Circulating Human Metabolites Resulting from TOTUM-070 Absorption (a Plant-Based, Polyphenol-Rich Ingredient) Improve Lipid Metabolism in Human Hepatocytes: Lessons from an Original Ex Vivo Clinical Trial.

TOTUM-070 is a patented polyphenol-rich blend of five different plant extracts showing separately a latent effect on lipid metabolism and potential synergistic properties. In this study, we investigated the health benefit of such a formula. Using a preclinical model of high fat diet, TOTUM-070 (3 g/kg of body weight) limited the HFD-induced hyperlipemia with a reduction in triglyceride (-32% after 6 weeks; -20.3% after 12 weeks) and non-HDL cholesterol levels (-21% after 6 weeks; -38.4% after 12 weeks). To further investigate such a benefit and its underlying mechanisms in humans, we designed an ex vivo clinical approach to collect the circulating bioactives resulting from TOTUM-070 ingestion and to determine their biological activities on human hepatocytes. Human serum was obtained from healthy subjects before and after intake of TOTUM-070 (4995 mg). The presence of circulating metabolites was assessed by UPLC-MS/MS. Serum containing metabolites was further incubated with hepatocytes cultured in a lipotoxic environment (palmitate, 250 µM). RNA sequencing analyses show that lipid metabolism was one of the most impacted processes. Using histologic, proteomic, and enzymatic assays, the effects of human TOTUM-070 bioactives on hepatocyte metabolism were characterized by (1) the inhibition of lipid storage, including both (2) triglycerides (-41%, p < 0.001) and (3) cholesterol (-50%, p < 0.001) intracellular content, (4) a reduced de novo cholesterol synthesis (HMG-CoA reductase activity -44%, p < 0.001), and (5) a lowered fatty acid synthase protein level (p < 0.001). Altogether, these data support the beneficial impact of TOTUM-070 on lipid metabolism and provide new biochemical insights in human mechanisms occurring in liver cells.

Enhanced Wound Healing Potential of Spirulina platensis Nanophytosomes: Metabolomic Profiling, Molecular Networking, and Modulation of HMGB-1 in an Excisional Wound Rat Model.

Excisional wounds are considered one of the most common physical injuries. This study aims to test the effect of a nanophytosomal formulation loaded with a dried hydroalcoholic extract of S. platensis on promoting excisional wound healing. The Spirulina platensis nanophytosomal formulation (SPNP) containing 100 mg PC and 50 mg CH exhibited optimum physicochemical characteristics regarding particle size (598.40 ± 9.68 nm), zeta potential (-19.8 ± 0.49 mV), entrapment efficiency (62.76 ± 1.75%), and Q6h (74.00 ± 1.90%). It was selected to prepare an HPMC gel (SPNP-gel). Through metabolomic profiling of the algal extract, thirteen compounds were identified. Molecular docking of the identified compounds on the active site of the HMGB-1 protein revealed that 12,13-DiHome had the highest docking score of -7.130 kcal/mol. SPNP-gel showed higher wound closure potential and enhanced histopathological alterations as compared to standard (MEBO® ointment) and S. platensis gel in wounded Sprague-Dawley rats. Collectively, NPS promoted the wound healing process by enhancing the autophagy process (LC3B/Beclin-1) and the NRF-2/HO-1antioxidant pathway and halting the inflammatory (TNF-, NF-κB, TlR-4 and VEGF), apoptotic processes (AIF, Caspase-3), and the downregulation of HGMB-1 protein expression. The present study's findings suggest that the topical application of SPNP-gel possesses a potential therapeutic effect in excisional wound healing, chiefly by downregulating HGMB-1 protein expression.

Coumarin biosynthesis genes are required after foliar pathogen infection for the creation of a microbial soil-borne legacy that primes plants for SA-dependent defenses.

Plants deposit photosynthetically-fixed carbon in the rhizosphere, the thin soil layer directly around the root, thereby creating a hospitable environment for microbes. To manage the inhabitants of this nutrient-rich environment, plant roots exude and dynamically adjust microbe-attracting and -repelling compounds to stimulate specific members of the microbiome. Previously, we demonstrated that foliar infection of Arabidopsis thaliana by the biotrophic downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) leads to a disease-induced modification of the rhizosphere microbiome. Soil conditioned with Hpa-infected plants provided enhanced protection against foliar downy mildew infection in a subsequent population of plants, a phenomenon dubbed the soil-borne legacy (SBL). Here, we show that for the creation of the SBL, plant-produced coumarins play a prominent role as coumarin-deficient myb72 and f6'h1 mutants were defective in creating a Hpa-induced SBL. Root exudation profiles changed significantly in Col-0 upon foliar Hpa infection, and this was accompanied by a compositional shift in the root microbiome that was significantly different from microbial shifts occurring on roots of Hpa-infected coumarin-deficient mutants. Our data further show that the Hpa-induced SBL primes Col-0 plants growing in SBL-conditioned soil for salicylic acid (SA)-dependent defenses. The SA-signaling mutants sid2 and npr1 were unresponsive to the Hpa-induced SBL, suggesting that the protective effect of the Hpa-induced shift in the root microbiome results from an induced systemic resistance that requires SA-signaling in the plant.

Verification: model-free phasing with enhanced predicted models in ARCIMBOLDO_SHREDDER.

Structure predictions have matched the accuracy of experimental structures from close homologues, providing suitable models for molecular replacement phasing. Even in predictions that present large differences due to the relative movement of domains or poorly predicted areas, very accurate regions tend to be present. These are suitable for successful fragment-based phasing as implemented in ARCIMBOLDO. The particularities of predicted models are inherently addressed in the new predicted_model mode, rendering preliminary treatment superfluous but also harmless. B-value conversion from predicted LDDT or error estimates, the removal of unstructured polypeptide, hierarchical decomposition of structural units from domains to local folds and systematically probing the model against the experimental data will ensure the optimal use of the model in phasing. Concomitantly, the exhaustive use of models and stereochemistry in phasing, refinement and validation raises the concern of crystallographic model bias and the need to critically establish the information contributed by the experiment. Therefore, in its predicted_model mode ARCIMBOLDO_SHREDDER will first determine whether the input model already constitutes a solution or provides a straightforward solution with Phaser. If not, extracted fragments will be located. If the landscape of solutions reveals numerous, clearly discriminated and consistent probes or if the input model already constitutes a solution, model-free verification will be activated. Expansions with SHELXE will omit the partial solution seeding phases and all traces outside their respective masks will be combined in ALIXE, as far as consistent. This procedure completely eliminates the molecular replacement search model in favour of the inferences derived from this model. In the case of fragments, an incorrect starting hypothesis impedes expansion. The predicted_model mode has been tested in different scenarios.

Elicitor-Induced VOC Emission by Grapevine Leaves: Characterisation in the Vineyard.

The present study is aimed at determining whether leaf volatile organic compounds (VOCs) are good markers of the grapevine response to defence elicitors in the field. It was carried out in two distinct French vineyards (Burgundy and Bordeaux) over 3 years. The commercial elicitor Bastid® (Syngenta, Saint-Sauveur, France) (COS-OGA) was first used to optimise the VOCs' capture in the field; by bagging stems together with a stir bar sorptive extraction (SBSE) sensor. Three elicitors (Bastid®, copper sulphate and methyl jasmonate) were assessed at three phenological stages of the grapevines by monitoring stilbene phytoalexins and VOCs. Stilbene production was low and variable between treatments and phenological stages. VOCs-particularly terpenes-were induced by all elicitors. However, the response profiles depended on the type of elicitor, the phenological stage and the vineyard, and no sole common VOC was found. The levels of VOC emissions discriminated between weak (Bastid® and copper sulphate) and strong (methyl jasmonate) inducers. Ocimene isomers were constitutively present in the overall blends of the vineyards and increased by the elicitors' treatments, whilst other VOCs were newly released throughout the growing seasons. Nonetheless, the plant development and climate factors undoubtedly influenced the release and profiles of the leaf VOCs.

VOCs Are Relevant Biomarkers of Elicitor-Induced Defences in Grapevine.

Grapevine is susceptible to fungal diseases generally controlled by numerous chemical fungicides. Elicitors of plant defence are a way of reducing the use of these chemicals, but still provide inconsistent efficiency. Easy-to-analyse markers of grapevine responses to elicitors are needed to determine the best conditions for their efficiency and position them in protection strategies. We previously reported that the elicitor sulphated laminarin induced the emission of volatile organic compounds (VOCs) by grapevine leaves. The present study was conducted to characterise and compare VOC emissions in response to other elicitors. Bastid® was first used to test the conditions of VOC collection and analysis. Using SBSE-GC-MS, we detected several VOCs, including the sesquiterpene α-farnesene, in a time-dependent manner. This was correlated with the induction of farnesene synthase gene expression, in parallel with stilbene synthesis (another defence response), and associated to resistance against downy mildew. The other elicitors (Redeli®, Romeo®, Bion®, chitosan, and an oligogalacturonide) induced VOC emission, but with qualitative and quantitative differences. VOC emission thus constitutes a response of grapevine to elicitors of various chemical structures. Therefore, VOC analysis is relevant for studying the impact of environmental factors on grapevine defence responses and optimising the performance of elicitors in vineyards.

External trigeminal nerve stimulation for drug resistant epilepsy: A randomized controlled trial.

BACKGROUND: External trigeminal nerve stimulation (ETNS) is an emergent, non-invasive neurostimulation therapy delivered bilaterally with adhesive skin electrodes. In previous studies, ETNS was associated to a decrease in seizure frequency in patients with focal drug-resistant epilepsy (DRE). OBJECTIVE: To determine the long-term efficacy and tolerability of ETNS in patients with focal DRE. Moreover, to explore whether its efficacy depends on the epileptogenic zone (frontal or temporal), and its impact on mood, cognitive function, quality of life, and trigeminal nerve excitability. METHODS: Forty consecutive patients with frontal or temporal DRE, unsuitable for surgery, were randomized to ETNS or usual medical treatment. Participants were evaluated at 3, 6 and 12 months for efficacy, side effects, mood scales, neuropsychological tests and trigeminal nerve excitability. RESULTS: Subjects had a median of 15 seizures per month and had tried a median of 12.5 antiepileptic drugs. At 12 months, percentage of responders was 50% in ETNS group and 0% in control group. Seizure frequency in ETNS group decreased by -43.5% from baseline. Temporal epilepsy subgroup responded better than frontal epilepsy subgroup (55.56% vs. 45.45%, respectively). Median stimulation intensity was 6.2 mA. ETNS improved quality of life, but not anxiety or depression. Long-term ETNS affected neither neuropsychological function, nor trigeminal nerve excitability. No relevant adverse events were observed. CONCLUSIONS: ETNS is an effective and well-tolerated therapy for focal DRE. Patients with temporal epilepsy showed a better response than those with frontal epilepsy. Future studies with larger populations may define its role compared to other neurostimulation techniques. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that ETNS reduces seizure frequency in patients with focal DRE.

Wood and roots of major grapevine cultivars and rootstocks: A comparative analysis of stilbenes by UHPLC-DAD-MS/MS and NMR.

INTRODUCTION: Grapevine wood and roots are by-products obtained during vineyard management. This plentiful biomass is known to be rich in stilbenes and can be used as a source of high-value compounds as well as active natural extracts. However, the stilbenes in grapevine wood and roots from different cultivars and rootstocks remain to be characterized. OBJECTIVE: The present study investigated the stilbene content of eight major Vitis vinifera cultivars and six different rootstocks. In addition, the distribution of stilbenes was established for each of seven parts into which the plants were sub-divided. METHODOLOGY: For stilbene characterization and quantification purposes, an ultra-high performance liquid chromatography-diode array detector-mass spectrometry (UHPLC-DAD-MS/MS) analysis of different samples was carried out. Moreover, structural data of stilbenes was unambiguously studied by nuclear magnetic resonance (NMR) spectra. RESULTS: Whatever the cultivar/rootstock combination, stilbenes were found to be oligomerized from the aerial part to the root system. Furthermore, stilbene content varied widely depending on the cultivars and rootstocks. For instance, the cultivars Merlot, Tannat and Gamay noir were the richest in stilbenes while the rootstocks Gravesac, Fercal and 3390C contained the highest amounts. CONCLUSION: These findings provide insight into the knowledge that major grapevine cultivars and rootstocks can be used as a potential source of complex stilbenes.

Chondroprotective Properties of Human-Enriched Serum Following Polyphenol Extract Absorption: Results from an Exploratory Clinical Trial.

Polyphenols are widely acknowledged for their health benefits, especially for the prevention of inflammatory and age-related diseases. We previously demonstrated that hydroxytyrosol (HT) and procyanidins (PCy), alone or in combination, drive preventive anti-osteoathritic effects in vivo. However, the lack of sufficient clinical evidences on the relationship between dietary phytochemicals and osteoarthritis remains. In this light, we investigated in humans the potential osteoarticular benefit of a grapeseed and olive extract (OPCO) characterized for its hydroxytyrosol (HT) and procyanidins (PCy) content. We first validated, in vitro, the anti-inflammatory and chondroprotective properties of the extract on primary cultured human articular chondrocytes stimulated by interleukin-1 beta (IL-1 ß). The sparing effect involved a molecular mechanism dependent on the nuclear transcription factor-kappa B (NF-κB) pathway. To confirm the clinical relevance of such a nutritional strategy, we designed an innovative clinical approach taking into account the metabolites that are formed during the digestion process and that appear in circulation after the ingestion of the OPCO extract. Blood samples from volunteers were collected following ingestion, absorption, and metabolization of the extract and then were processed and applied on human primary chondrocyte cultures. This original ex vivo methodology confirmed at a clinical level the chondroprotective properties previously observed in vitro and in vivo.

Variably protease-sensitive prionopathy presenting within ALS/FTD spectrum.

We report clinico-pathological features of a 65-year-old woman and a 56-year-old man with a 5-year clinical history who had clinical and neuropathological characteristics of upper and lower motor neuron disease consistent with amyotrophic lateral sclerosis, and a frontotemporal atrophy pattern in case 2 without TDP-43 pathology. Instead, spongiform change and pathological prion protein deposits were observed in several brain regions. No prion protein gene mutations were found. Western blot analysis showed a five-band profile compatible with variably protease-sensitive prionopathy. We conclude that this disease can display prolonged disease duration and clinico-pathological features within the ALS/FTLD spectrum.

Novel Types of Hypermodified Fluorescent Phyllobilins from Breakdown of Chlorophyll in Senescent Leaves of Grapevine (Vitis vinifera).

The tetrapyrrolic chlorophyll catabolites (or phyllobilins, PBs) were analyzed in yellow fall leaves of the grape Chardonnay, a common Vitis vinifera white wine cultivar. The major fractions in leaf extracts of V. vinifera, tentatively assigned to PBs, were isolated and their structures elucidated. The dominant fraction is a dioxobilin-type non-fluorescent Chl-catabolite of a previously observed type. Two less polar fluorescent PBs were characterized as a novel dioxobilin-type fluorescent Chl-catabolite with a bicyclo-1',6'-glycosyl architecture, and its new fluorescent formyloxobilin-type analogue. The discovery of persistent hypermodified fluorescent PBs with the architecture of bicyclo-[17.3.1]-PBs (bcPBs), suggests the activity of an unknown enzyme that forges the 20-membered macroring at the tetrapyrrolic core of a fluorescent PB. bcPBs may play specific physiological roles in grapevine plants and represent endogenous anti-infective agents, as found similarly for other organic bicyclo-[n.3.1]-1',6'-glycosyl derivatives.

A Plant Extract Acts Both as a Resistance Inducer and an Oomycide Against Grapevine Downy Mildew.

Protecting vineyards from cryptogamic diseases such as downy mildew, caused by Plasmopara viticola, generally requires a massive use of phytochemicals. However, the issues on unintentional secondary effects on environment and human health, and the occurrence of P. viticola resistant strains, are leading to the development of alternative strategies, such as the use of biocontrol products. In this paper, we evidenced the ability of a plant extract to protect grapevine from P. viticola. Further experiments carried out both on cell suspensions and on plants revealed that plant extract activates typical defense-related responses such as the production of H2O2, the up-regulation of genes encoding pathogenesis-related proteins and stilbene synthase, as well as the accumulation of resveratrol or its derivative piceid. We also brought to light a strong direct effect of PE on the release and motility of P. viticola zoospores. Furthermore, we found out that PE application left dried residues on leaf surface, impairing zoospores to reach stomata. Altogether, our results highlight the different modes of action of a new biocontrol product able to protect grapevine against downy mildew.

Plant-derived phenolics inhibit the accrual of structurally characterised protein and lipid oxidative modifications.

Epidemiological data suggest that plant-derived phenolics beneficial effects include an inhibition of LDL oxidation. After applying a screening method based on 2,4-dinitrophenyl hydrazine-protein carbonyl reaction to 21 different plant-derived phenolic acids, we selected the most antioxidant ones. Their effect was assessed in 5 different oxidation systems, as well as in other model proteins. Mass-spectrometry was then used, evidencing a heterogeneous effect on the accumulation of the structurally characterized protein carbonyl glutamic and aminoadipic semialdehydes as well as for malondialdehyde-lysine in LDL apoprotein. After TOF based lipidomics, we identified the most abundant differential lipids in Cu(++)-incubated LDL as 1-palmitoyllysophosphatidylcholine and 1-stearoyl-sn-glycero-3-phosphocholine. Most of selected phenolic compounds prevented the accumulation of those phospholipids and the cellular impairment induced by oxidized LDL. Finally, to validate these effects in vivo, we evaluated the effect of the intake of a phenolic-enriched extract in plasma protein and lipid modifications in a well-established model of atherosclerosis (diet-induced hypercholesterolemia in hamsters). This showed that a dietary supplement with a phenolic-enriched extract diminished plasma protein oxidative and lipid damage. Globally, these data show structural basis of antioxidant properties of plant-derived phenolic acids in protein oxidation that may be relevant for the health-promoting effects of its dietary intake.

Toxicology evaluation of a procyanidin-rich extract from grape skins and seeds.

The procyanidin-rich extract from grape seeds and skins (GSSE) has antioxidant properties which may have cardioprotective effects. Since it might be interesting to incorporate this extract into a functional food, toxicological tests need to be made to determine how safe it is. In this study we carried out a limit test to determine the acute oral toxicity and the lethal dose 50 (LD50) and some genotoxicity tests of the extract in rats. The LD50 was higher than 5000 mg/kg. Doses of up to 2000 mg/kg showed no increase in micronucleated erythrocytes 72 h after treatment. The bacterial reverse mutation test showed that the extract was weakly mutagenic to the dose of 5 mg/plate and 19.5 and 9.7 µg/ml of GSSE did not show significant differences in the frequency of aberrant metaphases in relation to negative controls. Our results indicated slight mutagenicity under the study conditions, so further studies should be conducted at lower doses to demonstrate that this extract is not toxic.

Rapid analysis of procyanidins and anthocyanins in plasma by microelution SPE and ultra-HPLC.

In the analysis of biological samples, such as plasma or serum, the quantity of sample available is a critical parameter in most cases. A good approach is the use of the microelution SPE (µSPE) plates as sample pre-treatment technique in which the loaded sample volume is low. An off-line µSPE and ultra-performance LC-ESI-MS/MS (UPLC-ESI-MS/MS) method was developed and validated to determine procyanidins and anthocyanins in spiked plasma samples. The sample pre-treatment µSPE allowed the simultaneous determination of procyanidins and anthocyanins from plasma by using a small sample volume (350 µL) and without an evaporation step previous to the chromatographic analysis. Moreover, the use of UPLC technique allowed to determine the studied compounds at low concentration levels in a short analysis time (12.5 min approximately). Then, the developed method was applied to determine the studied compounds, procyanidins and anthocyanins, and their metabolites in rat plasma samples. Previously, the rats had consumed 5000 mg/kg of a grape pomace extract and the plasma was extracted 4 h after administration. The procyanidins catechin and epicatechin glucuronide, methyl catechin and epicatechin glucuronide, and methyl catechin and epicatechin sulphate were detected at µM concentration level, and the parent anthocyanins at nM.

Bioavailability of procyanidin dimers and trimers and matrix food effects in in vitro and in vivo models.

Among procyanidins (PC), monomers, such as catechin and epicatechin, have been widely studied, whereas dimer and trimer oligomers have received much less attention, despite their abundance in our diet. Recent studies have showed that as dimers and trimers could be important in determining the biological effects of procyanidin-rich food, understanding their bioavailability and metabolism is fundamental. The purpose of the present work is to study the stability of PC under digestion conditions, the metabolism and the bioavailability by using a combination of in vitro and in vivo models. Simultaneously, the matrix effect of a carbohydrate-rich food on the digestibility and bioavailability of PC is investigated. The results show a high level of stability of PC under gastric and duodenal digestion conditions. However, the pharmacokinetic study revealed limited absorption. Free forms of dimers and trimers have been detected in rat plasma, reaching the maximum concentration 1 h after oral intake of a grape seed extract.

Advanced separation methods of food anthocyanins, isoflavones and flavanols.

In recent years, increasing knowledge of the positive health effects of food polyphenols has prompted the need to develop new separation techniques for their extraction, fractionation and analysis. This article provides an updated and exhaustive review of the application of counter-current chromatography, high performance liquid chromatography, capillary electrophoresis, and their hyphenation with mass spectrometry to the study of food polyphenols. Flavonoids constitute the largest class of polyphenols, widely spread in the plant kingdom and common in human diet which has been the most widely studied with respect to their antioxidant and biological activities. The main subgroups are anthocyanins, catechins, isoflavones, flavonols and flavones. They are reported to exhibit antioxidant, anti-carcinogenic, anti-inflammatory, anti-atherogenic, anti-thrombotic, and immune modulating functions, among others. Since red fruit anthocyanins, soy isoflavones and flavanols from grapes and teas are currently the most used phenolic compounds for producing new nutraceuticals and functional foods, this review is focused on these three flavonoid groups.

Relaxation induced by red wine polyphenolic compounds in rat pulmonary arteries: lack of inhibition by NO-synthase inhibitor.

Some red wine polyphenols exert nitric oxide (NO)-dependent relaxation in systemic arteries, following activation of endothelial NO synthase (eNOS). In this study, the effect of red wine polyphenols was determined in rat intrapulmonary arteries, and the effect of some of these compounds was compared with the responses obtained in rat aorta. In pulmonary arteries, red wine polyphenolic extract (> 300 microg/mL) exerted relaxation that was not inhibited by the NOS inhibitor N(omega)-nitro-L-arginine methylester (L-NAME) or endothelium removal. Among the several fractions obtained from the extract, the one enriched with anthocyanins was less active than fractions containing non-anthocyanins. Among the latter, the most active for relaxing pulmonary arteries was the one enriched in the stilbene derivative trans-resveratrol (relaxation for concentration >10 microg/mL). Trans-piceid, the glucoside derivative of trans-resveratrol, was almost inactive. Trans-resveratrol-induced relaxation, as well as relaxation to the anthocyanin delphinidin, was L-NAME-insensitive in pulmonary arteries. In aorta, trans-resveratrol and trans-piceid exerted similar effects to those in pulmonary arteries that were also not inhibited by L-NAME. However, red wine polyphenolic extract and delphinidin induced relaxation of aorta at much lower concentrations (about 10 microg/mL) than in pulmonary arteries, and their effects were inhibited by L-NAME. These data show differences between small intrapulmonary arteries and systemic conductance arteries in their responses to red wine polyphenols, the major difference being that the relaxant effect of these compounds is not blunted by NOS inhibitor in pulmonary arteries. They suggest that red wine polyphenols act directly on smooth muscle to promote pulmonary artery relaxation.

New polyphenols active on beta-amyloid aggregation.

New polyphenol classes have been tested against amyloid-beta peptide aggregation. We have identified four novel polyphenols which could be efficient fibril inhibitors in Alzheimer's disease: malvidin and its glucoside and curculigosides B and D. We suggest that molecules with the particular C(6)-linkers-C(6) structure could be potent inhibitors. From the results reported for the flavan-3-ol family, their anti-amyloidogenic effects against whole peptides (1-40 and 1-42) could involve several binding sites.