Benzodiazepine Interference with Fertility and Embryo Development: A Preliminary Survey in the Sea Urchin Paracentrotus lividus.

Psychotropic drugs and benzodiazepines are nowadays among the primary substances of abuse. This results in a large and constant release into aquatic environments where they have potentially harmful effects on non-target organisms and, eventually, human health. In the last decades, evidence has been collected on the possible interference of benzodiazepines with reproductive processes, but data are few and incomplete. In this study, the possible negative influence of delorazepam on fertilization and embryo development has been tested in Paracentrotus lividus, a key model organism in studies of reproduction and embryonic development. Sperm, eggs, or fertilized eggs have been exposed to delorazepam at three concentrations: 1 µg/L (environmentally realistic), 5 µg/L, and 10 µg/L. Results indicate that delorazepam reduces the fertilizing capacity of male and female gametes and interferes with fertilization and embryo development. Exposure causes anatomical anomalies in plutei, accelerates/delays development, and alters the presence and distribution of glycoconjugates such as N-Acetyl-glucosamine, α-linked fucose, and α-linked mannose in both morulae and plutei. These results should attract attention to the reproductive fitness of aquatic species exposed to benzodiazepines and pave the way for further investigation of the effects they may exert on human fertility. The presence of benzodiazepines in the aquatic environment raises concerns about the reproductive well-being of aquatic species. Additionally, it prompts worries regarding potential impacts on human fertility due to the excessive use of anxiolytics.

Antioxidant Properties of Pulp, Peel and Seeds of Phlegrean Mandarin (Citrus reticulata Blanco) at Different Stages of Fruit Ripening.

In this work, we assess the potential of waste products of Phlegrean mandarin (Citrus reticulata Blanco), namely seeds and peel, to be reutilized as a source of bioactive compounds beneficial for the human diet. Starting from the evidence that the by-products of this specific cultivar are the most powerful sources of antioxidants compared to pulp, we have investigated if and how the bioactive compounds in peel and seeds may be affected by fruit ripening. Three stages of fruit ripening have been considered in our study: unripe fruits = UF, semi-ripe fruits = SRF, ripe fruits = RF. The overall results indicated that RF showed the highest concentration of antioxidants. Among fruit components, peel was the richest in total antioxidant capacity, total polyphenol content, total flavonoids, total chlorophylls and carotenoids, while seeds exhibited the highest concentration of total condensed tannins and ascorbic acid. The liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay indicates the occurrence, in peel extracts, of 28 phenolic compounds, mainly flavonoids (FLs); in seeds, 34 derivatives were present in the first stage (UF), which diminish to 24 during the ripening process. Our data indicated that the content of phytochemicals in citrus strongly varies among the fruit components and depends on the ripening stage. The higher antioxidant activity of peel and seeds, especially in RF, encourage a potential use of by-products of this specific citrus cultivar for industrial or pharmacological applications. However, to maximize the occurrence of desired bioactive compounds, it is important also to consider the ripening stage at which fruits must be collected.

Comparative Studies on Different Citrus Cultivars: A Revaluation of Waste Mandarin Components.

Peel, pulp and seed extracts of three mandarin varieties, namely Phlegraean mandarin (Citrus reticulata), Kumquat (Citrus japonica), and Clementine (Citrus clementina) were compared and characterised in terms of photosynthetic pigment content, total polyphenols amount, antioxidant activity and vitamin C to assess the amount of functional compounds for each cultivar. The highest polyphenols content was found in the Phlegraean mandarin, especially in peel and seeds, whereas Kumquat exhibited the highest polyphenols amount in the pulp. The antioxidant activity was higher in the peel of Phlegraean mandarin and clementine compared to Kumquat, which showed the highest value in the pulp. The antioxidant activity peaked in the seeds of Phlegraean mandarin. The vitamin C in the Phlegraean mandarin was the highest in all parts of the fruit, especially in the seeds. Total chlorophyll content was comparable in the peel of different cultivars, in the pulp the highest amount was found in clementine, whereas kumquat seeds showed the greatest values. As regards total carotenoids, peel and pulp of clementine exhibited higher values than the other two cultivars, whereas the kumquat seeds were the richest in carotenoids. Among the analysed cultivars Phlegraean mandarin may be considered the most promising as a source of polyphenols and antioxidants, compared to the clementine and Kumquat, especially for the functional molecules found in the seeds. Moreover, regardless of cultivars this study also highlights important properties in the parts of the fruit generally considered wastes.

Allium ursinum and Allium oschaninii against Klebsiella pneumoniae and Candida albicans Mono- and Polymicrobic Biofilms in In Vitro Static and Dynamic Models.

The present study assesses the in vitro antibiofilm potential activity of extracts of wild Allium ursinum and Allium oschaninii. The active ingredients of the extracts were obtained with a technique named Naviglio (rapid solid-liquid dynamic extraction, RSLDE) which is based on an innovative and green solid-liquid extraction methodology. The extracts were tested against models of mono- and polymicrobial biofilm structures of clinically antibiotic-resistant pathogens, Klebsiella pneumoniae ATCC 10031 and Candida albicans ATCC 90028. Biofilms were studied using a static and a dynamic model (microtiter plates and a CDC reactor) on three different surfaces reproducing what happens on implantable medical devices. Antimicrobic activities were determined through minimum inhibitory concentration (MIC), while antibiofilm activity was assessed by minimum biofilm eradication concentration (MBEC) using a crystal violet (CV) biofilm assay and colony forming unit (CFU) counts. Results showed that both Allium extracts eradicated biofilms of the tested microorganisms well; biofilms on Teflon were more susceptible to extracts than those on polypropylene and polycarbonate, suggesting that when grown on a complex substrate, biofilms may be more tolerant to antibiotics. Our data provide significant advances on antibiotic susceptibility testing of biofilms grown on biologically relevant materials for future in vitro and in vivo applications.

Rapid Solid-Liquid Dynamic Extraction (RSLDE): A Powerful and Greener Alternative to the Latest Solid-Liquid Extraction Techniques.

Traditionally, solid-liquid extractions are performed using organic and/or inorganic liquids and their mixtures as extractant solvents in contact with an insoluble solid matrix (e.g., the Soxhlet method) or using sequential atmospheric pressure systems that require long procedures, such as maceration or percolation. The objective of this procedure is the extraction of any compounds that can be carried out from the inner solid material to the outlet, resulting in a solution containing colorants, bioactive compounds, odorous substances, etc. Over the years, in the extraction techniques sector, there have been many important changes from the points of view of production, quality, and human and environmental safety due to improvements in technology. In more recent times, the interest of the scientific community has been aimed at the study of sustainable processes for the valorization of extracts from vegetables and food by-products, through the use of non-conventional (innovative) technologies that represent a valid alternative to conventional methods, generally through saving time and energy and the formation of fewer by-products. Therefore, with the development of principles based on the prevention of pollution, on a lower risk for human health, and on a low environmental impact, new systems have been implemented to reduce extraction times and solvent consumption, to improve efficiency, and to increase the productivity of the extracts. From this point of view, rapid solid-liquid dynamic extraction (RSLDE), performed using the Naviglio extractor, compared to traditional applications, is a technique that is able to reduce extraction times, generally leads to higher yields, does not require heating of the system, allows one to extract the active ingredients, and avoids their degradation. This technique is based on a new solid-liquid extraction principle named Naviglio's principle. In this review, after reviewing the latest extraction techniques, an overview of RSLDE applications in various research and production sectors over the past two decades is provided.

Iron (II) Citrate Complex as a Food Supplement: Synthesis, Characterization and Complex Stability.

Iron deficiency represents a widespread problem for a large part of the population, especially for women, and has received increasing attention in food/supplement research. The contraindications of the iron supplements commercially available (e.g., imbalances in the levels of other essential nutrients, low bioavailability, etc.) led us to search for a possible alternative. In the present work, a rapid and easy method to synthetize a solid iron (II) citrate complex from iron filings and citric acid was developed to serve, eventually, as a food supplement or additive. In order to state its atomic composition and purity, an assortment of analytical techniques was employed (e.g., combustion analysis, thermogravimetry, X-ray diffractometry, UV/Vis spectrophotometry, etc.). Results demonstrate that the synthesized crystalline solid corresponds to the formula FeC6H6O7∙H2O and, by consequence, contains exclusively iron (II), which is an advantage with respect to existing commercial products, because iron (II) is better absorbed than iron (III) (high bioavailability of iron).

Determination of Egg Number Added to Special Pasta by Means of Cholesterol Contained in Extracted Fat Using GC-FID.

Pasta with eggs added (generally termed "special pasta" for Italian legislation) is made by adding no less than 4 eggs without shells (or no less than 200 g of liquid or lyophilized egg product) per kilogram of semolina, as provided by law. In this work, to determine the final content of eggs added to dough, an analytical procedure was developed for the rapid analysis of the cholesterol content in the finished pastas. The proposed procedure was simpler, faster, and more accurate than that of official methods of analysis based on the gravimetric determination of sterols. Moreover, the determination of the quality of fat content in the special pasta (egg pasta in this case) allowed the evaluation of its origin, avoiding possible fraud resulting from the addition of foreign fat as an alternative to fat derived from eggs. In this new gas chromatographic procedure, the internal standard squalene for the quantification of cholesterol was used because a more polar GC capillary column was used (RTX 65 TG-HT) for the separation of sterols, rather than 5% phenyl methylsilicone. The ratio between cholesterol and squalene allowed for the determination of the number of eggs added, while from analysis of the same gas chromatogram, it was also possible to evaluate the composition of triglycerides in the fat contained in the pasta, allowing discrimination of foreign fats with respect to fats contained in eggs and therefore avoiding adulteration of pasta. The same analytical procedure was applied to the determination of cholesterol content in lyophilized yolk.

The Sea Urchin Arbacia lixula: A Novel Natural Source of Astaxanthin.

Several echinoderms, including sea urchins, are valuable sources of bioactive compounds but their nutraceutical potential is largely unexplored. In fact, the gonads of some sea urchin species contain antioxidants including carotenoids and polyhydroxylated naphthoquinones (PHNQ's), such as echinochrome A. Astaxanthin is known to have particular bioactivity for the prevention of neurodegenerative diseases. This carotenoid is produced by microalgae, while several marine invertebrates can bioaccumulate or synthetize it from metabolic precursors. We determined the carotenoid content and analyzed the bioactivity potential of non-harvested Atlantic-Mediterranean sea urchin Arbacia lixula. The comparison of methanol crude extracts obtained from eggs of farmed and wild specimens revealed a higher bioactivity in farmed individuals fed with a customized fodder. HPLC-analysis revealed a high concentration of astaxanthin (27.0 µg/mg), which was the only pigment observed. This study highlights the potential of farmed A. lixula as a new source of the active stereoisomer of astaxanthin.

Immune and reproductive system impairment in adult sea urchin exposed to nanosized ZnO via food.

In marine environment the release and the consequent sedimentation of ZnO NPs, mainly used in sunscreens, could provoke toxic effects in particular in grazer organisms, like sea urchins. In this work, a first evaluation of DNA and cellular effects on adult sea urchins Paracentrotus lividus exposed through the diet to different sizes (100 and 14nm) ZnO NPs, was performed. Moreover, the consequent impact upon offspring quality was evaluated. Preliminarily results showed that the assumption of food containing ZnO NPs 100nm provoked in adult echinoids damages to immune cells (33% of damaged nucleus) and transmissible effects to offspring (75.5% of malformed larvae). Instead food with ZnO NPs 14nm provoked 64% of damaged nucleus in immune cells and 84.7% of malformed larvae.

Rapid Solid-Liquid Dynamic Extraction (RSLDE): a New Rapid and Greener Method for Extracting Two Steviol Glycosides (Stevioside and Rebaudioside A) from Stevia Leaves.

Stevioside and rebaudioside A are the main diterpene glycosides present in the leaves of the Stevia rebaudiana plant, which is used in the production of foods and low-calorie beverages. The difficulties associated with their extraction and purification are currently a problem for the food processing industries. The objective of this study was to develop an effective and economically viable method to obtain a high-quality product while trying to overcome the disadvantages derived from the conventional transformation processes. For this reason, extractions were carried out using a conventional maceration (CM) and a cyclically pressurized extraction known as rapid solid-liquid dynamic extraction (RSLDE) by the Naviglio extractor (NE). After only 20 min of extraction using the NE, a quantity of rebaudioside A and stevioside equal to 1197.8 and 413.6 mg/L was obtained, respectively, while for the CM, the optimum time was 90 min. From the results, it can be stated that the extraction process by NE and its subsequent purification developed in this study is a simple, economical, environmentally friendly method for producing steviol glycosides. Therefore, this method constitutes a valid alternative to conventional extraction by reducing the extraction time and the consumption of toxic solvents and favouring the use of the extracted metabolites as food additives and/or nutraceuticals. As an added value and of local interest, the experiment was carried out on stevia leaves from the Benevento area (Italy), where a high content of rebaudioside A was observed, which exhibits a sweet taste compared to stevioside, which has a significant bitter aftertaste.