The influence of copper-doped mesoporous bioactive nanospheres on the temperature rise during polymerization, polymer cross-linking density, monomer release and embryotoxicity of dental composites.

OBJECTIVES: Composites with copper-doped mesoporous bioactive nanospheres (Cu-MBGN) were developed to prevent secondary caries by imparting antimicrobial and ion-releasing/remineralizing properties. METHODS: Seven experimental composites containing 1, 5 or 10 wt% Cu-MBGN, the corresponding inert controls (silica) and bioactive controls (bioactive glass 45S5) were prepared. The temperature rise during light curing, cross-linking density by ethanol softening test, monomer elution and their potential adverse effects on the early development of zebrafish Danio rerio was investigated. RESULTS: Materials combining Cu-MBGN and silica showed the highest resistance to ethanol softening, as did the bioactive controls. Cu-MBGN composites showed significant temperature rise and reached maximum temperature in the shortest time. Bisphenol A was not detected, while bis-GMA was found only in the control materials and TEGDMA in the eluates of all materials. There was no increase in zebrafish mortality and abnormality rates during exposure to the eluates of any of the materials. CONCLUSIONS: The composite with 5 wt% Cu-MBGN combined with nanosilica fillers showed the lowest ethanol softening, indicating the polymer's highest durability and cross-linking density. Despite the TEGDMA released from all tested materials, no embryotoxic effect was observed.

Synthesis and Biological Evaluations of Granulatamide B and its Structural Analogues.

BACKGROUND: While granulatamides A and B have been previously isolated, their biological activities have been only partially examined. The aim of this study was to synthesize granulatamide B (4b), a tryptamine-derivative naturally occurring in Eunicella coral species, using the well-known procedure of Sun and Fürstner and its 12 structural analogues by modifying the side chain, which differs in length, degree of saturation as well as number and conjugation of double bonds. METHOD: The prepared library of compounds underwent comprehensive assessment for their biological activities, encompassing antioxidative, antiproliferative, and antibacterial properties, in addition to in vivo toxicity evaluation using a Zebrafish model. Compound 4i, which consists of a retinoic acid moiety, exhibited the strongest scavenging activity against ABTS radicals (IC50 = 36 ± 2 µM). In addition, 4b and some of the analogues (4a, 4c and 4i), mostly containing an unsaturated chain and conjugated double bonds, showed moderate but non-selective activity with certain IC50 values in the range of 20-40 µM. RESULT: In contrast, the analogue 4l, a derivative of alpha-linolenic acid, was the least toxic towards normal cell lines. Moreover, 4b was also highly active against Gram-positive Bacillus subtilis with an MIC of 125 µM. Nevertheless, both 4b and 4i, known for the best-observed effects, caused remarkable developmental abnormalities in the zebrafish model Danio rerio. CONCLUSION: Since modification of the side chain did not significantly alter the change in biological activities compared to the parent compound, granulatamide B (4b), the substitution of the indole ring needs to be considered. Our group is currently carrying out new syntheses focusing on the functionalization of the indole core.

Enhancement of Biomass Production of Diatom Nitzschia sp. S5 through Optimisation of Growth Medium Composition and Fed-Batch Cultivation.

The growing commercial application of microalgae in different industry sectors, including the production of bioenergy, pharmaceuticals, nutraceuticals, chemicals, feed, and food, demands large quantities of microalgal biomass with specific compositions produced at reasonable prices. Extensive studies have been carried out on the design of new and improvement of current cultivation systems and the optimisation of growth medium composition for high productivity of microalgal biomass. In this study, the concentrations of the main macronutrients, silicon, nitrogen and phosphorus, essential for the growth of diatom Nitzschia sp. S5 were optimised to obtain a high biomass concentration. The effect of main macronutrients on growth kinetics and cell composition was also studied. Silicon had the most significant effect on diatom growth during batch cultivation. The concentration of biomass increased 5.45-fold (0.49 g L-1) at 1 mM silicon concentration in modified growth medium compared to the original Guillard f/2 medium. Optimisation of silicon, nitrogen, and phosphorus quantities and ratios further increased biomass concentration. The molar ratio of Si:N:P = 7:23:1 mol:mol:mol yielded the highest biomass concentration of 0.73 g L-1. Finally, the fed-batch diatom cultivation of diatom using an optimised Guillard f/2 growth medium with four additions of concentrated macronutrient solution resulted in 1.63 g L-1 of microalgal biomass. The proteins were the most abundant macromolecules in microalgal biomass, with a lower content of carbohydrates and lipids under all studied conditions.

Bioprospecting of Five Ocimum sp. Cultivars from Croatia: New Potential for Dietary and Dermatological Application with Embryotoxicity Tests.

Ocimum basilicum L. is the most common Ocimum species, and it is used as an ornamental plant and in food condiments. This unique study examined the chemical composition and biological activities of six extracts from five basil cultivars, including their antimicrobial, antidiabetic, antilipidemic, neuroprotective, and anticollagenase activity. Moreover, their toxicological effects were studied using the zebrafish Danio rerio. Volatile components were determined using HS-SPME and GC-MS, while total polyphenols were detected using HPLC and the spectrophotometric Folin-Ciocalteu method. Spectrophotometric assays (DPPH, ABTS, ORAC, FRAP) were performed to determine antioxidant activity, collagenase inhibition, acetylcholinesterase inhibition, and pancreatic lipase inhibition. Antimicrobial activity was determined using the broth microdilution test. The study found that the biological activities of different basil cultivars varied depending on the proportion of active compounds, as determined by chemical analyses. All six basil extracts significantly inhibited α-amylase, while Purple basil extract most significantly inhibited the activity of collagenase, acetylcholinesterase, and pancreatic lipase. Purple basil and Dark Opal basil I extracts exhibited the highest antimicrobial activity, while the Dark Opal basil II extract had the most significant antioxidant potential. The findings in this study suggest that ethanolic basil extracts have the potential to be used as dietary drugs and implemented in antiaging products. This study is unique in its aims to compare the chemical composition and biological activities of basil cultivars from Croatia and to evaluate potential toxicological effects through embryotoxicity tests on zebrafish Danio rerio embryos, and it reports the first evidence of anticollagenase, antidiabetic, and antilipidemic activities for these cultivars.

Intra-Species Variations of Bioactive Compounds of Two Dictyota Species from the Adriatic Sea: Antioxidant, Antimicrobial, Dermatological, Dietary, and Neuroprotective Potential.

The marine environment has a significant impact on life on Earth. Organisms residing in it are vital for the ecosystem but also serve as an inexhaustible source of biologically active compounds. Herein, the biodiversity of two brown seaweeds, Dictyota dichotoma and Dictyota fasciola from the Adriatic Sea, was evaluated. The aim of the study was the determination of differences in compound composition while comparing their activities, including antioxidant, antimicrobial, and enzyme inhibition, in connection to human digestion, dermatology, and neurological disorders. Chemical analysis revealed several terpenoids and steroids as dominant molecules, while fucoxanthin was the main identified pigment in both algae. D. dichotoma had higher protein, carbohydrate, and pigment content. Omega-6 and omega-3 fatty acids were identified, with the highest amount of dihomo-γ-linolenic acid and α-linolenic acid in D. dichotoma. Antimicrobial testing revealed a dose-dependent inhibitory activity of methanolic fraction against Escherichia coli and Staphylococcus aureus. Moderate antioxidant activity was observed for both algae fractions, while the dietary potential was high, especially for the D. fasciola dichloromethane fraction, with inhibition percentages of around 92% for α-amylase and 57% for pancreatic lipase at 0.25 mg/mL. These results suggest that Dictyota species might be a potent source of naturally derived agents for obesity and diabetes.

Effect of seasonal temperature shifts on body homeostasis, biochemical parameters and chemical composition of green ormer (Haliotis tuberculata) in the Northern Adriatic.

Seasonal responses of green ormer in terms of antioxidant capacity and lipid peroxidation, proximate and fatty acid tissue composition, trace and macro elements concentrations over the seasons were studied in relation to temperature shifts in the Northern Adriatic Sea. Overall antioxidative defenses (SOD, TBARS, TAS, LDH) varied significantly (p < 0.001) according to seasons (primarily spring and summer). The proportions of overall SFA were highest in summer. The proportions of MUFA increased in autumn, with significant differences between genders in spring and summer, and spring, summer and autumn for C18:1n7 and C20:4n6. The only fatty acid lacking significant variation between seasons was C22:5n3. Significant overall differences were observed in summer vs. winter samples for As, Ba, Co, Ni, Mn, Pb, Sb, and Se content in soft tissues, however, gender variations were not significant. The data obtained in the study are of utmost importance for the management of this under-investigated species.

Fish liver damage related to the wastewater treatment plant effluents.

Wastewater treatment plants (WWTPs) continuously release a complex mixture of municipal, hospital, industrial, and runoff chemicals into the aquatic environment. These contaminants are both legacy contaminants and emerging-concern contaminants, affecting all tissues in a fish body, particularly the liver. The fish liver is the principal detoxifying organ and effects of consistent pollutant exposure can be evident on its cellular and tissue level. The objective of this paper is thus to provide an in-depth analysis of the WWTP contaminants' impact on the fish liver structure, physiology, and metabolism. The paper also gives an overview of the fish liver biotransformation enzymes, antioxidant enzymes, and non-enzymatic antioxidants, their role in metabolizing xenobiotic compounds and coping with oxidative damage. Emphasis has been placed on highlighting the vulnerability of fish to xenobiotic compounds, and on biomonitoring of exposed fish, generally involving observation of biomarkers in caged or native fish. Furthermore, the paper systematically assesses the most common contaminants with the potential to affect fish liver tissue.

Sample preparation and culture condition effects on MALDI-TOF MS identification of bacteria: A review.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an excellent tool for bacterial identification. It allows high throughput, sensitive and specific applications in clinical diagnostics and environmental research. Currently, there is no optimal standardized protocol for sample preparation and culture conditions to profile bacteria. The performance of MALDI-TOF MS is affected by several variables, such as sample preparation, culture media and culture conditions, incubation time/growth stage, incubation temperature, high salt content, blood in the culture media, and others. This review thus aims to clarify why a uniformed protocol is not plausible, to assess the effects these factors have on MALDI-TOF MS identification score, and discuss possible optimizations for its methodology, in relation to specific bacterial representatives and strain requirements.

Estimating risk of cardiovascular pharmaceuticals in freshwaters using zebrafish embryotoxicity test - statins threat revealed.

Cardiovascular pharmaceuticals (CVPs) are globally present in inland waters and have also been found in the sediment and plasma of fish from the Sava River, Croatia. Based on the previous research, CVPs amiodarone (AMI), ramipril (RAM), simvastatin (SIM), and verapamil (VER) have been selected for this study. Their effect has been investigated, individually and in a mixture, on the development of the zebrafish embryo Danio rerio (Hamilton, 1822) within the first 96 h of development. Upon exposure to environmentally relevant concentrations of tested CVPs (0.1, 1, and 10 µg/L) zebrafish survival and development as apparent from observed morphological abnormalities, heartbeat rates and changes in behavior, hatching success, larval length and oxidative stress level were monitored. The CVP causing the highest mortality and pathological changes was SIM (1 and 10 µg/L), which corresponds well with the observed effects during zebrafish exposure to CVPs' mixtures (4 and 40 µg/L). All pharmaceuticals affected cardiac function and decreased heart rate. SIM (1 µg/L), VER and RAM (10 µg/L) decreased larval length, while induced oxidative stress was recorded in the SIM- and VER-exposed specimens. Behavioral alterations of zebrafish were observed only in AMI-treated group (10 µg/L). Our amino acid sequence comparison and structural and docking analysis showed a highly conserved binding site between human and zebrafish HMG-CoA reductase for SIM and its main metabolite simvastatin acid. Using these ecotoxicological bioassays on a zebrafish model with particular emphasis on sublethal endpoints, the risk of CVPs, especially statins, for fish in inland waters has been identified.

Identification of environmental aquatic bacteria by mass spectrometry supported by biochemical differentiation.

In this study, the purposefulness of using the API20E biochemical identification system as a supportive tool for enhancing the discrimination of environmental bacteria by MALDI-TOF MS method was evaluated. The identification results of MALDI-TOF MS and API20E for 321 Gram-negative strains isolated from the riverine freshwater and its sediment, and from the tissues of fish from the same water body were compared. Of 190 isolates identified with probable to highly probable species-level identification, and secure genus to probable species identification, 14 isolates (7.37%) had identification score over 2.300, and from the same group 19 isolates (10%) had excellent or very good identification to the genus by API20E system. With regard to agreement at genus level, out of 231 strains with genus designation available by API20E at any level of identification reliability, MALDI-TOF MS genus identification agreed in 163 (70.6%) strains. Of these, 135 (82.8%) were Aeromonas species and the remaining isolates belonged to 7 different genera. Although API20E resulted in frequent misidentification due to a limited profile index, its individual biochemical reactions might contribute to overall characterization of isolates. For example, for all reliable A. hydrophila strain identifications with MALDI-TOF MS, ONPG, GLU and OX reactions were unarguably positive for all fish and water/sediment isolates, whereas only fish isolates yielded additional 100% positive TDA and VP reactions. Thus, after initial identification with MALDI-TOF MS, environmental isolates with lower identification scores should be further analyzed. Before commencing confirmatory testing with nucleic acid-based methods, biochemical API20E tests could be applied as a purposeful and inexpensive identification support in targeting better identification accuracy. In this study, this was particularly evident with A. hydrophila, Chryseobacterium sp. and Pseudomonas sp. This identification strategy could significantly resolve methodological and cost-related shortcomings frequently occurring with large number of environmental isolates.

In Vivo and In Vitro Antioxidant Activity of Less Polar Fractions of Dasycladus vermicularis (Scopoli) Krasser 1898 and the Chemical Composition of Fractions and Macroalga Volatilome.

The present research is a comprehensive investigation of Dasycladus vermicularis (Scopoli) Krasser 1898 from the Adriatic Sea (Croatia) regarding volatilome-volatile organic compounds (VOCs, mostly nonpolar compounds) and less polar nonvolatile compounds for the first time. Headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD) were used showing the great volatilome variability among fresh (HS-FrDV and HD-FrDV) and dried (HS-DrDV and HD-DrDV) samples after GC-MS analysis. Aromatic aldehydes were dominant in both fresh and air-dried HS samples with benzaldehyde as the most abundant in fresh samples and decreasing 2.7-3.7 times after drying together with 2-phenylbut-2-enal that was not present after drying. Aliphatic compounds (unsaturated hydrocarbons in HS-FrDV; saturated hydrocarbons in HS-DrDV) were also present. C11-hydrocarbons (dictyopterpene C' and dictyopterpene D') were detected in HS-FrDV. (E)-Phytol was the most dominant compound in HD-FrDV and HD-DrDV. Diterpene alcohols (cembra-4,7,11,15-tetraen-3-ol and (Z)-falcarinol) and sesquiterpene alcohol, cubenol, were dominant in HD-FrDV, and their abundance decreased after drying. C13-norisoprenoides (α-ionone and ß-ionone) increased after drying. Aliphatic compounds were present in both HD-FrDV and HD-DrDV samples. The less polar nonvolatile compounds in the obtained fractions F3 and F4 were analysed and identified by UHPLC-ESI(+)-HRMS. Identified compounds belonged to a group of pigments (7 compounds), fatty acid derivatives (13 compounds), as well as steroids and terpenes (10 compounds). Porphyrin-based compounds (C55H74N4O5-7), xanthophylls, sphingolipid compounds, fatty acid amides, and phytosterols represented the majority of identified compounds. By implementing both in vitro and in vivo assays for antioxidant activity determination, F3 showed a higher activity than F4. Inhibitory concentrations (IC50) for F3 and F4 were 498.00 ± 0.01 µg/mL and 798.00 ± 0.81 µg/mL, respectively, while a 1.5-fold reduction in the ROS level was observed after pre-treatment of zebrafish larvae with 45 µg/mL of F3.

Shewanella spp. from wastewater treatment plant-affected environment: isolation and characterization.

Bacteria from the genus Shewanella are inhabitants of marine and freshwater ecosystems, recognized fish spoilage bacteria, but less known as fish disease agents. Shewanella spp. isolated from fish living in waters close to effluents of a wastewater treatment plant (WWTP) were not previously characterized. We have tested Shewanella isolates from WWTP-affected waters and related fish. Genotypic characterization identified most strains as S. baltica and S. oneidensis. In order to investigate the sensibility and accuracy of their MALDI-TOF MS identification, they were grown on two culture media enriched by various NaCl concentrations, incubated at different temperatures and duration. We analyzed their antimicrobial susceptibility on a panel of antimicrobial drugs and capacity for biofilm production. With a view to demonstrate their capacity to produce fatty acids, we assessed the impact of different culture media on their lipid profile. We performed zebrafish embryotoxicity tests to simulate the environmental infection of the earliest life stages in S. baltica-contaminated waters. The best MALDI-TOF MS identification scores were for strains cultivated on TSA for 24 h at 22 °C and with supplementation of 1.5% NaCl. Less than 17% of isolates demonstrated antimicrobial resistance. Most isolates were weak biofilm producers. Strain-to-strain variation of MIC and MBC was low. The major fatty acids were C15:0, C16:0, C16:1, C17:1, and iC15:0. Exposure of Danio rerio to different S. baltica concentrations induced severe effects on zebrafish development: decreased heartbeat rate, locomotor activity, and melanin pigmentation. S. baltica passed through chorionic pores of zebrafish.

Biotechnological Enhancement of Probiotics through Co-Cultivation with Algae: Future or a Trend?

The diversity of algal species is a rich source of many different bioactive metabolites. The compounds extracted from algal biomass have various beneficial effects on health. Recently, co-culture systems between microalgae and bacteria have emerged as an interesting solution that can reduce the high contamination risk associated with axenic cultures and, consequently, increase biomass yield and synthesis of active compounds. Probiotic microorganisms also have numerous positive effects on various aspects of health and represent potent co-culture partners. Most studies consider algae as prebiotics that serve as enhancers of probiotics performance. However, the extreme diversity of algal organisms and their ability to produce a plethora of metabolites are leading to new experimental designs in which these organisms are cultivated together to derive maximum benefit from their synergistic interactions. The future success of these studies depends on the precise experimental design of these complex systems. In the last decade, the development of high-throughput approaches has enabled a deeper understanding of global changes in response to interspecies interactions. Several studies have shown that the addition of algae, along with probiotics, can influence the microbiota, and improve gut health and overall yield in fish, shrimp, and mussels aquaculture. In the future, such findings can be further explored and implemented for use as dietary supplements for humans.

Bioprospecting of Microalgae Isolated from the Adriatic Sea: Characterization of Biomass, Pigment, Lipid and Fatty Acid Composition, and Antioxidant and Antimicrobial Activity.

Marine microalgae and cyanobacteria are sources of diverse bioactive compounds with potential biotechnological applications in food, feed, nutraceutical, pharmaceutical, cosmetic and biofuel industries. In this study, five microalgae, Nitzschia sp. S5, Nanofrustulum shiloi D1, Picochlorum sp. D3, Tetraselmis sp. Z3 and Tetraselmis sp. C6, and the cyanobacterium Euhalothece sp. C1 were isolated from the Adriatic Sea and characterized regarding their growth kinetics, biomass composition and specific products content (fatty acids, pigments, antioxidants, neutral and polar lipids). The strain Picochlorum sp. D3, showing the highest specific growth rate (0.009 h-1), had biomass productivity of 33.98 ± 0.02 mg L-1 day-1. Proteins were the most abundant macromolecule in the biomass (32.83-57.94%, g g-1). Nanofrustulum shiloi D1 contained significant amounts of neutral lipids (68.36%), while the biomass of Picochlorum sp. D3, Tetraselmis sp. Z3, Tetraselmis sp. C6 and Euhalothece sp. C1 was rich in glycolipids and phospholipids (75%). The lipids of all studied microalgae predominantly contained unsaturated fatty acids. Carotenoids were the most abundant pigments with the highest content of lutein and neoxanthin in representatives of Chlorophyta and fucoxanthin in strains belonging to the Bacillariophyta. All microalgal extracts showed antioxidant activity and antimicrobial activity against Gram-negative E. coli and S. typhimurium and Gram-positive S. aureus.

Bioprospecting of Less-Polar Fractions of Ericaria crinita and Ericaria amentacea: Developmental Toxicity and Antioxidant Activity.

Ericaria crinita and Ericaria amentacea from the Adriatic Sea (Croatia) were investigated with respect to the presence of less-polar compounds for the first time after fractionation by solid-phase extraction (SPE). The composition of less-polar fractions of freeze-dried E. crinita (FdEc) and E. amentacea (FdEa) were analyzed by high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionization (UHPLC-ESI-HRMS). The major identified compounds were: amides of higher aliphatic acids (palmitoleamide, linoleamide, palmitamide, oleamide and erucamide) and related compounds, carotenoid (fucoxanthin), chlorophyll derivatives (pheophytin a and b and their derivatives) and higher terpenes (loliolide, isoamijiol with its oxidation product), ß-stigmasterol and (3ß,6α)-14-methylergosta-8,24(28)-diene-3,6-diol). The toxic effects observed on the less-polar fractions obtained from Ericaria species on zebrafish Danio rerio embryos could be associated with the high abundance of all five detected amides. The antioxidant activity of the fractions was evaluated by means of five independent assays, including the reduction of the radical cation (ABTS), the oxygen radical absorbance capacity (ORAC), ferric-reducing antioxidant power (FRAP), the 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assay and the Folin-Ciocalteu method. A higher antioxidant activity of E. amentacea in comparison to that of the E. crinita fractions was found with IC50 concentrations of 0.072 and 1.177 mg/mL, respectively. The correlation between the activity and the chemical composition revealed that the synergistic effect of different compounds impacted their antioxidant response.

Selenite as a Lipid Inductor in Marine Microalga Dunaliella tertiolecta: Comparison of One-Stage and Two-Stage Cultivation Strategies.

Microalgae have emerged as one of the most promising alternative sources of biofuels due to their high lipid accumulation ability. High lipid content is of pivotal importance for biodiesel production. In order to obtain high lipid content, modifications of culture conditions and development of an efficient lipid induction method are called for. In the present study, the possibility of using selenium in a form of sodium selenite as a lipid inductor in marine microalga Dunaliella tertiolecta was investigated during one- and two-stage cultivation modes. The effects of selenite on algal growth, pigment content, oxidative stress, and neutral lipid content were determined during both cultivation modes. The results revealed that the two-stage cultivation on 10.00-40.00 mg L-1 of selenite resulted in up to twofold higher algal cell density compared to the one-stage cultivation. Selenite concentrations from 2.50 to 20.00 mg L-1 increased lipid peroxidation during both cultivation modes, emphasizing the selenite-induced oxidative stress accompanied by the increased lipid accumulation in microalgae cells. During one- and two-stage cultivation on 20.00 mg L-1 of selenite, lipid content increased 2.39- and 5.73-fold at days 9 and 14 of cultivation, respectively. Moreover, the highest obtained neutral lipid content during the two-stage cultivation was 5.40-fold higher than lipid content obtained during the one-stage cultivation. Collectively, these results suggest that the two-stage cultivation strategy, initiated with optimal culture conditions for biomass production and followed by the addition of selenite as a stress inductor, can be successfully deployed to enhance the lipid content in D. tertiolecta.

Combining short-term bioassays using fish and crustacean model organisms with ToxCast in vitro data and broad-spectrum chemical analysis for environmental risk assessment of the river water (Sava, Croatia).

This study focused on the short-term whole organism bioassays (WOBs) on fish (Danio rerio) and crustaceans (Gammarus fossarum and Daphnia magna) to assess the negative biological effects of water from the major European River Sava and the comparison of the obtained results with in vitro toxicity data (ToxCast database) and Risk Quotient (RQ) methodology. Pollution profiles of five sampling sites along the River Sava were assessed by simultaneous chemical analysis of 562 organic contaminants (OCs) of which 476 were detected. At each sampling site, pharmaceuticals/illicit drugs category was mostly represented by their cumulative concentration, followed by categories industrial chemicals, pesticides and hormones. An exposure-activity ratio (EAR) approach based on ToxCast data highlighted steroidal anti-inflammatory drugs, antibiotics, antiepileptics/neuroleptics, industrial chemicals and hormones as compounds with the highest biological potential. Summed EAR-based prediction of toxicity showed a good correlation with the estimated toxicity of assessed sampling sites using WOBs. WOBs did not exhibit increased mortality but caused various sub-lethal biological responses that were dependant relative to the sampling site pollution intensity as well as species sensitivity. Exposure of G. fossarum and D. magna to river water-induced lower feeding rates increased GST activity and TBARS levels. Zebrafish D. rerio embryo exhibited a significant decrease in heartbeat rate, failure in pigmentation formation, as well as inhibition of ABC transporters. Nuclear receptor activation was indicated as the biological target of greatest concern based on the EAR approach. A combined approach of short-term WOBs, with a special emphasis on sub-lethal endpoints, and chemical characterization of water samples compared against in vitro toxicity data from the ToxCast database and RQs can provide a comprehensive insight into the negative effect of pollutants on aquatic organisms.

Chemical Evaluation, Antioxidant, Antiproliferative, Anti-Inflammatory and Antibacterial Activities of Organic Extract and Semi-Purified Fractions of the Adriatic Sea Fan, Eunicella cavolini.

Due to sedentary lifestyle and harsh environmental conditions, gorgonian coral extracts are recognized as a rich source of novel compounds with various biological activities, of interest to the pharmaceutical and cosmetic industries. The presented study aimed to perform chemical screening of organic extracts and semi-purified fractions obtained from the common Adriatic gorgonian, sea fan, Eunicella cavolini (Koch, 1887) and explore its abilities to exert different biological effects in vitro. Qualitative chemical evaluation revealed the presence of several classes of secondary metabolites extended with mass spectrometry analysis and tentative dereplication by using Global Natural Product Social Molecular Networking online platform (GNPS). Furthermore, fractions F4 and F3 showed the highest phenolic (3.28 ± 0.04 mg GAE/g sample) and carotene (23.11 ± 2.48 mg ß-CA/g sample) content, respectively. The fraction F3 inhibited 50% of DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) and ABTS (2,2'-azino-bis (3-ethylbenzthiazolin-6-yl) sulfonic acid) radicals at the concentrations of 767.09 ± 11.57 and 157.16 ± 10.83 µg/mL, respectively. The highest anti-inflammatory potential was exhibited by F2 (IC50 = 198.70 ± 28.77 µg/mL) regarding the inhibition of albumin denaturation and F1 (IC50 = 254.49 ± 49.17 µg/mL) in terms of soybean lipoxygenase inhibition. In addition, the most pronounced antiproliferative effects were observed for all samples (IC50 ranging from 0.82 ± 0.14-231.18 ± 46.13 µg/mL) against several carcinoma cell lines, but also towards non-transformed human fibroblasts pointing to a generally cytotoxic effect. In addition, the antibacterial activity was tested by broth microdilution assay against three human pathogenic bacteria: Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The latter was the most affected by fractions F2 and F3. Finally, further purification, isolation and characterization of pure compounds from the most active fractions are under investigation.

Supercritical Carbon Dioxide Extraction of Four Medicinal Mediterranean Plants: Investigation of Chemical Composition and Antioxidant Activity.

With everyday advances in the field of pharmaceuticals, medicinal plants have high priority regarding the introduction of novel synthetic compounds by the usage of environmentally friendly extraction technologies. Herein, a supercritical CO2 extraction method was implemented in the analysis of four plants (chamomile, St. John's wort, yarrow, and curry plant) after which the non-targeted analysis of the chemical composition, phenolic content, and antioxidant activity was evaluated. The extraction yield was the highest for the chamomile (5%), while moderate yields were obtained for the other three plants. The chemical composition analyzed by gas chromatography-high-resolution mass spectrometry (GC-HRMS) and liquid chromatography-high-resolution mass spectrometry (LC-HRMS) demonstrated extraction of diverse compounds including terpenes and terpenoids, fatty acids, flavonoids and coumarins, functionalized phytosterols, and polyphenols. Voltammetry of microfilm immobilized on a glassy carbon electrode using square-wave voltammetry (SWV) was applied in the analysis of extracts. It was found that antioxidant activity obtained by SWV correlates well to 1,1-diphenyl-2-picrylhidrazine (DPPH) radical assay (R2 = 0.818) and ferric reducing antioxidant power (FRAP) assay (R2 = 0.640), but not to the total phenolic content (R2 = 0.092). Effective results were obtained in terms of activity showing the potential usage of supercritical CO2 extraction to acquire bioactive compounds of interest.

Less Polar Compounds and Targeted Antioxidant Potential (In Vitro and In Vivo) of Codium adhaerens C. Agardh 1822.

Codium adhaerens from the Adriatic Sea (Croatia) was comprehensively investigated regarding less polar compounds for the first time. Although there are several phytochemical studies on C. adhaerens from other regions, this is the first report on volatile organic compounds (VOCs) from fresh (FrCa) and air-dried (DrCa) samples. The novelty is also related to its targeted antioxidant potential in vitro and in vivo. The main aims were to: (a) identify and compare VOCs of FrCa and DrCa obtained by headspace solid-phase microextraction (HS-SPME) and hydrodistillation (HD); (b) determine fatty acid (FA) composition of freeze-dried sample (FdCa); (c) determine the composition of less polar fractions of FdCa by high-performance liquid chromatography-high-resolution mass spectrometry with electrospray ionisation (UHPLC-ESI-HRMS); and (d) comprehensively evaluate the antioxidant activity of the fractions by four in vitro assays and in vivo zebrafish model (including embryotoxicity). Significant changes of VOCs were found after air drying. ω6 FAs were present in higher content than ω3 FAs indicating C. adhaerens as a good source of dietary polyunsaturated FAs. The results obtained in vivo correlate well with in vitro methods and both fractions exerted similar antioxidative responses which is in agreement with the high abundance of present biomolecules with known antioxidant properties (e.g., fucoxanthin, pheophytin a, and pheophorbide a). These results suggest that C. adhaerens might be a potent source of natural antioxidants that could be further used in the research of oxidative stress-related diseases.