Evaluation of an in vitro method to estimate trace elements bioavailability in edible seaweeds

Raw edible seaweed harvested in the Galician coast (Northwestern Spain), including two red seaweed types (Dulse and Nori), three brown seaweed (Kombu, Wakame and Sea Spaghetti), one green seaweed (Sea Lettuce) and one microalgae (Spirulina platensis) were studied to assess trace elements bioavailability using an in vitro method (simulated gastric and intestinal digestion/dialysis). Similarly, a cooked seaweed sample (canned in brine) consisting of a mixture of two brown seaweed (Sea Spaghetti and Furbelows) and a derived product (Agar-Agar) from the red seaweed Gelidiumm sesquipedale, were also included in the study. The total trace element content as well as the non-dialyzable fractions was carried out after a microwave acid digestion of the seaweed samples by inductively coupled plasma-mass spectrometry (ICP-MS). The dialyzable fraction was determined without any pre-treatment by ICP-MS.PIPES buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut off (MWCO) were used for intestinal digestion. Accuracy of the method was assessed by analyzing a NIES-09 certified reference material (Sargasso seaweed). The accuracy of the in vitro procedure was established by a mass balance study which led to good accuracy of the whole in vitro process, after statistical evaluation (95% confidence interval). The highest dialyzability percentages (100 ± 0.2%) were obtained for Dulse in Mn and V.     

In vitro and in vivo antitumor activity of novel 3D-organotin supramolecular coordination polymers based on CuCN and pyridine bases

Two novel organotin supramolecular coordination polymers (SCP), namely, ³_∞[Me₃SnCu(CN)₂·(EN)₂], 1 and ³_∞[Ph₃SnCu(CN)₂·(3-mpy)₂], 2 are obtained by the reactions of K₃[Cu(CN)₄], ethyl nicotinate (EN) or 3-methylpyridine (3-mpy) and Me₃SnCl or Ph₃SnCl in H₂O/acetonitrile solution at room temperature. 2D-layers are constructed via H-bonds between the parallel 1D-puckered chains which contain nanometer-sized [-CN(R₃Sn)NC-] spacers connecting the tetrahedral (T-4) copper sites. The network structures of 1 and 2 consist of infinite layers connected by interlayer H-bonds forming 3D-framworks. 2 is the first compound in this family containing the Ph₃Sn cation. The electronic absorption spectra of 1 and 2 reveal a broad band at 320 nm due to CT transition while the emission spectra exhibit high energy bands at 400-460 nm due to metal-centered (MC) transitions and low energy bands at 485-530 nm corresponding to MLCT. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine the in vitro antitumor effects of the SCP 1 and 2 on human breast cancer cell line, ZR-75-1. Cell cycle analysis revealed that the SCP 1 and 2 induced apoptosis in ZR-75-1 breast cancer cell line through activating caspase-3. Moreover, in vivo model, the compound SCP 2 suppressed tumor growth developed mammary carcinoma by 52.3%. Taken together, our novel compounds selectivity exhibit specific in vivo and in vitro antitumor effects.     

Use of an in vitro digestion method to evaluate the bioaccessibility of arsenic in edible seaweed by inductively coupled plasma-mass spectrometry

Different types of edible seaweeds (Kombu, Wakame, Nori and Sea Lettuce) harvested in the Galician coast (Northwestern Spain) were analyzed for total arsenic quantification before and after being cooked following manufacturer's instructions. Furthermore, the total arsenic in the bioaccessible fraction obtained after simulating a human digestion by an in vitro process was determined in those raw and cooked seaweeds. The detection of the target element was performed by inductively coupled plasma-mass spectrometry (ICP-MS) which was equipped with a collision cell to avoid polyatomic interferences. Piperazine-NN-bis (2-ethane-sulfonic acid) disodium (PIPES) buffer solution at a pH of 7.0 and dialysis membranes of 10 kDa molecular weight cut-off (MWCO) were used for intestinal digestion. Accuracy of the method was assessed by analyzing a BCR-279 certified reference material. The accuracy of the in vitro procedures was established by a mass balance study for Nori and Sea Lettuce which led to good accuracy of the whole in vitro process, after statistical evaluation (95% confidence interval). Results showed that the effect of cooking seaweed causes the removal of the element into the cooking water. Dialyzability percentages found in raw seaweed samples were comparable to those found in cooked seaweed, except in the case of Sea Lettuce sample for which a lower dialyzability percentage was found when it was cooked.     

In vitro degradation of poly(lactide-co-p-dioxanone)-based shape memory poly(urethane-urea)

The degradation of poly(lactide-co-p-dioxanone)-based shape memory poly(urethane-urea) (SMPU) in vitro was investigated by observing the changes of the pH value of incubation media, weight loss rate, molecular weight and scanning electron microscopy (SEM) during degradation duration of 12 weeks. Moreover, ¹H NMR was used to precisely study the degradation position by calculating the change of characteristic peaks value. The results revealed that the introduction of p-dioxanone (PDO) and -NH-(CO)- and -HN-(CO)-NH- would increase the hydrophilicity of polymer, so the degradation of SMPUs is higher than PDLLA control in the initial time, however, the degradation rate decreased in the anaphase of degradation, which can be attributed to the alkalic -NH₂ from the NH₂ and -NH-(CO), -NH-(CO)-NH-.     

Degradation and calcification in vitro of new bioresorbable terpolymers of lactides with an improved degradation pattern

Experimental implants produced from poly(l-lactide/?-caprolactone/glycolide) 80/10/10% (polymer 1), poly(l-lactide/dl-lactide/glycolide) 80/10/10% (polymer 2) and poly(l-lactide/dl-lactide/glycolide) 80/5/15% (polymer 3) were subjected to in vitro degradation in a buffer solution at 37 °C and pH = 7.4 and calcification in vitro was performed at 37 °C in a simulated body fluid for 4, 8, 12, 16, 24, 28, 32 and 36 weeks. In vitro degradation was performed in static and pseudodynamic modes. Samples from poly(l-lactide) were used as a control. The changes in the materials during the course of degradation were assessed from the measurements of molecular weight, mechanical properties and crystallinity. The changes in the appearance of the materials upon degradation and calcification were observed using a scanning electron microscope with an EDAX attachment. The decrease of molecular weight at 4 weeks was 66% for polymer 1, 56% for polymer 2 and 20% for polymer 3. Samples retained 55% of their bending strength at 4 weeks (polymer 1), 50% at 12 weeks (polymer 2) and 99% at 12 weeks (polymer 3). The bending modulus of polymer 3 remained practically unchanged during the first 12 weeks of degradation. Subsequently it increased by 44% at week 16 and remained unchanged up to 24 weeks and next decreased to 33% of the initial value at the end of the experiment at week 32. The bending modulus of polymer 2 decreased 35% at week 8 and subsequently increased to 44% of the initial value at week 16 and remained at this level until week 20. Next the modulus decreased to 84% of the initial value at week 24. The bending modulus of polymer 1 progressively decreased over the first 12 weeks of degradation to 40% of the initial value. The maximum crystallinity attained by the samples at the end of the experiments was 60% for polymer 1 and 38% for polymers 2 and 3. In the static mode the pH remained constant up to week 8 for polymer 1, week 20 for polymer 2 and week 28 for polymer 3. It decreased to 3.8 at weeks 12, 20 and 36 for polymers 1, 2 and 3, respectively. All the samples underwent calcification from week 16 of the experiments with the Ca/P ratio ranging from 0.92 to 1.20.     

Applications of in vivo and in vitro solid-phase microextraction techniques in plant analysis: A review

As a very popular sample preparation technique, solid-phase microextraction (SPME) coupled with various analytical instrumentation, has been widely used for the determination of trace levels of different plant compounds, such as volatile organic compounds (VOCs) emitted from the different plant organs, and environmental contaminants in plants. In this review, recent applications of in vitro and in vivo SPME in plant analysis are discussed and summarized according to the different organs of plants, including fruits, flowers, leaves, stems, roots and seeds, and the whole plant as well. Future developments and applications of SPME in plant analysis, especially in vivo sampling approaches, are also prospected.     

In vitro bioassays for the study of endocrine-disrupting food additives and contaminants

Endocrine-disrupting chemicals (EDCs) are capable of interfering with normal hormone homeostasis by acting on several targets and through a wide variety of mechanisms. Unwanted exposure to EDCs can lead to a wide spectrum of adverse health effects, especially when exposure is during critical windows of development. Feed and food are considered to be among the main routes of inadvertent exposure to EDCs, so there is an important need for efficient detection of EDCs in these matrices.We describe in vitro bioassays that can complement current analytical chemistry in order to detect unwanted EDCs and describe their action, emphasizing assays that can measure effects on nuclear receptor signaling or hormone production. We outline both validated and unvalidated in vitro assays currently available in the scientific community for detecting and studying the effects of EDCs, and discuss their possible role in the food-safety context. We conclude by identifying gaps in the current battery of in vitro assays available for EDCs and suggest future possibilities for development and validation.     

Synthesis and characterization of glycoconjugate tin(IV) complexes: In vitro DNA binding studies, cytotoxicity, and cell death

New glycosyl derived ligand and its complexes, with SnCl₄·5H₂O (1) and (CH₃)₂SnCl₂(2) were synthesized and characterized by spectroscopic (IR, ¹H, ¹³C, and ¹¹⁹Sn NMR, UV-vis, ESI-MS) and analytical methods. Interaction studies of 1 and 2 with CT DNA were studied by using various biophysical techniques, which showed high binding affinity of 2 with CT DNA. In vitro cytotoxicity of complexes 1 and 2 were evaluated against different human cancer cell lines of different histological origins by employing SRB Assay. The organotin(IV) complex 2 exhibited remarkable activity against DWD (oral cancer) cell lines with GI₅₀ values <10 μg/ml. Complex 2 induced apoptosis of DWD cell line at a very low concentration of 1-4 μg/mL.     

Preparation and in vitro degradation of porous three-dimensional silk fibroin/chitosan scaffold

Degradation behaviors of porous scaffolds play an important role in the engineering process of a new tissue. In this study, three-dimensional porous silk fibroin/chitosan (SFCS) scaffolds were successfully prepared by freeze-drying method. In vitro degradation behaviors of SFCS scaffolds have been systematically investigated up to 8 weeks in phosphate buffer saline (PBS) solution at 37 °C. The following properties of the scaffolds were measured as a function of degradation time: pore morphology, structure, weight loss, and wet/dry weight value. The pH value of the PBS solution during degradation was also detected. SFCS scaffolds maintained its porous structure till 6 weeks of degradation. During the first 2 weeks, the pH value fluctuated in a narrow range from 6.53 to 6.93. SFCS scaffolds degraded much more quickly during the first 2 weeks, and the weight loss reached 19.28 wt% after 8 weeks of degradation. The degradation process affects little SFCS scaffolds' swelling properties.     

Feasibility of using in situ fusion for the determination of Co,Cr and Mn in Portland cement by direct solid sampling graphite furnace atomic absorption spectrometry

In situ fusion on the boat-type graphite platform has been used as a sample pretreatment for the direct determination of Co, Cr and Mn in Portland cement by solid sampling graphite furnace atomic absorption spectrometry (SS-GF AAS). The 3-field Zeeman technique was adopted for background correction to decrease the sensitivity during measurements. This strategy allowed working with up to 200 µg of sample. The in situ fusion was accomplished using 10 µL of a flux mixture 4.0% m/v Na₂CO₃ + 4.0% m/v ZnO + 0.1% m/v Triton® X-100 added over the cement sample and heated at 800 °C for 20 s. The resulting mould was completely dissolved with 10 µL of 0.1% m/v HNO₃. Limits of detection were 0.11 µg g^− 1 for Co, 1.1 µg g^− 1 for Cr and 1.9 µg g^− 1 for Mn. The accuracy of the proposed method has been evaluated by the analysis of certified reference materials. The values found presented no statistically significant differences compared to the certified values (Student's t-test, p < 0.05). In general, the relative standard deviation was lower than 12% (n = 5).     

Synthesis and in vitro antitumor activity of phthalimide polymers containing podophyllotoxin

Polymer-linked PT (podophyllotoxin) conjugates have been designed to improve the therapeutic efficacy of PT. A new PT-conjugate, 3,6-endo-methylene-1,2,3,6-tetrahydrophthalimido-acetamidoglycinylglycine podophyllotoxin ester (ETPA-gly-gly-PT), was synthesized by covalently coupling its hydroxyl group onto the phthalimido monomer through a glycine-glycine-glycine spacer. Its homo- and copolymer with acrylic acid (AA) were prepared by photopolymerization using 2,2-dimethoxy-2-phenylacetophenone (DMP) as a photoinitiator. ETPA-gly-gly-PT and its polymers were characterized by IR and proton NMR spectra. The ETPA-gly-gly-PT content in the copolymer obtained by elemental analysis was 44 wt%. The number-average molecular weights of the polymers determined by gel permeation chromatography were as follows: M_n = 13,500 for poly(ETPA-gly-gly-PT), M_n = 17,000 for poly(ETPA-gly-gly-PT-co-AA). The in vitro antitumor activity of these conjugates and polymers were determined and used to evaluate the potential applications in antitumor drugs. The IC₅₀ values indicated that the synthesized ETPA-gly-gly-PT and its polymers against cancer cells were much better inhibitors than PT.     

Continuous sonoassisted digestion coupled to flow injection minicolumn separation for the determination of total and labile Cu and Fe in fresh waters by flame atomic absorption spectrometry

An automatic on-line system is developed for the trace determination of copper and iron species in fresh waters by flame atomic absorption spectrometry using only 5 and 2?mL of sample, for copper and iron determination, respectively. This system, which includes a home-made minicolumn of commercially available resin containing aminomethylphosphonic acid functional groups (Chelite P), comprises two operational modes. The first, used for the determination of the dissolved labile fraction (free copper and iron ions and their weak complexes) is based on the elution of this fraction from a minicolumn containing the chelating resin loaded in-situ with the sample. The second mode is used for the determination of total trace copper and iron concentrations. This last mode is based on the retention/preconcentration of total metals on the Chelite P resin after on-line sonoassisted digestion of water samples acidified with nitric acid (0.5?mol?L^?1 final concentration) to break down metal organic complexes present in fresh waters as river waters. The figures of merit for copper and iron determination in both fractions are given and the obtained values are discussed. The analytical method was characterized and the limit of detection and limit of quantification for the two metals were 0.5 and 1.6?µg?L^?1 for Cu and 2.3 and 6.1?µg?L^?1 for Fe, respectively. The repeatability, expressed as relative standard deviation, was in the range 1.0–2.1%. The speciation scheme was applied to the analysis of river surface water samples collected in Galicia (Northwest, Spain).     

In vitro methylation by methanol: Proteomic screening and prevalence investigation

It is assumed that much more functional importance for protein activity than expected may be granted by methylation that occurs at the side-chain of aspartate or glutamate residue. In vitro methylation mainly comes from the use of methanol in sample preparation prior to MS analysis. In this study, we first performed the methylation site-directed proteomic screening of bovine serum albumin, ovalbumin and 20S proteasome for gel staining using a meaningfully indicative MS-pattern of peak tag (termed as 4P tag) and manual inspection for mass spectral data. As a result, there were 17 proteolytic peptides with 20 modified sites confirmed to be in vitro methylated. Subsequently, the prevalence investigation was performed, focusing on the reaction kinetic behavior of in vitro methylation. This study provided a simple and robust approach for confirmation of in vitro methylation by methanol, as well as the precautious guide for the use of methanol in proteomic study.     

In-vivo and in-vitro testing to assess the bioaccessibility and the bioavailability of arsenic, selenium and mercury species in food samples

In-vivo and in-vitro gastrointestinal (GI) extractions, also known as oral bioaccessibility and bioavailability, are important approaches to assess chemical risk to humans. We give an overview of in-vivo and in-vitro bioaccessibility and bioavailability assays for testing arsenic, selenium and mercury (As, Se and Hg) species from food samples. We critically evaluate the parameters affecting in-vivo and in-vitro processes. In addition, we consider the effect of cooking food on bioaccessibility and bioavailability, and stability and transformation, of species during in-vivo or in-vitro processes. The bioaccessibility and bioavailability of As, Se and Hg species are affected by the sample matrix, cooking food and the experimental conditions applied (gastric and intestinal pH, incubation temperature and residence time). Regarding species degradation and transformation during in-vitro procedures, good stability has been observed for most As species, except for certain arsenosugars. Important transformations during in-vitro processes have been reported for Se species [e.g., conversion of γ-glu-Se-MeSeCys to Se-MeSeCys, and organic Se species (MeSeCys, SeCys2 and SeMet) degradation to inorganic Se]. Finally, we summarize speciation and detection conditions for As, Se and Hg speciation, and quality control to assure reliable measurements.     

α-Tocopherol-loaded Ca-pectinate microcapsules: Optimization, in vitro release, and bioavailability

We used response surface methodology to optimize microcapsule preparation conditions, including the ratio of pectin:α-tocopherol (TP) (X₁), emulsifier concentration (X₂), and CaCl₂ concentration (X₃) for maximal entrapment efficiency (EE) of TP-loaded Ca-pectinate microcapsules. The values of X₁, X₂, and X₃, optimized for maximal EE were a ratio of 9.7:6.3, and 1.33% and 5.09%, respectively. The experimental results obtained from the optimum formulation agreed with the predicted results, indicating the usefulness of models for EE. TP release from the Ca-pectinate microcapsules prepared according to the optimized conditions was slow and incomplete in simulated gastric fluid, whereas it was relatively rapid and considerably sustained in simulated intestinal fluid. An in vivo release study revealed that physical entrapment of TP within Ca-pectinate microcapsules can be a good technique to demonstrate the sustained release pattern of TP and to improve the bioavailability for TP following oral administration.     

In situ spectroeletrochemistry and cytotoxic activities of natural ubiquinone analogues

Quinones are a group of potent antineoplastic agents. Here we described effective and facile routes to synthesize a series of ubiquinone analogues (UQAs). These unique compounds have been investigated by electrochemistry and in situ UV-vis spectroelectrochemistry to explore their electron-transfer processes and radical properties in aprotic media. The structure-activities relationships of inhibiting cancer cell proliferation of UQAs were examined in murine melanoma B16F10 cells using a 72 h continuous exposure MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Our results revealed that UQAs had improved antiproliferative activity and displayed better inhibitory effects than natural ubiquinone 10. The cytotoxic activities of UQAs were correlated to the semiubiquinone radicals, which were confirmed by in situ electron spin resonance (ESR). In the cytotoxicity test, 6-vinylubiquinone 5 and 6-(4′-fluorophenyl) ubiquinone 7 that possess half maximal inhibitory concentration value (IC₅₀) of 6.1 μM and 6.2 μM. This would make them as valuable candidates for future pharmacological studies.     

Speciation of the bio-available iodine and bromine forms in edible seaweed by high performance liquid chromatography hyphenated with inductively coupled plasma-mass spectrometry

A bioavailability study based on an in vitro dialyzability approach has been applied to assess the bio-available fractions of iodine and bromine species from edible seaweed. Iodide, iodate, 3-iodo-tyrosine (MIT), 3,5-diiodo-tyrosine (DIT), bromide and bromate were separated by anion exchange chromatography under a gradient elution mode (175 mM ammonium nitrate plus 15% (v/v) methanol, pH 3.8, as a mobile phase, and flow rates within the 0.5–1.5 mL min⁻¹ range). Inductively coupled plasma-mass spectrometry (ICP-MS) was used as a selective detector for iodine (¹²⁷I) and bromine (⁷⁹Br). Low dialyzability ratios (within the 2.0–18% range) were found for iodine species; whereas, moderate dialyzability percentages (from 9.0 to 40%) were obtained for bromine species. Iodide and bromide were the major species found in the dialyzates from seaweed, although MIT and bromate were also found in the dialyzates from most of the seaweed samples analysed. However, DIT was only found in dialyzates from Wakame, Kombu, and NIES 09 (Sargasso) certified reference material; whereas, iodate was not found in any dialyzate. Iodine dialyzability was found to be dependent on the protein content (negative correlation), and on the carbohydrate and dietary fibre levels (positive correlation). However, bromine dialyzability was only dependent on the protein amount in seaweed (negative correlation).     

The application of an in vitro gastrointestinal extraction to assess the oral bioaccessibility of polycyclic aromatic hydrocarbons in soils from a former industrial site

The total and bioaccessible concentration of 16 polycyclic aromatic hydrocarbons (PAHs) in soil from a former industrial site was investigated. Typical total concentrations across the sampling sites ranged from 1.5 mg kg⁻¹ for acenaphthylene up to 243 mg kg⁻¹ for fluoranthene. The oral bioaccessibility of PAHs in soil was assessed using an in vitro gastrointestinal extraction (Fed Organic Estimation human Simulation Test, FOREhST method). The oral bioaccessibility data indicated that fluorene, phenanthrene, chrysene, indeno(1,2,3-cd)pyrene and dibenzo(a,h)anthracene had the highest % bioaccessible fraction (based on their upper 75th percentile values being >60%) while the other PAHs had lower % bioaccessible fractions (means ranging between 35 and 59%). Significantly lower bioaccessibilities were determined for naphthalene. With respect to method validation and inter-laboratory comparison, the total and bioaccessible concentrations of benzo(a)anthracene, benzo(b)anthracene, benzo(k)fluoranthene, benzo(a)pyrene, indeno(1,2,3-cd)pyrene and dibenzo(a,h)anthracene was compared to published data derived using the same samples. The total PAH concentrations at the site were compared with generic assessment criteria (GAC) using the residential land use scenario (with plant uptake at 6% soil organic matter). Concentrations of 7 of the PAHs investigated within the soils could lead to an unacceptable risk to human health at this site.     

Fingerprint quality control of Angelica sinensis (Oliv.) Diels by high-performance liquid chromatography coupled with discriminant analysis

High-performance liquid chromatography (HPLC) was employed in the fingerprint analysis of Angelica sinensis (Oliv.) Diels. A chromatographic profile of A. sinensis (Oliv.) Diels from the Dingxi District of Gansu province, China, was established as the characteristic fingerprint. The feasibility and advantages of employing chromatographic fingerprint combined with discriminant analysis were investigated and demonstrated for the evaluation of A. sinensis (Oliv.) Diels for the first time. Our results showed that the chromatographic fingerprint combining with discriminant analysis can efficiently distinguish A. sinensis (Oliv.) Diels from various areas.