Waste Citrus limon Leaves as Source of Essential Oil Rich in Limonene and Citral: Chemical Characterization, Antimicrobial and Antioxidant Properties, and Effects on Cancer Cell Viability.

This study investigated chemical composition, cytotoxicity in normal and cancer cells, and antimicrobial and antioxidant activity of the essential oil (EO) isolated by hydrodistillation from the discarded leaves of lemon (Citrus limon) plants cultivated in Sardinia (Italy). The volatile chemical composition of lemon leaf EO (LLEO) was analyzed with gas chromatography-mass spectrometry combined with flame ionization detection (GC/MS and GC/FID). The most abundant component of LLEO was limonene (260.7 mg/mL), followed by geranial (102.6 mg/mL) and neral (88.3 mg/mL). The antimicrobial activity of LLEO was tested using eight bacterial strains and two types of yeasts by a microdilution broth test. Candida albicans showed the greatest susceptibility (MIC = 0.625 µL/mL) and Listeria monocytogenes and Staphylococcus aureus were inhibited at low LLEO concentration (MIC values from 2.5 to 5 µL/mL). The C. limon leaf EO displayed radical scavenging ability (IC50 value of 10.24 mg/mL) in the 2,2-diphenyl-1-picryl-hydrazylhydrate (DPPH) assay. Furthermore, the LLEO impact on cell viability was explored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in cancer HeLa cells, A375 melanoma cell line, normal fibroblasts (3T3 cells), and keratinocytes (HaCaT cells). LLEO, at 24 h of incubation, significantly reduced viability from 25 µM in Hela cells (33% reduction) and A375 cells (27%), greatly affecting cell morphology, whereas this effect was found from 50 µM on 3T3 fibroblasts and keratinocytes. LLEO's pro-oxidant effect was also established in HeLa cells by 2',7'-dichlorodihydrofluorescein diacetate assay.

Exploring the DPP-IV Inhibitory, Antioxidant and Antibacterial Potential of Ovine "Scotta" Hydrolysates.

The aim of this work was to valorize the by-product derived from the ricotta cheese process (scotta). In this study, ovine scotta was concentrated by ultrafiltration and then subjected to enzymatic hydrolyses using proteases of both vegetable (4% E:S, 4 h, 50 °C) and animal origin (4% E:S, 4 h, 40 °C). The DPP-IV inhibitory, antioxidant, and antibacterial activities of hydrolysates from bromelain (BSPH) and pancreatin (PSPH) were measured in vitro. Both the obtained hydrolysates showed a significantly higher DPP-IV inhibitory activity compared to the control. In particular, BSPH proved to be more effective than PSPH (IC50 8.5 ± 0.2 vs. 13 ± 1 mg mL-1). Moreover, BSPH showed the best antioxidant power, while PSPH was more able to produce low-MW peptides. BSPH and PSPH hydrolysates showed a variable but slightly inhibitory effect depending on the species or strain of bacteria tested. BSPH and PSPH samples were separated by gel permeation chromatography (GPC). LC-MS/MS analysis of selected GPC fractions allowed identification of differential peptides. Among the peptides 388 were more abundant in BSPH than in the CTRL groups, 667 were more abundant in the PSPH group compared to CTRL, and 97 and 75 of them contained sequences with a reported biological activity, respectively.

Fibroblast Proliferation and Migration in Wound Healing by Phytochemicals: Evidence for a Novel Synergic Outcome.

Wound-healing is a dynamic skin reparative process that results in a sequence of events, including inflammation, proliferation, and migration of different cell types as fibroblasts. Fibroblasts play a crucial role in repairing processes, from the late inflammatory phase until the fully final epithelization of the injured tissue. Within this context, identifying tools able to implement cell proliferation and migration could improve tissue regeneration. Recently, plants species from all over the world are coming out as novel tools for therapeutic applications thanks to their phytochemicals, which have antioxidant properties and can promote wound healing. In this paper, we aimed at investigating antioxidant activity of waste extracts from different medicinal plants, endemic of the Mediterranean area, on fibroblast proliferation and wound healing. We determined the amount of total phenols and anti-oxidant activity by ABTS assay. We then evaluated the cytotoxicity of the compounds and the proliferative capabilities of fibroblasts by scratch assay. Our results showed that waste extracts retain antioxidant and regenerative properties, inducing tissue re-establishment after environmental stress exposure. Taken together, our findings suggest that waste material could be used in the future also in combinations to stimulate wound healing processes and antioxidant responses in damaged skin.

Direct-to-Consumer Nutrigenetics Testing: An Overview.

At present, specialized companies offering genetic testing services without the involvement of clinicians are growing; this development is a direct consequence of the significant decrease in genotyping and sequencing costs. Online companies offer predictions about the risk of developing complex diseases during one's life course, and they offer suggestions for personal lifestyle. Several companies have been created that provide nutrigenetics services; these companies suggest dietary indications-a central issue in the prevention and etiopathogenesis of specific diseases-based on one's personal genetic background. Dietary patterns are defined on the basis of a limited set of genetic markers. In this article, we analyze the online nutrigenetics services offered by 45 companies worldwide, to obtain an overall picture of the costs, the types of nutritional traits considered and the level of scientific precision of the services proposed. Our analysis clearly highlights the need for specific guidelines, in order to ensure a set of minimum quality standards for the nutrigenetics services offered to the customer.

Myrtus communis Liquor Byproduct as a Source of Bioactive Compounds.

The fatty acid (FA), polyphenol content and evaluation of the antioxidant capacity of exhausted Myrtus communis berries (EMB) resulting from the production of myrtle liqueur were assessed. All parts of the exhausted berries exhibited high concentrations of carbohydrates, proteins, lipids and phenolic compounds. The lipid fraction contained a high amount of poly unsaturated fatty acids (PUFA), mainly represented by linoleic acid (>70%). Of the phenolic acids evaluated by liquid chromatography/mass spectrometry, ellagic acid was the most predominant (>50%), followed by gallic and quinic acids. Quercetin and quercetin3-O-rhamnoside were the most abundant flavonoids. The seed extracts showed a higher antioxidant potential than the pericarp extracts; the same trend was observed for total phenolic compounds evaluated by spectrophotometric assay. The overall high content of bioactive compounds and the high antioxidant potential of this byproduct sustain its suitability for a number of industrial applications, such as a food ingredient in novel foods, an additive in cosmetic formulations or a component of animal feed formulations.

Metabolomic study of wild and cultivated caper (Capparis spinosa L.) from different areas of Sardinia and their comparative evaluation.

Capparis spinosa L. (Capparidaceae), also known as caper, is widely known for its very aromatic flower buds (capers),that are largely employed as a flavouring in cooking. Capparis species are regarded as a potential source of important bioactive compounds, in fact, due to their botanical relationship with Brassica species; they contain glucosinolates, secondary plant metabolites, that have been studied for their potential anticarcinogenic properties. In addition, the presence of other numerous beneficial compounds such as polyphenols, alkaloids, lipids, vitamins and minerals have been reported. The aim of this study was to individuate and determinate the principal bioactive compounds occurring in different part (leaves, buds and flowers) of wild and cultivated C. spinosa collected from different area of Sardinia (Italy). Ultra-high performance liquid chromatography-triple quadrupole/linear ion trap tandem mass spectrometry methods were used for identification and simultaneous determination of 27 bioactive molecules. Analysis of different samples revealed qualitative and quantitative differences in the content of flavonoids, glucosinolates, anthocyanins and phenolic acids. In particular, glucocapparin resulted the most abundant with values ranging from 112 to 364 mg/100 g Fresh Weight (FW); followed by rutin with highest value of 126 mg/100 g FW, 4-hydroxyglucobrassicin with highest value of 42 mg/100 g FW and isorhamnetin 3-O-rutinoside with highest value of 24 mg/100 g FW. Based on this metabolomic targeted approach, quantitative results were treated by principal component analysis to explore and visualise correlation and discrimination among collections of C. spinosa samples. Copyright © 2016 John Wiley & Sons, Ltd.

Variability of chemical composition and antioxidant activity of essential oils between Myrtus communis var. Leucocarpa DC and var. Melanocarpa DC.

Essential oils (EOs) from several individuals of Myrtus communis L. (M. communis) growing in different habitats in Sardinia have been studied. The analyses were focused on four groups of samples, namely cultivated and wild M. communis var. melanocarpa DC, characterized by red/purple berries, and cultivated and wild M. communis var. leucocarpa DC, characterized by white berries. Qualitative and quantitative analyses demonstrated different EO fingerprints among the studied samples: cultivated and wild leucocarpa variety differs mainly from the melanocarpa variety by a high amount of myrtenyl acetate (>200 mg/mL and 0.4 mg/mL in leucocarpa and melanocarpa varieties respectively). Conversely, the wild group is characterized by a higher amount, compared with the cultivated species, of linalool (about 110 mg/mL and 20 mg/mL respectively), linalyl acetate (about 24 mg/mL and about 6 mg/mL respectively) whereas EOs of the cultivated plants were rich in pinocarveol-cis compared with wild plants (about 2 mg/mL and about 0.5 mg/mL respectively). Principal component analysis applied to the chromatographic data confirm a differentiation and classification of EOs from the four groups of M. communis plants. Finally, antioxidant activity of the studied EOs shows differences between the various categories of samples.

Human umbilical endothelial cells (HUVECs) have a sex: characterisation of the phenotype of male and female cells.

BACKGROUND: Human umbilical endothelial cells (HUVECs) are widely used to study the endothelial physiology and pathology that might be involved in sex and gender differences detected at the cardiovascular level. This study evaluated whether HUVECs are sexually dimorphic in their morphological, proliferative and migratory properties and in the gene and protein expression of oestrogen and androgen receptors and nitric oxide synthase 3 (NOS3). Moreover, because autophagy is influenced by sex, its degree was analysed in male and female HUVECs (MHUVECs and FHUVECs). METHODS: Umbilical cords from healthy, normal weight male and female neonates born to healthy non-obese and non-smoking women were studied. HUVEC morphology was analysed by electron microscopy, and their function was investigated by proliferation, viability, wound healing and chemotaxis assays. Gene and protein expression for oestrogen and androgen receptors and for NOS3 were evaluated by real-time PCR and Western blotting, respectively, and the expression of the primary molecules involved in autophagy regulation [protein kinase B (Akt), mammalian target of rapamycin (mTOR), beclin-1 and microtubule-associated protein 1 light chain 3 (LC3)] were detected by Western blotting. RESULTS: Cell proliferation, migration NOS3 mRNA and protein expression were significantly higher in FHUVECs than in MHUVECs. Conversely, beclin-1 and the LC3-II/LC3-I ratio were higher in MHUVECs than in FHUVECs, indicating that male cells are more autophagic than female cells. The expression of oestrogen and androgen receptor genes and proteins, the protein expression of Akt and mTOR and cellular size and shape were not influenced by sex. Body weights of male and female neonates were not significantly different, but the weight of male babies positively correlated with the weight of the mother, suggesting that the mother's weight may exert a different influence on male and female babies. CONCLUSIONS: The results indicate that sex differences exist in prenatal life and are parameter-specific, suggesting that HUVECs of both sexes should be used as an in vitro model to increase the quality and the translational value of research. The sex differences observed in HUVECs could be relevant in explaining the diseases of adulthood because endothelial dysfunction has a crucial role in the pathogenesis of cardiovascular diseases, diabetes mellitus, neurodegeneration and immune disease.

Deregulation of microRNA-503 contributes to diabetes mellitus-induced impairment of endothelial function and reparative angiogenesis after limb ischemia.

BACKGROUND: Diabetes mellitus impairs endothelial cell (EC) function and postischemic reparative neovascularization by molecular mechanisms that are not fully understood. microRNAs negatively regulate the expression of target genes mainly by interaction in their 3' untranslated region. METHODS AND RESULTS: We found that microRNA-503 (miR-503) expression in ECs is upregulated in culture conditions mimicking diabetes mellitus (high D-glucose) and ischemia-associated starvation (low growth factors). Under normal culture conditions, lentivirus-mediated miR-503-forced expression inhibited EC proliferation, migration, and network formation on Matrigel (comparisons versus lentivirus.GFP control). Conversely, blocking miR-503 activity by either adenovirus-mediated transfer of a miR-503 decoy (Ad.decoymiR-503) or by antimiR-503 (antisense oligonucleotide) improved the functional capacities of ECs cultured under high D-glucose/low growth factors. We identified CCNE1 and cdc25A as direct miR-503 targets which are downregulated by high glucose/low growth factors in ECs. Next, we obtained evidence that miR-503 expression is increased in ischemic limb muscles of streptozotocin-diabetic mice and in ECs enriched from these muscles. Moreover, Ad.decoymiR-503 delivery to the ischemic adductor of diabetic mice corrected diabetes mellitus-induced impairment of postischemic angiogenesis and blood flow recovery. We finally investigated miR-503 and target gene expression in muscular specimens from the amputated ischemic legs of diabetic patients. As controls, calf biopsies of nondiabetic and nonischemic patients undergoing saphenous vein stripping were used. In diabetic muscles, miR-503 expression was remarkably higher, and it inversely correlated with cdc25 protein expression. Plasma miR-503 levels were also elevated in the diabetic individuals. CONCLUSIONS: Our data suggest miR-503 as a possible therapeutic target in diabetic patients with critical limb ischemia.