Antonio Francioso

Chemistry, stability and bioavailability of resveratrol.

Resveratrol is a bioactive polyphenol found in many vegetables. It is well known for its multiple pharmacological activities, such as anti-inflammatory, antioxidant, antimicrobial, anticancer, neuroprotective and cardioprotective effects. In vitro evidence of resveratrol efficacy is widespread, however, many concerns regarding its effectiveness in vivo arise from its poor stability in vitro and bioavailability following oral ingestion. This review focuses on the in vitro stability, with special focus on the photochemical stability of resveratrol, and on the therapeutic perspectives of this molecule due to its low bioavailability.

Improved Stability of trans-Resveratrol in Aqueous Solutions by Carboxymethylated (1,3/1,6)-β-d-Glucan

Resveratrol is a polyphenolic compound endowed with multiple health benefits. However, its limited bioavailability and poor stability in solution hamper its use in pharmaceutical applications. Due to its low solubility in water, solvents such as ethanol and dimethyl sulfoxide are often used to dissolve resveratrol. However, these solvents have adverse effects on cultured cells or in vivo. The purpose of this study was to develop an aqueous liquid formulation of resveratrol in combination with a modified glucan, the carboxymethylated (1,3/1,6)-β-D-glucan (CM-glucan). The proposed liquid formulation conferred stability to resveratrol without affecting its antioxidant capability. Shelf-life measurements revealed that resveratrol in aqueous solution is degraded within a few weeks, due to spontaneous oxidation. In contrast, the combination with CM-glucan matrix exerted a strong stabilizing effect in aqueous medium and increased resveratrol stability up to 12 months at 25 °C. These data provide evidence of a stable resveratrol formulation in liquid suspensions and support the possible development of pharmaceutical applications of this association in biopharmaceutics and drug delivery.

5‐S‐cysteinyldopamine neurotoxicity: Influence on the expression of α‐synuclein and ERp57 in cellular and animal models of Parkinson's disease

Parkinsons disease (PD) is a progressive neurodegenerative disorder whose etiology is still unclear in spite of extensive investigations. It has been hypothesized that 5‐S‐cysteinyldopamine (CysDA), a catechol‐thioether metabolite of dopamine (DA), could be an endogenous parkinsonian neurotoxin. To gain further insight into its role in the neurodegenerative process, both CD1 mice and SH‐SY5Y neuroblastoma cells were treated with CysDA, and the data were compared with those obtained by the use of 6‐hydroxydopamine, a well‐known parkinsonian mimetic. Intrastriatal injection of CysDA in CD1 mice caused a long‐lasting depletion of DA, providing evidence of in vivo neurotoxicity of CysDA. Both in mice and in SH‐SY5Y cells, CysDA treatment induced extensive oxidative stress, as evidenced by protein carbonylation and glutathione depletion, and affected the expression of two proteins, α‐synuclein (α‐Syn) and ERp57, whose levels are modulated by oxidative insult. Real‐time PCR experiments support these findings, indicating an upregulation of both ERp57 and α‐Syn expression. α‐Syn aggregation was also found to be modulated by CysDA treatment. The present work provides a solid background sustaining the hypothesis that CysDA is involved in parkinsonian neurodegeneration by inducing extensive oxidative stress and protein aggregation.

Transient increase in neuronal chloride concentration by neuroactive aminoacids released from glioma cells

Neuronal chloride concentration ([Cl−]i) is known to be dynamically modulated and alterations in Cl− homeostasis may occur in the brain at physiological and pathological conditions, being also likely involved in glioma-related seizures. However, the mechanism leading to changes in neuronal [Cl−]i during glioma invasion are still unclear. To characterize the potential effect of glioma released soluble factors on neuronal [Cl−]i, we used genetically encoded CFP/YFP-based ratiometric Cl-(apical) Sensor transiently expressed in cultured hippocampal neurons. Exposition of neurons to glioma conditioned medium (GCM) caused rapid and transient elevation of [Cl−]i, resulting in the increase of fluorescence ratio, which was strongly reduced by blockers of ionotropic glutamate receptors APV and NBQX. Furthermore, in HEK cells expressing GluR1-AMPA receptors, GCM activated ionic currents with efficacy similar to those caused by glutamate, supporting the notion that GCM contains glutamate or glutamatergic agonists, which cause neuronal depolarization, activation of NMDA and AMPA/KA receptors leading to elevation of [Cl−]i. Chromatographic analysis of the GCM showed that it contained several aminoacids, including glutamate, whose release from glioma cells did not occur via the most common glial mechanisms of transport, or in response to hypoosmotic stress. GCM also contained glycine, whose action contrasted the glutamate effect. Indeed, strychnine application significantly increased GCM-induced depolarization and [Cl−]i rise. GCM-evoked [Cl−]i elevation was not inhibited by antagonists of Cl− transporters and significantly reduced in the presence of anion channels blocker NPPB, suggesting that Cl− selective channels are a major route for GCM-induced Cl− influx. Altogether, these data show that glioma released aminoacids may dynamically alter Cl− equilibrium in surrounding neurons, deeply interfering with their inhibitory balance, likely leading to physiological and pathological consequences.

Resveratrol inhibits rhinovirus replication and expression of inflammatory mediators in nasal epithelia.

Human rhinoviruses (HRV), the cause of common colds, are the most frequent precipitants of acute exacerbation of asthma and chronic obstructive pulmonary disease, as well as causes of other serious respiratory diseases. No vaccine or antiviral agents are available for the prevention or treatment of HRV infection. Resveratrol exerts antiviral effect against different DNA and RNA viruses. The antiviral effect of a new resveratrol formulation containing carboxymethylated glucan was analyzed in H1HeLa cell monolayers and ex vivo nasal epithelia infected with HRV-16. Virus yield was evaluated by plaque assay and expression of viral capsid proteins by Western blot. IL-10, IFN-β, IL-6, IL-8 and RANTES levels were evaluated by ELISA assay. ICAM-1 was assessed by Western blot and immunofluorescence. Resveratrol exerted a high, dose-dependent, antiviral activity against HRV-16 replication and reduced virus-induced secretion of IL-6, IL-8 and RANTES to levels similar to that of uninfected nasal epithelia. Basal levels of IL-6 and RANTES were also significantly reduced in uninfected epithelia confirming an anti-inflammatory effect of the compound. HRV-induced expression of ICAM-1 was reversed by resveratrol. Resveratrol may be useful for a therapeutic approach to reduce HRV replication and virus-induced cytokine/chemokine production.

Isolation and identification of 2,4,6-trihydroxyphenanthrene as a byproduct of trans-resveratrol photochemical isomerization and electrocyclization

UV irradiation of trans-resveratrol leads to its photochemical isomerization and electrocyclization, giving rise to different byproducts. Preliminary attempts to purify and characterize these products were in the majority of cases unsuccessful. In the present work, the resveratrol photoreaction products were analyzed by HPLC, and one of these compounds, 2,4,6-trihydroxyphenanthrene (THP), was purified and unambiguously identified. The structure of THP was unequivocally characterized for the first time by combined GC-MS, ESI-MS/MS, NMR, and FT-IR analyses.

ATP release during cell swelling activates a Ca2+-dependent Cl - Current by autocrine mechanism in mouse hippocampal microglia

Microglia cells, resident immune cells of the brain, survey brain parenchyma by dynamically extending and retracting their processes. Cl− channels, activated in the cellular response to stretch/swelling, take part in several functions deeply connected with microglia physiology, including cell shape changes, proliferation, differentiation and migration. However, the molecular identity and functional properties of these Cl− channels are largely unknown. We investigated the properties of swelling-activated currents in microglial from acute hippocampal slices of Cx3cr1+/GFP mice by whole-cell patch-clamp and imaging techniques. The exposure of cells to a mild hypotonic medium, caused an outward rectifying current, developing in 5–10 minutes and reverting upon stimulus washout. This current, required for microglia ability to extend processes towards a damage signal, was carried mainly by Cl− ions and dependent on intracellular Ca2+. Moreover, it involved swelling-induced ATP release. We identified a purine-dependent mechanism, likely constituting an amplification pathway of current activation: under hypotonic conditions, ATP release triggered the Ca2+-dependent activation of anionic channels by autocrine purine receptors stimulation. Our study on native microglia describes for the first time the functional properties of stretch/swelling-activated currents, representing a key element in microglia ability to monitor the brain parenchyma.

Hyaluronan-cholesterol nanohydrogels: Characterisation and effectiveness in carrying alginate lyase

Although in recent years several methods have been studied and developed to obtain different types of nanosized drug delivery systems, the set up of suitable procedures and materials remains highly expensive, their preparation is time consuming and often not feasible for a scale-up process. Furthermore, the sterilisation and storage of nanocarrier formulations represents a complicated but mandatory step for their effective use. In our previous work we assessed the use of an autoclaving process to achieve, in one simple step, sterile self-assembled hyaluronan-cholesterol (HA-CH) and hyaluronan-riboflavin (HA-Rfv) nanohydrogels (NHs). In the present work, the effect of the high temperature on HA-CH has been studied in detail. HA-CH suspensions were characterised in terms of size and polydispersity by Dynamic Light Scattering at different temperatures and conditions; the HA-CH chemical structure and its molecular weight were assessed via FT-IR and GPC analysis after the sterilising cycle in an autoclave at 121°C for 20min. The obtained NHs were then observed with TEM and AFM microscopy, in both dry and liquid conditions. The Youngs modulus of the NHs was determined, evidencing the soft nature of these nanosystems; the critical aggregation concentration (c.a.c) of the nanosuspension was also assessed. Thereafter, alginate lyase (AL) was conjugated to NHs, with the aim of developing a useful system for therapies against bacterial infections producing alginate biofilms. The conjugation efficiency and the enzymatic activity of AL were determined after immobilisation. The AL-NHs system showed the ability to depolymerise alginate, offering an opportunity to be a useful nanosystem for the treatment of biofilm-associated infections.

Carbonate anion radical generated by the peroxidase activity of copper-zinc superoxide dismutase: scavenging of radical and protection of enzyme by hypotaurine and cysteine sulfinic acid

Copper-zinc superoxide dismutase (SOD) is considered one of the most important mammalian antioxidant defenses and plays a relevant role due to its main function in catalyzing the dismutation of superoxide anion to oxygen and hydrogen peroxide. However, interaction between SOD and H2O2 produced a strong copper-bound oxidant (Cu(II)•OH) that seems able to contrast the self-inactivation of the enzyme or oxidize other molecules through its peroxidase activity. The bicarbonate presence enhances the peroxidase activity and produces the carbonate anion radical (CO3•-). CO3•- is a freely diffusible reactive species capable of oxidizing several molecules that are unwieldy to access into the reactive site of the enzyme. Cu(II)•OH oxidizes bicarbonate to the CO3•-, which spreads out of the binding site and oxidizes hypotaurine and cysteine sulfinic acid to the respective sulfonates through an efficient reaction. These findings suggest a defense role for sulfinates against the damage caused by CO3•- . The effect of hypotaurine and cysteine sulfinic acid on the CO3•--mediated oxidation of the peroxidase probe ABTS to ABTS cation radical (ABTS•+) has been studied. Both sulfinates are able to inhibit the oxidation of ABTS mediated by CO3•-. The effect of hypotaurine and cysteine sulfinic acid against SOD inactivation by H2O2 (~42% protection of enzyme activity) has also been investigated. Interestingly, hypotaurine and cysteine sulfinic acid partially avoid the H2O2-mediated SOD inactivation, suggesting that the two sulfinates may have access to the SOD reactive site and preserve it by reacting with the copper-bound oxidant. In this way hypotaurine and cysteine sulfinic acid not only intercept CO3•- which could move out from the reactive site and cause oxidative damage, but also prevents the inactivation of SOD.

Hyaluronan‐Based Nanohydrogels for Targeting Intracellular S. Aureus in Human Keratinocytes

Staphylococcus aureus is one of the most significant human pathogens that is frequently isolated in a wide range of superficial and systemic infections. The ability of S. aureus to invade and survive within host cells such as keratinocytes and host immune cells has been increasingly recognized as a potential factor in persistent infections and treatment failures. The incorporation of antibiotics into hyaluronan-cholesterol nanohydrogels represents a novel paradigm in the delivery of therapeutic agents against intracellular bacteria. The work presented herein shows that NHs quickly enter human keratinocytes and accumulate into lysosomes. When used for targeting intracellular S. aureus the antimicrobial activity of loaded levofloxacin is enhanced, possibly changing the antibiotic intracellular fate from cytosol to lysosome. Indeed, gentamicin, an antibiotic that predominantly accumulates in lysosomes, shows significant and equal antibacterial activity when entrapped into NHs. These results strongly suggest that lysosomal formulations may display preferential activity toward intracellular S. aureus, opening new avenues for the use of HA-based NHs for treatment of such skin infections.