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The fungal genus Pyrenochaetopsis is commonly found in soil, terrestrial, and marine environments, however, has received little attention as a source of bioactive secondary metabolites so far. In a recent work, we reported the isolation and characterization of three new anticancer decalinoyltetramic acid derivatives, pyrenosetins A–C, from the Baltic Fucus vesiculosus-derived endophytic fungus Pyrenochaetopsis sp. FVE-001. Herein we report a new pentacyclic decalinoylspirotetramic acid derivative, pyrenosetin D (1), along with two known decalin derivatives wakodecalines A (2) and B (3) from another endophytic strain Pyrenochaetopsis FVE-087 isolated from the same seaweed and showed anticancer activity in initial screenings. The chemical structures of the purified compounds were elucidated by comprehensive analysis of HR-ESIMS, FT-IR, [α]D, 1D and 2D NMR data coupled with DFT calculations of NMR parameters and optical rotation. Compounds 1–3 were evaluated for their anticancer and toxic potentials against the human malignant melanoma cell line (A-375) and the non-cancerous keratinocyte cell line (HaCaT). Pyrenosetin D (1) showed toxicity towards both A-375 and HaCaT cells with IC50 values of 77.5 and 39.3 µM, respectively, while 2 and 3 were inactive. This is the third chemical study performed on the fungal genus Pyrenochaetopsis and the first report of a pentacyclic decalin ring system from the fungal genus Pyrenochaetopsis.

Introduction: The phytoalimurgic plants, common dandelion (Taraxacum officinale), corn poppy (Papaver rhoeas) and stinging nettle (Urtica dioica) are a source of nutraceuticals. Objectives: To apply a combined metabolomic fingerprinting approach by nuclear magnetic resonance (NMR) and gas chromatography–mass spectrometry (GC–MS) to common dandelion, corn poppy and stinging nettles to obtain simultaneous identification and quantitation of the major classes of organic compounds. Methodology: The whole plants collected in the Cilento National Park were dried and then extracted to obtain non‐polar and polar organic extracts. GC–MS was used for non‐polar extracts while 1 H‐NMR spectroscopy was used for polar extracts. In both cases, simultaneous identification and quantification of the bioactive metabolites was obtained. Results: Non‐polar organic extracts of all plants were mainly composed of palmitic, stearic and oleic acids. The two pentacyclic triterpenols α‐ and β‐amyrin were detected in nettle extract. The analysis of polar organic extracts allowed to detect and quantify organic acids and sugars as main metabolites along with amino acids, caffeoyl derivatives, flavonoids, and nucleotides. In particular, corn poppy leaves contained a huge amount of glyceric acid (55.7% of the total extract). Stinging nettles, instead, exhibited a large amount of choline (19.5%). Conclusion: Metabolomic approach coupling GC–MS with NMR spectroscopy allowed to provide a detailed metabolite profile of three alimurgic plants, common dandelion, corn poppy and stinging nettle, from both a qualitative and quantitative point of view

Vagiallene (1), a rearranged C 15 acetogenin with a molecular formula and a carbon skeleton unprecedented in natural products, was isolated as a trace constituent from the organic extract of the red alga Laurencia obtusa from Lefkada island. The planar structure and the relative configuration of 1 were established on the basis of extensive analysis of its spectroscopic data, while its absolute configuration was determined by comparison of its experimental and quantum-mechanically predicted electronic circular dichroism spectra. © 2019 American Chemical Society.

In the Mediterranean Sea, the biological invasion by the green algae Caulerpa racemosa var. cylindracea is maybe the most striking marine sea-bottom landscape change of the last decades. In this paper, we have synthesized the results of 5 years of studies about the C. cylindracea effects on fish of ecological and economic importance, such as the white sea bream Diplodus sargus. We found that a secondary metabolite of C. cylindracea has the potential to enter in trophic chains and accumulate in fish tissues. Ecotoxicological analyses made on tissues of the D. sargus specimens showed alterations of some of main antioxidant systems, such as catalase, glutathione peroxidases and total glutathione, and a significant induction of P450 biotransformation system. We found also that the C. cylindracea based-diet can alter muscle fatty acids composition in the white sea bream by reducing the percentage of polyunsaturated fatty acids (PUFA) of the n-3 and n-6 series, such as eicosapentaenoic (EPA, C20:5), docosahexaenoic (DHA, C22:6) and arachidonic acids (AA, C20:4). On the whole, this work sheds light on an unexplored and critical aspect of biological invasions with implication on the health of both humans and the environment.

The green alga Caulerpa racemosa is a non-native, invasive species in the Mediterranean, and an important stressor for several native organisms. The algal capacity to produce secondary metabolites has been suggested to modulate success of the C. racemosa invasion, although many of potentially involved biological pathways still remain unexplored. In this respect, the aim of the present study was to investigate some molecular and cellular effects in the white seabream Diplodus sargus , an ecologically key species, which included the alien C. racemosa in its diet. Organisms were sampled in 2 seasonal periods from 3 locations of Southern Italy, each characterized by different levels of algal abundance. The level of caulerpin, one of the main secondary algal metabolites, in fish tissues has been used as an indicator of the trophic exposure to the seaweed and related with molecular and cellular responses. Chemical analyses indicated that fish from invaded sites can accumulate caulerpin, with liver concentrations ranging from a few up to hundreds of μg/g. Biomarkers analyses revealed only limited alterations of the main antioxidant defences, such as glutathione reductase and levels of glutathione; on the other hand, increased enzymatic activities of cytochrome P450, glutathione S-transferases and acyl CoA oxidase, as well as enhanced gene transcription for peroxisome proliferator-activated receptor alpha, cytochrome P4501A and vitellogenin 1 were observed in fish more exposed to C. racemosa as indicated by liver concentrations of caulerpin higher than 50 μg/g. Despite a direct molecular relationship with this algal metabolite could not be established, our results suggest that a C. racemosa enriched diet can modulate biotransformation and fatty acids metabolism of D. sargus. Assessing whether similar effects represent short- or long-term effects will be of crucial importance to understand consequences on the general health status and reproductive performance of exposed key fish species in the Mediterranean region.

Two diarylheptanoid heterodimers, zosterabisphenones A (1) and B (2), were isolated from the seagrass Zostera marina. They feature unprecedented catechol keto tautomers, stable because of steric constraints. Their structure elucidation was based on extensive low-temperature NMR studies and ECD and MS data, with the essential aid of DFT prediction of NMR and ECD spectra. Zosterabisphenone B (2) was selectively cytotoxic against the adenocarcinoma colon cancer cell line HCT116 with IC50 3.6 ± 1.1 μM at 48 h.

BACKGROUND Chia (Salvia hispanica L.) is a functional food from Central America. Interest in it is growing rapidly due to the many health benefits from the seed. However, when chia is grown at high latitudes, seed yield may be low whereas a high stem biomass and immature inflorescences are produced. Little is known about the chemical composition and the properties of stems and flowers. In this work, the metabolite profile, the antioxidant activity, and the total polyphenol content of stems and inflorescences were evaluated in a factorial experiment with different chia populations (commercial black chia and long‐day flowering mutants G3, G8, and G17) and irrigation (100% and 50% of evapotranspiration). RESULTS The results show the influence of irrigation and seed source on the antioxidant activity and total polyphenol content of chia flower and stem. Inflorescences exhibit higher antioxidant activity, suggesting their potential use as natural antioxidant. The mutants G3 and G8, at 50% irrigation, contained the highest amounts of compounds with nutraceutical value, especially within the flower. The mutant G17 showed lower antioxidant activity and polyphenol content compared to other seed sources but exhibited high omega 3 content in flowers but low in stems. This indicates that chia varieties should be chosen according to the objective of cultivation. CONCLUSION These findings, indicating a close relation of metabolite content with irrigation and seed source, may provide the basis for the use of chia flower and stem for their nutraceutical value in the food, feed, and supplement industries. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Alexandrium ostenfeldii is a widespread toxic dinoflagellate that has recently bloomed across the Adriatic Sea, seriously threatening both shellfish consumers and aquacultures. In 2007 we reported on preliminary studies carried out on field samples and cultures of A. ostenfeldii. At the time, along with three major spirolides - among which 27-hydroxy-13,19-didesmethyl spirolide C (3) proved to be a novel compound - a number of new minor spirolides were detected. Unfortunately, for all of them only Mass Spectrometry-based structural hypotheses could be ventured due to their very small amount. In the present paper we report on isolation and High Resolution Mass Spectrometry- and NMR-based structural elucidation of two of those minor spirolides detected in our previous study.

Euphorbia myrsinites is one of the oldest spurges described and used in folk medicine. It is characterized by blue-grey stems similar to myrtle, and it is spread in the Mediterranean region, Asia, and the USA. Chemical analysis of E. myrsinites collected in Turkey afforded the isolation of 4 diterpenes based on the so-called myrsinane skeleton being tetraesters of the tetracyclic diterpene alcohol myrsinol. In this study, the phytochemical analysis of this species collected in Italy has been undertaken to afford the isolation of a new atisane diterpene, named myrsatisane, 3 ingenol derivatives, along with the 4 tetraester derivatives previously found. A triterpene compound based on the euphane skeleton has also been isolated. Structural elucidation of the new myrsatisane was based on spectroscopic techniques, including HR-MS and 1- and 2-dimensional NMR experiments. Its relative configuration was determined by NOE correlations, while absolute stereochemistry was obtained by quantum-mechanical DFT studies. While diterpenes with the atisane skeleton are relatively common in Euphorbia species, this is the first report of an atisane diterpene from E. myrsinites. All the isolated terpenes were tested for anti-inflammatory activity on J774A.1 macrophages stimulated with lipopolysaccharide by evaluation of nitrite and pro-inflammatory cytokine Il-1β levels. Among tested compounds, the 3 ingenol diterpenes exhibited a dose-dependent (0.001 – 3 µM) significant activity, thus showing their potential as anti-inflammatory drug candidates.

Zosteraphenols, two new tetracyclic diarylheptanoids were isolated from the seagrass Zostera marina. The rotameric equilibrium of the strained tetracyclic structures, involving a diastereomeric minor rotamer with opposite axial chirality, resulted in coalescent NMR spectra. Although the elusive minor rotamer was only characterized with 1H chemical shifts, the excellent agreement between experimental and DFT-calculated chemical shifts of both rotamers unequivocally supported this analysis. Absolute configuration of zosteraphenols was determined by DFT prediction of their ECD spectra.

Introduction Most secondary metabolites from plants have a prominent defensive role and repellency against predators and microbial pathogens. These properties largely vary among plant species and offer potential applications as biologically active compounds in medicine as well in agriculture. Objectives We propose a new procedure that combine different spectroscopic techniques and multivariate data analysis to determine the chemical composition and the relative amounts of each metabolites and/or each class of organic compounds. The approach was used for a rapid identification of secondary metabolites from leaf and root of eight Mediterranean plants species. Methodology The polar and the apolar extracts of two leaves and roots of each plant were analysed by proton nuclear magnetic resonance (1 H-NMR) and gas chromatography coupled to mass spectrometry (GC-MS), respectively. Multivariate data analysis was used for a faster interpretation of data. Results The metabolic fingerprint of the Mediterranean plants, Acanthus mollis, Dittrichia viscosa, Festuca drymeja, Fraxinus ornus, Fagus sylvatica, Hedera helix, Quercus ilex, and Typha latifolia, showed a complex chemical composition, being specific for each species and plant tissue. Two alditols, mannitol and quercitol, were found in manna ash (Fraxinus ornus) and holm oak (Q. ilex) polar leaf extracts, respectively. The highest levels of aromatic compounds were found in D. viscosa and T. latifolia. Fatty acids were the predominant class of compounds in all apolar extracts under investigation. Triterpene were almost exclusively found in roots, except for holm oak, where they constitute 58% of total extract. Steroids were widespread in leaf extracts. Conclusion The major advantages of the proposed approach are versatility and rapidity, thus making it suitable for a fast comparison among species and plant tissue types.

Papaver rhoeas L. (Papaveraceae), commonly known as corn poppy, is a cosmopolitan weed and edible plant capable of possessing biological properties, thus positive correlating with human health. The plant is also known as field poppy, flanders poppy or shirley poppy. It can be consumed raw or cooked, and has been traditionally used to treat nervousness, insomnia, digestive and respiratory disorders, baldness, eye infections, as well as measles treatment. This paper is aimed to contribute to the knowledge of corn poppy by providing a critical review of the botanic characteristics, the traditional uses, the plant chemistry and pharmacology.

The Red Palm Weevil (RPW), Rhynchophorus ferrugineus (Coleoptera: Curculionidae), is an insect pest native to Southeast Asia, which has become the major threat to palms in the Mediterranean Basin, mainly due to the movement of infested planting material. In case of infestation, the exotic weevil is difficult to control effectively and host plants often die. Chamaerops humilis (Arecales: Arecaceae) palms are reported as resistant to RPW, but little is known about the chemical basis of this resistance and about the identity of the endogenous metabolites that may be involved. In the present study, we focused on the potential toxic effect of catechin against RPW larvae. In laboratory bioassays, purified catechin from C. humilis leaves was provided to two-and five-week old RPW larvae at three different concentrations. We found that catechin can impair the survival of RPW, causing toxicity at concentrations as low as 0.03 mg g-1. Larval mortality was concentra-tion-dependent, and furthermore larval age influenced the effect of catechin, older larvae being more susceptible than younger ones. The observed toxicity of catechin on RPW larvae agrees with the hypothesis that this compound contributes to C. humilis being an unsuitable host for this insect pest.

A diketopiperazine has been purified from a culture filtrate of the endophytic fungus Paraphaeosphaeria sporulosa, isolated from healthy tissues of strawberry plants in a survey of microbes as sources of anti-bacterial metabolites. Its structure has been determined by nuclear magnetic resonance (NMR) and liquid chromatography–mass spectrometry (LC–MS) analyses and was found to be identical to cyclo(L-Pro-L-Phe) purified from species of other fungal genera. This secondary metabolite has been selected following bioguided-assay fractionation against two strains of Salmonella enterica, the causal agent of bovine gastroenteritis. The diketopiperazine cyclo(L-Pro-L-Phe), isolated for the first time from Paraphaeosphaeria species, showed minimum inhibitory concentration (MIC) values of 71.3 and 78.6 μg/mL against the two S. enterica strains. This finding may be significant in limiting the use of synthetic antibiotics in animal husbandry and reducing the emergence of bacterial multidrug resistance. Further in vivo experiments of P. sporulosa diketopiperazines are important for the future application of these metabolites.

Phytoalimurgy is a term that derives from Greek and Latin by combination of the words φυτόν, which meaning plant, and alimenta urgentia, indicating foods available in case of urgency and necessity. Nowadays, the lack of products from agriculture is fortunately no longer a problem, however, the interest in using the phytoalimurgic plants has increased in the last years due to their rediscovery for use of food ingredients for human health. Among these, common dandelion, Taraxacum officinale, is used since ancient times because of its curative properties. The aim of this review is the contribution to the knowledge of this worldwide-spread phytoalimurgic plant with its botanical, phytochemical and pharmacological description. In vitro and in vivo scientific investigations have confirmed its pharmacological potential by showing antioxidant, antibacterial, anti-inflammatory, antiviral, cytotoxic, diuretic and hepatoprotective properties. Responsible of these activities are bioactive metabolites belonging to different classes, including sesquiterpenoids, caffeoylquinic acids and flavonoids. This allows to include dandelion among the medicinal plants of commercial interest for nutraceutical and phytopharmaceutical industries.

Covering: up to 2019This review covers the current status of the quantum mechanical prediction of chiroptical properties, such as electronic CD and optical rotation, as needed for stereochemical assignments in new natural products. The reliability of the prediction of chiroptical properties is steadily increasing, with a parallel decrease in the required computational resources. Now, quantum mechanical calculations for a medium-sized natural product can be reliably performed by natural product chemists on a mainstream PC. This review is aimed to guide natural product chemists through the numerous steps involved in such calculations. Through a concise, but comprehensive, discussion of the current computational practice, enriched by a few illustrative examples, this review provides readers with the theoretical background and practical knowledge needed to select the most appropriate parameters for performing the calculations, to anticipate possible problems, and to critically evaluate the reliability of their computational results. Common reasons for mistakes are also discussed; in particular, the importance of the correct evaluation of conformational ensembles of flexible molecules (an aspect often overlooked in current research) is stressed.

In microbial cultures the production of secondary metabolites is affected by experimental conditions, and the discovery of novel compounds is often prevented by the re-isolation of known metabolites. To limit this, it is possible to cultivate microorganisms by simulating naturally occurring interactions, where microbes co-exist in complex communities. In this work, co-culturing experiments of the biocontrol agent Trichoderma harzianum M10 and the endophyte Talaromyces pinophilus F36CF have been performed to elicit the expression of genes which are not transcribed in standard laboratory assays. Metabolomic analysis revealed that the co-culture induced the accumulation of siderophores for both fungi, while production of M10 harzianic and iso-harzianic acids was not affected by F36CF. Conversely, metabolites of the latter strain, 3-O-methylfunicone and herquline B, were less abundant when M10 was present. A novel compound, hereby named harziaphilic acid, was isolated from fungal co-cultures, and fully characterized. Moreover, harzianic and harziaphilic acids did not affect viability of colorectal cancer and healthy colonic epithelial cells, but selectively reduced cancer cell proliferation. Our results demonstrated that the co-cultivation of plant beneficial fungi may represent an effective strategy to modulate the production of bioactive metabolites and possibly identify novel compounds.

Recent studies have shown that the Mediterranean white sea bream Diplodus sargus includes the invasive green alga Caulerpa cylindracea in its diet, with consequent metabolic and enzymatic alterations. As a result of this novel alimentary habit, the bioactive algal red pigment caulerpin has been detected in its tissues. However, this may not be an isolated case: other fish species have also been reported to feed on C. cylindracea, although the possible accumulation of caulerpin in their tissues has not yet been investigated. In this report, we analysed stomach con- tents and caulerpin levels in the native sparid species Spondyliosoma cantharus, Sarpa salpa, and Diplodus vulgaris, and in the scarid Sparisoma cretense, along with the Lessepsian siganid Siganus luridus. C. cylindracea was found in the stomachs of all but one fish species, the exception being S. cretense, in which prey items could not be determined due to the high degree of diges- tion. Chemical analysis of fish tissues revealed that only S. cantharus and S. salpa accumulated caulerpin, while no traces of the compound were detected in the other species. Despite intense research efforts on natural products obtained from C. cylindracea, a complete picture of the impacts caused by fish including this alga in their diet has not been elucidated. The identification of caulerpin in other Mediterranean native fish suggests a need for further research in order to assess the possible transfer of such molecules to humans through seafood consumption.

Four undescribed lignans and two undescribed sesquiterpenic acids, together with three known compounds (hypochoeroside C, hypochoeroside D, and 5- O -caffeoylshikimic acid) were isolated from the roots of Hypochaeris radicata subsp. neapolitana (Asteraceae, Cichorieae). The lignans were identified as 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy- O -β-D-glucopyranoside, 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy- O -β-D-glucopyranosyl-2′- O -methacrylate, (7 S ,8 R ,8′ R )-7-(3,4-dihydroxyphenyl)-3′,4′-dihydroxy-7,8,7′,8′-tetrahydronaphtho [8,8′-c]furan-1(3H)-one, and (7 S ,8 R ,8′ R )-7-(3,4-dihydroxyphenyl)-3′,4′-dihydroxy-8'-(hydroxymethyl)-7,8,7′,8′-tetrahydronaphthalen-8-carboxylic acid. The two sesquiterpenic acids were identified as the ring open precursors of hypochoerosides C and D. Structures were elucidated using NMR and HRMS. Absolute configurations of (7 S ,8 R ,8′ R )-7-(3,4-dihydroxyphenyl)-3′,4′-dihydroxy-7,8,7′,8′-tetrahydronaphtho [8,8′-c]furan-1(3H)-one and (7 S ,8 R ,8′ R )-7-(3,4-dihydroxyphenyl)-3′,4′-dihydroxy-8'-(hydroxymethyl)-7,8,7′,8′-tetrahydronaphthalen-8-carboxylic acid were determined using electronic circular dichroism (ECD) spectroscopy. 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy- O -β-D-glucopyranoside was evaluated for its anti-proliferative activity against myeloma cell lines MM1S, U266, and NCI-H929 and showed cytotoxicity at 100 mM against MM1S strain. No neurotoxicity was observed for major compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy- O -β-D-glucopyranoside, hypochoeroside C, and hypochoeroside D in a fluorescence assay measuring neurite outgrowth in dorsal root ganglion (DRG) neurons. Additionally, compounds 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy- O -β-D-glucopyranoside, hypochoeroside C, hypochoeroside D, and hypochoerosidic acid D were quantified in unstressed and drought-stressed plants using HPLC-DAD. Drought-stressed plants were found to contain lower concentrations of the lignan 4-(3,4-dihydroxybenzyl)-2-(3,4-dihydroxyphenyl)tetrahydrofuran-3-carboxy- O -β-D-glucopyranoside and sesquiterpene lactone hypochoeroside C.

Two oxazinins, namely oxazinin-5 and -6, along with a related linear precursor (preoxazinin-7) were isolated from toxic mussels collected along the Northern Adriatic coasts in October 2005. Determination of the planar structure of these novel compounds was achieved through extensive NMR spectroscopic analysis, whereas a synthetic approach was crucial for their absolute stereochemical elucidation.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Since 2005, the benthic dinoflagellate Ostreopsis cf. ovata has bloomed across the Mediterranean basin, provoking serious toxic outbreaks. LC/MS studies have identified a number of palytoxin-like compounds, termed ovatoxins, along with trace amounts of putative palytoxin as the causative agents of the O. cf. ovata -related human sufferings. So far, any risk assessment for ovatoxins as well as establishment of their allowance levels in seafood has been prevented by the lack of pure toxins. The present paper reports on the isolation, NMR-based structural determination, and preliminary mouse lethality evaluation of ovatoxin-a, the major toxic compound contained in O. cf. ovata extracts. Availability of pure ovatoxin-a will open the double prospect of fully evaluating its toxicity and preparing reference standards to be employed in LC/MS quantitative analyses. Elucidation of ovatoxin-a's complex structure will ultimately herald the understanding of the molecular bases of ovatoxins bioactivity.

High resolution (HR) LC-MS investigation of an Adriatic Ostreopsis ovata culture is reported herein. It highlighted the presence of putative palytoxin and ovatoxin-a in combination with four new ovatoxins. Elemental formulae and information about their structural features were gained.

In the last decades, the recurring presence of the benthic dinoflagellate Ostreopsis ovata has caused severe sanitary emergencies and economic losses along the whole Italian coastline. Liquid chromatography-mass spectrometry (LC-MS) technique played a key role in revealing the presence of putative palytoxin and much higher amounts of ovatoxin-a, a palytoxin-like compound, in field samples of O. ovata. In our ongoing research on O. ovata toxins we report herein on high resolution LC-MS and MS2 investigation of O. ovata cultures, which disclosed the occurrence of several new ovatoxins in algal extracts. Elemental formulae very close to palytoxin???s were assigned to the new compounds and information was gained about their structural features. A quantitative study of several O. ovata culture extracts indicated that ovatoxin-a generally represents the major component of the algal toxin profile and that the whole of the other ovatoxins constitutes up to 46% of the total toxin content of the analyzed strains; thus, their presence should be taken into account when LC-MS based monitoring programs of either plankton or contaminated seafood are carried out.

Over the past decades, Italian coastlines have been plagued by recurring presence of the benthic dinoflagellate Ostreopsis ovata. Such an alga has caused severe sanitary emergencies and economic losses due to its production of palytoxin-like compounds. Previous studies have confirmed the presence of ovatoxin-a (OVTX-a) as the major toxin of the algal toxin profile together with small amounts of putative palytoxin (PLTX). In our ongoing research on O. ovata toxins we report herein on in-depth investigation of an O. ovata culture carried out by high-resolution (HR) liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS2). Particularly, the presence of putative PLTX and OVTX-a was confirmed and the occurrence in the extract of four new palytoxin-like compounds, OVTX-b, -c, -d, and -e, was highlighted. Elemental formulae have been assigned to the new ovatoxins and information has been gained about their structural features. A quantitative study of the O. ovata culture extract indicated that the whole of the new ovatoxins represents about 46% of the total toxin content and, thus, their presence has to be taken into account when LC/MS-based monitoring programs of either plankton or contaminated seafood are carried out. Copyright © 2010 John Wiley & Sons, Ltd.

Since its isolation dated back to as far as 1971, palytoxin has all along drawn scientists' attention from across the world because of its high toxicity and fascinating chemical architecture. Commitment of the international scientific community to the study of this extremely potent non-proteic toxin has led to discover quite a number of palytoxin analogues. Once confined only to tropical and subtropical areas, palytoxins have recently spread also to more temperate regions, such as the Mediterranean Sea where they have caused severe human intoxications. Studies on the Mediterranean toxic outbreaks brought to light the existence of further palytoxin-like compounds, ovatoxins, never reported elsewhere in the world.

In the last decades, the Italian coastlines have been plagued by the recurring presence of the benthic dinoflagellate Ostreopsis ovata. Such an alga has caused severe sanitary emergencies and economic losses due to its production of palytoxin-like compounds. Previous studies evidenced the presence of ovatoxin-a, as the major toxin of the algal toxin profile together with small amounts of putative palytoxin. In our ongoing research on O. ovata toxins we report herein on high resolution LC-MS and MS2 investigation of an O. ovata culture, which revealed an algal toxin profile more complex than previously found. Particularly, the presence of putative palytoxin and ovatoxin-a was confirmed and the occurrence in the extract of four new palytoxin-like compounds, ovatoxin-b, -c, -d, and -e, was highlighted. Elemental formulae very close to palytoxin???s have been assigned to the new ovatoxins and information has been gained about their structural features. A quantitative study of the O. ovata culture extract indicated that ovatoxin-a represented almost 54% of the total toxin content with the remaining 46% being constituted by the whole of ovatoxin-b, -c, -d and -e. Thus, the presence of the new ovatoxins should be taken into account when LC-MS based monitoring programs of either plankton or contaminated seafood are carried out.

Tra le problematiche legate alle fioriture di microalghe nocive, un aspetto emergente che si inquadra nella tropicalizzazione del Mar Mediterraneo riguarda lo sviluppo di specie appartenenti al genere Ostreopsis, un fenomeno venuto prepotentemente alla luce negli ultimi anni lungo le coste Tirreniche, che ha ottenuto vasta risonanza da parte della stampa e dei media. Intossicazioni umane associate alla presenza di Ostreopsis spp. sono state riportate per la prima volta nel 1998 lungo le coste Toscane,1 ma gli episodi più gravi, per il numero di persone interessate, si sono verificati nell'estate 2005, lungo le coste Liguri, quando circa 200 persone, in seguito ad esposizione ad aerosol marino tossico, manifestarono i sintomi di una intossicazione caratterizzata da tosse, rinorrea, febbre alta e gravi congiuntiviti. Un'analisi accurata delle acque del mare, effettuata dall'Agenzia Regionale Protezione Ambiente della Liguria (ARPAL) portò alla luce la presenza di alte concentrazioni nel plankton di una microalga tropicale tossica, l'Ostreopsis ovata, che fu ritenuta il possibile agente causale delle intossicazioni umane verificatesi nei tratti di costa interessati. Studi condotti dal nostro gruppo di ricerca su campioni di plancton raccolti nel mar Ligure in coincidenza delle proliferazioni di O. ovata, hanno permesso di stabilire una relazione di causa-effetto tra la fioritura della microalga e gli episodi di intossicazione umana. Tale studio, che ha richiesto la messa a punto di un nuovo metodo analitico basato sull'accoppiamento cromatografia liquida/spettrometria di massa (LC/MS),2 ha dimostrato che tale microalga produce palitossina (stesso peso molecolare e stesso tempo di ritenzione nell'analisi cromatografica) insieme ad un'altra tossina, di gran lunga più abbondante, a cui abbiamo dato il nome di ovatossina-a.3 Sulla base della formula molecolare, del pattern di frammentazione e del comportamento cromatografico, la struttura dell'ovatossina-a appare strettamente correlata a quella della palitossina. Indagini LC/MS effettuate su colture di O. ovata hanno permesso di stabilire che le tossine ritrovate nei campioni di plancton analizzati sono reali prodotti del metabolismo della microalga. Studi LC/MS condotti dal nostro gruppo di ricerca nell'ambito di un piano di monitoraggio dell'Ostreopsis ovata lungo il litorale della Campania hanno dimostrato la presenza di palitossina e di ovatossina-a anche nei campioni raccolti lungo alcune coste campane. Inoltre, i nostri studi hanno evidenziato che entrambe le tossine si accumulano in animali utilizzati a scopo alimentare, in particolare ricci e mitili, a livelli tali da rappresentare un concreto rischio per la salute umana.

This paper reports on the analysis of the toxin content from Palythoa tuberculosa and Palythoa toxica samples collected off of the Hawaiian coast. Our work, based on in-depth high-resolution liquid chromatography-mass spectrometry analysis along with extensive NMR study, led us to structurally characterize 42-hydroxy-palytoxin, a new palytoxin congener. This toxin and palytoxin itself appeared to be the major components of toxic extract from a P. tuberculosa sample, while 42-hydroxy-palytoxin was proven by far to be the main palytoxin derivative in P. toxica. Functional studies on this new palytoxin-like compound suggest that the new palytoxin analogue and palytoxin itself present similar biological activities.

This paper reports on the determination of the toxin profile of batch cultures of Alexandrium ostenfeldii established from water samples collected along the Emilia-Romagna coasts (Italy) in November 2003. Combined use of LC-MS/ MS and 1D and 2D NMR techniques led us to identify 13,19-didesmethylspirolide C and 13-desmethylspirolide C as products of the alga and to highlight the occurrence of a new component, 27-hydroxy-13,19-didesmethylspirolide C, whose structure was fully elucidated by NMR. MS data for a number of minor spirolides are also reported.

Two new desulfated yessotoxin (YTX) analogues were isolated from a toxic batch of Adriatic mussels collected in October 2004. Their stereostructures were elucidated through extensive NMR and MS-based analysis. The finding of these desulfocarboxyhomoYTXs in shellfish poses additional problems to those institutions entitled to control seafood safety, since desulfated YTXs fail the European Union method currently in force for checking toxicity in mollusks.

Over the last few years a new treat due to harmful algal blooms has been impending in the Mediterranean Sea: microalgae belonging to Ostreopsis genus have spread along the Italian coasts, mostly in the Tyrrhenian Sea, causing serious concerns to both environment and public health. In fact, concurrently to the algal blooms, environmental sufferings involving mostly epibenthos were observed and symptoms of rhinorrhoea, conjunctivitis, dermatitis, cough, dyspnoea, wheezes and severe fever arouse in people exposed to marine aerosols by recreational or working activities. Such a phenomenon was recorded for the first time during summer 1998 along the north-western Tuscany coasts,1 but reached alarming proportions in late July 2005, when about 200 people exposed to marine aerosols on the beach and promenade of Genoa required extended hospitalization. Respiratory distress observed in humans was concurrent with a massive bloom of Ostreopsis ovata along Genoa coasts and disappeared when the O. ovata population decreased. Human illness were recorded also in summer 2006, when O. ovata populations spread along the whole Tirrenian coasts from Liguria to Sicily and in the Southern Adriatic Sea. In the present work, we report on the investigation of plankton samples collected along Liguria coasts during the 2005/2006 outbreaks. The application of LC-MS/MS methods we have developed so far for the analyses of a number of liposoluble and hydrosoluble phycotoxins2-5 prompted us to exclude the presence of such toxins in the samples. Then, since some Ostreopsis strains are regarded as the producing organisms of palytoxin,6 one of the most potent marine toxin so far known, a new LC-MS/MS method for detection of palytoxin was set up.7 It allows the rapid and specific determination of palytoxin in selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. The minimum detection levels for matrix-free toxin on-column were estimated to be 125 pg and 200 pg in MRM and SIM mode, respectively. Spiking experiments before and after extraction allowed to assess limits of detection and quantitation for palytoxin in matrix, accuracy, intra-day and inter-day reproducibility. Relying on the newly developed method, we were able to detect the presence of putative palytoxin and its analogue in the plankton samples and to infer them as the likely cause of the Genoa 2005/2006 outbreaks.

The toxin profile of massive cultures of Alexandrium ostenfeldii collected along the North-western Adriatic coasts of Italy in November 2003 was determined by liquid chromatography tandem mass spectrometry. The chromatographic separation was carried out by using a reversed phase column and a buffered mobile phase, as suggested by Quilliam et al. for the analysis of spirolides and various lipophilic toxins (1). MS detection was accomplished either by using a heated-ESI-linear ion trap-MS instrument, operating in Data Dependent (DD) scanning mode, or a ESI-triple quadrupole MS instrument, operating in full scan MS and MS/MS modes. The spirolide profile of the Adriatic strain was dominated by the recently reported 13,19-didesmethyl spirolide C (2) whose identity was assessed by the presence of three characteristic ion clusters in the LC-MS/MS spectrum of the [M+H]+ ion at m/z 678.5. The Adriatic strain was shown also to produce 13-desmethyl spirolide C, but unlike most of the worldwide strains of the dinoflagellate in which it accounts for >90% of the total spirolide content, this wasn???t the major component. Furthermore, a new spirolide was also isolated in remarkable amounts and its structure assigned to the novel 27-hydroxy-13,19-didesmethyl spirolide C by combined use of LC-MS/MS and NMR evidence. The occurrence of a number of potentially new spirolides was also highlighted and LC-MS/MS-based structural hypotheses are provided for most of them.

The relative stereochemistry of 13,19-didesmethyl spirolide C was determined through careful analysis of NMR parameters strongly dependent upon molecular conformations supported and extended by computational studies. This work has also shed light on the conformational behavior of spirolides in solution. An equilibrium between two possible conformers of the identified diastereoisomer was inferred, while the uncommon cyclic imine moiety of spirolides-the putative pharmacophore of this class of toxins-was interestingly found to adopt only a single dominant conformation. The insightful details provided on spirolide conformations may represent a key means to pharmacologists involved in clarifying the mechanism of action of spirolide, which is yet to be totally defined.