Bioavailability Assessment of an Iron Formulation Using Differentiated Human Intestinal Caco-2 Cells.

In recent years, there has been growing interest in exploring alternative and innovative delivery systems to improve the efficacy of iron supplements, satisfying iron needs and lowering side effects. To address this issue, this study aimed at demonstrating the advantages of Ferro Supremo formulation (composed of encapsulated iron, vitamins, and micronutrients), in terms of capacity to improve iron intestinal absorption, in comparison with standard FeSO4. Hence, differentiated Caco-2 cells have been used for assessing the in vitro bioavailability and safety of FS and FeSO4. MTT experiments demonstrated that both FS and FeSO4 are not able to impair the viability of Caco-2 cells. Furthermore, the quantitative and qualitative analysis, conducted by atomic absorption spectrometry and fluorescence determinations, revealed that FS can enter, accumulate in the cytoplasm, and be transported by intestinal cells four times more efficiently than FeSO4. Our findings indicate that this formulation can be considered a valuable and efficiently good choice as food supplements for improving iron deficiency.

Assessment of Electrospun Poly(ε-caprolactone) and Poly(lactic acid) Fiber Scaffolds to Generate 3D In Vitro Models of Colorectal Adenocarcinoma: A Preliminary Study.

Three-dimensional scaffold-based culture has been increasingly gaining influence in oncology as a therapeutic strategy for tumors with a high relapse percentage. This study aims to evaluate electrospun poly(ε-caprolactone) (PCL) and poly(lactic acid) (PLA) scaffolds to create a 3D model of colorectal adenocarcinoma. Specifically, the physico-mechanical and morphological properties of PCL and PLA electrospun fiber meshes collected at different drum velocities, i.e., 500 rpm, 1000 rpm and 2500 rpm, were assessed. Fiber size, mesh porosity, pore size distribution, water contact angle and tensile mechanical properties were investigated. Caco-2 cells were cultured on the produced PCL and PLA scaffolds for 7 days, demonstrating good cell viability and metabolic activity in all the scaffolds. A cross-analysis of the cell-scaffold interactions with morphological, mechanical and surface characterizations of the different electrospun fiber meshes was carried out, showing an opposite trend of cell metabolic activity in PLA and PCL scaffolds regardless of the fiber alignment, which increased in PLA and decreased in PCL. The best samples for Caco-2 cell culture were PCL500 (randomly oriented fibers) and PLA2500 (aligned fibers). Caco-2 cells had the highest metabolic activity in these scaffolds, with Young's moduli in the range of 8.6-21.9 MPa. PCL500 showed Young's modulus and strain at break close to those of the large intestine. Advancements in 3D in vitro models of colorectal adenocarcinoma could move forward the development of therapies for this cancer.

Antioxidant Effect Assessment and Trans Epithelial Analysis of New Hempseed Protein Hydrolysates.

Hempseed (Cannabis sativa) is one of the most promising sources of plant proteins. It contains approximately 24% (w/w) protein, and edestin accounts for approximately 60-80% (w/w) of its total proteins. In a framework of research aimed at fostering the proteins recovered from the press cake by-products generated after the extraction of hempseed oil, two hempseed protein hydrolysates (HH1 and HH2) were produced at an industrial level using a mixture of different enzymes from Aspergillus niger, Aspergillus oryzae, and Bacillus licheniformis for different times (5 h and 18 h). Using a combination of different direct antioxidant tests (DPPH, TEAC, FRAP, and ORAC assays, respectively), it has been demonstrated that HHs exert potent, direct antioxidant activity. A crucial feature of bioactive peptides is their intestinal bioavailability; for this reason, in order to solve this peculiar issue, the ability of HH peptides to be transported by differentiated human intestinal Caco-2 cells has been evaluated. Notably, by using mass spectrometry analysis (HPLC Chip ESI-MS/MS), the stable peptides transported by intestinal cells have been identified, and dedicated experiments confirmed that the trans-epithelial transported HH peptide mixtures retain their antioxidant activity, suggesting that these hempseed hydrolysates may be considered sustainable antioxidant ingredients to be exploited for further application, i.e., nutraceutical and/or food industries.

Ethyl Protocatechuate Encapsulation in Solid Lipid Nanoparticles: Assessment of Pharmacotechnical Parameters and Preliminary In Vitro Evaluation for Colorectal Cancer Treatment.

Colorectal cancer is one of the most diffused tumoral diseases. Since most medicaments employed for its treatment are debilitating, the use of naturally derived products, which can be effective against the mutated cells and, in addition, can reduce most inflammatory-related effects, could be extremely beneficial for the continued treatment of this disease. In this research, ethyl protocatechuate (PCAEE), a protocatechuic acid prodrug, was encapsulated in solid lipid nanoparticles (SLN) (prepared without and with Tween 80), which were characterized in terms of size, polydispersity index (PDI), zeta potential and thermotropic behavior. Encapsulation efficiency, release profile and interaction with a model of biomembrane were also assessed. The nanoparticles were tested in vitro on both healthy cells and on a model of tumoral cells. SLN prepared with Tween 80 was promising in terms of physicochemical properties (z-average of 190 nm, PDI 0.150 and zeta potential around -20 mV) and encapsulation efficiency (56%); they showed a desirable release profile, demonstrated an ability to penetrate and release the encapsulated PCAEE into a biomembrane model and were nontoxic on healthy cells. In addition, they caused a greater dose-dependent decrease in the viability of CaCo-2 cells than PCAEE alone. In conclusion, the formulation could be proposed for further studies to assess its suitability for the treatment of colorectal cancer.

Assessment of the Permeability of 3,4-Methylenedioxypyrovalerone (MDPV) across the Caco-2 Monolayer for Estimation of Intestinal Absorption and Enantioselectivity.

3,4-Methylenedioxypyrovalerone (MDPV) is a widely studied synthetic cathinone heterocycle mainly concerning its psychoactive effects. It is a chiral molecule and one of the most abused new psychoactive substances worldwide. Enantioselectivity studies for MDPV are still scarce and the extent to which it crosses the intestinal membrane is still unknown. Herein, an in vitro permeability study was performed to evaluate the passage of the enantiomers of MDPV across the Caco-2 monolayer. To detect and quantify MDPV, a UHPLC-UV method was developed and validated. Acceptable values within the recommended limits were obtained for all evaluated parameters (specificity, linearity, accuracy, limit of detection (LOD), limit of quantification (LOQ) and precision). The enantiomers of MDPV were found to be highly permeable across the Caco-2 monolayer, which can indicate a high intestinal permeability. Enantioselectivity was observed for the Papp values in the basolateral (BL) to apical (AP) direction. Furthermore, efflux ratios are indicative of efflux through a facilitated diffusion mechanism. To the best of our knowledge, determination of the permeability of MDPV across the intestinal epithelial cell monolayer is presented here for the first time.

Toxicological assessment of a bioactive extract from Triplaris gardneriana Wedd. seeds using alternative models.

The Triplaris gardneriana Wedd. seeds extract has great therapeutic potential due to numerous biological activities such as antioxidant, antibacterial and anti-inflammatory, which are associated with phenolic content. Although this herbal preparation has shown many benefits, recently their toxicity profile has begun to be explored. In this present study, the toxic effects of T. gardneriana seeds ethanolic extract (EETg) on biological systems of different taxonomical groups and levels of complexity (from cell culture to lower vertebrates) were assessed, through a variety of viability and toxicological assays. It was found that EETg did not impair the Saccharomyces cerevisiae growth at the highest tested concentration (200 µg/mL), and no toxicant evidence was observed in Aedes aegypti larvae or in Drosophila melanogaster adult stage. Contrarily, the extract reduced the viability of undifferentiated Caco-2 cells (250 µg/mL, 40% of viable cells), but did not affect differentiated ones. The embryotoxicity in Danio rerio model showed a LC50 of 7.41 mg/L (95% confidence interval, 4.78 - 11.49 mg/L). EETg did not show signs of toxicity in the majority of the models used, but lethality and malformations in zebrafish embryos occurred. Further analyses are needed to better understand the selective toxicity mechanism of EETg on zebrafish, as well as whether the toxic effects happen in higher vertebrates.

Evaluation of the increase of the thymoquinone permeability formulated in polymeric micelles: In vitro test and in vivo toxicity assessment in Zebrafish embryos.

Thymoquinone (TQ) is a natural compound present in the essential oil and in the fixed oil of Nigella sativa L. Like many natural substances, it is characterized by poor aqueous solubility and low stability which limit its bioavailability. Soluplus®-Solutol® HS15 polymeric micelles (TQ-MP) were developed to increase the permeability of TQ with particular attention to overcoming intestinal barrier and the blood brain barrier, for possible oral and parenteral administration. The optimized micelles have dimensions < 100 nm and PdI < 0.2 indicating that the formulation was homogeneous as confirmed also by TEM experiments. EE% was 92.4 ± 0.3%. Stability studies showed a stable formulation following subsequent dilutions and in the gastric-intestinal media. In vitro studies have revealed that the carrier enhances the permeability of TQ in the intestine and in the blood-brain barrier using Parallel Artificial Membrane Permeability Assay (PAMPA) assay and cellular tests with Caco-2 cells and hCMEC/D3 monolayer cells. Up-take study, cell viability and cytotoxicity studies were also conducted. Fluorescent micelles (FITC-MP), were also optimized to perform in vitro up-take study in Caco-2 cells and to study their toxicity in Zebrafish model. The toxicity was evaluated on three lines of Zebrafish: wild type, transgenic line Tg(Myl7:EGFP) in which cardiomyocytes are marked with green fluorescence protein and Tg(flk1-GFP) line which expresses GFP under the control of the vascular endothelial growth factor receptor 2 (vegfr2) promoter.

Enzymatic synthesis of hydrophilic phytosterol polyol esters and assessment of their bioaccessibility and uptake using an in vitro digestion/Caco-2 cell model.

A novel enzyme-catalyzed method was developed for the synthesis of phytosterol polyol esters from ß-sitosterol and polyols (sorbitol, mannitol and xylitol) by two-step transesterification using divinyl adipate (DVA) as a link. A high conversion (exceeding 94%) of ß-sitosterol with a vinyl group was achieved, in the presence of Candida rugosa lipase (CRL), at low temperature (35 °C) within 30 min. Subsequently, the maximum conversion of phytosterol polyol esters (>94%) was obtained using alkaline protease from Bacillus subtilis at 65 °C. Phytosterol polyol esters had enhanced thermal stability (up to an above 355 °C) and excellent water solubility (4.6-7.9 mM at 35 °C). Moreover, obvious increases in the bioaccessibility (41.5-63.6%) and intestinal uptake (5.2-6.5%) were observed using a simulated gastrointestinal digestion/Caco-2 cell model. These results highlighted the key role of hydrophilic structural modifications on physicochemical properties and absorption of phytosterols.

Trans-Epithelial Transport, Metabolism, and Biological Activity Assessment of the Multi-Target Lupin Peptide LILPKHSDAD (P5) and Its Metabolite LPKHSDAD (P5-Met).

P5 (LILPKHSDAD) is a hypocholesterolemic peptide from lupin protein with a multi-target activity, since it inhibits both 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoAR) and proprotein convertase subtilisin/kexin type-9 (PCSK9). This work shows that, during epithelial transport experiments, the metabolic transformation mediated by intestinal peptidases produces two main detected peptides, ILPKHSDAD (P5-frag) and LPKHSDAD (P5-met), and that both P5 and P5-met are linearly absorbed by differentiated human intestinal Caco-2 cells. Extensive comparative structural, biochemical, and cellular characterizations of P5-met and the parent peptide P5 demonstrate that both peptides have unique characteristics and share the same mechanisms of action. In fact, they exert an intrinsically multi-target behavior being able to regulate cholesterol metabolism by modulating different pathways. The results of this study also highlight the dynamic nature of bioactive peptides that may be modulated by the biological systems they get in contact with.

Oral Bioavailability Enhancement of Melanin Concentrating Hormone, Development and In Vitro Pharmaceutical Assessment of Novel Delivery Systems.

The rapid progress in biotechnology over the past few decades has accelerated the large-scale production of therapeutic peptides and proteins, making them available in medical practice. However, injections are the most common method of administration; these procedures might lead to inconvenience. Non-invasive medications, such as oral administration of bio-compounds, can reduce or eliminate pain and increase safety. The aim of this project was to develop and characterize novel melanin concentrating hormone (MCH) formulations for oral administration. As a drug delivery system, penetration enhancer combined alginate beads were formulated and characterized. The combination of alginate carriers with amphiphilic surfactants has not been described yet. Due to biosafety having high priority in the case of novel pharmaceutical formulations, the biocompatibility of selected auxiliary materials and their combinations was evaluated using different in vitro methods. Excipients were selected according to the performed toxicity measurements. Besides the cell viability tests, physical properties and complex bioavailability assessments were performed as well. Our results suggest that alginate beads are able to protect melanin concentrating hormones. It has been also demonstrated that penetration enhancer combined alginate beads might play a key role in bioavailability improvement. These formulations were found to be promising tools for oral peptide delivery. Applied excipients and the performed delivery systems are safe and highly tolerable; thus, they can improve patients' experience and promote adherence.

Assessment of intestinal absorption/metabolism of 3-chloro-1,2-propanediol (3-MCPD) and three 3-MCPD monoesters by Caco-2 cells.

3-chloro-1,2-propanediol (3-MCPD) and 3-MCPD esters are contaminants present in a variety of processed foods, including infant formulas. Toxicological data are unavailable in humans, but rodent studies have demonstrated renal and testicular toxicity from 3-MCPD and 3-MCPD esters. There is evidence that 3-MCPD esters are hydrolyzed in the digestive system, releasing 3-MCPD that would be absorbed and induce damage. We assessed absorption and metabolism of 3-MCPD and three 3-MCPD monoesters, 1-oleoyl (1-Ol), 1-linoleoyl (1-Li) and 1-palmitoyl (1-Pa) commonly found in U.S. infant formula using differentiated Caco-2 cells. After 1-hour incubation, all three monoesters released free 3-MCPD and free fatty acids (FFA) into Caco-2 cell supernatants. Free 3-MCPD had a high apparent permeability (Papp = 30.36 ± 1.31 cm/s × 10-6) suggesting that it is freely diffusible and highly absorbed by intestinal epithelium. 1-Li released 3-4-fold more 3-MCPD than 1-Ol and 1-Pa over 1 h, suggesting that this variable release rates might contribute to the overall in vivo exposure to 3-MCPD. None of the monoesters or FFA were detected in basolateral supernatants, suggesting that these compounds do not cross the intestinal wall without further transformation. In summary, this study provides relevant data to advance knowledge of in vivo intestinal absorption and metabolism of 3-MCPD monoesters.

Lemon Juice, Sesame Paste, and Autoclaving Influence Iron Bioavailability of Hummus: Assessment by an In Vitro Digestion/Caco-2 Cell Model.

Hummus, an iron-containing plant-based dish mainly made from chickpea purée, tahini, lemon juice and garlic, could be a valuable source of iron when bioavailable. Since the processing and formulation of food influence iron bioavailability, the present study investigated for the first time, their effects on hummus. Firstly, iron bioaccessibility was assessed on eight samples (prepared according to the screening Hadamard matrix) by in vitro digestion preceding iron dialysis. Then, iron bioavailability of four selected samples was estimated by the in vitro digestion/Caco-2 cell model. Total and dialyzable iron were determined by the atomic absorption spectrometry and ferritin formation was determined using an ELISA kit. Only autoclaving, among other processes, had a significant effect on iron bioaccessibility (+9.5, p < 0.05). Lemon juice had the highest positive effect (+15.9, p < 0.05). Consequently, the effect of its acidic components were investigated based on a full factorial 23 experimental design; no significant difference was detected. Garlic's effect was not significant, but tahini's effect was negative (-8.9, p < 0.05). Despite the latter, hummus had a higher iron bioavailability than only cooked chickpeas (30.4 and 7.23 ng ferritin/mg protein, respectively). In conclusion, hummus may be a promising source of iron; further in vivo studies are needed for confirmation.

Bioavailability Assessment of Copper, Iron, Manganese, Molybdenum, Selenium, and Zinc from Selenium-Enriched Lettuce.

Cu, Fe, Mn, Mo, Selenium (Se), and Zn bioavailability from selenate- and selenite-enriched lettuce plants was studied by in vitro gastrointestinal digestion followed by an assay with Caco-2 cells. The plants were cultivated in the absence and presence of two concentrations (25 and 40 µmol/L of Se). After 28 days of cultivation, the plants were harvested, dried, and evaluated regarding the total concentration, bioaccessibility, and bioavailability of the analytes. The results showed that biofortification with selenate leads to higher Se absorption by the plant than biofortification with selenite. For the other nutrients, Mo showed high accumulation in the plants of selenate assays, and the presence of any Se species led to a reduction of the plant uptake of Cu and Fe. The accumulation of Zn and Mn was not strongly influenced by the presence of any Se species. The bioaccessibility values were approximately 71%, 10%, 52%, 84%, 71%, and 86% for Cu, Fe, Mn, Mo, Se, and Zn, respectively, and the contribution of the biofortified lettuce to the ingestion of these minerals is very small (except for Se and Mo). Due to the low concentrations of elements from digested plants, it was not possible to estimate the bioavailability for some elements, and for Mo and Zn, the values are below 6.9% and 3.4% of the total concentration, respectively. For Se, the bioavailability was greater for selenite-enriched than selenate-enriched plants (22% and 6.0%, respectively), because selenite is biotransformed by the plant to organic forms that are better assimilated by the cells.

Exploring the mucoadhesive behavior of sucrose acetate isobutyrate: a novel excipient for oral delivery of biopharmaceuticals.

Oral drug delivery is an attractive noninvasive alternative to injectables. However, oral delivery of biopharmaceuticals is highly challenging due to low stability during transit in the gastrointestinal tract (GIT), resulting in low systemic bioavailability. Thus, novel formulation strategies are essential to overcome this challenge. An interesting approach is increasing retention in the GIT by utilizing mucoadhesive biomaterials as excipients. Here, we explored the potential of the GRAS excipient sucrose acetate isobutyrate (SAIB) to obtain mucoadhesion in vivo. Mucoadhesive properties of a 90% SAIB/10% EtOH (w/w) drug delivery system (DDS) were assessed using a biosimilar mucus model and evaluation of rheological behavior after immersion in biosimilar intestinal fluid. To ease readability of this manuscript, we will refer to this as SAIB DDS. The effect of SAIB DDS on cell viability and epithelial membrane integrity was tested in vitro prior to in vivo studies that were conducted using SPECT/CT imaging in rats. When combining SAIB DDS with biosimilar mucus, increased viscosity was observed due to secondary interactions between biosimilar mucus and sucrose ester predicting considerable mucoadhesion. Mucoadhesion was confirmed in vivo, as radiolabeled insulin entrapped in SAIB DDS, remained in the small intestine for up to 22 h after administration. Moreover, the integrity of the system was investigated using the dynamic gastric model under conditions simulating the chemical composition of stomach fluid and physical shear stress in the antrum under fasted conditions. In conclusion, SAIB is an interesting and safe biomaterial to promote high mucoadhesion in the GIT after oral administration.

In vitro assessment of CeO2 nanoparticles effects on intestinal microvilli morphology.

Some nanoparticles (NPs) have been shown to disrupt intestinal microvilli morphology in vitro, an alteration that could potentially affect nutrient absorption and barrier properties. This study aimed at evaluating the potential effect of CeO2 NPs (4-8 nm, citrate stabilized) on Caco-2 microvilli morphology. In addition to the standard Caco-2 cell clone, the C2BBe1 clone was used, as it is considered to develop a more homogeneous cellular morphology. Semiautomated microvilli density quantification and a new cell scoring approach were used to evaluate scanning electron microscopy (SEM) images. The quantification method made use of the whole micrograph surface, avoiding the need to choose subareas for analysis, and increasing the representativeness of the results when compared to previous studies. The main advantage of the scoring system is that it informs on the intercellular variability within a cell preparation. Benzalkonium was used as a positive control inducing toxicity and morphological alterations on microvilli. After three-week differentiation, Caco-2 cells were exposed to 100 µg/mL of CeO2 NPs for 24 h. The integrity of the membrane was evaluated by transepithelial electrical resistance (TEER) and thereafter processed for its observation by SEM. Results showed that both the standard Caco-2 clone and the C2BBe1 clone present notable morphological heterogeneity. The two evaluation approaches were able to identify morphological effects caused by the positive control, but did not detect statistically significant morphological alterations after exposure to CeO2 NPs.

Interaction between Different Pharmaceutical Excipients in Liquid Dosage Forms-Assessment of Cytotoxicity and Antimicrobial Activity.

Nowadays, the safety of parabens as pharmaceutical preservatives is debated. Recent studies investigated their interference with the oestrogen receptors, nevertheless their carcinogenic activity was also proved. That was the reason why the re-evaluation of the biocompatibility and antimicrobial activity of parabens is required using modern investigation methods. We aimed to test the cytotoxic, antifungal and antibacterial effect of parabens on Caco-2 cells, C. albicans, C. parapsilosis, C. glabrata, E. coli, P. aeruginosa and S. aureus. Two complex systems (glycerol-Polysorbate 20; ethanol-Capryol PGMC™) were formulated to study-with the MTT-assay and microdilution method, respectively-how other excipients may modify the biocompatibility and antimicrobial effect of parabens. In the case of cytotoxicity, the toxicity of these two systems was highly influenced by co-solvents and surfactants. The fungi and bacteria had significantly different resistance in the formulations and in some cases the excipients could highly modify the effectiveness of parabens both in an agonistic and in a counteractive way. These results indicate that with appropriate selection, non-preservative excipients can contribute to the antimicrobial safety of the products, thus they may decrease the required preservative concentration.

Bioaccessibility, cellular uptake and transport of luteins and assessment of their antioxidant activities.

A rapid method for producing 9Z- and 13'Z-isomers from all-E-lutein was developed using I-TiO2 as catalyst. In a simulated in vitro gastrointestinal digestion model, both trans-cis isomerization of all-E-lutein and cis-trans isomerization of Z-luteins occurred during the intestinal phase. The bioaccessibility of all isomers was between 14 and 23%, and it was higher for Z-luteins. In a Caco-2 cell monolayer model, all isomers were relatively stable during cellular uptake and transport across the membrane as no significant isomerization and degradation was detected, but all-E-lutein exhibited significantly higher cellular uptake and transport efficiencies. These results suggest that Z-luteins found in human plasma may likely be formed before intestinal absorption. 13'Z-Lutein also exhibited highest antioxidant activity in FRAP, DPPH and ORAC-L assays, but no significant difference in cell-based antioxidant assay compared with other isomers. Future studies on the different antioxidant activities of cis isomers of lutein in vivo will provide further explanation.

Assessment of Passive Intestinal Permeability Using an Artificial Membrane Insert System.

Despite reasonable predictive power of current cell-based and cell-free absorption models for the assessment of intestinal drug permeability, high costs and lengthy preparation steps hamper their use. The use of a simple artificial membrane (without any lipids present) as intestinal barrier substitute would overcome these hurdles. In the present study, a set of 14 poorly water-soluble drugs, dissolved in 2 different media (fasted state simulated/human intestinal fluids [FaSSIF/FaHIF]), were applied to the donor compartment of an artificial membrane insert system (AMI-system) containing a regenerated cellulose membrane. Furthermore, to investigate the predictive capacity of the AMI-system as substitute for the well-established Caco-2 system to assess intestinal permeability, the same set of 14 drugs dissolved in FaHIF were applied to the donor compartment of a Caco-2 system. For 14 drugs, covering a broad range of physicochemical parameters, a reasonable correlation between both absorption systems was observed, characterized by a Pearson correlation coefficient r of 0.95 (FaHIF). Using the AMI-system, an excellent predictive capacity of FaSSIF as surrogate medium for FaHIF was demonstrated (r = 0.96). Based on the acquired data, the AMI-system appears to be a time- and cost-effective tool for the early-stage estimation of passive intestinal permeability for poorly water-soluble drugs.

Advantages and limitations of in vitro and in vivo methods of iron and zinc bioavailability evaluation in the assessment of biofortification program effectiveness.

Biofortification aims to improve the micronutrient concentration of staple food crops through the best practices of breeding and modern biotechnology. However, increased zinc and iron concentrations in food crops may not always translate into proportional increases in absorbed zinc (Zn) and iron (Fe). Therefore, assessing iron and zinc bioavailability in biofortified crops is imperative to evaluate the efficacy of breeding programs. This review aimed to investigate the advantages and limitations of in vitro and in vivo methods of iron and zinc bioavailability evaluation in the assessment of biofortification program effectiveness. In vitro, animal and isotopic human studies have shown high iron and zinc bioavailability in biofortified staple food crops. Human studies provide direct knowledge regarding the effectiveness of biofortification, however, human studies are time consuming and are more expensive than in vitro and animal studies. Moreover, in vitro studies may be a useful preliminary screening method to identify promising plant cultivars, however, these studies cannot provide data that are directly applicable to humans. None of these methods provides complete information regarding mineral bioavailability, thus, a combination of these methods should be the most appropriate strategy to investigate the effectiveness of zinc and iron biofortification programs.

Self-Nanoemulsifying Drug Delivery Systems Containing Plantago lanceolata-An Assessment of Their Antioxidant and Antiinflammatory Effects.

The most important components of Plantago lanceolata L. leaves are catalpol, aucubin, and acteoside (=verbascoside). These bioactive compounds possess different pharmacological effects: anti-inflammatory, antioxidant, antineoplastic, and hepatoprotective. The aim of this study was to protect Plantago lanceolata extract from hydrolysis and to improve its antioxidant effect using self-nano-emulsifying drug delivery systems (SNEDDS). Eight SNEDDS compositions were prepared, and their physical properties, in vitro cytotoxicity, and in vivo AST/ALT values were investigated. MTT cell viability assay was performed on Caco-2 cells. The well-diluted samples (200 to 1000-fold dilutions) proved to be non-cytotoxic. The acute administration of PL-SNEDDS compositions resulted in minor changes in hepatic markers (AST, ALT), except for compositions 4 and 8 due to their high Transcutol contents (80%). The non-toxic compositions showed a significant increase in free radical scavenger activity measured by the DPPH test compared to the blank SNEDDS. An indirect dissolution test was performed, based on the result of the DPPH antioxidant assay; the dissolution profiles of Plantago lancolata extract were statistically different from each SNEDDS. The anti-inflammatory effect of PL-SNEDDS compositions was confirmed by the ear inflammation test. For the complete examination period, all compositions decreased ear edema as compared to the positive (untreated) control. It can be concluded that PL-SNEDDS compositions could be used to deliver active natural compounds in a stable, efficient, and safe manner.