Biomedical subjects

Elena Ibáñez

Publications and source records attributed to Elena Ibáñez.

17 recordsLinked to original sources

PubMed2007

Use of specially designed columns for antioxidants and antimicrobials enrichment by preparative supercritical fluid chromatography.

A new, specially designed column has been developed for fractionation of supercritical fluid extract of rosemary by using a preparative supercritical fluid chromatography system (Prep-SFC). The column evaluated in this work was prepared using a new packing method consisting of a combination of slurry and supercritical CO2 with commercial silica particles coated with a stationary phase commonly used in gas chromatography, such as SE-54 (5% phenyl-, 95% methylsilicone). The new packing procedure provided columns with reasonable efficiencies, with high stability and useful at high-pressure range. A 25 cm x 10 mm i.d. column packed with silica particles coated with 3% of SE-54 was prepared, and its separation power was tested for isolating fractions with high antioxidant and/or antimicrobial activity from a supercritical rosemary extract. The SFC conditions were selected based on a previous work done with a commercial LC-Diol packed column (130 bar, 80 degrees C), and different percentages of modifier in the mobile phase were tested (5 and 10%). Two cyclones were employed to collect the fractions which were then characterized by HPLC-diode array detection (DAD), GC, and in vitro antioxidant and antimicrobial assays. The use of coated packed columns allowed the fractionation of a complex mixture of rosemary supercritical extract with a minimum amount of modifier in the mobile phase (5% ethanol). At the optimum conditions it was possible to obtain two very active fractions in terms of antioxidant and antimicrobial activity, with no residual rosemary aroma and with improved activities compared to the original supercritical extract.

Anti-Infective Agents↗

PubMed2006

Optimization of the extraction of antioxidants from Dunaliella salina microalga by pressurized liquids.

In this work, extraction of antioxidant compounds from Dunaliella salina microalga is optimized by combining pressurized liquid extraction (PLE) and experimental design (three-level factorial design) with three different solvents (hexane, ethanol, and water). Two main factors were considered, the extraction temperature (40, 100, and 160 degrees C) and the extraction time (5, 17.5, and 30 min). As response variables, the extraction yield (percent dry weight/initial weight) and the antioxidant activity of the extracts (determined using the TEAC method) were used. The parameters of the model were estimated by multiple linear regression. Results showed that the extraction temperature was the factor having the strongest influence (positive) on the two response variables. The best yields were obtained with ethanol at the higher extraction temperature and time tested. Besides, although hexane extracts provided the best antioxidant activity, ethanol extracts were also very active. The chemical characterization of ethanol extracts was carried out using HPLC-DAD, and attempts have been made to correlate their chemical composition with the antioxidant activity measured. Results pointed out that the extracts contained, besides all-trans-beta-carotene and isomers, several different minor carotenoids that seemed to make a contribution to the antioxidant activity of the extracts.

Antioxidants↗

PubMed2006

Isolation of functional ingredients from rosemary by preparative-supercritical fluid chromatography (Prep-SFC).

A supercritical fluid extract of rosemary has been fractionated under supercritical conditions by using a preparative-SFC system. In this work, the optimum conditions have been evaluated to achieve a selective isolation of the compounds responsible for both, antioxidant and antimicrobial activities. A 25 cm x 10 mm i.d. LC-Diol packed column (dp=5 microm) has been used and the separation took place at 80 degrees C of column temperature, 130 bar of pressure, and 10% of ethanol as modifier of the mobile phase (CO(2)). Two cyclones were employed to collect the fractions which were subsequently characterized by HPLC-DAD, GC, and in vitro antioxidant and antimicrobial assays. By a careful selection of the separation conditions it is possible to obtain two different fractions, one enriched with antioxidant and antimicrobial compounds (with an improvement of about 20% and 40% of antioxidant and antimicrobial activity, respectively, compared to the original extract) collected in cyclone 2 and with no residual rosemary aroma and another one containing the essential oil.

Abietanes↗

PubMed2006

Expression of recombinant human follicle-stimulating hormone in the mammary gland of transgenic mice.

In this article, we describe the use of a caprine beta-lactoglobulin (betaLG) expression cassette previously obtained by our group to target the expression of the human follicle-stimulating hormone (hFSH) to the mammary gland of transgenic mice. The hFSH is a pituitary glycoprotein composed of two subunits (alpha and beta). At present, this protein is obtained from mammalian cellular fermentors, and it is extensively used in the treatment of human infertility. Four lines of double (hFSHalpha/beta) transgenic mice that stably transmitted the transgenes were obtained, and hFSHalpha and hFSHbeta mRNA was detected by reverse transcriptase-polymerase chain reaction in the mammary gland of lactating females from all four transgenic lines. The hFSH protein was present in the mammary gland of the lactating females, but could not be detected in the milk by Western blot, probably as a result of low levels of transgene expression.

Animals↗

PubMed2006

Dunaliella salina microalga pressurized liquid extracts as potential antimicrobials.

In the present work, the antimicrobial activity of different pressurized liquid extracts obtained from Dunaliella salina microalga was tested against several microorganisms of importance for the food industry (Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger). Different solvents (hexane, petroleum ether, hexane, and water) and extraction conditions (40, 100, and 160 degrees C) were tested. Results showed that the best antimicrobial activity was obtained for each solvent at the highest extraction temperature (160 degrees C). Likewise, the extraction yield followed the same trend, i.e., increasing with extraction temperature and was at a maximum when ethanol was used as an extraction solvent. Water extracts had the lowest extraction yields. In general, the best results in terms of antimicrobial activity were obtained using petroleum ether and hexane, although ethanolic extracts also showed good antimicrobial activity. Because the main antimicrobial activity of the extracts was against bacteria, the extracts can be considered to be specifically antibacterial. The extracts were analyzed by gas chromatography-mass spectrometry in order to identify the compounds responsible for activity. Fifteen different volatile compounds as well as several fatty acids (mainly palmitic, alpha-linolenic, and oleic acids) that could have been responsible for the antimicrobial activity were identified in the extracts. beta-Cyclocitral, alpha- and beta-ionone, neophytadiene, and phytol were identified among other volatile compounds; all of these compounds have previously been described as antimicrobial agents.

Anti-Bacterial Agents↗

PubMed2005

Pressurized liquid extraction-capillary electrophoresis-mass spectrometry for the analysis of polar antioxidants in rosemary extracts.

A method based on capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS) was developed to qualitatively characterize natural antioxidants from rosemary (Rosmarinus officinalis L.) in different fractions obtained by pressurized liquid extraction (PLE) using subcritical water. The parameters of CE-ESI-MS were adjusted allowing the separation and characterization of different compounds from rosemary in the PLE fractions. These parameters for CE are kind, pH and concentration of the separation buffer, parameters for ESI-MS are dry gas temperature and flow, nebulizing gas pressure, and make-up flow. The following analytical conditions were found most favorable: aqueous CE buffer (40 mM ammonium acetate/ammonium hydroxide, pH 9); sheath liquid containing 2-propanol-water (60:40, v/v) and 0.1% (v/v) triethylamine at a flow rate of 0.24 mL/h; drying gas flow rate equal to 7 L/min at 350 degrees C, nebulizing gas pressure of 13.8 kPa (2 psi), using a compound stability of 50%. Different antioxidant compounds (e.g., rosmarinic acid and carnosic acid) could be detected in the rosemary extracts by CE-ESI-MS without any additional treatment, enabling the determination of variations in the extract composition caused by the different PLE conditions (i.e., 60 and 100 degrees C). The results provide complementary information to HPLC analysis.

Abietanes↗

PubMed2005

Characterization of proteins from Spirulina platensis microalga using capillary electrophoresis-ion trap-mass spectrometry and capillary electrophoresis-time of flight-mass spectrometry.

In this work, a new capillary electrophoresis-mass spectrometry (CE-MS) procedure is developed to analyze proteins in Spirulina platensis microalgae. It is demonstrated that a fine optimization of several separation parameters is essential in order to achieve suitable CE-MS analysis of these proteins in natural extracts from microalgae. Namely, optimization of the composition of the separation buffer, electrospray conditions, and washing routine between runs are required in order to obtain reliable and reproducible CE-MS analyses of the main proteins found in this microalga (namely, allophycocyanin-alpha chain, allophycocyanin-beta, c-phycocyanin-alpha, and c-phycocyanin-beta). The relative molecular mass of these biopolymers is determined using two different MS instruments coupled to CE, i.e., CE-ion trap-MS and CE-time of flight-MS (CE-TOF-MS). A comparison between the results obtained with both instruments is carried out. The high resolution of the TOF-MS enables the distinction of small modifications in proteins and, thus, a more accurate mass determination. Interestingly, molecular mass values obtained by both CE-MS procedures agree very well while these experimental values are only in partial agreement with those theoretically expected (i.e., genetically derived masses). Some protein modifications due to amino acids exchange induced by nucleotide codon mutations are proposed to explain this difference.

Bacterial Proteins↗

PubMed2005

Capillary electrophoresis-mass spectrometry of Spirulina platensis proteins obtained by pressurized liquid extraction.

In this work, the usefulness of CE-MS to monitor and optimize the pressurized liquid extraction (PLE) of proteins from Spirulina platensis microalga is demonstrated. Crude and purified PLE extracts from microalga were analyzed by CE-MS. It was observed that the use of purification protocols of phycobiliproteins (namely, ultrafiltration or precipitation-dialysis-freeze drying) resulted in better CE resolution and MS signals, demonstrating that sample matrix plays an important role in CE-MS of proteins in real samples. Ultrafiltration was found less laborious and much faster than precipitation-dialysis-freeze drying (1 vs. 48 h). Direct analysis of crude extracts was demonstrated to be also possible by CE-MS, providing less-quality information but enough to characterize PLE extracts in a much faster way. Therefore, the latter protocol was selected to monitor and optimize the extraction process of phycobiliproteins from S. platensis. To do that, different extraction conditions were tested, including time, temperature and pressure of extraction, nature of pressurized liquid, distribution of microalga inside the extraction cell, type of packing, etc. It is demonstrated that the combined use of PLE and CE-MS allows the attainment of extracts rich in phycobiliproteins in short extraction times (namely, yields of 20% can be obtained in less than 2 h under the optimum PLE process in an automatic way). To our knowledge, this work shows for the first time the usefulness of CE-MS for monitoring and optimizing a PLE process.

Algal Proteins↗

PubMed2005

Separation and characterization of antioxidants from Spirulina platensis microalga combining pressurized liquid extraction, TLC, and HPLC-DAD.

A new procedure has been developed to separate and characterize antioxidant compounds from Spirulina platensis microalga based on the combination of pressurized liquid extraction (PLE) and different chromatographic procedures, such as TLC, at preparative scale, and HPLC with a diode array detector (DAD). Different solvents were tested for PLE extraction of antioxidants from S. platensis microalga. An optimized PLE process using ethanol (generally recognized as safe, GRAS) as extraction solvent has been obtained that provides natural extracts with high yields and good antioxidant properties. TLC analysis of this ethanolic extract obtained at 115 degrees C for 15 min was carried out and the silica layer was stained with a DPPH (diphenyl-pycril-hydrazyl) radical solution to determine the antioxidant activity of different chromatographic bands. Next, these colored bands were collected for their subsequent analysis by HPLC-DAD, revealing that the compounds with the most important antioxidant activity present in Spirulina extracts were carotenoids, as well as phenolic compounds and degradation products of chlorophylls.

Antioxidants↗

PubMed2005

Effect of genetic background and activating stimulus on the timing of meiotic cell cycle progression in parthenogenetically activated mouse oocytes.

With the aim of investigating the effects of oocyte genotype and activating stimulus on the timing of nuclear events after activation, oocytes collected from hybrid B6D2F1, inbred C57BL/6 and outbred CF-1 and immunodeficient nude (NU/+) females were activated using ethanol or strontium and fixed at various time-points. Meiotic status, spindle rotation and second polar body (PB2) extrusion were monitored by fluorescence microscopy using DNA-, microtubule- and microfilament-selective probes. Although activation efficiency was similar in all groups of oocytes, a significant percentage of CF-1 and NU/+ oocytes treated with ethanol and of C57BL/6 oocytes treated either with ethanol or strontium failed to complete activation and became arrested at a new metaphase stage (MIII) after PB2 extrusion. C57BL/6 oocytes also showed slower release from MII arrest but faster progression to telophase (TII) after ethanol exposure, and they exhibited the most rapid exit from TII under both activation treatments. Strontium caused delayed meiotic resumption, spindle rotation and PB2 extrusion, but rapid TII exit, in B6D2F1, CF-1 and NU/+ oocytes when compared with ethanol. Compared with all other strains, NU/+ oocytes were significantly slower in completing spindle rotation and PB2 extrusion, irrespective of the activating stimulus, and a significant decrease in activation rates and pace of meiotic progression was observed after strontium exposure. Thus, our findings demonstrated that the kinetics of meiosis resumption and completion, spindle rotation and PB2 extrusion following parthenogenetic activation depends on both genotype-specific factors and on the activation treatment applied.

Animals↗

PubMed2005

Genetic strain variations in the metaphase-II phenotype of mouse oocytes matured in vivo or in vitro.

The interplay between genetic and epigenetic factors plays a central role in mammalian embryo production strategies that superimpose ex vivo or in vivo manipulations upon strain background characteristics. In this study, we examined the relationship between genetic background and the phenotypic properties of mouse metaphase-II (M-II) oocytes that were matured under in vivo (IVO) or in vitro conditions, either in a basal (IVM) or a supplemented (IVM + ) medium. Differences existed amongst inbred (C57BL/6), outbred (CF-1, Black Swiss, NU/NU) and hybrid lines (B6D2F1) induced to superovulate with regard to cytoplasmic microtubule organizing center (MTOC) number but not spindle size or shape, except for larger and asymmetrical spindles in Black Swiss oocytes. When oocytes were matured in culture, meiotic spindle and cytoplasmic phenotypic properties of M-II oocytes were affected relative to in vivo conditions and between strains. Specifically, measures of meiotic spindle size, shape, polar pericentrin distribution and cytoplasmic MTOC number all revealed characteristic variations. Interestingly, the overall reduction in cytoplasmic MTOC number noted upon IVM was concomitant with an overall increase in spindle and polar body size. Maturation under IVM + conditions resulted in a further decrease in cytoplasmic MTOC number, but spindle and polar body characteristics were intermediate between IVO and IVM. How these oocyte phenotypic properties of maternal origin may be linked to predictive assessments of fecundity remains to be established.

Animals↗

PubMed2004

Disruption of the mouse phospholipase C-beta1 gene in a beta-lactoglobulin transgenic line affects viability, growth, and fertility in mice.

A recessive insertional mutation was identified in one line of transgenic mice for the caprine beta-lactoglobulin (betaLG) gene. High mortality after birth, a significant reduction in postnatal growth and adult body size, changes in the morphometric features of the head, and infertility are the most prominent phenotypic traits of the mutant animals. Molecular cloning and sequencing of the transgene insertion site showed that 22 copies of the betaLG transgene are inserted in an intronic region of the phospholipase C-beta1 (PLC-beta1) gene, which plays a pivotal role in modulating different cellular functions. As a result of the insertional mutation (PLC-beta1(betaLG) mutation), a hybrid messenger RNA (mRNA) between the mouse PLC-beta1 and the goat betaLG genes is transcribed. The tissue-specific pattern of expression of this hybrid mRNA in PLC-beta1(betaLG) homozygotes is equivalent to that of the endogenous PLC-beta1 mRNA in nontransgenic animals, which is reported for the first time in this species, but expression levels are significantly reduced. Although the hybrid PLCbeta1-betaLG mRNA contains all the essential information to produce a PLCbeta1 protein that could be activated, this protein was not detected by Western blot. The PLC-beta1(betaLG) mouse model described here represents a useful tool to investigate the role of the PLC-beta1 gene in the molecular mechanisms underlying growth and fertility.

Animals↗

PubMed2004

Pressurized liquid extracts from Spirulina platensis microalga. Determination of their antioxidant activity and preliminary analysis by micellar electrokinetic chromatography.

In this work, different extracts from the microalga Spirulina platensis are obtained using pressurized liquid extraction (PLE) and four different solvents (hexane, light petroleum, ethanol and water). Different extraction temperatures (115 and 170 degrees C) were tested using extraction times ranging from 9 to 15 min. The antioxidant activity of the different extracts is determined by means of an in vitro assay using a free radical method. Moreover, a new and fast method is developed using micellar electrokinetic chromatography with diode array detection (MEKC-DAD) to provide a preliminary analysis on the composition of the extracts. This combined application (i.e., in vitro assays plus MEKC-DAD) allowed the fast characterization of the extracts based on their antioxidant activity and the UV-vis spectra of the different compounds found in the extracts. To our knowledge, this work shows for the first time the great possibilities of the combined use of PLE-in vitro assay-MEKC-DAD to investigate natural sources of antioxidants.

Antioxidants↗

PubMed2004

Countercurrent supercritical fluid extraction and fractionation of high-added-value compounds from a hexane extract of olive leaves.

Countercurrent supercritical fluid extraction (CC-SFE) at a pilot scale plant was used for fractionation of high-added-value products from a raw extract of olive leaves in hexane. Compounds found in the raw extract were waxes, hydrocarbons, squalene, beta-carotene, triglycerides, alpha-tocopherol, beta-sitosterol, and alcohols. The CC-SFE extraction process was investigated according to a 2(3) full factorial experimental design using the following variables and ranges: extraction pressure, 75-200 bar; extraction temperature, 35-50 degrees C; and ethanol as modifier, 0-10%. Data were analyzed in terms of extraction yield, enrichment, recovery, and selectivity. Higher extraction yields were attained at 200 bar. For most of the compounds analyzed enrichment was attained at the same conditions, that is, 75 bar, 35 degrees C, and 10% ethanol. Hydrocarbons were usually recovered in the separators, whereas waxes and alpha-tocopherol remain in the raffinate. Selectivity data reveal that alpha-tocopherol is the most easily separable compound. The influence of the experimental factors on the recovery of all the compounds was studied by means of regression models. The best fitted model was attained for beta-sitosterol, with R2 = 99.25%.

Chemical Fractionation↗

PubMed2004

Real-time quantitative PCR-based system for determining transgene copy number in transgenic animals.

In this paper, we describe a rapid and accurate real-time quantitative PCR-based system to determine transgene copy number in transgenic animals. We used the 2(-deltadeltaCt) method to analyze different transgenic lines without the requirement of a control sample previously determined by Southern blot analysis. To determine the transgene copy number in several mouse lines carrying a goat beta-Lactoglobulin transgene, we developed a TaqMan assay in which a goat genomic DNA sample was used as a calibrator. Moreover, we used the glucagon gene as a reference control because this gene is highly conserved between species and amplifies with the same efficiency and sensitivity in goat as in mouse. With this assay, we provide an alternative simple method to determine the transgene copy number, avoiding the traditional and tedious blotting techniques. The assay's discrimination ability from our results is of at least six copies and, similar to the limitations of the blotting techniques, the accuracy of the quantification diminishes when the transgene copy number is high.

Animals↗

PubMed2003

New trends in food processing.

In this work some of the newest trends in food processing are reviewed. This revision intends to provide an updated overview (including works published until February 2001) on the newest food processes, including food manufacturing, preservation, and control. Modern processes for food and food ingredients manufacturing based on membrane technology, super-critical fluid technology, and some applications of biotechnology are presented, mainly applied to obtain functional foods, "all-natural" enriched foods, probiotics and prebiotics. Also included is a critical assessment concerning non-thermal preservation techniques used for food preservation, such as high hydrostatic pressure, pulsed electric fields, ultrasound, pulsed light, hurdle systems, etc. Finally, a group of new analytical techniques (i.e., molecular techniques such as Polymerase Chain Reaction (PCR), food image analysis, and biosensors) and their use for food and process control is reviewed.

Food Handling↗

PubMed2002

Demecolcine-induced oocyte enucleation for somatic cell cloning: coordination between cell-cycle egress, kinetics of cortical cytoskeletal interactions, and second polar body extrusion.

Studies were designed to further explore the use of pharmacological agents to produce developmentally competent enucleated mouse oocytes for animal cloning by somatic cell nuclear transfer. Metaphase II oocytes from CF-1 and B6D2F1 strains were activated with ethanol and subsequently exposed to demecolcine at various times postactivation. Chromosome segregation, spindle dynamics, and polar body (PB) extrusion were monitored by fluorescence microscopy using DNA-, microtubule-, and microfilament-selective probes. Exposure to demecolcine did not affect rates of oocyte activation induced by ethanol but did disrupt the coordination of cytokinesis and karyokinesis, suppressing the extent and completion of spindle rotation and second PB extrusion in a strain-dependent manner. Moreover, strain- and treatment-specific variations in the rate of oocyte enucleation were also detected. In particular, CF1 oocytes were more efficiently enucleated relative to B6D2F1 oocytes, and demecolcine treatments initiated early after activation resulted in higher enucleation rates than when treatment was delayed. The observed strain differences are possibly caused by a combination of factors, such as the time course of meiotic cell-cycle progression after ethanol activation, the degree of spindle rotation, and the extent of second PB extrusion. These results suggest that developmentally competent cytoplasts can be produced by timely exposure of activated oocytes to agents that disrupt spindle microtubules. However, the utility of the demecolcine-induced enucleation protocol will require further investigation into factors linking karyokinesis to cytokinesis at the levels of cell-cycle control and oocyte cytoskeletal remodeling following artificial or natural means of egg activation.

Animals↗