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This study was meant to determine the inhibitory activity of tannins and flavonoid compounds from Geranium robertianum, Helleborus purpurascens and Hyssopus officinale plant polyphenol rich extracts against urease and a-chymotrypsin. The G. robertianum, H. purpurascens and H. officinale extracts were purified and concentrated by microfiltration and ultrafiltration. Phenolic compounds including flavonoids and tannins have been linked to many pharmacological activities. Thus, the polyphenolic content of the extracts was assessed by UV-Vis spectroscopy and HPLC. The concentrated extracts enriched in polyphenolic compounds (flavonoids, tannins and phenolic acids) showed a significant inhibition against urease from jack bean (over 90%), whereas in case of the a-chymotrypsin, they proved to have an inhibition below 54%. The results of this support the use of G. robertianum, H. purpurascens and H. officinale polyphenolic extracts as potential sources of urease inhibitors. Among the three plant extracts tested, H. officinale polyphenolic extracts exhibited a high inhibitory activity (92.67%) against urease and low inhibition (19.6%) against a-chymotrypsin and could be considered as possible remedy in ulcer treatment.

A novel aptamer and surface plasmon resonance (SPR)-based sensor was developed for the label-free detection of lysozyme. The aptasensor is characterised by a detection limit of 1 μg mL(-1) and a linear range of 5-50 μg mL(-1). As an application, we examined the usefulness of the aptasensor for monitoring the early stages of the aggregation of lysozyme. It was surprisingly found that, despite a significant decrease in monomer content during aggregation, the response of the aptasensor for protein solutions aged for 12 hours was similar to that for the fresh protein. To correlate the results obtained with the aptasensor with the composition of lysozyme solutions at various time points, we examined them in detail by atomic force microscopy (AFM), thioflavin T fluorescence, size-exclusion chromatography (SEC) and Matrix Assisted Laser Desorption Ionisation Time of Flight Mass Spectrometry (MALDI-TOF-MS). All methods together indicated that during the initial hours of aggregation, the protein solutions contained small lysozyme oligomers (mainly dimers) and decreasing amounts of monomers. Our results thus suggest that the aptamer also recognizes lysozyme dimers/oligomers. A higher non-specific binding was observed for the aggregated lysozyme at the surface of the aptasensor as compared to the native protein. This was attributed to the hydrophobic patches which are exposed by the unfolded lysozyme and/or oligomer species, allowing for different adsorption and organisation at the surface of the aptasensor. This hypothesis is supported by square wave voltammetry (SWV) studies using solutions of aggregated lysozyme. A higher electrochemical signal due to the direct oxidation of tyrosine/tryptophan residues was observed for aged protein solutions as compared to the fresh solution, indicative of an increased number of such exposed electroactive residues and of overall increased surface hydrophobicity of the protein. Our work presents a label-free lysozyme aptasensor that is useful not only for the detection of the protein monomer but also for observing the onset of aggregation. The approach can be extended to other proteins which are prone to aggregation.

The association of magnetic nanoparticles, which could be controlled by a magnetic field and have dimensions which facilitate their penetration in cells/tissues, with hydrogel type biopolymeric shells confer them compatibility and the capacity to retain and deliver bioactive substances. The main objective of this work is the development of a new system based on a biocompatible polymer with organic-inorganic structure capable of vectoring support for biologic active agents (L: -asparaginase, e.g.). Characterization of size and morphology of the hydrogel-magnetic nanoparticles with entrapped L: -asparaginase was made using Dynamic Light Scattering method, Transmission Electron Microscopy and Confocal Microscopy. The structure of magnetic nanoparticles coated with hydrogel was characterized by Fourier Transformed Infrared Spectroscopy. The cytotoxicity of nanoparticles was evaluated and also the interactions with microorganisms. We obtained hydrogel-magnetic nanoparticles with L: -asparaginase entrapped, with sizes below 30 nm in dried stage, capable to penetrate the cells and tissues.

Context: Verbascum phlomoides L. (Scrophulariaceae) (mullein) used in the European folk medicine due to its anti-inflammatory and soothing action on the respiratory tract is thoroughly documented in handbooks and scientific literature. Nevertheless, information regarding the influence of the polyphenol content on pharmacological activity is scarce. Objective: This study explored the antioxidant and anti-inflammatory potential of V. phlomoides polyphenol-rich extract. Materials and methods: Dried mullein flowers (200 g) were subjected to water extraction (60 C, 2 h, herb/solvent ratio ¼ 1/10 m/v) and further to n-butanol partition. Total phenolics were spectrophotometrically determined and specific compounds were evaluated by HPLC. The antioxidant activity was assessed by the 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) assay. The anti-inflammatory potential of the extract (50-200 mg/mL) was evaluated in vitro by ELISA measurement of ICAM-1 expression in TNF-a-stimulated endothelial cells and in vivo by the rat paw edema assay. Results: The mullein extract contained 4.18% total polyphenols expressed as gallic acid. The main components identified by HPLC were: rosmarinic acid (14.93 mg/g), caffeic acid (39.96 mg/g), ferulic acid (29.61 mg/g) and quercetin (17.29 mg/g). Acteoside was not detected; aucubin was detected in traces (0.028 mg/g). Depending on concentration, the extract exerted scavenging activity on DPPH radical (EC 50 7.09 mg/mL), significantly inhibited TNF-a-induced ICAM-1 expression by 55-58.8% on human umbilical vein endothelial cells at 100 and 200 mg/mL, but failed to reduce egg-white-induced rat paw edema. Discussion and conclusion: Mullein polyphenols play an important role in exerting the antioxidant effect but have a weak influence on anti-inflammatory activity that is correlated, probably, to a higher content of iridoids and phenylethanoids.