Phytochemical evaluation and exploration of some biological activities of aqueous and ethanolic extracts of two species of the genus Plantago L.

Plantago major L. and Plantago lagopus L. are cosmopolitan species, belonging to the Plantaginaceae family, used in traditional and modern medicine. In this study, a phytochemical evaluation of different aqueous and ethanolic extracts of leaves and roots of both species from the region of Beja in Tunisia was performed. Some biological activities, including antioxidant, anticancer and antibacterial were also done. LC-MS qualitative analysis revealed that the aqueous extracts of the roots of P. lagopus were richer in polyphenols, mainly flavonoids (Luteoline 7-rutinoside, Luteoline 7-rhamnoside) and hydroxycinnamic acids including caffeic acid, than the hydro-ethanolic extracts. Additionally, we identified for the first time the presence of salicylic acid in the hot aqueous extracts of roots of P. lagopus and its absence in the roots of P. major. The antioxidant activity of the extracts was assessed using cyclic voltammetry (CV), revealing that the voltammograms of leaf and root extracts from P. lagopus exhibited a higher antioxidant capacity compared to those of P. major. Antiproliferative activity, was determined against two-colon cancer cell lines, demonstrated that only the 12 h treatments with P. lagopus leaf and root aqueous and hydro-ethanolic extracts at low concentration were able to significantly reduce the colon carcinoma coli-2 (CaCo-2) cells proliferation. The antibacterial /antibiofilm activity was performed on yeast, Gram- negative and +positive bacterial strains. We demonstrated for the first time that ethanolic extracts of leaves and roots of P. lagopus have an inhibitory activity against Escherichia coli and Klebsiella pneumonia at MIC = 2 µg/mL for leaves and 4 µg/mL for roots.

Low concentrations of Ambrosia maritima L. phenolic extract protect endothelial cells from oxidative cell death induced by H2O2 and sera from Crohn's disease patients.

ETHNOPHARMACOLOGICAL RELEVANCE: A rising resort to herbal therapies in Crohn's disease (CD) alternative treatments has been recently observed due to their remarkable natural efficiency. In this context, the weed plant Ambrosia maritima L., traditionally known as Hachich el Aouinet in Algeria and as Damsissa in Egypt and Sudan, is widely used in North African folk medicine to treat infections, inflammatory diseases, gastrointestinal and urinary tract disturbances, rheumatic pain, respiratory problems, diabetes, hypertension and cancer. AIM OF THE STUDY: To assess an Ambrosia maritima L. phenolic extract for its phenolic profile composition, its potential antioxidant activity in vitro, and its cytoprotective effect on cultured primary human endothelial cells (ECs) stressed with H2O2 and sera from CD patients. MATERIALS AND METHODS: Phenolic compound extraction was performed with a low-temperature method. Extract chemical profile was attained by HPLC-DAD/ESI-MS. The extract in vitro antioxidant activity was assessed using several methods including cupric ion reducing power, DPPH radical scavenging assay, O-Phenanthroline free radical reducing activity, ABTS cation radical decolourisation assay, Galvinoxyl free radicals scavenging assay. Intracellular reactive oxygen species levels were evaluated in human endothelial cells by H2DCFDA, while cell viability was assessed by MTT. RESULTS: The phenolic compounds extraction showed a yield of 17.66% with three di-caffeoylquinic acid isomers detected for the first time in Ambrosia maritima L. Using different analytical methods, a significant in vitro antioxidant activity was reported for the Ambrosia maritima L. extract, with an IC50 value of 14.33 ± 3.86 µg/mL for the Galvinoxyl antioxidant activity method. Challenged with ECs the Ambrosia maritima L. extract showed a biphasic dose-dependent effect on H2O2-treated cells, cytoprotective and antioxidant at low doses, and cytotoxic and prooxidant at high doses, respectively. Viability and ROS levels data also demonstrated a prooxidant and cytotoxic effect of CD sera on cultured ECs. Interestingly, 10 µg/mL of Ambrosia maritima L. extract was able to counteract both CD sera-induced oxidative stress and ECs death. CONCLUSION: Our data indicated Ambrosia maritima L. as a source of bioactive phenolics potentially employable as a natural alternative for CD treatment.

Anti- and Pro-Oxidant Activity of Polyphenols Extracts of Syrah and Chardonnay Grapevine Pomaces on Melanoma Cancer Cells.

The phenolic composition of Syrah and Chardonnay grape pomaces was studied to assess their antioxidant and prooxidant properties. Polyphenols were extracted by a "green" hydroalcoholic solvent (ethanol/water 1:1 v/v), and a detailed chemical and electrochemical characterization of the phenolic compounds was performed. The antioxidant and prooxidant capacity of the pomace was first studied by cyclic voltammetry (CV) and other reference analytical assays, then with biological tests on B16F10 metastatic melanoma cancer cells. Electrochemical data showed that, when a +0.5 V potential was applied, a low to moderate antioxidant capacity was observed. MTT test showed an increasing viability of melanoma cells, after treatments at low concentration (up to 100 µg/mL) and for a short time (6 h), but when cells were treated with higher doses of extract (≥250 µg/mL for 12/24 h), their viability decreased from 25 to 50% vs. control, depending on treatment time, dose, and extract origin. A stronger prooxidant activity resulted when 250 µg/mL of extract was combined with non-toxic doses of H2O2; this activity was correlated with the presence of copper in the extracts. This study shows the potential of winemaking by-products and suggests the opportunity to exploit them for the production of cosmeceuticals, or for combined therapies with approved anticancer drugs.

Antioxidant, Antimicrobial, and Other Biological Properties of Pompia Juice.

Pompia is a Citrus species belonging to Sardinian endemic biodiversity. Health benefits were attributed to its flavedo rind extracts and essential oils while the juice qualities have never been investigated. In this paper, the antioxidant, antimicrobial, and other biological properties of Pompia juice were studied. A combined LCMS/electrochemical/biological approach was used to clarify a still debated phylogeny of this species and to explain the role of its juice phenolic compounds. A closer phylogenetic relationship with lemon and citron, rather than oranges was suggested. Sensors-based electrochemical measures, together with LCMS qualitative and quantitative analyses, revealed a high contribution of ascorbic acid and phenolics with low redox potential, isorhamnetin 3-O-rutinoside, diosmin, and diosmetin 6,8-diglucoside, to antioxidant capacity. The biological assays demonstrated a marked effect of low concentration of Pompia juice against reactive oxygen species (ROS) starting from 50 µg mL-1, and a moderate capacity to reduce ROS damages on cell membrane. Treatments with Pompia juice also resulted in a significant reduction (20%) of the metabolic activity of SW48 colon cancer cells. Lastly, MIC, MBC, and MBIC antimicrobial assays demonstrated that Pompia and lemon juices have inhibitory and antibiofilm effects against the pathogenic bacteria Pseudomonas aeruginosa, Streptococcus aureus, and Enterococcus faecalis.

The Pharmaceutical Ability of Pistacia lentiscus L. Leaves Essential Oil Against Periodontal Bacteria and Candida sp. and Its Anti-Inflammatory Potential.

BACKGROUND: Given the increasing request for natural pharmacological molecules, this study assessed the antimicrobial capacity of Pistacia lentiscus L. essential oil (PLL-EO) obtained from the leaves of wild plants growing in North Sardinia (Italy) toward a wide range of periodontal bacteria and Candida, including laboratory and clinical isolates sp., together with its anti-inflammatory activity and safety. METHODS: PLL-EO was screened by gas chromatography/mass spectrometry. The minimal inhibitory concentration (MIC) was determined. The anti-inflammatory activity was measured by cyclooxygenase (COX-1/2) and lipoxygenase (LOX) inhibition, while the antioxidant capacity was determined electro-chemically and by the MTT assay. The WST-1 assay was used to ascertain cytotoxicity toward four lines of oral cells. RESULTS: According to the concentrations of terpens, PLL-EO is a pharmacologically-active phytocomplex. MICs against periodontal bacteria ranged between 3.13 and 12.5 µg/ml, while against Candida sp. they were between 6.25 and 12.5 µg/mL. Oxidation by COX-1/2 and LOX was inhibited by 80% and 20% µg/mL of the oil, respectively. Antioxidant activity seemed negligible, and no cytotoxicity arose. CONCLUSIONS: PLL-EO exhibits a broad-spectrum activity against periodontal bacteria and Candida, with an interesting dual inhibitory capacity toward COX-2 and LOX inflammatory enzymes, and without side effects against oral cells.

SUMOylation Protects FASN Against Proteasomal Degradation in Breast Cancer Cells Treated with Grape Leaf Extract.

Existing therapeutic strategies for breast cancer are limited by tumor recurrence and drug-resistance. Antioxidant plant-derived compounds such as flavonoids reduce adverse outcomes and have been identified as a potential source of antineoplastic agent with less undesirable side effects. Here, we describe the novel regulation of fatty-acid synthase (FASN), the key enzyme in de novo fatty-acid synthesis, whereby Vitis vinifera L. cv Vermentino leaf hydroalcoholic extract lowers its protein stability that is regulated by small ubiquitin-like modifier (SUMO)ylation. The phenolic compounds characterization was performed by liquid chromatography-mass spectrometry (LC-MS), whereas mass spectrometry (LC-MS/MS), Western blotting/co-immunoprecipitation (Co-IP) and RT-PCR, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), clonogenicity assays, and FACS analysis were used to measure the expression of targets and tumorigenicity. Vermentino extract exhibits antitumorigenic effects, and we went on to determine that FASN and ubiquitin-conjugating enzyme 9 (UBC9), the sole E2 enzyme required for SUMOylation, were significantly reduced. Moreover, FASN was found SUMOylated in human breast cancer tissues and cell lines, and lack of SUMOylation caused by SUMO2 silencing reduced FASN protein stability. These results suggest that SUMOylation protects FASN against proteasomal degradation and may exert oncogenic activity through alteration of lipid metabolism, whereas Vermentino extract inhibits these effects which supports the additional validation of the therapeutic value of this compound in breast cancer.

Influence of pH, buffers and role of quinolinic acid, a novel iron chelating agent, in the determination of hydroxyl radical scavenging activity of plant extracts by Electron Paramagnetic Resonance (EPR).

The Fenton reaction is used to produce hydroxyl radicals for the evaluation of the antioxidant activity of plant extracts. In this paper the parameters affecting the production of hydroxyl radicals and their spin trapping with DMPO were studied. The use of quinolinic acid (Quin) as an Fe(II) ligand was proposed for antioxidant activity determination of Green tea, orange juice and asparagus extracts. Quin, buffers and pH affect the DMPO-OH signal intensity of the EPR spectra. Quin/Fe(II) and low pH enhance the OH generation. Phosphate and Tris-HCl buffers decrease the signal intensity measured in Fe(II)-sulfate and Fe(II)-Quin systems. The extracts were analyzed with Fenton systems containing Fe(II)-sulfate and Fe(II)-Quin with and without buffer. The highest activity was shown with Fe(II)-Quin without buffer, this system being less influenced by pH and chelating agents present in the extracts. This paper will help researchers to better design spin trapping experiments for food matrices.

Real-time monitoring of glucose and phenols intestinal absorption through an integrated Caco-2TC7cells/biosensors telemetric device: Hypoglycemic effect of fruit phytochemicals.

An integrated device for real-time monitoring of glucose and phenols absorption, that consists of a sensors/biosensors system (SB) and a Caco-2TC7 human intestinal cell culture, is described in this study. The SB is composed of a glucose oxidase-based biosensor, a sentinel platinum sensor, a laccase/tyrosinase-based biosensor and a sentinel carbon sensor, all located in the basolateral compartment (BC) of a cell culture plate. Caco-2TC7 cells, differentiated on culture inserts, separated the apical compartment that simulates the intestinal lumen, from the BC which represented the bloodstream. The system recorded currents relative to glucose (1mM) absorption, obtaining bioavailability values (5.1%) comparable to HPLC analysis (4.8%). Phloridzin and phloretin, specific phenolic inhibitors of SGLT1 and GLUT2 glucose transporters, reduced the glucose transport of almost 10 times. They were minimally absorbed in the BC with a bioavailability of 0.13% and 0.49% respectively. The hypoglycemic potential of blueberry and pomegranate juices was also studied. In particular, the amount of glucose absorbed through the Caco-2TC7 monolayer was 8‰ for pomegranate and 1.7‰ for blueberry, demonstrating the potential hypoglycemic effect of the juices. Polyphenols absorption was also monitored by the SB and an increase was recorded during the first 50min in presence of both blueberry and pomegranate juices, then a constant decrease occurred. The proposed device has been developed as innovative tool for the dynamic monitoring of natural compounds effects on glucose absorption, in order to manage postprandial hyperglycemia.

Simultaneous amperometric detection of ascorbic acid and antioxidant capacity in orange, blueberry and kiwi juice, by a telemetric system coupled with a fullerene- or nanotubes-modified ascorbate subtractive biosensor.

Four fullerenes- or nanotubes-modified graphite sensor-biosensor systems (SBs), coupled with a dual-channel telemetric device, based on an ascorbate oxidase (AOx) biosensor, were developed for on line simultaneous amperometric detection of ascorbic acid (AA) and antioxidant capacity in blueberry, kiwi and orange juice. Fullerene C60 (FC60), fullerene C70 (FC70), single-walled carbon nanotubes (SWCN) and multi-walled carbon nanotubes (MWCN) increased the sensitivity of graphite toward AA and phenols 1.2, 1.5, 5.1 and 5.1 times respectively. Fullerenes combined with AOx improved the selectivity toward AA more than nanotubes, being able to hold a higher number of AOx molecules on the biosensor surface. The SBs work at an applied potential of +500 mV, in a concentration range between the LOD and 20 µM, with a response time of two minutes. The LOD is 0.10, 0.13, 0.20 and 0.22 µM for SBs modified with FC60, FC70, SWCN and MWCN respectively. Biosensors register lower AA currents than the sensors due to the enzyme capability to oxidize AA before it reaches the transductor surface. Phenols currents registered by sensors and biosensors did not differ. Based on the difference between sensor and biosensor recorded currents a AA selectivity index was developed as an indicator of specificity toward AA and of the capacity to distinguish between AA and phenols contribution to the antioxidant capacity. This value is almost zero for fullerene-modified SBs, 0.13 and 0.22 for SWCN- and MWCN-modified SBs respectively. The results of juices analysis performed with SBs were in accordance with reference methods.

Development and characterization of an ascorbate oxidase-based sensor-biosensor system for telemetric detection of AA and antioxidant capacity in fresh orange juice.

A new carbon ascorbate oxidase-based sensor-biosensor system (SB) was coupled to a dual-channel telemetric device for online simultaneous electrochemical detection of ascorbic acid (AA) and antioxidant capacity in Hamlin, Sanguinello, and Moro orange varieties. The electrocatalytic performances of the SB were investigated by cyclic voltammetry and amperometric techniques. The phenol composition of orange juice of each variety, and the cyclic voltammetries of the most represented phenols, were provided. The in vitro calibrations were performed in PBS (pH 5.6), applying a constant potential of +500 mV. A standard mixture of phenols, based on orange juice composition, was used as reference material for studying SB behavior. SB works at an applied potential of +500 mV, in a concentration range comprised between the LOD 0.26 µM and 20 µM. In this concentration range, limiting the data acquisition time to 2 min, the problems of electrode passivation due to phenols polymerization were overcome. AA calibration showed that the biosensor registered statistically lower currents than the sensor since the enzyme oxidized AA before it reached the electrode surface. Standard mixture calibration showed that currents registered by sensor and biosensor did not statistically differ. The difference between sensor and biosensor AA registered currents was used to calculate an AA selectivity index and, consequently, to determine the AA content and the antioxidant capacity in the juices. The novelty of the SB is its ability to distinguish between AA and phenols contribution to antioxidant capacity. The obtained results were in accordance with reference methods.