Liposomal Encapsulation of Citicoline for Ocular Drug Delivery.

Glaucoma represents a group of neurodegenerative diseases characterized by optic nerve damage and the slowly progressive death of retinal ganglion cells. Glaucoma is considered the second leading cause of irreversible blindness worldwide. Pharmaceutical treatment of glaucoma is critical because of the properties of the ocular barrier that limit the penetration of drugs, resulting in lower systemic bioavailability. This behavior causes the need of frequent drug administration, which leads to deposition of concentrated solutions on the eye, causing toxic effects and cellular damage to the eye. To overcome these drawbacks, novel drug-delivery systems, such as liposomes, can play an important role in improving the therapeutic efficacy of antiglaucomatous drugs. In this work, liposomes were synthesized to improve various aspects, such as ocular barrier penetration, bioavailability, sustained release of the drug, targeting of the tissue, and reduction in intraocular pressure. Citicoline (CDP-choline; cytidine 5'-diphosphocholine) is an important intermediate in the biosynthesis of cell membrane phospholipids, with neuroprotective and neuroenhancement properties, and it was used in the treatment on retinal function and neural conduction in the visual pathways of glaucoma patients. In this study, citicoline was loaded into the 1,2-dioleoyl-sn-glycerol-3-phosphocholine and cholesterol liposomal carrier to enhance its therapeutic effect. The citicoline encapsulation efficiency, drug release, and size analysis of the different liposome systems were investigated using dynamic light scattering, nuclear magnetic resonance, infrared spectroscopy, and ToF-SIMS experiments.

Green Hydrogels Loaded with Extracts from Solanaceae for the Controlled Disinfection of Agricultural Soils.

The UN 2030 Agenda for Sustainable Development established the goal of cutting the use of pesticides in the EU by 50% by 2030. However, a ban on pesticides could seriously affect the productivity of agriculture, resulting in severe issues due to global hunger and food deficiency. Controlled release (CR) of bioactive chemicals could play a valid alternative in this context. To this aim, two biodegradable polymers, namely sodium alginate (AL) and sodium carboxymethylcellulose (CMC), were employed to obtain crosslinked hydrogel beads for the encapsulation and CR of glycoalkaloids extracted from tomato and potato leaves to be used as biocompatible disinfectants for agricultural soils. The physico-chemical characterization of the controlled-release systems was carried out by means of Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy, Scanning Electron Microscopy (SEM), thermogravimetry (TGA), differential scanning calorimetry (DSC) (FWI > 80%) and drying kinetics. The plant extracts and the encapsulation efficiency (~84%) were, respectively, characterized and evaluated by High-performance Liquid Chromatography-Mass Spectrometry (HPLC-MS). Finally, preliminary microbiological tests were conducted to test the efficacy of the most promising systems as biocidal formulations both in the lab and on a model soil, and interesting results were obtained in the reduction of bacterial and fungal load, which could lead to sustainable perspectives in the field.

Structuring and De-Structuring of Nanovectors from Algal Lipids: Simulated Digestion, Preliminary Antioxidant Capacity and In Vitro Tests.

Biocompatible nanocarriers can be obtained by lipid extraction from natural sources such as algal biomasses, which accumulate different lipid classes depending on the employed culture media. Lipid aggregates can be distinguished according to supramolecular architecture into lamellar and nonlamellar structures. This distinction is mainly influenced by the lipid class and molecular packing parameter, which determine the possible values of interfacial curvature and thus the supramolecular symmetries that can be obtained. The nanosystems prepared from bio-sources are able to self-assemble into different compartmentalized structures due to their complex composition. They also present the advantage of increased carrier-target biocompatibility and are suitable to encapsulate and vehiculate poorly water-soluble compounds, e.g., natural antioxidants. Their functional properties stem from the interplay of several parameters. Following previous work, here the functionality of two series of structurally distinct lipid nanocarriers, namely liposomes and cubosomes deriving from algal biomasses with different lipid composition, is characterized. In the view of their possible use as pharmaceutical or nutraceutical formulations, both types of nanovectors were loaded with three well-known antioxidants, i.e., curcumin, α-tocopherol and piperine, and their carrier efficacy was compared considering their different structures. Firstly, carrier stability in biorelevant conditions was assessed by simulating a gastrointestinal tract model. Then, by using an integrated chemical and pharmacological approach, the functionality in terms of encapsulation efficiency, cargo bioaccessibility and kinetics of antioxidant capacity by UV-Visible spectroscopy was evaluated. Subsequently, in vitro cytotoxicity and viability tests after administration to model cell lines were performed. As a consequence of this investigation, it is possible to conclude that nanovectors from algal lipids, i.e., cubosomes and liposomes, can be efficient delivery agents for lipophilic antioxidants, being able to preserve and enhance their activity toward different targets while promoting sustained release.

Characterization of Extracts of Coffee Leaves (Coffea arabica L.) by Spectroscopic and Chromatographic/Spectrometric Techniques.

Coffea arabica L. leaves represent a viable alternative to the canonical matrices used for preparation of beverages, such as tea leaves and grounded coffee beans. Coffee leaves infusions are rich in antioxidant phenolic compounds and have a lower concentration of caffeine. Due to increasing interest in this field, a complete study of the bioactive compounds as chlorogenic acids, xanthones and alkaloids is noteworthy. C. arabica leaves were subjected to ultrasound-assisted extraction, and the extracts were studied via nuclear magnetic resonance spectroscopy (NMR) and chromatographic techniques coupled with mass spectrometry (HPLC-MSn) to identify and quantify the secondary metabolites profile through an untargeted data dependent approach. A quantitative analysis was performed for the major components-chlorogenic acids, mangiferin, caffeine and trigonelline-via HPLC-MS in Single Ion Monitoring (SIM) mode. In total, 39 compounds were identified. The presence of these bioactive compounds proved the strong potential of C. arabica leaves as functional food and as an alternative to classic infused beverages.

Bioactive Compounds and Their Impact on Protein Modification in Human Cells.

Reactive oxygen species (ROS) represent a group of molecules with a signaling role that are involved in regulating human cell proliferation and differentiation. Increased ROS concentrations are often associated with the local nonspecific oxidation of biological macromolecules, especially proteins and lipids. Free radicals, in general, may randomly damage protein molecules through the formation of protein-centered radicals as intermediates that, in turn, decay into several end oxidation products. Malondialdehyde (MDA), a marker of free-radical-mediated lipid oxidation and cell membrane damage, forms adducts with proteins in a nonspecific manner, leading to the loss of their function. In our study, we utilized U-937 cells as a model system to unveil the effect of four selected bioactive compounds (chlorogenic acid, oleuropein, tomatine, and tyrosol) to reduce oxidative stress associated with adduct formation in differentiating cells. The purity of the compounds under study was confirmed by an HPLC analysis. The cellular integrity and changes in the morphology of differentiated U-937 cells were confirmed with confocal microscopy, and no significant toxicity was found in the presence of bioactive compounds. From the Western blot analysis, a reduction in the MDA adduct formation was observed in cells treated with compounds that underlaid the beneficial effects of the compounds tested.

Effect of Flaking and Precooking Procedures on Antioxidant Potential of Selected Ancient Cereal and Legume Flours.

Consumption of cereals (and particularly ancient cereals) is considered the base of a healthy diet, and all current dietary guidelines have cereals at the bottom of the nutrition pyramid. Together with cereals, legumes are an excellent source of nutrients and nutraceuticals. The effects of agroindustrial pretreatments (flaking and precooking processes) on the antioxidant potential of flours from ancient cereals and legumes were studied. The extraction of free hydrophilic phenolic compounds was carried out in a hydroalcoholic solvent mixture via an ultrasound-assisted process. Furthermore, the solid residue was successively hydrolyzed by an alkaline solution to extract the bound phenolic fraction. Both free and bound extracted fractions were then quantitatively characterized for total polyphenolic and flavonoid contents, and the antioxidant potential was determined by carrying out the ABTS and DPPH radical scavenging assays, expressing the results (in both cases) as the Trolox equivalent antioxidant capacity (TEAC/ABTS and TEAC/DPPH, respectively). The samples were also extracted in organic apolar solvents (acetone or water-saturated iso-butanol) to quantitatively characterize lipophilic antioxidant compounds and pigments. A discussion on the comparison of these analytical parameters of flours obtained from raw, flaked, and precooked cereals and legumes is reported revealing that (i) phenolic compounds are mainly present in the post-hydrolysis extract (bound fraction), (ii) the precooking process significantly reduced the concentration of antioxidants, (iii) the flaking process slightly increased the phenolic content, (iv) legumes were less influenced by pretreatments, suggesting the possibility of using legumes to enrich cereal foods.

Determination of nano and microplastic particles in hypersaline lakes by multiple methods.

Microplastics and nanoplastics have a range of impacts on the aquatic environment and present major challenges to their mitigation and management. Their transport and fate depend on their composition, form, and the characteristics of the receiving environment. We explore the spatial and temporal dynamics of plastic particles in the world's second-largest hypersaline lake, combining information from microscopic, thermal gravimetric, and fractional methods. Studies on microplastic and nanoplastic pollution in these important environments are scarce, and there is limited understanding of their dynamics and fate. Our results for Urmia Lake (Iran) in 2016 and 2019 show a discrepancy in the composition and quantity of microplastics measured in river tributaries to the lake and the lake itself, suggesting an active microplastic sink. Potential sink mechanisms in hypersaline lakes are explored. The present study indicates that microplastics have different transport mechanisms and fate in these extreme environments, compared to lake and ocean environments.

Water content quantification by FTIR in carboxymethyl cellulose food additive.

Water content quantification of raw polysaccharide materials for food processing is generally performed by gravimetric analysis or titrimetric methods, which are time- and energy-consuming, non-eco-friendly and sample destructive. The present study develops and validates a new approach, based on the use of Fourier transform infrared (FTIR) spectroscopy, resulting in a model of the water content of carboxymethyl cellulose (CMC) polysaccharides. Samples of CMC were exposed to different relative humidity conditions. Water content was determined by standard gravimetric methods (OIV-Oeno 404-2010) and compared with the area of FTIR absorption in the range 3675-2980 cm-1, attributed to the stretching of OH groups. The strong correlation between gravimetric results and FTIR area (R2 = 0.88) showed no signs of bias across the water content range. A cross-validation technique to predict the water content by band area was assessed obtaining a general equation: y = 2.12 x + 2.80 with a high repetitively and good prediction of the tested models.

Varietal and Geographical Origin Characterization of Peaches and Nectarines by Combining Analytical Techniques and Statistical Approach.

Prunus persica L. is one of the most important fruit crops in European production, after grapes, apples, oranges and watermelons. Most varieties are rich in secondary metabolites, showing antioxidant properties for human health. The purpose of this study was to develop a chemical analysis methodology, which involves the use of different analytical-instrumental techniques to deepen the knowledge related to the profile of metabolites present in selected cultivars of peaches and nectarines cultivated in the Mediterranean area (Southern Italy). The comparative study was conducted by choosing yellow-fleshed peaches (RomeStar, ZeeLady) and yellow-fleshed nectarines (Nectaross, Venus) from two geographical areas (Piana di Sibari and Piana di Metaponto), and by determining the chemical parameters for the flesh and skin that allow for identification of any distinctive varietal and/or geographical characteristics. A combined analytical and chemometric approach was used, trough rheological, thermogravimetric (TGA), chromatographic (HPLC-ESI-MS), spectroscopic (UV-Vis, ATR-FTIR, NMR) and spectrometric (ToF-SIMS) analysis. This approach allowed us to identify the characterizing parameters for the analysis of a plant matrix so that the developed methodology could define an easily exportable and extendable model for the characterization of other types of vegetable matrices.

Combined Experimental and Multivariate Model Approaches for Glycoalkaloid Quantification in Tomatoes.

The intake of tomato glycoalkaloids can exert beneficial effects on human health. For this reason, methods for a rapid quantification of these compounds are required. Most of the methods for α-tomatine and dehydrotomatine quantification are based on chromatographic techniques. However, these techniques require complex and time-consuming sample pre-treatments. In this work, HPLC-ESI-QqQ-MS/MS was used as reference method. Subsequently, multiple linear regression (MLR) and partial least squares regression (PLSR) were employed to create two calibration models for the prediction of the tomatine content from thermogravimetric (TGA) and attenuated total reflectance (ATR) infrared spectroscopy (IR) analyses. These two fast techniques were proven to be suitable and effective in alkaloid quantification (R2 = 0.998 and 0.840, respectively), achieving low errors (0.11 and 0.27%, respectively) with the reference technique.

Solid Lipid Nanoparticles Produced via a Coacervation Method as Promising Carriers for Controlled Release of Quercetin.

Quercetin is a poorly water-soluble flavonoid with many benefits to human health. Besides the natural food resources that may provide Quercetin, the interest in delivery systems that could enhance its bioavailability in the human body has seen growth in recent years. Promising delivery system candidates are represented by Solid Lipid Nanoparticles (SLNs) which are composed of well-tolerated compounds and provide a relatively high encapsulation efficiency and suitable controlled release. In this study, Quercetin-loaded and negatively charged Solid Lipid Nanoparticles were synthesized based on a coacervation method, using stearic acid as a core lipid and Arabic Gum as a stabilizer. Samples were qualitatively characterized by Dynamic light scattering (DLS), Zeta Potential, Surface infrared spectroscopy (FTIR-ATR), and Time of flight secondary ion mass spectrometry (ToF-SIMS). Encapsulation efficiency, drug release, and antioxidant effect against ABTS•+ were evaluated in vitro by UV-VIS spectrophotometry.

Kinetics of glucosinolate hydrolysis by myrosinase in Brassicaceae tissues: A high-performance liquid chromatography approach.

Glucosinolates are a group of secondary metabolites occurring in all the vegetables belonging to the Brassicaceae family. Upon tissue damage, glucosinolates are hydrolyzed by myrosinase to a series of degradation products, including isothiocyanates, which are important for their health-promoting effects in humans. The glucosinolate-myrosinase system has been characterized in several Brassica species, of which white mustard (Sinapis alba) has been studied the most. In this study, a new HPLC-UV assay to evaluate the activities and kinetics of myrosinases in aqueous extracts, which closely represent the physiological conditions of plant tissues, was developed. This method was tested on myrosinases extracted from broccoli and cauliflower inflorescences, employing sinigrin and glucoraphanin as substrates. The results showed a strong inhibition of both enzymes at high substrate concentrations. The main issues related to kinetic analysis on the glucosinolate-myrosinase system were also elucidated.

Effects of Aqueous Extract of Lycopersicum esculentum L. var. "Camone" Tomato on Blood Pressure, Behavior and Brain Susceptibility to Oxidative Stress in Spontaneously Hypertensive Rats.

Behavioral disorders affect millions of people worldwide. Hypertension contributes to both the development and progression of brain damage and cognitive dysfunction and could represent the most powerful modifiable risk factor for cerebral vessel dysfunction and consequent behavioral impairment. Tomato contains antioxidants and bioactive molecules that might play an important role in the prevention of cardiovascular and brain diseases. The effects of the combined gel and serum from Lycopersicum esculentum L. var. "Camone" tomatoes and those of purified tomato glycoalkaloids (tomatine) and an antihypertensive drug (captopril) were investigated in male spontaneously hypertensive rats (SHRs) and compared with normotensive Wistar Kyoto (WKY) rats. Body weight, systolic blood pressure, behavioral parameters, as well as brain susceptibility to oxidative stress and brain cytokine contents, were assessed. Treating hypertensive rats with tomato gel/serum or captopril for four weeks caused a significant reduction in blood pressure, decreased locomotor activity and increased grooming behavior; the last two parameters were also significantly affected by tomatine treatment. Brain slices obtained from hypertensive rats treated with tomato gel/serum were more resistant to oxidative stress and contained lower levels of inflammatory cytokines than vehicle-treated ones. In contrast, tomatine treatment had no effect. In conclusion, the tomato-derived gel/serum can be considered a dietary supplement able to drive in vivo blood pressure towards healthier values and also control some central effects such as behavior and brain oxidative stress.

Chemical Characterisation and Antihypertensive Effects of Locular Gel and Serum of Lycopersicum esculentum L. var. "Camone" Tomato in Spontaneously Hypertensive Rats.

Blood pressure control in hypertensive subjects calls for changes in lifestyle, especially diet. Tomato is widely consumed and rich in healthy components (i.e., carotenoids, vitamins and polyphenols). The aim of this study was to evaluate the chemical composition and antihypertensive effects of locular gel reconstituted in serum of green tomatoes of "Camone" variety. Tomato serum and locular gel were chemically characterised. The antihypertensive effects of the locular gel in serum, pure tomatine, and captopril, administered by oral gavage, were investigated for 4 weeks in male spontaneously hypertensive and normotensive rats. Systolic blood pressure and heart rate were monitored using the tail cuff method. Body and heart weight, serum glucose, triglycerides and inflammatory cytokines, aorta thickness and liver metabolising activity were also assessed. Locular gel and serum showed good tomatine and polyphenols content. Significant reductions in blood pressure and heart rate, as well as in inflammatory blood cytokines and aorta thickness, were observed in spontaneously hypertensive rats treated both with locular gel in serum and captopril. No significant effects were observed in normotensive rats. Green tomatoes locular gel and serum, usually discarded during tomato industrial processing, are rich in bioactive compounds (i.e., chlorogenic acid, caffeic acid and rutin, as well as the glycoalkaloids, α-tomatine and dehydrotomatine) that can lower in vivo blood pressure towards healthier values, as observed in spontaneously hypertensive rats.

Tomatine Displays Antitumor Potential in In Vitro Models of Metastatic Melanoma.

There is a growing interest in the cytotoxic effects of bioactive glycoalkaloids, such as α-tomatine on tumor cells. Here, for the first time, we determine the antitumor potential of tomatine, a mixture of α-tomatine and dehydrotomatine, in metastatic melanoma (MM) cell lines harboring different BRAF and MC1R variants. We performed cytotoxicity experiments and annexin-V/propidium iodide staining to assess the apoptotic/necrotic status of the cells. ER stress and autophagy markers were revealed by Western Blot, whereas antiangiogenic and vascular-disrupting effects were evaluated through a capillary tube formation assay on matrigel and by ELISA kit for VEGF release determination. Cell invasion was determined by a Boyden chamber matrigel assay. Tomatine reduced 50% of cell viability and induced a concentration-dependent increase of apoptotic cells in the range of 0.5-1 µM in terms of α-tomatine. The extent of apoptosis was more than two-fold higher in V600BRAF-D184H/D184H MC1R cells than in BRAF wild-type cells and V600BRAF-MC1R wild-type cell lines. Additionally, tomatine increased the LC3I/II autophagy marker, p-eIF2α, and p-Erk1/2 levels in BRAF wild-type cells. Notably, tomatine strongly reduced cell invasion and melanoma-dependent angiogenesis by reducing VEGF release and tumor-stimulating effects on capillary tube formation. Collectively, our findings support tomatine as a potential antitumor agent in MM.

Enriched Gellan Gum hydrogel as visco-supplement.

Viscosupplementation, i.e. intra-articular injection of hyaluronic acid derivatives, is considered as the most effective treatment for patients with mild to moderate osteoarthritis. Even if hyaluronic acid is still considered as the gold standard, research is now focusing on the development of new products with enhanced injectability and yet reasonable viscoelastic behavior for OA treatment. A Gellan Gum (GG) hydrogel was synthesized and coated with crosslinked polyvinyl alcohol (PVA) to protect the polysaccharide from degradation during sterilization and improve its performance for the foreseen application. Thermal analyses indicated that mixed hydrogel showed a higher degree of structuring than the bare polysaccharide core without losing its swelling properties, thanks to the hydrophylicity of both coating and cross-linking agent. The PVA coating increased elastic and viscous moduli of the polysaccharide core conferring it a higher resistance to shear and compression and better thixotropic properties. Despite the double crosslinking, hydrogel was injectable. Cytocompatibility towards chondrocytes was verified.

Chemical characterization and antioxidant properties of products and by-products from Olea europaea L.

The products and by-products of Olea europaea L.: olive fruits (primary agricultural product), oils (primary agro-industrial product), pomaces (agro-industrial processing by-product), and leaves (agricultural practices by-product), are promising sources of bioactive compounds. In the present study, qualitative and quantitative analyses of selected bioactive components in olive fruits, oils, and pomaces were performed. Total polyphenol content and antioxidant activity were analyzed in all samples (humid pomaces 2015: TPP, 26.0 ± 1.5-43.7 ± 3.0 g(GAEq)/kg DW; TEAC/ABTS, 189.5 ± 3.7-388.1 ± 12.0 mmol(Trx)kg DW). Radical (DPPH) quenching potential was analyzed via photometric and EPR methods, obtaining Vis/EPR signal ratio by 1.05 ± 0.45 and 1.66 ± 0.39 for fruits and pomaces, respectively. Through HPLC-UV and HPLC-MS/MS techniques, oleuropein and hydroxytyrosol, as well as selected hydroxycinnamic acids and flavonoids, were identified and quantified in olive fruits and pomaces. The main components were rutin, luteolin, and chlorogenic acid. Cytotoxic assay on fibroblast cells revealed toxic effects for selected extracts at highest tested concentrations (5%).

Ordering effect of protein surfaces on water dynamics: NMR relaxation study.

Proteins in solution affect the structural and dynamic properties of the bulk water at the protein-water interface, resulting in a contribution to the order of the hydration water. Theoretical and experimental NMR relaxation methods were developed to study the dynamic properties of water molecules in the protein hydration shell. Water non-selective and selective relaxation rates, were shown to be sensitive to contributions from ordered solvent molecules at protein surface. The average rotational correlation time of water molecules in the protein hydration shell was determined for three protein systems of different size: ribonuclease A, human serum albumin and fibrinogen. The knowledge of these properties is an important step toward the determination of the size of the water ordering contributions originate in proteins systems.

Evaluation of in vitro cell and blood compatibility and in vivo analgesic activity of plant-derived dietary supplements.

OBJECTIVE: In vitro cell and blood compatibility of three dietary supplements, comprised of multiple plant extracts, Pneumo Go (PG), Green active (GA) and Equistasi (Eq), and their main component, the phytocomplex Matrix U.B.® (Union Bio S.r.l.) (M), were evaluated. Moreover, preliminary in vivo tests were performed on GA in order to assess its ability to reduce pain in an animal model. METHODS: Cell compatibility was determined using fibroblasts (NIH3T3) and primary adult human microvascular endothelial cells (HMVECad) and the neutral red uptake test. Blood compatibility was evaluated by analyzing blood parameters after incubation of the products with sodium citrate anticoagulated whole blood. Thrombin time was determined by adding thrombin to aliquots of human plasma containing the samples. Clotting time was revealed by an automatic coagulometer. The in vivo analgesic effect of GA was evaluated in Wistar rats using the formalin test. RESULTS: M and PG reduced the percentage of viable NIH3T3 cells, indicating their interference in the cell cycle. GA and Eq stimulated fibroblast proliferation and neutralized the toxic effect of M. M and PG reduced HMVECad cell viability. GA and Eq did not affect cell viability as well as negative control. The hemocompatibility tests indicated that all the samples did not interfere with fibrinogen. The in vivo test carried out in male rats showed a significant analgesic effect of GA in all formalin-induced pain behaviors. CONCLUSION: No hemotoxicity and good cell compatibility were found for all the tested samples. GA and Eq were the best candidates for further biocompatibility testing. Moreover, GA reduced pain in the animal model.

Distribution of Gadolinium in Rat Heart Studied by Fast Field Cycling Relaxometry and Imaging SIMS.

Research on microcirculatory alterations in human heart disease is essential to understand the genesis of myocardial contractile dysfunction and its evolution towards heart failure. The use of contrast agents in magnetic resonance imaging is an important tool in medical diagnostics related to this dysfunction. Contrast agents significantly improve the imaging by enhancing the nuclear magnetic relaxation rates of water protons in the tissues where they are distributed. Gadolinium complexes are widely employed in clinical practice due to their high magnetic moment and relatively long electronic relaxation time. In this study, the behavior of gadolinium ion as a contrast agent was investigated by two complementary methods, relaxometry and secondary ion mass spectrometry. The study examined the distribution of blood flow within the microvascular network in ex vivo Langendorff isolated rat heart models, perfused with Omniscan® contrast agent. The combined use of secondary ion mass spectrometry and relaxometry allowed for both a qualitative mapping of agent distribution as well as the quantification of gadolinium ion concentration and persistence. This combination of a chemical mapping and temporal analysis of the molar concentration of gadolinium ion in heart tissue allows for new insights on the biomolecular mechanisms underlying the microcirculatory alterations in heart disease.