Utilization of Olive Pomace in Green Synthesis of Selenium Nanoparticles: Physico-Chemical Characterization, Bioaccessibility and Biocompatibility.

Olive pomace extract (OPE) was investigated as a potential surface modifier for the development of the green synthesis process of selenium nanoparticles (SeNPs). In order to evaluate them as potential nutraceuticals, the obtained nanosystems were characterized in terms of size distribution, shape, zeta potential, stability in different media, gastrointestinal bioaccessibility and biocompatibility. Systems with a unimodal size distribution of spherical particles were obtained, with average diameters ranging from 53.3 nm to 181.7 nm, depending on the type of coating agent used and the presence of OPE in the reaction mixture. The nanosystems were significantly affected by the gastrointestinal conditions. Bioaccessibility ranged from 33.57% to 56.93% and it was significantly increased by functionalization of with OPE. Biocompatibility was investigated in the HepG2 and Caco2 cell models, proving that they had significantly lower toxicity in comparison to sodium selenite. Significant differences were observed in cellular responses depending on the type of cells used, indicating differences in the mechanisms of toxicity induced by SeNPs. The obtained results provide new insight into the possibilities for the utilization of valuable food-waste extracts in the sustainable development of nanonutraceuticals.

Cytokinin response in pepper plants (Capsicum annuum L.) exposed to silver nanoparticles.

The increasing development of different nanomaterials, such as silver nanoparticles (AgNPs), and their practical use in agriculture and biotechnology has created a strong need for elucidations of biological effects and risk assessments of AgNPs in plants. This study was aimed to investigate AgNPs effects on metal uptake and their biodistribution in pepper plants as well as on morphological parameters and hormonal responses of the isoprenoid cytokinin (CK) family. In addition, the comparison of effects silver form, nanoparticles vs. ionic, has also been examined. To the best of our knowledge, this is the first study describing CK responses in plants exposed to metallic NPs. The obtained results indicate that both AgNPs and Ag+ ions significantly increased total content of Ag+ in pepper tissues in a dose-dependent manner and affected on plant development by decreasing both plant height and biomass in a similar way. This study evidenced for the first time the role of CKs in abiotic stress in plants caused by AgNPs. The hormonal analysis, conducted by an ultra-high performance liquid chromatography-electrospray tandem mass spectrometry, revealed a significant increase in total CKs in the leaves and also highlighted the importance of cis-zeatin type CKs in plants treated with AgNPs. Our observations suggest potential risks of AgNPs on plant ecosystems upon their release into the environment.

Functionalization of selenium nanoparticles with olive polyphenols - impact on toxicity and antioxidative activity.

Selenium nanoparticles (SeNPs) represent novel selenium (Se) formulation characterized by improved biocompatibility and a wider therapeutic range in comparison to inorganic Se. The aim of this work was to investigate the possibilities of functionalization of SeNPs with olive pomace extract (OPE), rich in health-promoting polyphenols, and to obtain innovative forms of nutraceuticals. Cytotoxic and antioxidative activities of four types of SeNPs (polyvinylpyrrolidone stabilized (PVP SeNPs), polysorbate stabilized (PS SeNPs), polyvinylpyrrolidone stabilized and functionalized using OPE (f PVP SeNPs) and polysorbate stabilized and functionalized using OPE (f PS SeNPs) were investigated. SeNPs showed lower toxicity on human hepatocellular carcinoma (HepG2) and human colorectal adenocarcinoma (Caco2) cells compared to selenite. Functionalization with polyphenols significantly improved their direct antiradical (f PVP SeNPs: 24.4 ± 1.84 and f PS SeNPs: 30.9 ± 2.47 mg TE/mmol Se) and reducing properties (f PVP SeNPs: 50 ± 3.16 and f PS SeNPs: 53.6 ± 3.22 mg GAE/mmol) compared to non-functionalized SeNPs. The significant impact of tested SeNPs on intracellular antioxidative mechanisms has been observed and it was dependent on both cell type and physico-chemical properties of SeNPs, indicating the complexity of involved mechanisms.

Phyto-Assisted Synthesis of Nanoselenium-Surface Modification and Stabilization by Polyphenols and Pectins Derived from Agricultural Wastes.

Raw and purified mandarin peel-derived pectins were characterized and combined with olive pomace extract (OPE) in the green synthesis of selenium nanoparticles (SeNPs). SeNPs were characterized in terms of size distribution and zeta potential, and their stability was monitored during 30 days of storage. HepG2 and Caco-2 cell models were used for the assessment of biocompatibility, while antioxidant activity was investigated by the combination of chemical and cellular-based assays. SeNP average diameters ranged from 171.3 nm up to 216.9 nm; smaller SeNPs were obtained by the utilization of purified pectins, and functionalization with OPE slightly increased the average. At concentrations of 15 mg/L SeNPs were found to be biocompatible, and their toxicity was significantly lower in comparison to inorganic selenium forms. Functionalization of SeNPs with OPE increased their antioxidant activity in chemical models. The effect was not clear in cell-based models, even though all investigated SeNPs improved cell viability and protected intracellular reduced GSH under induced oxidative stress conditions in both investigated cell lines. Exposure of cell lines to SeNPs did not prevent ROS formation after exposure to prooxidant, probably due to low transepithelial permeability. Future studies should focus on further improving the bioavailability/permeability of SeNPs and enhancing the utilization of easily available secondary raw materials in the process of phyto-mediated SeNP synthesis.

Effect of natural biostimulants on yield and nutritional quality: an example of sweet yellow pepper (Capsicum annuum L.) plants.

BACKGROUND: Modifications in growing techniques can affect the yield and nutritional quality of various cultivated plant species. Owing to its high nutritional value, pepper (Capsicum annuum L.) was used in this study as a model plant to investigate the effect of natural biostimulants on yield and fruit quality parameters under conditions of reduced fertilisation. RESULTS: A positive influence of biostimulant treatment on yield parameters was observed. The overall increase in the pigment content of leaves after biostimulant application agreed well with the higher total and commercial yields of treated pepper cultivars compared with their controls. The results showed that natural biostimulants had a positive effect on the vitamin C and total phenolic contents in pepper fruits during the hot summer season. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) antioxidant activities were also significantly higher (P < 0.05) in treated plants and correlated strongly with all measured quality parameters except total phenolic content. CONCLUSION: Generally, biostimulants improved the antioxidant activity, vitamin C and phenolic contents in fruits as well as the pigment content in leaves of treated compared with non-treated pepper plants grown hydroponically. Thus the application of biostimulants could be considered as a good production strategy for obtaining high yields of nutritionally valuable vegetables with lower impact on the environment.

Assessment of the influence of different sample processing and cold storage duration on plant free proline content analyses.

INTRODUCTION: A method which is widely accepted for the analysis of free proline content in plant tissues is based on the use of 3% sulfosalicylic acid as an extractant, followed by spectrophotometric quantification of a proline-ninhydrin complex in toluene. However, sample preparation and storage may influence the proline actually measured. This may give misleading or difficult to compare data. OBJECTIVE AND METHODOLOGY: To evaluate free proline levels fresh and frozen strawberry (Fragaria × ananassa Duch.) leaves and soybean [Glycine max (L.) Merr.] hypocotyl tissues were used. These were ground with or without liquid nitrogen and proline extracted with sulfosalicylic acid. A particular focus was the influence of plant sample cold storage duration (1, 4 and 12 weeks at -20°C) on tissue proline levels measured. RESULTS: The free proline content analyses, carried out in leaves of Fragaria × ananassa Duch. as well as in hypocotyls of Glycine max (L.) Merr., showed a significant influence of the sample preparation method and cold storage period. Long-term storage of up to 12 weeks at -20°C led to a significant increase in the measured proline in all samples analysed. CONCLUSION: The observed changes in proline content in plant tissue samples stored at -20°C indicate the likelihood of the over-estimation of the proline content if the proline analyses are delayed. Plant sample processing and cold storage duration seem to have an important influence on results of proline analyses. Therefore it is recommended that samples should be ground fresh and analysed immediately.

Antifungal activities of silver and selenium nanoparticles stabilized with different surface coating agents.

BACKGROUND: Extensive and growing use of different chemical pesticides that affect both the environment and human health raises a need for new and more suitable methods to deal with plant pathogens. Nanotechnology has enabled the use of materials at the nanoscale with exceptional functionality in different economic domains including agricultural production. This study aimed to evaluate antifungal potential of selenium nanoparticles (SeNPs) and silver nanoparticles (AgNPs) stabilized with different surface coatings and characterized by different surface charge on plant pathogenic fungi Macrophomina phaseolina, Sclerotinia sclerotiorum and Diaporthe longicolla. RESULTS: AgNPs were coated with three different stabilizing agents: mono citrate (MC-AgNPs), cetyltrimethyl ammonium bromide (CTAB-AgNPs) and polyvinylpyrrolidon (PVP-AgNPs). SeNPs were coated with poly-l-lysine (PLL-SeNPs), polyacrylic acid (PAA-SeNPs), and polyvinylpyrrolidon (PVP-SeNPs). Seven different concentrations (0.1, 0.5, 1, 5, 10, 50 and 100 mg L-1 ) of nanoparticles were applied. All AgNPs and SeNPs significantly inhibited the growth of the tested fungi. Among the tested NPs, PVP-AgNPs showed the best inhibitory effect on the tested plant pathogenic fungi, especially against S. sclerotiorum. The similar inhibition of the sclerotia formation was observed for S. sclerotiorum treated with PLL-SeNPs. CONCLUSION: Obtained results provides new insights on fungicide effect of AgNPs and SeNPs stabilized with different coating agents on different plant pathogens. Further work should focus on detailed risk/benefit ratio assessment of using SeNPs or AgNPs in agriculture taking into account whole agroecosystem. © 2020 Society of Chemical Industry.

Superior protective effects of in vitro propagated green garlic against hydrogen peroxide-induced cytotoxicity in human hepatoma cells.

Garlic is a valuable source material for medicines due to its known antitumor, hypolipidaemic, antioxidant, and immunomodulatory effects. This study compares the protective effects of conventionally grown (CG) and in vitro propagated garlic (PG) against hydrogen peroxide-induced cytotoxicity in HepG2 cells and their antioxidant activity. Garlic used in this study was obtained by planting garlic cloves or by planting the transplants of PG directly in the field. At the end of the vegetation period, CG and PG were sampled and extracts prepared for the experiment. Compared to conventionally grown garlic bulbs, PG leafy part yielded significantly higher content of polyphenols, flavonoids and alliin, and also showed equal or higher antioxidant activity, measured by the cell viability test, GSH and ROS level. Moreover, PG can be produced in less time (shorter vegetation period) and with significantly less material (cloves). Significantly higher content of alliin, polyphenols, and flavonoids and significantly higher yield of plant biomass in PG has a great potential to become a new production model with improved garlic properties as a medicine material.

Stability and toxicity of differently coated selenium nanoparticles under model environmental exposure settings.

This study, motivated to fill the knowledge gap on environmental safety of selenium nanoparticles (SeNPs), provides information on the stability and environmental safety of four differently coated SeNPs rendering both positive and negative surface charges. The stability and dissolution behaviour of SeNPs were determined in an aquatic model media of different ionic strength to provide information regarding the environmental fate of SeNPs in different environmental conditions. The environmental safety of SeNPs was evaluated by acute regulatory toxicity tests using Daphina magna and Vibrio fischeri as model organisms. Agglomeration was observed for all studied SeNPs in test media with higher ionic strength caused by the disruption of surface charge leading to electrostatic instability. Toxicity of SeNPs on both aquatic species was dose-dependent and increased with exposure time. The obtained data indicated that all of the tested SeNPs could be classified as harmful to the natural bacteria V. fischeri and harmful to toxic to crustaceans D. magna, but dependent on the coating agent used for SeNPs stabilization. Although SeNPs have attracted great interest for use in biomedicine, this study demonstrated that their ecotoxicological effects should be considered during the design of new of SeNPs-based products.

[Ocular manifestations of Lyme borreliosis in northwest Croatia]. / Ocne manifestacije lajmske borelioze u sjeverozapadnoj Hrvatskoj.

Ocular manifestations in patients with Lyme borreliosis in northwest Croatia that occurred during the period between 1992 and 2001 were retrospectively analysed. The diagnosis of Lyme borreliosis was based on medical history, clinical manifestations, epidemiological data about tick contact, determinations of antibodies to Borrelia burgdorferi by serological assays, immunoblot analysis, therapeutic results and exclusion of other infectious agents of ocular disease. Of the eleven patients reported here with clinical manifestations of ocular Lyme borreliosis, 6 had chorioretinitis, 1 papillitis, 2 iridocyclitis, 1 occlusion of the arteriae centralis retinae, 1 neuritis retrobulbaris and 1 neuroretinitis. Diagnostic confirmation of LB was in most patients done by serological tests and/or by immunoblot method in serum. In the last few years we have also done culture and molecular diagnostic methods polymerase chain reaction (PCR)) in serum and cerebrospinal fluid. The patients were treated with 2 g of ceftriaxon i.v. per day for 14-21 days and/or with doxucyclin orally in a daily dose of 2 x 100 mg for 2-4 weeks. The therapeutic effect was followed up by regression of clinical symptoms (better visual acuity of the infected eye) and in changing of the specific antibody titer in serological tests. The authors emphasise the more detailed diagnostic proceedings of ocular Lyme borreliosis in patients with suspected Lyme borreliosis, an algorithm which contains data about residence in endemic area for LB, positive contact with ticks and/or the history of erythema migrans or any other Lyme borreliosis-like symptoms. For diagnostic confirmation it is necessary to use enzymeimmunoassay and immunoblot methods in serum, cerebrospinal fluid and aqueous humor, and isolation by culture or PCR can be used in the same diagnostic samples. Ocular Lyme borreliosis is an underdiagnosed disorder, because it is often unrecognised by ophthalmologists and due to weak seropositivity and seronegativity in the late ocular Lyme borreliosis. This is especially important in a highly endemic area for Lyme borreliosis like northwest Croatia, a border area of the central European andemic area for Lyme borreliosis.