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Alzheimer's disease (AD) is a multifactorial pathology that requires multifaceted agents able to address its peculiar nature. Increasing evidence has shown that aggregation of amyloid β (Aβ) and oxidative stress are strictly interconnected, and their modulation might have a positive and synergic effect in contrasting AD-related impairments. Herein, a new and efficient fragment-based approach towards tyrosol phosphodiester derivatives (TPDs) has been developed starting from suitable tyrosol building blocks and exploiting the well-established phosphoramidite chemistry. The antioxidant activity of new TPDs has been tested as well as their ability to inhibit Aβ protein aggregation. In addition, their metal chelating ability has been evaluated as a possible strategy to develop new natural-based entities for the prevention or therapy of AD. Interestingly, TPDs containing a catechol moiety have demonstrated highly promising activity in inhibiting the aggregation of Aβ40 and a strong ability to chelate biometals such as CuII and ZnII .

A reduced proteasome activity tiles excessive amyloid growth during the progress of protein conformational diseases (PCDs). Hence, the development of safe and effective proteasome enhancers represents an attractive target for the therapeutic treatment of these chronic disorders. Here we analyze two natural diastereoisomers belonging to the family of flavonolignans, Sil A and Sil B, by evaluating their capacity to increase proteasome activity. Enzyme assays carried out on yeast 20S (y20S) proteasome and in parallel on a permanently "open gate" mutant (α3ΔN) evidenced that Sil B is a more efficient 20S activator than Sil A. Conversely, in the case of human 20S proteasome (h20S) a higher affinity and more efficient activation is observed for Sil A. Driven by experimental data, computational studies further demonstrated that the taxifolin group of both diastereoisomers plays a crucial role in their anchoring to the α5/α6 groove of the outer α-ring. However, due to the different stereochemistry at C-7" and C-8" of ring D, only Sil A was able to reproduce the interactions responsible for h20S proteasome activation induced by their cognate regulatory particles. The provided silybins/h20S interaction models allowed us to rationalize their different ability to activate the peptidase activities of h20S and y20S. Our results provide structural details concerning the important role played by stereospecific interactions in driving Sil A and Sil B binding to the 20S proteasome and may support future rational design of proteasome enhancers.

Gymnema sylvestre is a commonly used herb in Ayurvedic medicine. The demand for its extracts in the commercial and pharmaceutical fields has been steadily increasing in recent years. Its extracts are used to treat various ailments as well as for their antimicrobial properties. This study has evaluated the antimicrobial effects of different G. sylvestre extracts and of eight triterpenes isolated from the most active extract on six bacterial poultry pathogens i.e. Bacillus subtilis, Enterococcus faecalis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterobacter aerogenes. In particular, it has been evaluated the minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of all extracts and isolated triterpenes. Finally, the cytotoxicity activity of triterpenes was evaluated by MTT assay and their antioxidant activity in basal and oxidant conditions by DCFH-DA assay.

Introduction: Artemisia annua is a small herbaceous plant belonging to the Asteraceae family declared therapeutic by the World Health Organisation, in particu- lar for its artemisinin content, an active ingredient at the base of most antimalarial treatments, used every year by over 300 million people. In the last years, owing to low artemisinin content, research of new ways to increase the yield of the plant matrix has led to the use of the total extract taking advantage from the synergic and stabilising effects of the other components. Objective: In this work we evaluated and compared the content of artemisinin and scopoletin in extracts of A. annua collected in the Campania Region (southern Italy), by two different extraction processes. Methodology: Artemisia annua plants were extracted by traditional maceration (TM) in hydroalcoholic solution as a mother tincture prepared according to the Euro- pean Pharmacopeia and by pressurised cyclic solid–liquid (PCSL) extraction, a new generation method using the Naviglio extractor. Results: The results showed that the PCSL extraction technique is more effective than traditional methods in extracting both phytochemicals, up to 15 times more, reducing the extraction times, without using solvents or having risks for the opera- tors, the environment and the users of the extracts. Conclusion: The Naviglio extractor provides extracts with an artemisinin and scopoletin content eight times higher than the daily therapeutic dose, which should be evaluated for its stability over time and biological properties for possible direct use for therapeutic purposes.

Curcumin is a bioactive natural compound with a wide range of pharmacological properties, including antitumor activity; however, its clinical application has been limited because of its low solubility, stability, and bioavailability. In this study, a solid phase approach was proposed for the combinatorial synthesis of a mini library of the mimics of curcumin in good purity and yield. The non-effective findings in pancreatic cancer cells switched to strong growth inhibition and cell death efficacy for PC3 prostate cancer cells, and mimic 9, in which tyrosol (TYR) and homovanillyl alcohol (HVA) units were linked by a phosphodiester bond, was quite effective not only in cell growth inhibition but also in causing strong cell death under the study conditions and treatments that were not effective in PANC1 cells. The results got more exciting when we also consider the findings in SW480 human colorectal carcinoma cell line, where the growth inhibitor effects were more in line with that of the PC3 cells, but the lack of cell death effect was more in line with the PANC1 cells.

The setup of an economic and sustainable method to increase the production and commercialization of products from microalgae, beyond niche markets, is a challenge. Here, a cascade approach has been designed to optimize the recovery of high valuable bioproducts starting from the wet biomass of Galdieria phlegrea. This unicellular thermo-acidophilic red alga can accumulate high-value compounds and can live under conditions considered hostile to most other species. Extractions were performed in two sequential steps: a conventional high-pressure procedure to recover phycocyanins and a solvent extraction to obtain fatty acids. Phycocyanins were purified to the highest purification grade reported so far and were active as antioxidants on a cell-based model. Fatty acids isolated from the residual biomass contained high amount of PUFAs, more than those recovered from the raw biomass. Thus, a simple, economic, and high effective procedure was set up to isolate phycocyanin at high purity levels and PUFAs.

The abnormal deposition of Aβ amyloid deposits in the brain is a hallmark of Alzheimer's disease (AD). Based on this evidence, many current therapeutic approaches focus on the development of small molecules halting Aβ aggregation. However, due to the temporary and elusive structures of amyloid assemblies, the rational design of aggregation inhibitors remains a challenging task. Here we combine ThT assays and MD simulations to study Aβ aggregation in the presence of the natural compounds tyrosol (TY), 3-hydroxytyrosol (HDT), and 3-methoxytyrosol (homovanillyl alcohol - HVA). We show that albeit HDT is a potent inhibitor of amyloid growth, TY and HVA catalyze fibril formation. An inspection of MD simulations trajectories revealed that the different effects of these three molecules on Aβ1-40 aggregation are ascribable to their capacity to arrange H-bonds network between the ligand (position C-3) and the peptide (Glu22). We believe that our results may contribute to the design of more effective and safe small molecules able to contrast pathogenic amyloid aggregation.

The subsurface drip irrigation (SDI) system is a micro-irrigation technique applied below the soil surface through drip lines buried at a depth depending on the characteristics of the soil and on the plants to be irrigated. SDI distributes precise amounts of water directly to the root area, with the possibility of leaving the soil surface dry and less subject to weeds. This system reduces the use of water, herbicides, and environmental pollution. Furthermore, SDI allows the use of urban wastewater, advantageous from the environmentalpoint of view since it reduces the consumption of ground water and energy costs required for its pumping. In addition, it reduces the use of chemical fertilizers through the enhancement of organic fertilizer content in the waste. However, there are issues related to the use of SDI systems, such as the elimination or reduction of roots that wrap the dripper thus blocking the water flow. It has been hypothesized that it would be useful to add a pure or blended phytotoxic mixture to plastic during the production of drippers, whose herbicidal action dissolves gradually with the passage of water. Five species of plants have been selected in this study: Vetch villosa, Brassica juncea, Secale cereale, Juncus effusus, and Vallisneria natans. The phytotoxicity has been tested in vivo on Lactuca sativa, Lycopersicon esculentum, and Allium cepa. The plants showed the same behavior but the aerial biomass of V. natans resulted the most active ones. The phytotoxicity of the hydroalcoholic extract of each plant was evaluated on the same test organisms, with peak inhibitions up to 60, 70, and 80% at concentrations ranging from 10−4 to 10−7 M. In general, the most active hydroalcoholic infusion was that of V. villosa. Finally, after some chromatographic steps and LC/GC-MS analyses, the most abundant metabolites of the hydroalcoholic extracts were identified.

Polyphenols are active compounds from medicinal plants widely distributed in nature. Many reports suggest that the mechanisms by which plant polyphenols exert their protective actions against various diseases are in part due to their redox properties [1]. Silibinin, belonging to flavonolignan class, is the major component of Silymarin complex extracted from the fruits of milk thistle (Silybum marianum) [2]. Silibinin is a diastereoisomeric mixture of two flavonolignans, Silybin A and Silybin B, in a ratio of approximately 1:1; this metabolite has long been studied for its large variety of pharmacological properties ranging from the antioxidant to neuroprotective [3] and antiviral activities. Unfortunately, many drug candidates including Silibinin, even if have been reported to possess a strong therapeutic potential in vitro, fail in vivo because of their poor bioavailability, often connected to a low water solubility. The design and synthesis of water-soluble pro-drugs are a powerful approach in order to overcome these limits and to improve the pharmacological potentialities of these compounds [4]. In this regard, here, we report the synthesis and characterization of new phosphodiester Silibinin conjugates by the insertion of a phosphate group at the 9” position. This, besides improving the Silibinin water solubility, allows conjugation of different molecules (Figure) able to recognize cell targets and to improve Silibinin pharmaceutical properties [5-8]. The new derivatives have been tested in different radical scavenging assays. A antiviral and neuroprotective activities have also been explored.

Gymnema sylvestre has been used in Asian traditional medicine for its anti-microbial, anti-hypercholesterolemic, hepatoprotective and sweet suppressing properties and activities. G. sylvestre has also been used extensively in chewing gum, as a health food for preventing obesity and diabetes, and as a tea. This study has evaluated the total phenolic content and antioxidant activity of the aqueous and organic G. sylvestre extracts and their sub-fractions for the initial characterization of the biological properties of the isolated compounds. An in vivo cell model was used to calculate the concentration inhibiting cell growth by 50% and the ability to exert antioxidant activity. All compounds inhibit cell growth in a dose-dependent manner, with an IC 50 value ranging between 29 and 1462 μM. The effects on intracellular ROS levels are extremely variable, but it is of interest that some of the compounds appear to display an antioxidant effect.

Recently, many studies highlighted the consistent finding of irbesartan in effluents from wastewater treatment plants (WWTPs) and in some rivers and lakes in both Europe and North America, suggesting that no N80% can be removed by specific treatments. The present investigation attempts to study the chemical fate of irbesartan in a simulated chlorination step, mimicking the conditions of a WWTP. A total of six disinfection by-products were identified, five were completely new, and separated on a C-18 column by employing a gradient HPLC method. Initially, a complete mass fragmentation pathway of the drug was established with the help of MS/ TOF, and subsequently, the disinfection by-products were subjected to MS/TOF mass studies to obtain their mass and fragment pattern. The MS results helped to assign tentative structures to the disinfection products, which were verified through 1D and 2D NMR experiments. The chemical structures of the new compounds have been justified by a proposed mechanism of formation. A preliminary ecotoxicity assessment with the crus- tacean Daphnia magna showed that some of the identified by-products were up to 12-times more toxic than irbesartan.

In the last decade, a large number of evidences points to the misfolding, aggregation and accumulation of structurally abnormal proteins, termed amyloid, as a common pathogenic mechanism of a range of increasingly common human disorders, including Alzheimer`s Disease (AD) and type II diabetes mellitus (T2DM). In particular, AD and T2DM are characterized by the accumulation of the amyloid-β (Aβ) peptide in neuronal tissues and islet amyloid polypeptide (IAPP) in pancreatic cells, respectively (1), implicitly suggesting that targeting protein misfolding and self-assembly would cure the diseases. However, the failure of all clinical trials focusing on anti-aggregating drugs has clearly demonstrated that a deeper understanding of the phenomena involved in proteome maintenance is needed. It is widely known that cells express an integrated array of proteolytic machineries that control protein homeostasis (proteostasis). As a matter of fact, the latest studies suggest that pathological conditions occur when the equilibrium between the production and the clearance of the involved protein results unbalanced. The ubiquitin–proteasome system (UPS) is the major quality control pathway responsible for cellular homeostasis; it is the primary proteolytic route for misfolded proteins and ubiquitination is utilized as a degradation signal (2). Many researchers have screened a number of molecules using the whole UPS as target (proteasome activators, inhibitors of deubiquitinating enzymes DUBs or ubistatins); however, very few molecules, if any, selected by using this strategy have shown to have the potential to be pipelined to clinical trials. But, due to the multifactorial nature of protein diseases, it may be difficult to find an effective drug by screening molecules on a single target. This study evaluates the ability of silybins A and B, components of the natural product silymarin, to rescue the proteostatic balance of the amyloidogenic peptides Aβ and IAPP against several targets: proteasome, polyubiquitination, membrane protection, amyloid aggregation and oxidative stress. The obtained results evidence the important role of the stereochemistry, demonstrating the high potential of these natural compounds in anti-AD therapeutics. Our multi-target strategy to rescue proteostasis opens the door to a new approach to face these important pathologies.

During the last years, the widespread occurrence of personal care products, pesticides and stimulating drugs in water has gained much attention, especially for the evaluation of the environmental risk based on the calculation of the Predicted Environmental Concentrations (PEC) obtained from consumption data [1]. Data on the occurrence of these emerging pollutants were reported by Xing et al. (2018) in sewage treatment plant influents, effluents and sludge in many countries of the world, where they were found until to hundreds ng/L concentrations [2]. Often the products obtained by disinfection treatment could be more toxic than parent compounds especially using the chlorination process [3]. Moreover, the potential risk posed by these pollutants could be increased by their susceptibility to produce photo-transformation products [4]. Tramadol is a synthetic, centrally acting analgesic agent used for the relief of acute and chronic pain, which has been used in both human and veterinary medicine. Recent papers have reported a study on the increasing tramadol utilization associated to the mortality over the past 15 years in the UK, highlighting the high toxicity of this drug [5]. Our research is focused on the evaluation of the environmental effects of tramadol and its transformation products, mimicking the most common experimental conditions in wastewater treatment plants. After disinfection treatment, the main transformation products have been isolated and characterized by NMR and ESI-MS analyses. Furthermore, in order to evaluate the environmental eects of products, aquatic acute and chronic toxicity have been tested on Brachionus calyciorus and Ceriodaphnia dubia as well as their mutagenesis and genotoxicity tested on bacterial strains.

In the last years, G-rich oligonucleotides (GROs) able to form G-quadruplexes, have attracted considerable interest in biological and therapeutic fields. G-quadruplexes are among the most studied DNA structures because of the large variety of their biological properties ranging from anticancer to antiviral activities.(1) Recently, new findings on the anti-HIV-1 activity of ODNs forming G-quadruplex have been reported.(2) In this frame, here it is reported the synthesis of a mini-library of 5' and 3'-modified oligonucleotides with the Riboflavin (Vitamin B). The choice to insert the Riboflavin into lead sequence, has been inspired by the similarity with the hydrogen bond pattern of guanine, in addition to its excellent fluorescence properties that may be an useful tools for in vitro and in vivo studies. In order to allow the Riboflavin moiety similar to a nucleoside unit, the ribityl chain is rigidified by 2',4'-O-benzylidene group. The synthetic strategy is focused on the synthesis of two key intermidiates, the Riboflavin-Nucleoside phosphoramidite (I), useful for the synthesis of the 5'-end modified sequences and the CPG (Control Pore Glass) support anchoring the 5'-O-DMT-2',4'-O-benzyldene-riboflavin unit (II), fruitful for the synthesis of 3'-modified sequences. Using the phosphoramidite coupling automated protocol, the building block I was incorporated into oligonucleotides with a good yield. The new modified ODNs were assembled by standard automated DNA synthesis and after RP-HPLC purifications, they were analyzed by MALDI-TOF. Preliminary biophysical studies (CD, DSC and fluorescence studies) were carried out in order to evaluate the ability of modified ODNs to fold into G-quadruplex structures. Finally, their anti-HIV activity were evaluated.

The synthesis of (1'S,2'S,4'R) and (1'S,2'R,4'R) stereoisomers of 2'-fluoro-3'-thiacytidine [(2' S)-F-3TC and (2' R)-F-3TC], the earliest examples of oxathiolanyl nucleosides with a fluorine atom in the 'sugar' backbone, is herein reported. From of a variety of synthetic routes devised for their preparation, the Pummerer rearrangement of protected lamivudine sulfoxides was successfully exploited for fluorine atom introduction. Despite the presence of three potentially labile stereocenters in such a small molecule, the (2'R)-isomer of F-3TC exhibited good chemical stability after protective group cleavage. Conversely, the (2'S)-epimer suffered from weak stability, owing to the formation of an undesired cyclization product. Based on the remarkable antiviral efficacy of the parent drugs, the access to 2'-fluorinated oxathiolanyl nucleosides (and more generally, 2'-fluorinated heterocyclic nucleosides) may provide a new source of candidates with antiviral potential.

Sideritis species have been used in folk medicine for their antimicrobial, antiulcerogenic, digestive and anti-inflammatory properties. Over the years, the phytochemistry of the genus Sideritis has been studied, and various terpenoids, sterols, coumarins and especially flavonoid aglycones and glycosides have been identified. In particular, species from the Balkan Peninsula have been studied and were found to be rich in flavonoids, with valuable antioxidant activity. In the folk medicine of the Balkan countries, Sideritis raeseri is used as a herbal tea in the treatment of inflammation, gastrointestinal disorders and coughs, and also as a tonic, whereas extracts are used as a component of dietary supplements for anaemia. Its dried inflorescences are used to prepare a beverage called 'mountain tea'. In light of the considerable interest generated in the chemistry, pharmacological properties and commercial value of S. raeseri Boiss. & Heldr., we review and summarise the available literature on these plants. The review details the chemical composition of the essential oil, its mineral and polyphenol contents, the naming of these plants and their physicochemical characterisation, and the nuclear magnetic resonance spectral data and biological properties associated with the plant extracts, with a focus on their potential chemotherapeutic applications. © 2016 Society of Chemical Industry.

Phenylpropanoids are secondary metabolites widely distributed in plants. They are believed to protect human cells against oxidative stress and in turn, to prevent various diseases associated with it, such as cancer, cardiovascular, and neurodegenerative disorders as well [2,3]. Recently, many natural phenylpropanoids have emerged as a particularly promising class of small molecules able to inhibit β-amyloid aggregation and disrupt preformed amyloid fibrils that represent important targets in the development of pharmacological treatments of Alzheimer’s disease (AD) [4]. Unfortunately, natural phenylpropanoids often have poor pharmacokinetic properties and low bioavailability. In this frame an interesting approach in the design of new bioactive compounds could be the combination of two or more polyphenolic "fragments". This "natural-fragment-based drug-discovery" approach would allow the assembly, also in a combinatorial manner, of libraries based on complex polyphenols in a few steps. This strategy provides us with the possibility of easily modifying both scaffold and decorations and modulating pharmacodynamic and pharmacokinetic properties [5]. Therefore, we planned to join two "tyrosol-fragments" by a phosphodiester bridge to obtain a new class of phosphate-linked compounds [6], which are potentially more bioactive than the corresponding precursors. We report here the general synthetic strategy to obtain new phosphate-linked tyrosol dimers in good yield and their full characterization. Since oxidative stress, as well as metal ion dyshomeostasis, are known to play important roles in AD pathogenesis, preliminary studies are focused on the evaluation of their antioxidant activity, metal chelating ability and their ability to inhibit β-amyloid aggregation.

Outer membrane vesicles (OMVs) are nanostructures of 20– 200 nm diameter deriving from the surface of several Gram- negative bacteria. OMVs are emerging as shuttles involved in several mechanisms of communication and environmental adaptation. In this work, OMVs were isolated and charac- terized from Novosphingobium sp. PP1Y, a Gram-negative non-pathogenic microorganism lacking LPS on the outer membrane surface and whose genome was sequenced and annotated. Scanning electron microscopy performed on sam- ples obtained from a culture in minimal medium highlighted the presence of PP1Y cells embedded in an extracellular ma- trix rich in vesicular structures. OMVs were collected from the exhausted growth medium during the mid-exponential phase, and purified by ultracentrifugation on a sucrose gra- dient. Atomic force microscopy, dynamic light scattering and nanoparticle tracking analysis showed that purified PP1Y OMVs had a spherical morphology with a diameter of ca. 150 nm and were homogenous in size and shape. Moreover, pro- teomic and fatty acid analysis of purified OMVs revealed a specific biochemical “fingerprint”, suggesting interesting de- tails concerning their biogenesis and physiological role. More- over, these extracellular nanostructures do not appear to be cytotoxic on HaCaT cell line, thus paving the way to their future use as novel drug delivery systems.

Silibinin is the major component of an extract, known as Silymarin, obtained from the seeds of the milk thistle (Silybum marianum). Structurally, Silibinin is diastereoisomeric equimolar mixture of two flavonolignans (Silybin A and Silybin B). As extensively reported in literature, Silibinin displays multiple biological activities, most of them related to its radical scavenging activity. In the past two decades, in addition to hepatoprotective effects, Silibinin has demonstrated remarkable anti-cancer as well as cancer chemopreventive efficacy in pre-clinical cell culture and animal models of several epithelial cancers, including skin, bladder, colon, prostate, and lung [2]. Oral administration of chemotherapeutic agents is the mainstay for the treatment of disease. Sustained release formulations have been crucial for the safe and effective dosing of orally administered drugs [3]. In this frame, the synthesis of phosphodiester derivatives can improve the oral bioavailability of poorly water-soluble metabolites maintaining their pharmacologic activity [4,5]. Here, we reported the synthesis of new Silibinin conjugates with 3'-ribonucleotide units. The phosphodiester junction is typically used in pro-drug strategies, in which the phosphate group is susceptible to hydrolysis by endogenous phosphatases, allowing the release of the active compound. The new conjugates were prepared in few steps and in good yields, starting from the suitable Silibinin or Silybin building blocks and nucleotide phosphoramidites. All compounds were full characterized by NMR and Maldi-TOF and their serum stability was evaluated by HPLC analyses.

BACKGROUND Epidemiological studies have proved that tomato consumption is associated with a lower risk of developing several diseases (for example, certain types of cancers, cardiovascular diseases, macular degeneration, age-related eye disease). Many micronutrients and bioactive compounds are mainly present in peel and seeds and are lost during the processing into sauce, puree, paste and juice. RESULTS The addition of lyophilized and powdered tomato pomace enhances the properties of puree. In this paper we report the chemical and physicochemical characterization of a puree enriched with 2% dry pomace. Comparison of the analytical data of starting puree with the enriched puree showed a significant increase of all micronutrients, without the taste and appearance being compromised or altered negatively. CONCLUSION The product obtained is an example of a functional food rich in health-promoting phytochemicals, with the significant aspect of recovering a waste fraction of the tomato processing that would normally be disposed of in landfill, with associated costs and environmental impact. © 2015 Society of Chemical Industry

Polyphenols are active compounds of the medicinal plants widely distributed in nature. Despite the few and common biosynthetic origins, polyphenols encompass many subclasses of structurally different entities with a variety of pharmacological properties. Many studies suggests that the mechanisms by which plant polyphenols exert their protective actions against various diseases, in part are due to their redox properties. Silibinin belongs to flavonolignan class and is a major component (approximately 30%) of the Silymarin complex extracted from milk thistle [Silybum marianum (L.) Gaertn. Carduus marianus L., Asteraceae]. Silibinin exists as a mixture of two diastereomers: Silybin A and Silybin B. Silibinin has long been studied for a large variety of pharmacological properties ranging from its antioxidant activity to neuroprotective and antiviral activities. Unfortunately, its in vivo applications are rather hampered by its very low bioavailability mainly due to low water solubility. In an attempt to improve its biological properties and facilitate the in vivo applications, we have developed an efficient strategy for the synthesis of Silibinin modified namely Phosphate-Linked Silibinin dimers (PLSd) in which the Silibinin monomer units are linked by phosphodiester bond (Figure). Exploiting the selective protection of Silibinin’s hydroxyl groups, we carried out a synthetic approach using the phosphoramidite chemistry. Water solubility of dimers, radical scavenger activity as well as their ability to react with reactive oxygen species (ROS) were determined. In particular, dimers reactivity with 1O2 and HO∙ was determined by use of Rose Bengal (RB) and hydrogen peroxide (H2O2) as respective ROS sources. For this purpose, laser flash photolysis (LFP) experiments were coupled with a kinetic competition approach. In addition to scavenging activity, the serum stability and cytoprotective (X/XO assay on HepG2 cells) behavior of dimers were evaluated.

Many drug candidates have been reported to possess a strong therapeutic potential in vitro but they have failed in vivo because of their poor pharmacokinetic behaviour, very often due to the low water solubility. There are many formulations and drug design strategies that can be used to overcome solubility issues. The design and synthesis of soluble pro-drugs is one of the most common approach used. In this frame, the phosphate group is a useful tool for the enhancement of aqueous solubility of phenolic and other metabolites, in addition it displayed excellent chemical stability and rapid bioconversion in vivo to the parent drug by phosphatases. On the other hand, also the conjugation of specific molecules recognized by a receptor on the target cell could be a successful strategy. In our studies, we have combined both aspects through the synthesis of new phosphodiester modified Silibinin with a good water solubility, as well as, attractive antioxidant properties. In the past decade, Silibinin has received more attention due to its large variety of activities ranging from anticancer and chemopreventive actions to hypocholesterolemic, cardioprotective and neuroprotective activities. Unfortunately, the bioavailability and the therapeutic efficiency of Silibinin are rather limited by its low water-solubility. In this work, we present the synthesis of a new library of modified Silibinins and related studies of their redox behaviour. Exploiting the selective protection of the hydroxyl groups of the Silibinin, we developed an efficient strategy for the synthesis of Silibinin phosphate-based flavonolignans consisting of phosphodiester glycoconjugates and dimers of Silibinin. The water solubility, the radical scavenger efficiency and the ability to scavenge different reactive oxygen species (ROS) have been evaluated for the new phosphodiester modified Silibinins in comparison to Silibinin. Moreover, the serum stability, and their cytoprotective (X/XO assay on HepG2 cells) behaviours have been studied. The remarkable antioxidant activity and the high water solubility (compared to Silibinin) make Silibinin phosphatebased flavonolignans promising molecules for future studies.

In the last years, G-rich oligonucleotides (GROs) able to form G-quadruplexes, have attracted considerable interest in biological and therapeutic fields. G-quadruplex are among the most studied DNA structures because they are thought to be involved in important biological processes such as the modulation of gene expression; in addition they present a large variety of biological properties ranging from anticancer to antiviral activities. In all cases, the G-quadruplex formation is a crucial prerequisite for the biological effects. Recently, we reported the synthesis and full characterization of a mini-library of G quadruplex ODNs carrying aryl groups at the 5’-end through a phosphodiester bond and endowed with prominent anti-HIV activity. In this frame, we report here, the synthesis of new Riboflavin-Nucleoside (RF, Vitamin B) building block in which the ribityl chain is rigidified by 2',4' benzylidene group, the 5'-OH function is protected with DMT group and the 3’ position is functionalized with phosphoramidite. The new Riboflavin building block is incorporated into different ODN sequences able to form Gquadruplex structures using the well known phosphoramidite chemistry. This research project has been encouraged by recent studies on the use of RF as useful tool for both in vivo and in vitro studies: the Riboflavin is internalized through RF transporters and is one of the most efficient natural photosensitizers. Several modified ODN sequences have been synthesized with a solid phase synthetic approach in good yields; their biophysical and biological characterization has been carried out in order to evaluate the effect of RF-nucleotide insertion into G-quadruplex arrangements and their anti-HIV activity.

The biological relevance of tetramolecular G-quadruplexes especially as anti-HIV agents has been extensively reported in the literature over the last years. In the light of our recent results regarding the slow G-quadruplex folding kinetics of ODNs based on d(TGGGAG) sequence, here we report a systematic anti-HIV screening to investigate the impact of the G-quadruplex folding on their anti-HIV activity. In particular, varying the single stranded concentrations of ODNs, it has been tested a pool of ODN sample solutions with different G-quadruplex concentrations. The anti-HIV assays have been designed favouring the limited kinetics involved in the tetramolecular G4-association based on the d(TGGGAG) sequence. Aiming to determine the stoichiometry of G-quadruplex structures in the same experimental conditions of the anti-HIV assays, a native gel electrophoresis was performed. The gel confirmed the G-quadruplex formation for almost all sample solutions while showing the formation of high order G4 structures for the more concentrated ODNs solutions. The most significant result is the discovery of a potent anti-HIV activity of the G-quadruplex formed by the natural d(TGGGAG) sequence (IC50 = 14 nM) that, until now, has been reported to be completely inactive against HIV infection.

Potatoes are a staple food for over a billion people worldwide, a primary dietary source of carbohydrates, and a vital crop to the agricultural economy of South America, Africa, East Asia and Central Asia. Potatoes occupy third place (after rice and wheat) on the list of foods on which the world depends for food security. In particular, glyco-alkaloids contents in potatoes are potentially toxic and secondary metabolites such as α-solanine and α-chaconine are reported to be dangerous to human health. This paper describes new methods for α-solanine and α-chaconine reduction in the potato cultivar known as Marabel. The potatoes were incubated at room temperature in the dark for 24 h and the optimal experimental condition was achieved with NaOH solution at pH 12. In the assayed samples, α-solanine and α-chaconine reduction was 43% and 27% respectively. The process proposed here allows to minimize the total content of glyco-alkaloids, with respect to mode of collection, storage and cleaning of Marabel potatoes.

New silibinin phosphodiester glyco-conjugates were synthesized by efficient phosphoramidite chemistry and were fully characterized by 2D-NMR. A wide-ranging study focused on the determination of their pKa and E° values as well as on their radical scavenging activities by different assays (DPPH, ABTS+ and HRSA) was conducted. The new glyco-conjugates are more water-soluble than silibinin, and their radical scavenging activities are higher than those of silibinin. The conjugation therefore improves both the water solubilities and antioxidant activities of the flavonolignan moieties. The serum stability was evaluated under physiological conditions, and the glyco-conjugates degraded with half-lives of 40-70 h, making them useful in pro-drug approaches. We started by treating androgen-dependent prostate cancer (PCa) LNCaP cells and then expanded our studies to androgen-independent PCa PC3 and DU145 cells. In most cases, the new derivatives significantly reduced both total and live cell numbers, albeit at different levels. Anti-HIV activities were evaluated and the glucosamine-phosphate silibinin derivative showed higher activity (IC50 = 73 μM) than silibinin.

International audience; By exploiting the regioselective protection of the hydroxyl groups of silibinin along with the well-known phosphoramidite chemistry, we have developed an efficient strategy for the synthesis of new silibinin-modified species, which we have named Phosphate-Linked Silibinin Dimers (PLSd), in which the monomer units are linked by phosphodiester bonds. The antioxidant abilities of the new PLSd were estimated on HepG2 cells using DPPH free radical scavenging and xanthine/xanthine oxidase assays. The new phosphate-metabolites showed a higher anti-oxidant activity than the silibinin, as well as very low toxicity. The ability to scavenge reactive oxygen species (ROS) such as singlet oxygen () and hydroxyl radical () reveals that the two dimers are able to scavenge about two times more effectively than silibinin. Finally, solubility studies have shown that the PLSd present good water solubility (more than 20 mg·L−1) under circumneutral pH values, whereas the silibinin was found to be very poorly soluble (less than 0.4 mg·L−1) and not stable under alkaline conditions. Together, the above promising results warrant further investigation of the future potential of the PLSd as anti-oxidant metabolites within the large synthetic polyphenols field.

In ‘90s, Hotoda et al. identified a variety of tetramolecular G-quadruplexes based on the sequence d(TGGGAG) and modified at the 5’-end with aromatic groups, targeting the glycoprotein of HIV-1, gp120. In 2011 Di Fabio et al. reported the synthesis and characterization of a new mini-library of d(TGGGAG) ODNs carrying aryl groups at the 5’-end through a phosphodiester bond and endowed with prominent anti-HIV activity[2]. In order to have more information on the G-quadruplex folding depending on the modifications at 5’-end of d(TGGGAG), we have explored the kinetic aspects involved into G-quadruplex folding using electrospray mass spectrometry (ESI-MS). The studies are conducted on more active 5’-end modified ODNs in comparison with unmodified sequence d(TGGGAG). By ESI-MS experiments, we could obtain information about the number of strands, and the inner cations for each complex, moreover it is possible to detect all possible intermediate complexes and to determine their equilibrium binding constants. We followed the kinetics of G-quadruplex formation during 14 days in the presence of an internal reference (dT6), selecting some time points after addition of different buffers (150mM NH4OAc and 150mM TMAA/1mM KCl). Overall data, have reported a rapid conversion of the single strand into dimer and trimer, and slower conversion in a tetramer and into an unexpected G4-supramolecular structure: the octamer complex. The formation of octamer is confirmed also in solution by native PAGE.

Introduction: Recently, we reported the synthesis and characterization of a new mini-library of tetramolecular G quadruplexes based on the d(TGGGAG) sequence, which was previously shown to target the HIV-1 glycoprotein gp120. Oligodeoxynucleotides (ODNs) carrying aryl groups at the 5’-end through a phosphodiester bond were endowed with prominent anti-HIV activity. Biophysical studies on these aptamers demonstrated that there is no relationship between thermal stability of G4 structures and their anti-HIV activity. To identify the properties that make these tetramolecular G-quadruplexes good anti-HIV aptamers, we studied by ESI-MS the stoichiometry and the self-assembly kinetics of G-quadruplex structures based on the most active 5’-end modified d(TGGGAG) aptamers [5]. ESI-MS results showed that the conjugation of d(TGGGAG) at the 5’-end does not necessarily increase the folding rate of the G-quadruplex structure. Unexpectedly, the folding kinetics of the inactive G4 (unmodified sequence) was similar to that of the 5’-end modified sequences. In addition to the slow G4-kinetics revealed by ESI-MS studies, PAGE experiments on the modified d(TGGGAG) highlighted the strong effect of ODN concentration (single stranded) on the G4-folding kinetics. Results and Discussion: In order to clarify the role of G-quadruplex structures in the anti-HIV activity on the basis of the above kinetic studies, we investigated the anti-HIV activity of ODNs at different concentrations of G-quadruplex structure. All ODNs, as well as the unmodified sequence, were evaluated against a variety of HIV-1 viruses and resistant strains. SPR experiments were also carried out to evaluate their binding to the HIV envelope gp120 and gp41 proteins. Almost all compounds showed a good anti-HIV activity, suggesting the absence of a straight correlation between the amount of available G-quadruplex and anti-HIV activity of ODNs. Surprisingly, we found a strong anti-HIV activity of the unmodified sequence d(TGGGAG), in contrast to what has been extensively reported by Hotoda and others, who showed a lack of anti-HIV activity of the d(TGGGAG) sequence without 5’-end modifications. Also, by SPR experiments no straight correlation between gp120 binding and anti HIV activity was observed. Conclusions: The anti-HIV activity results highlight the absence of a straight correlation between the formation of G-quadruplex structures and the antiviral potential of ODNs, suggesting instead that the G4 is not the specie providing anti-HIV activity. In addition, anti-HIV data do not strictly correlate with the SPR results, suggesting that gp120 and gp41 is not the primary target of these aptamers.

Polyphenols are active compounds of the medicinal plants widely distributed in nature. Despite the few and common biosynthetic origins, polyphenols encompass many subclasses of structurally different entities with a variety of pharmacological properties. Many studies suggest that the mechanisms by which plant polyphenols exert their protective actions against various diseases, are in part due to their redox properties.1Silibinin belongs to flavonolignan class, it is a major component of the Silymarin complex extracted from the milk thistle (Silybum marianum). Silibinin has long been studied for its large variety of pharmacological properties ranging from the antioxidant activity to neuroprotective and antiviral activities. In order to expand the repertoire of the modified Silibinins with high molecular diversity and good water solubility, here, we present a new and efficient method for the synthesis of dimers and trimers of the Silibinin, in which the monomer units are linked by phosphodiester bond Exploiting the selective protection of the Silibinin hydroxyl groups, we carried out a new synthetic approach using the well-established phosphoramidite chemistry. Aiming at the discovery of new pharmacological activities of these molecules, the water solubility, the radical scavenger activity by DPPH test, as well as, their ability to scavenge different reactive oxygen species (1O2 and HO●) have been studied. Additionally, the serum stability and cytoprotective behaviour (X/XO assay on HepG2 cells) of dimers have been evaluated. The combination of both striking antioxidant ability and high water solubility, makes these molecules, promising synthetic metabolites for future therapeutic applications.