The Influence of Pedo-Climatic Conditions on the Micromorphological, Phytochemical Features, and Biological Properties of Leaves of Saponaria sicula Raf.

Saponaria sicula Raf. grows in Sicily, Sardinia, and Algeria on limestone cliffs and volcanic sands 1300-2500 m above sea level. The aim of the present study was to investigate how the pedo-climatic conditions influence the micromorphological, phytochemical, and biological properties of Sicilian S. sicula leaves collected in the Madonie Mountains (SsM) and on Etna Mt (SsE). Micromorphological investigations revealed that leaves from SsM had a higher amount of calcium oxalate druses in the mesophyll and a more intense blue-green staining with Toluidine blue O, indicating a higher content of polyphenols. These data were confirmed by phytochemical analyses carried out on hydroalcoholic extracts, which showed a higher content of total phenols (8.56 ± 0.57 g GAE/100 g DE) and flavonoids (6.09 ± 0.17 g RE/100 g DE) in SsM. Sixty-four compounds were identified by LC-DAD-ESI-MS analysis with propelargonidin dimer as the most abundant compound (10.49% and 10.19% in SsM and SsE, respectively). The higher polyphenol content of SsM leaves matches also with their biological activity, identifying SsM extract as the strongest plant complex (IC50 2.75-477.30 µg/mL). In conclusion, the present study experimentally demonstrates that not only climatic differences but also soil characteristics affect the micromorphological, phytochemical, and biological features of this plant species.

Nano-Hybrid Ag@LCCs Systems with Potential Wound-Healing Properties.

The synthesis of contaminant-free silver@linear carbon chains (Ag@LCCs) nanohybrid systems, at different Ag/LCCs ratios, by pulsed laser ablation was studied. The ablation products were first characterized by several diagnostic techniques: conventional UV-Vis optical absorption and micro-Raman spectroscopies, as well as scanning electron microscopy, operating in transmission mode. The experimental evidence was confirmed by the theoretical simulations' data. Furthermore, to gain a deeper insight into the factors influencing metal@LCCs biological responses in relation to their physical properties, in this work, we investigated the bioproperties of the Ag@LCCs nanosystems towards a wound-healing activity. We found that Ag@LCC nanohybrids maintain good antibacterial properties and possess a better capability, in comparison with Ag NPs, of interacting with mammalian cells, allowing us to hypothesize that mainly the Ag@LCCs 3:1 might be suitable for topical application in wound healing, independent of (or in addition to) the antibacterial effect.

Punica granatum Peel and Leaf Extracts as Promising Strategies for HSV-1 Treatment.

Punica granatum is a rich source of bioactive compounds which exhibit various biological effects. In this study, pomegranate peel and leaf ethanolic crude extracts (PPE and PLE, respectively) were phytochemically characterized and screened for antioxidant, antimicrobial and antiviral activity. LC-PDA-ESI-MS analysis led to the identification of different compounds, including ellagitannins, flavonoids and phenolic acids. The low IC50 values, obtained by DPPH and FRAP assays, showed a noticeable antioxidant effect of PPE and PLE comparable to the reference standards. Both crude extracts and their main compounds (gallic acid, ellagic acid and punicalagin) were not toxic on Vero cells and exhibited a remarkable inhibitory effect on herpes simplex type 1 (HSV-1) viral plaques formation. Specifically, PPE inhibited HSV-1 adsorption to the cell surface more than PLE. Indeed, the viral DNA accumulation, the transcription of viral genes and the expression of viral proteins were significantly affected by PPE treatment. Amongst the compounds, punicalagin, which is abundant in PPE crude extract, inhibited HSV-1 replication, reducing viral DNA and transcripts accumulation, as well as proteins of all three phases of the viral replication cascade. In contrast, no antibacterial activity was detected. In conclusion, our findings indicate that Punica granatum peel and leaf extracts, especially punicalagin, could be a promising therapeutic candidate against HSV-1.

Antioxidant Potential and Enhancement of Bioactive Metabolite Production in In Vitro Cultures of Scutellaria lateriflora L. by Biotechnological Methods.

Studies carried out using three different in vitro assays and a biological setting (Escherichia coil) demonstrated the antioxidant activity of Scutellaria lateriflora microshoot extract. Moreover, the extract exhibited no toxicity in a brine shrimp lethality bioassay. These results indicated that microshoots are a rich, safe source of antioxidants, which encouraged us to enhance their production in vitro. In agar and agitated cultures, two biotechnological strategies were applied: feeding the cultures with the biogenetic precursors of the phenolics-phenylalanine and tyrosine, and eliciting them with methyl jasmonate. Specific Scutellaria flavonoids and verbascoside were analysed by HPLC. Feeding with precursors (1 g/L) in agar cultures decreased the production of the metabolites. In agitated cultures, different concentrations of precursors (1.0-2.5 g/L) and the elicitor (10; 50; 100 µM) were tested. Additionally, parallel feeding with the precursor and elicitor in a concentration of 50 µM were applied. The best strategy for total flavonoid and verbascoside production was phenylalanine feeding (1.5 g/L), max. 3765 and 475 mg/100 g DW, respectively, after 7 days. This is the first report documenting the high antioxidant production in S. lateriflora microshoots after feeding with phenylalanine. Moreover, for the first time, bioreactor cultures were successfully maintained, obtaining attractive results (max. total flavonoid content 2348 and verbascoside 485 mg/100 g DW).

Phytochemical Characterization of Olea europea Leaf Extracts and Assessment of Their Anti-Microbial and Anti-HSV-1 Activity.

Owing to the richness of bioactive compounds, Olea europea leaf extracts exhibit a range of health effects. The present research evaluated the antibacterial and antiviral effect of leaf extracts obtained from Olea europea L. var. sativa (OESA) and Olea europea var. sylvestris (OESY) from Tunisia. LC-DAD-ESI-MS analysis allowed the identification of different compounds that contributed to the observed biological properties. Both OESA and OESY were active against Gram-positive bacteria (MIC values between 7.81 and 15.61 µg/mL and between 15.61 and 31.25 µg/mL against Staphylococcus aureus ATCC 6538 for OESY and OESA, respectively). The antiviral activity against the herpes simplex type 1 (HSV-1) was assessed on Vero cells. The results of cell viability indicated that Olea europea leaf extracts were not toxic to cultured Vero cells. The half maximal cytotoxic concentration (CC50) values for OESA and OESY were 0.2 mg/mL and 0.82 mg/mL, respectively. Furthermore, both a plaque reduction assay and viral entry assay were used to demonstrate the antiviral activity. In conclusion, Olea europea leaf extracts demonstrated a bacteriostatic effect, as well as remarkable antiviral activity, which could provide an alternative treatment against resistant strains.

Flexible mats as promising antimicrobial systems via integration of Thymus capitatus (L.) essential oil into PLA.

Aim: To develop electrospun mats loaded with Thymus capitatus (L.) essential oil (ThymEO) and to study their morpho-mechanical and antimicrobial properties. Materials & methods: Poly(lactic acid) (PLA) mats containing ThymEO were prepared by electrospinning. The effect of ThymEO on the morpho-mechanical properties of fibers was assayed by scanning electron microscopy and dynamometer measurements. The antimicrobial activity of ThymEO delivered either in liquid or vapor phase was assessed through killing curves and invert Petri dishes method. The cytotoxicity was also investigated. Results: The mechanical properties were enhanced by integrating ThymEO into PLA. Both liquid and vapors of ThymEO released from mats caused reductions of microbial viable cells. Negligible cytotoxicity was demonstrated. Conclusion: PLA/ThymEO delivery systems could be suitable for treating microbial infections.

Brassica incana Ten. (Brassicaceae): Phenolic Constituents, Antioxidant and Cytotoxic Properties of the Leaf and Flowering Top Extracts.

Brassica incana Ten. is an edible plant belonging to the Brassicaceae family. In this work, the phenolic composition and the antioxidant and cytotoxic properties of the hydroalcoholic extracts obtained from the leaves and the flowering tops of B. incana grown wild in Sicily (Italy) were studied for the first time. A total of 17 and 20 polyphenolic compounds were identified in the leaf and in the flowering top extracts, respectively, by HPLC-PDA-ESI-MS analysis. Brassica incana extracts showed in vitro antioxidant properties; the leaf extract displayed greater radical scavenging activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test than the flowering top extract (IC50 = 1.306 ± 0.049 mg/mL and 2.077 ± 0.011 mg/mL), which in turn had a stronger ferrous ion chelating ability than the other (IC50 = 0.232 ± 0.002 mg/mL and 1.147 ± 0.016 mg/mL). The cytotoxicity of the extracts against human colorectal adenocarcinoma (CaCo-2) and breast cancer (MCF-7) cell lines was evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and the lactic dehydrogenase (LDH) release determination. The extracts showed cytotoxic efficacy against Caco-2 cells, with the flowering top extract being the most effective (about 90% activity at the highest concentration tested). In the brine shrimp lethality bioassay, the extracts exhibited no toxicity, indicating their potential safety.

In vitro evaluation of the activity of an essential oil from Pistacia vera L. variety Bronte hull against Candida sp.

BACKGROUND: Candida sp. represent the most common cause of fungal infections worldwide. In the present work, we have evaluated the activity of an essential oil extracted from pistachio hulls against a number of standard and clinical strains of Candida sp. METHODS: C. albicans ATCC 64550, C. parapsilosis ATCC 22019, 4 clinical strains of C. albicans, 3 clinical strains of C. parapsilosis and 3 clinical strains of C. glabrata were used. All clinical isolates were identified by species-specific PCR-based methods. Susceptibility studies were performed using pistachio hull essential oil alone or in combination with antifungal compounds. The interactions between pistachio hull essential oil and selected antifungal compounds were also evaluated using the checkerboard method and the mechanisms of interaction investigated by droplet size distribution. RESULTS: Pistachio hull essential oil was fungicidal at the concentrations between 2.50 and 5.0 mg/ml. D-limonene and 3-Carene were the components with major activity. An antagonistic effect was observed with all combinations tested. CONCLUSION: The antifungal activity of pistachio hull essential oil could be used to help control resistance in Candida species. More studies need to be performed to elucidate the mechanisms responsible for the activity of pistachio hull essential oil.

Phenolic profile and biological properties of the leaves of Ficus vasta Forssk. (Moraceae) growing in Egypt.

BACKGROUND: Ficus vasta Forssk. (Moraceae) is traditionally used for the treatment of various ailments; nonetheless, this species has been poorly studied to date. This work aimed to characterize the phenolic profile and to evaluate the antioxidant and antimicrobial properties of a hydroalcoholic extract obtained from F. vasta leaves collected in Egypt. METHODS: The phenolic profile of the extract was characterized by HPLC-PDA/ESI-MS. The antioxidant properties were examined by different in vitro systems: DPPH test, reducing power and metal chelating activity assays. Moreover, the ability of the extract to protect Escherichia coli growth and survival from H2O2-induced oxidative stress was evaluated. The potential toxicity was investigated using Artemia salina lethality bioassay. Finally, the antimicrobial properties against a representative set of Gram-positive and Gram-negative bacterial strains and the yeast C. albicans were assayed by standard methods. RESULTS: By HPLC-PDA/ESI-MS analysis 12 compounds belonging to the groups of phenolic acids and flavonoids were identified. The extract exhibited strong radical scavenging activity in DPPH test (IC50 = 0.0672 ± 0.0038 mg/mL), reducing power (3.65 ± 0.48 ASE/mL) and chelating activity (IC50 = 0.801 ± 0.007 mg/mL). A total protection against H2O2-induced damage on E. coli was observed. No toxicity against A. salina was found (LC50 > 1000 µg/mL). The extract exhibited bacteriostatic activity against almost all the bacteria tested (MICs: 250-62.5 µg/mL). CONCLUSIONS: The obtained results demonstrate the potential of F. vasta leaves as safe sources of natural antioxidant and antimicrobial compounds.

Efficacy of poly(lactic acid)/carvacrol electrospun membranes against Staphylococcus aureus and Candida albicans in single and mixed cultures.

Carvacrol (CAR) is one of the most promising essential oil components with antimicrobial activity. New technologies aimed to incorporate this active molecule into carrier matrix to improve the stability and prolong the biological activity. The goal of this study was to investigate the feasibility of incorporating CAR into electrospun membranes of poly(lactic acid) (PLA) for potential applications as active antimicrobial system. To this end, PLA membranes containing homogeneously dispersed CAR were successfully prepared and a series of systematic tests including morpho-mechanical properties, in vitro release rate, and antimicrobial/antibiofilm activities against Staphylococcus aureus and Candida albicans were carried out. The results revealed that CAR has a good compatibility with PLA and acts as a plasticizer, improving flexibility and extensibility of the matrix. The gradual release of CAR from PLA membranes warranted a significant antimicrobial activity up to 144 h and reduced the biofilm production by 92-96 and 88-95% of S. aureus and C. albicans in single and mixed cultures. A strong decrease of cell count, biomass, metabolic activity, and vitality of established 24- and 48-h biofilms were also demonstrated. In conclusion, this work highlights the potential of electrospun nanofibrous membranes as efficient stabilizers-carriers of CAR and opens up interesting perspectives on the use of this system as new tool for skin and wound bacterial-fungal infections.

Design, Synthesis, and Antibacterial Activity of a Multivalent Polycationic Calix[4]arene-NO Photodonor Conjugate.

The role of nitric oxide (NO) as an antimicrobial and anticancer agent continues to stimulate the search of compounds generating NO in a controlled fashion. Photochemical generators of NO are particularly appealing due to the accurate spatiotemporal control that light-triggering offers. This contribution reports a novel molecular construct in which multiple units of 3-(trifluoromethyl)-4-nitrobenzenamine NO photodonor are clustered and spatially organized by covalent linkage to a calix[4]arene scaffold bearing two quaternary ammonium groups at the lower rim. This multivalent calix[4]arene-NO donor conjugate is soluble in hydro-alcoholic solvent where it forms nanoaggregates able to release NO under the exclusive control of visible light inputs. The light-stimulated antibacterial activity of the nanoconstruct is demonstrated by the effective bacterial load reduction of Gram-positive Staphylococcus aureus ATCC 6538 and Gram-negative Escherichia coli ATCC 10536.

Efficacy of a Fixed Combination of Tetracycline, Chloramphenicol, and Colistimethate Sodium for Treatment of Candida albicans Keratitis.

Purpose: To evaluate the antifungal activity of a fixed antibiotic combination (AC) containing tetracycline (TET), chloramphenicol (CAF), and colistimethate sodium (CS). Methods: In vitro: Candida ATCC and clinical strains were used. The minimum inhibitory concentrations (MICs) of AC and of each antibiotic were determined. Fluconazole (FLC) was tested for comparison. Time-killing curves of selected strains were performed. Ex vivo keratitis: corneas were injected intrastromally with the selected strains. After the injection, corneas were divided into groups of treatments: AC, FLC, or saline. Then, the tissues were analyzed for colony-forming units per gram (CFU/g). Propidium iodide (PI) and MitoTracker (MTR) staining were used to investigate the mode of action. Results: Values of MIC required to inhibit the growth of 90% of organisms for the antibiotics alone were higher than FLC. However, their activity was enhanced when used in combination against Candida yeasts. Time-killing curves showed that at 24 hours, AC reduced the load of both strains of approximately 1 Log10 CFU/g compared with the initial inoculum (P < 0.0001). This effect was also significant versus FLC. In ex vivo, AC was effective in decreasing the loads of both strains by 4 Log10 CFU/g with respect to the control. Moreover, it showed higher activity than FLC against Candida albicans ATCC 10231 (1 Log10 CFU/g, P < 0.01 versus control). PI staining demonstrated that CS changed the membrane's permeability, whereas MTR staining demonstrated that TET or CAF altered mitochondrial function. The cells treated with AC and stained showed both effects. Conclusions: In this study, AC showed antifungal efficacy versus Candida spp.; this activity can be due to the synergistic effects of antibiotics in it.

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.

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.

Phytocomplexes from liquorice (Glycyrrhiza glabra L.) leaves--chemical characterization and evaluation of their antioxidant, anti-genotoxic and anti-inflammatory activity.

Three extracts of different polarities of Glycyrrhiza glabra L. leaves were characterized and evaluated for their antioxidant, anti-genotoxic and anti-inflammatory activity. In total, thirty components have been identified and quantified through the use of liquid chromatography (LC) with ultraviolet-visible diode-array-detector (UV-vis-DAD) and mass spectrometry (MS). The main components belong to the polyphenols family, being flavonoid and dihydrostilbene derivatives. The extracts have been investigated for their antioxidant, anti-genotoxic and anti-inflammatory activities, which are fundamental requirements of efficacious chemo-preventive agents. The ethyl acetate extract proved to be the most valuable, evidently for the conspicuous presence of several polyphenols, namely flavonoids and dihydrostilbenes.

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.

Synergism and postantibiotic effect of tobramycin and Melaleuca alternifolia (tea tree) oil against Staphylococcus aureus and Escherichia coli.

The application of antimicrobial combinations may address the rising resistance to established classes of both systemic and topical agents and their clinical relevance is related to the presence of a significant postantibiotic effect (PAE). We investigated the effectiveness in vitro of the association between tobramycin and tea tree oil (TTO) against Gram-positive and Gram-negative bacteria. The minimal inhibitory concentrations, the bacterial killing and the PAE of tobramycin and TTO were determined both singly and in combination against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213. A synergistic interaction was observed against both strains tested: the mean PAEs were 1.3 and 1.7h for tobramycin against E. coli and S. aureus respectively, 10.8h for tobramycin and TTO (0.05%) against E. coli, 10.4h and 17.4h against S. aureus for tobramycin and TTO (0.25 and 0.50%, respectively). Longer PASMEs were observed with S. aureus after TTO/tobramycin exposure. In vitro interactions can improve the antimicrobial effectiveness of the antibiotic and may contribute for the development of novel topical agents for the treatment of skin lesions including conjunctiva and respiratory infections by inhalation.

Chemical composition and antimicrobial activity of Momordica charantia seed essential oil.

The essential oil obtained from the seeds of Momordica charantia was analyzed by GC/MS. Twenty-five components, representing 90.9% of the oil, were identified. The main constituents were trans-nerolidol, apiole, cis-dihydrocarveol and germacrene D. Furthermore, the oil was tested for its antibacterial and antifungal activities. Staphylococcus aureus was found to be the most sensitive microorganism with MIC values <500 microg/ml.

Interaction of four monoterpenes contained in essential oils with model membranes: implications for their antibacterial activity.

The present article reports the antimicrobial efficacy of four monoterpenes (thymol, carvacrol, p-cymene, and gamma-terpinene) against the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Escherichia coli. For a better understanding of their mechanism of action, the damage caused by these four monoterpenes on biomembranes was evaluated by monitoring the release, following exposure to the compounds under study, of the water-soluble fluorescent marker carboxyfluorescein (CF) from large unilamellar vesicles (LUVs) with different lipidic composition (phosphatidylcholine, PC, phosphatidylcholine/phosphatidylserine, PC/PS, 9:1; phosphatidylcholine/stearylamine, PC/SA, 9:1). Furthermore, the interaction of these terpenes with dimyristoylphosphatidylcholine multilamellar vesicles as model membranes was monitored by means of differential scanning calorimetry (DSC) technique. Finally, the results were related also with the relative lipophilicity and water solubility of the compounds examined. We observed that thymol is considerably more toxic against S. aureus than the other three terpenes, while carvacrol and p-cymene are the most inhibitory against E. coli. Thymol and carvacrol, but not gamma-terpinene and p-cymene, caused a concentration-dependent CF leakage from all kinds of LUVs employed; in particular, thymol was more effective on PC and PC/SA LUVS than on PC/PS vesicles, while carvacrol challenge evoked a CF leakage from PC/PS LUVs similar to that induced from PC/SA LUVs, and lower than that measured with PC vesicles. Concerning DSC experiments, these four terpenes caused a decrease in Tm and (especially carvacrol and p-cymene) DeltaH values, very likely acting as substitutional impurities. Taken together, our findings lead us to speculate that the antimicrobial effect of thymol, carvacrol, p-cymene, and gamma-terpinene may result, partially at least, from a gross perturbation of the lipidic fraction of the plasmic membrane of the microorganism. In addition to being related to the physicochemical characteristics of the compounds (such as lipophilicity and water solubility), this effect seems to be dependent on the lipidic composition and net surface charge of the microbic membranes. Furthermore, the compounds might cross the cell membranes, thus penetrating into the interior of the cell and interacting with intracellular sites critical for antibacterial activity.