Emmanuelle Meudec

Interspecific variation in leaf litter tannins drives decomposition in a tropical rain forest of French Guiana

Tannins are believed to be particularly abundant in tropical tree foliage and are mainly associated with plant herbivore defense. Very little is known of the quantity, variation, and potential role of tannins in tropical leaf litter. Here we report on the interspecific variability of litter condensed tannin (CT) concentration among 16 co-occurring tropical rain forest tree species of French Guiana and explore the functional significance of variable litter CT concentration for litter decomposition. We compared some classical methods in the ecological literature to a method based on high-performance liquid chromatography (HPLC), coupled with CT degradation by phloroglucinolysis. The same litter was allowed to decompose in the field in the presence or absence of soil fauna. We found large interspecific differences in the average polymerization degree (2.7 to 21.3, for non-extractable CT) and concentration of litter CT (0-3.7% dry mass, for total CT) determined by HPLC, which did not correlate with Folin total phenolics but correlated reasonably well with acid butanol CT. The concentration and polymerization degree of HPLC-determined CT were the only variables of the multitude of measured initial litter quality parameters that explained a significant amount of variation in litter mass loss among species, irrespective of animal presence. However, animal presence increased mean litter mass loss by a factor of 1.5, and the fauna effect on decomposition was best explained by a negative correlation with total HPLC CT and by a positive correlation with hemicellulose. Our results suggest that the commonly used acid butanol assay yields a reliable estimate of interspecific variation in CT concentration. However, the chemical structure of CTs, such as the polymerization degree, adds important information for the understanding of the functional role of CTs in litter decomposition. We conclude that the wide variation in structure and concentration of leaf litter CTs among tropical tree species is an important driver of decomposition in this nutrient-poor Amazonian rain forest.

Direct mass spectrometry approaches to characterize polyphenol composition of complex samples

Lower molecular weight polyphenols including proanthocyanidin oligomers can be analyzed after HPLC separation on either reversed-phase or normal phase columns. However, these techniques are time consuming and can have poor resolution as polymer chain length and structural diversity increase. The detection of higher molecular weight compounds, as well as the determination of molecular weight distributions, remain major challenges in polyphenol analysis. Approaches based on direct mass spectrometry (MS) analysis that are proposed to help overcome these problems are reviewed. Thus, direct flow injection electrospray ionization mass spectrometry analysis can be used to establish polyphenol fingerprints of complex extracts such as in wine. This technique enabled discrimination of samples on the basis of their phenolic (i.e. anthocyanin, phenolic acid and flavan-3-ol) compositions, but larger oligomers and polymers were poorly detectable. Detection of higher molecular weight proanthocyanidins was also restricted with matrix-assisted laser desorption ionization (MALDI) MS, suggesting that they are difficult to desorb as gas-phase ions. The mass distribution of polymeric fractions could, however, be determined by analyzing the mass distributions of bovine serum albumin/proanthocyanidin complexes using MALDI-TOF-MS.

Identification and functional characterization of cDNAs coding for hydroxybenzoate/hydroxycinnamate glucosyltransferases co-expressed with genes related to proanthocyanidin biosynthesis

Grape proanthocyanidins (PAs) play a major role in the organoleptic properties of wine. They are accumulated mainly in grape skin and seeds during the early stages of berry development. Despite the recent progress in the identification of genes involved in PA biosynthesis, the mechanisms involved in subunit condensation, galloylation, or fine regulation of the spatio-temporal composition of grape berries in PAs are still not elucidated. Two Myb transcription factors, VvMybPA1 and VvMybPA2, controlling the PA pathway have recently been identified and ectopically over-expressed in an homologous system. In addition to already known PA genes, three genes coding for glucosyltransferases were significantly differentially expressed between hairy roots over-expressing VvMybPA1 or VvMybPA2 and control lines. The involvement of these genes in PA biosynthesis metabolism is unclear. The three glucosyltransferases display high sequence similarities with other plant glucosyltransferases able to catalyse the formation of glucose esters, which are important intermediate actors for the synthesis of different phenolic compounds. Studies of the in vitro properties of these three enzymes (Km, Vmax, substrate specificity, pH sensitivity) were performed through production of recombinant proteins in E. coli and demonstrated that they are able to catalyse the formation of 1-O-acyl-Glc esters of phenolic acids but are not active on flavonoids and stilbenes. The transcripts are expressed in the early stages of grape berry development, mainly in the berry skins and seeds. The results presented here suggest that these enzymes could be involved in vivo in PA galloylation or in the synthesis of hydroxycinnamic esters.

Characterization, stoichiometry, and stability of salivary protein–tannin complexes by ESI-MS and ESI-MS/MS

AbstractNumerous protein–polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein–ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP–tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP–tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5·EgCG complexes are maintained intact in the gas phase.n FigureSchematic illustrating the interaction between IB5 and EgCG, leading to IB5·EgCG complexes that remain intact in the gas phase during ESI-MS analysis. This system provides a model of biological interest with regards to astringency

Polyphenolic compounds in date fruit seed (Phoenix dactylifera): characterisation and quantification by using UPLC-DAD-ESI-MS.

BACKGROUNDnDate fruit seeds have been demonstrated to possess high antioxidant activities due to their high content of flavonoids and phenolic compounds. The objective of this work was to identify and quantify the phenolic composition of date seeds.nnnMETHODSnTwo UPLC-DAD-ESI-MS analyses were performed on the seed of the Khalas variety as follows: (1) an analysis of simple phenolic compounds [phenolic acids, hydroxycinnamic acids, flavonols, flavones, flavan-3-ols (monomers, dimers and trimers)]; and (2) an analysis of all flavan-3-ols (monomers, and proanthocyanidin oligomers and polymers) after depolymerisation.nnnRESULTSnThe amount of total phenolic compounds before depolymerisation was found to be 2.194u2009±u20090.040 g kg(-1) date seed. The analysis of flavan-3-ol monomers and constitutive units of proanthocyanidins after depolymerisation revealed 50.180u2009±u20091.360 g kg(-1) flavan-3-ols with 46.800u2009±u20091.012 g kg(-1) epicatechin and 3.380u2009±u20090.349 g kg(-1) catechin.nnnCONCLUSIONnThe results indicate that date seeds are a very rich source of bioactive compounds, thus constituting strong candidates for functional food additives and nutraceuticals.

Silencing of the chalcone synthase gene in Casuarina glauca highlights the important role of flavonoids during nodulation

Nitrogen-fixing root nodulation is confined to four plant orders, including > 14,000 Leguminosae, one nonlegume genus Parasponia and c. 200 actinorhizal species that form symbioses with rhizobia and Frankia bacterial species, respectively. Flavonoids have been identified as plant signals and developmental regulators for nodulation in legumes and have long been hypothesized to play a critical role during actinorhizal nodulation. However, direct evidence of their involvement in actinorhizal symbiosis is lacking. Here, we used RNA interference to silence chalcone synthase, which is involved in the first committed step of the flavonoid biosynthetic pathway, in the actinorhizal tropical tree Casuarina glauca. Transformed flavonoid-deficient hairy roots were generated and used to study flavonoid accumulation and further nodulation. Knockdown of chalcone synthase expression reduced the level of specific flavonoids and resulted in severely impaired nodulation. Nodule formation was rescued by supplementing the plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of an important role for flavonoids during the early stages of actinorhizal nodulation.

Qualitative and Semi-quantitative Analysis of Phenolics in Eucalyptus globulus Leaves by High-performance Liquid Chromatography Coupled with Diode Array Detection and Electrospray Ionisation Mass Spectrometry

INTRODUCTIONnEucalyptus species are widely cultivated in Mediterranean regions. Moreover, plants of this family have been utilized for medicinal purposes. A number of studies have been devoted to the identification of eucalypt phenolics, all of them have focused on specific families of compounds, and no exhaustive profiling has been reported in leaves of this plant.nnnOBJECTIVEnTo develop methods that allows the identification and quantification of different classes of phenolics in Eucalyptus globulus leaf.nnnMETHODOLOGYnAcetonic extract was fractionated by chromatography on a Sephadex LH-20 column using consecutive elution with ethanol, methanol and aqueous acetone (60%). High-performance liquid chromatography coupled with diode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESI/MS) were applied to determine the structure of different compounds. Quantities were evaluated from peak areas in the HPLC profile, using external calibration curves.nnnRESULTSnFractionation of acetonic extract yielded three fractions: F1, F2 and F3. In total 39 phenolic compounds are detected. Among them: 16 hydrolyzable tannins, 3 terpenyl derivatives, 12 ellagic acid derivatives, 5 flavonols, 2 hydroxybenzoic acids and 1 formylated phloroglucinol. 26 compounds described in this study have not previously detected in leaves of this plant and this is the first report of quercetin 3-O-β-galactoside-6-O-gallate and cypellogin A and B, in E. globulus plant. Quantitatively, ellagic acid derivatives and sideroxylonal A or B are largely predominant.nnnCONCLUSIONnFractionation of crude extract by chromatography on Sephadex LH-20 was efficient to separate different molecular weight compounds. HPLC-DAD-ESI/MS enabled detection of gallotannin, ellagitannin and flavonol derivatives, in leaves of E. globulus.

Anthocyanin Characterization of Pilot Plant Water Extracts of Delonix regia Flowers

Following the development of new applications of pilot plant scale extraction and formulation processes for natural active bioproducts obtained from various under-utilized tropical plants and herbs, we have manufactured water-extracts from Delonix regia flowers, grown in Ivory Coast. These extracts, which contain polyphenols, are traditionally home made and used as healthy bioproducts. They are reddish-coloured due to the presence of anthocyanins. The three major anthocyanins in these extracts have been characterized. The molecular structures were confirmed by LC-SM analysis. Amongst them, two are described for the first time in Delonix regia.

Combination of several mass spectrometry ionization modes: a multiblock analysis for a rapid characterization of the red wine polyphenolic composition.

In the present study, direct flow injection mass spectrometry was investigated for rapid characterization of the polyphenolic composition of red wines. Atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) (in both positive and negative ion modes) have been simultaneously used for a more comprehensive analysis of the samples studied. In this way, four mass spectra have been recorded for each wine. Each spectrum was considered as a fingerprint related to the chemical composition. This methodology was applied to a large number of Beaujolais wines from different grades and different vintages. This data set was processed using a chemometrical multiblock analysis, which allowed to synthesize the whole information collected. The results obtained showed that the wine fingerprints address the composition of the main polyphenolic compounds present in the red wines and can discriminate groups of wines showing different polyphenolic compositions. Multiblock analysis appears as a very promising tool to deal with several data tables of multivariate signals in order to define, by combining the whole information, the best operating protocol according to the desired analytical objectives.

Moderate consumption of wine, through both its phenolic compounds and alcohol content, promotes hydroxytyrosol endogenous generation in humans. A randomized controlled trial

In humans, urinary hydroxytyrosol (OHTyr) concentrations have been associated to alcohol and wine consumption. To explore the role of wine components on promoting an endogenous OHTyr generation we performed a cross-over, double-blind, randomized controlled clinical trial (n = 28 healthy volunteers). Ethanol (wine and vodka), dealcoholized wine, and placebo were administered. Alcohol, dealcoholized wine, and particularly wine promoted a de novo OHTyr generation in vivo in humans. Potential OHTyr precursors (tyrosine, tyrosol, tyramine) were investigated in rats. Tyrosol was metabolized to OHTyr. Collating both studies, it is postulated that an increased Tyr bioavailability, a shift to a reductive pathway in dopamine and tyramine oxidative metabolism, and the biotransformation of Tyr to OHTyr were mechanisms involved in the OHTyr endogenous generation.