Targeting of Antifungal Metabolites from Grapevine Byproducts by UPLC-HRMS/MS Approaches Using Bioactivity-Based Molecular Networking.

This study focuses on countering Fusarium graminearum, a harmful fungal pathogen impacting cereal crops and human health through mycotoxin production. These mycotoxins, categorized as type B trichothecenes, pose significant health risks. Research explores natural alternatives to synthetic fungicides, particularly investigating phenolics in grapevine byproducts. Thirteen eco-extracts from five French grape varieties (Merlot, Cabernet Sauvignon, Sauvignon blanc, Tannat, and Artaban) exhibited substantial antifungal properties, with ten extracts displaying remarkable effects. Extracts from grapevine stems and roots notably reduced fungal growth by over 91% after five days. Through UHPLC-HRMS/MS analysis and metabolomics, the study identified potent antifungal compounds such as ampelopsin A and cyphostemmin B, among other oligomeric stilbenes. Interestingly, this approach showed that flavan-3-ols have been identified as markers for extracts that induce fungal growth. Root extracts from rootstocks, rich in oligostilbenes, demonstrated the highest antifungal activity. This research underscores grapevine byproducts' potential both as a sustainable approach to control F. graminearum and mycotoxin contamination in cereal crops and the presence of different metabolites from the cultivars of grapevine, suggesting different activities.

Semi-synthesis, α-amylase inhibition, and kinetic and molecular docking studies of arylidene-based sesquiterpene coumarins isolated from Ferula tunetana Pomel ex Batt.

Despite all the significant progresses made to enhance the efficacy of the existing bank of drugs used to manage and cure type II diabetes mellitus, there is still a need to search and develop novel bioactive compounds with superior efficacy and minimal adverse effects. This study describes the valorization of the natural bioactive sesquiterpene coumarin via the semi-synthesis of new analogs and the study of their α-amylase inhibition activity. The sesquiterpene coumarin named coladonin (1) was quantitatively isolated from the chloroform extract of endemic Ferula tunetana roots. Subsequently, the oxidation of 1via the Jones oxidation reaction, used as a key reaction, afforded precursor 2. The condensation of oxidized coladonin (2) with various aryl aldehydes provided a series of new arylidene-based sesquiterpene coumarin derivatives (3a-m), which were characterized by NMR and ESI-HRMS experiments. All derivatives evaluated in vitro for their α-amylase inhibitory potential showed interesting α-amylase inhibition with IC50 values ranging from 7.24 to 28.98 µM. Notably, compounds 3k and 3m exhibited lower IC50 values (7.24 µM and 8.38 µM, respectively) compared to the standard (acarbose: IC50 = 9.83 µM). In addition, the structure-activity relationship (SAR) for all the compounds was studied. The most active compounds were found to be mixed-type inhibitors, which was revealed by kinetic studies. Furthermore, molecular in silico docking studies were established for all synthesized analogs with the binding site for the α-amylase enzyme.

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.

Chiral analysis of E-ε-viniferin enantiomers, towards a new chemotaxonomic marker of the vine.

BACKGROUND: The accurate characterization of grapevine cultivars (Vitis vinifera) is crucial for grape growers, winemakers, wine sellers, consumers and authorities, considering that mistakes could involve significant damage to the wine economic system. To avoid any misunderstanding, morphological, molecular and chemical tools are developed to positively identify grape varieties. RESULTS: E-ε-viniferin is a stilbene dimer mainly present in the woody part of grapevine and present as a mixture of two enantiomers: (7aR, 8aR)-(-)-E-ε-viniferin (1) and (7aS, 8aS)-(+)-E-ε-viniferin (2). In addition to phenotypic and genotypic approaches, a chemotaxonomic method using E-ε-viniferin enantiomers as chemical markers of grapevine cultivars was investigated. The isolation and purification of E-ε-viniferin enantiomers by preparative high-performance liquid chromatography (HPLC) and chiral HPLC from 14 red and eight white grapevine cane cultivars enabled us to determine the proportion of each enantiomer and therefore to calculate the enantiomeric excess for each variety. The relative abundance of each E-ε-viniferin enantiomer permitted us to distinguish grape varieties, as well as to establish cultivar relationships and patterns through statistical analysis. CONCLUSION: This pioneering work highlighting the enantiomeric excess of E-ε-viniferin as a chemical marker of grapevine paves the way for further studies to understand what mechanisms are involved in the production of these enantiomers in grapevine. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Arundinosides I-IX and graminifolosides A-B: 2R-benzylmalate and 2R-isobutylmalates derivatives from Arundina graminifolia (D.Don) Hochr. with antioxidant, cytocompatibility and cytoprotective properties.

Phytochemical investigation of the underground parts of Arundina graminifolia D.Don Hochr was conducted leading to the isolation of nine new glucosyloxybenzyl 2R-benzylmalate and two new glucosyloxybenzyl 2R-isobutylmalate derivatives. The compounds were purified using chromatographic techniques and their structures were deduced based on spectroscopic techniques including nuclear magnetic resonance and high-resolution mass spectrometry as well as comparing with previous literature. The antioxidant activities of the isolated compounds were also evaluated. The compounds showed potent antioxidant activities in the ABTS radical scavenging, DPPH radical scavenging and FRAP activities. Furthermore, the isolated compounds were observed to exert minimal cytotoxic effects against RAW 264.7 cell, suggesting biocompatibility as well as cytoprotective effects against hydrogen peroxide induced cell toxicity.

Chiroptical and potential in vitro anti-inflammatory properties of viniferin stereoisomers from grapevine (Vitis vinifera L.).

Determination of stereochemistry and enantiomeric excess in chiral natural molecules is a research of great interest because enantiomers can exhibit different biological activities. Viniferin stilbene dimers are natural molecules present in grape berries and wine but also, in larger amount, in stalks of grapevine. Four stereoisomers of viniferin stilbene dimers (7aS,8aS)-E-ε-viniferin (1a), (7aR,8aR)-E-ε-viniferin (1b), (7aS,8aR)-E-ω-viniferin (2a), and (7aR,8aS)-E-ω-viniferin (2b) were isolated from grapevine stalks of Cabernet Sauvignon, Merlot and Sauvignon Blanc, using a combination of centrifugal partition chromatography (CPC), preparative and chiral HPLC. The structure elucidation of these molecules was achieved by NMR whereas the absolute configurations of the four stereoisomers were investigated by vibrational circular dichroism spectroscopy in combination with density functional theory (DFT) calculations. This study unambiguously established the (+)-(7aS,8aS) and (+)-(7aR,8aS) configurations for E-ε-viniferin and E-ω-viniferin, respectively. Finally, we show that Cabernet Sauvignon provided the quasi enantiopure (+)-(7aS,8aS)-E-ε-viniferin compound which presents the best anti-inflammatory and anti-oxidant activities.

Naturally occurring phenolic compounds as promising antimycotoxin agents: Where are we now?

Mycotoxins are metabolites produced by molds that contaminate food commodities, are harmful to both humans and animals, as well as cause economic losses. Many countries have set regulatory limits and strict thresholds to control the level of mycotoxins in food and feedstuffs. New technologies and strategies have been developed to inhibit toxigenic fungal invasion and to decontaminate mycotoxins. However, many of these strategies do not sufficiently detoxify mycotoxins and leave residual toxic by-products. This review focuses on the use of phenolic compounds obtained from botanical extracts as promising bioagents to inhibit fungal growth and/or to limit mycotoxin yields. The mechanism of these botanicals, legislation concerning their use, and their safety are also discussed. In addition, recent strategies to overcome stability and solubility constraints of phenolic compounds to be used in food and feed stuffs are also mentioned.

Anthocyanins Promote Learning through Modulation of Synaptic Plasticity Related Proteins in an Animal Model of Ageing.

Anthocyanin-rich foods, such as berries, reportedly ameliorate age-related cognitive deficits in both animals and humans. Despite this, investigation into the mechanisms which underpin anthocyanin-mediated learning and memory benefits remains relatively limited. The present study investigates the effects of anthocyanin intake on a spatial working memory paradigm, assessed via the cross-maze apparatus, and relates behavioural test performance to underlying molecular mechanisms. Six-week supplementation with pure anthocyanins (2% w/w), administered throughout the learning phase of the task, improved both spatial and psychomotor performances in aged rats. Behavioural outputs were accompanied by changes in the expression profile of key proteins integral to synaptic function/maintenance, with upregulation of dystrophin, protein kinase B (PKB/Akt) and tyrosine hydroxylase, and downregulation of apoptotic proteins B-cell lymphoma-extra-large (Bcl-xL) and the phosphorylated rapidly accelerated fibrosarcoma (p-Raf). Separate immunoblot analysis supported these observations, indicating increased activation of extracellular signal-related kinase (ERK1), Akt Ser473, mammalian target of rapamycin (mTOR) Ser2448, activity-regulated cytoskeleton-associated protein (Arc/Arg 3.1) and brain-derived neurotrophic factor (BDNF) in response to anthocyanin treatment, whilst α-E-catenin, c-Jun N-terminal kinase (JNK1) and p38 protein levels decreased. Together, these findings suggest that purified anthocyanin consumption enhances spatial learning and motor coordination in aged animals and can be attributed to the modulation of key synaptic proteins, which support integrity and maintenance of synaptic function.

Untargeted LC-HRMS profiling followed by targeted fractionation to discover new taste-active compounds in spirits.

Taste is a key driver of food and beverage acceptability due to its role in consumers' pleasure. The great interest that natural food and beverages now arouse lies notably in the complexity of their taste, which in turn is related to a wide range of taste-active compounds. Going beyond the classic divide between targeted and untargeted strategies, an integrative methodology to spirits was applied. Untargeted profiling of several cognac spirits was implemented by LC-HRMS to identify compounds of interest among hundreds of ions. A targeted fractionation protocol was then developed. By using HRMS and NMR, dihydrodehydrodiconiferyl alcohol was identified and described for the first time in spirits and oak wood. It was characterized as sweet at 2 mg/L in two matrices and was quantified in spirits up to 4 mg/L. These findings demonstrated how this methodology is relevant and effective to discover new taste-active compounds.

Understanding sweetness of dry wines: First evidence of astilbin isomers in red wines and quantitation in a one-century range of vintages.

Astilbin (2R, 3R) was recently reported to contribute to wine sweetness. As its aglycon contains two stereogenic centers, three other stereoisomers may be present: neoisoastilbin (2S, 3R), isoastilbin (2R, 3S), and neoastilbin (2S, 3S). This work aimed at assaying their presence for the first time in wines as well as their taste properties. The isomers were synthesized from astilbin and purified by semi-preparative HPLC. With the four stereoisomers, a sweet taste was perceived whose intensity varied with the configuration. Their content was assayed by developing a UHPLC-Q-Exactive method. The method was applied to screen astilbin and isomers in various wines, especially in different vintages from the same estate. While young wines contained higher concentrations of astilbin than the old ones, the concentrations of the other isomers, mainly neoastilbin, were higher in the old wines, suggesting their formation over time.

Screening of Wood/Forest and Vine By-Products as Sources of New Drugs for Sustainable Strategies to Control Fusarium graminearum and the Production of Mycotoxins.

Fusarium graminearum is a fungal pathogen that can colonize small-grain cereals and maize and secrete type B trichothecene (TCTB) mycotoxins. The development of environmental-friendly strategies guaranteeing the safety of food and feed is a key challenge facing agriculture today. One of these strategies lies on the promising capacity of products issued from natural sources to counteract crop pests. In this work, the in vitro efficiency of sixteen extracts obtained from eight natural sources using subcritical water extraction at two temperatures was assessed against fungal growth and TCTB production by F. graminearum. Maritime pine sawdust extract was shown to be extremely efficient, leading to a significant inhibition of up to 89% of the fungal growth and up to 65% reduction of the mycotoxin production by F. graminearum. Liquid chromatography/mass spectrometry analysis of this active extract revealed the presence of three families of phenolics with a predominance of methylated compounds and suggested that the abundance of methylated structures, and therefore of hydrophobic compounds, could be a primary factor underpinning the activity of the maritime pine sawdust extract. Altogether, our data support that wood/forest by-products could be promising sources of bioactive compounds for controlling F. graminearum and its production of mycotoxins.

Characterization of Stilbene Composition in Grape Berries from Wild Vitis Species in Year-To-Year Harvest.

Asian and American Vitis species possess a strong potential for crossbreeding programs, owing to their several resistance properties. Stilbenes are phenolic compounds present in grape berries and are well-known for their main role as phytoalexins and resistance to biotic stresses in plants. However, their identification and quantification in grape berries from wild Vitis remains unexplored. A mass spectrometry multiple reaction monitoring method combined with the analysis of pure standards allowed for the unambiguous characterization of 20 stilbenes in the grape berry skin extracts of nine native Vitis species and one cultivated Vitis vinifera species (cv. Cabernet Sauvignon). A main occurrence of monomeric (Z-piceid, E-piceid, E-isorhapontin, and E-astringin), dimeric (E-ε-viniferin, Z-ε-viniferin, and pallidol), and oligomeric (isohopeaphenol and r-viniferin) stilbenes was highlighted. Some stilbenes were clearly characterized for the first time in grape berries, such as the dimers ampelopsin A, E-vitisinol C, and parthenocissin A as well as the tetramers r2-viniferin and r-viniferin. Stilbene composition and content varied widely among several Vitis species and vintage years.

Anti-tyrosinase, anti-cholinesterase and cytotoxic activities of extracts and phytochemicals from the Tunisian Citharexylum spinosum L.: Molecular docking and SAR analysis.

Previously phytochemical investigations carried out on the flowers and trunk bark extracts of Citharexylum spinosum L. tree, allowed the isolation of twenty molecules belonging to several families of natural substances [triterpene acids, iridoid glycosides, phenylethanoid glycosides, 8,3'-neolignan glycosides, together with other phenolic compounds]. In the present work, a biological evaluation (anti-tyrosinase, anticholinesterase and cytotoxic activities) was performed on the prepared extracts and the isolated secondary metabolites. The results showed that the EtOAc extract of the trunk bark displayed the highest anti-tyrosinase effect with a percent inhibition of 55.0 ± 1.8% at a concentration of 100 µg/mL. The highest anticholinesterase activity was presented by the same extract with an IC50 value of 99.97 ± 3.01 µg/mL. The EtOAc extract of flowers and that of the trunk bark displayed the best cytotoxic property with IC50 values of 96.00 ± 2.85 and 88.75 ± 2.00 µg/mL, respectively, against the human cervical cancer cell line (HeLa), and IC50 values of 188.23 ± 3.88 and 197.00 ± 4.25 µg/mL, respectively, against the human lung cancer (A549) cell lines. Biological investigation of the pure compounds showed that the two 8,3'-neolignan glycosides, plucheosides D1-D2, generate the highest anti-tyrosinase potency with a percent inhibition of 61.4 ± 2.0 and 79.5 ± 2.3%, respectively, at a concentration of 100 µM. The iridoid glycosides exhibited a significant anticholinesterase activity with IC50 values ranging from 17.19 ± 1.02 to 52.24 ± 2.50 µM. Triterpene pentacyclic acids and iridoid glycosides exerted encouraging cytotoxic effects against HeLa with IC50 values ranging from 9.00 ± 1.10 to 25.00 ± 1.00 µM. The study of the structure-activity relationship (SAR) has been sufficiently and widely discussed. The natural compounds that exhibited the significant bioactivities were docked.

Isolation of Taste-Active Triterpenoids from Quercus robur: Sensory Assessment and Identification in Wines and Spirit.

Six new triterpenoids (1-6), two known genins (7 and 8), and five known functionalized triterpenoids (9-13) were isolated from a Quercus robur heartwood extract. The purification protocol was guided by LC-HRMS by searching for structural analogues of bartogenic acid on the basis of their putative empirical formula. The structures of the new compounds were unequivocally elucidated using HRESIMS and 1D/2D NMR experiments. Sensory analyses were performed in water and in a non-oaked white wine on the pure compounds 1-13 at 5 mg/L. All molecules were perceived as bitter in water and wine, but they were mostly reported as modifying the wine taste balance. Using LC-HRMS, compounds 1-13 were observed in oaked red wine and cognac and were semiquantified in oak wood extracts. The influence of two cooperage parameters, oak species and toasting process, on compounds 1-13 content was studied. All compounds were found in quantities significantly higher in pedunculate than in sessile oak wood. Toasting is a key step in barrel manufacture and modulates the concentration of the discussed compounds. Significantly higher quantities were observed in untoasted wood compared to medium or highly toasted wood. These findings provide new insights into the molecular origin of taste changes due to oak aging.

Separation and isolation of major polyphenols from maritime pine (Pinus pinaster) knots by two-step centrifugal partition chromatography monitored by LC-MS and NMR spectroscopy.

Pine knots are a rich source of lignans, flavonoids, and stilbenes. These bioactive compounds are widely known for their roles to combat human disorders but also to protect plants against pathogens. In order to gain knowledge inside their potential activities, a suitable isolation and purification of these high-added value compounds is required. To this end, centrifugal partition chromatography, as a rapid and useful methodology of separation, was employed and developed. The coefficient partition values (KD ) of six major compounds in nine biphasic solvent systems were determined to evaluate the most appropriate system. Two-step centrifugal partition chromatography was required to separate lignans using ARIZONA system K (n-heptane/ethyl acetate/methanol/water 1:2:1:2, v:v) and to isolate stilbenes and flavonoids using ARIZONA system P (n-heptane/ethyl acetate/methanol/water 6:5:6:5, v:v). Eight one-compound enriched-fractions were obtained as follows: nortrachelogenin (70.1%), secoisolariciresinol (53.7%), isolariciresinol (61.1%), taxifolin (48.4%), pinocembrin (91.3%), pinobanksin (91.1%), pinosylvin (91.4%), and pinosylvin monomethyl ether (91.1%). Additionally, the centrifugal partition chromatography allowed to unravel the composition of pine knot owing to the several fractions generated. Twenty-two compounds were characterized by liquid chromatography-mass spectrometry and NMR spectroscopy, some of which are described for the first time in literature.

New glucosyloxybenzyl 2R-benzylmalate derivatives from the undergrounds parts of Arundina graminifolia (Orchidaceae).

Phytochemical investigation of the underground part of the blossoming tropical orchid Arundina graminifolia led to the isolation of six new glucosyloxybenzyl 2R-benzylmalate derivatives named arundinosides L-Q (1-6) together with 5 known compounds arundinosides D-F, J and K (7-11). The structures of the isolated compounds were determined by extensive spectroscopic data analysis. The anti-α-glucosidase and antioxidant activities of the isolated compounds were determined. The result indicated that compounds 4-6 and 9 showed moderate to weak α-glucosidase inhibitory effects as well as moderate antioxidant effect.

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.

Triterpenoids from Quercus petraea: Identification in Wines and Spirits and Sensory Assessment.

Eight new triterpenoids (1-8), the known genin (9), and two known functionalized triterpenoids (10 and 11) were isolated from a Quercus petraea heartwood extract. The structures of the new compounds were unequivocally elucidated using HRESIMS and 1D/2D NMR experiments. Sensory analyses were performed in a non-oaked wine on the pure compounds 1-11. Except compounds 1 and 11, all molecules exhibited a sweet taste at 5 mg/L that was particularly intense for compounds 3 and 9. Using LC-HRMS, compounds 1-11 were observed in an oak wood extract and in oaked red wine and cognac. They were also semiquantified in several samples of sessile ( Q. petraea) and pedunculate ( Q. robur) oak wood extract. All compounds were found in quantities significantly higher in sessile than in pedunculate oak wood. These results support the hypothesis of their contribution to the increase in sweetness during oak aging and show that they can be used as chemical markers to identify the species of oak used for cooperage.

Taste-guided isolation of sweet-tasting compounds from grape seeds, structural elucidation and identification in wines.

This work aimed at improving knowledge about sweetness in dry wines. Following on from the empirical observations of winegrowers, we assessed the contribution of grape seeds to wine sensory properties. An inductive fractionation method guided by gustatometry was used to isolate and characterize sweet-tasting compounds from grapes. Fractionation of grape seed macerates was achieved by liquid-liquid extraction, centrifugal partition chromatography (CPC) and preparative HPLC. Then, the structures of the purified compounds were elucidated by use of FTMS and NMR. Five compounds were identified: two new compounds, 2-hydroxy-3-methylpentanoic-2-O-ß-glucopyranoside (H3MP-G) and 2-hydroxy-4-methylpentanoic-2-O-ß-glucopyranoside acids (H4MP-G), along with gallic-4-O-ß-glucopyranoside acid (AG-G), 3-indolyl-(2R)-O-ß-d-glycoside lactic acid (ILA-G) and epi-DPA-3'-O-ß-glucopyranoside acid (epi-DPA-G). These compounds exhibited various levels of sweetness in a hydro-ethanolic solution and in white and red wines. Additionally, H3MP-G, H4MP-G and epi-DPA-G were identified for the first time in grapes and wines, whereas AG-G has already been reported in white grapes but never in wine.