Vito Rizzi

α-Cyclodextrin encapsulation of supercritical CO2 extracted oleoresins from different plant matrices: A stability study

Here we describe the encapsulation in α-cyclodextrins (α-CDs) of wheat bran, pumpkin and tomato oleoresins, extracted by supercritical carbon dioxide, to obtain freeze-dried powders useful as ready-to-mix ingredients for novel functional food formulation. The stability of tocochromanols, carotenoids and fatty acids in the oleoresin/α-CD complexes, compared to the corresponding free oleoresins, was also monitored over time in different combinations of storage conditions. Regardless of light, storage at 25°C of free oleoresins determined a rapid decrease in carotenoids, tocochromanols and PUFAs. α-CD encapsulation improved the stability of most bioactive compounds. Storage at 4°C synergized with encapsulation in preventing degradation of bioactives. Unlike all other antioxidants, lycopene in tomato oleoresin/α-CD complex resulted to be more susceptible to oxidation than in free oleoresin, likely due to its selective sequestration from the interaction with other lipophilic molecules of the oleoresin.

Rose Bengal-photosensitized oxidation of 4-thiothymidine in aqueous medium: evidence for the reaction of the nucleoside with singlet state oxygen

The photoreactivity of 4-thiothymidine (S(4)TdR) under visible light in the presence of Rose Bengal (RB), acting as a photosensitizer, was investigated in aqueous solutions at pH 7 and 12, using UV-vis, FTIR-ATR and (1)H-NMR spectroscopic techniques, time resolved absorption spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Evidence for the generation of thymidine (TdR) as the main product, after one hour of irradiation, was obtained from UV-Vis data, that suggested 4-thiothymidine photodegradation to be faster at basic pH, and confirmed by FTIR-ATR and (1)H-NMR data. Clues for the presence of a further product, likely corresponding to a dimeric form of S(4)TdR, were obtained from the latter techniques. Besides indicating the presence of thymidine, the ESI-MS and MS/MS spectra of the reaction mixtures enabled the identification of the additional product as a S-S bridged covalent dimer of 4-thiothymidine. The concentration of the dimeric species could be estimated with the aid of (1)H-NMR data and was found to be lower than that of thymidine in pH 7 reaction mixtures and almost negligible in the pH 12 ones. From a mechanistic point of view, time-resolved absorption spectroscopy measurements provided direct evidence that the formation of the two products cannot be ascribed to a photoinduced electron transfer involving S(4)TdR and the excited triplet state of RB. Rather, their generation can be interpreted as the result of a bimolecular reaction occurring between singlet state oxygen ((1)O2), photogenerated by RB, and S(4)TdR, as demonstrated by the direct detection of (1)O2 through IR luminescence spectroscopy. More specifically, a sequential reaction pathway, consisting in the generation of an electrophilic hydroxylated form of S(4)TdR and its subsequent, rapid reaction with S(4)TdR, was hypothesized to explain the presence of the S-S bridged covalent dimer of 4-thiothymidine in the reaction mixtures. The described processes make S(4)TdR an interesting candidate in the role of molecular probe for the detection of (1)O2 under different pH conditions.

Detailed investigation of ROS arisen from chlorophyll a/Chitosan based-biofilm.

The aim of this work is to study the nature of reactive oxygen species, ROS, arisen from Chitosan/2-HP-β-Cyclodextrin/Chlorophyll a (CH/CD/Chla) blended biofilm under a photodynamic activity. Suitable molecules, called primary acceptors, able to react selectively with ROS, in turn generated by the photosensitizer (PS), herein Chla, are used to attempt this purpose. The changes of the absorption and the emission spectra of these acceptors after the irradiation of aqueous solution containing the active biofilm have provided the specific nature of ROS and thus the main pathway of reaction followed by PS, in our condition. The (1)O2 formation was unveiled using Uric Acid (UA) and 9,10-diphenilanthracene (DPA). On the other hand, 2,7- dichlorofluorescin and Ferricytochrome c (Cyt-c) were used to detect the formation of hydrogen peroxide and superoxide radical anion, respectively. Results suggest that among the possible pathways of reaction, namely Type I and Type II, potentially followed by PSs, in our condition the hybrid biofilm CH/CD/Chla follows mainly Type II mechanism with the formation of (1)O2. However, the latter is involved in subsequent pathway of reaction involving Chla inducing, in addition, the formation of O2(-) and H2O2.

pH-related features and photostability of 4-thiothymidine in aqueous solution: an investigation by UV-visible, NMR and FTIR-ATR spectroscopies and by electrospray ionization mass spectrometry

The pH-related characteristics of 4-thiothymidine and its stability during prolonged exposure, at room temperature, to a neon lamp emitting in the 400–700 nm wavelength range were investigated by different spectroscopic techniques (UV-Vis, FTIR-ATR, 1H-NMR) and by ElectroSpray Ionization Mass Spectrometry (ESI-MS). The evaluation of the nucleoside photostability was performed as a control, with the perspective of studying its reactivity under the same conditions but in the presence of visible light-absorbing photosensitizers, able to generate reactive oxygen species. The comparison between UV-Vis spectra recorded at different pH values in the 7–12 range suggested the presence of an equilibrium related to the deprotonation of the N3–H group of 4-thiothymidine, with a pKa estimated to be close to 9. Some effects of the deprotonation occurring at alkaline pH were observed also in FTIR-ATR spectra, the main feature being the appearance of a band related to CN stretching, interpreted with the assumption of a partial double bond character by C2–N3, N3–C4 and C2–O− bonds, as a consequence of negative charge delocalization on the pyrimidine ring. As for photostability, UV-Vis, FTIR-ATR and NMR measurements suggested the generation of thymidine as a by-product but only after a prolonged (48 hours) irradiation time, whereas no significant alteration occurred in a shorter time range (1–2 hours), i.e. the one that will be considered in future studies involving the presence of photosensitizers. The nucleoside stability up to 2 hours of irradiation was confirmed by ESI-MS analyses; furthermore, on the other hand, the latter indicated the presence of three additional by-products, besides thymidine, after 48 hours of irradiation. In particular, an hydroxylated form of 4-thiothymidine and two dimeric species, characterized by S–S and S–O covalent bridges between two 4-thiothymidine and a 4-thiothymidine and a thymidine molecule, respectively, were detected.

One pot environmental friendly synthesis of gold nanoparticles using Punica Granatum Juice: A novel antioxidant agent for future dermatological and cosmetic applications

HYPOTHESIS The interesting properties of Gold Nanoparticles (AuNPs) make them attractive for different application fields such as cosmetology, medicine and clinical nanotechnologies. In this work a fast, easy and eco-friendly method for the AuNPs synthesis is proposed by using the Punica Granatum Juice (PGJ) with potential dermatological and cosmetic applications. The AuNPs antioxidant activity, due to the presence of phenols from the juice, and their use as booster for improving the Sun Protection Factor (SPF) in commercial sunscreen formulations, are thus expounded. EXPERIMENTS By using appropriate amounts of PGJ and HAuCl4, under mild work conditions, AuNPs with a mean size of 100 ± 40 nm are observed and carefully characterized. Solution pH, temperature, and volume were also changed for optimizing the AuNPs formation and features. The antioxidant activity was studied, by evaluating the AuNP ability of scavenging the radical 2,2-diphenyl-1-picrylhydrazylhydrate (DPPH). This finding was confirmed performing special experiments focused on the reaction between AuNPs and H2O2, by using suitable probes, such as 4-thiothymidine (S4TdR) and Cytochrome-c (Cyt-c). The SPF value was also calculated. FINDINGS The synthetized AuNPs showed a surface plasmon in visible range at 577 nm and resulted stable for long time in aqueous medium, also changing the pH values in the range 2-12. The studied antioxidant activity, confirmed also by performing special experiments with suitable probes, demonstrated the high performance of AuNPs. The AuNP photostability under sun irradiation is also shown. The calculated SPF values were in the range 3-18, related to AuNPs concentration in the range 1.80 × 10-12-1.00 × 10-11 M. The same AuNPs concentrations were used for cellular experiments. Indeed, since the AuNPs-PGJ mediated will be potentially introduced by dermal contact, dermal fibroblasts (Human Dermal Fibroblasts, HDF) and Human Microvascular Endothelial Cells (HMVEC) were used to evaluate the possible effects of these nanoparticles as a preliminary step. The results indicated that an AuNP concentrations in the range 1.80 × 10-12-3.60 × 10-12 M could be adopted since they do not appeared cyctotoxic.

Interactions between 4-thiothymidine and water-soluble cyclodextrins: Evidence for supramolecular structures in aqueous solutions

Summary Since several years the inclusion of organic compounds (guests) within the hydrophobic cavity (host) of cyclodextrins (CDs) has been the subject of many investigations. Interestingly, the formation of inclusion complexes could affect the properties of the guest molecules and, for example, the influence of the delivery system can be a method to improve/change the photochemical behavior of the guest. In particular, very recent studies have shown the protective role of CDs preventing the degradation of the encapsulated guest. Starting from this consideration, in this work, only the structure and complexation mode of the inclusion complexes involving 4-thiothymidine (S4TdR, a known photosensitizer) and five CDs, namely 2-hydroxypropyl-α-cyclodextrin (2-HP-α-CD), 2-hydroxypropyl-β-cyclodextrin (2-HP-β-CD), 2-hydroxypropyl-γ-cyclodextrin (2-HP-γ-CD), heptakis-(2,6-di-O-methyl)-β-cyclodextrin (DIMEB CD) and heptakis-(2,3,6-tri-O-methyl)-β-cyclodextrin (TRIMEB CD) were investigated by different spectroscopic techniques (UV–vis, FTIR–ATR, 1H NMR) and cyclic voltammetry analysis (CV). This work is necessary for a prospective research on the photoreactivity of S4TdR in aqueous environment and in the presence of CDs to prevent its degradation under irradiation. UV–vis, FTIR–ATR and CV measurements suggested the formation of supramolecular structures involving the employed CDs and mainly the pyrimidine ring of S4TdR. 1H NMR analyses confirmed such indication, unveiling the presence of inclusion complexes. The strongest and deepest interactions were suggested when TRIMEB and DIMEB CDs were studied. The S4TdR affinity towards CDs was also evaluated by using the Benesi–Hildebrand (B–H) equation at 25 °C employing CV and 1H NMR methods. The stoichiometry of the interaction was also inferred and it appears to be 1:1 for all examined CDs.

Chlorophyll a in cyclodextrin supramolecular complexes as a natural photosensitizer for photodynamic therapy (PDT) applications

Chlorophyll a (Chl a), an amphipathic porphyrin, was employed as natural photosensitizer for photodynamic therapy applications. Due to its lacking solubility in water and high tendency to aggregate, Chl a was included into different modified cyclodextrins (CDs) to form stable water-soluble supramolecular complexes. To achieve this aim, 2-Hydroxypropyl-β-cyclodextrin (2-HP-β-CD), 2-Hydroxypropyl-γ-cyclodextrin (2-HP-γ-CD), Heptakis(2,6-di-o-methyl)-β-cyclodextrin (DIMEB) and Heptakis(2,3,6-tri-o-methyl)-β-cyclodextrin (TRIMEB) were used. The chemical physical properties of Chl a/CD complexes in cellular medium were studied by means of UV-Vis absorption spectroscopy. Results demonstrated the good aptitude of 2-HP-γ-CD, and more particularly of 2-HP-β-CD, to solubilize the Chl a in cell culture medium in monomeric and photoactive form. Then, Chl a/2-HP-β-CD and Chl a/2-HP-γ-CD complexes were evaluated in vitro on human colorectal adenocarcinoma HT-29 cell line, and cytotoxicity and intracellular localization were respectively assessed. Further tests, such as phototoxicity, ROS generation, intracellular localization and mechanism of cell death were then focused exclusively on Chl a/2-HP-β-CD system. This complex exhibited no dark toxicity and a high phototoxicity toward HT-29 cells inducing cell death via necrotic mechanism. Therefore, it is possible to affirm that Chl a/2-HP-β-CD supramolecular complex could be a promising and potential formulation for applications in photodynamic therapy.

Gold-chlorophyll a-hybrid nanoparticles and chlorophyll a/cetyltrimethylammonium chloride self-assembled-suprastructures as novel carriers for chlorophyll a delivery in water medium: Photoactivity and photostability

The stability of Chlorophyll a in water during prolonged exposure, at room temperature, to a neon lamp has been investigated by means of UV-vis and fluorescence spectroscopies. In addition, the Chlorophyll a (photo)stability evaluation in presence of suitable carriers has been performed in order to investigate its reactivity under the same conditions, for possible and future applications in Antimicrobial Photodynamic Therapy. Cetyltrimethylammonium chloride was chosen to solubilize Chlorophyll a in water. While, cetyltrimethylammonium chloride-capped gold nanoparticles offer a great opportunity because combine the Chlorophyll a action, used as a photosensitizer in Antimicrobial Photodynamic Therapy, with gold nanoparticles effect used in photothermal therapy. Indeed, the latter ones have exhibited an interesting rise of temperature if irradiated with visible light. Overall, both examined systems, cetyltrimethylammonium chloride/Chlorophyll a and gold nanoparticles/Chlorophyll a, were able to induce the Reactive Oxygen Species formation fundamental for a potential application in Antimicrobial Photodynamic Therapy.