Liposomal integration method for assessing antioxidative activity of water insoluble compounds towards biologically relevant free radicals: example of avarol.

The liposomal integration method, in conjunction with electron paramagnetic resonance (EPR) spectroscopy, has been presented for the investigation of antioxidant activity of selected water-insoluble compound towards biologically relevant free radicals. This method was applied to avarol, a sesquiterpenoid hydroquinone isolated from the marine sponge Dysidea avara. The antioxidant activity of water-insoluble avarol towards •OH, O2•- and NO• radicals was attained by its incorporation into the DPPC liposomes bilayer, and towards ascorbyl radicals in the organic solvent. Avarol's activity towards •OH, O2•-, NO• and ascorbyl radicals was 86.2%, 50.9%, 23.6% and 61.8%, respectively, showing its significant radical scavenging potential.

Antioxidant Activity of Selected Polyphenolics in Yeast Cells: The Case Study of Montenegrin Merlot Wine.

Screens of antioxidant activity (AA) of various natural products have been a focus of the research community worldwide. This work aimed to differentiate selected samples of Merlot wines originated from Montenegro, with regard to phenolic profile and antioxidant capacity studied by survival rate, total sulfhydryl groups and activities of glutathione peroxidase (GPx), glutathione reductase and catalase in H2O2⁻stressed Saccharomyces cerevisiae cells. In this study, DPPH assay was also performed. Higher total phenolic content leads to an enhanced AA under both conditions. The same trend was observed for catechin and gallic acid, the most abundant phenolics in the examined wine samples. Finally, the findings of an Artificial Neural Network (ANN) model were in a good agreement (r² = 0.978) with the experimental data. All tested samples exhibited a protective effect in H2O2⁻stressed yeast cells. Pre-treatment with examined wines increased survival in H2O2⁻stressed cells and shifted antioxidative defense towards GPx⁻mediated defense. Finally, sensitivity analysis of obtained ANN model highlights the complexity of the impact that variations in the concentrations of specific phenolic components have on the antioxidant defense system.

The lignicolous fungus Trametes versicolor (L.) Lloyd (1920): a promising natural source of antiradical and AChE inhibitory agents.

This study aimed to determine antiradical (DPPH• and •OH) and acetylcholinesterase (AChE) inhibitory activities along with chemical composition of autochtonous fungal species Trametes versicolor (Serbia). A total of 38 phenolic compounds with notable presence of phenolic acids were identified using HPLC/MS-MS. Its water extract exhibited the highest antiradical activity against •OH (3.21 µg/mL), among the rest due to the presence of gallic, p-coumaric and caffeic acids. At the concentration of 100 µg/mL, the same extract displayed a profound AChE inhibitory activity (60.53%) in liquid, compared to donepezil (89.05%), a drug in clinical practice used as positive control. The flavonoids baicalein and quercetin may be responsible compounds for the AChE inhibitory activity observed. These findings have demonstrated considerable potential of T. versicolor water extract as a natural source of antioxidant(s) and/or AChE inhibitor(s) to be eventually used as drug-like compounds or food supplements in the treatment of Alzheimer's disease.

Viticultural and chemical characteristics of Muscat Hamburg preselected clones grown for table grapes.

BACKGROUND: Clonal selection is one of the tools used for grapevine improvement and therefore is very important for obtaining clones with better characteristics than the variety population. The aim of this study was to select superior grapevines of Vitis vinifera L. cv. Muscat Hamburg grown for fresh consumption. RESULTS: The viticultural parameters and fruit composition of 35 selected vines were determined during a 5-year period. The evaluated parameters showed high variability among selected vines. The significant effect of vintage was observed for all descriptors with the exception of the number of seeds per berry and sugar concentration. Additionally, all vines were examined for their tolerance to low temperatures and the results showed 73% and 90% of primary bud injury at -20 and -25 °C, respectively. In relation to berry classification, the percentage of first-class grapes ranged from 60% to 69% for all selected grapevines. Multivariate statistical analysis was performed to classify grapevines based on their performance. CONCLUSION: Fourteen grapevines were identified as the most promising among the 35 vines initially planted, based on high yield, bunch and berry weight, sugar content and percentage of first-grade grapes. Those grapevines were selected for the next phase of the clonal selection. This study highlighted the importance of clonal selection for improvement of the variety population. © 2016 Society of Chemical Industry.

Further insight into the bioactivity of the freshwater sponge Ochridaspongia rotunda.

CONTEXT: Bioprospection has become a dynamic scientific field that explores novel possibilities for the implementation of natural products in medicine and pharmacy. Compared to marine species from all kingdoms, freshwater species have been highly neglected. OBJECTIVE: This work focuses on the screening of acetylcholinesterase inhibitory (AChE) and mutagenic activities of the acetone extract (obtained by maceration) of the freshwater sponge Ochridaspongia rotunda Arndt (Malawispongiidae) in vitro. MATERIALS AND METHODS: AChE inhibitory activity was evaluated both in liquid (five different concentrations of the extract, from 1 to 100 µg/mL) and in solid (seven different concentrations of the extract, from 0.5 to 10.0 µg) by methods well described in literature, while mutagenicity was estimated using the Ames test (four different concentrations of the extract, from 0.106 to 1.328 mg/plate). RESULTS: Ochridaspongia rotunda acetone extract exhibited promising AChE inhibitory activity in a dose-dependent manner both in liquid (IC50 23.07 µg/mL) and in solid (1.50 µg). Furthermore, the Ames test revealed no sign of mutagenicity at any concentration tested. Its FTIR spectrum coupled with the positive Liebermann?Burchard, Salkowski and Zak color reactions (tests) indicated the presence of sterol compounds. DISCUSSION AND CONCLUSION: The screened extract may inspire a search for novel anticholinesterase therapeutic agent(s) potentially used in the treatment of Alzheimer's disease. Further research will be directed toward its detailed chemical analysis along with addressing the issue of a real producer of the natural product(s) responsible for the AChE activity observed.

A comparative overview of antioxidative properties and phenolic profiles of different fungal origins: fruiting bodies and submerged cultures of Coprinus comatus and Coprinellus truncorum.

Bioactive properties of fungi considerably differ between the fruiting body (FB) and the submerged culture as regards mycelia (M) and the fermentation broth (F). Antioxidant properties of hot-water extracts obtained from three different fungal origins: FB, M and F of two autochthonous fungal species (Northern Serbia), Coprinus comatus and Coprinellus truncorum were investigated. Free radical scavenging capacity (RSC) was evaluated in vitro by the DPPH assay and reducing power ability (FRAP assay). Considering possible bioactive properties of different compounds present in fungal extracts, the content of total proteins (TP), phenols (TC) and flavonoids (TF) were investigated colorimetrically. The chemical characterisation of the examined extracts was evaluated using the HPLC-MS/MS method. C. comatus showed the strongest RSC activity; more precisely, fermentation broth extract (FCc) on DPPH radicals (IC50 = 5.06 µg mL-1) and fruiting body extract (FBCc) for the FRAP assay (42.86 mg ascorbic acid equivalents (AAE)/g). Submerged M extract of both species showed the highest TC (MCc 81.95 mg gallic acid eq (GAE)/g d.w.; MCt 81.64 mg GAE/g d.w.), while FB extracts contained the highest content of TP. Comparing LC-MS phenolic profiles between species-interspecifically and among different fungal origins-intraspecifically (fruiting bodies and submerged cultures), high variations were noticed. In submerged M or F extracts of C. comatus, vanillic, gallic, gentisic and cinnamic acids were detected, as opposed to FB. Considering that diverse phenolic profiles of detected antioxidant compounds were obtained by submerged cultivation, this type of cultivation is promising for the production of antioxidant substances.

Further in vitro biological activity evaluation of amino-, thio- and ester-derivatives of avarol.

The acetylcholinesterase inhibitory and/or antitumour activities of amino-, thio- and ester-derivatives of avarol selected were evaluated for the first time at in vitro conditions. Avarol-3',4'-dithioglycol (1) and avarol-4'-(3)mercaptopropionic acid (3) were shown to be the best inhibitors of the enzyme tested (0.50 µg and IC50 0.05 mM and 0.50 µg and IC50 0.12 mM, respectively), while 4'-tryptamine-avarone (9) and avarol-3'-(3)mercaptopropionic acid (2) exhibited the highest cytotoxicity against the human breast T-47D cancer cell line (IC50 0.66 µg/mL and 1.25 µg/mL, respectively). According to experimental data obtained, the sesquiterpenoid hydroquinone structure of bioactive avarol derivatives may inspire development of new pharmacologically useful substances to be used in the treatment of Alzheimer's disease and/or human breast tumour.

A computational insight into acetylcholinesterase inhibitory activity of a new lichen depsidone.

Acetylcholinesterase (AChE) inhibitors are yet the best drugs currently available for the management of Alzheimer's disease. The recent phytochemical investigation has led to the isolation of a new depsidone 1 with moderate AChE activity (1 µg). This work was focused on its electronic properties analysed using commercially available programs. Both the active depsidone molecule 1 and galanthamine showed to have higher HOMO energies than the inactive depsidones 2-4, isolated from the same lichen species. However, the amino depsidone derivative 7, whose structure was proposed using computational approaches, is expected to be more active AChE inhibitor than the depsidone 1, due to the improved HOMO energy value. In addition, the molecular docking study indicated that the compound 7 has ability to make the well-known interactions of potent AChE inhibitors with the enzyme active site. The data presented herein support the design of novel AChE inhibitors based on the depsidone scaffold.

Further in vitro biological activity evaluation of amino-, thio- and ester-derivatives of avarol.

The acetylcholinesterase inhibitory and/or antitumour activities of amino-, thio- and ester-derivatives of avarol selected were evaluated for the first time at in vitro conditions. Avarol-3',4'-dithioglycol (1) and avarol-4'-(3)mercaptopropionic acid (3) were shown to be the best inhibitors of the enzyme tested (0.50 µg and IC50 0.05 mM and 0.50 µg and IC50 0.12 mM, respectively), while 4'-tryptamine-avarone (9) and avarol-3'-(3)mercaptopropionic acid (2) exhibited the highest cytotoxicity against the human breast T-47D cancer cell line (IC50 0.66 µg/mL and 1.25 µg/mL, respectively). According to experimental data obtained, the sesquiterpenoid hydroquinone structure of bioactive avarol derivatives may inspire development of new pharmacologically useful substances to be used in the treatment of Alzheimer's disease and/or human breast tumour.

A new depsidone of Lobaria pulmonaria with acetylcholinesterase inhibition activity.

The phytochemical investigation conducted on a foliose lichen, Lobaria pulmonaria(L.) Hoffm. (Lobariaceae), led to the isolation of a new depsidone in the form of its diacetate derivative which showed a moderate acetylcholinesterase inhibition activity (1 µg) in vitro. This is the first record of identified depsidone structure in searching for these inhibitors.

Cytotoxic activity of the crude polysaccharides/exopolysaccharides of Coprinus comatus and Coprinellus truncorum.

Fungi are an important source of polysaccharides (PSH) and phenolic compounds (PC). Numerous studies have highlighted the beneficial effects of fungal consumption, but the impact of submerged cultivated mycelia (M) and filtrate (F) has not been fully investigated. We aimed to investigate the cytotoxic activity of isolated crude PSH and exopolysaccharides (ePSH) of submerged cultivated M and F of edible Coprinus comatus and Coprinellus truncorum species. Both PSH and ePSH exhibited significant cytotoxic activity towards HepG2 cancer cells of human origin (three-way ANOVA). The C. truncorum PSH/ePSH was more efficient inducing maximal reduction in cell viability (≈50% at 450 µg/mL) after 24 h while C. comatus PSH/ePSH needed 72 h to reach similar effect (≈60% at 450 µg/mL). Partial least square regression (PLSR) analysis indicated that specific phenolic composition of the PSH/ePSH could be responsible for the difference in their activity.

3.5-scFv-ß-lactamase, a new protein detector of picric acid, the highly nitrated organic explosive.

A new protein detector of 2,4,6-trinitrophenol (TNP, picric acid) is concisely reported herein for the first time. In brief, the gene of a specific scFv fragment, namely 3.5, was fused separately with the gene of ß-lactamase and, subsequently, expressed in the periplasmic space of Escherichia coli, affording in such a way the recombinant fusion protein 3.5-scFv-ß-lactamase. This bifunctional immunoreagent tool was further convincingly demonstrated for being capable to directly detect trinitrophenol-tris(hydroxymethyl)aminomethane (TNP-Tris), a less toxic TNP derivative of choice compared to TNP itself, with competitive sensitivity (50 ± 2 fmol or 175 ± 6 pg/mL).[Formula: see text].

A novel and effective natural product-based immunodetection tool for TNT-like compounds.

This study aimed to develop a fast and reliable protocol for Trinitrophenol-Tris(hydroxymethyl)aminomethane (TNP-Tris) detection applying a ß-lactamase-fusion protein of choice, the natural product-based immunoreagent tool of competitive sensitivity developed herein for the first time. Since the fusion protein 11B3-scFv-ß-lactamase is constructed from a scFv-antibody (11B3) linked to an enzyme (ß-lactamase), the step related to the use of secondary antibody in the enzyme-linked immunosorbent assay (ELISA) is completely omitted. Indeed, this fusion protein itself serves both as binding mean of the antigen model and detecting agent, due to the presence of the naturally occurring enzyme. In such a way, it actually affords the one-step TNP-Tris detection reaching a promising LOD value of 45 ± 2 fmol or 157 ± 6 pg/mL. Taken all together, the current protocol does represent much cheaper and significantly less-time consuming alternative compared both to the recombinant antibodies and recombinant phages, previously designed means in our labs for the same purpose.

Low sugar jellies of berry fruits: the impact of low vs. high temperature regime on their chemical composition and antioxidativity.

This study focuses on the impact of low and high temperature regimes on the chemical composition and antioxidativity of low sugar berry fruits jellies. High quality fruits (strawberry, raspberry and blackberry) were collected from Western Serbia region, quite well recognised both nationally and internationally due to an extremely well developed practice in growing berry fruits. The obtained results have clearly indicated the importance of low temperature regime for enriched contents of both total phenolics and anthocyanins followed by an enhanced antioxidativity. Rubus fruticosus L. Cacak Thornless cultivar, the only autochthonous berry fruit variety screened herein, may be firmly recommended as a raw material for industrial production of low sugar blackberry jellies with exceptional characteristics. This innovative procedure of preparing berry fruit jellies have encompassed the application of low temperature regime (55 °C), lower content of sugar (40%), seeds separation from jellies followed by no use of pectin throughout the whole process.

Raspberry seeds extract selectively inhibits the growth of human lung cancer cells in vitro.

This study was focused on in vitro cytotoxicity screening of the raspberry seeds methanol extract towards a number of cancer cell lines of human origin. The tested extract at the preferred concentrations (IC50 <30 µg/mL) inhibited only the growth of the lung cancer A-549 cells (IC50 = 14.07 ± 0.96 µg/mL). At the same time, it was practically inactive (IC50 >300 µg/mL) and non-mutagenic towards normal MRC-5 lung cells. Finally, the extract potently scavenged both OH· (IC50 = 20.11 ± 1.77 µg/mL) and O2-· (IC50 = 47.23 ± 3.82 µg/mL), the free radicals of proved relevance for cancer pathophysiology. Though seeds were enriched with phenolic compounds (TPC = 5.21 ± 0.07 mg GAE/g), anthocyanins were present in traces only (TAC = 0.07 ± 0.003 mg cyn-3-glu/g), while flavonoids were not detected at all. This is the first report on anti-lung cancer potential of the seeds of any soft fruit.

A neglected natural source for targeting glioblastoma.

The cytotoxicity of the methanol extract of the freshwater sponge Ochridaspongia rotunda (Arndt, 1937) (Malawispongiidae) was evaluated by MTT assay at in vitro conditions against three brain tumour cell lines (Neuro-2A, U-251 MG and U-87 MG). The extract was actually found to be most effective against the malignant glioma U-251 MG cells reaching a promising IC50 value of 1.87 ± 0.09 µg/mL at 96 h. However, it exhibited only a bit of cytotoxicity (IC50 321.14 ± 11.29 µg/mL, 96 h) towards the normal cells. Also, this sponge extract was 5-fold more selective for U-251 MG versus U-87 MG cells. Finally, monitoring genotoxicity at chromosomal level using the micronucleus test practically revealed lack of any significant toxicity of O. rotunda extract, compared to doxorubicin.

Polarography as a technique of choice for the evaluation of total antioxidant activity: The case study of selected Coprinus Comatus extracts and quinic acid, their antidiabetic ingredient.

This study was focused on in vitro screening of the total antioxidant activity of the selected extracts of the mushroom Coprinus comatus and quinic acid, one of their antidiabetic ingredients, by an uncommon electrochemical assay. Indeed, direct current (DC) polarographic HydroxoPerhydroxo Mercury(II) Complex (HPMC) assay based on decrease of anodic limiting current originating from HPMC formation in alkaline solutions of hydrogen peroxide at potential of mercury dissolution, observed upon gradual addition of antioxidants, was applied herein for the estimation of the natural products' antioxidativity. Quinic acid was found to exhibit most promising antioxidant potential (4.0 ± 0.2%µL-1) being ≈ 2-fold more active than the screened C. comatus extract samples. Actually, such a finding puts some light on the antioxidativity of cyclic polyols, well understimated class of organic compounds, compared to aromatic (poly)phenolics. As a low cost, easy-to handle and accurate this polarographic assay may be thoroughly recommended for much broader use. [Formula: see text].

Coprinus comatus filtrate extract, a novel neuroprotective agent of natural origin.

In vitro acetylholinesterase (AChE) inhibitory activity of an autochthonous sample of the mushroom Coprinus comatus (encompassing fruiting body FB, mycelia M and filtrate F from the submerged cultivation) was the subject of this study. C. comatus F extract exhibited rather potent anti-AChE activity (73.0 ± 1.5%) at in liquid conditions, comparable to those of the conventional drug donepezil (80.6 ± 1.4%). Also, the same extract exhibited high anti-AChE activity (1 µg) in solid. While its FTIR spectrum indicated the presence of phenolic compounds, quercetin (28.1 µg g-1 d.w.) was found to affect the observed bioactivity (59.8 ± 0.9%). This is the first report of profound anti-AChE activity of any C. comatus extract, a medicinal mushroom that has been successfully cultivated in P.R. China, due to the demanding needs of food industry.

An insight into chemical composition and bioactivity of 'Prokupac' red wine.

Autochthonous variety 'Prokupac' (Vitis vinifera L.) red wines with the addition of selected aromatic herbs (Artemisia absinthium, Cinnamomum zeylanicum, Glycyrrhiza glabra and Pimpinella anisum L.) were evaluated for chemical composition and biological activity. Furthermore, their polyphenolic profiles were determined by HPLC-DAD. While total phenolic (TPC) and flavonoid (TFC) contents, along with antioxidant activity, were significantly higher in all examined wine samples, the content of total anthocyanins was decreased, compared to a pure 'Prokupac' wine, used as the control one. On the other hand, 'Prokupac' wines with the addition of cinnamon (S. zeylanicum) and wormwood (A. absinthium) displayed highest antioxidant (EC50, 0.021 ± 0.001 mg/mL) and antibacterial (Enterococcus faecalis, MIC/MBC 15.63/62.50 µL/mL) activities, respectively. The study showed that 'Prokupac' wine with the addition of cinnamon was significantly enriched both with TPC and TFC, compared to control wine (p < 0.05), which may contribute to its market potential.

Identification and first insights into the structure of chitin from the endemic freshwater demosponge Ochridaspongia rotunda (Arndt, 1937).

Studies on the identification, properties and function of chitin in sponges (Porifera), which are recognized as the first multicellular organisms on Earth, continue to be of fundamental scientific interest. The occurrence of chitin has so far been reported in 21 marine sponge species and only in two inhabiting fresh water. In this study, we present the discovery of α-chitin in the endemic demosponge Ochridaspongia rotunda, found in Lake Ohrid, which dates from the Tertiary. The presence of chitin in this species was confirmed using special staining, a chitinase test, FTIR, Raman and NEXAFS spectroscopy, and electrospray ionization mass spectrometry (ESI-MS). In contrast to the case of marine sponges, chitin in O. rotunda has been found only within its holdfast, suggesting a role of chitin in the attachment of the sponge to the hard substratum. Isolated fibrous matter strongly resemble the shape and size of the sponge holdfast with membrane-like structure.