A smartphone aptasensor for fipronil detection in honey samples.

In this study, an electrochemical smartphone-based aptasensor for the determination of fipronil was developed by modifying a screen-printed carbon electrode (SPCE). Fipronil is a broad-spectrum insecticide that has been widely used in various applications such as agriculture, veterinary, and household pest control. Recently, its use has raised concerns over the potential impact on the environment and human health. The absence of effective methods for this purpose poses a significant obstacle. To tackle this problem, we have developed a cutting-edge aptamer-based portable sensor capable of rapidly and conveniently detecting fipronil in situ. Considering that the detection of small molecules, such as fipronil, can be a challenging task, a competitive replacement assay was set up based on the aptamer's preference for the free form of fipronil over the immobilized one on the electrode. The analytical performance provided by the sensor on standard solutions of a known fipronil content made it possible to estimate a limit of detection (LOD) equal to 1.07 µg kg-1 and a limit of quantification (LOQ) of 3.21 µg kg-1. Selectivity tests were conducted using atrazine as a possible interferent. The use and performance of the developed portable aptasensor was assessed on honey samples, which were simultaneously analyzed using an HPLC-MS method. This aptasensor could be an affordable and effective tool for accurately quantifying fipronil not only in honey samples but also in other food products.

Nrf2-Mediated Pathway Activated by Prunus spinosa L. (Rosaceae) Fruit Extract: Bioinformatics Analyses and Experimental Validation.

In our previous studies, Prunus spinosa fruit (PSF) ethanol extract was showed to exert antioxidant, antimicrobial, anti-inflammatory and wound healing activities. In the present study, an integrated bioinformatics analysis combined with experimental validation was carried out to investigate the biological mechanism(s) that are responsible for the reported PSF beneficial effects as an antioxidant during a pro-inflammatory TLR4 insult. Bioinformatics analysis using miRNet 2.0 was carried out to address which biological process(es) the extract could be involved in. In addition, Chemprop was employed to identify the key targets of nuclear receptor (NR) signaling and stress response (SR) pathways potentially modulated. The miRNet analysis suggested that the PSF extract mostly activates the biological process of cellular senescence. The Chemprop analysis predicted three possible targets for nine phytochemicals found in the extract: (i) ARE signaling, (ii) mitochondrial membrane potential (MMP) and (iii) p53 SR pathways. The PSF extract antioxidant effect was also experimentally validated in vitro using the human monocyte U937 cell line. Our findings showed that Nrf2 is modulated by the extract with a consequent reduction of the oxidative stress level. This was confirmed by a strong decrease in the amount of reactive oxygen species (ROS) observed in the PSF-treated cells subjected to lipopolysaccharide (LPS) (6 h treatment, 1 µg/mL). No visible effects were observed on p53 and MMP modulation.

Design and advanced characterization of quercetin-loaded nano-liposomes prepared by high-pressure homogenization.

Quercetin-loaded nano-liposomes were prepared by high-pressure homogenization (HPH) at different pressures (up to 150 MPa) and number of passes (up to 3) to define the best processing conditions allowing the lowest particle size and the highest encapsulation efficiency (EE). The process at 150 MPa for 1 pass was the best, producing quercetin-loaded liposomes with the lowest particle size and 42% EE. Advanced techniques (multi-detector asymmetrical-flow field flow fractionation and analytical ultracentrifugation combined with transmission electron microscopy) were further used for the characterization of the liposomes which were oblong in shape (ca. 30 nm). Results highlight the need for several techniques to study nano-sized, polydisperse samples. The potential of quercetin-loaded liposomes against colon cancer cells was demonstrated. Results prove that HPH is an efficient and sustainable method for liposome preparation and highlight the remarkable role of process optimisation as well as the powerfulness of advanced methodologies for the characterisation of nano-structures.

Cydonia oblonga Mill. Pulp Callus Inhibits Oxidative Stress and Inflammation in Injured Cells.

The pharmacological activity of a callus extract from the pulp of Cydonia oblonga Mill., also known as quince, was investigated in murine macrophage (RAW 264.7) and human keratinocyte (HaCaT) cell lines. In particular, the anti-inflammatory activity of C. oblonga Mill. pulp callus extract was assessed in lipopolysaccharides (LPS)-treated RAW 264.7 by the Griess test and in LPS-treated HaCaT human keratinocytes by examining the expression of genes involved in the inflammatory process, including nitric oxide synthase (iNOS), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), nuclear factor-kappa-B inhibitor alfa (ikBα), and intercellular adhesion molecule (ICAM). The antioxidant activity was evaluated by quantizing the reactive oxygen species (ROS) production in the hydrogen peroxide and tert-butyl hydroperoxide-injured HaCaT cell line. The obtained results indicate that C. oblonga callus from fruit pulp extract has anti-inflammatory and antioxidant activities, suggesting its possible application in delaying and preventing acute or chronic diseases associated with aging or in the treatment of wound dressing.

APE1 interacts with the nuclear exosome complex protein MTR4 and is involved in cisplatin- and 5-fluorouracil-induced RNA damage response.

The nuclear RNA surveillance mechanism is essential for cancer cell survival and is ensured by the RNA nuclear exosome including some co-factors, such as the RNA helicase MTR4. Recent studies suggest an involvement of DNA repair proteins such as apurinic/apyrimidinic (AP) endodeoxyribonuclease 1 (APE1), a major endodeoxyribonuclease of Base Excision Repair (BER), in RNA metabolism and RNA decay of oxidized and abasic RNA. Cisplatin (CDDP) and 5-fluorouracil (5-FU) are commonly used for the treatment of solid tumours. Whether APE1 is involved in the elimination of CDDP- or 5-FU-damaged RNA is unknown, as is its possible interaction with the nuclear exosome complex. Here, by using different human cancer cell models, we demonstrated that: (a) APE1 is involved in the elimination of damaged-RNA, upon CDDP- and 5-FU-treatments, in a MTR4-independent manner; (b) the interaction between APE1 and MTR4 is stimulated by CDDP- and 5-FU-treatments through lysine residues in the APE1 N-terminal region and is, in part, mediated by nucleic acids and (c) APE1- and MTR4-depletion lead to the generation of R-loop formation causing the activation of the DNA damage response (DDR) pathway through the ATM-p53-p21 axis. Our data demonstrate a role of MTR4 in DDR underpinning the function of APE1 in controlling the RNA quality upon genotoxic treatments with possible implications in chemoresistance.

Characterization of the Biological Activity of the Ethanolic Extract from the Roots of Cannabis sativa L. Grown in Aeroponics.

Cannabis sativa var. Kompolti, a variety routinely used for food production purposes, is characterized by a low concentration of psychoactive molecules, although containing many other biologically attractive metabolites in all parts of the plant, including the roots. In the present work, we evaluate the specific biological activities of the roots' extract from plants cultivated through aeroponics, an affordable and reliable method facilitating the isolation and processing of roots, with the advantage of being suitable for industrial scale-up. Furthermore, aeroponics results in an increased net accumulation of the most biologically attractive constituents (ß-sitosterol, friedelin and epi-friedelanol) found in the roots. The ethanolic extract of the aeroponic roots of C. sativa (APEX) and its separate components are studied to evaluate their anti-inflammatory (modulation of the expression level of specific markers upon LPS stimulation in U937 cells, such as IL-6, IL-8, TNF-α, IkB-α, iNOS, IRAK-1 and miR-146a) and antioxidant (in either acellular or cellular settings) activities. The APEX anti-inflammatory and antioxidant capacities are also functionally benchmarked using the wound-healing assay. On the whole, the data obtained show that APEX and its main components showed significant anti-inflammatory and antioxidant activities, which may render the exploitation of roots as a source of natural antioxidants and anti-inflammatory agents highly attractive, with the additional technical and economic advantages of aeroponics compared to soil cultivation.

Yield, Characterization, and Possible Exploitation of Cannabis Sativa L. Roots Grown under Aeroponics Cultivation.

Cannabis sativa L. has been used for a long time to obtain food, fiber, and as a medicinal and psychoactive plant. Today, the nutraceutical potential of C.sativa is being increasingly reappraised; however, C. sativa roots remain poorly studied, despite citations in the scientific literature. In this direction, we identified and quantified the presence of valuable bioactives (namely, ß-sitosterol, stigmasterol, campesterol, friedelin, and epi-friedelanol) in the root extracts of C. sativa, a finding which might pave the way to the exploitation of the therapeutic potential of all parts of the C. sativa plant. To facilitate root harvesting and processing, aeroponic (AP) and aeroponic-elicited cultures (AEP) were established and compared to soil-cultivated plants (SP). Interestingly, considerably increased plant growth-particularly of the roots-and a significant increase (up to 20-fold in the case of ß-sitosterol) in the total content of the aforementioned roots' bioactive molecules were observed in AP and AEP. In conclusion, aeroponics, an easy, standardized, contaminant-free cultivation technique, facilitates the harvesting/processing of roots along with a greater production of their secondary bioactive metabolites, which could be utilized in the formulation of health-promoting and health-care products.

Reformulation and food combination as strategies to modulate glycaemia: the case of apple pomace containing biscuits administered with apple juice to healthy rats.

Conventional (CB) and apple-pomace-reformulated (RB) biscuits were administered to healthy rats. Although the areas under curve (AUC) of glucose concentration were comparable between samples, differences in the glycaemic profile of CB and RB were observed. RB caused an initial steeper increase in glycaemia but a shift in the glycaemic peak from 45 to 60 min, as compared to CB. When CB or RB was ingested with apple juice (AJ) no differences were observed as compared to their ingestion with a soft drink (SD) simulating AJ sugar content, indicating that reformulation, more than the presence of AJ, was crucial in affecting the glycaemic response. Consumer acceptability towards reformulation was assessed through conjoint analysis, by simulating labels reporting information on reformulation. Consumers preferred information generally referring to the health-promoting effect (i.e. "low sugar" and "high fibre" contents), despite directly relating to a specific disease (i.e. "suitable for diabetics" and "low glycaemic index").

Prunus spinosa Extract Loaded in Biomimetic Nanoparticles Evokes In Vitro Anti-Inflammatory and Wound Healing Activities.

Prunus spinosa fruits (PSF) contain different phenolic compounds showing antioxidant and anti-inflammatory activities. Innovative drug delivery systems such as biomimetic nanoparticles could improve the activity of PSF extract by promoting (i) the protection of payload into the lipidic bilayer, (ii) increased accumulation to the diseased tissue due to specific targeting properties, (iii) improved biocompatibility, (iv) low toxicity and increased bioavailability. Using membrane proteins extracted from human monocyte cell line THP-1 cells and a mixture of phospholipids, we formulated two types of PSF-extract-loaded biomimetic vesicles differing from each other for the presence of either 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG). The biological activity of free extract (PSF), compared to both types of extract-loaded vesicles (PSF-DOPCs and PSF-DOPGs) and empty vesicles (DOPCs and DOPGs), was evaluated in vitro on HUVEC cells. PSF-DOPCs showed preferential incorporation of the extract. When enriched into the nanovesicles, the extract showed a significantly increased anti-inflammatory activity, and a pronounced wound-healing effect (with PSF-DOPCs more efficient than PSF-DOPG) compared to free PSF. This innovative drug delivery system, combining nutraceutical active ingredients into a biomimetic formulation, represents a possible adjuvant therapy for the treatment of wound healing. This nanoplatform could be useful for the encapsulation/enrichment of other nutraceutical products with short stability and low bioavailability.

Phenolic content and potential bioactivity of apple juice as affected by thermal and ultrasound pasteurization.

Thermal (T) and ultrasound (US) pasteurization processes were applied to apple juice and the phenolic compounds (TPC) were quantified before and after in vitro digestion by HPLC-DAD-ESI-MSn, with their bioaccessibility ascertained. Digested samples were analysed for their inhibitory capacity against α-glucosidase. Since some of the compounds exhibit fluorescence, both steady state and time-resolved fluorescence methods were used to investigate the binding to a blood transport protein, human serum albumin (HSA). It was found that processing induced an increase in the TPC content, which was more pronounced when US was applied. In contrast, digestion reduced the TPC content, evening out the overall effect. Still T and US pasteurized juices exhibited a higher quantity of TPC upon digestion as compared to the raw sample. No correlation was found between the TPC content and α-glucosidase inhibition, as the T and US pasteurized juices showed the highest and lowest inhibitory capacities against the enzyme, respectively. This is indicative that other compounds, such as those formed upon thermal treatment, may be involved in the antidiabetic effect of apple juice. The fluorescence study showed that binding occurred to HSA, at slightly different rates for different species present in the US treated extract. Considering energy consumption, US pasteurization is the most power consuming treatment despite its shorter duration. Overall, no univocal indication on the best pasteurization process can be gathered. Thus, it is necessary to define the desired target in order to drive technological interventions by a customized approach.

Polyphenol-enriched spelt husk extracts improve growth and stress-related biochemical parameters under moderate salt stress in maize plants.

Biostimulants improve yield, quality, and stress acclimation in crops. In this work, we tested the possibility of using phenolics-rich extracts from spelt (Triticum dicoccum L.) husks to attenuate the effects of salt stress (100-200 mM NaCl) in maize. Two methanolic extracts were prepared from the soluble-conjugated (SC), and the insoluble-bound (IB) phenolic acid fractions of the spelt husk, and their effects were investigated on several stress-associated biochemical parameters, such as proline, lipid peroxidation, H2O2, GSH levels, and ion content. Results show that SC and IB fractions of husk extracts behaved very differently, no doubt due to their greatly divergent chemical composition, as revealed by both GC-MS and HPLC analyses. The efficacy of treatments in mitigating salt stress was also dose- and timing-dependent. IB, even at the lower concentration tested, was able to recover the performance of stressed plants in terms of growth, photosynthetic pigments content, and levels of salt stress markers. Recovery of shoot growth to control levels and reduction of stress-induced proline accumulation occurred regardless of whether plants were pre-treated or post-treated with IB, whereas only pre-treatment with the higher dose of IB was effective in mitigating oxidative stress. Although in some cases SC and even methanol alone exerted some positive effects, they could also be deleterious whereas IB never was. Overall, results indicate that a polyphenol-containing extract obtained from spelt by-products can behave as biostimulant in maize plants and can mitigate their response to salt stress, by acting on different biochemical targets.

New triterpenic acids produced in callus culture from fruit pulp of Acca sellowiana (O. Berg) Burret.

The aim of this work was the study of the best conditions for obtaining a callus culture from the pulp of Acca sellowiana, and to perform a quali-quantitative analysis of the secondary metabolites yielded by the in vitro callus culture. To this end, callus was induced on both Murashige and Skoog and Gamborg B5 media containing various combinations of growth regulators. Three previously undescribed ursane-type triterpenoids, 2α,3ß,6α,23-tetrahydroxy-18α,19α-urs-20-en-28-oic acid, 2α,3ß,23-trihydroxy-18α,19α-urs-20-en-28-oic acid and 2α,3ß,6ß,23-tetrahydroxy-18α,19α-urs-20-en-28-oic acid were isolated from the methanolic extract of A. sellowiana culture and characterized by 1D and 2D NMR experiments. Moreover, the quali-quantitative analysis (ESI-MSn and GC-MS) also showed the presence of ß-sitosterol, phloridzin, oleanolic, ursolic, 3ß-hydroxy-18α,19α-urs-20-en-28-oic, maslinic, corosolic, 2α,3ß-dihydroxy-18α,19α-urs-20-en-28-oic, and tormentic acid.

Rapid and selective screening for toxic phorbol esters in Jatropha curcas seed oil using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry.

Jatropha curcas L. is an inedible plant whose seed oil is an interesting source for biodiesel production. Seed cake, the main byproduct remaining (about 70% w/w) after the oil extraction process, has a high nutritional value but the presence in Jatropha curcas seed of phorbol esters (PEs), a family of toxic compounds with a tigliane skeleton, prevents application of seed cake and other byproducts (e.g. glycerin) in animal feed without an efficient detoxification. Considering the high toxicity of PEs, it is important to have a sensitive analytical method to evaluate the presence of these compounds in Jatropha curcas derivatives. In this paper we present the study of the ESI-MS/MS fragmentation pattern of the [M+Na]+ ion at m/z 733.5 of the six known PEs, namely Jatropha factors (JFs) C1-C6, which allowed to tentatively identify a series of characteristic and specific fragment ions useful to reveal the presence of JFs in Jatropha curcas seed oil, distinguish them from each other, and identify new PEs (J1-J4). Moreover, the substitution of the usual acetonitrile/water as mobile phase with a mixture of methanol/water (85:15, v/v) allowed to increase the signal of the sodium adduct of about 50-fold during the HPLC-ESI-MS/MS analysis.

Polymeric reversed phase and small particle size silica gel solid phase extractions for rapid analysis of sterols and triterpene dialcohols in olive oils by GC-FID.

The sterols and triterpene dialcohols composition is an important parameter to assess the authenticity of the high value and prone to adulteration extra virgin olive oil. The official methods used to carry out this analysis are time-consuming, labor-intensive and require a high amount of solvents. In this work a simple and time-saving method, based on two solid phase extraction (SPE) steps was developed. After oil saponification, the unsaponifiable matter was purified by polymeric SPE and then the sterols and triterpene dialcohols were isolated by an in-house packed small particle silica gel SPE and analyzed by GC-FID. Results obtained analyzing a sample of extra virgin olive oil, olive oil and refined olive pomace oil with the proposed method showed a good agreement with those obtained with the International Olive Council (IOC) official method. Thus, the use of the proposed method allows a rapid screening for extra virgin olive oils authentication.

Rapid determination of cholesterol oxidation products in milk powder based products by reversed phase SPE and HPLC-APCI-MS/MS.

A rapid and sensitive HPLC-APCI-MS/MS method for the determination of cholesterol oxidation products (COPs) in milk powder based foods is reported. The method consists in the direct saponification of the sample and purification of oxysterols by reversed phase C18-SPE followed by HPLC-MS/MS analysis. By this procedure, the extraction and enrichment of oxysterols are combined in a unique step, reducing sample manipulation and the possible formation of artifacts. LOD and LOQ were in the concentration ranges of 2-8ngg-1 and 8-30ngg-1, respectively. The precision (CV%) was in the range 10-36% in fresh samples with a total COPs amount from 212 to 645ngg-1 and 6-14% for an oxidized sample with a higher amount (3651ngg-1). The recovery ranged from 74±8% for 7-ketocholesterol to 101±12% for 7α-hydroxycholesterol at 200ngg-1 and from 82±2% for 7-ketocholesterol to 117±10% for 5α,6α-epoxycholesterol at 500ngg-1 spiked levels, respectively.

High Triterpenic Acids Production in Callus Cultures from Fruit Pulp of Two Apple Varieties.

INTRODUCTION: Very rarely fruit pulp has been used in in vitro culture to produce secondary metabolites useful in promoting health. OBJECTIVES: The aims of this work were the study of the best conditions to obtain the callus cultures from the pulp of two varieties of apples, Golden Delicious (GD) and "Mela Rosa Marchigiana" (MRM), and the quali-quantitative analysis of secondary metabolites produced by the two in vitro callus cultures. METHODOLOGY: Callus was induced on both Murashige and Skoog and Gamborg B5 media containing various combinations of supplements. To achieve the maximum recovery of secondary metabolites produced, preliminary extraction tests were carried out on GD apple culture using two different organic solvents (MeOH and EtOAc). The quali-quantitative analysis of the methanolic extract of both cultures was carried out by ESI-MSn and GC-MS techniques. RESULTS: The GC-MS analysis revealed the presence of triterpenic acids, in particular, oleanolic, ursolic, maslinic, pomolic, tormentic, corosolic and annurcoic acid along with a phytosterol, ß-sitosterol. In addition, GD callus culture produced phloridzin, absent in the MRM culture. In this last culture, however, the total amount of secondary metabolites was markedly higher. The in vivo production of these bioactive compounds were also quantified in the GD and MRM apple pulps. CONCLUSION: Apple pulps produced higher amounts of triterpenic acids in vitro than in vivo. The present work can be considered a method to amplify the production of important secondary metabolites which exert beneficial effects on human health. Copyright © 2016 John Wiley & Sons, Ltd.

Activity of Vitis vinifera Tendrils Extract Against Phytopathogenic Fungi.

The in vitro antifungal activity was determined of an ethanolic extract of Vitis vinifera L. tendrils (TVV) against ten plant pathogenic fungi, using the agar dilution method; activity was shown against all tested fungi. Fusarium species were the most sensitive with MIC values ranging from 250 to 300 ppm, while the basidiomycete fungus Rhizoctonia solani was the most resistant, with a MIC value of 500 ppm. Electrospray ionization tandem mass spectrometry (ESI-MS(n)) was used to obtain qualitative information on the main components of TVV. The high amount of polyphenolic compounds contained in TVV is likely to contribute significantly to its antifungal activity.

Characterization of N-Boc/Fmoc/Z-N'-formyl-gem-diaminoalkyl derivatives using electrospray ionization multi-stage mass spectrometry.

N-Boc/Fmoc/Z-N'-formyl-gem-diaminoalkyl derivatives, intermediates particularly useful in the synthesis of partially modified retro-inverso peptides, have been characterized by both positive and negative ion electrospray ionization (ESI) ion-trap multi-stage mass spectrometry (MS(n)). The MS(2) collision induced dissociation (CID) spectra of the sodium adduct of the formamides derived from the corresponding N-Fmoc/Z-amino acids, dipeptide and tripeptide acids show the [M + Na-NH2CHO](+) ion, arising from the loss of formamide, as the base peak. Differently, the MS(2) CID spectra of [M + Na](+) ion of all the N-Boc derivatives yield the abundant [M + Na-C4H8](+) and [M + Na-Boc + H](+) ions because of the loss of isobutylene and CO2 from the Boc protecting function. Useful information on the type of amino acids and their sequence in the N-protected dipeptidyl and tripeptidyl-N'-formamides is provided by MS(2) and subsequent MS(n) experiments on the respective precursor ions. The negative ion ESI mass spectra of these oligomers show, in addition to [M-H](-), [M + HCOO](-) and [M + Cl](-) ions, the presence of in-source CID fragment ions deriving from the involvement of the N-protecting group. Furthermore, MS(n) spectra of [M + Cl](-) ion of N-protected dipeptide and tripeptide derivatives show characteristic fragmentations that are useful for determining the nature of the C-terminal gem-diamino residue. The present paper represents an initial attempt to study the ESI-MS behavior of these important intermediates and lays the groundwork for structural-based studies on more complex partially modified retro-inverso peptides.

Chemical and physical analysis for the improvement of the dry mounting method for fragile documents.

Paper lamination is a widely used method to consolidate fragile documents. Previous studies have presented a new method of lamination that allows the consolidation of documents before undertaking aqueous treatments. In this method a thin Japanese paper coated with an acrylic resin is applied on the fragile document by means of a heated press. In this work we optimised the preparation of the lamination sheets as well as the working procedures, and we were able to establish that our laboratory-made specimens are chemically stable, easily reversible and permeable to aqueous solutions. The latest property is of particular importance, since it allows a subsequent aqueous deacidification even for fragile documents. The laminated paper documents were further analysed by means of colorimetry and FTIR spectroscopy before and after accelerated ageing as well as by determination of the alkali reserve left on the coated paper, in order to compare our lamination method with other commercially available lamination sheets.

Lamination of fragile documents as preliminary intervention to aqueous mass deacidification. A study on reversibility effects.

Paper lamination is a widely known method to consolidate fragile and brittle documents. Since the loss of mechanical strength of paper documents is almost completely due to their acidity, the procedure generally adopted by restorers consists of an aqueous treatment of deacidification followed by consolidation. The drawback of this method is due to the loss of paper fragments during the deacidification step, that must be therefore accurately controlled. Some preliminary experiments showed that a better and faster practice would be the lamination of paper documents with a water-resistant sheet before their immersion in the deacidification bath. The aim of this work is to evaluate the permeability, chemical stability and reversibility of the lamination sheets coated with two commercially available acrylic resins. From both gravimetric and FTIR analyses it appeared that slight modifications of the acrylic monomers led to different properties, and this behaviour can be exploited to improve the properties of lamination sheets in order to set up a reliable method of aqueous mass deacidification.