Innovative techniques for the extraction and quantification of bioactive compounds from food and agroindustrial by-products

The present PhD thesis is the result of my three-year research in the fields of green extraction and analytical chemistry, carried out mainly in the laboratory of Food and Dietary Products of the Department of Pharmacy (DIFAR) of Genoa University, and taking advantage of collaborations among other research groups of Genoa University, the Chemometric and Qualimetry Group of University of Burgos and the Green Extraction Team of Avignon University. The thread that runs through the entire thesis are innovative techniques both for the extraction and for the analysis of bioactive compounds, endowed with health-promoting activities or potentially harmful contaminants, occurring in different foods and agroindustrial by-products. Several green extraction techniques have been performed, with a view towards environmental sustainability as well as economic and safety considerations, in order to optimize the extraction of antioxidant compounds from Oryza sativa L. \u2018Violet Nori\u2019 and pomegranate by-products. \u2018Violet Nori\u2019 rice is particularly rich in anthocyanins. The effect of different cooking conditions on this polyphenolic content have been also investigated. Pomegranate peels and marcs can be valorised thanks to their high content in ellagic acid and ellagitannins. To improve ellagic acid bioavailability, its encapsulation in dendrimeric nanocarriers has been proposed too. With respect to the analytical methods, the innovative second order calibration of excitation-emission fluorescence matrices (EEMs) coupled with the chemometric tool PARAFAC has been applied also to another food matrix. Thanks to this technique, in fact, it has been possible to evaluate the content of carcinogenic polycyclic aromatic hydrocarbons in a commercial smoked tuna sample

Biodegradable and biocompatible spherical dendrimer nanoparticles with a gallic acid shell and a double-acting strong antioxidant activity as potential device to fight diseases from \uaboxidative stress\ubb

Gallic acid (GA) is a natural polyphenol with remarkable antioxidant power present in several vegetables and fruits. A normal feeding regime leads to a daily intake of GA which is reasonably regarded as \u2018\u2018natural\u2019\u2019 and \u2018\u2018safe\u2019\u2019 for humans. It owns strong potentials as alternative to traditional drugs to treat several diseases triggered by oxidative stress (OS), but poor gastrointestinal absorbability, pharmacokinetic drawbacks and fast metabolism limit its clinical application. In this work, a fifth generation polyester-based dendrimer was firstly prepared as a better absorbable carrier to protect and deliver GA. Then, by its peripheral esterification with GA units, a GA-enriched delivering system (GAD) with remarkable antioxidant power and high potential against diseases from OS, was achieved. Scanning Electron Microscopy results and Dynamic Light Scattering analysis, revealed particles with an average size around 387 and 375 nm respectively and an extraordinarily spherical morphology. These properties, by determining a large particles surface area, typically favor higher systemic residence time and bio-efficiency. Z-potential of -25 mV suggests satisfactory stability in solution with tendency to form megamers and low PDI. GAD showed intrinsic antioxidant power, higher than GA by 4 times and like prodrugs, it can carry contemporary several bioactive GA units versus cells. In physiological condition, the action of Pig Liver Esterase (PLE), selected as a model of cells esterase, hydrolyzes GAD to non-cytotoxic small molecules, thus setting free the bioactive GA units, for further antioxidant effects. Cytotoxicity studies performed on two cell lines demonstrated a high cell viability

By connecting a synthetic scaffold and a natural shell of Gallic acid: an innovative double-acting antioxidant device to figh \uaboxidative stress\ubb.

Oxidative stress (OS) is involved in the onset and developing of most degenerative diseases hard to fight with exsisting synthetic drugs without adverse side effects. Gallic acid (GA) (Figure 1), a natural triphenolic acid present in several plants, fruits and common foodstuffs, is provided both with the basic nutritional values and with several extra health benefits such as a remarkable antioxidant power. GA exhibited abilities in protecting cells from OS via a number of pathways without triggering unpleasant side effects.1 GA several potentials could be exploited both in industry and in medicine but unfortunately its clinical application is limited by its pharmacokinetic drawbacks, poor bioavailability, slow GIT absorption, fast metabolism and short half-life. Dendrimer nanoparticles, thanks to their nonpareil physicochemical properties are extensively exploited in nanomedicine to control molecular weight, hydrophilicity, solubility,2-4 bioavailability and pharmacokinetic behaviour of drugs as well as to protect them from early degradation or fast metabolism. With the aim at minimizing GA's limitations for medical purposes, in this study, a G5 polyester-based dendrimer was prepared and subsequently, it was peripherally further esterified with bioactive GA

SYNTHESIS OF WATER-SOLUBLE, POLYESTER-BASED DENDRIMER PRODRUGS FOR EXPLOITING THERAPEUTIC PROPERTIES OF TWO TRITERPENOID ACIDS

Dendrimers are macromolecules characterized by high controlled size, shape and architecture, presence of inner cavities able to accommodate small molecules and many peripheral functional groups to bind target entities and are of eminent interest for biomedical applications, including gene transfection, tissue engineering, imaging, drug delivery. The well-known pharmacological activities of Ursolic and Oleanolic acids are limited by their small water solubility, non-specific cells distribution, low bioavailability, poor pharmacokinetics and their direct administration could result in the release of thrombi. To overcome such problems, in this paper we described their physical incorporation inside amino acids-modified polyester-based dendrimers which made them highly water-soluble. IR, NMR, zeta potential, mean size of particles, buffer capacity and drugs release profiles of prepared materials were reported. The achieved water-soluble complexes harmonize a polycationic character and a buffer capacity which presuppose efficient cells penetration and increased residence time with a biodegradable cells respectful scaffold thus appearing a promising team of not toxic prodrugs for safe administration of Ursolic and Oleanolic acids

Traditional decoction and PUAE aqueous extracts of pomegranate peels as potential low-cost anti-tyrosinase ingredients

open6The aim of the study is to evaluate the anti-tyrosinase activity of dierent aqueous extracts obtained from pomegranate juice processing by-products. External pomegranate peels of two certified cultivars (Akko and Wonderful), were extracted using only water as the extraction solvent. A traditional decoction and a pulsed ultrasound-assisted extraction (PUAE), both 10 min long, were performed and compared. All the aqueous extracts proved to be rich in bioactive compounds. In particular, the total phenolic content (TPC) ranged from 148 to 237 mg gallic acid equivalent (GAE)/g of dried peels (DW), the radical-scavenging ability (RSA) ranged from 307 to 472 mg ascorbic acid equivalent (AAE)/g DW, the free ellagic acid content (EA) ranged from 49 to 94 g/mL, and the ellagitannins (ETs) ranged from 242 to 340 g/mL. For both cultivars, PUAE extracts had higher ET content and a lower EC50, while the decoctions had slightly higher TPC, RSA, and free EA amounts. Principal component analysis (PCA) highlighted the direct correlation between the ET content and the tyrosinase enzyme inhibition (lower values of EC50). These findings suggest the potential use of both these natural extracts as low-cost lightening and/or anti-browning ingredients exploitable in several formulations (e.g., cosmetics) or extemporarily usable.openFEDERICA TURRINI; PAOLA MALASPINA; PAOLO GIORDANI; SILVIA CATENA; PAOLA ZUNIN; RAFFAELLA BOGGIA;Turrini, Federica; Malaspina, Paola; Giordani, Paolo; Catena, Silvia; Zunin, Paola; Boggia, Raffaell

Non-PAMAM amino acids-modified dendrimers nanoparticles for enhancing water-solubility of insoluble bioactive molecules: our state of the art

Non-PAMAM amino acids-modified dendrimers nanoparticles for enhancing water-solubility of insoluble bioactive molecules: our state of the art Silvana Alfei,* Andrea Spallarossa, Silvia Catena, Federica Turrini, Guendalina Zuccari, Anna Pittaluga, Raffaella Boggia Dipartimento di Farmacia, Universit\ue0 di Genova, Viale Cembrano 4, I-16148 Genova, Italy E-mail: [email protected] ABSTRACT Water-solubility is essential for GIT absorbability or parenteral administration of drugs, therefore it is a key parameter to achieve the systemic drug concentration necessary for an effective therapeutic activity. Unfortunately, low aqueous solubility is the major problem with bioactive chemical entities (BCEs), in fact, more than 40% BCEs developed in pharmaceutical industry are practically water-insoluble. As a consequence, great are the research efforts focused on the development of new techniques aiming at enhancing it. Toxic excipients and harmful solubilizing agents were also extensively used for solubilizing and delivering non water-soluble drugs, despite the resulting unpleasant side effects complained of by patients. Nowadays, safer strategies, such as drugs physicochemical modifications or particle size reduction, crystal engineering, salt formation, solid dispersion, use of surfactant and complexation are being exploited. As far as what regards dispersion/complexation techniques, nanoparticles, including dendrimers, are intensely utilized for this purpose, thus in parallel achieving drugs protection from early degradation, more efficient target delivery into cells and tissues and lower systemic toxicity. Synthetic thiocarbamate (O-TC 1) (Fig. 1) is a non-nucleoside HIV-1 reverse transcriptase inhibitor [1] while Ellagic Acid (EA 2) (Fig. 2) is a polyphenol present in some fruits, nuts and seeds endowed with strong antioxidant, anti-inflammatory and other several healthy properties. Unfortunately, both of them are practically insoluble (Table 1), non orally bioavailable, non parenteral administrable, then non usable for therapeutic purposes in their free forms. Fig. 1: Structure of O-TC 1 Fig. 2: Structure of EA 2 Fig. 3: Examples of hydrophilic (left) and amphiphilic (right) dendrimers structure During the last year, these problems have been addressed and successfully resolved by us, and in this communication, the reached promising outcomes have been summarized and the current state of the art provided. Afar from commercially high cytotoxic PAMAM, five non cells-damaging amino acid-modified hydrophilic (3, 4) [2] and amphiphilic (5-7) [3] dendrimers (Fig. 3) have been synthetized and then used as polymer nano-containers to improve 1 and 2 water-solubility. Five (8-12) [4] and two (13, 14) [5] structurally different drugs-loaded nanodispersions (DPXs) were obtained respectively. The structures were confirmed by FT-IR and NMR analysis and all the samples have resulted in being endowed with very good Drug Loading (DL %). Compound 1, totally insoluble except for in highly diluted DMSO when free, once entrapped in dendrimers, shown to be well soluble both in water and in ethanol. In the case of 2, water-solubility was increased even up to 1000 times compared to the free form. For the prerogatives demonstrated in the performed routine analyses, the prepared DPXs could be considered eligible for biomedical and therapeutic applications thus allowing to exploit 1 and 2 pharmacological properties. REFERENCES: 1. A. Spallarossa et al., Eur. J. Med. Chem., 44, 2190 (2009). 2. S. Alfei & S. Catena, Polym. Advan. Technol., 29, 2735 (2018). 3. S. Alfei & S. Catena, Polym. Int., 67, 1572 (2018). 3. S. Alfei et al., Eur. J. Pharm. Sci., 124, 153 (2018). 4. S. Alfei et al., New J. Chem., 2019, DOI: 10.1039/c8nj05657a

Characterization data of water-soluble hydrophilic and amphiphilic dendrimers prodrugs for delivering bioactive chemical entities otherwise non soluble.

More than 40% of bioactive chemical entities (BCEs) developed in pharmaceutical industry are almost water-insoluble, poorly orally bioavailable and/or not via parenteral administrable, and this strongly limits their clinical applications. Drug Delivery (DD) is an engineered technology dealing with the development of delivery systems (DDSs) able to solubilize, transport, target release and maintain therapeutic drugs concentration where needed for long periods. DD frequently makes use of nanosized carriers, often positive charged, including dendrimer such as commercially available and strongly cationic PAMAM and PEI. Nowadays, uncharged dendrimer scaffolds modified with amino acids-modified in their cationic form, are preferred because a more controlled number of nitrogen atoms causes less damage to cells. Then, two hydrophilic (1, 2) [1] (Fig. 1) and three amphiphilic (3-5) [2] (Fig. 2) water-soluble dendrimers were prepared and completely characterized. Once established through proper routine investigations, that these materials could work well as DDSs, they have been used to physically entrap two completely insoluble BCEs i.e. the thiocarbamate (O-TC) 6 [3] and Ellagic Acid (EA) 7 (Fig. 3) with the aim at improving their solubility and in parallel at protecting them from early degradation, at promoting their fast cellular up-take and thus reducing eventual systemic toxicity. Without resorting to toxic excipients and harmful solubilizing agents often used despite the resulting unpleasant side effects, five structurally different nanodispersions (DPXs) loaded with 6 [4] and two with 7 [5] were achieved and completely characterized to confirm their structure and to evaluate their potentiality in biomedical applications

Accelerated amyloid deposition, neurofibrillary degeneration and neuronal loss in double mutant APP/tau transgenic mice

Even though the idea that amyloid β peptide accumulation is the primary event in the pathogenesis of Alzheimer's disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein processing and tau alterations in this type of dementia remains controversial. We further investigated the role of β-amyloid production/deposition in tau pathology and neuronal cell death in the mouse brain by crossing Tg2576 and VLW lines expressing human mutant amyloid precursor protein and human mutant tau, respectively. The resulting double transgenic mice showed enhanced amyloid deposition accompanied by neurofibrillary degeneration and overt neuronal loss in selectively vulnerable brain limbic areas. These findings challenge the idea that tau pathology in Alzheimer's disease is merely a downstream effect of amyloid production/deposition and suggest that reciprocal interactions between β-amyloid and tau alterations may take place in vivo.This project was funded in part by EC grant DIADEM QRLT-2000-026362, SAF2004-07802 and UTE project CIM

Accelerated amyloid deposition, neurofibrillary degeneration and neuronal loss in double mutant APP/tau transgenic mice

Even though the idea that amyloid beta peptide accumulation is the primary event in the pathogenesis of Alzheimer's disease has become the leading hypothesis, the causal link between aberrant amyloid precursor protein processing and tau alterations in this type of dementia remains controversial. We further investigated the role of beta-amyloid production/deposition in tau pathology and neuronal cell death in the mouse brain by crossing Tg2576 and VLW lines expressing human mutant amyloid precursor protein and human mutant tau, respectively. The resulting double transgenic mice showed enhanced amyloid deposition accompanied by neurofibrillary degeneration and overt neuronal loss in selectively vulnerable brain limbic areas. These findings challenge the idea that tau pathology in Alzheimer's disease is merely a downstream effect of amyloid production/deposition and suggest that reciprocal interactions between beta-amyloid and tau alterations may take place in vivo

Association of COVID-19 Vaccinations With Intensive Care Unit Admissions and Outcome of Critically Ill Patients With COVID-19 Pneumonia in Lombardy, Italy

IMPORTANCE Data on the association of COVID-19 vaccination with intensive care unit (ICU) admission and outcomes of patients with SARS-CoV-2-related pneumonia are scarce. OBJECTIVE To evaluate whether COVID-19 vaccination is associated with preventing ICU admission for COVID-19 pneumonia and to compare baseline characteristics and outcomes of vaccinated and unvaccinated patients admitted to an ICU. DESIGN, SETTING, AND PARTICIPANTS This retrospective cohort study on regional data sets reports: (1) daily number of administered vaccines and (2) data of all consecutive patients admitted to an ICU in Lombardy, Italy, from August 1 to December 15, 2021 (Delta variant predominant). Vaccinated patients received either mRNA vaccines (BNT162b2 or mRNA-1273) or adenoviral vector vaccines (ChAdOx1-S or Ad26.COV2). Incident rate ratios (IRRs) were computed from August 1, 2021, to January 31, 2022; ICU and baseline characteristics and outcomes of vaccinated and unvaccinated patients admitted to an ICU were analyzed from August 1 to December 15, 2021. EXPOSURES COVID-19 vaccination status (no vaccination, mRNA vaccine, adenoviral vector vaccine). MAIN OUTCOMES AND MEASURES The incidence IRR of ICU admission was evaluated, comparing vaccinated people with unvaccinated, adjusted for age and sex. The baseline characteristics at ICU admission of vaccinated and unvaccinated patients were investigated. The association between vaccination status at ICU admission and mortality at ICU and hospital discharge were also studied, adjusting for possible confounders. RESULTS Among the 10 107 674 inhabitants of Lombardy, Italy, at the time of this study, the median [IQR] agewas 48 [28-64] years and 5 154 914 (51.0%) were female. Of the 7 863 417 individuals who were vaccinated (median [IQR] age: 53 [33-68] years; 4 010 343 [51.4%] female), 6 251 417 (79.5%) received an mRNA vaccine, 550 439 (7.0%) received an adenoviral vector vaccine, and 1 061 561 (13.5%) received a mix of vaccines and 4 497 875 (57.2%) were boosted. Compared with unvaccinated people, IRR of individuals who received an mRNA vaccine within 120 days from the last dosewas 0.03 (95% CI, 0.03-0.04; P <.001), whereas IRR of individuals who received an adenoviral vector vaccine after 120 days was 0.21 (95% CI, 0.19-0.24; P <.001). There were 553 patients admitted to an ICU for COVID-19 pneumonia during the study period: 139 patients (25.1%) were vaccinated and 414 (74.9%) were unvaccinated. Compared with unvaccinated patients, vaccinated patients were older (median [IQR]: 72 [66-76] vs 60 [51-69] years; P <.001), primarily male individuals (110 patients [ 79.1%] vs 252 patients [60.9%]; P <.001), with more comorbidities (median [IQR]: 2 [1-3] vs 0 [0-1] comorbidities; P <.001) and had higher ratio of arterial partial pressure of oxygen (PaO2) and fraction of inspiratory oxygen (FiO(2)) at ICU admission (median [IQR]: 138 [100-180] vs 120 [90-158] mm Hg; P =.007). Factors associated with ICU and hospital mortality were higher age, premorbid heart disease, lower PaO2/FiO(2) at ICU admission, and female sex (this factor only for ICU mortality). ICU and hospital mortality were similar between vaccinated and unvaccinated patients. CONCLUSIONS AND RELEVANCE In this cohort study, mRNA and adenoviral vector vaccines were associated with significantly lower risk of ICU admission for COVID-19 pneumonia. ICU and hospital mortality were not associated with vaccinated status.These findings suggest a substantial reduction of the risk of developing COVID-19-related severe acute respiratory failure requiring ICU admission among vaccinated people