Selection and characterization of DNA aptamers for highly selective recognition of the major allergen of olive pollen Ole e 1.

In this study, single-stranded DNA aptamers with binding affinity to Ole e 1, the major allergen of olive pollen, were selected using systematic evolution of ligands by exponential enrichment (SELEX) method. Binding of the aptamers was firstly established by enzyme-linked oligonucleotide assay (ELONA) and aptaprecipitation assays. Additionally, aptamer-modified monolithic capillary chromatography was used in order to evaluate the recognition of this allergenic protein against other non-target proteins. The results indicated that AptOle1#6 was the aptamer that provided the highest affinity for Ole e 1. The selected aptamer showed good selective recognition of this protein, being not able to retain other non-target proteins (HSA, cyt c, and other pollen protein such as Ole e 9). The feasibility of the affinity monolithic column was demonstrated by selective recognition of Ole e 1 in an allergy skin test. The stability and reproducibility of this monolithic column was suitable, with relative standard deviations (RSDs) in retention times and peak area values of 7.8 and 9.3%, respectively (column-to-column reproducibility). This is the first study that describes the design of an efficient DNA aptamer for this relevant allergen.

Weak anion-exchange mixed-mode materials to selectively extract acidic compounds by stir bar sorptive extraction from environmental waters.

In this study, the first example of a polytetrafluoroethylene (PTFE)-based magnet coated with weak anion exchange (WAX) monolith as novel support for stir bar sorptive extraction (SBSE) is presented. Firstly, the PTFE magnets were properly modified and vinylized in order to immobilize polymer monoliths onto its surface. Then, a glycidyl methacrylate monolith was prepared and modified with ethylenediamine (EDA) to create weak anion exchanger via ring opening reaction of epoxy groups. The prepared covalently immobilized EDA-modified monoliths onto PTFE magnet exhibited good stability and reusability. Application of resulting material as stir bar for SBSE was investigated for a series of acidic compounds that includes acesulfame, saccharin, diclofenac or ibuprofen, among others as target compounds. Firstly, the SBSE conditions were optimized to promote the WAX interactions with the target compounds achieving recoveries from 37 to 75% and enable the selective extraction of these compounds as it provided values of% matrix effect from 17 to -13% when they were determined by SBSE followed by liquid chromatography - tandem mass spectrometry. The analytical methodology, was then validated and applied for the determination of the target solutes in environmental water samples, which were found at concentration up to 2500 ng L-1 in river waters.

Extraction of aflatoxins by using mesoporous silica (type UVM-7), and their quantitation by HPLC-MS.

A solid-phase extraction procedure has been developed by using a sorbent derived from UVM-7 mesoporous silica. The sorbent was applied to the extraction of aflatoxins B1, B2, G1 and G2 from tea samples followed by HPLC with mass spectrometric detection. The sorbent was characterized by transmission electron microscopy, nuclear magnetic resonance, X-ray diffraction and nitrogen adsorption-desorption. UVM-7 is found to be the best solid phase. The amount of solid-phase, type and volume of eluent, pH value and ionic strength and breakthrough volume were optimized. Following the recommended procedure, recoveries between 96.0 and 98.2% were achieved, with RSD values of <5.1%, and the limits of detection are in the range from 0.14 to 0.7 µg·kg-1. The material is reusable. The method was applied to the analysis of real tea samples. A low matrix effect is found, and recoveries are >88%. The results were compared with those obtained by immunoaffinity columns as a reference method. Only low concentrations of aflatoxin G2 were found in some samples, and results obtained with both methods are shown to be statistically sound and comparable. Graphical abstractSchematic representation of a mesoporous silica sorbent (type UVM-7) for the extraction of aflatoxins (AF) from tea by solid-phase extraction (SPE), and its determination by liquid chromatography. The morphology of the material allows to retain the analytes very well.

Proteomic fingerprinting of mistletoe (Viscum album L.) via combinatorial peptide ligand libraries and mass spectrometry analysis.

Combinatorial peptide ligand libraries (CPLLs), coupled to mass spectrometry (MS) analysis, have been used to investigate in depth the proteome of Viscum album L. (VA), commonly named European mistletoe, in order to provide a first proteomic fingerprinting. For this purpose, the proteins were captured via CPLLs at two different pH values (acidic and neutral). A total of 648 non-redundant proteins were identified by using two different databases. The two pH values, chosen for bead incubations, have contributed to increment the capture ability: 56% and 31% of CPLLs species were respectively recognized at pH7.2 and at pH2.2. Finally the biological function of identified proteins was evaluated in order to understand their role on human health and the potential benefits of mistletoe extracts in medicine. SIGNIFICANCE: Viscum album L. (VA) extracts are recently used as supporting medicine for cancer therapy, improving patients' survival and increasing their quality of life in medicine. These anticancer effects are investigated and they are probably due to mistletoe's capability to favor tumor cell's death and to modulate the immune system. Although the increasing interest in VA medical benefits, the role of its components in human health remains unclear. In order to exploit this aspect, it is important to comprehensively study proteins present in Viscum album L. (VA) extracts. Nevertheless, since plant proteomics analysis is in most cases handicapped by the presence of high-abundance proteins masking the detection of the low-abundance ones, it is important to overcome this challenge. In this sense, combinatorial peptide ligand libraries (CPLLs) have been used to reduce the dynamic protein concentration range to enable the identification of a higher amount of proteins than employing conventional methods. In this work, a total of 648 non-redundant proteins were identified: 56% and 31% of CPLLs species were respectively recognized at pH7.2 and at pH2.2. This deep proteome identification was useful to investigate the biological functions of proteins in order to evaluate their potential role in human health.

Simultaneous determination of main phenolic acids and flavonoids in tomato by micellar electrokinetic capillary electrophoresis.

A methodology for the determination of tomato phenolic acids and flavonoids has been developed combining MEKC and DAD detection. The influence on polyphenol separation of pH and background electrolyte, BGE (borax, acetonitrile, methanol and SDS concentrations), was studied and optimized using response surface methodology and weighted desirability function. Separation of polyphenols was achieved within 20min at 15°C using 11.3mM borax and 11.2mM SDS adjusted to pH 8.5 as BGE. Validation was performed using standards and tomato extracts. Recoveries ranged from 77 to 106%. Acceptable repeatabilities were obtained for peak area (%RSD <3.1% and <3.7%) and migration times (%RSD <0.2% and <1.4%) for intra- and inter-day respectively. Detection limits ranged between 0.8 and 3.8mgkg-1. Five and seven of these polyphenols were determined in samples of tomato and related species. This methodology will be valuable tool in breeding programs, analyzing a large number of samples.

Chemical analysis and antioxidant activity of the essential oils of three Piperaceae species growing in the central region of Cuba.

The present study describes the phytochemical profile and antioxidant activity of the essential oils of three Piperaceae species collected in the central region of Cuba. The essential oils of Piper aduncum, P. auritum and P. umbellatum leaves, obtained by hydrodistillation, were analyzed by gas chromatography-mass spectrometry. The main components of P. aduncum oil were piperitone (34%), camphor (17.1%), camphene (10.9%), 1,8-cineol (8.7%) and viridiflorol (7.4%), whereas that of P. auritum and P. umbellatum was safrole (71.8 and 26.4%, respectively). The antioxidant properties of the essential oils were also evaluated using several assays for radical scavenging ability (DPPH test and reducing power) and inhibition of lipid oxidation (ferric thiocyanate method and evaluation against Cucurbita seed oil by peroxide, thiobarbituric acid and p-anisidine methods). P. auritum showed the strongest antioxidant activity among the Piper species investigated, but lower than those of butylated hydroxyanisol and propyl gallate.

Chemical composition, antioxidant properties and antimicrobial activity of the essential oil of Murraya paniculata leaves from the mountains of Central Cuba.

The essential oil of Murraya paniculata L leaves from the mountains of the Central Region of Cuba, obtained by hydrodistillation, was analyzed by gas chromatography-mass spectrometry. Eighteen compounds, accounting for 95.1% of the oil were identified. The major component was beta-caryophyllene (ca. 30%). The antioxidant activity of essential oil was evaluated against Cucurbita seed oil by peroxide, thiobarbituric acid and p-anisidine methods. The essential oil showed stronger antioxidant activity than that of butylated hydroxyanisole and butylated hydroxytoluene, but lower than that of propyl gallate. Moreover, this antioxidant activity was supported by the complementary antioxidant assay in the linoleic acid system and 2, 2'-diphenyl-1-picrylhydrazyl. The essential oil also showed good to moderate inhibitory effects against Klebsiellapneumoniae and Bacillus subtilis.

Capillary electrophoresis of free fatty acids by indirect ultraviolet detection: application to the classification of vegetable oils according to their botanical origin.

A method for the determination of fatty acids in vegetable oils by capillary electrophoresis with indirect UV-vis detection has been developed. The separation of fatty acids was optimized in terms of Brij surfactant nature and concentration and organic modifier (2-propanol) percentage. The optimal background electrolyte consisted of 10 mM p-hydroxybenzoate, 5 mM Tris at pH 8.8, 80 mM Brij 98, 40% acetonitrile, and 10% 2-propanol. Under these conditions, vegetable oils from five botanical origins (avocado, corn, extra virgin olive, hazelnut, and soybean) were analyzed and the fatty acid contents established. Linear discriminant analysis (LDA) models were constructed using fatty acid peak areas as predictors. An excellent resolution among all category pairs was obtained, and all samples were correctly classified with assignment probabilities of >95%.

Classification of pumpkin seed oils according to their species and genetic variety by attenuated total reflection Fourier-transform infrared spectroscopy.

Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR), followed by multivariate treatment of the spectral data, was used to classify seed oils of the genus Cucurbita (pumpkins) according to their species as C. maxima, C. pepo, and C. moschata. Also, C. moschata seed oils were classified according to their genetic variety as RG, Inivit C-88, and Inivit C-2000. Up to 23 wavelength regions were selected on the spectra, each region corresponding to a peak or shoulder. The normalized absorbance peak areas within these regions were used as predictors. Using linear discriminant analysis (LDA), an excellent resolution among all categories concerning both Cucurbita species and C. moschata varieties was achieved. The proposed method was straightforward and quick and can be easily implemented. Quality control of pumpkin seed oils is important because Cucurbita species and genetic variety are both related to the pharmaceutical properties of the oils.

Methacrylate ester-based monolithic columns for nano-LC separation of tocopherols in vegetable oils.

The separation and determination of tocopherols (Ts) in vegetable oils by nano-LC chromatography with UV-vis detection using lauryl methacrylate ester-based monolithic columns has been developed. The separation of Ts was optimized in terms of mobile phase composition on the basis of the best compromise among efficiency, resolution and analysis time. Using a mobile phase composed of ACN/methanol/water, an excellent resolution between Ts was achieved within 18 min. The LODs were lower than 0.26 µg/mL, being repeatability values of retention time and peak area below 0.15 and 3.1%, respectively. The method was applied to the quantification of Ts and tocotrienols present in several vegetable oils from different botanical origins.

Fast separation and determination of sterols in vegetable oils by ultraperformance liquid chromatography with atmospheric pressure chemical ionization mass spectrometry detection.

A method for the determination of sterols in vegetable oils by ultraperformance liquid chromatography (UPLC) with atmospheric pressure chemical ionization mass spectrometry detection has been developed. The separation of sterols was optimized in terms of mobile phase composition, column temperature and flow rate. The optimal conditions were achieved using an Acquity UPLC BEH C18 column (50 x 2.1 mm, 1.7 microm) with a mobile phase consistent of acetonitrile/water (0.01% acetic acid) using a linear gradient, at a flow rate of 0.8 mL min(-1) and column temperature of 10 degrees C, giving a total analysis time below 5 min. The determination was performed in selective ion recording mode. The limits of detection were in all cases below 0.07 microg mL(-1), with relative standard deviation values of retention times and peak areas below 0.4 and 5%, respectively. The content of main sterols present in several vegetable oils with different botanical origins was also established.

Determination of tocopherols and tocotrienols in vegetable oils by nanoliquid chromatography with ultraviolet-visible detection using a silica monolithic column.

A method for the determination of tocopherols and tocotrienols in vegetable oils by nanoliquid chromatography with UV-vis detection has been developed. The separation of tocopherols was optimized in terms of mobile phase composition on the basis of the best compromise between efficiency, resolution, and analysis time. The optimal conditions were achieved using a C18 silica monolithic column (150 mm x 0.1 mm) with an isocratic elution of acetonitrile/methanol/water (acidified with 0.2% acetic acid) at a flow rate of 0.5 microL min(-1), giving a total analysis time below 18 min. The method has been applied to the quantification of tocopherols and tocotrienols present in several vegetable oils with different botanical origins.

Classification of extra virgin olive oils produced at La Comunitat Valenciana according to their genetic variety using sterol profiles established by high-performance liquid chromatography with mass spectrometry detection.

A method to classify extra virgin olive oils (EVOOs) according to their genetic variety using sterol profiles obtained by high-performance liquid chromatography (HPLC) with mass spectrometry (MS) detection has been developed. Sterol extracts were chromatographed on a dC18 Atlantis column (100x3 mm, 3 microm) with a gradient of acetonitrile/water (0.01% acetic acid) at a flow rate of 1.0 mL min(-1) and positive-ion mode MS detection. Using linear discriminant analysis of the HPLC-MS data (extracted ion chromatograms), EVOO samples belonging to six genetic varieties cultivated at La Comunitat Valenciana, Spain (Arbequina, Borriolenca, Canetera, Farga, Picual, and Serrana), were correctly classified with an excellent resolution among all of the categories.

Prediction of the genetic variety of extra virgin olive oils produced at La Comunitat Valenciana, Spain, by fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy (FTIR), followed by multivariate treatment of the spectral data, was used to classify extra virgin olive oils (EVOOs) according to their genetic variety. EVOO samples corresponding to seven different genetic varieties (Arbequina, Borriolenca, Canetera, Farga, Hojiblanca, Picual, and Serrana) were analyzed. The wavelength scale of the FTIR spectra of the oils was divided into 20 regions. The normalized absorbance peak areas within these regions were used as predictor variables. Classification of the EVOO samples according to their genetic variety was achieved by linear discriminant analysis (LDA). A good resolution among all categories was achieved using a LDA model constructed with only nine predictor variables. With this LDA model, 88% of the EVOOs were correctly classified, with assignment probabilities higher than 95%. This method is helpful for olive oil producers because it provides useful information related to the genetic variety of EVOOs, which is required by European Community regulations.