Strawberry Fragaria x ananassa cv. Festival: A Polyphenol-Based Phytochemical Characterization in Fruit and Leaf Extracts.

Berry fruits are an important dietary source of health-promoting antioxidant polyphenols. Interestingly, berry leaves of diverse species, including strawberries, have shown higher bioactive phytochemical content in the leaves than in the fruit. Moreover, the vegetative part of the plants is usually discarded, representing a presumably large source of underutilized bioactive biomass. In this investigation, the polyphenol profiles of tropical highland strawberry (Fragaria x ananassa cv. Festival) leaves and fruits were compared by high-performance liquid chromatography coupled with a diode array detector (UHPLC-DAD) and mass spectrometry (HPLC-MS). The total polyphenol strawberry leaf extracts exhibited a 122-fold-higher total polyphenol content and 13-fold higher antioxidant activity (ORAC) than strawberry fruits, and they showed evidence of possible photoprotective effects against UV damage in human melanoma cells (SK-MEL-28) and in murine embryo fibroblasts (NIH/3T3), together with promising anti-proliferative activities against the same melanoma cells. Seven polyphenols were confirmed by HPLC-DAD in the leaf extracts, with differences depending on fraction solubility. Moreover, three substituted quercetin derivatives, three substituted kaempferol derivatives, two anthocyanins, and catechin were confirmed in the soluble fraction by HPLC-MS. Given their higher total polyphenol content and bioactive activities, underutilized strawberry Festival leaves are a potential source of apparently abundant biomass with prospective bioactive applications.

Evaluation of Salicylic Acid and Methyl Jasmonate as Elicitors in Phyllanthus acuminatus Hairy Roots by Non-Targeted Analysis Using High-Resolution Mass Spectrometry.

Phyllanthus acuminatus has been studied for its vast medical and industrial potential. Phytochemical investigations reveal that the genus is a rich source of lignans, flavonoids, phenolics, terpenoids, and other metabolites. However, the phytochemical profile elucidation of this species still needs further research. The use of eliciting compounds such as salicylic acid and methyl jasmonate has managed to increase the production of secondary metabolites in plant cell cultures. Hairy roots of Phyllanthus acuminatus were produced in 250 mL flasks with a 16 h light/8 h darkness photoperiod under diffused light with a culture time of four weeks. The elicitors salicylic acid and methyl jasmonate were tested in 50 µM and 200 µM concentrations. Non-targeted analysis was done for the different treatments using HR-MS. Identified metabolites were grouped in phenylpropanoids, phenols, and mucic acids, and statistical analysis of relative concentrations was achieved. A significant change in phenols' relative concentrations appeared in the elicitations with salicylic acid. Because of the elicitation treatment, specific compounds increased their concentrations, some of which have known pharmacological effects and are used in treating chronic diseases. The best elicitation treatment was salicylic acid 50 µM as it increased by more than 100% the general content of phenols and phenylpropanoid derivates and triplicates the concentration of mucic acid derivates in treated hairy root extracts. The application of non-targeted analysis showed interesting changes in phytochemical concentration due to elicitation in Phyllanthus acuminatus hairy roots.

QTOF-ESI MS Characterization and Antioxidant Activity of Physalis peruviana L. (Cape Gooseberry) Husks and Fruits from Costa Rica.

There is increasing interest in research of secondary metabolites from Physalis peruviana (Cape gooseberry) because of their potential bioactivities. In this study, the profile of compounds found in fruits and husks from Costa Rica was determined through ultra-performance liquid chromatography coupled with high-resolution mass spectrometry using a quadrupole time-of-flight analyzer (UPLC-ESI-QTOF MS) on extracts (n = 10) obtained through pressurized liquid extraction (PLE) conditions. In total, 66 different compounds were identified, comprising 34 withanolides, 23 sucrose ester derivatives and 9 flavonoids. UPLC-DAD analysis was performed to determine the ß-carotene in fruits and to quantify the flavonoids in all 10 samples, with the results showing higher contents in samples from the Dota region (58.6−60.1 µg/g of dry material versus 1.6−2.8 mg/g of dry material). The Folin−Ciocalteau total polyphenolic content (FC) and antioxidant activity using the DPPH method showed better results for the husk extracts, with the ones from the Dota region holding the best values (4.3−5.1 mg GAE/g of dry material versus IC50 = 1.6−2.3 mg of dry material/mL). In addition, a significant negative correlation was found between the RU, FC and DPPH values (r = −0.902, p < 0.05), aligning with previous reports on the role of polyphenols in antioxidant activity. Principal correlation analysis (PCoA) and hierarchical clustering (HC) analysis were performed on HRMS results, and they indicated that the D1 and D2 fruit samples from the Dota region were clustered with husks related to a higher presence of the analyzed metabolites. In turn, principal component analysis (PCA) performed on the flavonoid content and antioxidant activity yielded results indicating that the D1 and D2 husks and fruit samples from the Dota region stood out significantly, showing the highest antioxidant activity. In summation, our findings suggest that P. peruviana husks and fruits from Costa Rica constitute a substrate of interest for further studies on their potential health benefits.

Hybrid Nanoparticles of Proanthocyanidins from Uncaria tomentosa Leaves: QTOF-ESI MS Characterization, Antioxidant Activity and Immune Cellular Response.

Previous studies in Uncaria tomentosa have shown promising results concerning the characterization of polyphenols with leaves yielding more diverse proanthocyanidins and higher bioactivities values. However, the polyphenols-microbiota interaction at the colonic level and their catabolites avoid the beneficial effects that can be exerted by this medicinal plant when consumed. In this regard, a new generation of hybrid nanoparticles has demonstrated improvements in natural compounds' activity by increasing their bioavailability. In this line, we report a detailed study of the characterization of a proanthocyanidin-enriched extract (PA-E) from U. tomentosa leaves from Costa Rica using UPLC-QTOF-ESI MS. Moreover, two types of hybrid nanoparticles, a polymeric-lipid (F-1) and a protein-lipid (F-2) loaded with PA-E were synthesized and their characterization was conducted by dynamic light scattering (DLS), attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FT-IR), high-resolution transmission electron microscopy (HR-TEM), and encapsulation efficiency (%EE). In addition, in vitro release, antioxidant activity through 2,2-diphenyl-1-picrylhidrazyl (DPPH) as well as in vivo delayed-type hypersensitivity (DTH) reaction was evaluated. Results allowed the identification of 50 different compounds. The PA-E loaded nanoparticles F-1 and F-2 achieved encapsulation efficiency of ≥92%. The formulations exhibited porosity and spherical shapes with a size average of 26.1 ± 0.8 and 11.8 ± 3.3 nm for F-1 and F-2, respectively. PA-E increased its release rate from the nanoparticles compared to the free extract in water and antioxidant activity in an aqueous solution. In vivo, the delayed-type hypersensitive test shows the higher immune stimulation of the flavan-3-ols with higher molecular weight from U. tomentosa when administered as a nanoformulation, resulting in augmented antigen-specific responses. The present work constitutes to our knowledge, the first report on these bioactivities for proanthocyanidins from Uncaria tomentosa leaves when administrated by nanosystems, hence, enhancing the cellular response in mice, confirming their role in immune modulation.

Polyphenolic HRMS Characterization, Contents and Antioxidant Activity of Curcuma longa Rhizomes from Costa Rica.

Curcuma longa constitutes an important source of secondary metabolites that have been associated with multiple health benefits. For instance, curcumin, demethoxycurcumin and bisdemethoxycurcumin, have been found to perform important biological activities, such as anti-inflammatory, antioxidant, anticancer, antimicrobial, antihypertensive and anticoagulant. These promising results prompted this research to evaluate the polyphenols of C. longa rhizomes in Costa Rica. The present work reports a comprehensive study on the polyphenolic profile and the contents of the three main curcuminoids as well as the antioxidant activity of extracts from C. longa rhizomes (n = 12) produced in Costa Rica. Through UPLC-QTOF-ESI MS, a total of 33 polyphenols were identified, grouped in eight types of structures. In addition, our findings on the main curcuminoids using UPLC-DAD show all rhizomes complying with total curcuminoids (TC) content established by the United States Pharmacopeia (USP). At an individual level, samples NW-3 and NE-1 show the higher contents (118.7 and 125.0 mg/g dry material), representing more than twice the average values of the lowest samples. These samples also exhibit the highest Folin−Ciocalteu (FC) reducing capacity results as well as the best DPPH (IC50 15.21 and 16.07 µg extract/mL) and NO (IC50 between 52.5 and 54.3 µg extract/mL) antioxidant values. Further, Pearson correlation analysis findings indicated positive correlation (p < 0.05) between TC, CUR with FC results (r = 0.833 and r = 0.867 respectively) and negative correlation (p < 0.05) between CUR, TC and FC with DPPH results (r = −0.898, r = −0.911, and r = −0.890, respectively) and between NO results and DPPH (r = −0.805, p < 0.05). Finally, results for Principal Component Analysis (PCA) showed composition variability associated with their region of origin with products from the Northeastern (NE) region exhibiting higher average values for FC, TC and antioxidant activities. Further, PCA confirmed that two samples, namely NE-1 and NW-3, stand out by presenting the highest PC1 due to their particularly high TC, CUR and antioxidant activities. Consequently, our findings agree with previous results indicating the importance of C. longa extracts to elaborate products with potential benefits for health, while delivering extracts with higher levels of curcuminoids than previous reports and exhibiting high antioxidant activity.

HRMS Characterization, Antioxidant and Cytotoxic Activities of Polyphenols in Malus domestica Cultivars from Costa Rica.

There is increasing interest in research into fruits as sources of secondary metabolites because of their potential bioactivities. In this study, the phenolic profiles of Malus domestica Anna and Jonagold cultivars from Costa Rica were determined by Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry (HRMS) using a quadrupole-time-of-flight analyzer (UPLC-QTOF-ESI MS), on enriched-phenolic extracts from skins and flesh, obtained through Pressurized Liquid Extraction (PLE). In total, 48 different phenolic compounds were identified in the skin and flesh extracts, comprising 17 flavan-3-ols, 12 flavonoids, 4 chalcones, 1 glycosylated isoprenoid and 14 hydroxycinnamic acids and derivatives. Among extracts, the flesh of Jonagold exhibits a larger number of polyphenols and is especially rich in procyanidin trimers, tetramers and pentamers. Evaluating total phenolic content (TPC) and antioxidant activities using ORAC and DPPH procedures yields higher values for this extract (608.8 mg GAE/g extract; 14.80 mmol TE/g extract and IC50 = 3.96 µg/mL, respectively). In addition, cytotoxicity evaluated against SW620 colon cancer cell lines and AGS gastric cancer cell lines also delivered better effects for Jonagold flesh (IC50 = 62.4 and 60.0 µg/mL, respectively). In addition, a significant negative correlation (p < 0.05) was found between TPC and cytotoxicity values against SW620 and AGS adenocarcinoma (r = -0.908, and -0.902, respectively). Furthermore, a significant negative correlation (p < 0.05) was also found between the number of procyanidins and both antioxidant activities and cytotoxicity towards SW620 (r = -0.978) and AGS (r = -0.894) cell lines. These results align with Jonagold flesh exhibiting the highest abundance in procyanidin oligomers and yielding better cytotoxic and antioxidant results. In sum, our findings suggest the need for further studies on these Costa Rican apple extracts-and particularly on the extracts from Jonagold flesh-to increase the knowledge on their potential benefits for health.

Polyphenolic QTOF-ESI MS Characterization and the Antioxidant and Cytotoxic Activities of Prunus domestica Commercial Cultivars from Costa Rica.

There is an increased interest in plum research because of their metabolites' potential bioactivities. In this study, the phenolic profiles of Prunus domestica commercial cultivars (Methley, Pisardii and Satsuma) in Costa Rica were determined by Ultra Performance Liquid Chromatography coupled with High Resolution Mass Spectrometry using a quadrupole-time-of-flight analyzer (UPLC-ESI-QTOF MS) on enriched phenolic extracts obtained through Pressurized Liquid Extraction (PLE) under acidic and neutral extraction conditions. In total, 41 different phenolic compounds were identified in the skin and flesh extracts, comprising 11 flavan-3-ols, 14 flavonoids and 16 hydroxycinnamic acids and derivatives. Neutral extractions for the skins and flesh from all of the cultivars yielded a larger number of compounds, and were particularly rich in the number of procyanidin trimers and tetramers when compared to the acid extractions. The total phenolic content (TPC) and antioxidant potential using the DPPH and ORAC methods exhibited better results for neutral extracts with Satsuma skins and Methley flesh, which showed the best values (685.0 and 801.6 mg GAE/g extract; IC50 = 4.85 and 4.39 µg/mL; and 12.55 and 12.22 mmol TE/g extract, respectively). A Two-Way ANOVA for cytotoxicity towards AGS gastric adenocarcinoma and SW620 colon adenocarcinoma indicated a significant difference (p < 0.05) for PLE conditions, with better results for neutral extractions, with Satsuma skin delivering the best results (IC50 = 60.7 and 46.7 µg/mL respectively) along with Methley flesh (IC50 = 76.3 and 60.9 µg/mL, respectively). In addition, a significant positive correlation was found between TPC and ORAC (r = 0.929, p < 0.05), as well as a significant negative correlation (p < 0.05) between TPC and cytotoxicity towards AGS and SW620 cell lines (r = -0.776, and -0.751, respectively). A particularly high, significant, negative correlation (p < 0.05) was found between the number of procyanidins and cytotoxicity against the AGS (r = -0.868) and SW620 (r = -0.855) cell lines. Finally, the PCA clearly corroborated that neutral extracts are a more homogenous group exhibiting higher antioxidant and cytotoxic results regardless of the part or cultivar; therefore, our findings suggest that PLE extracts under neutral conditions would be of interest for further studies on their potential health benefits.

Photolysis of the nonsteroidal anti-inflammatory drug sulindac: elucidation of kinetic behaviour and photodegradation pathways in water.

Non-steroidal anti-inflammatory drugs are recognized widely as emerging contaminants. Sulindac has received additional attention as a prodrug in cancer treatment and because of its detection in drinking water and wastewaters. Nevertheless, there is limited knowledge about its kinetic behaviour and fate in the aquatic environment. In this work, the direct photolysis of sulindac, in which photochemical reactions were monitored and phototransformation products identified, was investigated under prolonged periods using UV-A and UV-B radiation and pH conditions (2 and 7) to evaluate the effect of the protonation state and the efficiency of the photolytic process. A novel kinetic mechanism has been proposed in which sulindac exhibits a consecutive reaction pathway, with pseudo-first order kinetics for rapid and reversible Z to E isomerization. Once photoequilibrium was reached, second-order degradation of the isomers in the presence of the new photodegradation products was observed. Photochemical transformation was faster under UV-B irradiation and lower pH, which suggests greater persistence of sulindac at more relevant environmental conditions of UV-A and pH 7. Two novel and major byproducts were identified, corresponding to the oxidative cleavage of the alkene exo to the indene system. The degradation pathway is mainly photoinduced, enhanced by acidic conditions and presumes the double bond as the most reactive site for the parent compound. This research demonstrates an approach for determining kinetics of compounds under challenging conditions, including, absorption from multiple electronic transitions, photoinduced products with unknown extinction coefficients, concentration dependence, photoinduced sensitizing intermediates, and speciation effects. Our work greatly improves our understanding of the degradation process of sulindac and will contribute to exposure assessments and treatment methodologies for this compound in impacted waters.

Polysaccharide κ-Carrageenan as Doping Agent in Conductive Coatings for Electrochemical Controlled Release of Dexamethasone at Therapeutic Doses.

Smart conductive materials are developed in regenerative medicine to promote a controlled release profile of charged bioactive agents in the vicinity of implants. The incorporation and the active electrochemical release of the charged compounds into the organic conductive coating is achieved due to its intrinsic electrical properties. The anti-inflammatory drug dexamethasone was added during the polymerization, and its subsequent release at therapeutic doses was reached by electrical stimulation. In this work, a Poly (3,4-ethylenedioxythiophene): κ-carrageenan: dexamethasone film was prepared, and κ-carrageenan was incorporated to keep the electrochemical and physical stability of the electroactive matrix. The presence of κ-carrageenan and dexamethasone in the conductive film was confirmed by µ-Raman spectroscopy and their effect in the topographic was studied using profilometry. The dexamethasone release process was evaluated by cyclic voltammetry and High-Resolution mass spectrometry. In conclusion, κ-carrageenan as a doping agent improves the electrical properties of the conductive layer allowing the release of dexamethasone at therapeutic levels by electrochemical stimulation, providing a stable system to be used in organic bioelectronics systems.

Electrochemical Characterization of Mancozeb Degradation for Wastewater Treatment Using a Sensor Based on Poly (3,4-ethylenedioxythiophene) (PEDOT) Modified with Carbon Nanotubes and Gold Nanoparticles.

Mancozeb is a worldwide fungicide used on a large scale in agriculture. The active component and its main metabolite, ethylene thiourea, has been related to health issues. Robust, fast, and reliable methodologies to quantify its presence in water are of great importance for environmental and health reasons. The electrochemical evaluation of mancozeb using a low-cost electrochemical electrode modified with poly (3,4-ethylene dioxythiophene), multi-walled carbon nanotubes, and gold nanoparticles is a novel strategy to provide an in-situ response for water pollution from agriculture. Additionally, the thermal-, electrochemical-, and photo-degradation of mancozeb and the production of ethylene thiourea under controlled conditions were evaluated in this research. The mancozeb solutions were characterized by electrochemical oxidation and ultraviolet-visible spectrophotometry, and the ethylene thiourea concentration was measured using ultra-high-performance liquid chromatography high-resolution mass spectrometry. The degradation study of mancozeb may provide routes for treatment in wastewater treatment plants. Therefore, a low-cost electrochemical electrode was fabricated to detect mancozeb in water with a robust electrochemical response in the linear range as well as a quick response at a reduced volume. Hence, our novel modified electrode provides a potential technique to be used in environmental monitoring for pesticide detection.

Site-Specific Cleavage of Ribosomal RNA in Escherichia coli-Based Cell-Free Protein Synthesis Systems.

Cell-free protein synthesis, which mimics the biological protein production system, allows rapid expression of proteins without the need to maintain a viable cell. Nevertheless, cell-free protein expression relies on active in vivo translation machinery including ribosomes and translation factors. Here, we examined the integrity of the protein synthesis machinery, namely the functionality of ribosomes, during (i) the cell-free extract preparation and (ii) the performance of in vitro protein synthesis by analyzing crucial components involved in translation. Monitoring the 16S rRNA, 23S rRNA, elongation factors and ribosomal protein S1, we show that processing of a cell-free extract results in no substantial alteration of the translation machinery. Moreover, we reveal that the 16S rRNA is specifically cleaved at helix 44 during in vitro translation reactions, resulting in the removal of the anti-Shine-Dalgarno sequence. These defective ribosomes accumulate in the cell-free system. We demonstrate that the specific cleavage of the 16S rRNA is triggered by the decreased concentrations of Mg2+. In addition, we provide evidence that helix 44 of the 30S ribosomal subunit serves as a point-of-entry for ribosome degradation in Escherichia coli. Our results suggest that Mg2+ homeostasis is fundamental to preserving functional ribosomes in cell-free protein synthesis systems, which is of major importance for cell-free protein synthesis at preparative scale, in order to create highly efficient technical in vitro systems.

Tracking dipeptides at work-uptake and intracellular fate in CHO culture.

Market demands for monoclonal antibodies (mAbs) are steadily increasing worldwide. As a result, production processes using Chinese hamster ovary cells (CHO) are in the focus of ongoing intensification studies for maximizing cell-specific and volumetric productivities. This includes the optimization of animal-derived component free (ADCF) cultivation media as part of good cell culture practice. Dipeptides are known to improve CHO culture performance. However, little or even conflicting assumptions exist about their putative import and functionality inside the cells. A set of well-known performance boosters and new dipeptide prospects was evaluated. The present study revealed that dipeptides are indeed imported in the cells, where they are decomposed to the amino acids building blocks. Subsequently, they are metabolized or, unexpectedly, secreted to the medium. Monoclonal antibody production boosting additives like L-alanine-L-glutamine (AQ) or glycyl-L-glutamine (GQ) can be assigned to fast or slow dipeptide uptake, respectively, thus pinpointing to the need to study dipeptide kinetics and to adjust their feeding individually for optimizing mAb production.

Identification of the agr Peptide of Listeria monocytogenes.

Listeria monocytogenes (Lm) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (agr) system was shown to impact on biofilm formation and virulence and has been proposed as one of the regulatory mechanisms involved in adaptation to these changing environments. The Lm agr operon is homologous to the Staphylococcus aureus system, which includes an agrD-encoded autoinducing peptide that stimulates expression of the agr genes via the AgrCA two-component system and is required for regulation of target genes. The aim of the present study was to identify the native autoinducing peptide (AIP) of Lm using a luciferase reporter system in wildtype and agrD deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of agrD, luciferase reporter activity driven by the PII promoter of the agr operon was completely abolished and this defect was restored by co-cultivation of the agrD-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential Lm AIPs was designed and tested for PII-activation. This led to the identification of a cyclic pentapeptide that was able to induce PII-driven luciferase reporter activity and restore defective invasion of the agrD deletion mutant into Caco-2 cells. Analysis of supernatants of a recombinant Escherichia coli strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The Lm agr system is specific for this pentapeptide since the AIP of Lactobacillus plantarum, which also is a pentapeptide yet with different amino acid sequence, did not induce PII activity. In summary, the presented results provide further evidence for the hypothesis that the agrD gene of Lm encodes a secreted AIP responsible for autoregulation of the agr system of Lm. Additionally, the structure of the native Lm AIP was identified.

Alkaline conditions in hydrophilic interaction liquid chromatography for intracellular metabolite quantification using tandem mass spectrometry.

Modeling of metabolic networks as part of systems metabolic engineering requires reliable quantitative experimental data of intracellular concentrations. The hydrophilic interaction liquid chromatography-electrospray ionization-tandem mass spectrometry (HILIC-ESI-MS/MS) method was used for quantitative profiling of more than 50 hydrophilic key metabolites of cellular metabolism. Without prior derivatization, sugar phosphates, organic acids, nucleotides, and amino acids were measured under alkaline and acidic mobile phase conditions with pre-optimized multiple reaction monitoring (MRM) transitions. Irrespective of the polarity mode of the acquisition method used, alkaline conditions achieved the best quantification limits and linear dynamic ranges. Fully 90% of the analyzed metabolites presented detection limits better than 0.5pmol (on column), and 70% presented 1.5-fold higher signal intensities under alkaline mobile phase conditions. The quality of the method was further demonstrated by absolute quantification of selected metabolites in intracellular extracts of Escherichia coli. In addition, quantification bias caused by matrix effects was investigated by comparison of calibration strategies: standard-based external calibration, isotope dilution, and standard addition with internal standards. Here, we recommend the use of alkaline mobile phase with polymer-based zwitterionic hydrophilic interaction chromatography (ZIC-pHILIC) as the most sensitive scenario for absolute quantification for a broad range of metabolites.