Maria Halabalaki

Walnut extract (Juglans regia L.) and its component ellagic acid exhibit anti-inflammatory activity in human aorta endothelial cells and osteoblastic activity in the cell line KS483

Epidemiological studies suggest that the incidence of CVD and postmenopausal osteoporosis is low in the Mediterranean area, where herbs and nuts, among others, play an important role in nutrition. In the present study, we sought a role of walnuts (Juglans regia L.) in endothelial and bone-cell function. As the endothelial cell expression of adhesion molecules has been recognised as an early step in inflammation and atherogenesis, we examined the effect of walnut methanolic extract and ellagic acid, one of its major polyphenolic components (as shown by HPLC analysis), on the expression of vascular cell adhesion molecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1 in human aortic endothelial cells. After incubating the cells with TNF-alpha (1 ng/ml) in the absence and in the presence of walnut extract (10-200 microg/ml) or ellagic acid (10- 7-10- 5 m), the VCAM-1 and ICAM-1 expression was quantified by cell-ELISA. We further evaluated the effect of walnut extract (10-50 microg/ml), in comparison with ellagic acid (10- 9-10- 6m), on nodule formation in the osteoblastic cell line KS483. Walnut extract and ellagic acid decreased significantly the TNF-alpha-induced endothelial expression of both VCAM-1 and ICAM-1 (P < 0.01; P < 0.001). Both walnut extract (at 10-25 microg/ml) and ellagic acid (at 10- 9-10- 8 m) induced nodule formation in KS483 osteoblasts. The present results suggest that the walnut extract has a high anti-atherogenic potential and a remarkable osteoblastic activity, an effect mediated, at least in part, by its major component ellagic acid. Such findings implicate the beneficial effect of a walnut-enriched diet on cardioprotection and bone loss.

Recent advances and new strategies in the NMR-based identification of natural products.

Nature comprises an untapped pool of unique compounds with high structural uniqueness and exceptional properties. At the core of natural products (NPs) discovery is the identification procedure and NMR remains the most efficient method. Technical improvements such as miniaturized and crycogenic NMR probes along with hyphenation capabilities and computational support are at the center of evolution. Concepts such as dereplication and metabolomics are increasingly adopted in NPs using the power of databases, currently fragmented. The introduction and utilization of these technical and computational implements could lead NPs research to more comprehensive structure identification and new holistic perspectives.

From Olive Drupes to Olive Oil. An HPLC-Orbitrap-based Qualitative and Quantitative Exploration of Olive Key Metabolites

The aim of the current study was the qualitative exploration and quantitative monitoring of key olive secondary metabolites in different production steps (drupes, paste, first and final oil) throughout a virgin olive oil production line. The Greek variety Koroneiki was selected as one of the most representative olives, which is rich in biological active compounds. For the first time, an HPLC-Orbitrap platform was employed for both qualitative and quantitative purposes. Fifty-two components belonging to phenyl alcohols, secoiridoids, flavonoids, triterpenes, and lactones were identified based on HRMS and HRMS/MS data. Nine biologically and chemically significant metabolites were quantitatively determined throughout the four production steps. Drupes and paste were found to be rich in several components, which are not present in the final oil. The current study discloses the chemical nature of different olive materials in a successive and integrated way and reveals new sources of high added value constituents of olives.

New Concepts, Experimental Approaches, and Dereplication Strategies for the Discovery of Novel Phytoestrogens from Natural Sources

Phytoestrogens constitute an attractive research topic due to their estrogenic profile and their biological involvement in womans health. Therefore, numerous studies are currently performed in natural products chemistry area aiming at the discovery of novel phytoestrogens. The main classes of phytoestrogens are flavonoids (flavonols, flavanones), isoflavonoids (isoflavones, coumestans), lignans, stilbenoids as well as miscellaneous chemical groups abundant in several edible and/or medicinal plants, belonging mostly to the Leguminosae family. As for other bioactives, the detection of new structures and more potent plant-derived phytoestrogens typically follows the general approaches currently available in the natural product discovery process. Plant-based approaches selected from traditional medicine knowledge and bioguided concepts are routinely employed. However, these approaches are associated with serious disadvantages such as time-consuming, repeated, and labor intensive processes as well as lack of specificity and reproducibility. In recent years, the natural products chemistry became more technology-driven, and several different strategies have been developed. Structure-oriented procedures and miniaturized approaches employing advanced hyphenated analytical platforms have recently emerged. They facilitate significantly not only the discovery of novel phytoestrogens but also the dereplication procedure leading to the anticipation of major drawbacks in natural products discovery. In this review, apart from the traditional concepts followed in phytochemistry for the discovery of novel biologically active compounds, recent applications in the field of extraction, analysis, fractionation, and identification of phytoestrogens will be discussed. Moreover, specific methodologies combining identification of actives and biological evaluation in parallel, such as liquid chromatography-biochemical detection, frontal affinity chromatography-mass spectrometry and pulsed ultrafiltration-MS will also be presented. Finally, miniaturized methods (microchip and biosensor) will be also discussed.With the current review, we attempt to give a wide and holistic overview of the different approaches which could be employed in the discovery of new phytoestrogens. On the other hand, we anticipate to attract more scientists to the area of phytoestrogens and to indicate the need of multidisciplinary concepts.

Influence of extraction procedures on phenolic content and antioxidant activity of Cretan barberry herb

The main goal of present study was the development, optimization and application of different extraction protocols, especially those employing green technologies, in order to obtain from Berberis cretica extracts with high antioxidant capacity. For this purpose, the applied methods: maceration, ASE and SFE coupled with ASE were incorporated. The antioxidant assessment was carried out using DPPH and total phenolic content (Folin-Ciocalteu) assays. Major constituents were elucidated using HPLC-DAD and UHPLC-HRMS/MS (hybrid IT-Orbital trap spectrometer) equipped with an ESI probe. The chromatographic and spectral data revealed the presence of several simple phenolic acids, derivatives of both caffeic and benzoic acids, and flavonoids in the produced extracts. It was clearly evidenced that the extraction method and solvents used affected both the activity and the chemical content of the results, significantly. The most beneficial conditions were calculated for methanol and water:ethanol (50:50) extracts derived from the combination of SFE and ASE methodologies. Obtained results classify Cretan barberry as a strong antioxidant agent.

Structure-oriented UHPLC-LTQ Orbitrap-based approach as a dereplication strategy for the identification of isoflavonoids from Amphimas pterocarpoides crude extract

Hyphenated techniques and especially ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) are nowadays widely employed in natural products research. However, the complex nature of plant extracts complicates considerably the analysis and the identification of their constituents. Nevertheless, new MS analyzers with increased resolving power and accuracy such as the orbital trap (Orbitrap) could facilitate drastically this process. The objective of this study is the development of a new structure-oriented approach based on fast UHPLC-high-resolution (HR)MS and HRMS/MS methodologies for the identification of isoflavonoids in crude extracts. In addition, aims to assist dereplication procedures, to decrease the laborious isolation steps and orient the focused isolation of compounds of interest. As a proof of concept, the methanol extract of the stem bark of Amphimas pterocarpoides (Leguminosae) was selected. Based on chromatographic (retention time, polarity) and spectrometric features (ultraviolet spectra, accurate m/z, proposed elemental composition, ring double bond equivalent, and relative isotopic abundance) as well as HRMS/MS spectra, several isoflavonoids were identified. In order to verify the proposed structures, 11 isoflavonoids were selectively isolated and unambiguously identified using 1&2D nuclear magnetic resonance techniques. Moreover, the isolated isoflavonoids were studied in HRMS/MS level, employing electrospray ionization and atmospheric pressure chemical ionization sources, in both modes. Useful information regarding their fragmentation patterns was obtained, and characteristic diagnostic ions were defined for the identification of methoxylated isoflavones, dihydroisoflavones and 5-hydroxylated isoflavonoids. Based on the current results, the proposed dereplication strategy was verified and could comprise a novel approach for the analysis of crude extracts in the future not only for isoflavonoids but also for other chemical classes of natural products.

The Antioxidant Effects of a Polyphenol-Rich Grape Pomace Extract In Vitro Do Not Correspond In Vivo Using Exercise as an Oxidant Stimulus

Fruits, such as grapes, are essential food of the Mediterranean diet. Grape extracts have potent antioxidant and chemopreventive properties in vitro. Numerous studies have examined the effects of plant extract administration on redox status at rest in animals and humans but their results are controversial. However, there are no studies comparing the in vitro and in vivo effects of plant extracts on oxidative stress using exercise as an oxidant stimulus. Thus, the aim of this study was to investigate whether a polyphenol-rich grape pomace extract of the Vitis vinifera species possesses in vitro antioxidant properties and to examine whether these properties apply in an in vivo model at rest and during exercise. Our findings indicate that the tested extract exhibits potent in vitro antioxidant properties because it scavenges the DPPH• and ABTS•+ radicals and inhibits DNA damage induced by peroxyl and hydroxyl radicals. Administration of the extract in rats generally induced oxidative stress at rest and after exercise whereas exercise performance was not affected. Our findings suggest that the grape pomace extract does not behave with the same way in vitro and in vivo.

One-step semisynthesis of oleacein and the determination as a 5-lipoxygenase inhibitor.

The dialdehydes oleacein (2) and oleocanthal (4) are closely related to oleuropein (1) and ligstroside (3), the two latter compounds being abundant iridoids of Olea europaea. By exploiting oleuropein isolated from the plant leaf extract, an efficient procedure has been developed for a one-step semisynthesis of oleacein under Krapcho decarbomethoxylation conditions. Highlighted is the fact that 5-lipoxygenase is a direct target for oleacein with an inhibitory potential (IC50: 2 μM) more potent than oleocanthal (4) and oleuropein (1). This enzyme catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes. Taken together, the methodology presented here offers an alternative solution to isolation or total synthesis for the procurement of oleacein, thus facilitating the further development as a potential anti-inflammatory agent.

Ebenfurans IV-VIII from Onobrychis ebenoides: Evidence that C-Prenylation is the Key Determinant of the Cytotoxicity of 3-Formyl-2-arylbenzofurans

Phytochemical investigation of a methanol extract of Onobrychis ebenoides yielded five new 3-formyl-2-arylbenzofurans, namely, ebenfurans IV-VIII (1-5), together with the known compounds ebenfurans I, II (6), and III (7). Only 1 and 7 exhibited growth inhibitory activity against MCF-7 and Ishikawa cells, suggesting that the prenyl moiety at position C-5 is the key determinant of the cytotoxic activity of this group of compounds.

Cytotoxic effects of 2-arylbenzofuran phytoestrogens on human cancer cells: Modulation by adrenal and gonadal steroids☆

Although 2-arylbenzofuran phytoalexins are known for decades, their anticancer activity has not been studied systematically. We have previously reported on the isolation and the estrogen receptor (ER) modulation properties of three new 2-arylbenzofurans from Onobrychis ebenoides, ebenfuran I [2-(2,4-dihydroxyphenyl)-5-hydroxy-6-methoxy-benzofuran], ebenfuran II [2-(2,4-dihydroxyphenyl)-3-formyl-4-hydroxy-6-methoxy-benzofuran] and ebenfuran III [2-(2,4-dihydroxyphenyl)-3-formyl-4-hydroxy-6-methoxy-5-(3-methyl-buten-2-yl)-benzofuran]. We now show that, while I and II could stimulate the proliferation of MCF-7 cells, III was inhibitory in a proliferation-dependent manner. III inhibited the growth of all human cancer cells examined, regardless of ER or multidrug resistance status. Estradiol rendered MCF-7 cells more sensitive to III, and this coincided with the ability of the hormone at concentrations > or = 0.1 nM to bind to the ER of the cells and stimulate their proliferation in the presence of III. Cell proliferation stimulating concentrations of I and II also enhanced the effect of III on MCF-7 cells. However, dehydroepiandrosterone and dihydrotestosterone were ineffective in this respect. III-treated MCF-7 cells exhibited G1 phase arrest followed by detachment-induced cell death and/or apoptosis in the adherent fraction, pronounced induction of Bax and suppression of estradiol induction of Bcl-2. Our data indicate that the largely unexplored pool of benzofuran phytoalexins includes entities potentially suitable for chemoprevention and treatment of human cancer.