Methanolic Extracts of D. viscosa Specifically Affect the Cytoskeleton and Exert an Antiproliferative Effect on Human Colorectal Cancer Cell Lines, According to Their Proliferation Rate.

Numerous studies have reported the pharmacological effects exhibited by Dittrichia viscosa, (D. viscosa) including antioxidant, cytotoxic, antiproliferative, and anticancer properties. In our research, our primary objective was to validate a prescreening methodology aimed at identifying the fraction that demonstrates the most potent antiproliferative and anticancer effects. Specifically, we investigated the impact of various extract fractions on the cytoskeleton using a screening method involving transgenic plants. Tumors are inherently heterogeneous, and the components of the cytoskeleton, particularly tubulin, are considered a strategic target for antitumor agents. To take heterogeneity into account, we used different lines of colorectal cancer, specifically one of the most common cancers regardless of gender. In patients with metastasis, the effectiveness of chemotherapy has been limited by severe side effects and by the development of resistance. Additional therapies and antiproliferative molecules are therefore needed. In our study, we used colon-like cell lines characterized by the expression of gastrointestinal differentiation markers (such as the HT-29 cell line) and undifferentiated cell lines showing the positive regulation of epithelial-mesenchymal transition and TGFß signatures (such as the DLD-1, SW480, and SW620 cell lines). We showed that all three of the D. viscosa extract fractions have an antiproliferative effect but the pre-screening on transgenic plants anticipated that the methanolic fraction may be the most promising, targeting the cytoskeleton specifically and possibly resulting in fewer side effects. Here, we show that the preliminary use of screening in transgenic plants expressing subcellular markers can significantly reduce costs and focus the advanced characterization only on the most promising therapeutic molecules.

1H-NMR metabolomic profile of healthy and osteoarthritic canine synovial fluid before and after UC-II supplementation.

The aim of the study was to compare the metabolomic synovial fluid (SF) profile of dogs affected by spontaneous osteoarthritis (OA) and supplemented with undenatured type II collagen (UC-II), with that of healthy control dogs. Client-owned dogs were enrolled in the study and randomized in two different groups, based on the presence/absence of OA (OA group and OA-free group). All dogs were clinically evaluated and underwent SF sampling for 1H-Nuclear Magnetic Resonance spectroscopy (1H-NMR) analysis at time of presentation. All dogs included in OA group were supplemented with UC-II orally administered for 30 days. After this period, they were reassessed (OA-T30). The differences in the 1H-NMR metabolic SFs profiles between groups (OA-free, OA-T0 and OA-T30) were studied. The multivariate statistical analysis performed on SFs under different conditions (OA-T0 vs OA-T30 SFs; OA-T0 vs OA-free SFs and OA-T30 vs OA-free SFs) gave models with excellent goodness of fit and predictive parameters, revealed by a marked separation between groups. ß-Hydroxybutyrate was identified as a characteristic compound of osteoarthritic joints, showing the important role of fat metabolism during OA. The absence of ß-hydroxybutyrate after UC-II supplementation suggests the supplement's effectiveness in rebalancing the metabolism inside the joint. The unexpectedly high level of lactate in the OA-free group suggests that lactate could not be considered a good marker for OA. These results prove that 1H-NMR-based metabolomic analysis is a valid tool to study and monitor OA and that UC-II improves clinical symptoms and the SF metabolic profile in OA dogs.

Agro-active endo-therapy treated Xylella fastidiosa subsp. pauca-infected olive trees assessed by the first 1H-NMR-based metabolomic study.

Xylella fastidiosa is a xylem-limited bacterium causing a range of economically important plant diseases in hundreds of crops. Over the last decade, a severe threat due to Olive Quick Decline Syndrome (OQDS), caused by Xylella fastidiosa subspecies pauca, affected the Salento olive groves (Apulia, South-East Italy). Very few phyto-therapeutics, including a Zn/Cu citric acid biocomplex foliar treatment, were evaluated to mitigate this disease. However, the traditional foliar applications result in the agro-actives reaching only partially their target. Therefore the development of novel endo-therapeutic systems was suggested. Metabolite fingerprinting is a powerful method for monitoring both, disease progression and treatment effects on the plant metabolism, allowing biomarkers detection. We performed, for the first time, short-term monitoring of metabolic pathways reprogramming for infected Ogliarola salentina and Cima di Melfi olive trees after precision intravascular biocomplex delivery using a novel injection system. Upon endo therapy, we observed specific variations in the leaf content of some metabolites. In particular, the 1H NMR-based metabolomics approach showed, after the injection, a significant decrease of both the disease biomarker quinic acid and mannitol with simultaneous increase of polyphenols and oleuropein related compounds in the leaf's extracts. This combined metabolomics/endo-therapeutic methodology provided useful information in the comprehension of plant physiology for future applications in OQDS control.

Analysis of the Phytochemical Composition of Pomegranate Fruit Juices, Peels and Kernels: A Comparative Study on Four Cultivars Grown in Southern Italy.

The increasing popularity of pomegranate (Punica granatum L.), driven by the awareness of its nutraceutical properties and excellent environmental adaptability, is promoting a global expansion of its production area. This investigation reports the variability in the weight, moisture, pH, total soluble solids, carbohydrates, organic acids, phenolic compounds, fatty acids, antioxidant activities, and element composition of different fruit parts (juices, peels, and kernels) from four (Ako, Emek, Kamel, and Wonderful One) of the most widely cultivated Israeli pomegranate varieties in Salento (South Italy). To the best of our knowledge, this is the first systematic characterization of different fruit parts from pomegranate cultivars grown simultaneously in the same orchard and subjected to identical agronomic and environmental conditions. Significant genotype-dependent variability was observed for many of the investigated parameters, though without any correlation among fruit parts. The levels of phenols, flavonoids, anthocyanins, and ascorbic and dehydroascorbic acids of all samples were higher than the literature-reported data, as was the antioxidant activity. This is likely due to positive interactions among genotypes, the environment, and good agricultural practices. This study also confirms that pomegranate kernels and peels are, respectively, rich sources of punicic acid and phenols together, with several other bioactive molecules. However, the variability in their levels emphasizes the need for further research to better exploit their agro-industrial potential and thereby increase juice-production chain sustainability. This study will help to assist breeders and growers to respond to consumer and industrial preferences and encourage the development of biorefinery strategies for the utilization of pomegranate by-products as nutraceuticals or value-added ingredients for custom-tailored supplemented foods.

First Insight into Nutraceutical Properties of Local Salento Cichorium intybus Varieties: NMR-Based Metabolomic Approach.

BACKGROUND: Plants of genus Cichorium are known for their therapeutic and nutraceutical properties determined by a wealth of phytochemical substances contained in the whole plant. The aim of this paper was to characterize the metabolic profiles of local Salento chicory (Cichorium intybus L.) varieties ("Bianca", "Galatina", "Leccese", and "Otranto") in order to describe their metabolites composition together with possible bioactivity and health beneficial properties. METHODS: The investigation was performed by 1H-NMR spectroscopy and Multivariate Analysis (MVA), by which the metabolic profiles of the samples were easily obtained and compared. RESULTS: The supervised Partial Least Squares Discriminant Analysis (PLS-DA) analysis showed as "Bianca" and "Galatina" samples grouped together separated by "Leccese" and "Otranto" varieties. A different content of free amino acids and organic acids was observed among the varieties. In particular a high content of cichoric and monocaffeoyl tartaric acid was observed for the "Leccese" variety. The presence of secondary metabolites adds significant interest in the investigation of Cichorium inthybus, as this vegetable may benefit human health when incorporated into the diet. CONCLUSIONS: The 1H-NMR (Nuclear Magnetic Resonance Spectroscopy) based characterization of Salento chicory varieties allowed us to determine the potential usefulness and nutraceutical properties of the product, also providing a method to guarantee its authenticity on a molecular scale.

1H-NMR metabolomics reveals a multitarget action of Crithmum maritimum ethyl acetate extract in inhibiting hepatocellular carcinoma cell growth.

Hepatocellular carcinoma (HCC) is nowadays the sixth cause of tumour-related deceases worldwide, estimated to become the third in Western countries by 2030. New drugs for HCC treatment still have many adverse effects. Several lines of evidence indicate that plant metabolites offer concrete opportunities for developing new therapeutic strategies for many diseases, including cancer. We previously reported that ethyl acetate extract of a spontaneous edible plant harvested in Apulia, Crithmum maritimum, significantly inhibited cell growth in HCC cells. By 1H-NMR spectroscopy, here we show that Crithmum maritimum ethyl acetate extract counteracts the Warburg effect, by reducing intracellular lactate, inhibits protein anabolism, by decreasing amino acid level, and affects membrane biosynthesis by lowering choline and phosphocholine. Also, we observed an effect on lipid homeostasis, with a reduction in triglycerides, cholesterol, monounsaturated fatty acids (MUFA), and diunsaturated fatty acids (DUFA), and an increase in polyunsaturated fatty acids (PUFA). Taken together, these data demonstrate that Crithmum maritimum-induced cytostasis is exerted through a multi-effect action, targeting key metabolic processes in HCC cells. Overall, our findings highlight the role of Crithmum maritimum as a promising tool for the prevention and the improvement of the therapeutic options for HCC and other types of tumours.

[Pt(O,O'-acac)(γ-acac)(DMS)]: Alternative Strategies to Overcome Cisplatin-Induced Side Effects and Resistance in T98G Glioma Cells.

Cisplatin (CDDP) is one of the most effective chemotherapeutic agents, used for the treatment of diverse tumors, including neuroblastoma and glioblastoma. CDDP induces cell death through different apoptotic pathways. Despite its clinical benefits, CDDP causes several side effects and drug resistance.[Pt(O,O'-acac)(γ-acac)(DMS)], namely PtAcacDMS, a new platinum(II) complex containing two acetylacetonate (acac) and a dimethylsulphide (DMS) in the coordination sphere of metal, has been recently synthesized and showed 100 times higher cytotoxicity than CDDP. Additionally, PtAcacDMS was associated to a decreased neurotoxicity in developing rat central nervous system, also displaying great antitumor and antiangiogenic activity both in vivo and in vitro. Thus, based on the knowledge that several chemotherapeutics induce cancer cell death through an aberrant increase in [Ca2+]i, in the present in vitro study we compared CDDP and PtAcacDMS effects on apoptosis and intracellular Ca2+ dynamics in human glioblastoma T98G cells, applying a battery of complementary techniques, i.e., flow cytometry, immunocytochemistry, electron microscopy, Western blotting, qRT-PCR, and epifluorescent Ca2+ imaging. The results confirmed that (i) platinum compounds may induce cell death through an aberrant increase in [Ca2+]i and (ii) PtAcacDMS exerted stronger cytotoxic effect than CDDP, associated to a larger increase in resting [Ca2+]i. These findings corroborate the use of PtAcacDMS as a promising approach to improve Pt-based chemotherapy against gliomas, either by inducing a chemosensitization or reducing chemoresistance in cell lineages resilient to CDDP treatment.

1H NMR Metabolic Profile of Scyphomedusa Rhizostoma pulmo (Scyphozoa, Cnidaria) in Female Gonads and Somatic Tissues: Preliminary Results.

The Mediterranean basin is one of the regions heavily affected by jellyfish bloom phenomena, mainly due to the presence of scyphozoans, such as Rhizostoma pulmo. The jellyfish have few natural predators, and their bodies represent an organic-rich substrate that can support rapid bacterial growth with great impact on the structure of marine food webs. In Asiatic countries, jellyfish are widely studied for their health benefits, but their nutritional and nutraceutical values still remain poorly characterized. In this study, the differences in the 1H NMR spectroscopy metabolic profiles of R. pulmo female gonads and body fractions (including umbrella and oral arms), in different sampling periods, were studied. For each body compartment both lipid and aqueous extracts were characterized and their 1H NMR metabolic profiles subjected to multivariate analysis. From a statistical analysis of the extracts, a higher contents of ω-3 polyunsaturated fatty acids (PUFAs), amino acid and osmolytes (homarine, betaine, taurine) with important roles in marine invertebrates were observed in female gonads, whereas umbrella and oral arms showed similar metabolic profiles. These results support a sustainable exploitation of the jellyfish for the extraction of bioactive compounds useful in nutraceutical, nutricosmetics, and functional food fields.

Biostimulants from food processing by-products: agronomic, quality and metabolic impacts on organic tomato (Solanum lycopersicum L.).

BACKGROUND: Biostimulants have recently gained increased attention due to their multiple benefits for sustainable agriculture. In this study, three food processing by-products - fennel processing residues (FPR), lemon processing residues (LPR) and brewer's spent grain (BSG) - were investigated as potential sources of biostimulants. Their aqueous extracts as individual and associated applications were assessed for their effects on agronomic, quality and metabolic performance of organic tomato in comparison to extract of humic substances (HS) and untreated control (CTRL). RESULTS: Only FPR extracts stimulated shoot growth and tomato dry matter content, whereas all candidates improved tomato yield. FPR and BSG increased fruit mineral content and BSG-FPR-LPR in combination enhanced titratable acidity. FPR-treated fruits had also 20% more vitamin C than CTRL, and higher phenol content was obtained in those of BSG-LPR. Fruit metabolomic profile showed the tendency of all extracts, except BSG-LPR, to increase tomato citric acid and to decrease ß-glucose and methanol concentrations. The analysis revealed accordingly the indispensable role of FPR in combined applications for inducing an HS-like response in fruits. CONCLUSION: The results were indicative of the biostimulant activity of these extracts and demonstrated them, particularly FPR, as promising candidates for enhancing plant productivity and fruit quality. © 2017 Society of Chemical Industry.

Comparative analysis of the proximate and elemental composition of the blue crab Callinectes sapidus, the warty crab Eriphia verrucosa, and the edible crab Cancer pagurus.

The proximate composition and element contents of claw muscle tissue of Atlantic blue crabs (Callinectes sapidus) were compared with the native warty crab (Eriphia verrucosa) and the commercially edible crab (Cancer pagurus). The scope of the analysis was to profile the chemical characteristics and nutritive value of the three crab species. Elemental fingerprints showed significant inter-specific differences, whereas non-significant variations in the moisture and ash contents were observed. In the blue crab, protein content was significantly lower than in the other two species, while its carbon content resulted lower than that characterizing only the warty crab. Among micro-elements, Ba, Cr, Cu, Li, Mn, Ni, and Pb showed extremely low concentrations and negligible among-species differences. Significant inter-specific differences were observed for Na, Sr, V, Ba, Cd and Zn; in particular, cadmium and zinc were characterized in the blue crab by concentrations significantly lower than in the other two species. The analysis of the available literature on the three species indicated a general lack of comparable information on their elemental composition. The need to implement extended elemental fingerprinting techniques for shellfish quality assessment is discussed, in view of other complementary profiling methods such as NMR-based metabolomics.

Robustness of NMR-based metabolomics to generate comparable data sets for olive oil cultivar classification. An inter-laboratory study on Apulian olive oils.

Nuclear Magnetic Resonance (NMR) spectroscopy is emerging as a powerful technique in olive oil fingerprinting, but its analytical robustness has to be proved. Here, we report a comparative study between two laboratories on olive oil (1)H NMR fingerprinting, aiming to demonstrate the robustness of NMR-based metabolomics in generating comparable data sets for cultivar classification. Sample preparation and data acquisition were performed independently in two laboratories, equipped with different resolution spectrometers (400 and 500 MHz), using two identical sets of mono-varietal olive oils. Partial Least Squares (PLS)-based techniques were applied to compare the data sets produced by the two laboratories. Despite differences in spectrum baseline, and in intensity and shape of peaks, the amount of shared information was significant (almost 70%) and related to cultivar (same metabolites discriminated between cultivars). In conclusion, regardless of the variability due to operator and machine, the data sets from the two participating units were comparable for the purpose of classification.

First-time comparison of the in vitro antimalarial activity of Artemisia annua herbal tea and artemisinin.

Artemisia annua tea has been proven to be a very effective treatment for malaria in various clinical trials, but to date its efficacy has not been investigated in vitro. A study was therefore performed to evaluate the effects of A. annua tea on Plasmodium falciparum cultures in vitro. The concentration of artemisinin in the herbal tea preparation was also determined. The herbal tea extract was tested against chloroquine (CQ)-sensitive D10 and CQ-resistant W2 strains of P. falciparum using the parasite lactate dehydrogenase assay. Quantification of artemisinin in the extract of leaves of A. annua was performed using proton nuclear magnetic resonance ((1)H-NMR). Results of the in vitro tests were consistent with the clinical efficacy of A. annua tea [50% inhibitory concentration (IC(50)) for strain D10=1.11±0.21 µg/ml; IC(50) for strain W2=0.88±0.35 µg/ml]. The concentration of artemisinin in A. annua tea (0.18±0.02% of dry weight) was far too low to be responsible for the antimalarial activity. The artemisinin present in the tea is probably co-solubilised with other ingredients, some of which also have antimalarial activity and act synergistically with it. These compounds also merit further research to determine whether their presence hinders the development of parasite resistance compared with pure artemisinin.

1H nuclear magnetic resonance study of olive oils commercially available as Italian products in the United States of America.

Multivariate analysis of (1)H NMR data has been used for the characterization of 12 blended olive oils commercially available in the U.S. as Italian products. Chemometric methods such as unsupervised Principal Component Analysis (PCA) allowed good discrimination and gave some affinity indications for the U.S. market olive oils compared to other single cultivars of extra virgin olive oil such as Coratina and Ogliarola from Apulia, one of Italy's leading olive oil producers, Picual (Spain), Kalamata (Greece) and Sfax (Tunisia). The olive oils commercially available as Italian products in the U.S. market clustered into 3 groups. Among them only the first (7 samples) and the second group (2 samples) showed PCA ranges similar to European references. Two oils of the third group (3 samples) were more similar to Tunisian references. In conclusion, our study revealed that most EVOO (extra virgin olive oils) tested were closer to Greek (in particular) and Spanish olive oils than Apulia EVOO. The PCA loadings disclose the components responsible for the discrimination as unsaturated (oleic, linoleic, linolenic) and saturated fatty acids. All are of great importance because of their nutritional value and differential effects on the oxidative stability of oils. It is evident that this approach has the potential to reveal the origin of EVOO, although the results support the need for a larger database, including EVOO from other Italian regions.

Phytochemical analysis of a herbal tea from Artemisia annua L.

Strategies to control diffusion of malaria needs to account for the increase of resistance of the parasite to the conventional antimalarial drugs. It has been proposed that a traditional aqueous preparation from Artemisia annua, with a low content of the active compound, artemisinin, may reduce the risk of resistance of the protozoa and be relatively more effective in the treatment of the disease. The solubility properties of the molecule have been the matter of concern about the therapeutic usefulness of herbal teas from A. annua. The present study aimed at analysing the chemical profile of a tea infusion from A. annua. Tea from A. annua was prepared through infusion of the plant aerial parts in water for 1, 24 and 48 h. Content of artemisinin was determined by HPLC-ELSD. Overall chemical characterization of the extracts was carried out by a combination of metabolomic techniques. The artemisinin content varied only slightly in the three different extracts (about 0.12%). A series of mono-caffeoyl- and mono-feruloyl-quinic acids, di-caffeoyl- and di-feruloyl-quinic acids was identified as main components of the tea infusion, together with some flavonoids. Reconstitution of the same extracts in less polar or apolar solvents resulted in a different composition with no phenolics and a much lower concentration of artemisinin.

Glucosinolates profile of "mugnolo", a variety of Brassica oleracea L. native to southern Italy (Salento).

Glucosinolates (GLSs) from a variety of Brassica oleracea, known locally as "mugnolo" and widely distributed in southern Italy, were studied. It was found that "mugnolo" inflorescences are characterized by the presence of the aliphatic GLSs glucoraphanin (1.79 µmol/g), glucoiberin, glucoerucin, and sinigrin, of the aromatic GLSs glucobarberin (0.56 µmol/g) and gluconasturtin, and of the indole GLSs glucobrassicin (3.51 µmol/g), neoglucobrassicin, 4-methoxyglucobrassicin, and 4-hydroxyglucobrassicin. Indole GLSs were predominant, while aliphatic and aromatic GLS were found in lower quantities. The metabolic profile of "mugnolo" leaves was also studied. It was found that 4-hydroxyglucobrassicin (0.13 µmol/g) and glucoraphanin (0.11 µmol/g) are the predominant GLSs. Vegetables of the Brassicaceae family are seldom consumed raw; therefore we also analyzed the GLS profile of "mugnolo" after cooking in water. The results showed variations in the GLSs content with a fall in concentration of 50%.