An alternative approach of TUNEL assay to specifically characterize DNA fragmentation in cell model systems.

DNA damage is one of the most important effects induced by chemical agents. We report a comparative analysis of DNA fragmentation on three different cell lines using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, generally applied to detect apoptosis. Our approach combines cytogenetic techniques and investigation in detached cellular structures, recovered from the culture medium with the aim to compare the DNA fragmentation of three different cell line even beyond the cells adherent to substrate. Consequently, we detect any fragmentation points on single chromosomes, whole nuclei and other cellular structures. Cells were exposed to resveratrol (RSV) and doxorubicin (Doxo), in single and combined treatments. Control and treated astrocytes showed DNA damage in condensed nuclei and detached structures. Caco-2 cells showed fragmented DNA only after Doxo-treatment, while controls showed fragmented chromosomes, indicating DNA damage in replicating cells. MDA-MB-231 cells showed nuclear condensation and DNA fragmentation above all after RSV-treatment and related to detached structures. This model proved to perform a grading of genomic instability (GI). Astrocytes show a hybrid level of GI. Caco-2 cells showed fragmented metaphase chromosomes, proving that the DNA damage was transmitted to the daughter cells probably due to an absence of DNA repair mechanisms. Instead, MDA-MB-231 cells showed few or no fragmented metaphase, suggesting a probable activation of DNA repair mechanisms. By applying this alternative approach of TUNEL test, we obtained data that can more specifically characterize DNA fragmentation for a suitable application in various fields.

New Bioactive Peptides from the Mediterranean Seagrass Posidonia oceanica (L.) Delile and Their Impact on Antimicrobial Activity and Apoptosis of Human Cancer Cells.

The demand for new molecules to counter bacterial resistance to antibiotics and tumor cell resistance is increasingly pressing. The Mediterranean seagrass Posidonia oceanica is considered a promising source of new bioactive molecules. Polypeptide-enriched fractions of rhizomes and green leaves of the seagrass were tested against Gram-positive (e.g., Staphylococcus aureus, Enterococcus faecalis) and Gram-negative bacteria (e.g., Pseudomonas aeruginosa, Escherichia coli), as well as towards the yeast Candida albicans. The aforementioned extracts showed indicative MIC values, ranging from 1.61 µg/mL to 7.5 µg/mL, against the selected pathogens. Peptide fractions were further analyzed through a high-resolution mass spectrometry and database search, which identified nine novel peptides. Some discovered peptides and their derivatives were chemically synthesized and tested in vitro. The assays identified two synthetic peptides, derived from green leaves and rhizomes of P. oceanica, which revealed interesting antibiofilm activity towards S. aureus, E. coli, and P. aeruginosa (BIC50 equal to 17.7 µg/mL and 70.7 µg/mL). In addition, the natural and derivative peptides were also tested for potential cytotoxic and apoptosis-promoting effects on HepG2 cells, derived from human hepatocellular carcinomas. One natural and two synthetic peptides were proven to be effective against the "in vitro" liver cancer cell model. These novel peptides could be considered a good chemical platform for developing potential therapeutics.

Chemical Characterization and Cytotoxic and Antioxidant Activity Evaluation of the Ethanol Extract from the Bulbs of Pancratium maritimun Collected in Sicily.

P. maritimum L., belonging to the Amaryllidaceae family, is a species that grows on beaches and coastal sand dunes mainly on both sides of the Mediterranean Sea and Black Sea, the Middle East, and up to the Caucasus region. It has been largely investigated due to its several interesting biological properties. With the aim of providing new insights into the phytochemistry and pharmacology of this species, the ethanolic extract of the bulbs from a local accession, not previously studied, growing in Sicily (Italy), was investigated. This chemical analysis, performed by mono- and bi-dimensional NMR spectroscopy, as well as LC-DAD-MSn, allowed to identify several alkaloids, three of which were never detected in the genus Pancratium. Furthermore, the cytotoxicity of the preparation was assessed in differentiated human Caco-2 intestinal cells by trypan blue exclusion assay, and its antioxidant potential was evaluated using the DCFH-DA radical scavenging method. The results obtained demonstrate that P. maritimum bulbs' extract exerts no cytotoxic effect and is able to remove free radicals at all the concentrations tested.

Nutrigenetics, nutrigenomics and phenotypic outcomes of dietary low-dose alcohol consumption in the suppression and induction of cancer development: evidence from in vitro studies.

It is known that the intake of alcoholic beverages may impair genetic and epigenetic regulatory events with consequent crucial effects on cell phenotypes and that its association with selected genotypes can lead to a different risk of cancer in the population. The aim of this review is to pick up selected studies on this topic and recapitulate some of the biochemical and nutrigenetic/nutrigenomic aspects involved in the impact of dietary low-dose alcohol consumption on the switching-on or -off of tumorigenic pathways. These include i) the existence of predisposing or protective human genotypes and the relationship between dietary compounds and alcohol in the promotion or inhibition of carcinogenesis; ii) the effects of other components of alcoholic drinks in the modulation of the expression of oncogenes and oncosuppressors, the autophagic flux and the onset of apoptosis, with examples of their cytospecificity; and iii) the role of alcoholic beverage consumption within particular dietary regimens, including the Mediterranean diet. Taking all the data into account, several alcohol-associated bioactive dietary compounds appear capable to modulate peculiar intracellular pathways predisposing to or protecting from cancer. Advances in the nutrigenetic, nutrigenomic and nutriepigenetic knowledge complementing the biochemical and molecular approaches will help in unveiling their impact on health outcome.

Genotoxicity and Epigenotoxicity of Carbazole-Derived Molecules on MCF-7 Breast Cancer Cells.

The carbazole compounds PK9320 (1-(9-ethyl-7-(furan-2-yl)-9H-carbazol-3-yl)-N-methylmethanamine) and PK9323 (1-(9-ethyl-7-(thiazol-4-yl)-9H-carbazol-3-yl)-N-methylmethanamine), second-generation analogues of PK083 (1-(9-ethyl-9H-carbazol-3-yl)-N-methylmethanamine), restore p53 signaling in Y220C p53-mutated cancer cells by binding to a mutation-induced surface crevice and acting as molecular chaperones. In the present paper, these three molecules have been tested for mutant p53-independent genotoxic and epigenomic effects on wild-type p53 MCF-7 breast adenocarcinoma cells, employing a combination of Western blot for phospho-γH2AX histone, Comet assay and methylation-sensitive arbitrarily primed PCR to analyze their intrinsic DNA damage-inducing and DNA methylation-changing abilities. We demonstrate that small modifications in the substitution patterns of carbazoles can have profound effects on their intrinsic genotoxic and epigenetic properties, with PK9320 and PK9323 being eligible candidates as "anticancer compounds" and "anticancer epi-compounds" and PK083 a "damage-corrective" compound on human breast adenocarcinoma cells. Such different properties may be exploited for their use as anticancer agents and chemical probes.

Collective Locomotion of Human Cells, Wound Healing and Their Control by Extracts and Isolated Compounds from Marine Invertebrates.

The collective migration of cells is a complex integrated process that represents a common theme joining morphogenesis, tissue regeneration, and tumor biology. It is known that a remarkable amount of secondary metabolites produced by aquatic invertebrates displays active pharmacological properties against a variety of diseases. The aim of this review is to pick up selected studies that report the extraction and identification of crude extracts or isolated compounds that exert a modulatory effect on collective cell locomotion and/or skin tissue reconstitution and recapitulate the molecular, biochemical, and/or physiological aspects, where available, which are associated to the substances under examination, grouping the producing species according to their taxonomic hierarchy. Taken all of the collected data into account, marine invertebrates emerge as a still poorly-exploited valuable resource of natural products that may significantly improve the process of skin regeneration and restrain tumor cell migration, as documented by in vitro and in vivo studies. Therefore, the identification of the most promising invertebrate-derived extracts/molecules for the utilization as new targets for biomedical translation merits further and more detailed investigations.

Bright Spots in The Darkness of Cancer: A Review of Starfishes-Derived Compounds and Their Anti-Tumor Action.

The fight against cancer represents a great challenge for researchers and, for this reason, the search for new promising drugs to improve cancer treatments has become inevitable. Oceans, due to their wide diversity of marine species and environmental conditions have proven to be precious sources of potential natural drugs with active properties. As an example, in this context several studies performed on sponges, tunicates, mollusks, and soft corals have brought evidence of the interesting biological activities of the molecules derived from these species. Also, echinoderms constitute an important phylum, whose members produce a huge number of compounds with diverse biological activities. In particular, this review is the first attempt to summarize the knowledge about starfishes and their secondary metabolites that exhibited a significant anticancer effect against different human tumor cell lines. For each species of starfish, the extracted molecules, their effects, and mechanisms of action are described.

DNA fragmentation index, pAKT and pERK1/2 in cumulus cells are related to oocyte competence in patients undergoing in vitro fertilization programme.

Activated pERK1/2 and pAKT are key players in supporting cell survival and proliferation pathways. Translocation of pERK1/2 into the nucleus, where it interacts with transcription factors and DNA itself, is instrumental in exerting an anti-apoptotic effect. In this study, pAKT levels, pERK1/2 nuclear localization and DNA fragmentation index (DFI) in cumulus cells of single cumulus-oocyte complexes of patients undergoing in vitro fertilization programmes were evaluated and correlated with the clinical outcome of the related embryos. For a positive clinical outcome of blastocyst development, pERK1/2 nuclear localization and DFI value had a significant inverse relationship, whereas the former and the intracellular accumulation of pAKT had a significant direct relationship. This relationship was not observed for the negative clinical outcome of the arrested embryos. Moreover, intracellular accumulation of pAKT and DFI value had a significant inverse relationship in all samples examined. The obtained data suggest that the intranuclear relocation of pERK1/2, along with an enhanced intracellular accumulation of pAKT, may exert a survival effect and increase cell viability, therefore providing a novel marker tool to choose the best oocyte to be fertilized and submitted to an intracytoplasmic sperm injection cycle.

Cytotoxic Activity of the Histone Deacetylase 3-Selective Inhibitor Pojamide on MDA-MB-231 Triple-Negative Breast Cancer Cells.

We examined the effects of the ferrocene-based histone deacetylase-3 inhibitor Pojamide (N¹-(2-aminophenyl)-N8-ferrocenyloctanediamide) and its two derivatives N¹-(2-aminophenyl)-N6-ferrocenyladipamide and N¹-(2-aminophenyl)-N8-ferroceniumoctanediamide tetrafluoroborate on triple-negative MDA-MB-231 breast cancer cells. Viability/growth assays indicated that only the first two compounds at 70 µM concentration caused an approximate halving of cell number after 24 h of exposure, whereas the tetrafluoroborate derivative exerted no effect on cell survival nor proliferation. Flow cytometric and protein blot analyses were performed on cells exposed to both Pojamide and the ferrocenyladipamide derivative to evaluate cell cycle distribution, apoptosis/autophagy modulation, and mitochondrial metabolic state in order to assess the cellular basis of the cytotoxic effect. The data obtained show that the cytotoxic effect of the two deacetylase inhibitors may be ascribed to the onset of non-apoptotic cell death conceivably linked to a down-regulation of autophagic processes and an impairment of mitochondrial function with an increase in intracellular reactive oxygen species. Our work expands the list of autophagy-regulating drugs and also provides a further example of the role played by the inhibition of autophagy in breast cancer cell death. Moreover, the compounds studied may represent attractive and promising targets for subsequent molecular modeling for anti-neoplastic agents in malignant breast cancer.

Effect of Manganese Chloride and of Cotreatment with Cadmium Chloride on the In Vitro Proliferative, Motile and Invasive Behavior of MDA-MB231 Breast Cancer Cells.

We examined the dose⁻response effect of MnCl2 on the proliferative behavior of triple-negative breast cancer MDA-M231 cells vs. immortalized HB2 cells from breast epithelium taken as nontumoral counterparts. We also tested the effect of MnCl2 on tumor cell invasiveness in vitro by evaluating the relative invasion indexes through Boyden chamber assays. Moreover, we checked whether cotreatment with both MnCl2 and CdCl2 could modify the observed biological response by MDA-MB231 cells. Our results show a promotional impact of MnCl2 on cell proliferation, with 5 µM concentration inducing the more pronounced increase after 96-h exposure, which is not shared by HB2 cells. Exposure to 5 µM MnCl2 induced also an elevation of the relative invasion index of cancer cells. The Mn-mediated stimulatory effects were counteracted by cotreatment with CdCl2. These data support the concept that human exposure to high environmental concentrations of Mn may increase the risk of carcinogenesis and metastasis by prompting the expansion and dissemination of triple-negative breast cancer cells. On the other hand, the Mn-counteracting anticancer property of Cd looks promising and deserves a more detailed characterization of the involved intracellular targets aimed to the molecular modeling of specific antineoplastic agents against malignant breast cancer spreading.

Gene Expression and Apoptosis Levels in Cumulus Cells of Patients with Polymorphisms of FSHR and LHB Undergoing in Vitro Fertilization Program.

BACKGROUND/AIMS: FSH receptor (FSHR) Ala307Thr and Asn680Ser and LHß chain (LHB) Trp28Arg and Ile35Thr polymorphisms affect the response to pharmacological ovarian stimulation with r-FSH in women undergoing assisted reproductive treatment (ART). Here, we evaluated the expression level of selected genes involved in follicle maturation and the possible onset of apoptosis in cumulus cells of patients with single and double FSHR and LHB polymorphisms, as potential markers of oocyte competence. METHODS: Cumulus cells from 36 stimulated patients were collected and SNP genotyping performed by PCR. Gene expression was evaluated through real-time PCR, and apoptosis estimated via TUNEL assay, and cleaved caspase-3 and pAKT immunostaining. RESULTS: The cumulative data show significant correlations indicating that the genetic alteration of FSHR and/or LHB genes may lead to perturbations of the signaling network programmed to granulosa cell survival and follicle development. Notably, when double heterozygotes were compared to the rest of the patients, a higher level of apoptosis in terms of both DNA fragmentation index and amount of active caspase-3 was observed in cumulus cells. CONCLUSIONS: These results may help to define personalized stimulation protocols in ART programs, to increase the success rate of ICSI procedures in accordance with the polymorphic condition of the individual patient.

Molecular Signatures Associated with Treatment of Triple-Negative MDA-MB231 Breast Cancer Cells with Histone Deacetylase Inhibitors JAHA and SAHA.

Jay Amin hydroxamic acid (JAHA; N8-ferrocenylN1-hydroxy-octanediamide) is a ferrocene-containing analogue of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA). JAHA's cytotoxic activity on MDA-MB231 triple negative breast cancer (TNBC) cells at 72 h has been previously demonstrated with an IC50 of 8.45 µM. JAHA's lethal effect was found linked to perturbations of cell cycle, mitochondrial activity, signal transduction, and autophagy mechanisms. To glean novel insights on how MDA-MB231 breast cancer cells respond to the cytotoxic effect induced by JAHA, and to compare the biological effect with the related compound SAHA, we have employed a combination of differential display-PCR, proteome analysis, and COMET assay techniques and shown some differences in the molecular signature profiles induced by exposure to either HDACis. In particular, in contrast to the more numerous and diversified changes induced by SAHA, JAHA has shown a more selective impact on expression of molecular signatures involved in antioxidant activity and DNA repair. Besides expanding the biological knowledge of the effect exerted by the modifications in compound structures on cell phenotype, the molecular elements put in evidence in our study may provide promising targets for therapeutic interventions on TNBCs.

The conditioned medium from osteo-differentiating human mesenchymal stem cells affects the viability of triple negative MDA-MB231 breast cancer cells.

This study aimed to investigate the effect of conditioned media (CM) from osteo-differentiating and adipo-differentiating human mesenchymal stem cells (MSCs) isolated from lipoaspirates of healthy female donors on the viability of triple-negative breast cancer cells MDA-MB231. The CM of undifferentiated and differentiating MSCs were collected after 7, 14, 21 and 28 days of culture. The effects of MSC CM on cell proliferation were assessed using an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after 24 h. The effects of osteo-differentiating cell CM on apoptotic promotion, cell cycle impairment, mitochondrial transmembrane potential dissipation, production of reactive oxygen species and autophagosome accumulation were analysed by flow cytometry and Western blot. MTT assay showed that only CM collected from osteo-induced cells at day 28 (d28O-CM) reduced tumour cell viability. Treatment with d28O-CM restrained cell cycle progression through G2 phase, elicited a caspase-8-driven apoptotic effect already after 5 h of culture, and down-regulated autophagosome accumulation and beclin-1 expression. The finding that factor(s) secreted by osteo-differentiating MSCs shows properties of an apoptotic inducer and autophagy inhibitor on triple-negative breast cancer cells may have an important applicative potential that deserves further investigation.

Biological Effect of a Hybrid Anticancer Agent Based on Kinase and Histone Deacetylase Inhibitors on Triple-Negative (MDA-MB231) Breast Cancer Cells.

We examined the effects of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) combined with the vascular endothelial growth factor receptor-1/2 inhibitor (3Z)-5-hydroxy-3-(1H-pyrrol-2-ylmethylidene)-2,3-dihydro-1H-indol-2-one on MDA-MB-231 breast cancer cells (triple-negative) in the form of both a cocktail of the separate compounds and a chemically synthesized hybrid (N-hydroxy-N'-[(3Z)-2-oxo-3-(1H-pyrrol-2-ylmethylidene)-2,3-dihydro-1H-indol-5-yl]octanediamide). Comparative flow cytometric and Western blot analyses were performed on cocktail- and hybrid-treated cells to evaluate cell cycle distribution, autophagy/apoptosis modulation, and mitochondrial metabolic state in order to understand the cellular basis of the cytotoxic effect. Cell cycle analysis showed a perturbation of the rate of progression through the cycle, with aspects of redistribution of cells over different cycle phases for the two treatments. In addition, the results suggest that the two distinct classes of compounds under investigation could induce cell death by different preferential pathways, i.e., autophagy inhibition (the cocktail) or apoptosis promotion (the hybrid), thus confirming the enhanced potential of the hybrid approach vs. the combination approach in finely tuning the biological activities of target cells and also showing the hybrid compound as an additional promising drug-like molecule for the prevention or therapy of "aggressive" breast carcinoma.

Cytotoxicity of the Urokinase-Plasminogen Activator Inhibitor Carbamimidothioic Acid (4-Boronophenyl) Methyl Ester Hydrobromide (BC-11) on Triple-Negative MDA-MB231 Breast Cancer Cells.

BC-11 is an easily synthesized simple thiouronium-substituted phenylboronic acid, which has been shown to be cytotoxic on triple negative MDA-MB231 breast cancer cells by inducing a perturbation of cell cycle when administered at a concentration equal to its ED50 at 72 h (117 µM). Exposure of cells to BC-11, either pre-absorbed with a soluble preparation of the N-terminal fragment of urokinase-plasminogen activator (uPa), or in co-treatment with two different EGFR inhibitors, indicated that: (i) BC-11 acts via binding to the N-terminus of the enzyme where uPa- and EGF receptor-recognizing sites are present, thereby abrogating the growth-sustaining effect resulting from receptor binding; and (ii) the co-presence of the EGFR inhibitor PD153035 potentiates BC-11's cytotoxicity. Exposure of cells to a higher concentration of BC-11 corresponding to its ED75 at 72 h (250 µM) caused additional impairment of mitochondrial activity, the production of reactive oxygen species and promotion of apoptosis. Therefore, BC-11 treatment appears to show potential for the development of this class of compounds in the prevention and/or therapy of "aggressive" breast carcinoma.

Type V collagen counteracts osteo-differentiation of human mesenchymal stem cells.

In search of novel gene signatures for osteo-differentiation of mesenchymal stem cells (MSCs), we submitted cDNA preparations from undifferentiated and differentiating MSCs to differential display- and semiquantitative-PCR and found down-regulation of COL5A1 in osteo-induced cultures at days 21 and 28, when the mineralized matrix accumulates. We also cultured osteo-differentiating MSCs onto type V collagen substrates and found a decrease in the accumulation of extracellular calcium compared to those grown in uncoated flasks. To our knowledge, this is first evidence that type V collagen might represent a stromal component that impairs osteogenesis.

The contribution of mitochondria to age-related skeletal muscle wasting: A sex-specific perspective.

As people age, their skeletal muscle (SkM) experiences a decline in mitochondrial functionality and density, which leads to decreased energy production and increased generation of reactive oxygen species. This cascade of events, in turn, might determine the loss of SkM mass, strength and quality. Even though the mitochondrial processes dysregulated by aging, such as oxidative phosphorylation, mitophagy, antioxidant defenses and mtDNA transcription, are the same in both sexes, mitochondria age differently in the SkM of men and women. Indeed, the onset and magnitude of the impairment of these processes seem to be influenced by sex-specific factors. Sexual hormones play a pivotal role in the regulation of SkM mass through both genomic and non-genomic mechanisms. However, the precise mechanisms by which these hormones regulate mitochondrial plasticity in SkM are not fully understood. Although the presence of estrogen receptors in mitochondria is recognized, it remains unclear whether androgen receptors affect mitochondrial function. This comprehensive review critically dissects the current knowledge on the interplay of sex in the aging of SkM, focusing on the role of sex hormones and the corresponding signaling pathways in shaping mitochondrial plasticity. Improved knowledge on the sex dimorphism of mitochondrial aging may lead to sex-tailored interventions that target mitochondrial health, which could be effective in slowing or preventing age-related muscle loss.

Modulation of Glucose Consumption and Uptake in HepG2 Cells by Aqueous Extracts from the Coelomic Fluid of the Edible Holothuria tubulosa Sea Cucumber.

The cell-free aqueous extract from the coelomic fluid of Holothuria tubulosa was prepared and examined for its glucose-lowering effect on HepG2 cells in vitro. In particular, employing a combination of cytochemical, flow cytometric, PCR, and protein blot techniques, we evaluated its role on glucose internalization and storage and on the upregulation and surface translocation of the two glucose transporters GLUT-2 and -4. The changes in expression, synthesis, and/or activation of the GLUT2-related transcription factor hepatocyte nuclear factor-1 alpha (HNF1α) and the GLUT-4-translocation regulatory factors insulin receptor substrate-1 (IRS-1) and AKT were also studied. Our results showed the improved glucose response by HepG2 cells, leading to an evident increase in glucose consumption/uptake and glycogen storage upon exposure. Moreover, the extract induced molecular reprogramming involving the upregulation of (i) IRS1 gene expression, (ii) the transcription and translation levels of HNF1α, AKT, and GLUT-4, (iii) the phosphorylation level of AKT, (iv) the synthesis of GLUT-2 protein, and (v) the translocation of GLUT-2 and -4 transporters onto the plasma membrane. Cumulatively, our results suggest that the coelomic fluid extract from H. tubulosa can be taken into consideration for the development of novel treatment agents against diabetes mellitus.

Mitochondrial remodeling underlying age-induced skeletal muscle wasting: let's talk about sex.

Sarcopenia is associated with reduced quality of life and premature mortality. The sex disparities in the processes underlying sarcopenia pathogenesis, which include mitochondrial dysfunction, are ill-understood and can be decisive for the optimization of sarcopenia-related interventions. To improve the knowledge regarding the sex differences in skeletal muscle aging, the gastrocnemius muscle of young and old female and male rats was analyzed with a focus on mitochondrial remodeling through the proteome profiling of mitochondria-enriched fractions. To the best of our knowledge, this is the first study analyzing sex differences in skeletal muscle mitochondrial proteome remodeling. Data demonstrated that age induced skeletal muscle atrophy and fibrosis in both sexes. In females, however, this adverse skeletal muscle remodeling was more accentuated than in males and might be attributed to an age-related reduction of 17beta-estradiol signaling through its estrogen receptor alpha located in mitochondria. The females-specific mitochondrial remodeling encompassed increased abundance of proteins involved in fatty acid oxidation, decreased abundance of the complexes subunits, and enhanced proneness to oxidative posttranslational modifications. This conceivable accretion of damaged mitochondria in old females might be ascribed to low levels of Parkin, a key mediator of mitophagy. Despite skeletal muscle atrophy and fibrosis, males maintained their testosterone levels throughout aging, as well as their androgen receptor content, and the age-induced mitochondrial remodeling was limited to increased abundance of pyruvate dehydrogenase E1 component subunit beta and electron transfer flavoprotein subunit beta. Herein, for the first time, it was demonstrated that age affects more severely the skeletal muscle mitochondrial proteome of females, reinforcing the necessity of sex-personalized approaches towards sarcopenia management, and the inevitability of the assessment of mitochondrion-related therapeutics.

Chemical Characterization, Free Radical Scavenging, and Cellular Antioxidant Properties of the Egadi Island Endemic Brassica macrocarpa Guss Leaf Extract.

The genus Brassica is an important source of food in the Mediterranean diet with documented nutritional and medicinal properties. However, few studies have investigated the phytochemical composition and the biological activity of wild Sicilian taxa. Thus, we aimed to study the chemical profile and the antioxidant potential, in vitro and in LPS-stimulated RAW 264.7 cells, of a methanolic extract of leaves of wild Brassica macrocarpa Guss (B. macrocarpa) (Egadi Islands; Sicily-Italy). B. macrocarpa methanolic extract showed a large amount of glucosinolates and different phenolic compounds. It exhibited antioxidant activity in the DPPH assay and in LPS-stimulated RAW 264.7 cells, being able to reduce NO and ROS levels and NOS2 mRNA expression. Our study demonstrated that Sicilian B. macrocarpa methanolic extract, in LPS-stimulated macrophages, efficiently counteracts oxidative stress and displays radical scavenging activity. Future studies are required to identify the contribution of the single phytocomponents, to characterize the action mechanism, and to reveal possible applications in human health.