Ex vivo and in vitro poultry intestinal models to evaluate antimycotoxins additives

ABSTRACT: In vitro tests are performed to evaluate the efficacy of antimycotoxins additives (AMAs); nevertheless, such assays show a low correlation with in vivo trials, which are also required to determine AMAs’ efficacy. In search of an alternative method, the current study investigated the use of an ex vivo technique. Six AMAs (AMA1 to AMA6) had their ability to reduce intestinal absorption of aflatoxin B1 (AFB1) evaluated. Jejunal explants were obtained from broilers and subjected to two treatments per AMA in Ussing chambers: T1 (control) - 2.8 mg/L AFB1, and T2 - 2.8 mg/L AFB1 + 0.5% AMA. AMAs were also tested in vitro to assess adsorption of AFB1 in artificial intestinal fluid. In the ex vivo studies, AMA1 to AMA6 decreased intestinal absorption of AFB1 by 67.11%, 73.82%, 80.70%, 85.86%, 86.28% and 82.32%, respectively. As for the in vitro results, AMA1 to AMA6 presented an adsorption of 99.72%, 99.37%, 99.67%, 99.53%, 99.04% and 99.15%, respectively. The evaluated ex vivo model proved useful in the assessment of AMAs. No correlation was reported between ex vivo and in vitro findings. Further studies are needed to elucidate the correlation between ex vivo and in vivo results seeking to reduce animal testing.

Synthesis, Stability, and Bioavailability of Nicotinamide Riboside Trioleate Chloride

Nicotinamide riboside chloride (NRCl) is an effective form of vitamin B3. However, it cannot be used in ready-to-drink (RTD) beverages or high-water activity foods because of its intrinsic instability in water. To address this issue, we synthesized nicotinamide riboside trioleate chloride (NRTOCl) as a new hydrophobic nicotinamide riboside (NR) derivative. Contrary to NRCl, NRTOCl is soluble in an oil phase. The results of stability studies showed that NRTOCl was much more stable than NRCl both in water and in oil-in-water emulsions at 25 °C and 35 °C. Finally, we evaluated the bioavailability of NRTOCl by studying its digestibility in simulated intestinal fluid. The results demonstrated that NRTOCl was partially digestible and released NR in the presence of porcine pancreatin in a simulated intestinal fluid. This study showed that NRTOCl has the potential to be used as an NR derivative in ready-to-drink (RTD) beverages and other foods and supplement applications.

An Enhanced Water Solubility and Stability of Anthocyanins in Mulberry Processed with Hot Melt Extrusion

Mulberry fruits are rich sources of anthocyanins that exhibit beneficial biological activity. These anthocyanins become instable in an aqueous media, leading to their low bioavailability. In this study, a colloidal dispersion was produced by processing mulberry samples with hot-melt extrusion. In this process, hydrophilic polymer matrices were used to disperse the compound in an aqueous media. Mulberry samples were processed with hot-melt extrusion and in the presence of an ionization agent and sodium alginate to form mulberry-extrudate solid formulations. The particle size of mulberry-extrudate solid formulations decreased, while the total phenol content, the total anthocyanin content, and solubility increased. Fourier transform infrared spectroscopy (FT-IR) revealed that mulberry-extrudate solid formulations now contained new functional groups, such as –COOH group. We investigated whether mulberry-extrudate solid formulations had a positive impact on the stability of anthocyanins. The non-extrudate mulberry sample and mulberry-extrudate solid formulations were incubated with a simulated gastric fluid system and an intestinal fluid system. The number of released anthocyanins was determined with HPLC. We found that anthocyanins were released rapidly from non-extrudate mulberry extract. Mulberry-extrudate solid formulations contained a large number of available anthocyanins even after being incubated for 180 min in the intestinal fluid system. Thus, hot-melt extrusion enhanced water solubility and stability of anthocyanins with the prolonged release.

Continuous On-Line Leaching To Evaluate The Effect Of Toasting On Bioaccessibility, And Pb Source Apportionment Of Alternative Breads

Laser-induced with the increasing popularity of alternative breads, investigating their health benefits and risks is vitally important. The bioaccessibility of potentially toxic elements in these breads could indicate a potential risk to consumers. The continuous online leaching method (COLM) involves the sequential leaching of a mini-column of food by artificial saliva, gastric juice, and intestinal fluid with real time monitoring of elements being released. Because the dissolution equilibrium is shifted to the right from continuous exposure to fresh reagent, it assesses bioaccessibility in three gastrointestinal matrices in less than 30 min and includes valuable kinetic extraction information. In this study, a gluten-free (GF) and a rye bread were analyzed for As, Cd, and Pb before and after toasting. Toasting lowered all PTE concentrations by a factor of 1.5 (As in rye bread) to 2.5 (Cd in rye bread). Most of the PTEs present were bioaccessible whether the bread was toasted or not. In the case of As, a significant portion is in the form of As(III) and As(V). COLM analysis allowed for Pb sourcing, revealing two potential sources of Pb being released separately in gastric juice from rye bread based on their significantly different 206Pb/207Pb and 208Pb/206Pb isotope ratios. Comparison with Pb ratios reported in previous literature revealed that some of the gastric-mobile Pb in rye bread came from the Pb historically added to gasoline in North America. This source completely vanished upon toasting rye bread.

A conserved β-bulge glycine residue facilitates folding and increases stability of the mouse α-defensin cryptdin-4

Defensins are key components of both innate and adaptive immune responses to pathogens. Cryptdins are mouse alpha-defensins that are secreted from Paneth cells in the small intestine and have disulfide-stabilised structures and antibacterial activities against both Gram-positive and Gram-negative bacteria. The folding and three-dimensional structures of alpha-defensins are thought to depend on a conserved glycine residue that forms a β-bulge. Here we investigated the role of this conserved glycine at position 19 of cryptdin-4 (Crp4) in terms of the folding, structure and stability. A Crp4 variant with D-Ala at position 19 folded efficiently, was stabilised by a large number of hydrogen bonds, and resisted proteolysis in simulated intestinal fluid. Although a variant with L-Ala at position 19 was able to adopt the correct fold, it showed less efficient folding and was degraded more rapidly than the D-Ala variant. These results demonstrate the key role that glycine residues can have in folding of bioactive peptides and can provide insights to guide design of stable antimicrobial peptides that fold efficiently.

In vitro Cholesterol Assimilation by Bifidobacterium animalis subsp. lactis (BPL1) Probiotic Bacteria under Intestinal Conditions.

Background: Hypercholesterolemia is one of the principal causes of the development of cardiovascular diseases. Recently, probiotics consumption has been also proposed as a non-pharmacological intervention to control cholesterol concentrations. Objective: To evaluate in vitro assimilation of cholesterol by Bifidobacterium animalis subsp. lactis (BPL1) under simulated intestinal environment in anaerobic conditions and to review and discuss potential physiological mechanisms in this context. Methods: Bacterial viability and cholesterol assimilation was evaluated in both standard MRS and stimulated intestinal fluid (SIF) medium under anaerobic conditions, and in presence or absence of cholesterol. For assimilation assays, cholesterol concentrations in the different suspensions, containing the probiotic or not, was determined by chromatography coupled to mass spectrometry. Results: Results showed that the growth of B. lactis BPL1 under intestinal conditions is favored when cholesterol is present in the culture medium. In addition, a cholesterol assimilation of up to 44.4% under intestinal and anaerobic conditions was observed. Conclusion: Taking into account the revised literature and the experimental results herein presented, administration of functional foodstuffs together with probiotic bacteria such as B. lactis BPL1 could be a potential effective option to decrease hypercholesterolemia, thus preventing the development of cardiovascular diseases. Nevertheless, further studies on mechanisms of effectiveness in animals and clinical trials are still needed.

Box-Behnken Design Optimization of Etoposide Loaded Nanoemulsion: Formulation Development, Cellular Uptake Analysis and Pharmacokinetic Evaluation

Etoposide is widely used in the management of different solid tumors as well as leukemias but low aqueous solubility and poor intestinal permeability limit its oral efficacy. The present investigation aimed to enhance the oral bioavailability of etoposide through formation of nanoemulsion employing Box-Behnken design for formulation optimization. The effect of nanoemulsion composition (concentration of labrasol and solutol HS 15) and process parameters (sonication time) on globule size and polydispersity index were investigated. The optimized formulation was found to be with globule size <200 nm and PDI of 0.129. The zeta potential value −35.8 mV for nanoemulsion indicated the formation of a stable colloidal system. The in-vitro release of etoposide from optimized nanoemulsion was conducted with the help of USP dissolution apparatus type-II in 250 ml of gastric fluid of pH = 1.2 and intestinal fluid of pH 6.8 at 37±0.5 °C using dialysis bag. High drug release was achieved in case of nanoemulsion (47.127±0.82%) as compared to the marketed capsule (25.877±1.33%) and drug suspension (21.374±1.69%) in the simulated intestinal fluid of pH 6.8 after 4 h. The developed delivery system exhibited pH-independent dissolution profile of the loaded etoposide. Confocal laser scanning microscopy (CLSM) study of developed nanoemulsion system was done on intestinal tissue of Wistar rats and optical cross sections revealed deeper penetration of Rhodamine B compared to the dye solution. A comparative in-vivo bioavailability profile of developed formulation, marketed product, and API suspension was also investigated after oral administration in Wistar rats. The relative bioavailability of etoposide loaded nanoemulsion system was 2.5 times higher than the drug suspension and 1.7 times higher than the marketed capsule (Posid®). The findings of the current investigation proved that the developed nanoemulsion system is helpful to improve the bioavailability of P-gp substrate drugs like etoposide that have low oral absorption.