Screening for pancreatic lipase natural modulators by capillary electrophoresis hyphenated to spectrophotometric and conductometric dual detection.

The search for novel pancreatic lipase (PL) inhibitors has gained increasing attention in recent years. For the first time, a dual detection capillary electrophoresis (CE)-based homogeneous lipase assay was developed employing both the offline and online reaction modes. The hydrolysis of 4-nitrophenyl butyrate (4-NPB) catalyzed by PL into 4-nitrophenol and butyrate was monitored by spectrophotometric and conductimetric detection, respectively. The assays presented several advantages such as economy in consumption (few tens of nanoliters for online assays to few tens of microliters for offline assays), no modification of lipase, rapidity (<10 min) and versatility. Tris/MOPS (10 mM, pH 6.6) was used as the background electrolyte and the incubation buffer for enzymatic reactions. We confirmed that in the conditions of the study (small substrate 4-NPB, 37 °C, pH 6.6), the PL was active even in the absence of dipalmitoylphosphatidylcholine (DPPC) vesicles, generally used to mimic the lipid-water interface. This was confirmed by the maximum velocity (Vmax) and the Michaelis-Menten constant (Km) values that were the same order of magnitude in the absence and presence of DPPC. The developed method was used to screen crude aqueous plant extracts and purified compounds. We were able to identify the promising PL inhibition of hawthorn leaf herbal infusions at 1 mg mL-1 (37%) and PL activation by fresh and dry hawthorn flowers (∼24%). Additionally, two triterpenoids purified from extracts of oakwood were identified for the first time as potent PL inhibitors demonstrating 51 and 58% inhibition at 1 mg mL-1, respectively.

Investigation of lipase-ligand interactions in porcine pancreatic extracts by microscale thermophoresis.

The evaluation of binding affinities between large biomolecules and small ligands is challenging and requires highly sensitive techniques. Microscale thermophoresis (MST) is an emerging biophysical technique used to overcome this limitation. This work describes the first MST binding method to evaluate binding affinities of small ligands to lipases from crude porcine pancreatic extracts. The conditions of the MST assay were thoroughly optimized to successfully evaluate the dissociation constant (Kd) between pancreatic lipases (PL) and triterpenoid compounds purified from oakwood. More precisely, the fluorescent labeling of PL (PL*) using RED-NHS dye was achieved via a buffer exchange procedure. The MST buffer was composed of 20 mM NaH2PO4 + 77 mM NaCl (pH 6.6) with 0.05% Triton-X added to efficiently prevent protein aggregation and adsorption, even when using only standard, uncoated MST capillaries. Storage at -20 °C ensured stability of PL* and its fluorescent signal. MST results showed that crude pancreatic extracts were suitable as a source of PL for the evaluation of binding affinities of small ligands. Quercotriterpenoside-I (QTT-I) demonstrated high PL* binding affinity (31 nM) followed by 3-O-galloylbarrinic acid (3-GBA) (500 nM) and bartogenic acid (BA) (1327 nM). To enrich the 50 kDa lipase responsible for the majority of hydrolysis activity in the crude pancreatic extracts, ammonium sulfate precipitation was attempted and its efficiency confirmed using capillary electrophoresis (CE)-based activity assays and HRMS. Moreover, to accurately explain enzyme modulation mechanism, it is imperative to complement binding assays with catalytic activity ones.

Polyethylene glycol crowding effect on hyaluronidase activity monitored by capillary electrophoresis.

To mimic the activity of hyaluronidase in natural environment, the hydrolysis of hyaluronic acid (HA) by hyaluronidase was investigated for the first time in the presence of crowding agents using capillary electrophoresis (CE) as a simple and reliable technique for conducting enzymatic assay. Polyethylene glycol (PEG) 6000 was selected as a model crowder and the hyaluronic acid degradation catalyzed by bovine testes hyaluronidase (BTH) was carried out at different PEG concentrations (0%, 10%, and 17%). After optimization of the CE analytical method and enzymatic assay, the degradation products were monitored at different HA concentrations. At 10% of PEG and 0.3 mg mL-1 of HA, the activity of the enzyme was significantly reduced showing inconvenient interactions of PEG with the hyaluronidase blocking the release of hydrolysis products. A similar reduction of hyaluronidase activity was observed at 1 mg mL-1 of HA due to the presumable formation of the BTH-substrate complex. The experimental curves obtained by CE also evidence that the overall kinetics are governed by the hydrolysis of hexasaccharide intermediates. Finally, the effect of PEG on hyaluronidase activity was evaluated in the presence of natural or synthetic inhibitors. Our results show a significant difference of the inhibitors' affinity toward hyaluronidase in the presence of PEG. Surprisingly, the presence of the crowding agent results in a loss of the inhibition effect of small polycyclic inhibitors, while larger charged inhibitors were less affected. In this work, CE analyses confirm the importance of mimicking the cellular environment for the discovery and development of reliable inhibitors. Graphical abstract.

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.

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.

The complexity of wine: clarifying the role of microorganisms.

The concept of wine complexity has gained considerable interest in recent years, both for wine consumers and wine scientists. As a consequence, some research programs concentrate on the factors that could improve the perceived complexity of a wine. Notably, the possible influence of microbiological factors is particularly investigated. However, wine complexity is a multicomponent concept not easily defined. In this review, we first describe the actual knowledge regarding wine complexity, its perception, and wine chemical composition. In particular, we emphasize that, contrary to expectations, the perception of wine complexity is not related to wine chemical complexity. Then, we review the impact of wine microorganisms on wine complexity, with a specific focus on publications including sensory analyses. While microorganisms definitively can impact wine complexity, the underlying mechanisms and molecules are far from being deciphered. Finally, we discuss some prospective research fields that will help improving our understanding of wine complexity, including perceptive interactions, microbial interactions, and other challenging phenomena.

Lignans in Spirits: Chemical Diversity, Quantification, and Sensory Impact of (±)-Lyoniresinol.

During barrel aging, spirits undergo organoleptic changes caused by the release of aroma and taste compounds. Recently, studies have revealed the bitter properties of oak wood lignans, such as (±)-lyoniresinol, and their contribution to wine taste. To evaluate the impact of lignans in spirits, a targeted screening of 11 compounds was set up and served to validate their presence in this matrix, implying their release by oak wood during aging. After development and validation of a quantification method, the most abundant and the bitterest lignan, (±)-lyoniresinol, was assayed by liquid chromatography⁻high resolution mass spectrometry (LC-HRMS) in spirits. Its gustatory detection threshold was established at 2.6 mg/L in spirits. A large number of samples quantified were above this detection threshold, which suggests its effect of increased bitterness in spirit taste. Significant variations were observed in commercial spirits, with concentrations ranging from 0.2 to 11.8 mg/L, which could be related to differences in barrel aging processes. In "eaux-de-vie" of cognac, concentrations of (±)-lyoniresinol were observed in the range from 1.6 mg/L to 12 mg/L. Lower concentrations were measured for older vintages.

Development of a quantitation method to assay both lyoniresinol enantiomers in wines, spirits, and oak wood by liquid chromatography-high resolution mass spectrometry.

Wine taste balance evolves during oak aging by the release of volatile and non-volatile compounds from wood. Among them, an enantiomer of lyoniresinol, (+)-lyoniresinol, has been shown to exhibit bitterness. To evaluate the impact of (+)-lyoniresinol on wine taste, a two-step quantitation method was developed and validated. First, (±)-lyoniresinol was assayed in wines, spirits, and oak wood macerates by C-18 liquid chromatography-high resolution mass spectrometry (LC-HRMS). Then, the lyoniresinol enantiomeric ratio was determined by chiral LC-HRMS in order to calculate the (+)-lyoniresinol content. In red and white wines, the average concentrations of (+)-lyoniresinol were 1.9 and 0.8 mg/L, respectively. The enantiomer proportions were not affected by bottle aging, and lyoniresinol appeared to remain stable over time. The sensory study of (+)-lyoniresinol established its perception threshold at 0.46 mg/L in wine. All the commercial wines quantitated were above this perception threshold, demonstrating its impact on wine taste by an increase in bitterness. In spirits, (+)-lyoniresinol ranged from 2.0 to 10.0 mg/L and was found to be released continuously during oak aging. Finally, neither botanical origin nor toasting was found to significantly affect the (+)-lyoniresinol content of oak wood. Graphical abstract From oak wood to wine: evaluation of the influence of (+)-lyoniresinol on the bitterness of wines and spirits.

Taste-Guided Isolation of Bitter Lignans from Quercus petraea and Their Identification in Wine.

Quercoresinosides A and B (1 and 2), two new lignans, were isolated from a toasted Quercus petraea heartwood extract along with a known compound, 3-methoxy-4-hydroxyphenol 1-O-ß-d-(6'-O-galloyl)glucopyranoside (3). The purification protocol was based on a taste-guided approach that sought to reveal new bitter compounds released from oak wood into wines and spirits. HRMS and NMR data were used to establish that compounds 1 and 2 are lignan derivatives bearing a glucosyl unit and a galloyl unit at the same positions. Hydrolysis of these compounds showed that they could be distinguished by the absolute configuration of their respective lyoniresinol genin as determined by chiral LC-HRMS in comparison with (+)- and (-)-lyoniresinol standards. Sensory analyses were performed in a non-oaked wine on the pure compounds 1-3. The three molecules exhibited a bitter taste at 2 mg/L that was particularly intense for compounds 2 and 3. Finally, LC-HRMS demonstrated the occurrence of compounds 1-3 in oaked wine and brandy, which supports the hypothesis of their contributions to the increase in bitterness during oak aging.

Isolation and structure elucidation of bioactive compounds from the roots of the Tunisian Ononis angustissima L.

A phytochemical investigation of the roots of Ononis angustissima L. (Fabaceae) offered to the bio-guided isolation of new isoflavone 3-(4-(glucopyranosyloxy)-5-hydroxy-2-methoxyphenyl)-7-hydroxy-4H-chromen-4-one 1, together with nine known compounds, ononin 2, formononetin 3, (+)-puerol A-2'-O-ß-D-glucose 4, (-)-puerol B-2'-O-ß-D-glucopyranose ((-)-sophoraside A) 5, (+)-puerol A 6, (-)-trifolirhizin 7, (-)-trifolirhizin-6'-O-malonate 8, (-)-maackiain 9 and (-)-medicarpin 10. Compounds 2-10 were isolated and identified for the first time in Ononis angustissima. We investigated antioxidant capacities of isolated molecules and results showed that compound 6 exhibited the highest antioxidant activity with IC50 values of 19.53 µg/mL, 28.29 µg/mL and 38.53 µg/mL by DPPH radical, ABTS radical cation and reducing power assay, respectively, and an interesting IC50 (20.45 µg/mL) of 1 against DPPH. In addition, the neuroprotective activity of six isolated molecules (4-7, 9, 10) were evaluated. Following the exposure of PC12 cells to Aß25-35, compounds 9 and 10 triggered a significant increase of cell viability and in a dose dependent manner.

Unambiguous Determination of the Absolute Configuration of Dimeric Stilbene Glucosides from the Rhizomes of Gnetum africanum.

Dimeric stilbene glucosides 1-3 [two diastereomers of (-)-gnemonoside A (1a and 1b), (-)-gnemonoside C (2), and (-)-gnemonoside D (3)] as well as a mixture of the two enantiomers of gnetin C (4) were isolated from the rhizomes of Gnetum africanum. The two enantiomers of gnetin C, (+)-4 and (-)-4, were obtained from the aglycones of 1a and 1b, respectively. The configurations of these stilbenoids were investigated by NMR and vibrational circular dichroism (VCD) experiments. The absolute configurations of (-)-1a, (-)-2, (-)-3, and (-)-4 were established as 7aS,8aS by VCD spectroscopy in combination with density functional theory calculations. The antiamyloidogenic activity of the isolated stilbenes was also evaluated versus beta-amyloid fibrils. The four glucosides of gnetin C (1a, 1b, 2, and 3) were found to be the most active compounds, with inhibition percentages of 56, 56, 58, and 54 at 10 µM, respectively.

Towards an understanding of bitterness in white wines: Contribution of 27 compounds assessed by LC-HRMS and sensory analysis.

Whereas bitterness perception can modify the taste balance of white wines, its molecular origin remains largely unclear. This work aimed at determining the influence of a selection of the most cited bitter compounds on the bitterness of commercial dry white wines. Forty-two wines were sensorially characterized by a trained panel and divided into two statistically different groups depending on their bitterness. Twenty-seven bitter compounds were selected and five quantitation methods were developed and validated. The methods were used to measure the levels of all the 27 compounds in dry wine, 25 of them in sweet wine and 22 of them in grape juice. The detected concentrations were generally below the taste detection thresholds. No significant positive correlation between the bitterness intensity of the tasted samples and the concentration of the assayed bitter compounds was observed, suggesting the existence of other markers of bitterness in white wines.

Optimization of extraction and development of an LC-HRMS method to quantify glutathione and glutathione disulfide in white wine lees and yeast derivatives.

The antioxidant capacity of wine depends on its quality and aging potential. Aging on lees can improve this capacity thanks to the release of glutathione (GSH), as can the addition of yeast derivatives (YD). Therefore, the GSH potential of wine lees (WL) and YD requires investigation. We propose an optimized method to extract and quantify GSH from WL and YD. First, a method was developed to detect and quantify GSH and glutathione disulfide (GSSG) using LC-HRMS. Second, Box-Behnken response surface methodologies (RSM) were applied to both matrices. Results showed that the main parameter affecting GSH extraction efficiency was ethanol concentration. Quantitation of various samples revealed GSH concentrations of up to 900 µg/g for WL and 40 mg/g for YD. To our knowledge, the absolute quantitation of GSH/GSSG in these matrices has not been reported until now.

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.

Isolation of a new taste-active brandy tannin A: Structural elucidation, quantitation and sensory assessment.

Enjoying a glass of spirits can be one of the delights of life. While it is well known that their taste improves during barrel aging, the molecular explanations of this phenomenon remain largely unknown. The present work aimed at searching for taste-active compounds formed in spirits during aging. An untargeted metabolomic approach using HRMS was applied on "eau-de-vie" of cognac. A fractionation protocol was then performed on brandies to isolate a targeted compound. By using HRMS and NMR, its structure was elucidated for the first time. This new ellagitannin, called brandy tannin A, considerably increased the sweetness of spirits at 2 mg/L. After development of an LC-HRMS quantitation method, it was assayed in various spirits and was detected mainly in cognacs up to 7 mg/L. These findings demonstrate the sensory contribution of this compound and more generally the relevance of combining metabolomics and separative techniques to purify new taste-active compounds.

Identification of new natural sweet compounds in wine using centrifugal partition chromatography-gustatometry and Fourier transform mass spectrometry.

Sweetness contributes notably to the taste-balance of dry wines and increases during oak-barrel aging owing to the release of natural sweeteners from wood. The search for such taste-active molecules, which are sometimes present at very low concentrations in wine or other complex matrixes, requires both reliable purification tools and powerful identification techniques. Here, we report the development of an original inductive method using centrifugal partition chromatography (CPC) and sensorial analysis. This method, called CPC-gustatometry, was implemented to isolate a sweet fraction with only four compounds from a complex oak wood extract. The recently developed Fourier transform mass spectrometry (FT-MS, Orbitrap analyzer) was used jointly with two-dimensional nuclear magnetic resonance (2D (1)H and (13)C NMR) to obtain the structural elucidation of the purified compounds. The tandem mass spectrometry (MS/MS) spectra obtained with resonant and nonresonant fragmentation modes were compared, thus providing complementary information about the molecular structure. Two oleanane-type triterpenoids substituted with galloyl and glucosyl moieties were identified, one of which exhibits sweet properties. We term these compounds which have never been reported, Quercotriterpenoside I and II.

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.

Contribution of Grapes and Oak Wood Barrels to Pyrrole Contents in Chardonnay Wines: The Influence of Several Cooperage Parameters.

The influence of some enological parameters on pyrrole concentrations in Chardonnay wines was studied. First, a quantitative method to assay five pyrroles was optimized and applied to determine their content in wines produced in different containers. All pyrroles were observed in wines aged in a stainless-steel tank, which indicated that they have a varietal or fermentative origin. However, their concentrations were significantly higher in wines made in new barrels than in older barrels or in a stainless-steel tank, so oak wood may largely contribute during the winemaking process. A quantitative method to assay pyrroles in oak wood extract was also developed to study the influence of several cooperage parameters such as different types of traditional toasting, as well as the temperature and the time of toasting. Significant differences were observed on pyrrole concentrations in oak wood extracts according to these different cooperage parameters. These findings bring new perspectives to the monitoring of winemaking and the aging of Chardonnay wines.

Trans-ε-Viniferin Encapsulation in Multi-Lamellar Liposomes: Consequences on Pharmacokinetic Parameters, Biodistribution and Glucuronide Formation in Rats.

Trans-ε-viniferin (εVin) is a resveratrol dimer exhibiting promising biological activities for human health. Its bioavailability being low, the development of encapsulation methods would be used to overcome this issue. The aim of this study was to measure the consequences of the encapsulation of εVin in multilamellar liposomes on its pharmacokinetic parameters, metabolism and tissue distribution in rats. After oral administration of εVin (20 mg/kg body weight), either as free or encapsulated forms, plasmas were sequentially collected (from 0 to 4 h) as well as liver, kidneys and adipose tissues (4 h after administration) and analyzed by LC-HRMS. The glucuronide metabolites (εVG) were also produced by hemisynthesis for their quantification in plasma and tissues. The encapsulation process did not significantly modify the pharmacokinetic parameters of εVin itself. However, a significant increase of the T1/2 was noticed for εVG after administration of the encapsulated form as compared to the free form. An accumulation of εVin and εVG in adipose tissues was noticed, and interestingly a significant increase of the latter in the mesenteric one after administration of the encapsulated form was highlighted. Since adipose tissues could represent storage depots, and encapsulation allows for prolonging the exposure time of glucuronide metabolites in the organism, this could be of interest to promote their potential biological activities.