Isolation and Structure Elucidation of Bioactive Secondary Metabolites from Marine and Terrestrial Organisms.

Natural products have historically been a rich source of “lead compounds” in drug discovery. The investigation of terrestrial plants and marine organisms aimed at searching new biologically active compounds is a central issue of this kind of studies, trough structure elucidation combined with biological tests. My research work, described in this PhD thesis, is included in this research topic and was addressed at three different topics: - cannabinoids from Cannabis sativa and from the Indonesian sponge Dasychalina sp.; - isolation and synthesis of antimalarial compounds; - metabolites isolated from plants belonging to the Euphorbiaceae family (Jatropha curcas, Euphorbia macroclada and Euphorbia bungei). A phytochemical investigation of the fibre cultivar of Cannabis sativa derived from the historical Carmagnola variety led to isolation of the novel spiranic stilbenoid isocannabispiradienone and the biphenyl-type cannabinoid cannabioxepane (CBX), a tetracyclic compound characterized by an unprecedented C-5/C-8’ oxygen bridge and devoid of cannabinoid activity. In the same area, from the polar organic extract of the Indonesian sponge Dasychalina sp., I have isolated haplosamate A, a unique C28 sterol containing a sulfate group at C-3 and a methyl phosphate at C-15, along with its new desulfo analogue. Both compounds, as well as their semi-synthetic analogues, have been evaluated for interaction with CB1 and CB2 receptors through a binding test. Desulfohaplosamate showed a selective affinity for CB2 receptors in the low μM range. Haplosamate derivatives represent the first CB receptor ligands belonging to the class of steroids. The search for antimalarial lead compounds is another main topics of my Ph.D. activity discussed in this thesis. On the bases of the previously developed pharmacophore of plakortin antimalarials, in this thesis I report a synthesis of a new series of simple endoperoxides, characterized by a 3-methoxy-1,2-dioxane scaffold. In particular, I have contributed to the design, the synthesis, the biological evaluation of a novel series of compounds obtained by means of an efficient one-pot three-component Mn(III)-mediated synthesis which utilizes cheap starting materials. The obtained molecules are 3,6,6-trisubstituted 3-methoxy-1,2-dioxanes with simple alkyl chains and bearing an ester group at position 4, which were tested against Pf. As part of the ongoing research aimed at finding new antimalarial leads from natural sources, I have carried out a detailed phytochemical investigation of the fruit of an African sample of Azadirachta indica, collected in Burkina Faso. Eight known and two new triterpenoid derivatives, named neemfruitins A and B, have been isolated from the fruits of neem, Azadirachta indica A. Juss., a traditional antimalarial plant used by Asian and African populations. In vitro antiplasmodial tests carried out with the isolated metabolites evidenced a significant activity of the known gedunin and azadirone and the new neemfruitin A, and provided useful information about the structure-antimalarial activity relationships in the limonoid class. Finally, the chemical investigation of Euforbiaceae plants carried out during my Ph.D., and described in this thesis, led to isolation of many diterpenoids. From the root barks of Jatropha curcas I isolated spirocurcasone, a diterpenoid possessing the unprecedented “spirorhamnofolane” skeleton, along with 11 known and two other new diterpenoids. The absolute configuration of spirocurcasone was established using quantum mechanical calculation of the electronic circular dichroic (ECD) spectrum. Some of the isolated diterpenoids showed a potent activity against L5178Y, a mouse lymphoma cell line. The phytochemical investigation of Euphorbia macroclada Boiss. and Euphorbia bungei, led to isolation of many new diterpenoids belonging to the jatrophane and pre-myrsinane classes. The main interest in these compounds is related to their biological activity as glycoprotein-P inhibitors and so modulators of multidrug resistance in cancer chemotherapy. The structures of all new compounds were elucidated using modern spectroscopic methods including 2D NMR and HRMS

Linear Aminolipids with Moderate Antimicrobial Activity from the Antarctic Gram-Negative Bacterium Aequorivita sp.

The combination of LC-MS/MS based metabolomics approach and anti-MRSA activity-guided fractionation scheme was applied on the Gram-negative bacterium Aequorivita sp. isolated from shallow Antarctic sea sediment using a miniaturized culture chip technique. This methodology afforded the isolation of three new (1⁻3) and four known (4⁻7) N-terminal glycine- or serine-bearing iso-fatty acid amides esterified with another iso-fatty acid through their C-3 hydroxy groups. The chemical structures of the new compounds were elucidated using a set of spectroscopic (NMR, [α]D and FT-IR) and spectrometric (HRMS, HRMS/MS) methods. The aminolipids possessing an N-terminal glycine unit (1, 2, 4, 5) showed moderate in vitro antimicrobial activity against MRSA (IC50 values 22⁻145 μg/mL). This is the first in-depth chemistry and biological activity study performed on the microbial genus Aequorivita

Occurrence of quantitative genetic polymorphism at the caprine beta-CN locus, as determined by a proteomic approach

Genetic screening of caseins in caprine milk samples enabled the identification of two novel b-casein (b- CN) phenotypes, C2 and F1, expressed at lower levels (3.3 and 2.7 g L_1 per allele, respectively) than reference b-CN C (4.7 g L_1 per allele), and another lacking in aS-complex. The b-CN C2 and F1 primary structures, determined by MS analysis, corresponded to b-CN C and F, respectively. The reasonable hypothesis supporting the low expression of both variants considers the C2 and F1 alleles as deriving from b-CN C1 and b-CN A1 precursors, respectively, and characterised by a genic transition C/T negatively affecting the mRNA stability. Further, phosphorylation of Thr41 responsible for the 7P b-CN component was determined by a proteomic approach for the first time. Caprine milk containing low levels of antigenic aS1-, aS2- and b-CN can be used for infant formula production and drinking milk for people with cows' milk protein allergy

Phenolic Profiles of Red Wine Relate to Vascular Endothelial Benefits Mediated by SIRT1 and SIRT6

Dietary phenolic compounds possess potent bioactivity against inflammatory pathways of chronic inflammatory conditions, such as type 2 diabetes. Here, the phenolic profile and bioac-tivity of Italian red wines Gaglioppo, Magliocco, and Nerello Mascalese were characterized. NMR, HPLC/UV-Vis and spectrophotometric characterization showed that Magliocco was the richest wine in monomeric anthocyanins (two-fold), catechins, and low molecular weight phenolics (LMWP). A positive correlation was observed between the polyphenolic content and antioxidant capacity (p < 0.05), with Magliocco displaying the highest antioxidant capacity (p < 0.01). In vitro evidence on the endothelial cell models of insulin resistance and hyperglycemia showed the ability of Magliocco to reduce reactive oxygen species (ROS) (p < 0.01) and cytokine release (p < 0.01) and to upregulate SIRT1 and SIRT6 (p < 0.01). On the whole, the results indicated that the quantitative and qualitative phenolic profiles of red wines influence their in vitro beneficial effects on oxidative and proinflammatory milieu in endothelial cells, showing a positive modulation of SIRT1 and SIRT6, both implied in vascular aging

Impact of repeated NeemAzal®-treated blood meals on the fitness of Anopheles stephensi mosquitoes

Background: Herbal remedies are widely used in many malaria endemic countries to treat patients, in particular in the absence of anti-malarial drugs and in some settings to prevent the disease. Herbal medicines may be specifically designed for prophylaxis and/or for blocking malaria transmission to benefit both, the individual consumer and the community at large. Neem represents a good candidate for this purpose due to its inhibitory effects on the parasite stages that cause the clinical manifestations of malaria and on those responsible for infection in the vector. Furthermore, neem secondary metabolites have been shown to interfere with various physiological processes in insect vectors. This study was undertaken to assess the impact of the standardised neem extract NeemAzal® on the fitness of the malaria vector Anopheles stephensi following repeated exposure to the product through consecutive blood meals on treated mice. Methods: Batches of An. stephensi mosquitoes were offered 5 consecutive blood meals on female BALB/c mice treated with NeemAzal® at an azadirachtin A concentration of 60, 105 or 150 mg/kg. The blood feeding capacity was estimated by measuring the haematin content of the rectal fluid excreted by the mosquitoes during feeding. The number of eggs laid was estimated by image analysis and their hatchability assessed by direct observations. Results: A dose and frequency dependent impact of NeemAzal® treatment on the mosquito feeding capacity, oviposition and egg hatchability was demonstrated. In the 150 mg/kg treatment group, the mosquito feeding capacity was reduced by 50% already at the second blood meal and by 50 to 80% in all treatment groups at the fifth blood meal. Consequently, a 50 – 65% reduction in the number of eggs laid per female mosquito was observed after the fifth blood meal in all treatment groups. Similarly, after the fifth treated blood meal exposure, hatchability was found to be reduced by 62% and 70% in the 105 and 150 mg/kg group respectively. Conclusions: The findings of this study, taken together with the accumulated knowledge on neem open the challenging prospects of designing neem-based formulations as multi-target phytomedicines exhibiting preventive, parasite transmission-blocking as well as anti-vectorial properties. Keywords: Malaria, Vectors, Neem, Azadirachtin, Transmission-blocking, Anti-vectoria

Mapping the Surface Microbiome and Metabolome of Brown Seaweed Fucus vesiculosus by Amplicon Sequencing, Integrated Metabolomics and Imaging Techniques

the brown alga Fucus vesiculosus is a keystone marine species, which is subject to heavy surface colonisation. this study was designed to analyse the surface epibiome of F. vesiculosus in conjunction with the composition and spatial distribution of its surface metabolome. the amplicon sequencing, seM and CARD-FIsH imaging studies showed Alphaproteobacteria to predominate the epibiotic bacteria. Fungi of the class Eurotiomycetes were visualised for the first time on an algal surface. An untargeted metabolomics approach using molecular networks, in silico prediction and manual dereplication showed the differential metabolome of the surface and the whole tissue extracts. In total, 50 compounds were putatively dereplicated by UPLC-MS/MS, 37 of which were previously reported from both seaweeds and microorganisms. Untargeted spatial metabolomics by DESI-Imaging MS identified the specific localisation and distribution of various primary and secondary metabolites on surface imprints and in algal cross sections. The UPLC-MS, DESI-IMS and NMR analyses failed to confirm the presence of any surface-associated metabolite, except for mannitol, which were previously reported from F. vesiculosus. This is the first study analysing the seaweed surface microbiome in conjunction with untargeted surface metabolomics and spatial metabolomics approaches

Antiviral Activity of Vitis vinifera Leaf Extract against SARS-CoV-2 and HSV-1

Vitis vinifera represents an important and renowned source of compounds with significant biological activity. Wines and winery bioproducts, such as grape pomace, skins, and seeds, are rich in bioactive compounds against a wide range of human pathogens, including bacteria, fungi, and viruses. However, little is known about the biological properties of vine leaves. The aim of this study was the evaluation of phenolic composition and antiviral activity of Vitis vinifera leaf extract against two human viruses: the Herpes simplex virus type 1 (HSV-1) and the pandemic and currently widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). About 40 phenolic compounds were identified in the extract by HPLC-MS/MS analysis: most of them were quercetin derivatives, others included derivatives of luteolin, kaempferol, apigenin, isorhamnetin, myricetin, chrysoeriol, biochanin, isookanin, and scutellarein. Leaf extract was able to inhibit both HSV-1 and SARS-CoV-2 replication in the early stages of infection by directly blocking the proteins enriched on the viral surface, at a very low concentration of 10 μg/mL. These results are very promising and highlight how natural extracts could be used in the design of antiviral drugs and the development of future vaccines

Successful surgical repair of left ventricular pseudoaneurysm in a patient with subacute ST-elevation myocardial infarction.

We report a successful surgical repair of left ventricular pseudoaneurysm in a patient with subacute ST-elevation myocardial infarction (STEMI). In the case of expansion of the infarct related ventricular wall, early (within 24 hours) or late (3-5 days after STEMI) cardiac rupture should be always borne in mind in order to proceed to life saving prompt surgical repair

Aurantoside J: a New Tetramic Acid Glycoside from Theonella swinhoei. Insights into the Antifungal Potential of Aurantosides

The chemical investigation of an Indonesian specimen of Theonella swinhoei afforded four aurantosides, one of which, aurantoside J (5), is a new compound. The structure of this metabolite, exhibiting the unprecedented N-α-glycosidic linkage between the pentose and the tetramate units, has been determined through detailed spectroscopic analysis. The four obtained aurantosides have been tested against five fungal strains (four Candida and one Fusarium) responsible of invasive infections in immuno-compromised patients. The non-cytotoxic aurantoside I (4) was the single compound to show an excellent potency against all the tested strains, thus providing valuable insights about the antifungal potential of this class of compounds and the structure-activity relationships