No abstract

subsp is a wild plant distributed in subtropical and east Africa extending from the north to the southern parts of Egypt. Widely used in folk medicine, it is the source of the diuretic sesquiterpene proximadiol. Nuclear magnetic resonance metabolomic analysis of polar extracts of shoots from wild, greenhouse, somatic embryos, and direct and indirect organogenic in vitro cultures was carried out. Metabolic profiling yielded 39 compounds, of which common metabolites were 15 (38.4%). Unique metabolites were trehalose (2.5%) in the wild plants, 2-hydroxylisobutyrate, galactarate and tyrosine (7.6%) in indirect organogenic shoots. Tartrate was found only in direct regenerated shoots (2.5%). Metabolites identified in greenhouse and embryogenic shoots showed no unique compounds. Multivariate analysis revealed significant differences between all tested shoots. 4-aminobutyrate, alanine, glutamine, glucose, fructose, and sucrose were the most significantly different metabolites. Proximadiol was identified and quantitatively measured from the non-polar extract of different types of shoots using gas chromatography and mass spectrometry (GC-MS). Concentrations ranged from 3.6 ± 0.03 to 198.6 ± 7.2 µg/100 mg dry weight in regenerated shoots from somatic embryogenesis and in wild plant shoots, respectively. Direct organogenesis yielded the highest in vitro concentration (20.3 ± 0.5 µg/100 mg dry weight). This study reported the metabolic profiling of polar extract and identified primary metabolites that are unique to the wild type and shoots regenerated from different in vitro cultures. Proximadiol was quantified and the in vitro culture system yielding the highest concentration relative to the wild plant was identified.

The inhibition of the β-glucuronidase released from gut bacteria is associated with specific health-related benefits. Though a number of β-glucuronidase inhibition assays are currently in use, none of them can directly measure the relevant activity of each single constituent in a complex mixture, without prior separation and tedious isolation of the pure compounds. Thus, the hyphenation of the high performance thin layer chromatography (HPTLC) technique with a β-glucuronidase inhibition assay was investigated and successfully demonstrated for the first time. A colorimetric as well as fluorometric detection of the inhibitors was achieved using 5-bromo-4-chloro-3-indolyl-β-D-glucuronide as a substrate. Hence, β-glucuronidase inhibitors were detected as bright zones against an indigo blue or fluorescent background. The established method was optimized and validated employing the well-known inhibitor d -saccharic acid 1,4-lactone monohydrate. As proof of concept, the suitability of the new workflow was verified through analysis of two botanical extracts, Primula boveana and silymarin flavonolignans from Silybum marianum fruits. The found inhibitors were identified by spectroscopic methods; one of them, 3ʹ- O -(β-galactopyranosyl)-flavone, is here described as a newly isolated natural compound. The new hyphenation HPTLC-UV/Vis/FLD-β-glucuronidase inhibition assay-HRMS covers four orthogonal dimensions, i.e. separation, spectral detection, biochemical activity and structural characterization, in a highly targeted time- and material-saving workflow for analysis of complex or costly mixtures.