Assunta Napolitano

Scalaradial, a Dialdehyde-Containing Marine Metabolite That Causes an Unexpected Noncovalent PLA2 Inactivation

Several marine terpenoids that contain at least one reactive aldehyde group, such as manoalide and its congeners, possess interesting anti‐inflammatory activities that are mediated by the covalent inactivation of secretory phospholipase A2 (sPLA2). Scalaradial, a 1,4‐dialdehyde marine terpenoid that was isolated from the sponge Cacospongia mollior, is endowed with a relevant anti‐inflammatory profile, both in vitro and in vivo, through selective sPLA2 inhibition. Due to its peculiar dialdehyde structural feature, it has been proposed that scalaradial exerts its enzymatic inactivation by means of an irreversible covalent modification of its target. In the context of our on‐going research on anti‐PLA2 natural products and their interaction at a molecular level, we studied scalaradial in an attempt to shed more light on the molecular mechanism of its PLA2 inhibition. A detailed analysis of the reaction profile between scalaradial and bee venom PLA2, a model sPLA2 that shares a high structural homology with the human synovial enzyme, was performed by a combination of spectroscopic techniques, chemical reactions (selective modifications, biomimetic reactions), and classical protein chemistry (such as proteolytic digestion, HPLC and mass spectrometry), along with molecular modeling studies. Unexpectedly, our data clearly indicated the noncovalent forces to be the leading event in the PLA2 inactivation process; thus, the covalent modification of the enzyme emerges as only a minor side event in the ligand–enzyme interaction. The overall picture might be useful in the design of SLD analogues as new potential anti‐inflammatory compounds that target sPLA2 enzymes.

Synthesis, structural aspects and bioactivity of the marine cyclopeptide hymenamide C.

Abstract Head-to-tail proline containing cyclopeptide hymenamide C [cyclo(Leu-Trp-Pro 3 -Phe-Gly-Pro 6 -Glu); 1 ], isolated from a marine sponge and for which a preliminary immunomodulating activity was reported, was efficiently synthesized by a three-dimensional orthogonal solid-phase strategy (Fmoc/tBu/Allyl) via anchoring the ω -carboxyl function of the glutamic acid to the solid support (PAC–PEG–PS). The linear precursor was entirely assembled and subsequently cyclized on resin, yielding a major product identical to the natural hymenamide C and a minor one, a geometric isomer of hymenamide C ( 2 ), differing for the geometry of peptide linkages at Pro residues. Both the ‘proline-rich’ cyclopeptides were submitted to a multiparametric in vitro screening on immune cells in order to acquire additional information on their biological activity. Indeed, compounds 1 and 2 were shown to exert inhibitory effect on human neutrophil elastase degranulation release at micromolar concentration.

Oxylipins from Dracontium loretense

Four novel oxylipins (1-4) were isolated from the n-butanol extract of the corms of Dracontium loretense. Their structures were assigned by 1D and 2D NMR analyses and electrospray ionization multistage ion trap mass spectrometry (ESI-ITMS(n)) data. Relative configurations were assigned on the basis of combined analysis of homonuclear and heteronuclear (2,3)J couplings, along with ROE data. Oxylipin 2 exhibited an immunostimulatory effect on human PBMC proliferation.

Oleanane glycosides from Astragalus tauricolus: Isolation and structural elucidation based on a preliminary liquid chromatography-electrospray ionization tandem mass spectrometry profiling

As a part of our ongoing research for bioactive compounds from Turkish Astragalus species, the investigation of Astragalus tauricolus has been carried out. An approach based on HPLC-ESIMS(n) experiments has been used to profile the triterpene glycosides occurring in the butanol extract of the whole plant. On the basis of the results of the online screening by HPLC-ESIMS(n), 22 oleanane-type triterpene glycosides, including ten compounds never reported before, were isolated, and their structures were established by the extensive use of 1D and 2D-NMR experiments along with ESIMS and HRMS analysis. Noteworthy, cycloartane-type triterpene glycosides, the main constituents of Astragalus spp., were not found. This peculiar feature characterizes a very limited group of Astragalus spp. The antiproliferative activity of the isolated compounds 1-12, 15, 17-19 was evaluated against a small panel of cancer cell lines. Only compound 11 showed an IC(50) of 22 μM against human leukemia cell line (U937). The other tested compounds, in a range of concentrations between 1 and 50 μM, did not cause any significant reduction of the cell number.

Steroidal glycosides from the leaves of Ruscus colchicus: Isolation and structural elucidation based on a preliminary liquid chromatography−electrospray ionization tandem mass spectrometry profiling

An HPLC-ESIMS(n) method, based on high-performance liquid chromatography coupled to electrospray positive ionisation multistage ion trap mass spectrometry, has been used as an effective tool to rapidly identify and guide the isolation of target saponins from the ethanol extract of the leaves of Ruscus colchicus Y. Yeo. Twenty-two steroidal glycosides, including seventeen furostanol, four spirostanol and one cholestane glycosides, were online identified. Subsequently, compounds were isolated and their structures were established by the extensive use of 1D- and 2D-NMR experiments. The structures identified by MS were fully consistent with those elucidated by NMR data. Sixteen steroidal glycosides, including thirteen furostanol, two spirostanol and one cholestane glycosides, were identified along with four known furostanol and two spirostanol glycosides. The saponin profile shows that the furostanol glycosides are the main constituents of R. colchicus extract, unlike the other Ruscus species, for which the spirostanol derivatives generally are reported as the major compounds. Moreover, for the first time a cholestane glycoside has been isolated from R. colchicus.

An analytical approach based on ESI-MS, LC–MS and PCA for the quali–quantitative analysis of cycloartane derivatives in Astragalus spp.

Astragalus species are widely used as health foods and dietary supplements, as well as drugs in traditional medicine. To rapidly evaluate metabolite similarities and differences among the EtOH extracts of the roots of eight commercial Astragalus spp., an approach based on direct analyses by ESI-MS followed by PCA of ESI-MS data, was carried out. Successively, quali-quantitative analyses of cycloartane derivatives in the eight Astragalus spp. by LC-ESI-MS(n) and PCA of LC-ESI-MS data were performed. This approach allowed to promptly highlighting metabolite similarities and differences among the various Astragalus spp. PCA results from LC-ESI-MS data of Astragalus samples were in reasonable agreement with both PCA results of ESI-MS data and quantitative results. This study affords an analytical method for the quali-quantitative determination of cycloartane derivatives in herbal preparations used as health and food supplements.

HPLC-ESIMSn Profiling, Isolation, Structural Elucidation, and Evaluation of the Antioxidant Potential of Phenolics from Paepalanthus geniculatus

The methanol extract of the flowers of Paepalanthus geniculatus Kunth. showed radical-scavenging activity in the TEAC assay. An analytical approach based on HPLC-ESIMS(n) was applied to obtain the metabolite profile of this extract and led to the rapid identification of 19 polyphenolic compounds comprising flavonoids and naphthopyranones. The new naphthopyranone (10, 16), quercetagetin (1, 5, 7, 13), and galetine derivatives (9, 11, 17, 19), and a flavonol glucoside cyclodimer in the truxillate form (12), were identified. Compounds 2, 6, and 7 showed the highest antioxidant capacity and ability to affect the levels of intracellular ROS in human prostate cancer cells (PC3).

Identification and quantitative determination of the polar constituents in Helichrysum italicum flowers and derived food supplements.

Helichrysum italicum is widely used in traditional medicine, in cosmetic, in food and pharmaceutical field. In spite of this, very little is known about the chemical composition of its polar extracts. Therefore this study was addressed to the determination of the metabolite profile of the methanol extract of H. italicum flowers, by using LC-ESI(IT)MSMS. This approach oriented the isolation of 14 compounds, whose structures were unambiguously elucidated by NMR as belonging to flavonoid, phenylpropanoid and acylbenzofuran classes. In addition, one novel drimane sesquiterpene was identified. The quantitative determination of the main compounds occurring in the methanol extract of H. italicum flowers was carried out and their content was compared with that of three selected commercial food supplements based on H. italicum, by using LC-ESI(QqQ)MS. In conclusion the wide occurrence, in high amounts, of quinic acid derivatives in all the analyzed samples was highlighted, showing these compounds as chemical markers of the species for standardization procedures.

Qualitative on-line profiling of ceramides and cerebrosides by high performance liquid chromatography coupled with electrospray ionization ion trap tandem mass spectrometry: The case of Dracontium loretense

Ceramides and cerebrosides are key compounds in the metabolism of sphingolipids. Produced in response to a variety of apoptotic stimuli, these metabolites mediate either mitogenic or apoptotic responses, depending on cell type and nature of stimulus. Novel strategies using these selective targets for a therapeutic intervention, e.g. in cancer, cardiovascular and neurodegenerative diseases, and HIV, have been developed, along with anticancer approaches using controlled delivery of exogenous natural ceramides from ceramide-based liposomes. Thus, great is the need to find selective and sensitive analytical methods allowing a prompt detection of ceramides and cerebrosides in natural matrices. Here we report an analytical study carried out on the Amazonian plant Dracontium loretense, resulted in a preliminary analysis a rich source of this class of natural compounds. A handy, selective, and sensitive methodology based on high-performance liquid chromatography coupled to electrospray negative ionization multistage ion trap mass spectrometry (HPLC-ESI/ITMS(n)) was developed. Analysis of fingerprint multistage mass spectra allowed the rapid identification of 3 major long-chain bases and their exact pairing with 11 different fatty acids and with carbohydrate headgroups. Thus, the structures of 21 ceramide and cerebroside species, among which 7 molecules never reported before, were unambiguously assigned. Results obtained in this study demonstrated that this analytical approach could provide a reliable and sensitive method to obtain the qualitative on-line profiling of ceramides and cerebrosides in new medicinal plant matrices.

Characterization of gallotannins from Astronium species by flow injection analysis-electrospray ionization-ion trap-tandem mass spectrometry and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The species Astronium urundeuva (Allemao) Engl. and Astronium graveolens Jacq., which are used in Brazilian folk medicine to treat allergies, inflammation, diarrhea and ulcers, were investigated for their composition. The aim of this study was to define a rapid and reliable analytical approach, based on the flow-injection analysis–electrospray ionization–ion trap–tandem mass spectrometry (FIA-ESI-IT-MS-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-ToF-MS), to investigate the full range of hydrolyzable tannins present in the extracts of these Astronium species. The MALDI-ToF-MS analysis allowed us to ascertain the presence of hydrolysable tannins in both Astronium species as a series of gallotannins with degrees of polymerization of 7 to 13 galloyl units. Moreover, the analysis by FIA-ESI-IT-MS-MS, as well as confirming this result and chemically defining gallotannins as galloylglucose compounds, highlighted the presence of further classes of hydrolysable tannins, such as hexahydrodiphenoyl esters of glucose and some gallic acid derivatives, providing information about their structure by a careful study of their fragmentation patterns. Finally, the evaluation of the number of positional isomers of gallotannins occurring in both Astronium species was obtained by high-performance liquid chromatography–electrospray ionization–ion trap mass spectrometry (HPLC/ESI-IT-MS). This is the first mass spectrometric evidence relating to the existence of gallotannins in Astronium genus.