealed significant dock score of the flavonols 1 and 2, i.e. −41.122 and −42.735 kcal/mol, respectively, in comparison with the known ligand to this enzyme HST -[4-Carboxy-5-(1-Pentyl) Hexylsulfanyl-1, 2, 3-Triazole] −44.643 kcal/mol. High protein sequence similarities and ligand binding specificities of the enzyme 1al7 from spinach and 2rdt from human encouraged us to extrapolate the results obtained with spinach GO to that of human GO enzyme. From this study it is concluded that kaempherol is a potential agent for optimization in effective management of urolithiasis in near future.Including a plant cell suspension culture first involves transferring the callus into liquid media. However, there are no objective criteria for selecting the location of the callus to be transferred. In this study, the inner and outer cells of Catharanthus roseus with various elicitors in solid-state cultures were differentiated by 1H NMR (nuclear magnetic resonance) spectrometry and principalcomponents analysis (PCA), with the aim of selecting high-yield cell lines. It was found that the samples of various elicitors and relative locations could be separated in PCA-derived score plots. Especially, there was a clear separation between nontreated samples and those cotreated with silver nitrate and methyl jasmonate. Loading-plot analysis was therefore applied to data obtained from nontreated samples and those cotreated with silver nitrate and methyl jasmonate to investigate the separation of major metabolites on score plots. The levels of valine, lactic acid/threonine, alanine, arginine, acetic acid, malic acid, succinic acid, citric acid, asparagine, choline, fumaric acid, phenylalanine, tryptophan, and formic acid were higher in the inner callus than in the outer callus, whereas glutamic acid, 2-oxoglutaric acid, oxalacetic acid, malonic acid, sucrose, lactose, and glucose dominated in the outer callus. The results obtained in this study suggest that inner and outer calli can be differentiated by 1H NMR-based metabolomic analysis, which also supplies basic information for selecting of adequate calli for use in suspension culture systems.For several years the main interest of our group is a cucumber (Cucumis sativus var. Borschagowski) chloroplast genome. Recently we have attempted to characterize plastosome expression using tiling microarray and to detect its response to chemically induced light stress. To induce the stress response we have used the ele...