Carmen Socaciu

Total and individual carotenoids and phenolic acids content in fresh, refrigerated and processed spinach (Spinacia oleracea L.).

The carotenoid and phenolic acid contents in fresh, stored and processed (blanched, frozen and boiled) spinach were comparatively determined by spectrophotometric and HPLC analyses. The major carotenoids identified after HPLC analysis in saponified samples were lutein (37-53μg/kg), β-carotene (18-31μg/kg), violaxanthin (9-23μg/kg) and neoxanthin (10-22μg/kg). These carotenoids were all affected by storage and/or heating. The content of carotenoids was best preserved after storage for one day at 4°C. The total phenolic content in the fresh spinach was 2088mg GAE/kg FW. After LC-MS analysis three phenolic acids were identified and quantified. These being ortho-coumaric acid (28-60mg/kg FW), ferulic acid (10-35mg/kg) and para-coumaric acid (1-30mg/kg) depending on the sample type. After storage of spinach at different temperatures (4°C or -18°C) the amount of total phenolic compounds decreased by around 20%, while the amount of individual phenolic acids increased by four times on average.

Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells

We investigated the time‐dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X‐rays) and we looked at various markers to follow the early‐to‐late apoptotic events: phospholipid translocation (identified through annexin V‐fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence‐labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X‐rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter‐related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of “early‐to‐late” apoptosis appears to be a fixed program.

Anthocyanin determination in blueberry extracts from various cultivars and their antiproliferative and apoptotic properties in B16-F10 metastatic murine melanoma cells

Blueberry consumption is associated with health benefits contributing to a reduced risk for cardiovascular disease, diabetes and cancer. The aim of this study was to determine the anthocyanin profile of blueberry extracts and to evaluate their effects on B16-F10 metastatic melanoma murine cells. Seven blueberry cultivars cultivated in Romania were used. The blueberry extracts were purified over an Amberlite XAD-7 resin and a Sephadex LH-20 column, in order to obtain the anthocyanin rich fractions (ARF). The antioxidant activity of the ARF of all cultivars was evaluated by ABTS, CUPRAC and ORAC assays. High performance liquid chromatography followed by electrospray ionization mass spectrometry (HPLC-ESI-MS) was used to identify and quantify individual anthocyanins. The anthocyanin content of tested cultivars ranged from 101.88 to 195.01 mg malvidin-3-glucoside/100g fresh weight. The anthocyanin rich-fraction obtained from cultivar Torro (ARF-T) was shown to have the highest anthocyanin content and antioxidant activity, and inhibited B16-F10 melanoma murine cells proliferation at concentrations higher than 500 μg/ml. In addition, ARF-T stimulated apoptosis and increased total LDH activity in metastatic B16-F10 melanoma murine cells. These results indicate that the anthocyanins from blueberry cultivar could be used as a chemopreventive or adjuvant treatment for metastasis control.

Antiproliferative and Antioxidant Properties of Anthocyanin Rich Extracts from Blueberry and Blackcurrant Juice

The present study was aimed at evaluating the antiproliferative potential of anthocyanin-rich fractions (ARFs) obtained from two commercially available juices (blueberry and blackcurrant juices) on three tumor cell lines; B16F10 (murine melanoma), A2780 (ovarian cancer) and HeLa (cervical cancer). Individual anthocyanin determination, identification and quantification were done using HPLC-MS. Antioxidant activity of the juices was determined through different mechanism methods such as DPPH and ORAC. For biological testing, the juices were purified through C18 cartridges in order to obtain fractions rich in anthocyanins. The major anthocyanins identified were glycosylated cyanidin derivatives. The antiproliferative activity of the fractions was tested using the MTT assay. The antiproliferative potential of ARF was found to be associated with those bioactive molecules, anthocyanins due to their antioxidant potential. The results obtained indicated that both blueberry and blackcurrants are rich sources of antioxidants including anthocyanins and therefore these fruits are highly recommended for daily consumption to prevent numerous degenerative diseases.

Different ways to insert carotenoids into liposomes affect structure and dynamics of the bilayer differently.

We apply and quantify two techniques to incorporate carotenoids into liposomes: (i). preparation of unilamellar liposomes from mixtures of phospholipids and a carotenoid or cholesterol; (ii). insertion of carotenoids into prepared liposomes. Homogeneous liposomal fractions with a vesicle size diameter of approximately 50 nm were obtained by an extrusion method. The resulting vesicles were subjected to a three-dimensional light scattering cross-correlation measurement in order to evaluate their size distribution. The fluorescent dyes Laurdan, DiI-C(18), C(6)-NBD-PC were used to label the liposomes and to evaluate modulations of ordering, hydrophobicity and permeability to water molecules adjacent to the bilayer in the presence of carotenoids and/or cholesterol. Zeaxanthin incorporation (up to 0.1-1 mol%) attributes to the symmetric and ordered structure of the bilayer, causing both a strong hydrophobicity and a lower water permeability at the polar region of the membrane. The incorporation of lutein has similar effects, but its ordering effect is inferior in the polar region and superior in the non-polar region of the membrane. beta-Carotene, which can be incorporated at lower effective concentrations only, distributes in a more disordered way in the membrane, but locates preferentially in the non-polar region and, compared to lutein and zeaxanthin, it induces a less ordered structure, a higher hydrophobicity and a lower water permeability on the bilayer.

Carotenoid composition of Rosa canina fruits determined by thin-layer chromatography and high-performance liquid chromatography.

The carotenoid composition of fruits of Rosa canina (Rosaceae) was determined comparatively by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) in total extracts and in three different fractions derived from previous separation of the total fruit extract on alumina columns. Both chromatographic analyses revealed as major carotenoids: beta-carotene, lycopene, beta-chryptoxanthin, rubixanthin, zeaxanthin and lutein. The distribution of these compounds was reproducible by TLC and by HPLC. The I-III fractions eluted successively from alumina columns by increasing the polarity of the solvents were analysed also by TLC and HPLC. In all situations, carotenoids were better separated and identified by gradient HPLC systems than by isocratic HPLC or TLC.

Carotenoid incorporation into natural membranes from artificial carriers: liposomes and β-cyclodextrins

Liposomes and beta-cyclodextrin (beta-CD) have been used as carriers for the incorporation of three dietary carotenoids (beta-carotene (BC), lutein (LUT) and canthaxanthin (CTX)) into plasma, mitochondrial, microsomal and nuclear membrane fractions from pig liver cells or the retinal epithelial cell line D407. The uptake dynamics of the carotenoids from the carriers to the organelle membranes and their incorporation yield (IY) was followed by incubations at pH 7.4 for up to 3 h. The mean IYs saturated between 0.1 and 0.9 after 10-30 min of incubation, depending on membrane characteristics (cholesterol to phospholipid ratio) and carotenoid specificity. Mitochondrial membranes (more fluid) favour the incorporation of BC (non-polar), while plasma membranes (more rigid) facilitate the incorporation of lutein, the most polar carotenoid. A high susceptibility of BC to degradation in the microsomal suspension was observed by parallel incubations with/without 2,6-di-t-buthyl-p-cresol (BHT) as antioxidant additive. The beta-CD carrier showed to be more effective for the incorporation of lutein while BC was incorporated equally into natural membranes either from liposomes or from cyclodextrins. The presence of cytosol in the incubation mixture had no significant effects on the carotenoid incorporations.

Effects of solid-state fermentation with two filamentous fungi on the total phenolic contents, flavonoids, antioxidant activities and lipid fractions of plum fruit (Prunus domestica L.) by-products

Evolutions of phenolic contents and antioxidant activities during solid-state fermentation (SSF) of plum pomaces (from the juice industry) and brandy distillery wastes with Aspergillus niger and Rhizopus oligosporus were investigated. The effect of fermentation time on the oil content and major lipid classes in the plum kernels was also studied. Results showed that total phenolic (TP) amounts increased by over 30% for SSF with Rhizopus oligosporus and by >21% for SSF with A. niger. The total flavonoid contents presented similar tendencies to those of the TPs. The free radical scavenging activities of methanolic extracts were also significantly enhanced. The HPLC-MS analysis showed that quercetin-3-glucoside was the major phenolic compound in both fermented plum by-products. The results also demonstrated that SSF not only helped to achieve higher lipid recovery from plum kernels, but also resulted in oils with better quality attributes (high sterol ester and n-3 PUFA-rich polar lipid contents).

Carotenoids in DPPC vesicles: membrane dynamics

Abstract Incorporation of carotenoids into membranes is supposed to change their physical properties with consequences to signal transduction and membrane protein activities. Here the physical parameters membrane fluidity, micropolarity and anisotropy are considered and measured in multilamellar and unilamellar vesicles of dipalmitoylphosphatidyicholine (DPPC) after incorporation of 1, 2.5 and 5 mol% β-carotene, lutein, zeaxanthin, canthaxanthin, or astaxhanthin using 4 mol% pyrene or 1 μM 1,6-diphenyl-1,3,5-hexatriene (DPH) as fluorescent labels. Contrary to other investigations, no significant change in membrane fluidity (as evaluated by the pyrene excimer method) can be found. But a change of micropolarity in the pyrene label environment is obtained from the pyrene monomer fluorescence emission fine structure after incorporation of carotenoids. The membrane anisotropy is enhanced significantly only by those carotenoids which incorporate worst into the membrane. This leads to the hypothesis that carotenoid incorporation into membranes is governed not only by carotenoid polarity hut also by their ability to change membrane anisotropy.

Quantification of carbohydrates in fruit juices using FTIR spectroscopy and multivariate analysis

A combination of Fourier-transform infrared spectroscopy (FTIR) and multivariate statistics was applied as screening tool for the quantitative determination of carbohydrates, such as glucose, fructose and sucrose, in 28 processed commercial fruit juices and 5 genuine juices obtained from squeezed fruits. A number of 13 mixtures of glucose, fructose and sucrose standard solutions were prepared at different concentrations, scanned by attenuated total reflectance (ATR) FTIR spectroscopy and analyzed in the 900 and 1400 cm−1 spectral range. Principal component analysis (PCA) of the standard carbohydrate solutions enabled a better understanding of the main sources of variability affecting the FTIR spectra. Also, PCA enabled the grouping of apple, orange and peach juices. Calibration models for each carbohydrate, using partial least squares (PLS) regression were developed and used for prediction purposes. Cross-validation procedures indicated correlations of 0.88, 0.92 and 0.98 for glucose, fructose and sucrose, respectively, between HPLC measured values and FTIR first derivative spectra estimates. Carbohydrates in the expected concentration ranges were found for most of the pure fruit labelled juices. The samples with 4–50% pure fruit juice content showed discrepancies from average concentration values of authentic juices, mainly a high sucrose concentration can flag sucrose addition to maintain the juice sweetness intensity. The present results confirmed the efficiency of FTIR spectroscopy, in combination with multivariate statistics, as a rapid, reliable and cost-effective tool for routine monitoring of multiple constituents in fruit juices, as quality indicators.