Chemotaxonomic and biosynthetic relationships between flavonolignans produced by Silybum marianum populations.

Flavonolignans constitute an important class of plant secondary metabolites formed by oxidative coupling of one flavonoid and one phenylpropanoid moiety. The standardized flavonolignan-rich extract prepared from the fruits of Silybum marianum is known as silymarin and has long been used medicinally, prominently as an antihepatotoxic and as a chemopreventive agent. Principal component analysis of the variation in flavonolignan content in S. marianum samples collected from different locations in Egypt revealed biosynthetic relationships between the flavonolignans. Silybin A, silybin B, and silychristin are positively correlated as are silydianin, isosilychristin, and isosilybin B. The detection of silyamandin in the extracts of S. marianum correlates with isosilychristin and silydianin content. The positive correlation between silydianin, isosilychristin, and silyamandin was demonstrated using quantitative 1H nuclear magnetic resonance spectroscopy (qHNMR). These correlations can be interpreted as evidence for the involvement of a flavonoid radical in the biosynthesis of the flavonolignans in S. marianum. The predominance of silybins A & B over isosilybin A & B in the silybin-rich samples is discussed in light of the relative stabilities of their respective radical flavonoid biosynthetic intermediates.

Proteome alterations monitored by DIGE analysis in Silybum marianum cell cultures elicited with methyl jasmonate and methyl B cyclodextrin.

Elicitation with methyl jasmonate (MeJA) or/and cyclodextrin (CD) strongly induced silymarin (Sm) accumulation in suspensions of Silybum marianum, with most of Sm isomers being detected in the culture medium. This induction provides a model platform to characterize the regulation of flavonolignan accumulation and release in response to elicitors and, with this aim, changes in the S. marianum cell proteome were investigated. The DIGE technique was used to detect statistically significant changes in the cell's proteome. A total number of 1269 unique spots were detected, 67 of which were de-regulated upon elicitation. Nineteen spots were identified by nLC-MS/MS database search analysis. Identified proteins belong to a few categories, including metabolism, stress and defense responses and transport processes. The most abundant group was represented by pathogenesis-related (PR) proteins and heat shock proteins. Two proteins related to transport process were identified and both were upregulated by elicitation. One was identified as Ras-related protein Rab11C of the Rab family of small ATPase superfamily. A second protein was identified as an ABC transporter. Some of the identified proteins are discussed with respect to their putative role in the extracellular flavonolignan accumulation in S. marianum cultures. BIOLOGICAL SIGNIFICANCE: Most approaches to increase secondary metabolite yields using plant cell cultures have been focused on the optimization of its biosynthesis. The study of other post biosynthetic events, like chemical or enzymatic modifications, transport, storage/secretion and catabolism/degradation are also biotechnologically relevant. Secretion is of particular interest since if cell cultures are to be used routinely for the commercial production, they must release the targeted metabolites into the extracellular medium. Elicitor-induced silymarin accumulation and release in S. marianum cell cultures provide a responsive model system to profile both alterations in proteins related to monolignol/flavonoid biosynthesis and to identify potential systems involved in secretion of secondary metabolites. The proteomic approach undertaken in this work has permitted identify some of the events occurring in elicited S. marianum cell cultures. One attainment of this study is that a vesicular transport mechanism could be involved in the release of this class of secondary metabolites to the extracellular compartment. This finding forms a baseline for future research on a non-sequenced medicinal plant S. marianum at molecular level.

Pyrazinecarboxamides as potential elicitors of flavonolignan and flavonoid production in Silybum marianum and Ononis arvensis cultures in vitro.

The effect of new synthetic pyrazinecarboxamide derivatives as potential elicitors of flavonolignan and flavonoid production in Silybum marianum and Ononis arvensis cultures in vitro was investigated. Both tested elicitors increased the production of flavonolignans in S. marianum callus and suspension cultures and flavonoids in O. arvensis callus and suspension cultures. Compound I, 5-(2-hydroxybenzoyl)-pyrazine-2-carboxamide, has shown to be an effective elicitor of flavonolignans and taxifoline production in Silybum marianum culture in vitro. The maximum content of silydianin (0.11%) in S. marianum suspension culture was induced by 24 h elicitor application in concentration of 1.159 × 10⁻³ mol/L. The maximum content of silymarin complex (0.08%) in callus culture of S. marianum was induced by 168 h elicitor application of a concentration 1.159 × 10⁻4 mol/L, which represents contents of silydianin (0.03%), silychristin (0.01%) and isosilybin A (0.04%) compared with control. All three tested concentrations of compound II, N-(2-bromo-3-methylphenyl)-5-tert-butylpyrazin-2-carboxamide increased the flavonoid production in callus culture of O. arvensis in a statistically significant way. The best elicitation effect of all elicitor concentrations had the weakest c3 concentration (8.36 × 10⁻6 mol/L) after 168 h time of duration. The maximum content of flavonoids (about 5,900%) in suspension culture of O. arvensis was induced by 48 h application of c3 concentration (8.36 × 10⁻6 mol/L).