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Il Rapporto Tecnico fornisce un quadro dettagliato delle competenze tecnico-scientifiche presenti nell'Istituto di Scienze dell'Alimentazione, al fine di costituire una nuova Infrastruttura di Servizi alla Ricerca (ISR) che possa favorire la collaborazione con le istituzioni accademiche e gli enti di ricerca, sia nazionali che stranieri, operanti nell'ambito delle scienze dell'alimentazione.

Our work aimed to investigate the phenolic composition and antioxidant capacity of the de-alcoholized extract of cider obtained from the Annurca apple (Malus domestica var. Annurca). The antimicrobial effect of the extract against different pathogens, including Chronobacter sakazakii, was also examined. The extract's potential anti-quorum-sensing (AQS) activity was assessed using the test microorganism Chromobacterium violaceum. Biochemical analysis of the extract using ultra-performance liquid chromatography revealed catechin and caffeic acid as the most abundant polyphenols present, which represented about 35.5% and 36.6% of the total phenolics identified, respectively. An antioxidant capacity was also found (50% effective concentraiton=10 μL). The extract exhibited clear antimicrobial activity against all strains used in the experiments. Escherichia coli and Bacillus cereus were the most sensitive bacteria to the antimicrobial activity. The extract also inhibited the growth of the emergent pathogen strain C. sakazakii. The AQS activity of apple cider is reported here for the first time. In conclusion, our results demonstrate some biological properties of the apple cider and contribute to reinforcing the potential of the apple and its derivatives as functional components of the diet.

Three exopolysaccharides, EPS(1), EPS(2), and EPS(3), were isolated from suspension-cultured cells of tomato (Lycopersicon esculentum, var. San Marzano). The partial primary structures were determined on the basis of spectroscopic analyses. EPS(2) was a heteropolysaccharide with a tetrasaccharide repeating unit constituted by sugars having one residue in alpha-manno, one residue in beta-manno, and two different residues in beta-gluco/galacto configurations. EPS(3) was a heteropolysaccharide with a pentasaccharide repeating unit with sugars having three residues in alpha-manno, one residue in alpha-gluco/galacto, and one residue in beta-gluco/galacto configurations. The anticytotoxic activities of exopolysaccharides were tested in a brine shrimp bioassay.

Together with the previously described cacospongionolide B (1) and cacospongionolide F (2) a new C21 terpene (3), related to cacospongionolide B has been isolated from the sponge Fasciospongia cavernosa collected in the Aegean Sea. The structure and relative stereochemistry of the new compound, were proposed on the basis of Spectroscopic data.

Exopolysaccharide (EPS) production and chemical composition of heterocystous and non-heterocystous cyanobacteria of subgroups 3, 4, and 5 together with four new species isolated from Pantelleria (Italy) hard sands and from a lake in the proximity of Edmonson Point in Antarctica were studied. The yield of extracellular soluble polysaccharides (RPS) and slime layers (CPS) showed a significant variation from one strain to another; generally RPS were more abundant than CPS with the exception of Anabaena torulosa and Scytonema hoffmanni. Phormidium sp. was the best producer of EPS. Among heterocystous cyanobacteria belonging to Nostocaceae the new species Anabaena WSAF and A. torulosa were found to produce the highest level of EPS. Among heterocystous cyanobacteria other than Nostocaceae, the best producer of EPS was Chlorogloeopsis sp. 6912. Four species were chosen for studying the effects of growth conditions, nutritional, physical and chemical, on total EPS formed. Glucose and galactose were the neutral sugars widely present in the species examined, although other sugars such as mannose and xylose were plentiful in some species. Amino-sugar, as glucosamine, and uronic acids were also found in EPS of some cyanobacteria. Finally the inhibition of avarol, a sesquiterpene hydroquinone, toxicity on Artemia salina by EPS of some species was analysed.

Cacospongionolide B is a novel marine metabolite isolated from the sponge Fasciospongia cavernosa. In in vitro studies, this compound inhibited phospholipase A2 (PLA2), showing selectivity for secretory PLA2 (sPLA2) versus cytosolic PLA2 (cPLA2), and its potency on the human synovial enzyme (group II) was similar to that of manoalide. This activity was confirmed in vivo in the 8 h zymosan-injected rat air pouch, on the secretory enzyme accumulating in the pouch exudate. Cacospongionolide B, that is bioavailable when is given orally, reduced the elevated levels of sPLA2 present in paw homogenates of rats with adjuvant arthritis. This marine metabolite showed topical anti-inflammatory activity on the mouse ear oedema induced by 12-O-tetradecanoylphorbol acetate (TPA) and decreased carrageenin paw oedema in mice after oral administration of 5, 10 or 20 mg kg−1. In the mouse air pouch injected with zymosan, cacospongionolide B administered into the pouch, induced a dose-dependent reduction in the levels of eicosanoids and tumour necrosis factor α (TNFα) in the exudates 4 h after the stimulus. It also had a weak effect on cell migration. The inflammatory response of adjuvant arthritis was reduced by cacospongionolide B, which did not significantly affect eicosanoid levels in serum, paw or stomach homogenates and did not induce toxic effects. Cacospongionolide B is a new inhibitor of sPLA2 in vitro and in vivo, with anti-inflammatory properties in acute and chronic inflammation. This marine metabolite was active after oral administration and able to modify TNFα levels, and may offer an interesting approach in the search for new anti-inflammatory agents.

A new inhibitor of human secretory phospholipase A2 (PLA2), cacospongionolide E (4a), has been isolated from the Tyrrhenian sponge Fasciospongia cavernosa. The structure was proposed on the basis of spectroscopic data and by chemical transformations. The absolute configuration of cacospongionolides 2a-4a was established using the modified Mosher's method. Cacospongionolide E was the most potent inhibitor toward human synovial PLA2, showing higher potency than the reference compound manoalide and exerting no signs of toxicity on human neutrophils. It showed high activity in the Artemia salina bioassay and moderate toxicity in the fish (Gambusia affinis) lethality assay.

A new 23,24-bishomoscalarane sesterterpene ( 2 ) was isolated from the northern Adriatic sponge Cacospongia scalaris . The structure was proposed on the basis of spectral data. The absolute stereochemistry was determined using the modified Mosher's method. Compound 2 is the first alkylated scalarane sesterterpenoid with methylation at C-23, to have been identified in nature.

Lupeol, cycloartenol, isofucosterol and the new β- d -glucosyl isofucosterol, have been isolated from the cell cultures of Lycopersicon esculentum . The structures were elucided from chemical and spectroscopic evidence.

25-Deoxycacospongionolide B (3), a new bioactive sesterterpene, and a diterpene, cacospongionolide C (4), have been isolated as minor components from the Adriatic sponge Fasciospongia cavernosa. The structures were proposed on the basis of spectroscopic data and defined for the former on the basis of X-ray analysis. Brine shrimp and fish lethalities of 25-deoxycacospongionolide B are reported.

From the brown alga Taonia atomaria; collected in the North Adriatic sea were isolated six sesquiterpenes: the new compound 4-cadinene, cadinane-4(14),5-diene, (-)-germacrene D, axenol, (-)-cubebol and 4-epi-cubebol, isolated for the first time from a natural source. The structure of the new compound was established on the basis of its spectral data, including 2D-NMR spectroscope. All 1H and 13C chemical shifts of known compounds, not reported in literature, are assigned.

Avarol, a sesquiterpenoid hydroquinone, its quinone avarone, both main secondary metabolites from the marine sponge Dysidea avara and nine of their natural and synthetic derivatives were tested for ability to interact selectively with rat liver microsomal phenobarbital (PB)- or 3-methylcholanthrene (3-MC)-induced cytochrome (cyt.) P-450 isoenzymatic forms. Ethoxy- and pentoxyresorufin, aminopyrine and ethoxycoumarin were the specific substrates used for assaying cyt.P-450-dependent mono-oxygenase activities. All compounds were more effective in inhibiting the enzymatic activities measured in microsomes from PB-induced rat liver than those measured in microsomes from 3-MC-treated rats. Avarol and avarone, which were the most active inhibitors, act as mixed-type inhibitors of pentoxyresorufin- O -dealkylase activity. Mono- and diacetyl-derivatives of avarol, being deacetylated by rat liver microsomes, were almost as effective as the parent compound. Conversely, avarone derivatives, where one or two hydrogen atoms of the quinone ring have been substituted, were less effective inhibitors than the parent compound. The selective inhibition of PB-inducible cyt.P-45 0IIB by avarol and avarone was confirmed by their ability to inhibit the mutagenic activation of cyclophosphamide, as opposed to that of benzo[ a ]py rene, which is activated mainly by the 3-MC-inducible cyt.P-450IA.

Bulbs of Pancratium maritimum were found to contain a new glucosyloxy phenolic metabolise as well as 14 known Amaryllidaceae alkaloids. The structure of the phenolic metabolite was elucidated by spectroscopic and chemical methods, and it was identified as narciclasine-4- O -β- d -glucopyranoside. When tested in Artemia salina and potato disc assays the new glucoside showed cytotoxic and antitumour activity very similar to narciclasine.

A mixture of acylglucosyl isofucosterols has been isolated from the cell culture of Lycopersicon esculentum. The structures were elucidated from chemical and spectroscopic evidence.

A series of prenyl hydroquinone derivatives synthesized as structural analogs of marine products were tested for their effects on inflammatory responses in vitro and in vivo. 2-Prenyl-1,4-hydroquinone (H1), 2-diprenyl-1,4-hydroquinone (H2), 2-triprenyl-1,4-hydroquinone (H3) and 2-tetraprenyl-1,4-hydroquinone (H4) scavenged reactive oxygen species and inhibited 5-lipoxygenase (5-LO) activity in human neutrophils. The inhibition of 5-LO activity was demonstrated in vivo in the mouse air pouch injected with zymosan and arachidonic acid-induced ear inflammation. The four compounds suppressed the production of tumour necrosis factor alpha (TNFalpha) in J774 cells stimulated with lipopolysaccharide (LPS) and also in vivo in the mouse air pouch injected with zymosan. In addition, all prenyl-hydroquinones inhibited the release of nitrite and PGE2 in LPS-stimulated J774 cells, without direct effects on cyclo-oxygenase-1 (COX-1), cyclo-oxygenase-2 (COX-2) or inducible nitric oxide synthase (iNOS) activities in several cell-free systems. The reduction in the length of the lateral chain in prenyl-hydroquinones (1-4 isoprene units) with respect to their marine analogs (7-8 isoprene units) has improved the anti-inflammatory activity of this class of compounds. Marine natural products may be a model to design new anti-inflammatory agents.

Three 2-polyprenyl-1,4-hydroquinone derivatives (2-heptaprenyl-1,4-hydroquinone: IS1, 2-octaprenyl-1,4-hydroquinone: IS2 and 2-[24-hydroxy]-octaprenyl-1,4-hydroquinone: IS3) isolated from the Mediterranean sponge Ircinia spinosula, were evaluated for effects on phospholipase A2 activity of different origin (Naja naja venom, human recombinant synovial fluid and bee venom), as well as on human neutrophil function and mouse ear oedema induced by 12-O-tetradecanoylphorbol 13-acetate (TPA). IS1 interacted minimally with these responses. In contrast, IS2 and IS3 inhibited human recombinant synovial phospholipase A2 in a concentration-dependent manner, with minor effects on the rest of the enzymes. Both compounds slightly affected superoxide generation and degranulation in human neutrophils, whereas they decreased thromboxane B2 and leukotriene B4 synthesis and release in a mixed suspension of human platelets and neutrophils stimulated by ionophore A23187, with IC50 values in the microM range. IS3 was the most effective inhibitor of the synthesis of thromboxane B2 by human platelet microsomes and of leukotriene B4 by high speed supernatants from human neutrophils. IS2 and IS3 showed topical anti-inflammatory activity against the TPA-induced ear inflammation in mice, with similar effects on oedema and a higher inhibition of IS3 on leukocyte migration, estimated as myeloperoxidase activity in supernatants of ear homogenates. Some structure-activity relationships were established since differences in the prenylated chain attached to the hydroquinone moiety result in important modifications of these inflammatory responses.

The isolation and the structural determination of six new 7′-(chrysophanol-4-yl)-chrysophanol-10′-anthrone 10′-C-glycosides 1-6 from Asphodelus ramosus tubers are reported.

2,6-Dimethylheptyl sulfate (1) was isolated for the first time from a marine ascidian from Policitor adriaticus Drasche (class Ascidiacea, order Aplousobranchiata, family Polycitoridae). In this work, the absolute stereochemistry at C-2 of 1 was determined by 1 H-NMR analysis of its (+)- and (-)-methoxy(trifluoromethyl)phenylacetic acid (MTPA) esters (Mosher's method). The distribution of 1 was investigated among the three orders (Aplousobranchiata, Phlebobranchiata, and Stolidobranchiata) of the class Ascidiacea. Compound 1 showed cytotoxicity (Lc 50 = 17.8 μg/mL) in the Artemia salina bioassay and was not toxic (not toxicity at 100 μg/ mL) in a fish (Gambusia affinis) lethality assay.