Bio-evaluation of Untapped Alkaloids from Vinca minor Enriched by Methyl-jasmonate-induced Stress: an Integrated Approach.

The low amount of metabolites isolated from natural products is one of the challenges preventing their biological evaluation. The modulation of biosynthetic pathways by stimulating stress-induced responses in plants was proven to be a valuable tool for diversification of already known natural products. Recently, we reported the dramatic effect of methyl jasmonate (MeJA) on Vinca minor alkaloids distribution. In this study, three compounds identified as 9-methoxyvincamine, minovincinine, and minovincine are successfully isolated in good yield and subjected to several bioassays based on a network pharmacology study. The extracts and isolated compounds show weak to moderate antimicrobial and cytotoxic activities. Also, they are found to significantly promote wound healing in scratch assay, and transforming growth factor-ß (TGF-ß) modulation is suggested to be the potential pathway based on bioinformatic analysis. Hence, Western blotting is used to assess the expression of several markers related to this pathway and wound healing. The extracts and isolated compounds are able to increase the expression of Smad3 and Phosphatidylinositol-3-kinase (PI3K), while downregulating the levels of cyclin D1 and the mammalian target of rapamycin (mTOR) except for minovincine, which increases the mTOR expression, inferring that it might act through a different mechanism. Molecular docking is used to give insights on the ability of isolated compounds to bind with different active sites in mTOR. Collectively, the integrated phytochemical, in silico, and molecular biology approach reveal that V. minor and its metabolite could be repurposed for the management of dermatological disorders where these markers are dysregulated, which opens the gate to develop new therapeutics in the future.

Novel Cognitions in Allelopathy: Implications from the "Horizontal Natural Product Transfer".

Whereas the translocation of allelochemicals between plants is well established, a related general transfer of genuine specialized metabolites has not been considered so far. The elucidation of the so-called "Horizontal Natural Product Transfer" revealed that alkaloids, such as nicotine and pyrrolizidine alkaloids, which are leached out from decomposing alkaloid-containing plants (donor plants), are indeed taken up by the roots of plants growing in the vicinity (acceptor plants). Further studies demonstrated that phenolic compounds, such as coumarins or stilbenes, are also taken up by acceptor plants. Contemporary analyses from co-cultivation experiments outlined that natural products are not exclusively transferred from dead and rotting donor plant materials, but also from vital plants. In analogy to xenobiotics, the imported specialized metabolites might also be modified within the acceptor plants. As known from the uptake of xenobiotics, the import of specialized metabolites is also generally due to a simple diffusion of the substances across the biomembranes and does not require a carrier. The uptake depends in stricto sensu on the physicochemical properties of the certain compound. This article presents a current overview of the phenomenon of "Horizontal Natural Product Transfer" and discusses its relevance for our understanding of allelopathic interactions. The knowledge that specialized metabolites might in general be readily translocated from one plant into others should significantly contribute to our understanding of plant-plant interactions and-in particular-to the evolution of typical allelopathic effects, such as inhibition of growth and germination of potential competitors.

Pilot study on the uptake and modification of harmaline in acceptor plants: An innovative approach to visualize the interspecific transfer of natural products.

Substances which have been leached out from decomposing plant parts or exuded from vital plants (donor plants), are taken up by acceptor plants and subsequently modified. This phenomenon was likewise established for harmala alkaloids. Employing hydroponically grown barley seedlings, it becomes evident that harmaline and harmine are taken up by the roots of the acceptor plants. Furthermore, based on HPLC and GC-MS analyses, it was demonstrated that these alkaloids also are present in Setaria viridis plants, which grew in the direct vicinity of the alkaloid containing Peganum harmala plants. Since harmaline exhibits a bright green fluorescence, this alkaloid was employed to visualize the uptake into the acceptor plants by feeding it to roots of barley seedlings. In the further course, the imported harmaline was converted in the leaves to yield harmine, which exhibits a dark blue fluorescence. This conversion was also verified by HPLC and GC-MS analyses. Based on the massive differences in the fluorescence properties, both processes, uptake and modification in the acceptor plants, could be monitored by macroscopical studies as well as by confocal laser scanning microscopical analyses. As result, for the first time, the phenomenon of "Horizontal Natural Product Transfer" is visualized vividly.

The genuine localization of indole alkaloids in Vinca minor and Catharanthus roseus.

Based on the occurrence of indole alkaloids in so-called "chloroform leaf surface extracts", it was previously deduced that these alkaloids are present in the cuticle at the leaf surface of Catharanthus roseus and Vinca minor. As no symplastic markers were found in these extracts this deduction seemed to be sound. However, since chloroform is known to destroy biomembranes very rapidly, these data have to be judged with scepticism. We reanalyzed the alleged apoplastic localization of indole alkaloids by employing slightly acidic aqueous surface extracts and comparing the corresponding alkaloid patterns with those of aqueous total leaf extracts. Whereas in the "chloroform leaf surface extracts" all alkaloids are present in the same manner as in the total leaf extracts, no alkaloids occur in the aqueous leaf surface extracts. These results clearly show that chloroform had rapidly destroyed cell integrity, and the related extracts also contain the alkaloids genuinely accumulated within the protoplasm. The related decompartmentation was verified by the massively enhanced concentration of amino acids in aqueous surface extracts of chloroform treated leaves. Furthermore, the chloroform-induced cell disintegration was vividly visualized by confocal laser scanning microscopical analyses, which clearly displayed a strong decrease in the chlorophyll fluorescence in chloroform treated leaves. These findings unequivocally display that the indole alkaloids are not located in the apoplastic space, but exclusively are present symplastically within the cells of V. minor and C. roseus leaves. Accordingly, we have to presume that also other leaf surface extracts employing organic solvents have to be re-investigated.

Horizontal Natural Product Transfer: Intriguing Insights into a Newly Discovered Phenomenon.

Just recently, the "horizontal natural product transfer" was unveiled: alkaloids, which have been leached out from decomposing alkaloidal donor plants, are taken up by the roots of acceptor plants. In the same manner, many other natural products, such as coumarins or stilbenes, are also taken up from the soil. Recent research outlined that alkaloids are transferred also from a living donor plant to plants growing in their vicinity. In the acceptor plants, the imported natural products might be modified by hydroxylation and glucosylation. These insights will strongly impact our understanding of contamination of plant-derived commodities as well as plant-plant interactions.

Transfer of pyrrolizidine alkaloids between living plants: A disregarded source of contaminations.

To elucidate the origin of the wide-spread contaminations of plant derived commodities with various alkaloids, we employed co-cultures of pyrrolizidine alkaloid (PA) containing Senecio jacobaea plants with various alkaloid free acceptor plants. Our analyses revealed that all plants grown in the vicinity of the Senecio donor plants indeed contain significant amounts of the PAs, which previously had been synthesized in the Senecio plants. These findings illustrate that typical secondary metabolites, such as pyrrolizidine alkaloids, are commonly transferred and exchanged between living plants. In contrast to the broad spectrum of alkaloids in Senecio, in the acceptor plants nearly exclusively jacobine is accumulated. This indicates that this alkaloid is exuded specifically by the Senecio roots. Although the path of alkaloid transfer from living donor plants is not yet fully elucidated, these novel insights will extend and change our understanding of plant-plant interactions and reveal a high relevance with respect to the widespread alkaloidal contaminations of plant-derived commodities. Moreover, they could be the basis for the understanding of various so far not fully understood phenomena in cultivation of various crops, e.g. the beneficial effects of crop rotations or the co-cultivation of certain vegetables.