Artemisia annua L. (AA) has shown for many centuries important therapeutic virtues associated with the presence of artemisinin (ART). The aim of this study was to identify and quantify ART and other secondary metabolites in ethanolic extracts of AA and evaluate the biological activity in the presence of an inflammatory stimulus. In this work, after the extraction of the aerial parts of AA with different concentrations of ethanol, ART was quantified by HPLC and HPLC-MS. In addition, anthocyanins, flavanols, flavanones, flavonols, lignans, low-molecular-weight phenolics, phenolic acids, stilbenes, and terpenes were identified and semi-quantitatively determined by UHPLC-QTOF-MS untargeted metabolomics. Finally, the viability of human neuroblastoma cells (SH-SY5Y) was evaluated in the presence of the different ethanolic extracts and in the presence of lipopolysaccharide (LPS). The results show that ART is more concentrated in AA samples extracted with 90% ethanol. Regarding the other metabolites, only the anthocyanins are more concentrated in the samples extracted with 90% ethanol. Finally, ART and all AA samples showed a protective action towards the pro-inflammatory stimulus of LPS. In particular, the anti-inflammatory effect of the leaf extract of AA with 90% ethanol was also confirmed at the molecular level since a reduction in TNF-α mRNA gene expression was observed in SH-SY5Y treated with LPS.

The use of plant biostimulants contributes to more sustainable and environmentally friendly farming techniques and offers a sustainable alternative to mitigate the adverse effects of stress. Protein hydrolysate-based biostimulants have been described to promote plant growth and reduce the negative effect of abiotic stresses in different crops. However, limited information is available about their mechanism of action, how plants perceive their application, and which metabolic pathways are activating. Here we used a multi-trait high-throughput screening approach based on simple RGB imaging and combined with untargeted metabolomics to screen and unravel the mode of action/mechanism of protein hydrolysates in Arabidopsis plants grown in optimal and in salt-stress conditions (0, 75, or 150 mM NaCl). Eleven protein hydrolysates from different protein sources were used as priming agents in Arabidopsis seeds in three different concentrations (0.001, 0.01, or 0.1 μl ml^–1). Growth and development-related traits as early seedling establishment, growth response under stress and photosynthetic performance of the plants were dynamically scored throughout and at the end of the growth period. To effectively classify the functional properties of the 11 products a Plant Biostimulant Characterization (PBC) index was used, which helped to characterize the activity of a protein hydrolysate based on its ability to promote plant growth and mitigate stress, and to categorize the products as plant growth promoters, growth inhibitors and/or stress alleviator. Out of 11 products, two were identified as highly effective growth regulators and stress alleviators because they showed a PBC index always above 0.51. Using the untargeted metabolomics approach, we showed that plants primed with these best performing biostimulants had reduced contents of stress-related molecules (such as flavonoids and terpenoids, and some degradation/conjugation compounds of phytohormones such as cytokinins, auxins, gibberellins, etc.), which alleviated the salt stress response-related growth inhibition.