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.

Antibacterial and Antibiofilm Effects of Allelopathic Compounds Identified in Medicago sativa L. Seedling Exudate against Escherichia coli.

In this study, the allelopathic properties of Medicago sativa L. (alfalfa) seedling exudates on the germination of seeds of various species were investigated. The compounds responsible for the allelopathic effects of alfalfa were identified and characterized by employing liquid chromatography ion mobility high-resolution mass spectrometry. Crude exudates inhibited the germination of seeds of all various plant species tested. Overall, nine compounds in alfalfa were identified and quantified. The most predominant compounds were a hyperoside representing a flavonoid glucoside, the non-proteinogenic amino acid canavanine, and two dipeptides, identified as H-Glu-Tyr-OH and H-Phe-Glu-OH. The latter corresponds to the first finding that dipeptides are exuded from alfalfa seedlings. In addition, the antibacterial and antibiofilm activities of alfalfa exudate and its identified compounds were elucidated. Both hyperoside and canavanine revealed the best antibacterial activity with minimum inhibitory concentration (MIC) values that ranged from 8 to 32 and 32 to 256 µg/mL, respectively. Regarding the antibiofilm action, hyperoside and canavanine caused a decline in the percentage of E. coli isolates that possessed a strong and moderate biofilm-forming potential from 68.42% to 21.05% and 31.58%, respectively. Studies on their inhibiting effects exhibit that these major substances are predominantly responsible for the allelopathic and antimicrobial effects of the crude exudates.

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.

Biochemistry and Molecular Basis of Intracellular Flavonoid Transport in Plants.

Flavonoids are a biochemically diverse group of specialized metabolites in plants that are derived from phenylalanine. While the biosynthesis of the flavonoid aglycone is highly conserved across species and well characterized, numerous species-specific decoration steps and their relevance remained largely unexplored. The flavonoid biosynthesis takes place at the cytosolic side of the endoplasmatic reticulum (ER), but accumulation of various flavonoids was observed in the central vacuole. A universal explanation for the subcellular transport of flavonoids has eluded researchers for decades. Current knowledge suggests that a glutathione S-transferase-like protein (ligandin) protects anthocyanins and potentially proanthocyanidin precursors during the transport to the central vacuole. ABCC transporters and to a lower extend MATE transporters sequester anthocyanins into the vacuole. Glycosides of specific proanthocyanidin precursors are sequestered through MATE transporters. A P-ATPase in the tonoplast and potentially other proteins generate the proton gradient that is required for the MATE-mediated antiport. Vesicle-mediated transport of flavonoids from the ER to the vacuole is considered as an alternative or additional route.

The complexity of sound quantification of specialized metabolite biosynthesis: The stress related impact on the alkaloid content of Catharanthus roseus.

Medicinal plants grown under stress conditions reveal higher concentrations of relevant specialized metabolites than well-watered plants, putatively due to an enhanced biosynthesis. Yet, stress also reduced the biomass gain. Accordingly, the concentration increase in comparison to control plants could also be due to lesser biomass employed as the reference value, whereas the rate of biosynthesis may remain unchanged. For an unequivocal proof that stress indeed enhances the biosynthesis, the total amount of the substances per plant has to be determined. In this study, we investigated the stress-induced impact on the alkaloids accumulated in Catharanthus roseus and quantified both, the changes in concentration and in the entire amount of alkaloids. At any time, all Catharanthus roseus plants grown under drought stress exhibited a markedly higher alkaloid concentration compared to the well-watered controls. However, by calculating the entire alkaloid content per plant, a corresponding increment occurred only within the first two weeks of drought stress. Thereafter, no significant differences among drought treatments and control were detected. Finally, within the last week, the alkaloid content per plant decreased markedly, although there was a meaningfully higher concentration of alkaloids in the drought-stressed plants. In contrast, when plants had been exposed to high salt concentrations, the alkaloid concentrations were quite the same in stressed and control plants. The related total contents were significantly lower in plants exposed to salt stress. These results display that both phenomena, an increased rate of biosynthesis and lesser reference values, i.e., the biomass, contribute to the stress-related increase in the concentration of natural product. Moreover, it has to be considered that the enhancement of biosynthesis could be due to either an "active" up-regulation of biosynthetic capacity or a "passive" shift caused by the over-reduced status as a result of the stress-induced stomatal closure.

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.

Stress-induced modification of indole alkaloids:Phytomodificines as a new category of specialized metabolites.

This study focuses on the elucidation of the stress-induced reverse changes of major indole alkaloids in Vinca minor, primarily on the postulated conversion of vincamine and vincadifformine to yield 9-methoxyvincamine, minovincine, and minovincinine, respectively. By applying the P450 enzyme inhibitors, naproxen and resveratrol, it was shown that the oxidative reaction involved in the postulated conversion of vincamine and vincadifformine is catalyzed by cytochrome P450 enzymes. In combination with the identification of 9-hydroxyvincamine as a postulated intermediate, this result confirms that the observed stress-induced reverse changes in the alkaloid pattern are caused by modifications of the alkaloids which regularly accumulate in the healthy Vinca minor plants. Up to now, just two main types of defense compounds are distinguished: phytoalexins, which are synthesized de novo from primary metabolites and phytoanticipins, which are constitutively present in plants - either intrinsically active or are activated after cell death by hydrolysis or oxidation of the precursors. In contrast, the results presented in this paper demonstrate that indole alkaloids, representing typical phytoanticipins, are just slightly modified in response to a stress-related elicitation. Accordingly, these modified alkaloids neither represent classical phytoalexins (being synthesized de novo), nor can they be classified as phytoanticipins, since modification does not occur postmortem. Consequently, we propose a new category for these modified alkaloids that we call phytomodificines.

Uptake and modification of umbelliferone by various seedlings.

Inspired by the recently discovered phenomenon of "horizontal natural product transfer" we investigated the putative uptake of phenolic specialized metabolites. Umbelliferone was chosen for this case study, since this coumarin as well as its derivatives can easily be determined by HPLC analyses. Barley (Hordeum vulgare L.), radish (Raphanus sativus L.), pea (Pisum sativum L.), flax (Linum usitatissimum L.), and garden cress (Lepidium sativum L.) were cultivated in hydroponic media, to which the coumarin was applied. Uptake of umbelliferone was verified by corresponding HPLC analyses of extracts obtained from the aerial parts of the seedlings. In all cases, a tremendous uptake of umbelliferone was observed. In plants that genuinely contain coumarins, the umbelliferone taken up was modified: in garden cress, it was hydroxylated and glucosylated to yield esculin, while in barley seedlings, the imported umbelliferone was modified by methoxylation to yield scopoletin. Corresponding reactions are known from modifications of xenobiotics to be catalyzed by cytochrome P450 enzymes. Accordingly, in an additional approach, umbelliferone was applied together with naproxen, which is reported to reduce enzyme activity of P450 enzymes. As predicted, the conversion of umbelliferone to scopoletin in barley as well as the modification to esculin in garden cress was strongly reduced by the addition of naproxen. These data for the first time demonstrate that - in addition to alkaloids - also phenolic compounds are taken up by various acceptor plants. Apart from the leaching of rotting plants, coumarins are known to be exuded by many plants. Accordingly, these compounds are frequently present in soils and will be taken up. These coherences imply that the horizontal natural product transfer might represent a more general phenomenon in plant ecology. Moreover, this study outlines that - in analogy to the modification of xenobiotics - also natural products taken up are modified in the acceptor plants.

Impact of drought and salt stress on the biosynthesis of alkaloids in Chelidonium majus L.

When plants are exposed to various stress situations, their alkaloid concentration frequently is enhanced. This well-known phenomenon is presumably due to a passively enhanced rate of biosynthesis, caused by greatly elevated concentrations of NADPH in stressed plants. Here, we used Chelidonium majus L. plants, which accumulate high concentrations of dihydrocoptisine in their leaves, to study the impact of drought and salt stress on the biosynthesis and accumulation of alkaloids. In comparison to well-watered controls, in the transcriptome of the gene encoding the key enzyme in alkaloid biosynthesis, stylopine synthase, is enhanced in stressed C. majus plants. If we presuppose that increased transcript levels correlate with increased enzymatic activity of the gene products, these data indicate, for the first time, that stress-related increases in alkaloid concentration might not only be caused by the well-known stress-related passive shift, but may also be due to an enhancement of enzymatic capacity.

Uptake of nicotine from discarded cigarette butts - A so far unconsidered path of contamination of plant-derived commodities.

This study aimed to elucidate the origin of the widespread nicotine contamination of plant-derived commodities, by conducting field experiments with various herbs and spice plants. By scattering tobacco and cigarette butts on the field and subsequent nicotine analyses of the acceptor plants, we verified that the alkaloid is leached out into the soil and is taken up by the crop plants. This path of contamination pertains even when there is only one cigarette butt per square meter. Even such minor pollution results - at least in the case of basil and peppermint - in considerable high nicotine contaminations, which exceed the maximum residue level by more than 20-fold. The data reported here clearly outline the large practical relevance of this soil-borne contamination path and imply that unthoughtful disposal of cigarette butts in the field by farm workers may be the reason for the widespread occurrence of nicotine contamination in plant-derived commodities. Therefore, such misbehavior needs to be prevented using education and sensitization, and by including this issue into the guidelines of good agricultural practice.

Crystal structure of akuammicine, an indole alkaloid from Catharanthus roseus.

The title compound, C20H22N2O2, an alkaloid isolated from the Madagascar periwinkle, crystallizes in P1 with two independent but closely similar mol-ecules in the unit cell. The mol-ecules are linked into pairs by two N-H⋯O=C hydrogen bonds. The absolute configuration was confirmed by anomalous dispersion effects as S at the 3 and 15 positions, and R at the 7 position.

Treatment of Vinca minor Leaves with Methyl Jasmonate Extensively Alters the Pattern and Composition of Indole Alkaloids.

Alkaloids extracted from mature Vinca minor leaves were fractionated by preparative HPLC. By means of HRMS and NMR data, the main alkaloids were identified as vincamine, strictamine, 10-hydroxycathofoline, and vincadifformine. Upon treatment with methyl jasmonate (MeJA), the pattern and composition of the indole alkaloids changed extensively. While 10-hydroxycathofoline and strictamine concentrations remained unaltered, vincamine and vincadifformine levels showed a dramatic reduction. Upon MeJA treatment, four other indole alkaloids were detected in high quantities. Three of these alkaloids have been identified as minovincinine, minovincine, and 9-methoxyvincamine. Whereas minovincinine and minovincine are known to occur in trace amounts in V. minor, 9-methoxyvincamine represents a novel natural product. Based on the high similarities of vincamine and 9-methoxyvincamine and their inverse changes in concentrations, it is postulated that vincamine is a precursor of 9-methoxyvincamine. Similarly, vincadifformine seems to be converted first to minovincinine and finally to minovincine. Because MeJA treatment greatly altered the alkaloidal composition of V. minor, it could be used as a potential elicitor of alkaloids that are not produced under normal conditions.

Horizontal natural product transfer: A potential source of alkaloidal contaminants in phytopharmaceuticals.

BACKGROUND: It was recently shown that nicotine and pyrrolizidine alkaloids that leach out from decomposing plant material (donor plants) are subsequently taken up by the roots of acceptor plants and translocated into their leaves. Furthermore, it is well established that plant roots take up xenobiotics, generally by simple diffusion, and that this passive import depends on the physico-chemical properties of the substances. HYPOTHESIS: Based on the well-known uptake of xenobiotics, we assumed that in analogy, the uptake of alkaloids, which are leached out from plant material (donor plants) represents a quite general feature of plant biology. METHODS: Using barley as a model plant, we analyzed the uptake of alkaloids by applying them to Hordeum vulgare seedlings. Based on HPLC analyses, the presence of the particular alkaloids in the acceptor plants was determined. RESULTS: We demonstrated that numerous alkaloids of different structural types are able to diffuse through biomembranes and are taken up by acceptor plants. In contrast, an uptake of quaternary alkaloids, with a permanent positive charge, could not be detected. CONCLUSION: As most alkaloidal plants generally die back afield, and the corresponding natural products are leached out into the soil. Our findings have substantial relevance for all plant-derived commodities, especially for the production of phytopharmaceuticals and the related safety issues. Moreover, the evidence that plants are inherently able to take up alkaloids from the soil, which are derived from other plants, will alter our appraisal of plant-plant interactions. In this context, the classical definition of xenobiotics, which are considered as "non-natural" substances, might be also extended by including natural products leached out into the soil.

Cloning and Characterization of Cheilanthifoline and Stylopine Synthase Genes from Chelidonium majus.

The most prominent alkaloid of Chelidonium majus is dihydrocoptisine, revealing the characteristic benzophenanthridine skeleton. To date, any informationon on the enzymes responsible for its biosynthesis and the related genes in C. majus is lacking. Based on sequence similarities to the corresponding methylenedioxy bridge-forming Cyt P450 enzymes involved in isoquinoline alkaloid biosynthesis in Eschscholzia californica, genes for a cheilanthifoline synthase and a stylopine synthase from C. majus were isolated, sequenced and heterologously expressed in yeast. The activity of the heterologously expressed Cyt P450 enzymes was determined in situ as well as on the basis of microsomal fractions. It was shown that cheilanthifoline synthase (c8931) converts scoulerine into cheilanthifoline, the latter subsequently being converted to stylopine by the action of a stylopine synthase (c1128). Based on the well-known instability of stylopine, it can be assumed that in vivo-under the acidic conditions in the vacuole-this alkaloid is converted to dihydrocoptisine, which accumulates in C. majus leaves. Both methylenedioxy bridge-forming Cyt P450 enzymes from C. majus are characterized by their high substrate specificity. Apart from their genuine substrates, i.e. scoulerine and cheilanthifoline, cheilanthifoline synthase and stylopine synthase do not accept other substrates tested; the only alternative substrate identified was scoulerine, which is converted by stylopine synthase to yield minor amounts of nandinine. Quantitative real-time PCR revealed that the expression of cheilanthifoline synthase and stylopine synthase genes is very similar in both roots and leaves from C. majus, although the alkaloid accumulation patterns in these organs are quite different.

Impact of drought stress on specialised metabolism: Biosynthesis and the expression of monoterpene synthases in sage (Salvia officinalis).

In previous experiments, we demonstrated that the amount of monoterpenes in sage is increased massively by drought stress. Our current study is aimed to elucidate whether this increase is due, at least in part, to elevated activity of the monoterpene synthases responsible for the biosynthesis of essential oils in sage. Accordingly, the transcription rates of the monoterpene synthases were analyzed. Salvia officinalis plants were cultivated under moderate drought stress. The concentrations of monoterpenes as well as the expression of the monoterpene synthases were analyzed. The amount of monoterpenes massively increased in response to drought stress; it doubled after just two days of drought stress. The observed changes in monoterpene content mostly match with the patterns of monoterpene synthase expressions. The expression of bornyl diphosphate synthase was strongly up-regulated; its maximum level was reached after two days. Sabinene synthase increased gradually and reached a maximum after two weeks. In contrast, the transcript level of cineole synthase continuously declined. This study revealed that the stress related increase of biosynthesis is not only due to a "passive" shift caused by the stress related over-reduced status, but also is due - at least in part-to an "active" up-regulation of the enzymes involved.

Interspecific transfer of pyrrolizidine alkaloids: An unconsidered source of contaminations of phytopharmaceuticals and plant derived commodities.

Many plant derived commodities contain traces of toxic pyrrolizidine alkaloids (PAs). The main source of these contaminations seems to be the accidental co-harvest of PA-containing weeds. Yet, based on the insights of the newly described phenomenon of the horizontal transfer of natural products, it is very likely that the PA-contaminations may also be due to an uptake of the alkaloids from the soil, previously being leached out from rotting PA-plants. The transfer of PAs was investigated using various herbs, which had been mulched with dried plant material from Senecio jacobaea. All of the acceptor plants exhibited marked concentrations of PAs. The extent and the composition of the imported PAs was dependent on the acceptor plant species. These results demonstrate that PAs indeed are leached out from dried Senecio material into the soil and confirm their uptake by the roots of the acceptor plants and the translocation into the leaves.

13,14-dihydrocoptisine--the genuine alkaloid from Chelidonium majus.

The genuine major benzylisoquinoline alkaloid occurring in the traditional medicinal plant greater celandine (Chelidonium majus L.) is 13,14-dihydrocoptisine and not - as described previously - coptisine. Structure of 13,14-dihydrocoptisine was elucidated. The discrepancy between the alkaloid pattern of the living plants and that of detached and dried leaves is due to the rapid and prompt conversion of 13,14-dihydrocoptisine to coptisine in the course of tissue injuries. Indeed, apart from the major alkaloid, some minor alkaloids might also be converted; this however is not in the centre of focus of this paper. This conversion is initiated by the change of pH. In vivo 13,14-dihydrocoptisine is localized in the acidic vacuoles, where it is stable. In contrast, in the neutral milieu, which results when vacuoles are destroyed in the course of tissue injuries, the genuine alkaloid is oxidized to yield coptisine. Accordingly, when alkaloids from C.majus should be analyzed, any postmortal conversion of 13,14-dihydrocoptisine has to be prevented.