Neem (Azadirachta indica) leaves have been widely proposed as a traditional medicine and many bioactive compounds have been isolated and characterized. Taking into account the high variability of the constituents content, which is influenced by many factors such as the environment and the crop production technology, a proper and fast analytical method is important for monitoring the chemical composition of the product. Here, we report, for the first time, the NMR-based metabolic fingerprinting of Neem leaves extracts obtained using two different extraction techniques, namely microwave-assisted and conventional extractions. To optimize the recovery of a large range of metabolites, three extraction times (10, 30 and 60 minutes) and three solvents (methanol, chloroform and acetone) were used. The microwave-assisted extraction gave the highest recovery in the case of methanol and acetone, whereas conventional extraction was the best procedure for chloroform.

Background The construction of efficient cell factories for the production of metabolites requires the rational improvement/engineering of the metabolism of microorganisms. The subject of this paper is directed towards the quantitative understanding of the respiratory/fermentative Kluyveromyces lactis yeast metabolism and its rag8 casein kinase mutant, taken as a model for all rag gene mutations. Methods 13C NMR spectroscopy and [1,2-13C2]glucose were used as metabolic stable-isotope tracer to define the metabolic profiling of a K. lactis yeast and its derivative mutants. Results Rag8 showed a decrease of all ¹³C glutamate fractional enrichments, except for [4-¹³C]glutamate that was higher than wild type ones. A decrease of TCA cycle flux in rag8 mutants and a contribution of a [4-¹³C]ketoglutarate pool not originating from mitochondria were suggested. ¹³C lysine enrichments confirmed the presence of two compartmentalized ?-ketoglutarate (?-KG) pools participating to glutamate and lysine synthesis. Moreover, an increased transaldolase, as compared to transketolase activity, was observed in the rag8 mutant by ¹³C NMR isotopomer analysis of alanine. Conclusions ¹³C NMR-based isotopomer analysis showed the existence of different ?-KG metabolic pools for glutamate and lysine biosynthesis. In the rag8 mutant, ¹³C labeled pentose phosphate intermediates participated in the synthesis of this compartmentalized ?-KG pool. General significance A compartmentalization of the ?-KG pools involved in lysine biosynthesis has been revealed for the first time in K. lactis. Given its great impact in metabolic engineering field, its existence should be validated/compared with other yeasts and/or fungal species.

In 2008 a Slow Food Presidium was launched in Sicily (Italy) for an early warning of the risk of extinction of the Sicilian native breed of black honeybee (Apis mellifera L. ssp sicula). Today, the honey produced by these honeybees is the only Sicilian honey produced entirely by the black honeybees. In view of few available data regarding the chemical composition of A. mellifera ssp. sicula honeys, in the present investigation the chemical compositions of sulla honey (Hedysarum coronarium L.) and dill honey (Anethum graveolens L.) were studied with a multimethodological approach, which consists of HPLC-PDA-ESI-MSn and NMR spectroscopy. Moreover, three unifloral honeys (lemon honey (obtained from Citrus limon (L.) Osbeck), orange honey (Citrus arantium L.), and medlar honey (Eriobotrya japonica (Thunb.) Lindl)), with known phenol and polyphenol compositions, were studied with NMR spectroscopy to deepen the knowledge about sugar and amino acid compositions.

In this review, fundamental aspects of the untargeted NMR-based methodology applied to fruit characterization are described. The strategy to perform the structure elucidation of fruit metabolites is discussed with some examples of spectral assignments by 2D experiments. Primary ubiquitous metabolites as well as secondary species-specific metabolites, identified in different fruits using an untargeted 1H-NMR approach, are summarized in a comprehensive way. Crucial aspects regarding the quantitative elaboration of spectral data are also discussed. The usefulness of the NMR-based metabolic profiling was highlighted using some results regarding quality, adulteration, varieties and geographical origin of fruits and fruit-derived products such as juices.

Ring-closing metathesis (RCM) catalyzed by a second-generation Grubbs catalyst has been used to synthesize resorc[4]arenes 2b - 5b starting from undecenyl resorc[4]arene 1b fi xed in the cone conformation. X-ray diffraction analysis of the major metathesis product, 3b (50% yield), revealed a cavity-shaped architecture resembling a basket, endowed with a large intramolecular space (~10 Å) and a strong propensity to self-assemble as a supramolecular trio of heterochiral dimers. This prompted us to investigate the aggregation propensity of basket 3b in THF/water solution by UV - visible spectroscopy. The cavitation Gibbs free-energy change ( ?? Gcav = 4.78 kcal mol-1) associated with the self-assembly of macrocycle 3b was calculated as a measure of the solvophobic interactions involved in the process.

Commercial flavoured olive and olive-sunflower oils and seed oils with particular nutritional properties (e.g. linseed, safflower, sunflower, sesame and rice oils) were analysed using a widespread analytical protocol to have information on their quality and chemical composition. The protocol involved traditional determinations (free acidity, peroxide value, UV and VIS spectrophotometric indices, and fatty acid composition) along with 1H and 13C NMR analyses. Most of flavoured olive oils turned out to be lampante olive oils and not extra virgin as declared in the label on the bottle. In the case of olive-sunflower oils only a minor fraction of olive oil was revealed although these products are particularly expensive and the presence of olive oil is emphasized on the label. In some seed oils, refinement processes, not indicated on the bottle, were highlighted. Some compounds characteristic of specific seed oils were identified in the 1H spectra.

The chemical composition of tea beverage is very complex and not yet completely elucidated. Many factors contribute to the chemical complexity of tea, from plant growth conditions (soil, climate, growth altitude and agricultural practices) and manufacturing processes (drying, steaming, fermentation and storage), to preparation methods (quality of water, infusion time and, not least, the time between tea preparation and consumption). Besides primary metabolites, tea leaves contain a number of secondary metabolites, belonging to many different classes of compounds (such as polyphenols, xanthines, proteic and nonproteic amino acids, sugars, volatile compounds) that are extracted during the infusion and transferred into the beverage. Epidemiological studies have suggested that tea consumption is inversely associated with the risk of developing chronic diseases such as cardiovascular diseases, stroke, some forms of cancer and diabetes. Thus, increasing interest in the health properties of tea resulted in a significant rise in scientific investigation on tea chemical composition. This review article highlights the recent results obtained in the tea beverage characterization using different analytical methodologies. The analytical approaches have been subdivided into two groups: targeted chromatographic and NMR techniques and untargeted NMR analytical approaches. Several examples of untargeted chromatographic techniques coupled with mass spectrometry methodologies are also reported. Advantages, drawbacks and significant applications of the different analytical approaches are discussed.

La spettroscopia NMR è una delle principali metodologie analitiche per stabilire l'identità, la tracciabilità e la valorizzazione di prodotti agroalimentari, permettendo di avere una visione completa dei metaboliti presenti nell'alimento. [1-3] Nell'ambito del progetto CISIA VISP-LP sono stati analizzati i profili metabolici di indivia, scarola e puntarelle (Cichorium spp.) del Lazio e della Puglia Lo studio NMR ha permesso di studiare il profilo metabolico di cultivar commerciali (E02, Myrna) di indivia (Cichorium endivia var. crispum) e cultivar commerciali (Flester, Confiance) di scarola (C.endivia var. latifolium) allo stadio di trapianto (22 giorni dalla semina) e alla raccolta (86 giorni) e di cultivar commerciali (Galatina , Molfettese) di puntarelle (C. Intybus var. Catalogna) nell'arco di due annate (2011, 2012). Sono stati identificati grazie a esperimenti 2D NMR e aggiunte di opportuni standard, oltre trenta metaboliti solubili in acqua quali amminoacidi (alanina, aspartato, aspargina, isoleucina, glutammato, glutammina, fenilalanina, tirosina, valina,GABA), acidi organici (citrico, fumarico, lattico, malico, quinico, succinico, tartarico), zuccheri e polialcoli (glucosio, fruttosio, saccarosio, inulina, kestosio, myo-inositolo, scyllo-inositolo, chiro-inositolo), acidi fenolici (acido cicorico, acido mono-caffeoil-tartarico), ed altri metaboliti quali colina ed etanolammina. L'analisi statistica dei profili metabolici ha evidenziato che lo stato di maturazione e le stagioni di produzione sono i fattori principali che incidono sul contenuto dei metaboliti.

An NMR analytical protocol is proposed to characterize saffron samples of different geographical origin (Greece, Spain, Hungary, Turkey and Italy). A microwave-assisted extraction procedure was developed to obtain a comparable recovery of metabolites with respect to the ISO specifications, reducing the solvent volume and the extraction time needed. Metabolite profiles of geographically different saffron extracts were compared showing significant differences in the content of some metabolites.

The antistaphylococcal activity as well as the metabolic profiling and polyphenols content of green tea (Camellia sinensis) before and after in vitro simulated gastric, duodenal and gastroduodenal digestion were investigated. Gastric and duodenal digested samples showed antistaphylococcal activity, whereas gastroduodenal digested samples did not show any antibacterial activity. Metabolite analysis, carried out using an explorative untargeted NMR-based approach and a RP-HPLC-PAD-ESI-MSn method, showed that green tea polyphenols are stable under gastric conditions. Duodenal digested sample maintained the antibacterial activity, even if some polyphenols are widely degraded. Epicatechin 3-gallate, under duodenal digestive conditions, is hydrolyzed to produce epicatechin, whereas epigallocatechin 3-gallate reacts with digestive enzymes and a galloyl-high molecular weight derivative is produced. Gastroduodenal digestion results in degradation of polyphenols, especially gallocatechins, considered the main responsible for the antibacterial activity. These results explain the loss of activity of gastroduodenal digested samples and why in vivo green tea has neither protective nor therapeutic effects against intestinal and systemic bacterial infections.

Exploring the effect that substituents on the cycloaliphatic ring had on the inhibitory activity against human monoamine oxidase B of a series of 4-aryl-2-cycloalkylidenhydrazinylthiazoles led to the synthesis of a new series of 2-methylcyclopentyl and 3-methylcyclopentyl derivatives which were tested in vitro as mixtures of diastereoisomers. In fact, due to the presence of a chiral center on the cycloaliphatic ring and a trisubstituted CN bond, they exist as four diastereoisomers ((E)-(R), (E)-(S), (Z)-(R), (Z)-(S)). 4-(2,4- Difluorophenyl)-2-(2-(3-methylcyclopentylidene)hydrazinyl)thiazole was chosen as a model to investigate the influence of stereochemical requirements on the inhibitory activity against hMAO-B of these derivatives after a stereoconservative synthesis and semi-preparative HPLC diastereoseparation. (R)-(Z) isomer of this compound was endowed with a potent and selective hMAO-B inhibition higher than that of reference drugs as also corroborated by molecular modeling studies.

Italy is the leader in the production of leafy crops such as endive and escarole (Cichorium endivia var. latifolium and crispum), which are consumed as fresh or minimally processed food. The valorisation of "made in Italy" produce was achieved by assessing cultivar identity and traceability and by enhancing the nutritive and economic value using genomic, transcriptomic and metabolomic approaches in collaboration with public and private enterprises. Within the CISIA project [1], the NMR spectroscopy was chosen to monitor the metabolic profiles of endive and escarole cultivars. Thirty hydro-soluble metabolites (amino acids, organic acids, sugars, polyols, phenols) from leaves of curly- and smooth- leafed endives (seven cultivars) were identified and quantified at transplanting and harvesting (22 and 86 days after sowing, respectively). Four cultivars ('Domari', 'Myrna', 'Flester', 'Confiance') were monitored in the Lazio region during two production cycles (2011-2012) and two of them ('Domari' and 'Flester') further tested in Puglia for a one year cycle (2013). The multivariate statistical analysis of NMR profiles pointed at the leaf developmental stages and environmental conditions as the major factors of metabolite variability, although cultivar-specific profiles could be assessed.

The tender stems (turions) of chicory (Cichorium intybus L. Catalogna group) are widely consumed as fresh or processed food in central and southern Italy. They are also known as 'puntarelle' and considered as elite groceries, cultivated as local varieties in Apulia and Lazio. The project CISIA has developed markers based on metabolic, transcriptional and allelic variations aimed to identity, traceability and valorisation of the Galatina and Molfettese Apulian landraces propagated by local enterprises. A de novo reference transcriptome was assembled using RNA of several organs (borne on young and adult plants of Galatina) and Illumina HiSeq2000 technology. Ca. 170M reads were generated (100bp paired-end) and assembled into 96,514 contigs of 1,168bp average length (N50 = 1,435bp). Among them, 68,260 (70.7%) showed significant similarity (Evalue <1e-5) to proteins in public databases (NCBI, TAIR, SwissProt, TrEMBL); 61,343 (63.6%) were classified based on gene ontology criteria and 13,536 (14.0%) were mapped onto 130 KEGG pathways. RNA-seq experiments (50bp single-end reads) were performed to collect gene transcriptional variation and polymorphism data using stems of Galatina and Molfettese grown in the two regions. Ca. 1,700 genes were found to maintain the differential expression patterns, suggesting that they may be putative markers as being feebly dependent on the environment. Polymorphic SSR (ca. 100) and high-quality SNPs (ca. 71,000) were effective to discriminate the two varieties, representing potential tools for fingerprinting and map enrichment in assisted breeding.

The effects of methanol (M) and acetonitrile (A) on the stability of cycloserine (1) have been studied. InfraRed Multiphoton PhotoDissociation (IRMPD) spectroscopy of the ionic species from electrospray ionization tandem mass spectrometry (ESI-MS) of 1/M and 1/A solutions points to extensive dimerization of 1 to cis-3,6-bis(aminooxymethyl)-2,5-piperidinedione (2), while the same process is not observed in the ESI-MS of 1/M solutions. 1D and 2D nuclear magnetic resonance experiments confirmed these findings by showing that partial dimerization of 1 actually takes place at room temperature in acetonitrile even before ESI-MS analysis. Comparison of nuclear magnetic resonance and IRMPD spectroscopic data from the same 1/A solution suggests that dimerization of cycloserine is enhanced in the ESI source.

Endive and escarole (C. endivia var. crispum and var. latifolium) are consumed as fresh or minimally processed food; Italy is a major producer in Europe and the CISIA project focuses on developing knowledge and tools to enhance their traceability and value. The profiles of carbohydrate content, including that of inulin (a prebiotic), gene transcription and allelic variability were used to fingerprint cultivars of broad-leafed ('Confiance' and 'Flester', a.k.a. escaroles) and curly-leafed endive ('E02.7162' and 'Myrna') at harvest. NMR profiling of over thirty hydro-soluble metabolites allowed statistically significant discrimination of the broad- from the curly- leafed genotypes. As for carbohydrates, significant differences were observed for sucrose, glucose and fructose among the four cultivars. Moreover, the inulin content (g kg-1 dry weight) of the curlyleafed varieties was ca. two fold higher than that of escaroles (11.9± 4.3 vs 6.23±1.4). Subsequently, inulin synthesis (fructosyl-fructosyl transferase, FFT; sucrose sucrosyl transferase, SST) and catabolic (Fructan 1-exohydrolases I and II, FEHI and FEHIIa) gene expression was assayed. Both SST and FFT transcription levels varied in the four genotypes, being maximal in 'Myrna', followed by 'E02.7162', 'Confiance' and 'Flester'. The FEHI transcription was steady in all cultivars, whereas that of FEHIIa was maximal and minimal in 'Confiance' and 'Flester' escaroles, respectively, and was comparable in the two curly-leafed endives. Overall, the mRNA abundance of the two catabolic genes was significantly higher than those of synthesis. Finally, structural diversity (single nucleotide polymorphism, SNP) of the aforesaid inulin genes was studied and SNPs in 1- SST were effective to univocally fingerprint 'Flester' and 'E02.7162' among the four genotypes.

Italy is leader in the production of leafy vegetables such as endive and escarole (C. endivia var. latifolium and var. crispum), which are consumed as fresh or minimally processed food. The project CISIA aims at assessing the identity and traceability of some patented varieties. The profiles of carbohydrate content, which included that of the prebiotic inulin, gene transcription and allelic variability were used to fingerprint and valorise cultivars of broad-leafed ('Confiance' and 'Flester', a.k.a. escaroles) and curly-leafed endive ('E02.7162' and 'Myrna') at harvest. Thirty eight hydro-soluble metabolites were profiled by NMR and statistical analyses significantly discriminated the broad- from the curly- leafed genotypes. Concerning carbohydrates, minimal (though significant) differences were observed for sucrose, glucose and fructose among the four cultivars. Most interestingly, the inulin content (g kg-1 dry weight) of the curly-leafed varieties was ca. two fold higher than that of escaroles (11.9± 4.3 vs 6.23±1.4). The expression of major inulin synthesis (fructosyl-fructosyl transferase, FFT; sucrose sucrosyl transferase, SST) and catabolic (Fructan 1-exohydrolases I and II, FEHI and FEHIIa) genes was monitored. The transcription levels of both SST and FFT significantly differed in the four varieties, being maximal in 'Myrna', followed by 'E02.7162', 'Confiance' and 'Flester'. The FEHI transcription did not vary in the four cultivars, whereas the FEHIIa expression was maximal and minimal in 'Confiance' and 'Flester' escaroles, respectively, and was comparable in the two curly-leafed endives. Overall, the mRNA abundance of the two catabolic genes was significantly higher than those of synthesis. Finally, the structural diversity (single nucleotide polymorphism) of the aforesaid inulin genes was studied to discriminate the four genotypes.

Chicory (C. intybus L.) of the Catalogna group produces tender stems (located in the middle of the leaf rosette) which are exquisite and nutritious food1 and traditionally eaten as raw ("puntarelle") or cooked products in Lazio and Puglia, respectively. The CISIA project, sustained by governmental and SMEs, falls in a context of valorisation of "made in Italy" produce and aims at assessing the identity and traceability of typical varieties and at enhancing their nutritive and economic value by holistic approaches2. Thirty seven hydro-soluble metabolites (amino acids, organic acids, sugars, polyols, phenols) from stems of two Apulian genotypes ('Galatina' and 'Molfettese') were monitored by NMR3 at harvesting (ca 85 days after sowing in a winter cycle). Overall, the 'Molfettese' stems showed a higher content of alanine, asparagine, glutamine, ?-aminobutirric acid, fumaric acid, fructose and glucose than those of 'Galatina'. On the contrary, the latter contained higher amounts of aspartic acid and inulin, which is an important prebiotic sugar stored by several Asteraceae plants. Interestingly, both varieties produced abundant amounts of scyllo-inositol, which has beneficial effects for memory deficits. Finally, statistical analyses applied to the NMR profiles significantly discriminated the two genotypes.

Freshness and authenticity of Chinese and Italian royal jelly samples were investigated by means of HPLC determination of furosine and (E)-10-hydroxy-2-decenoic acid. In addition, High Resolution Magic Angle Spinning Nuclear Magnetic Resonance (HR-MAS NMR) spectroscopy was used to determine the metabolic profile of the same royal jelly samples. Water-soluble metabolites such as amino acids and sugars as well as fatty acids were identified in the same experiment. A combined approach including NMR analytical profile together with HPLC measured markers was used to discriminate between Italian and Chinese samples by means of Principal Component Analysis.