"Mix and match" auto-assembly of glycosyltransferase domains delivers biocatalysts with improved substrate promiscuity.

Glycosyltransferases (GT) catalyze the glycosylation of bioactive natural products, including peptides and proteins, flavonoids, and sterols, and have been extensively used as biocatalysts to generate glycosides. However, the often narrow substrate specificity of wild-type GTs requires engineering strategies to expand it. The GT-B structural family is constituted by GTs that share a highly conserved tertiary structure in which the sugar donor and acceptor substrates bind in dedicated domains. Here, we have used this selective binding feature to design an engineering process to generate chimeric glycosyltransferases that combine auto-assembled domains from two different GT-B enzymes. Our approach enabled the generation of a stable dimer with broader substrate promiscuity than the parent enzymes that were related to relaxed interactions between domains in the dimeric GT-B. Our findings provide a basis for the development of a novel class of heterodimeric GTs with improved substrate promiscuity for applications in biotechnology and natural product synthesis.

Evaluation and Comparison of Dermo-Cosmetic Activities of Three Oak Species by Targeting Antioxidant Metabolites and Skin Enzyme Inhibitors.

The two main species, sessile oak (Quercus petraea Liebl.) and pedunculate oak (Quercus robur L.), predominant in French forests, are mainly used for aging wines and spirits; however, the potential of oak wood extract as a source of natural antioxidants, due to its high polyphenol content, could be more widely exploited. This study focuses on three oak species, the two that are well-known, namely, sessile and pedunculate oak, and a third that has seldom been described and valorized, namely, pubescent oak (Quercus pubescens). Water extracts of these three species were fractionated by semi-preparative HPLC. The antioxidant activities of crude extracts and fractions were measured by colorimetric and enzymatic tests. The anti-elastase and anti-collagenase activities of the extracts and their fractions were also evaluated. In parallel, samples were analyzed by UHPLC-HRMS to correlate the activity with the molecular composition using molecular networks. The results obtained for the total extract of the three species were compared to determine if the activity depended on the species. The results within the same species were also compared to highlight which fraction and, therefore, which molecular family was involved in the activity of the total extract. The various antioxidant tests showed good activity of the total extract for the three species of oak and a very good anti-collagenase activity. The antioxidant activity of oak extract has already been proven in the literature and this is correlated with its richness in polyphenols. This study shows that each molecular family of the extract contributes to the activities of the total extract. Oak extract can be used to neutralize the ROS produced during oxidative stress and to prevent the degradation of collagen and elastase during skin aging. Its complementary properties make oak extract a valuable ingredient to act against skin aging.

Characterization of Corn Silk Extract Using HPLC/HRMS/MS Analyses and Bioinformatic Data Processing.

In addition to having different biological activities of interest, corn silks play a role in the defense of plants. While benzoxamines and flavonoids have already been identified as molecules of plant defense and growth mechanisms, knowledge on the phytochemical composition of corn silk is lacking. Such knowledge would make it possible to better select the most effective varieties to improve resistance or bioactive properties. In this article, an approach was implemented to map a corn silk extract in two complementary ways. The first one involved working with UHPLC/HRMS data and Kendrick and van Krevelen plots to highlight a homologous series of compounds, such as lipids from 17 to 23 carbons, monoglycosylated flavonoids from 21 to 24 carbons, diglycosylated flavonoids of 26 to 28 carbons and organic acids of 14 to 19 carbons. The second way was to analyze the sample in UHPLC/HRMS2 and to plot mass spectral similarity networks with the GNPS platform and Cytoscape software to refine identification. By combining the information obtained, we were able to propose an identification for 104 detected molecules, including 7 nitrogenous, 28 lipidic and 67 phenolic compounds, leading to the first detailed phytochemical analysis of corn silk extract.

An Aminopyrimidone and Aminoimidazoles Alkaloids from the Rodrigues Calcareous Marine Sponge Ernsta naturalis.

A chemical study of the CH2Cl2-MeOH (1:1) extract from the sponge Ernsta naturalis collected in Rodrigues (Mauritius) based on a molecular networking dereplication strategy highlighted one novel aminopyrimidone alkaloid compound, ernstine A (1), seven new aminoimidazole alkaloid compounds, phorbatopsins D-E (2, 3), calcaridine C (4), naamines H-I (5, 7), naamidines J-K (6, 8), along with the known thymidine (9). Their structures were established by spectroscopic analysis (1D and 2D NMR spectra and HRESIMS data). To improve the investigation of this unstudied calcareous marine sponge, a metabolomic study by molecular networking was conducted. The isolated molecules are distributed in two clusters of interest. Naamine and naamidine derivatives are grouped together with ernstine in the first cluster of twenty-three molecules. Phorbatopsin derivatives and calcaridine C are grouped together in a cluster of twenty-one molecules. Interpretation of the MS/MS spectra of other compounds of these clusters with structural features close to the isolated ones allowed us to propose a structural hypothesis for 16 compounds, 5 known and 11 potentially new.

Changes in secoiridoids content and chemical characteristics of cultivated and wild Algerian olive oil, in term of fruit maturation.

Wild varieties in nature are known to be better adapted to climate change and more resistant to arid conditions common in some regions of the world. Oil samples of two cultivated varieties, Chemlal and Lemli, and one sylvestris variety were collected at four different harvesting periods in the semi-arid region of Bouira, Algeria. The aim of this study was to determine the influence of the genetic and maturity factors on the quality indices (acidity, peroxides value, and the parameters K232, K270), fatty acids profile, phenolic composition, and antioxidant activity of monovarietal olive oils. The study showed that early harvest dates of the fruits produced oils richer in pigments and phenolic compounds, with high antioxidant activity registered in both wild and cultivated varieties. Moreover, all oil samples showed high values of secoiridoids exceeding 60-90% of total biophenols, with higher values found in oleaster oils, which are correlated with high resistance to oxidation attacks. UHPLC-DAD and UHPLC-HRMS analyses showed that the secoiridoids composition is dominated by a profile rich in several isomers of oleuropein and ligstroside aglycons, which in turn represent more than 60% of the total secoiridoids in olive and Oleaster oils. Furthermore, chemometric analysis on the data allowed a better appreciation of the sensitivity of the virgin olive oil composition to the changes in genetic and ripening factors. According to the principal component analysis, phenolic and fatty acid profiles were the most important components contributing to the discrimination between olive oil samples.

Oak Species Quercus robur L. and Quercus petraea Liebl. Identification Based on UHPLC-HRMS/MS Molecular Networks.

Two species of oak are dominant in French forests: pedunculate oak (Quercus robur L.) and sessile oak (Quercus petraea Liebl.). Their differentiation is not straightforward but is essential to better understand their respective molecular content in order to better valorize them. Thus, to improve oak species identification, an untargeted UHPLC-HRMS/MS method associated with a two-step data treatment was developed to analyze a wide range of specialized metabolites enabling the comparison of both species of oak extracts. Pooled extracts from sessile and pedunculate oaks, composed of extracts from several trees of pure species from various origins, were compared using first the Venn diagram, as a quick way to get an initial idea of how close the extracts are, and then using a molecular network to visualize, on the one hand, the ions shared between the two species and, on the other hand, the compounds specific to one species. The molecular network showed that the two species shared common clusters mainly representative of tannins derivatives and that each species has specific molecules with similar fragmentation patterns, associated in specific clusters. This methodology was then applied to compare these two pooled extracts to unknown individuals in order to determine the species. The Venn diagram allowed for the quick presumption of the species of the individual and then the species could be assigned more precisely with the molecular network, at the level of specific clusters. This method, developed for the first time, has several interests. First, it makes it possible to discriminate the species and to correctly assign the species of unknown samples. Moreover, it gave an overview of the metabolite composition of each sample to better target oak tree utilization and valorization.

The rhizosphere of Salix viminalis plants after a phytostabilization process assisted by biochar, compost, and iron grit: chemical and (micro)-biological analyses.

Amendments, such as biochar, compost, and iron grit, used in phytostabilization studies, showed positive effects on soil physico-chemical properties, plant growth, and the microbial community. However, assisted phytostabilization studies do not always focus on the rhizosphere area where soil, plants, and microorganisms are affected by the amendments and plants and microorganisms can also interact with each other. The aims of this study were to evaluate the effects of amendment application on the exudation of organic acids by Salix viminalis plant roots, as well as the effects of amendments and plant development on the soil CHNS contents and the microbial community activity and diversity, assessed by measuring enzyme activities and using Biolog EcoPlatesTM tests and next-generation sequencing analyses. The results of the mesocosm experiment showed that soil C, H, and N contents were increased by amendment application, especially biochar and compost, while the one of S decreased. Enzyme activities, microbial activity, and diversity were also increased by the addition of amendments, except iron grit alone. Finally, the quantity of organic acids exuded by roots were little affected by amendments, which could in part explain the reduced effect of plant development on soil chemical and microbiological parameters. In conclusion, this study showed in particular that biochar and compost were beneficial for the soil CHN contents and the microbial community while affecting poorly Salix viminalis root exudates.

Untargeted metabolomic and molecular network approaches to reveal tomato root secondary metabolites.

INTRODUCTION: The tomato plant, Solanum lycopersicum L. (Solanaceae), is one of the most widely consumed vegetables in the world and plays an important role in human diet. Tomato cultivars are hosts for diverse types of pests, implying diverse chemical defence strategies. Glycoalkaloids are the main specialised metabolites produced by tomato leaves and fruits to protect against pests. However, the roots have received little attention, leading to limited knowledge about their phytochemical content. OBJECTIVE: The main goal of the current study was the development of an untargeted ultra-high-performance liquid chromatography high-resolution mass spectrometry (UHPLC-HRMS) based metabolomic approach to study phytochemical variations in tomato roots at two different development stages (i.e. 34th and 62nd day after sowing). METHODS: UHPLC-HRMS was used to establish the fingerprint of 24 batches of tomato roots. Statistical analyses were performed to highlight the compounds that discriminated between young and mature tomato roots. A dereplication strategy using molecular networking and HRMS/MS data was set up to identify the metabolites regulated during early root development. KEY FINDINGS: The main biomarkers were guanidine and adenosine derivatives associated with tryptophan. Secondary metabolites such as glycoalkaloids and steroidal alkaloids were also characterised. Most of the metabolites were up-regulated in young tomato roots (34 days old) while tryptophan was up-regulated in the older roots (62 days old). CONCLUSION: The metabolic changes observed in this work contribute to a deeper understanding of early-stage root development and may help our understanding of the complex processes involved in the tomato root defence arsenal.

Untargeted UHPLC-Q-TOF-HRMS based determination of discrimating compounds for oak species Quercus robur L. and Quercus petraea Liebl. identification.

INTRODUCTION: Two species of oak are dominant in French forests: pedunculate oak (Quercus robur L.) and sessile oak (Quercus petraea Liebl.). Differentiating oak species is difficult, since features such as morphological characters, geographical origin and grain are not always relevant. Even if the former is generally richer in tannin compounds while the latter is often richer in aromatic compounds, the intra-species variability is high. The characterisation of the oak species remains a suitable indicator of the molecular composition and quality of the wood. OBJECTIVES: The aim of this study was to determine differentiating molecules allowing oak species identification in order to assist in a suitable wood selection for a better oak tree valorisation since the selection of the oak wood to be used in the production of barrels plays an essential role in wine ageing. MATERIALS AND METHODS: Oak wood samples were collected both in forests and in cooperage timber yards. An untargeted metabolomic approach using ultra-high-pressure liquid chromatography qualitative time-of-flight high-resolution mass spectrometry (UHPLC-Q-TOF-HRMS) associated to multivariate statistical analyses (hierarchical ascendant clustering and partial least squares discriminant analysis) was implemented to determine molecular markers of oak species. RESULTS: Heartwood was identified as the suitable wood part to distinguish oak species. Discriminating molecules did not depend on the sample set. The pedunculate species showed overexpression of bartogenic derivatives while sessile oak presented a higher content in oak lactone precursors and in quercotriterpenosids. CONCLUSION: The developed method allowed the identification of relevant compounds for oak species identification to a better wood valorisation and selection.

Use of a clay mineral and its nonionic and cationic organoclay derivatives for the removal of pharmaceuticals from rural wastewater effluents.

A Na+ exchanged montmorillonite clay (Na-Mt) and its organoclay derivatives prepared with benzyldimethyltetradecylammonium (BDTA) cationic and polyoxyethylene (20)oleyl-ether (Brij-O20) non-ionic surfactants were used for first time at our knowledge as adsorbents the removal diverse pharmaceuticals (PPs) from samples collected in a rural wastewater facility (town of Josnes in France). The selected facility showed a poor efficiency for the elimination of PPs that were permanently release to the environment. Although involving different interactional mechanisms, the whole adsorbents Na-Mt, nonionic Brij-Mt and cationic BDTA-Mt organoclays, could remove the entire PPs of various chemical nature in a low concentration regime (ng L-1), where electrostatic interactions mainly controlled the adsorption. Thus, the organic PPs cations were preferentially adsorbed onto Na-Mt and Brij0.4-Mt (with its dual hydrophilic-hydrophobic nature) while anionic PPs showed a bold affinity to BDTA-Mt. The hydrophobic environment generated by the intercalation of surfactants within the interlayer space of organoclays conferred a versatility for the adsorption of numerous PPs through weak molecular forces (Van der Waals and/or pi-pi interactions). The study confirmed the proper efficiency of the studied layered materials including organoclays and emphasized about their promising interests in water remediation strategy.

Analysis of flavonoids with unified chromatography-electrospray ionization mass spectrometry-method development and application to compounds of pharmaceutical and cosmetic interest.

In this project, we aimed at analysing flavonoid-type compounds with unified chromatography (joining supercritical fluid chromatography and enhanced fluidity liquid chromatography with carbon dioxide-methanol mobile phases covering a wide range of compositions) and diode-array and electrospray ionization mass spectrometric detection (UC-DAD-ESI-MS). First, the chromatographic method was developed for 9 standard flavonoid molecules from three different families (flavanols, flavanones and flavonols, glycosylated or not), with a strong focus on mobile phase composition to achieve the elution of a wide range of flavonoids with good chromatographic quality (efficiency and resolution). For this purpose, two stationary phases were selected (ACQUITY UPC2 DEA and Diol), and five different additives (formic acid, citric acid, phosphoric acid, methanesulfonic acid and ammonium hydroxide) were successively introduced in the methanol co-solvent. The composition containing 0.1% methanesulfonic acid in methanol was retained as it provided the best chromatographic quality together with the possibility of hyphenating the chromatography to mass spectrometry. The DEA column appeared to provide the best efficiency and was retained for further method development. The gradient method was then optimized to achieve a fast analysis, which involved elution with a wide range of mobile phase composition (from 20 to 100% co-solvent in methanol) together with reversed flow rate and reversed pressure gradients at fixed temperature. The final gradient lasted 10 min, followed by 2.5 min of re-equilibration. Then, ESI-MS detection was optimized. Because the single-quadrupole mass spectrometer employed (ACQUITY UPC2 QDa) allowed the variation of only a few parameters, a design of experiments was used to define the best compromise for three parameters (probe temperature, cone voltage and capillary voltage). The make-up fluid introduced before entering the MS was also varied: different compositions of methanol-water containing either formic acid, ammonium hydroxide or sodium chloride were tested. The best results in terms of signal-to-noise ratio were obtained with methanol containing 20 mM ammonium hydroxide and 2% water. The optimal UC-DAD-ESI-MS method was then applied to two different flavonoid formulation ingredients. The first one, hidrosmin (5-O-(ß-hydroxyethyl)diosmin), is known for its vasoprotective properties and therefore employed in pharmaceutical formulations. The second one, α-glucosyl-hesperidin (sometimes referred to as vitamin P), is employed in cosmetic formulations. Identification of the major compounds in each sample was achieved with the help of MS detection. Graphical abstract.

Comparison of the Flavonoid Profiles of Corn Silks to Select Efficient Varieties as Trap Plants for Helicoverpa zea.

In Martinique, Helicoverpa zea is a common pest of tomato and is responsible for significant economic losses. To fight against H. zea proliferation and damage, corn could be used as a trap crop since H. zea larvae growth in the corn silk was inhibited by the presence of some flavonoids. However, only some corn varieties show an efficient inhibitory activity against H. zea depending on their flavonoid composition. In order to be able to select corn varieties with inhibition potential to be tested as a trap plant, a metabolomic approach was developed to compare the flavonoid composition of corn silks from resistant and nonresistant varieties. Quantitative analysis using UHPLC/TQ MRM MS associated with statistical treatments allowed the determination of the most concentrated and discriminant flavonoids of the resistant Java variety that clearly stood out, presenting a higher content in several C-glycosyl-O-glycosyl luteolin and apigenin derivatives such as maysin molecules.

Combination of molecular network and centrifugal partition chromatography fractionation for targeting and identifying Artemisia annua L. antioxidant compounds.

Artemisia annua L. is an annual weedy herb belonging to the Asteraceae family. As a traditional Chinese herb, Artemisia annua is a major source of artemisinin, an antimalarial drug. In addition to artemisinin, this plant contains several other molecular families presenting a wide range of biological properties. To facilitate the screening and the identification of active compounds, the present study describes their targeting by combining the dereplication information obtained by means of Molecular Networks and a crude extract fractionation by Centrifugal Partition Chromatography to obtain and test simplified fractions. This simple and fast approach was developed focused on the antioxidant activity of Artemisia annua with the aim of screening and identifying the antioxidant molecules for further cosmetic uses. Firstly, the aerial parts of Artemisia annua were extracted and their antioxidant activity was evaluated by DPPH, ABTS, CUPRAC, FRAP and iron (II) chelating assays. Extract with a positive response was subjected to UHPLC-HRMS with autoMS/MS experiments in order to build a Molecular Network using the GNPS (Global Natural Products Social Molecular Networking) platform. Secondly, the crude extract was fractionated using CPC with an adapted Arizona solvent system. The fractions obtained were evaluated for antioxidant activity to focus on active compounds, which were located on the Molecular Network and identified thanks to their MS/MS spectra. Using this approach, the major phenolic compound contributing to the antioxidant activity of Artemisia annua extract was identified.

Impact of meteorological and social events on human-excreted contaminant loads in raw wastewater: From daily to weekly dynamics.

The temporal dynamics of the wastewater influent loads of 25 drug target residues (DTR, both pharmaceuticals and illicit drugs) was assessed during 84 consecutive days. This monitoring scale enables longer temporal patterns than weekday/weekend patterns to be explored. In this study, we focus on day to day variations and the potential statistical correlation of each DTR analyzed in order to better understand the potential forcings that lead to the load variation of DTRs (alone or in clusters). The weekly patterns based on the weekly loads of DTRs were also analyzed and the impact of social and meteorological events on their variations was investigated. Two cold events occurred during the monitoring period and were associated with the highest loads of analgesics and non-steroidal anti-inflammatory drugs, as well as the lowest loads of stimulants. During the Easter holidays, a significant decrease in some year-long medication as well as analgesics was found, consistent with the demographic decrease within the catchment during this period. Lastly, a good correlation between the academic calendar and the loads of stimulants was found, emphasizing the overrepresentation of students in the consumption of recreational drugs. This study furnishes new insights in order to better understand the variations in DTR loads in wastewater influents, beyond the weekday/weekend pattern and the seasonal effect. Further investigations remain necessary, especially a real-time monitoring of the population figures within the catchment in order to improve our understanding of these results.

TLC-MALDI-TOF-MS-based identification of flavonoid compounds using an inorganic matrix.

INTRODUCTION: Thin layer chromatography (TLC) is frequently used to obtain the fingerprint of a plant extract. Although the retardation factor and the response to visualisation give primary information about compound identification, the direct TLC-mass spectrometry (MS) coupling allows a more detailed characterisation of samples. OBJECTIVES: To demonstrate the potential for the flavonoid dereplication using an inorganic matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) method with and without TLC separation. MATERIAL AND METHODS: Samples derived from wine, apple or rose were deposited on an aluminium-backed silica gel TLC sheet compatible with the MS adapter. Unlike the wine sample, for apple and rose samples compound derivatisation was necessary. These two samples were deposited twice and the plate was cut in two parts. One half was oversprayed with Neu-Peg reagent to visualise flavonoids while the inorganic matrix was deposited on each flavonoid zone on the second half for MS ionisation. RESULTS: Mass spectra obtained for samples without plate development showed numerous ions corresponding to glycosylated flavonoids. The lower m/z observed could be due either to aglycone flavonoids or to in-source fragment ions. After plate development, a separation of many spots was observed and each spot was analysed separately leading to a deeper identification of the present flavonoids. Moreover, isobaric flavonoids with different hRf values could be differentiated. CONCLUSION: TLC-MALDI-TOF-MS using an inorganic matrix enabled the analysis of anthocyanins in positive mode and of flavonols, flavanols, dihydrochalcones and phenolic acids in negative mode, reducing adduct, aggregate forms giving thus simple and reliable spectra for the dereplication approach of flavonoids in complex samples.

Competitive Association of Antibiotics with a Clay Mineral and Organoclay Derivatives as a Control of Their Lifetimes in the Environment.

A Na-smectite clay mineral (Na-Mt) was exchanged with two concentrations of benzyldimethyltetradecyl ammonium chloride cationic surfactant up to one time the cation exchange capacity. Nonionic organoclay was prepared with polyoxyethylene (20) oleyl ether (Brij-O20) nonionic surfactant at one concentration. The resulting organoclays displayed lateral layer organization of the surfactants within their interlayer space.. The adsorption properties of these organoclays and the starting raw clay mineral were evaluated for three extensively used antibiotic pharmaceutical products: the amoxicillin (AMX), the sulfamethoxazole (SMX), and the trimethoprim (TRI), recognized as recalcitrant compounds to conventional water treatments and to display a complex behavior for different pH and temperature experimental conditions. Besides showing short half-life time with possible degradation by UV radiation, these antibiotics associated with mineral phases cause serious environmental issues of which the toxic effect can be exacerbated in the presence of other chemical compounds. From the set of data obtained by complementary techniques: UV and Fourier transform infrared spectroscopy, high-performance liquid chromatography coupled with mass spectrometry, and X-ray diffraction, it appears that the nonionic organoclay shows its versatility for the adsorption of individual molecules as well as a pool of antibiotics. The mixing of the three antibiotics showing different electric charged species (cations, anions, and zwitterions) mimics the natural context drives to a deep modification of the adsorption behavior onto the different materials that can act as possible carrier mineral phases in aquatic environment. These competition effects can be measured through the significant decrease of the K F Freundlich constants for AMX in the presence of other molecules (or electrolytes), whereas TRI and SMX, by their possible association, create a synergistic effect that favors their adsorption on the whole layered materials.

Correction to "Competitive Association of Antibiotics with a Clay Mineral and Organoclay Derivatives as a Control of Their Lifetimes in the Environment".

[This corrects the article DOI: 10.1021/acsomega.8b02049.].

Temporal dynamics of human-excreted pollutants in wastewater treatment plant influents: Toward a better knowledge of mass load fluctuations.

The occurrence of 25 drug target residues (illicit drugs or pharmaceutically active compounds) was investigated during 85 consecutive days in the influents of a wastewater treatment plant in the Region Centre-Val de Loire, France. This long tracking period allowed a better understanding of the patterns affecting the occurrence of this type of contaminants. Among them, 2 were never detected (i.e. heroin and amphetamine). Concerning illicit drugs two patterns were found. Cocaine and ecstasy median loads varied considerably between weekdays and weekend days (i.e. 18.3 and 35.9% respectively) whereas cannabis and heroin (based on 6-mono-acetylmorphine loads) loads were within the same order of magnitude with a significant statistical correlation with pharmaceuticals such as acetaminophen or ketoprofen. The consumption of selected drugs was back-calculated from the loads. Among illicit drugs the highest consumption was found for cannabis with a median consumption of 51mg·day-1·inhabitant-1 (inh) whereas the median consumption for cocaine (based on benzoylecgonine loads) and ecstasy was 32 and 6mg·day-1·103·inh-1 respectively. The highest consumption values of pharmaceutically active compounds (PACs) were found for acetaminophen and acetylsalicylic acid with 108.8 and 34.1mg·day-1·inh-1 respectively, in good agreement with national sales data. A statistically significant weekly pattern was found for several PACs such as metoprolol and trimethoprim, but with the opposite pattern to that of illicit drugs. The variations in daily PAC loads could provide information about the mobility of people in the catchment, especially on the basis of daily taken PACs (i.e. to treat chronicle diseases).

Treatment with KLEPTOSE® CRYSMEB reduces mouse atherogenesis by impacting on lipid profile and Th1 lymphocyte response.

The ability of pharmacological agents to target both "classical" risk factors and inflammation may be key for successful outcomes in the prevention and treatment of atherogenesis. Among the promising drugs interfering with cholesterol metabolism, we investigated whether methyl beta-cyclodextrin (KLEPTOSE® CRYSMEB) could positively impact on atherogenesis, lipid profile and atherosclerotic plaque inflammation in ApoE-/- mice. Eleven-week old ApoE-/- mice were fed either a normal diet (N.D.) or a high-cholesterol diet (H.D.), resulting in different levels of hypercholesterolemia. KLEPTOSE® CRYSMEB (40mg/kg) or vehicle was intraperitoneally administrated 3 times per week in the last 16weeks before euthanasia in mice under N.D. and in the last 11weeks under H.D. Treatment with KLEPTOSE® CRYSMEB reduced triglyceride serum levels in both atherogenesis mouse models. In H.D. mice, treatment with KLEPTOSE® CRYSMEB increased HDL-cholesterol levels and reduced free fatty acids and spleen weight. In both mouse models, treatment with KLEPTOSE® CRYSMEB reduced atherosclerotic plaque size in thoraco-abdominal aortas and intraplaque T lymphocyte content, but did not induce relevant improvements in other histological parameters of vulnerability (macrophage, neutrophil, MMP-9 and collagen content). Conversely and more markedly in H.D. mice, treatment with KLEPTOSE® CRYSMEB was associated with a reduction in genetic markers of Th1-mediated immune response. In vitro, KLEPTOSE® CRYSMEB dose-dependently abrogated Th1 proliferation and IFNγ release. In conclusion, treatment with KLEPTOSE® CRYSMEB reduced atherosclerotic plaque size by improving triglyceride serum levels and Th1-mediated response. These results indicate this drug as a potential tool for blocking atheroprogression associated with different severity degrees of hypercholesterolemia.

Isolation by pressurised fluid extraction (PFE) and identification using CPC and HPLC/ESI/MS of phenolic compounds from Brazilian cherry seeds (Eugenia uniflora L.).

Brazilian cherry seeds are a waste product from juice and frozen pulp production and, the seeds composition was investigated to valorize this by-product. Compounds separation was performed with ethanol by pressurised fluid extraction (PFE). Here we determine the effect of temperature (T), static time (ST), number of cycles (C), and flush volume (VF) on the yield, composition and total phenolic content (TPC) of the seed extracts. T, ST and their interaction positively influenced yield and TPC. Extracts were fractionated by high performance liquid chromatography (HPLC) and centrifugal partition chromatography (CPC). The collected fractions characterizations were made by electrospray ionisation mass spectrometry (ESI/MS) and high resolution mass spectrometry (HRMS) indicated the presence of ellagic acid pentoside and deoxyhexose, quercitrin and kaempferol pentoside. All of these compounds have antioxidant properties and normally are found in plant extracts. These results confirm that Brazilian cherry seed extract is a potentially valuable source of antioxidants.