Bacterial responses to plant antimicrobials: the case of alkannin and shikonin derivatives.

Alkannin, shikonin and their derivatives (A/S) are secondary metabolites produced in the roots of certain plants of the Boraginaceae family such as Lithospermum erythrorhizon Siebold & Zucc. and Alkanna tinctoria (L.) Tausch. These naphthoquinones express anti-cancer, wound healing, and antimicrobial activities. To study the interactions between endophytic bacteria isolated from A. tinctoria and the antimicrobials A/S, endophytic bacteria known to be resistant to the compounds were screened for their effect on A/S in liquid medium. Thereafter, the strain Pseudomonas sp. R-72008, was selected and tested for its ability to modify A/S in nutrient medium and minimal medium with A/S as sole carbon source. Bacterial growth was recorded, and high performance liquid chromatography-diode array and ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry analyses were performed to detect and quantify metabolites. In nutrient medium inoculated with R-72008, a decrease in the amount of A/S monomers initially present was observed and correlated with an increase of A/S oligomers. Moreover, a significant decrease of initial A/S monomers in minimal medium was correlated with bacterial growth, showing for the first time that a bacterial strain, Pseudomonas sp. R-72008, was able to use the naphthoquinones A/S as sole carbon source. This study opens new perspectives on the interactions between bacteria and plant antimicrobials.

Dynamics of alkannin/shikonin biosynthesis in response to jasmonate and salicylic acid in Lithospermum officinale.

Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.

Transcriptional dynamics of Chitinophaga sp. strain R-73072-mediated alkannin/shikonin biosynthesis in Lithospermum officinale.

Plants are colonized by a wide range of bacteria, several of which are known to confer benefits to their hosts such as enhancing plant growth and the biosynthesis of secondary metabolites (SMs). Recently, it has been shown that Chitinophaga sp. strain R-73072 enhances the production of alkannin/shikonin, SMs of pharmaceutical and ecological importance. However, the mechanisms by which this bacterial strain increases these SMs in plants are not yet understood. To gain insight into these mechanisms, we analyzed the molecular responses of Lithospermum officinale, an alkannin/shikonin producing member of Boraginaceae, to inoculation with R-73072 in a gnotobiotic system using comparative transcriptomics and targeted metabolite profiling of root samples. We found that R-73072 modulated the expression of 1,328 genes, of which the majority appeared to be involved in plant defense and SMs biosynthesis including alkannin/shikonin derivatives. Importantly, bacterial inoculation induced the expression of genes that predominately participate in jasmonate and ethylene biosynthesis and signaling, suggesting an important role of these phytohormones in R-73072-mediated alkannin/shikonin biosynthesis. A detached leaf bioassay further showed that R-73072 confers systemic protection against Botrytis cinerea. Finally, R-73072-mediated coregulation of genes involved in plant defense and the enhanced production of alkannin/shikonin esters further suggest that these SMs could be important components of the plant defense machinery in alkannin/shikonin producing species.

Gelatin nanoparticles for NSAID systemic administration: Quality by design and artificial neural networks implementation.

The present study evaluates the preparation of systemic administrated NSAID gelatin nanoparticles with the aid of quality by design and artificial neural networks (ANNs). Specifically, two different preparation techniques (i.e. nanoprecipitation and two-step desolvation) were implemented for the formulation of diclofenac sodium (DLC) gelatin nanoparticles (GNs). Preliminary screening experiments showed that in the case of nanoprecipitation the best compromise (in terms of achieving both small particle size and high encapsulation efficiency) was the use of poloxamer 407 (as stabilizer) and acetone (as non-solvent), while in the case of two-step desolvation significant effect had the use of acetone, gelatin type and bloom number (type B with bloom 150 was selected for further evaluation). Implementation of a central composite experimental design (CCD), showed that in the case of nanoprecipitation the optimum formulation can be achieved at high poloxamer, high gelatin and moderate to high glutaraldehyde (GTA used for crosslinking) concentrations, while in the case of two-step desolvation high gelatin and GTA concentrations are needed. Artificial neural networks (ANN) implementation showed significantly improved prediction ability compared to MLR, while verification experiments showed good agreement between the ANN predicted and the experimentally obtained results. SEM analysis of the optimum suggested formulations showed nanoparticles with smooth surface, while powder X-ray diffraction (XRD) analysis showed the formation of amorphously dispersed systems, and Fourier transform infrared spectroscopy (FTIR) revealed the presence of molecular interactions irrespectively of the preparation method followed. A slightly faster release profile was observed in the case of nanoprecipitation based GNs, while all formulations followed biphasic release profile.