MYB30 and MYB14 form a repressor-activator module with WRKY8 that controls stilbene biosynthesis in grapevine.

When exposed to pathogen infection or ultraviolet (UV) radiation, grapevine (Vitis vinifera) plants rapidly accumulate the stilbenoid resveratrol (Res) with concomitant increase of stilbene synthase (STS), the key enzyme in stilbene biosynthesis. Although a few transcription factors have been shown to regulate STSs, the molecular mechanism governing the regulation of STSs is not well elucidated. Our previous work showed that a VvMYB14-VvWRKY8 regulatory loop fine-tunes stilbene biosynthesis in grapevine through protein-protein interaction; overexpression of VvWRKY8 down-regulates VvMYB14 and VvSTS15/21; and application of exogenous Res up-regulates WRKY8 expression. Here, we identified an R2R3-MYB repressor, VvMYB30, which competes with the activator VvMYB14 for binding to the common binding sites in the VvSTS15/21 promoter. Similar to VvMYB14, VvMYB30 physically interacts with VvWRKY8 through their N-termini, forming a complex that does not bind DNA. Exposure to UV-B/C stress induces VvMYB14, VvWRKY8, and VvSTS15/21, but represses VvMYB30 in grapevine leaves. In addition, MYB30 expression is up-regulated by VvWRKY8-overexpression or exogenous Res. These findings suggest that the VvMYB14-VvWRKY8-VvMYB30 regulatory circuit allows grapevine to respond to UV stress by producing Res and prevents over-accumulation of Res to balance metabolic costs. Our work highlights the stress-mediated induction and feedback inhibition of stilbene biosynthesis through a complex regulatory network involving multiple positive and negative transcriptional regulators.

NAC61 regulates late- and post-ripening osmotic, oxidative, and biotic stress responses in grapevine.

During late- and post-ripening stages, grape berry undergoes profound biochemical and physiological changes whose molecular control is poorly understood. Here, we report the role of NAC61, a grapevine NAC transcription factor, in regulating different processes involved in berry ripening progression. NAC61 is highly expressed during post-harvest berry dehydration and its expression pattern is closely related to sugar concentration. The ectopic expression of NAC61 in Nicotiana benthamiana leaves resulted in low stomatal conductance, high leaf temperature, tissue collapse and a higher relative water content. Transcriptome analysis of grapevine leaves transiently overexpressing NAC61 and DNA affinity purification and sequencing analyses allowed us to narrow down a list of NAC61-regulated genes. Direct regulation of the stilbene synthase regulator MYB14, the osmotic stress-related gene DHN1b, the Botrytis cinerea susceptibility gene WRKY52, and NAC61 itself was validated. We also demonstrate that NAC61 interacts with NAC60, a proposed master regulator of grapevine organ maturation, in the activation of MYB14 and NAC61 expression. Overall, our findings establish NAC61 as a key player in a regulatory network that governs stilbenoid metabolism and osmotic, oxidative, and biotic stress responses that are the hallmark of late- and post-ripening grape stages.

Isolation and characterization of piceatannol producing bacteria from soil in Erzurum, Turkey.

Piceatannol, resveratrol's derivative, and a valuable polyphenol has managed to become one of the most remarkable candidate molecules for drug development research, with its high bioactive properties and higher stability. On the other hand, the very low amount of piceatannol in plants which are its natural source increases the cost and limits the commercialization possibilities of the product. To overcome this bottleneck, a limited number of studies have recently shown that it is possible to produce piceatannol from the resveratrol precursor much cheaper by regioselective hydroxylation catalyzed by bacteria isolated from the soil, and the search for new bacteria of similar nature in new ecosystems has gained popularity. The aim of our study, which was prepared within this framework, is the bacterial isolate with regioselective hydroxylation potential obtained as a result of selective isolation steps; determination of resveratrol hydroxylation potentials and piceatannol product yields, investigation of possibilities to increase piceatannol yield with optimization trials and identification of isolates with the highest yield. For this purpose, 200 bacterial isolates capable of resveratrol hydroxylation were obtained from soil samples taken from Erzurum (Turkey) and its surroundings by using selective media. In the continuation of the study; resveratrol hydroxylation trials were carried out with these isolates and 55 active isolates capable of producing piceatannol by regioselective hydroxylation were selected. Then, yield improvement studies of active isolates were carried out by using different carbon sources and optimizing the culture conditions. As a result, a culture collection was created by identifying the 6 most active bacterial isolates with commercialization potential using conventional and molecular methods. These are 4 Gram-positive (Rhodococcus sp., Rhodococcus erythropolis, Paeniglutamicibacter sp., Arthrobacter sp.) and 2 Gram-negative (Shinella sp., Ensifer adhaerens) bacterial isolates. As a result of the optimization studies, three of these isolates used phenol as a biocatalyst, while the other three increased the production yield of piceatannol by using 4-hydroxyphenylacetic acid.

Prenylation of aromatic amino acids and plant phenolics by an aromatic prenyltransferase from Rasamsonia emersonii.

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii. Among a variety of aromatic substrates, RePT showed the highest substrate conversion for L-tryptophan and L-tyrosine (> 90%), yielding two mono-prenylated products in both cases. Nine phenolics from diverse phenolic subclasses were notably converted (> 10%), of which the stilbenes oxyresveratrol, piceatannol, pinostilbene, and resveratrol were the best acceptors (37-55% conversion). The position of prenylation was determined using NMR spectroscopy or annotated using MS2 fragmentation patterns, demonstrating that RePT mainly catalyzed mono-O-prenylation on the hydroxylated aromatic substrates. On L-tryptophan, a non-hydroxylated substrate, it preferentially catalyzed C7 prenylation with reverse N1 prenylation as a secondary reaction. Moreover, RePT also possessed substrate-dependent organic solvent tolerance in the presence of 20% (v/v) methanol or DMSO, where a significant conversion (> 90%) was maintained. Our study demonstrates the potential of RePT as a biocatalyst for the production of bioactive prenylated aromatic amino acids, stilbenes, and various phenolic compounds. KEY POINTS: • RePT catalyzes prenylation of diverse aromatic substrates. • RePT enables O-prenylation of phenolics, especially stilbenes. • The novel RePT remains active in 20% methanol or DMSO.

Indole Alkaloid Production by the Halo Blight Bacterium Treated with the Phytoalexin Genistein.

When Pseudomonas savastanoi pv. phaseolicola, the bacterium that causes halo blight, induces hypersensitive immunity in common bean leaves, salicylic acid and phytoalexins accumulate at the site of infection. Both salicylic acid and the phytoalexin resveratrol exert antibiotic activities and toxicities in vitro, adversely disrupting the P. savastanoi pv. phaseolicola proteome and metabolism and stalling replication and motility. These efficacious properties likely contribute to the cessation of bacterial spread in beans. Genistein is an isoflavonoid phytoalexin that also accumulates during bean immunity, so we tested its antibiotic potential in vitro. Quantitative proteomics revealed that genistein did not induce proteomic changes in P. savastanoi pv. phaseolicola in the same way that salicylic acid or resveratrol did. Rather, a dioxygenase that could function to metabolize genistein was among the most highly induced enzymes. Indeed, high-throughput metabolomics provided direct evidence for genistein catabolism. Metabolomics also revealed that genistein induced the bacterium to produce indole compounds, several of which had structural similarity to auxin. Additional mass spectrometry analyses proved that the bacterium produced an isomer of the auxin indole-3-acetic acid but not indole-3-acetic acid proper. These results reveal that P. savastanoi pv. phaseolicola can tolerate bean genistein and that the bacterium likely responds to bean-produced genistein during infection, using it as a signal to increase pathogenicity, possibly by altering host cell physiology or metabolism through the production of potential auxin mimics.

Metabolic Engineering of Corynebacterium glutamicum for the Production of Flavonoids and Stilbenoids.

Flavonoids and stilbenoids, crucial secondary metabolites abundant in plants and fungi, display diverse biological and pharmaceutical activities, including potent antioxidant, anti-inflammatory, and antimicrobial effects. However, conventional production methods, such as chemical synthesis and plant extraction, face challenges in sustainability and yield. Hence, there is a notable shift towards biological production using microorganisms like Escherichia coli and yeast. Yet, the drawbacks of using E. coli and yeast as hosts for these compounds persist. For instance, yeast's complex glycosylation profile can lead to intricate protein production scenarios, including hyperglycosylation issues. Consequently, Corynebacterium glutamicum emerges as a promising alternative, given its adaptability and recent advances in metabolic engineering. Although extensively used in biotechnological applications, the potential production of flavonoid and stilbenoid in engineered C. glutamicum remains largely untapped compared to E. coli. This review explores the potential of metabolic engineering in C. glutamicum for biosynthesis, highlighting its versatility as a cell factory and assessing optimization strategies for these pathways. Additionally, various metabolic engineering methods, including genomic editing and biosensors, and cofactor regeneration are evaluated, with a focus on C. glutamicum. Through comprehensive discussion, the review offers insights into future perspectives in production, aiding researchers and industry professionals in the field.

Direct regulation of shikimate, early phenylpropanoid, and stilbenoid pathways by Subgroup 2 R2R3-MYBs in grapevine.

The stilbenoid pathway is responsible for the production of resveratrol in grapevine (Vitis vinifera L.). A few transcription factors (TFs) have been identified as regulators of this pathway but the extent of this control has not been deeply studied. Here we show how DNA affinity purification sequencing (DAP-Seq) allows for the genome-wide TF-binding site interrogation in grape. We obtained 5190 and 4443 binding events assigned to 4041 and 3626 genes for MYB14 and MYB15, respectively (approximately 40% of peaks located within −10 kb of transcription start sites). DAP-Seq of MYB14/MYB15 was combined with aggregate gene co-expression networks (GCNs) built from more than 1400 transcriptomic datasets from leaves, fruits, and flowers to narrow down bound genes to a set of high confidence targets. The analysis of MYB14, MYB15, and MYB13, a third uncharacterized member of Subgroup 2 (S2), showed that in addition to the few previously known stilbene synthase (STS) targets, these regulators bind to 30 of 47 STS family genes. Moreover, all three MYBs bind to several PAL, C4H, and 4CL genes, in addition to shikimate pathway genes, the WRKY03 stilbenoid co-regulator and resveratrol-modifying gene candidates among which ROMT2-3 were validated enzymatically. A high proportion of DAP-Seq bound genes were induced in the activated transcriptomes of transient MYB15-overexpressing grapevine leaves, validating our methodological approach for delimiting TF targets. Overall, Subgroup 2 R2R3-MYBs appear to play a key role in binding and directly regulating several primary and secondary metabolic steps leading to an increased flux towards stilbenoid production. The integration of DAP-Seq and reciprocal GCNs offers a rapid framework for gene function characterization using genome-wide approaches in the context of non-model plant species and stands up as a valid first approach for identifying gene regulatory networks of specialized metabolism.

Transcriptomic Analysis Reveals Novel Regulators of the Scots Pine Stilbene Pathway.

Stilbenes accumulate in Scots pine heartwood where they have important roles in protecting wood from decaying fungi. They are also part of active defense responses, and their production is induced by different (a)biotic stressors. The specific transcriptional regulators as well as the enzyme responsible for activating the stilbene precursor cinnamate in the pathway are still unknown. UV-C radiation was the first discovered artificial stress activator of the pathway. Here, we describe a large-scale transcriptomic analysis of pine needles in response to UV-C and treatment with translational inhibitors, both activating the transcription of stilbene pathway genes. We used the data to identify putative candidates for the missing CoA ligase and for pathway regulators. We further showed that the pathway is transcriptionally activated by phosphatase inhibitor, ethylene and jasmonate treatments, as in grapevine, and that the stilbene synthase promoter retains its inducibility in some of the tested conditions in Arabidopsis, a species that normally does not synthesize stilbenes. Shared features between gymnosperm and angiosperm regulation and partially retained inducibility in Arabidopsis suggest that pathway regulation occurs not only via ancient stress-response pathway(s) but also via species-specific regulators. Understanding which genes control the biosynthesis of stilbenes in Scots pine aids breeding of more resistant trees.

A Pseudomonas taiwanensis malonyl-CoA platform strain for polyketide synthesis.

Malonyl-CoA is a central precursor for biosynthesis of a wide range of complex secondary metabolites. The development of platform strains with increased malonyl-CoA supply can contribute to the efficient production of secondary metabolites, especially if such strains exhibit high tolerance towards these chemicals. In this study, Pseudomonas taiwanensis VLB120 was engineered for increased malonyl-CoA availability to produce bacterial and plant-derived polyketides. A multi-target metabolic engineering strategy focusing on decreasing the malonyl-CoA drain and increasing malonyl-CoA precursor availability, led to an increased production of various malonyl-CoA-derived products, including pinosylvin, resveratrol and flaviolin. The production of flaviolin, a molecule deriving from five malonyl-CoA molecules, was doubled compared to the parental strain by this malonyl-CoA increasing strategy. Additionally, the engineered platform strain enabled production of up to 84 mg L-1 resveratrol from supplemented p-coumarate. One key finding of this study was that acetyl-CoA carboxylase overexpression majorly contributed to an increased malonyl-CoA availability for polyketide production in dependence on the used strain-background and whether downstream fatty acid synthesis was impaired, reflecting its complexity in metabolism. Hence, malonyl-CoA availability is primarily determined by competition of the production pathway with downstream fatty acid synthesis, while supply reactions are of secondary importance for compounds that derive directly from malonyl-CoA in Pseudomonas.

Tissue-specific stilbene accumulation is an early response to wounding/grafting as revealed by using spatial and temporal metabolomics.

Grafting is widely used in horticulture. Shortly after grafting, callus tissues appear at the graft interface and the vascular tissues of the scion and rootstock connect. The graft interface contains a complex mix of tissues, we hypothesised that each tissue has its own metabolic response to wounding/grafting and accumulates different metabolites at different rates. We made intact and wounded cuttings and grafts of grapevine, and then measured changes in bulk flavonoid, phenolic acid and stilbenoid concentration and used metabolite imaging to study tissue-specific responses. We show that some metabolites rapidly accumulate in specific tissues after grafting, for example, stilbene monomers accumulate in necrotic tissues surrounding mature xylem vessels. Whereas other metabolites, such as complex stilbenes, accumulate in the same tissues at later stages. We also observe that other metabolites accumulate in the newly formed callus tissue and identify genotype-specific responses. In addition, exogenous resveratrol application did not modify grafting success rate, potentially suggesting that the accumulation of resveratrol at the graft interface is not linked to graft union formation. The increasing concentration of complex stilbenes often occurs in response to plant stresses (via unknown mechanisms), and potentially increases antioxidant activity and antifungal capacities.

Involvement of the Calmodulin-like Protein Gene VaCML92 in Grapevine Abiotic Stress Response and Stilbene Production.

Calmodulin-like proteins (CMLs) are an important family of plant calcium sensor proteins that sense and decode changes in the intracellular calcium concentration in response to environmental and developmental stimuli. Nonetheless, the specific functions of individual CML family members remain largely unknown. This study aims to explore the role of the Vitis amurensis VaCML92 gene in the development of its high stress resistance and the production of stilbenes. The expression of VaCML92 was sharply induced in V. amurensis cuttings after cold stress. The VaCML92 gene was cloned and its role in the abiotic stress responses and stilbene production in grapevine was further investigated. The VaCML92-overexpressing callus cell cultures of V. amurensis and soil-grown plants of Arabidopsis thaliana exhibited enhanced tolerance to cold stress and, to a lesser extent, to the drought, while their tolerance to heat stress and high salinity was not affected. In addition, the overexpression of VaCML92 increased stilbene production in the V. amurensis cell cultures by 7.8-8.7-fold. Taken together, the data indicate that the VaCML92 gene is involved as a strong positive regulator in the rapid response to cold stress, the induction of cold stress resistance and in stilbene production in wild grapevine.

Between the Devil and the Deep Blue Sea-Resveratrol, Sulfotransferases and Sulfatases-A Long and Turbulent Journey from Intestinal Absorption to Target Cells.

For nearly 30 years, resveratrol has attracted the scientific community's interest. This has happened thanks to the so-called French paradox, that is, the paradoxically low mortality from cardiovascular causes in the French population despite a diet rich in saturated fat. This phenomenon has been linked to the consumption of red wine, which contains a relatively high level of resveratrol. Currently, resveratrol is valued for its versatile, beneficial properties. Apart from its anti-atherosclerotic activity, resveratrol's antioxidant and antitumor properties deserve attention. It was shown that resveratrol inhibits tumour growth at all three stages: initiation, promotion, and progression. Moreover, resveratrol delays the ageing process and has anti-inflammatory, antiviral, antibacterial, and phytoestrogenic properties. These favorable biological properties have been demonstrated in vitro and in vivo in animal and human models. Since the beginning of the research on resveratrol, its low bioavailability, mainly due to its rapid metabolism, especially the first-pass effect that leaves almost no free resveratrol in the peripheral circulation, has been indicated as a drawback that has hindered its use. The elucidation of such issues as pharmacokinetics, stability, and the biological activity of resveratrol metabolites is therefore crucial for understanding the biological activity of resveratrol. Second-phase metabolism enzymes are mainly involved in RSV metabolism, e.g., UDP-glucuronyl transferases and sulfotransferases. In the present paper, we took a closer look at the available data on the activity of resveratrol sulfate metabolites and the role of sulfatases in releasing active resveratrol in target cells.

Characterization of Cell Death Induced by Imine Analogs of Trans-Resveratrol: Induction of Mitochondrial Dysfunction and Overproduction of Reactive Oxygen Species Leading to, or Not, Apoptosis without the Increase in the S-Phase of the Cell Cycle.

Trans-resveratrol (RSV) is a non-flavonoid polyphenol (stilbene) with numerous biological activities, such as anti-tumor activities. However, RSV is rapidly metabolized, which limits its therapeutic use. The availability of RSV analogues with similar activities for use in vivo is therefore a major challenge. For this purpose, several isomeric analogues of RSV, aza-stilbenes (AZA-ST 1a-g), were synthesized, and their toxicities were characterized and compared to those of RSV on murine N2a neuronal cells using especially flow cytometric methods. All AZA-ST 1a-g have an inhibitory concentration 50 (IC50) between 11.3 and 25 µM when determined by the crystal violet assay, while that of RSV is 14.5 µM. This led to the characterization of AZA-ST 1a-g-induced cell death, compared to RSV, using three concentrations encompassing the IC50s (6.25, 12.5 and 25 µM). For AZA-ST 1a-g and RSV, an increase in plasma membrane permeability to propidium iodide was observed, and the proportion of cells with depolarized mitochondria measured with DiOC6(3) was increased. An overproduction of reactive oxygen species (ROS) was also observed on whole cells and at the mitochondrial level using dihydroethidium and MitoSox Red, respectively. However, only RSV induced a mode of cell death by apoptosis associated with a marked increase in the proportion of cells with condensed and/or fragmented nuclei (12.5 µM: 22 ± 9%; 25 µM: 80 ± 10%) identified after staining with Hoechst 33342 and which are characteristic of apoptotic cells. With AZA-ST, a slight but significant increase in the percentage of apoptotic cells was only detected with AZA-ST 1b (25 µM: 17 ± 1%) and AZA-ST 1d (25 µM: 26 ± 4%). Furthermore, only RSV induced significant cell cycle modifications associated with an increase in the percentage of cells in the S phase. Thus, AZA-ST 1a-g-induced cell death is characterized by an alteration of the plasma membrane, an induction of mitochondrial depolarization (loss of ΔΨm), and an overproduction of ROS, which may or may not result in a weak induction of apoptosis without modification of the distribution of the cells in the different phases of the cell cycle.

Influence of peanut hairy root cultivars on prenylated stilbenoid production and the response mechanism for combining the elicitors of chitosan, methyl jasmonate, and cyclodextrin.

MAIN CONCLUSION: Peanut cultivars are known to produce stilbene compounds. Transcriptional control plays a key role in the early stages of the stress response mechanism, involving both PR-proteins and stilbene compounds. In this study, the production of stilbenoid compounds, especially prenylated, was investigated in two cultivars of peanut hairy root lines, designated as K2-K599 and T9-K599 elicited with a combination of chitosan (CHT), methyl jasmonate (MeJA), and cyclodextrin (CD): CHT + MeJA + CD. The antioxidant activities and stilbenoid content of both K2-K599 and T9-K599 hairy root lines increased significantly during the elicitation period. The T9-K599 hairy root line expressed higher ABTS and FRAP antioxidant activities than the K2-K599 line while the latter exhibited greater total phenolic content than the former at all-time points. Additionally, the K2-K599 line exhibited more stilbene compounds, including trans-resveratrol, trans-arachidin-1, and trans-arachidin-3 than the T9-K599 line, which showed statistically significant differences at all-time points. Gene expression of the enzyme involved in the stilbene biosynthesis pathway (PAL, RS, RS3) was observed, responding early to elicitor treatment and the metabolic production of a high level of stilbenoid compounds at a later stage. The antioxidant enzyme (CuZn-SOD, APX, GPX) and pathogenesis-related protein (PR; PR4A, PR5, PR10, chitinase) genes were strongly expressed after elicitor treatment at 24 h and decreased with an increasing elicitation time. Investigation of the response mechanism illustrates that the elicitor treatment can affect various plant responses, including plant cell wall structure and integrity, antioxidant system, PR-proteins, and secondary plant metabolites at different time points after facing external environmental stimuli.

Effects of resveratrol supplementation on nitric oxide-mediated vascular outcomes in hypertension: A systematic review.

Hypertension is associated with endothelial dysfunction and decreased nitric oxide (NO). It has been proposed that decreasing oxidative stress may help regulate blood pressure by increasing NO concentration. Therefore, the purpose of this systematic review was to determine whether the antioxidant resveratrol effects NO-mediated vascular outcomes in hypertension. A comprehensive literature search of PubMed and EBSCOhost databases was conducted using the terms: "resveratrol" and "nitric oxide or NO" and "hypertension or high blood pressure." Searches were not restricted for year of publication or study design but limited to full-text studies from scholarly, peer-reviewed journals. Ten animal studies published between 2005 and 2017 were identified. Human studies did not meet criteria and were not included. Articles were critically assessed using the Academy of Nutrition and Dietetics' Evidence Analysis Library Quality Criteria Worksheet. All studies evaluated resveratrol supplementation and at least one NO outcome measure including: circulating NO metabolites, endothelial nitric oxide synthase (eNOS) expression, eNOS phosphorylation, and eNOS uncoupling. All but one study assessed blood pressure. Nine of ten studies reported positive significant results of resveratrol supplementation on NO outcomes, and in all but one study, this was seen concomitantly with decreases in blood pressure. Resveratrol supplementation shows promise for improving NO-mediated vascular outcomes and improving blood pressure. Translation to human studies is warranted, with dose of resveratrol considered, as the human equivalency doses are not consistent amongst animal studies. Additionally, a standard battery of tests examining NO-mediated vascular outcomes is needed to ensure generalizability among studies to determine dose-duration effects.

Recent Overview of Resveratrol's Beneficial Effects and Its Nano-Delivery Systems.

Natural polyphenols have a wide variety of biological activities and are taken into account as healthcare materials. Resveratrol is one such natural polyphenol, belonging to a group known as stilbenoids (STBs). Resveratrol (3,5,4'-trihydroxy-trans-stilbene) is mainly found in grapes, wine, nuts, and berries. A wide range of biological activities has been demonstrated by resveratrol, including antimicrobial, antioxidant, antiviral, antifungal, and antiaging effects, and many more are still under research. However, as with many other plant-based polyphenol products, resveratrol suffers from low bioavailability once administered in vivo due to its susceptibility to rapid enzyme degradation by the body's innate immune system before it can exercise its therapeutic influence. Therefore, it is of the utmost importance to ensure the best use of resveratrol by creating a proper resveratrol delivery system. Nanomedicine and nanodelivery systems utilize nanoscale materials as diagnostic tools or to deliver therapeutic agents in a controlled manner to specifically targeted locations. After a brief introduction about polyphenols, this review overviews the physicochemical characteristics of resveratrol, its beneficial effects, and recent advances on novel nanotechnological approaches for its delivery according to the type of nanocarrier utilized. Furthermore, the article summarizes the different potential applications of resveratrol as, for example, a therapeutic and disease-preventing anticancer and antiviral agent.

Separation of Dihydro-Isocoumarins and Dihydro-Stilbenoids from Hydrangea macrophylla ssp. serrata by Use of Counter-Current Chromatography.

Previously, different Hydrangea macrophylla ssp. serrata cultivars were investigated by untargeted LC-MS analysis. From this, a list of tentatively identified and unknown compounds that differ significantly between these cultivars was obtained. Due to the lack of reference compounds, especially for dihydro-isocoumarins, we aimed to isolate and structurally characterise these compounds from the cultivar 'Yae-no-amacha' using NMR and LC-MS methods. For purification and isolation, counter-current chromatography was used in combination with reversed-phase preparative HPLC as an orthogonal and enhanced purification workflow. Thirteen dihydro-isocoumarins in combination with other metabolites could be isolated and structurally identified. Particularly interesting was the clarification of dihydrostilbenoid glycosides, which were described for the first time in H. macrophylla ssp. serrata. These results will help us in further studies on the biological interpretation of our data.

LC-HRMS/MS-Based Metabolomics Approaches Applied to the Detection of Antifungal Compounds and a Metabolic Dynamic Assessment of Orchidaceae.

The liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach is a powerful technology for discovering novel biologically active molecules. In this study, we investigated the metabolic profiling of Orchidaceae species using LC-HRMS/MS data combined with chemometric methods and dereplication tools to discover antifungal compounds. We analyze twenty ethanolic plant extracts from Vanda and Cattleya (Orchidaceae) genera. Molecular networking and chemometric methods were used to discriminate ions that differentiate healthy and fungal-infected plant samples. Fifty-three metabolites were rapidly annotated through spectral library matching and in silico fragmentation tools. The metabolomic profiling showed a large production of polyphenols, including flavonoids, phenolic acids, chromones, stilbenoids, and tannins, which varied in relative abundance across species. Considering the presence and abundance of metabolites in both groups of samples, we can infer that these constituents are associated with biochemical responses to microbial attacks. In addition, we evaluated the metabolic dynamic through the synthesis of stilbenoids in fungal-infected plants. The tricin derivative flavonoid- and the loliolide terpenoidfound only in healthy plant samples, are promising antifungal metabolites. LC-HRMS/MS, combined with state-of-the-art tools, proved to be a rapid and reliable technique for fingerprinting medicinal plants and discovering new hits and leads.

Grape Pomace Polyphenols as a Source of Compounds for Management of Oxidative Stress and Inflammation-A Possible Alternative for Non-Steroidal Anti-Inflammatory Drugs?

Flavonoids, stilbenes, lignans, and phenolic acids, classes of polyphenols found in grape pomace (GP), were investigated as an important alternative source for active substances that could be used in the management of oxidative stress and inflammation. The benefic antioxidant and anti-inflammatory actions of GP are presented in the literature, but they are derived from a large variety of experimental in vitro and in vivo settings. In these in vitro works, the decrease in reactive oxygen species, malondialdehyde, and thiobarbituric acid reactive substances levels and the increase in glutathione levels show the antioxidant effects. The inhibition of nuclear factor kappa B and prostaglandin E2 inflammatory pathways and the decrease of some inflammatory markers such as interleukin-8 (IL-8) demonstrate the anti-inflammatory actions of GP polyphenols. The in vivo studies further confirmed the antioxidant (increase in catalase, superoxide dismutase and glutathione peroxidase levels and a stimulation of endothelial nitric oxide synthase -eNOS gene expression) and anti-inflammatory (inhibition of IL-1𝛼, IL-1ß, IL-6, interferon-𝛾, TNF-α and C-reactive protein release) activities. Grape pomace as a whole extract, but also different individual polyphenols that are contained in GP can modulate the endogenous pathway responsible in reducing oxidative stress and chronic inflammation. The present review analyzed the effects of GP in oxidative stress and inflammation, suggesting that it could become a valuable therapeutic candidate capable to reduce the aforementioned pathological processes. Grape pomace extract could become an adjuvant treatment in the attempt to reduce the side effects of the classical anti-inflammatory medication like non-steroidal anti-inflammatory drugs (NSAIDs).

Molecular and cellular dissection of the oxysterol-binding protein cycle through a fluorescent inhibitor.

ORPphilins are bioactive natural products that strongly and selectively inhibit the growth of some cancer cell lines and are proposed to target intracellular lipid-transfer proteins of the oxysterol-binding protein (OSBP) family. These conserved proteins exchange key lipids, such as cholesterol and phosphatidylinositol 4-phosphate (PI(4)P), between organelle membranes. Among ORPphilins, molecules of the schweinfurthin family interfere with intracellular lipid distribution and metabolism, but their functioning at the molecular level is poorly understood. We report here that cell line sensitivity to schweinfurthin G (SWG) is inversely proportional to cellular OSBP levels. By taking advantage of the intrinsic fluorescence of SWG, we followed its fate in cell cultures and show that its incorporation at the trans-Golgi network depends on cellular abundance of OSBP. Using in vitro membrane reconstitution systems and cellular imaging approaches, we also report that SWG inhibits specifically the lipid transfer activity of OSBP. As a consequence, post-Golgi trafficking, membrane cholesterol levels, and PI(4)P turnover were affected. Finally, using intermolecular FRET analysis, we demonstrate that SWG directly binds to the lipid-binding cavity of OSBP. Collectively these results describe SWG as a specific and intrinsically fluorescent pharmacological tool for dissecting OSBP properties at the cellular and molecular levels. Our findings indicate that SWG binds OSBP with nanomolar affinity, that this binding is sensitive to the membrane environment, and that SWG inhibits the OSBP-catalyzed lipid exchange cycle.