Contribution of the Glucosinolate Fraction to the Overall Antioxidant Potential, Cytoprotection against Oxidative Insult and Antimicrobial Activity of Eruca sativa Mill. Leaves Extract.

BACKGROUND: Eruca sativa Mill. (Brassicaceae) is commonly utilized as an ingredient in salads and also as a folk remedy to treat various diseases. OBJECTIVE: The objective of this study was to establish the contribution of the glucosinolate (GLS) fraction to the overall antioxidant, cytoprotection against oxidative insult and antimicrobial properties of the hydro-alcoholic extract of E. sativa leaves from Sicily (Italy), characterized phytochemically. MATERIALS AND METHODS: The antioxidant activity was evaluated by different in vitro systems. The cytoprotective effect against hydrogen peroxide (H2O2)-induced oxidative stress was tested in human peripheral blood mononuclear cells (PBMCs). The antimicrobial potential against bacteria and fungi was assayed by standard methods. RESULTS: E. sativa extract exhibited both radical scavenging (50% inhibitory concentration [IC50] 1.04 ± 0.04 mg/mL) and ferrous ions-chelating activity (IC50 0.327 ± 0.0032 mg/mL) and mild reducing power; the GLS fraction showed chelating ability only (IC50 0.225 ± 0.009 mg/mL). In the experimental model of H2O2-induced oxidative stress in human PBMCs, a significant cytoprotective effect and a suppression of reactive oxygen species production by both extract and GLS fraction were observed (P < 0.001). E. sativa extract displayed moderate antimicrobial activity against Gram-positive bacteria, and Staphylococcus aureus was the most sensitive strain (minimum inhibitory concentration 0.125 mg/mL), whereas the GLS fraction was not active. CONCLUSION: GLSs are not involved in the primary antioxidant activity of E. sativa leaf extract but they are, almost in part, responsible for its ferrous ion-chelating properties. Iron-chelating compounds in E. sativa extract may protect cells under conditions of oxidative stress, and GLSs might play a chief role in this effect. SUMMARY: Eruca sativa Mill. leaf extract exhibited antioxidant activity in different in vitro systems, whereas the glucosinolate (GLS) fraction showed Fe2+-chelating ability onlyA significant cytoprotective effect and a suppression of intracellular reactive oxygen species production by both extract and GLS fraction were observed in human peripheral blood mononuclear cellsE. sativa extract displayed moderate antimicrobial activity against Gram-positive bacteria, whereas the GLS fraction was not active. Abbreviations used: GLS: Glucosinolate; H2O2: Hydrogen peroxide; PBMCs: Peripheral blood mononuclear cells; IC50: 50% inhibitory concentration; MIC: Minimum inhibitory concentration.

Effects of adaptation to carvacrol on Staphylococcus aureus in the planktonic and biofilm phases.

The effect of exposure to sub-minimum inhibitory concentrations of carvacrol, for either 3-10 days, on direct (carvacrol) or cross-protection (cinnamaldehyde, eugenol, antibiotics) and the influence on planktonic and biofilm growth of four Staphylococcus aureus strains were reported. The sequential exposure to carvacrol resulted in a direct protection that was more evident in two of the four strains after 10 days. No significant cross-protection against cinnamaldehyde, eugenol and antibiotics was detected. An adaptive response was associated with a prolonged lag phase, a lower yield of bacteria, a colony phenotype likely to be associated to small colony variants and an increase in biofilm production. Generally, the biofilm of the adapted strains was less susceptible to subMICs of carvacrol compared to the biofilms of non-adapted strains. In contrast, it was demonstrated that in the case of mature biofilms the susceptibility was similar. The exposure of S. aureus to carvacrol at concentrations above the MIC resulted in a very low mutation frequency.

In vitro activity of plant extracts against biofilm-producing food-related bacteria.

The identification of effective antimicrobial agents also active on biofilms is a topic of crucial importance in food and industrial environment. For that purpose methanol extracts of Turkish plants, Ficus carica L., Juglans regia L., Olea europaea L., Punica granatum L. and Rhus coriaria L., were investigated. Among the extracts, P. granatum L. and R. coriaria L. showed the best antibacterial activity with minimum inhibitory concentrations (MIC) of 78-625µg/ml for Listeria monocytogenes and Staphylococcus aureus and 312-1250µg/ml for Escherichia coli and Pseudomonas aeruginosa. SubMICs produced a significant biofilm inhibition equal to 80-60% for L. monocytogenes and 90-80% for S. aureus. The extracts showed also the highest polyphenol content and the strongest antioxidant activity. Bioassay-guided and HPLC procedures demonstrated the presence of apigenin 4'-O-ß-glucoside in P. granatum L. and myricetrin and quercitrin in R. coriaria L. Antigenotoxicity of plant extracts was also observed The present findings promote the value-adding of P. granatum L. and R. coriaria L. leaves as natural antimicrobial/antioxidant agents for control of food-related bacterial biofilms.

Ex vivo efficacy of gemifloxacin in experimental keratitis induced by methicillin-resistant Staphylococcus aureus.

In recent years, the emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains has been observed in ocular infections. Resistance of MRSA to second- and third-generation fluoroquinolones has increased interest in the fourth-generation fluoroquinolones. In this study, the antibacterial activity of gemifloxacin against MRSA ocular isolates in vitro and in a modified ex vivo rabbit keratitis model was investigated. In vitro susceptibility test results indicated that the minimum inhibitory concentrations (MICs) of gemifloxacin were lower than the MICs of other fluoroquinolones, including moxifloxacin (MIC50 range, 0.016-0.032 µg/mL; MIC90 range, 0.047-0.094 µg/mL). Results from the ex vivo keratitis model showed a statistically significant decrease in MRSA counts (0.5-2 log10 CFU/g; P <0.05) in corneas treated with 0.3% gemifloxacin every 30 min for 7 h. Moreover, the dose-response effect of different concentrations of gemifloxacin (3-3000 µg/mL) demonstrated that a dose of 30 µg/mL had the same efficacy as the highest dose of 3000 µg/mL against all S. aureus strains. Possibly, gemifloxacin reached a steady-state level in the cornea, as the fourth-generation fluoroquinolones have better anterior chamber penetration. This study demonstrated that 0.3% gemifloxacin ophthalmic solution may be an effective topical therapy for the treatment of MRSA keratitis. In addition, this reproducible, ethical and economic ex vivo infection model can be used as a mechanistically-based alternative to in vivo animal testing, bridging the gap between in vitro and in vivo results.

Antimicrobial activities, toxicity and phenolic composition of Asphodeline anatolica E. Tuzlaci leaf extracts from Turkey.

The antimicrobial activity of acetone, methanol and aqueous extracts of Asphodeline anatolica E. Tuzlaci leaves was evaluated against American type culture collection, food and clinical isolates (Listeria monocytogenes and Staphylococcus aureus including methicillin-resistant strains-MRSA). Biofilm formation, toxicity and characterisation of the polyphenolic content were analysed. The acetone extract demonstrated a higher antibacterial activity against S. aureus including MRSA strains, L. monocytogenes and Pseudomonas aeruginosa than against other extracts. No effect was observed in biofilm formation. The extracts resulted non-toxic against Artemia salina Leach. The phytochemical screening of extracts indicated that they mainly contained six polyphenols identified as catechin 3-O-gallate, protocatechuic acid, diosmin, rutin, cirsimaritin and kaempferol glucoside. This study is the first report on antimicrobial activity and phenolic content of A. anatolica and contributes to enrich the literature data on the biological properties of this plant. A. anatolica leaves have a potential as source of natural antimicrobial compounds.

TLR2 activation in corneal stromal cells by Staphylococcus aureus-induced keratitis.

The Toll-Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus-induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air-interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub-groups exposed to UV-killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL-8 mRNA expressions were analyzed by quantitative real-time RT-PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL-8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial-corneal tissue interactions and their inflammatory consequences.

Staphylococcal biofilm formation as affected by type acidulant.

Staphylococcal growth and biofilm formation in culture medium where pH was lowered with weak organic (acetic and lactic) or strong inorganic (hydrochloric) acids were studied. The effects were evaluated by biomass measurements, cell-surface hydrophobicity, scanning electron microscopy (SEM), and confocal laser scanning microscopy (CLSM). The results demonstrated that the inhibition was related to type of acidulant and pH value. At pH 5.0, the antibacterial effect was more pronounced in the presence of acetic acid (58-60% growth reduction) compared with that in the presence of lactic (7-16% growth reduction) and hydrochloric acids (23-24% reduction). The biofilm biomass of Staphylococcus aureus and Staphylococcus epidermidis was reduced by 92, 85, 63, and 93, 87, 81% after exposition to acetic, lactic, and hydrochloric acids, respectively. Increasing the pH from 5.0 to 6.0 resulted in a noticeable reduction in the effectiveness of acids. A minor cells hydrophobic character was also documented. The SEM and CLSM revealed a poorly structured and thinner biofilm compared with the dense and multilayered control. Acidic environment could have important implications for food-processing system to prevent bacterial colonization and control biofilm formation. The findings of this study lead to consider the rational use of the type of acid to achieve acidic environments.

Polymethoxylated, C- and O-glycosyl flavonoids in tangelo (Citrus reticulata×Citrus paradisi) juice and their influence on antioxidant properties.

A separation/identification protocol based on RP-LC-DAD-ESI-MS-MS has been employed for the characterisation of the flavonoid fraction of the juice from tangelos (Citrus reticulata×Citrus paradisi) grown in Southern Italy. Eleven compounds were identified in a single chromatographic course. Of these, two C-glycosyl flavones (lucenin-2 and vicenin-2) and an O-triglycosyl flavanone (narirutin 4'-O-glucoside) were identified for the first time. Fruit juice antioxidant activity was evaluated on the basis of its ability to scavenge DPPH, O2(-), OH and ABTS(+) radicals, and to reduce iron (FRAP). Moreover, the influence of the identified polymethoxylated, C- and O-glycosyl flavonoids on the total antioxidant activity has been elucidated. We also checked the antimicrobial activity of a broad fraction, containing all the detected flavonoids obtained by preparative HPLC, in terms of MICs for Staphylococcus aureus.

Resveratrol role in Staphylococcus aureus-induced corneal inflammation.

The aim of this study was to evaluate the role of trans-resveratrol on Staphylococcus aureus-induced keratitis. Rabbit corneas (intact corneas, abraded corneas and abraded corneas exposed to inactivated S. aureus strains) were placed in an ex vivo culture model. The abraded corneas exposed to S. aureus were divided into two 1-h-treatment sub-groups: corneas treated with trans-resveratrol and corneas treated with vehicle. The tissues were examined by immunohistochemical analyses and quantitative real-time RT-PCR to determine whether resveratrol could reduce TLR2-mediated recognition of S. aureus on epithelial cells and, if so, whether this reduction repressed the expression of inflammatory cytokines. The results demonstrated that resveratrol treatment effectively downregulated cell surface TLR2 on cells stimulated by S. aureus and reduced the expression of interleukin-8 gene. In addition, the corneal culture model tested, which is simple and reproducible, could be an alternative to in vivo animal testing for the development of novel specific therapies.

Enhanced activity of carvacrol against biofilm of Staphylococcus aureus and Staphylococcus epidermidis in an acidic environment.

Carvacrol is an antimicrobial monoterpenic phenol which occurs in many plant essential oils. The aim of this study was to investigate its activity at acidic pH on staphylococcal forming and yet established biofilms, with particular focus to improve its effectiveness on Staphylococcus epidermidis biofilm. The results showed that the subinhibitory doses (1/2, 1/4 and 1/8 MIC) of carvacrol determined a higher reduction of S. epidermidis biofilm formation than that observed at neutral pH. A potentiated inhibitory effect was also observed on established biofilm, carvacrol caused either a strong reduction of biomass (>50%) and bacteria attached to polystyrene (>7 log units). The images of scanning electron microscopy and the gas-chromatographic analysis support these results. The development of acidic formulations containing carvacrol could be an important tool to control the staphylococcal biofilm in the medical and food environment.

Effect of alkaline pH on staphylococcal biofilm formation.

Biofilms are a serious problem, cause of severe inconvenience in the biomedical, food and industrial environment. Staphylococcus aureus and S. epidermidis are important pathogenic bacteria able to form thick and resistant biofilms on various surfaces. Therefore, strategies aimed at preventing or at least interfering with the initial adhesion and subsequent biofilm formation are a considerable achievement. The aim of this study was to evaluate the effect of alkaline pH on bacterial adhesion and further biofilm formation of S. aureus and S. epidermidis strains by biofilm biomass, cell-surface hydrophobicity, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) analysis. The results demonstrated that the amount of biofilm biomass formed and the surface hydrophobicity were significantly less than what were observed at higher levels of pH. SEM and CLSM images revealed a poorly structured and very thin biofilm (2.5-3 times thinner than that of the controls). The inhibiting effect of the alkaline pH on the bacterial attachment impaired the normal development of biofilm that arrested at the microcolony stage. Alkaline formulations could be promising towards the control of bacterial colonization and therefore the reduction of the biofilm-related hazard. In the clinical setting, alkaline solutions or cleaners could be promising to prevent the bacterial colonization, by treating surfaces such as catheters or indwelling medical devices, reducing the risk of biofilm related infections.

In vitro antimycoplasmal activity of Citrus bergamia essential oil and its major components.

Forty-two strains of Mycoplasma hominis (including PG21), 2 strain of Mycoplasma fermentans (Pg18 and K7), 1 strain of Mycoplasma pneumoniae (strain m129) were investigated for their susceptibilities to Citrus bergamia essential oil and to its major components (limonene, linalyl acetate and linalool). C. bergamia essential oil inhibited mycoplasmas at concentrations from 0.5 to 1% (MIC value as % v/v). M. hominis showed MIC(50) values of 0.5% and MIC(90) values of 1%; M. pneumoniae showed a MIC value of 0.5% while M. fermentans strains were inhibited by MIC values of 1%. M. pneumoniae and M. hominis shared the same susceptibility to linalyl acetate, with MIC values of 0.015% (corresponding to MIC(50) and MIC(90) for M. hominis); M. fermentans strains were less susceptible with MIC values of 0.12%. Among the major components tested, linalool showed higher activity against M. pneumoniae and M. fermentans (MIC values of 0.015 and 0.06%, respectively) but was less active against M. hominis (MIC(50) and MIC(90) values of both 1%); limonene was active against M. pneumoniae (MIC value of 0.03%) but was less active against M. fermentans (MIC values of 1%) and M. hominis (both MIC(50) and MIC(90) values of ≥4%). The results indicated that C. bergamia essential oil and its major components had shown an interesting in vitro antimycoplasmal activity.

Antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben plant extracts.

This study reported the antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben inflorescences. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanolic extracts were determined according to the Clinical Laboratory Standards Institute guidelines. Individual phenolic acids and flavonoids were detected by a high performance liquid chromatography (HPLC-DAD) method. The samples showed a comparable antimicrobial activity, although the natural site extract possessed the lower MIC values. The best activity was detected against Gram-positive bacteria, such as Staphylococcus aureus including methicillin resistant strains (MIC and MBC values ranging from 7.81 to 62.50 µg mL(-1) and from 500 to 2000 µg mL(-1) respectively). In contrast, a low activity was found on Gram-negative bacteria and Candida albicans. The HPLC-DAD analysis revealed ten phenolic acids and four flavonoids with a major amount of m-coumaric acid, naringin and quercetin in the natural site extract.

Characterization of bacterial lipid profiles by using rapid sample preparation and fast comprehensive two-dimensional gas chromatography in combination with mass spectrometry.

The bacteria fatty acid profile has been extensively studied for taxonomic classification purposes, since bacteria, in general, contain particular and rare fatty acids, compared with animal and plant tissues. As for any real-world sample type, the development of rapid and reliable methods for (i) sample identification (in this case, bacterium type), and (ii) constituent identification (in this instance, the fatty acid profile) is desirable. In this research, a half-an-hour procedure, to analyze bacteria, was developed: a 2-min one-step sample preparation step was followed by a relatively fast comprehensive 2D GC-MS separation (25 min). Furthermore, dedicated MS libraries were constructed for the identification of bacteria and fatty acids. Finally, data processing, only qualitative at this stage, was carried out with the support of a novel comprehensive 2D GC software.

In vitro effect of branch extracts of Juniperus species from Turkey on Staphylococcus aureus biofilm.

Methanol and aqueous branch extracts of five Juniperus species were examined for their effects on Staphylococcus aureus ATCC 6538P and S. aureus 810 biofilm. The Turkish plant material was Juniperus communis L. var. communis, J. communis L. var. saxatilis Pall., Juniperus drupacea Labill., Juniperus oxycedrus L. ssp. oxycedrus, J. oxycedrus L. ssp. macrocarpa (Sibth. & Sm.) Ball. The Juniperus extracts were subjected to preliminary phytochemical analysis by thin-layer chromatography. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The effects of the extracts on biofilm formation and preformed biofilm were quantified by both biomass OD and the CFU counting method. The phytochemical screening revealed the presence of polyphenols, coumarins, lignans, steroids, alkaloids and terpenes. For both strains, the MICs of all extracts were in the range of 4.88-78.12 microg mL(-1). On S. aureus ATCC 6538P, the effects of subinhibitory concentration (0.5 MIC) of the extracts were minimal on planktonic growth and on adhering cells, whereas they were greater on biofilm formation. Differently, on S. aureus 810, they showed only a rather low efficacy on biofilm formation. The extracts at 2 MIC demonstrated a good activity on a preformed biofilm of S. aureus ATCC 6538P.

In vitro protective effects of two extracts from bergamot peels on human endothelial cells exposed to tumor necrosis factor-alpha (TNF-alpha).

Bergamot ( Citrus bergamia Risso) is a less commercialized Citrus fruit, mainly used for its essential oil extracted from the peel. Bergamot peel (BP) represents about 60% of the processed fruits and is regarded as primary waste. However, it contains good amounts of useful compounds, such as pectins and flavonoids. Many of the bioactivities of Citrus flavonoids appear to impact vascular endothelial cells. Herein, we report the protective effect of two flavonoid-rich extracts from BP (endowed with radical-scavenging properties and lacking genotoxic activity) against alterations in cell modifications induced by the pleiotropic inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) on human umbilical vein endothelial cells (HUVECs), as demonstrated by monitoring intracellular levels of malondialdehyde/4-hydroxynonenal, reduced and oxidized glutathione and superoxide dismutase activity, and the activation status of nuclear factor-kappaB (NF-kappaB). Thus, BP appears to be a potential source of natural antioxidant/anti-inflammatory phytocomplexes to be employed as ingredients of nutraceutical products or functional foods.

Immunomodulatory and antiviral activity of almond skins.

The elimination of a viral infection requires a proinflammatory host response (type 1 immunity), characterized by activation of mononuclear cells and production of proinflammatory cytokines, such as interferons (IFNs), tumor necrosis factor (TNF)-alpha and interleukin (IL)-12. On the other hand, IL-4 and IL-10 play a role in decreasing the inflammatory response supported by helper T (Th)1 cells. In this study we evaluated the effects of almond skins on the release of cytokines by peripheral blood mononuclear cells (PBMC), either infected or not with herpes simplex virus type 2 (HSV-2). Natural (NS) and blanched almond skins (BS) were subjected to simulated gastric and duodenal digestion and used at not cytotoxic concentrations. NS induced a significant decrease in HSV-2 replication, whereas extracts obtained from BS did not significantly influence the viral replication. High levels of cytokines production, such as IFN-alpha (38+/-5.3 pg/ml), IL-12 (215+/-17.1 pg/ml), IFN-gamma (5+/-0.7 IU/ml), TNF-alpha (3940+/-201.0 pg/ml), were detected. Moreover, IL-10 (210+/-12.2 pg/ml) and IL-4 (170+/-21.4 pg/ml), representative of Th2 responses, were found. Our data suggest that almond skins improve the immune surveillance of PBMC towards viral infection, both by triggering the Th1 and Th2 subsets.

Control of biofilm formation by poly-ethylene-co-vinyl acetate films incorporating nisin.

The aim of this study was to evaluate the effect of poly-ethylene-co-vinyl acetate (EVA) films incorporating different concentrations (0.1%, 0.5% and 1%) of nisin on the biofilm-forming ability of Listeria monocytogenes ATCC 7644, Staphylococcus aureus 815 and Staphylococcus epidermidis ATCC 35984. Nisin was incorporated into two grades of EVA (EVA14 and EVA28) in the melt during a common film-blowing operation. The efficacy of EVA/nisin films was evaluated by biofilm biomass measurements and Live/Dead staining in combination with fluorescence microscopy. In order to evaluate whether the nisin incorporation could modify the film surface properties, contact angle measurements and scanning electron microscopy were performed. The results revealed the efficacy of EVA14/nisin films in reducing biofilm formation on their surfaces with more evident effect for S. epidermidis than L. monocytogenes and S. aureus strains. In contrast, EVA28/nisin films showed unsatisfactory activity. Fluorescence microscopy confirmed poor biofilm formation on EVA14/nisin films, also characterised by the presence of dead cells. The data presented in this study offer new potential applications for developing strategies aimed to improve the effect of antimicrobial agents.

Anatomical, chemical, and biochemical characterization of cladodes from prickly pear [Opuntia ficus-indica (L.) Mill.].

Opuntia ficus-indica cladodes represent the green stem of the plant and are generally used as animal feed or disposed of in landfills. The present work investigated the anatomical and chemical composition of Opuntia cladodes, which form the basis of their pharmacological effects. Glucose and galacturonic acid were the main sugars of Opuntia cladodes, whereas high-performance liquid chromatography (HPLC) analysis showed the presence of mainly kaempherol and isorhamnetin glycosides (glucoside and rhamnoside). The presence of high amounts of calcium oxalate crystals was demonstrated by light microscopy on fresh and lyophilized cladodes. No antimicrobial activity was observed even after enzymatic treatment. O. ficus-indica cladodes may retain material tightly associated with cell-wall components, and this property will have the potential to greatly reduce the bioavailability of bioactive compounds.

In vitro activity of carvacrol against staphylococcal preformed biofilm by liquid and vapour contact.

Carvacrol is an important component of essential oils and recently has attracted much attention as a result of its biological properties, such as a wide spectrum of antimicrobial activity. The aim of this study was to evaluate the effect of carvacrol in liquid and vapour phase on preformed biofilms of Staphylococcus aureus and Staphylococcus epidermidis by determining biofilm biomass and cultivable cell numbers, and by using epifluorescence and scanning electron microscopy. Carvacrol was able to reduce biofilm biomass and cell viability more effectively when used with liquid contact rather than with vapour phase. The efficacy of treatment with carvacrol vapour was found to be dependent on exposure time. The predominance of red fluorescence using a LIVE/DEAD BacLight Viability kit (Molecular Probes) and the partially destroyed biofilm architecture as determined by microscopy in treated samples provided evidence for the efficacy of carvacrol. The findings of this investigation suggest a potential application for carvacrol in the inactivation of staphylococcal biofilms.