Ferulated Poly(vinyl alcohol) based hydrogels.

New graft copolymers were prepared by reaction of poly (vinyl alcohol) (PVA) with mono-imidazolide or bis-imidazolide derivatives of ferulic acid (FA) with the formation of ester bonds. The obtained graft copolymers, thanks to the crosslinking capability of FA, formed in water strong gels as verified by rheological analyses. The resulting hydrogels were characterized to evaluate their applicability as wound dressing. In this perspective, their capability to absorb and retain a large amount of fluid without dissolving was verified by swelling kinetics and Moisture Vapour Transmission Rate measurements. Their stability towards mechanical solicitations was assessed by quantifying elasticity, compliance, stress-relaxation, and adhesivity properties. The analyses pointed out that hydrogel PVA-FA2-3 obtained by feruloylation of PVA with bis-imidazole derivative of ferulic acid using an acylation agent/polymer molar ratio 0.03/1 resulted the best candidate for the foreseen application.

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.

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.

Click-Chemistry Cross-Linking of Hyaluronan Graft Copolymers.

An easy and viable crosslinking procedure by click-chemistry (click-crosslinking) of hyaluronic acid (HA) was developed. In particular, the clickable propargyl groups of hyaluronane-based HA-FA-Pg graft copolymers showing low and medium molecular weight values were exploited in crosslinking by click-chemistry by using a hexa(ethylene glycol) spacer. The resulting HA-FA-HEG-CL materials showed an apparent lack of in vitro cytotoxic effects, tuneable water affinity, and rheological properties according to the crosslinking degree that suggests their applicability in different biomedical fields.

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.

The Diagnostic-Therapeutic Care Pathway in Psoriasis: Towards ISO 9001:2015 Certification.

Background and objectives: Psoriasis (Pso) is a common skin condition characterized by a strong psychosocial impact, and is nowadays accepted as a systemic immune-mediated inflammatory disease. Diagnostic-Therapeutic Care Pathways (DTCPs) represent a predefined sequence of diagnostic, therapeutic, and assistance activities that integrate the participation of several specialists to obtain, for each patient, the correct diagnosis and thus the most appropriate therapy. A DTCP was validated in our dermatology clinic (AOU Maggiore della Carità, Novara, Italy). The validation process included the detailed elaboration of a protocol of diagnosis, staging of care, therapies, and follow-up of the patient with Pso. The formalization and adaptation of our DTCP resulted in ISO 9001: 2015 certification in May 2019. Materials and methods: This process involved several stages, including analysis of context and the identification of (i) targets, (ii) indicators, and (iii) service providers. The evaluation was based on a cohort of over 200 patients affected by moderate to severe Pso, who were treated and followed-up at our institution from September 2017 to April 2019. Results: The ISO 9001:2015 quality certification process allowed us to identify our weaknesses, i.e., the long waiting times for the first visit and the reduced physician-patient ratio, but also our strengths, such as the commitment to clinical research, effective collaboration with other specialists, the efficient use of technological and human resources, and attention to ensuring patient follow-up. Conclusions: In qualifying for and achieving the ISO Quality Management System (QMS) certification we were heartened to realize that our basic methodology and approach were fit for purpose. The implementation of the ISO QMS helped us to reorganize our priorities by placing the patient at the center of the process and raising awareness that Pso is not just a skin disease.

Targeting dopamine D3 and serotonin 5-HT1A and 5-HT2A receptors for developing effective antipsychotics: synthesis, biological characterization, and behavioral studies.

Combination of dopamine D3 antagonism, serotonin 5-HT1A partial agonism, and antagonism at 5-HT2A leads to a novel approach to potent atypical antipsychotics. Exploitation of the original structure-activity relationships resulted in the identification of safe and effective antipsychotics devoid of extrapyramidal symptoms liability, sedation, and catalepsy. The potential atypical antipsychotic 5bb was selected for further pharmacological investigation. The distribution of c-fos positive cells in the ventral striatum confirmed the atypical antipsychotic profile of 5bb in agreement with behavioral rodent studies. 5bb administered orally demonstrated a biphasic effect on the MK801-induced hyperactivity at dose levels not able to induce sedation, catalepsy, or learning impairment in passive avoidance. In microdialysis studies, 5bb increased the dopamine efflux in the medial prefrontal cortex. Thus, 5bb represents a valuable lead for the development of atypical antipsychotics endowed with a unique pharmacological profile for addressing negative symptoms and cognitive deficits in schizophrenia.

Disease-modifying anti-Alzheimer's drugs: inhibitors of human cholinesterases interfering with ß-amyloid aggregation.

AIMS: We recently described multifunctional tools (2a-c) as potent inhibitors of human Cholinesterases (ChEs) also able to modulate events correlated with Aß aggregation. We herein propose a thorough biological and computational analysis aiming at understanding their mechanism of action at the molecular level. METHODS: We determined the inhibitory potency of 2a-c on Aß1-42 self-aggregation, the interference of 2a with the toxic Aß oligomeric species and with the postaggregation states by capillary electrophoresis analysis and transmission electron microscopy. The modulation of Aß toxicity was assessed for 2a and 2b on human neuroblastoma cells. The key interactions of 2a with Aß and with the Aß-preformed fibrils were computationally analyzed. 2a-c toxicity profile was also assessed (human hepatocytes and mouse fibroblasts). RESULTS: Our prototypical pluripotent analogue 2a interferes with Aß oligomerization process thus reducing Aß oligomers-mediated toxicity in human neuroblastoma cells. 2a also disrupts preformed fibrils. Computational studies highlighted the bases governing the diversified activities of 2a. CONCLUSION: Converging analytical, biological, and in silico data explained the mechanism of action of 2a on Aß1-42 oligomers formation and against Aß-preformed fibrils. This evidence, combined with toxicity data, will orient the future design of safer analogues.

Lycopene phytocomplex, but not pure lycopene, is able to trigger apoptosis and improve the efficacy of photodynamic therapy in HL60 human leukemia cells.

We compared the ability of pure lycopene (Lyco) versus lycopene phytocomplex (LycoC) to induce apoptosis in vitro. We found that LycoC, but not Lyco, was able to trigger apoptosis in HL60 cells, as documented by subdiploid DNA content and phosphatidylserine exposure. LycoC-induced apoptosis was associated with reactive oxygen species (ROS) generation and loss of mitochondrial transmembrane potential, suggesting that LycoC triggered apoptosis via a mitochondrial pathway. We also verified the redox state of cells by measuring glutathione (GSH) content, but only a small percentage of cells showed GSH depletion, suggesting that the loss of GSH may be a secondary consequence of ROS generation. Moreover, LycoC pretreatment effectively increased apoptosis induced by photodynamic therapy (PDT), a mode of cancer treatment using a photosensitizer and visible light. LycoC pretreatment was even more potent in improving PDT than pretreatment with ascorbic acid or alpha-tocopherol (or the two combined). Our results demonstrate that LycoC has a stronger cytotoxic effect than Lyco and is a better source of agents able to trigger apoptosis in HL60 cells and improve the efficacy of PDT in vitro.

Non-covalent inclusion of ferulic acid with alpha-cyclodextrin improves photo-stability and delivery: NMR and modeling studies.

Ferulic acid (FA) is a highly effective antioxidant and photo-protective agent, already approved in Japan as a sunscreen, but it is poorly suited for cosmetic application because of its low physicochemical stability. We prepared the inclusion complex of FA with alpha-cyclodextrin by co-precipitation from an aqueous solution, and used (1)H NMR and molecular dynamics to investigate the most probable structure of the inclusion complex. In rotating frame nuclear Overhouser effect spectroscopy (ROESY) experiments FA penetrated the alpha-CD hydrophobic cavity with the alpha,beta-unsaturated part of the molecule and some of its aromatic skeleton. In proton chemical shift measurements of FA and alpha-cyclodextrins we determined the stoichiometry of the association complex (1:1) by Job's method, and its stability constant (K(1:1) 1162+/-140 M(-1)) and described the molecular dynamics of the complex on the basis of theoretical studies. Encapsulation with alpha-cyclodextrin improves (i) the chemical stability of FA against UVB stress (10 MED [Minimal Erythemal Dose: 1 MED=25 mJ/cm(2) for skin phototype II: 30]), since no degradation products are formed after irradiation, and (ii) the bioavailability of FA on the skin, slowing its delivery (Strainer cell model).

Conformational analysis: a tool for the elucidation of the antioxidant properties of ferulic acid derivatives in membrane models.

With the aim to search and design more effective and safe antioxidant molecules to be used as functional ingredients in cosmetic formulations for UV protection, we evaluated the antioxidant/radical scavenging activities of ferulic acid and of some alkyl ferulates in both acellular and cellular systems. Ferulic acid esters, equipotent as antioxidant in homogeneous phase, showed when tested in membranous systems (rat liver microsomes, rat erythrocytes) marked differences in antioxidant potency. The n-C(12) derivative was the most potent, followed by n-C(8), n-C(16) and branched C(8), and then by ferulic acid. A conformational study carried out by NMR and modelling, indicates that the different antioxidant activity of ferulates in membrane models is due to the different spatial conformation and arrangement of the side chain of the molecule, which governs the access and binding to the phospholipid bilayer, the modality of orientation of the scavenging/quenching nucleus (phenol moiety), and hence the overall antioxidant potency of the derivative. These results emphasize the need of analytical studies (NMR and molecular modelling) addressed to the knowledge of the conformational parameters in combination with conventional antioxidant testings for understanding the antioxidant behaviour of a molecule in a biological membrane/system.

Involvement of oxidative stress in apoptosis induced by a mixture of isothiazolinones in normal human keratinocytes.

A 3:1 combination of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) is widely used to preserve cosmetic products. We show here that CMI/MI induced apoptosis in normal human keratinocytes (NHK) as at low concentrations (0.001-0.05% documented by subdiploid DNA content and phosphatidylserine exposure, while at the highest concentration (0.1% as supplied, 15 p.p.m.) the response was necrosis. Various molecular events accompanied the cytotoxic effects of CMI/MI. Generation of ROS and hyperpolarization of mitochondrial transmembrane potential (DeltaPsim) were early events, followed by increased Fas expression and activation of caspase-8, and then activation of caspase-3 and -9. The drop in DeltaPsim occurred only later in the cell death pathway, when NHK showed signs of apoptosis. Pretreatment of cells for 2 h with the redox-active agent N-acetyl-L-cysteine conferred complete protection against the CMI/MI-induced cytotoxic effects, DeltaPsim loss, and apoptosis. The pan-caspase inhibitor Z-Val-Ala-Asp(OMe)-CH2F blocked the CMI/MI-induced apoptosis without preventing ROS generation and the drop in DeltaPsim. These results indicate that the generation of ROS plays an important part in mediating apoptosis and necrosis associated with CMI/MI treatment. This new aspect of the in vitro toxicity of CMI/MI may provide important information about the relationship between the preservative's in vitro apoptotic activity and its in vivo toxicity.

In vitro induction of apoptosis vs. necrosis by widely used preservatives: 2-phenoxyethanol, a mixture of isothiazolinones, imidazolidinyl urea and 1,2-pentanediol.

Preservatives are added to many final products, such as detergents, cosmetics, pharmaceuticals and vaccines. We conducted an in vitro investigation of the apoptosis- and necrosis-inducing potential of brief applications (10 min) of four common preservatives: ethylene glycol monophenyl ether, 2-phenoxyethanol (EGPE), imidazolidinyl urea (IMU), a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one and 2-methyl-4-isothiazolin-3-one (CMI/MI), and 1,2-pentanediol, a "preservative-non-preservative" best known as pentylene glycol. Using HL60 cells, we monitored the kinetics of cell toxicity with the MTT test and analysed extranuclear end points of apoptosis, i.e. phosphatidylserine exposure and nuclear fragmentation. Preservative treatment resulted in a dose-dependent decrease of cell viability. The mode of cell death was dose-dependent: necrosis occurred at high concentrations while apoptosis, shown by DNA laddering, DNA sub-diploid peak and caspase-3 activation, occurred at lower concentrations 0-24hr after exposure to a single dose: CMI/MI induced apoptosis at low concentrations (0.001-0.01%) and necrosis at high concentrations (0.5-0.1%); IMU and EGPE required higher concentrations to induce apoptosis (IMU 0.01-0.1% and EGPE 0.01-0.5%) or necrosis (IMU 0.5-1% and EGPE only at 1%). PG induced apoptosis only at 5%. Externalization of PS, a hallmark of apoptosis, occurred early in HL60 treated with low concentrations of CMI/MI and EGPE and was concomitant with the subdiploid peak in HL60 treated with PG. However, it did not occur in HL60 treated with IMU. In conclusion, at appropriate concentrations, each of the four preservatives modulates the apoptotic machinery by a caspase-dependent mechanism. Thus, apoptosis could be a good parameter to evaluate the cytoxicity of these chemical compounds.