The Composition of Volatiles and the Role of Non-Traditional LOX on Target Metabolites in Virgin Olive Oil from Autochthonous Dalmatian Cultivars.

The lipoxygenase pathway has a significant influence on the composition of the volatile components of virgin olive oil (VOO). In this work, the influence of the maturity index (MI) on the activity of the lipoxygenase enzyme (LOX) in the fruits of the autochthonous Dalmatian olive cultivars Oblica, Levantinka and Lastovka was studied. The analysis of the primary oxidation products of linoleic acid in the studied cultivars showed that LOX synthesises a mixture of 9- and 13-hydroperoxides of octadecenoic acid in a ratio of about 1:2, which makes it a non-traditional plant LOX. By processing the fruits of MI~3, we obtained VOOs with the highest concentration of desirable C6 volatile compounds among the cultivars studied. We confirmed a positive correlation between MI, the enzyme activity LOX and the concentration of hexyl acetate and hexanol in cultivars Oblica and Lastovka, while no positive correlation with hexanol was observed in the cultivar Levantinka. A significant negative correlation was found between total phenolic compounds in VOO and LOX enzyme activity, followed by an increase in the MI of fruits. This article contributes to the selection of the optimal harvest time for the production of VOOs with the desired aromatic properties and to the knowledge of the varietal characteristics of VOOs.

Quantitatively Unraveling Hierarchy of Factors Impacting Virgin Olive Oil Phenolic Profile and Oxidative Stability.

A single phenolic group and even a compound play different roles in the sensory properties and stability of virgin olive oil (VOO), which in turn are strongly influenced by several factors. Understanding the causes of differences in phenolic compound composition and oxidative stability (OS) in VOOs is essential for targeted and timely harvest and processing while maintaining desired oil quality. The phenolic profile and OS of two monocultivar VOOs (Oblica and Leccino) grown in two geographical sites of different altitudes (coastal plain and hilly hinterland) were analyzed throughout the ripening period over two years. Concentration of secoiridoids was 30% higher in the Oblica than in the Leccino VOOs, which in turn had significantly higher values of OS. Both cultivars had more than twice as high concentrations of the two most abundant phenolic compounds, the dialdehyde form of decarboxymethyl oleuropein aglycone and the dialdehyde form of decarboxymethyl ligstroside aglycone, and OS values in a colder growing site of higher altitude. Among the studied monocultivar VOOs, the secoiridoid group did not behave equally during ripening. The hierarchy of different influencing factors was investigated using multivariate statistics and revealed: cultivar > geographical site > harvest period > growing season. In addition, the possibility of traceability of VOO using molecular markers was investigated by establishing SSR profiles of oils of the studied cultivars and comparing them with SSR profiles of leaves.

Not Only a Weed Plant-Biological Activities of Essential Oil and Hydrosol of Dittrichia viscosa (L.) Greuter.

With the increasing interest in obtaining biologically active compounds from natural sources, Dittrichia viscosa (L.) Greuter (Asteraceae) came into our focus as a readily available and aromatic wild shrub widely distributed in the Mediterranean region. This work provides a phytochemical profile of D. viscosa in terms of parallel chemical composition in the lipophilic fraction (essential oil) and the water fraction (hydrosol). GC-MS analysis identified 1,8-cineole, caryophyllene oxide, α-terpenyl acetate, and α-muurolol as the major components of the essential oil, while in the hydrosol p-menth-1-en-9-ol, 1,8-cineole, linalool, cis-sabinene hydrate, and α-muurolol were the major volatile components. 3,4-Dihydroxybenzoic acid was found to be the predominant compound in the hydrosol composition by HPLC analysis. The antimicrobial potential of both extracts was evaluated against thirteen opportunistic pathogens associated with common skin and wound infections and emerging food spoilage microorganisms. The antimicrobial activity of the essential oil suggests that the volatiles of D. viscosa could be used as novel antimicrobial agents. The antiproliferative results of D. viscosa volatiles are also new findings, which showed promising activity against three cancer cell lines: HeLa (cervical cancer cell line), HCT116 (human colon cancer cell line), and U2OS (human osteosarcoma cell line). The decrease in GSH level observed in hydrosol-treated HeLa cells suggests oxidative stress as a possible mechanism of the antiproliferative effect of hydrosol on tumor cells. The presented results are also the first report of significant antiphytoviral activity of hydrosol against tobacco mosaic virus (TMV) infection. Based on the results, D. viscosa might have the potential to be used in crop protection, as a natural disinfectant and natural anticancer agent.

Chemical Composition and New Biological Activities of Essential Oil and Hydrosol of Hypericum perforatum L. ssp. veronense (Schrank) H. Lindb.

The chemical profile, antiproliferative, antioxidant and antiphytoviral activities of the species Hypericum perforatum ssp. veronense (Schrank) H. Lindb. (Clusiaceae) were investigated. Free volatiles were isolated and the chemical composition was determined in the lipophilic fraction (essential oil) and for the first time in the water fraction (hydrosol). The aim is to provide phytochemical data for H. perforatum ssp. veronense useful for distinguishing ssp. veronense from ssp. angustifolium, as there are taxonomic disagreements between them and the composition of the secretory products may be helpful in this respect. In the essential oil, the most abundant compounds identified were α-pinene and n-nonane, while in the hydrosol, myrtenol, carvacrol and α-pinene were the most abundant. Overall, the class of monoterpenes and oxygenated monoterpenes dominated in the EO and hydrosol samples. The essential oil showed high antioxidant activity, in contrast to the antiproliferative activity, where the hydrosol showed exceptional activity against three cancer cell lines: Hela (cervical cancer cell line), HCT116 (human colon cancer cell line) and U2OS (human osteosarcoma cell line). Both the essential oil and hydrosol showed antiphytoviral activity against tobacco mosaic virus infection on the local host plants. This is the first report dealing with biological activities of hydrosol of H. perforatum ssp. veronense, and the obtained results suggest that this traditional medicinal plant is a valuable source of volatiles with promising antiproliferative, antioxidant and antiphytoviral activities.

Bioactive Phenolic Metabolites from Adriatic Brown Algae Dictyota dichotoma and Padina pavonica (Dictyotaceae).

In this study, the influences of temperature (20, 40 and 60 °C) and extraction solvents (water, ethanol) on the ultrasound-assisted extraction of phenolics from the Adriatic macroalgae Dictyota dichotoma and Padina pavonica were studied. The extracts were analysed for major phenolic sub-groups (total phenolics, flavonoids and tannins) using spectrometric methods, while the individual phenolics were detected by HPLC. The antioxidant activities were evaluated using three methods: Ferric Reducing/Antioxidant Power (FRAP), scavenging of the stabile 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and Oxygen Radical Antioxidant Capacity (ORAC). The aim of the study was also to find the connection between the chemical composition of the extracts and their biological activity. Therefore, principal component analysis (PCA), which permits simple representation of different sample data and better visualisation of their correlations, was used. Higher extraction yields of the total phenolics, flavonoids and tannins were obtained using an alcoholic solvent, while a general conclusion about the applied temperature was not established. These extracts also showed good antioxidant activity, especially D. dichotoma extracts, with high reducing capacity (690-792 mM TE) and ORAC values (38.7-40.8 mM TE in 400-fold diluted extracts). The PCA pointed out the significant influence of flavonoids and tannins on the investigated properties. The results of this investigation could be interesting for future studies dealing with the application of these two algae in foods, cosmetics and pharmaceuticals.

Fruit quality and volatile compound composition of processing tomato as affected by fertilisation practices and arbuscular mycorrhizal fungi application.

The effects of different fertilisation treatments with arbuscular mycorrhizal fungi (AMF) inoculation on AMF root colonisation, fruit yield, nutrient and total phenol contents, volatile compound composition, and sensory attributes of tomato (Solanum lycopersicum L.) were investigated. Mineral, organic, and mineral + organic fertiliser application positively affected tomato yield (35%-50%) and phosphorus concentration (24%-29%) compared with controls. AMF application had a significant impact on the total nitrogen (+9%), manganese (+12%), and hydrophilic phenol (+8%) contents in the fruit. Volatile compounds were affected by the interactive effects of fertilisation and AMF application. The response of tomato fruit sensory quality indicators was relatively modest, with only a few sensory characteristics affected to a lesser extent. Although tomato showed susceptibility to field-native AMF, particular combinations of fertilisation and AMF inoculation were more effective at improving the quality parameters of tomatoes under field conditions applied in this study.

The mode of antibacterial action of quaternary N-benzylimidazole salts against emerging opportunistic pathogens.

Quaternary ammonium compounds (QACs) are antimicrobial agents displaying a broad spectrum of activity due to their mechanism of action targeting the bacterial membrane. The emergence of bacterial resistance to QACs, especially in times of pandemics, requires the continuous search for new and potent QACs structures. Here we report the synthesis and biological evaluation of QACs based on imidazole derivative, N-benzylimidazole. The antimicrobial activity was tested against a range of pathogenic bacteria and fungi, both ATCC and clinical isolates, showing varying activities ranging in minimal inhibitory concentrations (MICs) from as low as 7 ng/mL. The most promising compound, N-tetradecyl derivative (BnI-14), proved to be very potent against bacterial biofilms, even at sub-MIC doses, suggesting interference with the bacterial growth and/or division process. The BnI-14 treatment induces bacterial membrane disruption, as observed by fluorescence spectroscopy and atomic force microscopy and it also binds to DNA indicating that bacterial membrane might not be the only cellular target of QACs. Most importantly, BnI-14 exhibits low toxicity to healthy human cell lines, suggesting that N-benzylimidazolium-based QACs may be promising new antimicrobial agents.

Phytochemical Characterization and Screening of Antioxidant, Antimicrobial and Antiproliferative Properties of Allium × cornutum Clementi and Two Varieties of Allium cepa L. Peel Extracts.

Onions are one of the most widely grown vegetable crops. As production increases, so does the generation of waste from various parts of the onion, raising the need for efficient ecological disposal and use of such waste products. However, onion waste products are a rich source of antioxidants with a range of biological properties, therefore, they could potentially be used in food and pharmaceutical industries. In the present study, we identified the main flavonols and anthocyanins in peel extracts of Allium × cornutum Clement ex Visiani, 1842, and two varieties of Allium cepa L. and tested their antioxidant, antimicrobial and antiproliferative properties. Quercetin 3,4'-diglucolside, quercetin 4'-monoglucoside and quercetin are the most abundant flavonols in all onion extracts detected by high-performance liquid chromatography (HPLC) method. The composition of anthocyanins varied in all extracts. 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC) assays showed that the triploid onion A. × cornutum had the highest antioxidant power. Evaluation of antimicrobial activity by broth microdilution assay also showed that A. × cornutum had higher antimicrobial activity compared to the red and yellow onion varieties. Comparable antiproliferative activity was confirmed for all onion extracts tested on three cancer cell lines: Hela (cervical cancer cell line), HCT116 (human colon cancer cell line) and U2OS (human osteosarcoma cell line). The most abundant onion flavonols (quercetin 3,4'-diglucoside and quercetin 4'-monoglucoside) showed weaker antimicrobial as well as antiproliferative properties compared to the extracts, leading to the conclusion that other phytochemicals besides flavonols contribute to the biological activity of onion peel extracts. The results demonstrate the antioxidant and antimicrobial properties of onion peels, which have promising potential as cancer cell proliferation inhibitors.

Endemic Veronica saturejoides Vis. ssp. saturejoides-Chemical Composition and Antioxidant Activity of Free Volatile Compounds.

Chemical profile and antioxidant activity of the species Veronica saturejoides Vis. ssp. saturejoides (Plantaginaceae)-which is endemic to Croatia, Bosnia and Herzegovina and Montenegro -were investigated. Volatile compounds produced by glandular trichomes (composed of one stalk cell and two elliptically formed head cells according to scanning electron microscope investigation) were isolated from the plants collected in two locations. Additionally, as a part of specialized metabolites, total polyphenols, total tannins, total flavonoids and total phenolic acids were determined spectrophotometrically. In the lipophilic volatile fractions-essential oils, the most abundant compounds identified were hexahydrofarnesyl acetone, caryophyllene oxide and hexadecanoic acid. In total, the class of oxygenated sesquiterpenes and the group of fatty aldehydes, acids and alcoholic compounds dominated in the essential oils. In the hydrophilic volatile fractions-hydrosols, the most abundant compounds identified were trans-p-mentha-1(7),8-dien-2-ol, allo-aromadendrene and (E)-caryophyllene. A group of oxygenated monoterpenes and the sesquiterpene hydrocarbons dominated in the hydrosols. Antioxidant activity of essential oils and hydrosols was tested with two methods: 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and oxygen radical absorbance capacity (ORAC). Essential oils showed higher antioxidant activity than hydrosols and showed similar antioxidant activity to Rosmarinus officinalis essential oil. Obtained results demonstrate that this genus is a potential source of volatiles with antioxidant activity.

Comparison of Organosulfur and Amino Acid Composition between Triploid Onion Allium cornutum Clementi ex Visiani, 1842, and Common Onion Allium cepa L., and Evidences for Antiproliferative Activity of Their Extracts.

Species that belong to the genus Allium have been widely used for human food and traditional medicine. Their beneficial health effects, as well as the specific aroma, are associated with their bioactive chemical compounds, such as sulfur compounds and flavonoids. Gas chromatography and mass spectrometry (GC-MS) and reverse-phase high-performance liquid chromatography (reverse-phase HPLC) were used to identify organosulfur and amino acid content of triploid hybrid onion, Allium cornutum Clement ex Visiani, 1842, and common onion, Allium cepa L. Allium extracts were tested for their antiproliferative activity in three human cancer cell lines (HeLa, HCT116, and U2OS). DNA fragmentation and DAPI staining analysis were performed on HeLa cells to evaluate the effect of extracts on DNA damage and cell morphology. The mRNA expression of p53, Bax, and Caspase-3 genes involved in apoptosis were analyzed by real-time PCR. Using GC-MS, 27 compounds were found in two Allium species headspaces. Differences were noted among the main compound abundance in the headspace (although the major thiols and disulfides were qualitatively identic in both Allium species) and dipropyl disulfide, diisopropyl trisulfide, and (Z)-prop-1-enyl propyl trisulfide were predominant sulfides. Identification of amino acids and their quantities were determined by reverse-phase HPLC. Most abundant amino acids in both onions were arginine (Arg) and glutamic acid (Glu). The results of cytotoxicity testing confirmed antiproliferative effects of both species. The DNA fragmentation assay, DAPI staining and real time PCR analysis confirmed that A. cornutum and A. cepa extracts induced apoptosis in HeLa cells. This study presents the evidence for possible therapeutic use of A. cornutum and A. cepa extracts against human cervical carcinoma cell line.

Modification of the Sensory Profile and Volatile Aroma Compounds of Tomato Fruits by the Scion × Rootstock Interactive Effect.

Sensory quality is of increasing importance to consumer decisions in choosing a product, and it is certainly an important factor in repurchasing in terms of meeting the necessary aroma quality and taste properties. To better understand the effects of rootstocks and scions on fruit quality, the sensory profile and volatile aroma composition of the fruits of hydroponically grown tomato plants were evaluated. Experiments were established using the tomato cultivars Clarabella and Estatio as scions during two spring-summer seasons. In both experiments, the scion plants were self-grafted or grafted onto rootstocks of cultivars Arnold, Buffon, Emperador, and Maxifort, with the exception that in experiment 1, the Estatio scion was not grafted onto Buffon. The scions and rootstocks caused differences in observed sensory properties in both experiments. For most of the sensory traits, interaction effects between scion and rootstock were observed. Compared to those obtained from self-grafted Clarabella, the fruits obtained from Clarabella grafted onto Buffon in the first experiment and Clarabella grafted onto Arnold in the second experiment were sweeter by one measurement unit. The contents of seven aldehydes, six alcohols, five terpenes and two ketones were determined. A lower accumulation of total aldehydes, 22-45%, due to lower amounts of pentanal, (E)-2-heptanal and (E,E)-2,4-decadienal, was found in the fruits from plants where Estatio was rootstock compared with the other rootstocks treatments. Clarabella as a rootstock increased (Z)-3-hexenal + (E)-2-hexenal accumulation from 35 to 65%. Grafting Clarabella onto the tested rootstocks led to a change in the composition of volatile compounds, while differences between the combinations with Estatio as a scion were generally not recorded. Fruits from self-grafted Clarabella had higher (Z)-3-hexenal + (E)-2-hexenal concentrations than did fruits from Clarabella grafted onto Arnold (for 54%) and Emperador (for 68%), and in the second experiment, grafting onto all commercial rootstocks reduced (Z)-3-hexenal + (E)-2-hexenal concentrations, from 25 to 74%, compared to those from self-grafted Clarabella. Higher (+)-2-carene and (-)-caryophyllene oxide concentrations were attained in plants in which Clarabella was grafted onto Maxifort (by 56%) and plants in which Estatio was grafted onto Arnold (by 36%) compared to self-grafted plants. This study showed the possibility of altering the composition of volatile aroma compounds and sensory properties of tomato fruits by the use of grafting techniques.

Differentiation Between Unfiltered and Filtered Oblica and Leccino cv. Virgin Olive Oils.

The quality parameters, a variety of microcomponents, and the sensory characteristics of Oblica and Leccino cv. virgin olive oils (VOOs) were evaluated before and after filtration process adopted in order to estimate the individual varietal compositional changes. The dynamics of the formation of hydrolytic and oxidative changes in unfiltered (UF) and filtered (F) oils was asses by comparing level of free fatty acids (FFA), peroxide value (PV), and spectrophotometric indices periodically during 1 year of oil storage. An analysis of phenolics, tocopherols, and fatty acids was determined by chromatographic (HPLC and GC) and spectrometric methods, oxidative stability by Rancimat method while sensory analyses of obtained olive oils were performed by a trained professional panel. Single monovarietal VOO loses phenols in different rate with the applied filtration. Total secoiridoids decreases significantly in "Oblica" VOOs while no changes in their concentrations were found between unfiltered and filtered "Leccino" oils. Intensity of desired sensory properties decreases with filtration. In "Leccino" VOOs decrease of oxidative stability was more pronounced. After 12 months of storage, filtered "Leccino" VOOs had significantly lower FFA values than observed for the unfiltered counterparts. Further, there were no significant changes in PV and K270 values between unfiltered and filtered oils of both studied varietal oils. Storage time influenced more studied quality parameters than filtration, during which PV of unfiltered oils faster deteriorate. The highest changes between stored and corresponding fresh samples were exhibited in unfiltered "Oblica" VOOs. PRACTICAL APPLICATION: Quality enhancement of olive oil is constantly being done professionally and scientifically. The information provided in this study can be used in the industry of olive oil for improve the phenolic content, oxidative stability, and the sensory quality of virgin olive oils. The findings of stability test could be guidelines for mindful leading of the oil finishing up to bottling.

Production and Refinement of Omega-3 Rich Oils from Processing By-Products of Farmed Fish Species.

In this study, the effect of a four-stage chemical refining process (degumming, neutralization, bleaching, deodorization) on the quality parameters, fatty acid composition and volatile compounds of crude oils produced from processing by-products of farmed fish species (tuna, seabass and gilthead seabream) was evaluated. The quality of the oils was compared to commercially available cod liver oil on the basis of free fatty acid, peroxide value, p-anisidine, total oxidation (TOTOX), thiobarbituric acid reactive species (TBARS), oxidative stability at 80, 100 and 120 °C, tocopherol content, and volatile components, while the fatty acid profile and the proportion of polyunsaturated fatty acids (PUFAs) were used as an indicator of the nutritional values of fish oils. Quality parameters of the studied oils and oil oxidative stability were enhanced with refining and were within the limits recommended for fish oils without the loss of PUFAs. In tuna by-product refined oils, the proportion of PUFAs was over 40%, with 30% of eicosapentaenoic and docosahexaenoic fatty acids. The volatile compounds of the oils were quantified (in mg/kg) and major components were 2,4-heptadienal, pentadecane, 2,4-decadienal, 2,4-nonadienal and dodecane. The use of aquaculture by-products as an alternative source for fish oil production could contribute to a more sustainable and profitable aquaculture production, providing economic benefits for the producers and setting new standards for a fish by-product disposal strategy.

Effect of winemaking on phenolic profile, colour components and antioxidants in Crljenak kastelanski (sin. Zinfandel, Primitivo, Tribidrag) wine.

Crljenak kastelanski or Tribidrag is a Croatian autochthonous grape variety which is a parent of the America's most popular variety Zinfandel or Italian Primitivo. The aim of this study was to investigate the dynamics of extraction of phenolics and changes in antioxidant activity of the samples collected during maceration of C. kastelanski grapes processed using two different macerating enzymes. According to the obtained results it can be concluded that wines from C. kastelanski grapes are great source of bioactive phenolics although differences in phenolic profiles between control and enzyme-treated wines were detected. The highest content of total phenolics was detected in control wine (2691 mg GAE/L). Use of pectolitic enzyme Vinozym Vintage improved anthocyanin extraction, while higher colour parameters were observed for wine samples produced using Sihazym Extro. The statistical analysis confirmed great influence of total phenolics and concentrations of some individual phenolic compounds (e.g. catechin, gallic acid, epicatechin) on reducing and free radical scavenging activity of wine samples while the impact of anthocyanins was not detected. According to the obtained results it can be concluded that use of enzymes has slightly negative effect on total phenolics and wine antioxidant properties, but it increases the extraction yield of wine colour components what enables shorter maceration and prevents colour losses during the aging process.

Manganese soil and foliar fertilization of olive plantlets: the effect on leaf mineral and phenolic content and root mycorrhizal colonization.

BACKGROUND: The present study aimed to examine the effect of foliar (Mn_fol) and soil Zeolite-Mn (Mn_ZA) application on leaf mineral, total phenolic and oleuropein content, and mycorrhizae colonization of self-rooted cv. Leccino plantlets grown on calcareous soil. RESULTS: The dissolution of zeolite was 97% when citric acid was applied at 0.05 mM dm-3 , suggesting that organic acids excreted by roots can dissolve modified zeolite (Mn_ZA), making Mn available for plant uptake. The leaf Mn concentration was the highest for Mn_fol treatment at 90 days after transplanting (DAT) (172 mg kg-1 ) and 150 DAT (70 mg kg-1 ) compared to other treatments. Mn_ZA soil application increased leaf Mn concentration at 150 DAT compared to control and NPK treatments. The oleuropein leaf content was highest for Mn_fol compared to other treatments at 90 DAT and lowest at 150 DAT. Arbuscular mycorrhizal colonization was higher for Mn_fol treatment at 150 DAT compared to all other treatments. CONCLUSION: Changes in the arbuscular colonization percentage and oleuropein content may be connected to stress conditions provoked by a high leaf Mn concentration in the Mn_fol treatment at 90 DAT. Mn_ZA application increased leaf Mn concentration at 150 DAT compared to control and NPK treatments. It can be assumed that the dominant mechanism in Mn uptake from modified zeolite is Mn_ZA dissolution through root exudates. © 2018 Society of Chemical Industry.

Discovery of novel quaternary ammonium compounds based on quinuclidine-3-ol as new potential antimicrobial candidates.

Quaternary ammonium compounds (QACs) are amphiphilic molecules displaying a broad-spectrum of antibacterial activity. QACs are commonly used antiseptics in industrial, home and hospital settings. Given the emergence of the QAC-resistant bacteria, there is an urgent need to design new QACs with good antimicrobial activity, able to escape the host resistance mechanism. Therefore, a series of QACs derived from quinuclidine-3-ol and an alkyl chain of variable length (QOH-C3 to -C14), was designed and synthesized. The antimicrobial potential of the new monoquaternary QACs was surveyed against seventeen strains of emerging food spoilage and pathogenic microorganisms, including clinical multidrug-resistant ESKAPE isolates. The QOH-C14 proved to have the strongest antimicrobial activity. It was highly active against all pathogens tested, particularly against the Gram-positive bacteria with minimal inhibitory concentrations (MICs) ranging from 0.06 to 3.9 µg/mL, and fungi exerting the MIC90 between 0.12 and 3.9 µg/mL. The potency of QOH-C14, confirmed that alkyl chains are an important part of the structure with their lengths playing a critical role in bioactivity of these compounds. The atomic force microscopy images show the disruption of a cell membrane upon the treatment with QOH-C14. These results were additionally confirmed by flow cytometry and fluorescence microscopy. A relatively low toxicity toward healthy human cells underline that QOH-C14 has a potential as new QAC antimicrobial candidate.

Substrate-Induced Conformational Changes of the Tyrocidine Synthetase 1 Adenylation Domain Probed by Intrinsic Trp Fluorescence.

Nonribosomal peptide synthetases (NRPS) are multifunctional proteins that catalyze the synthesis of the peptide products with enormous biological potential. The process of biosynthesis starts with the adenylation (A) domain, which during the catalytic cycle undergoes extensive structural rearrangements. In this paper, we present the first study of the tyrocidine synthetase 1 A-domain (TycA-A) fluorescence properties. The TycA-A protein contains five potentially fluorescent Trp residues at positions 227, 301, 323, 376 and 406. The contribution of each Trp to the TycA-A emission was determined using protein variants bearing single Trp to Phe substitutions. The accessibility of the Trp side chains during adenylation showed that only W227 is affected by substrate binding. The protein variant containing solely fluorescent W227 residue was constructed and further used as a probe to explore the binding effect of different non-cognate amino acid substrates. The results indicate a different accessibility of W227 residue in the presence of non-cognate amino acids, which might offer an explanation for the higher aminoacyl-adenenylate leakage. Overall, our results suggest that intrinsic tryptophan fluorescence could be used as a method to probe the effect of substrate binding on the local structure in NRPS adenylation domains.

Chemical Composition and Biological Activity of Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842) Methanolic Extracts.

Here, we report a comparative study of the phytochemical profile and the biological activity of two onion extracts, namely Allium cepa L. and Allium × cornutum (Clementi ex Visiani 1842), members of the family Amaryllidaceae. The identification of flavonoids and anthocyanins, and their individual quantities, was determined by high-performance liquid chromatography (HPLC). The potency of both extracts to scavenge free radicals was determined by the DPPH (2,2'-diphenyl-1-picrylhydrazyl) radical-scavenging activity and oxygen radical absorbance capacity (ORAC) methods. The DNA protective role was further tested by the single-cell gel electrophoresis (COMET) assay and by Fenton's reagent causing double-strand breaks on the closed circular high copy pUC19 plasmid isolated from Escherichia coli. In the presence of both extracts, a significant decrease in DNA damage was observed, which indicates a protective role of Allium cepa and Allium × cornutum on DNA strand breaks. Additionally, cytotoxicity was tested on glioblastoma and breast cancer cell lines. The results showed that both extracts had antiproliferative effects, but the most prominent decrease in cellular growth was observed in glioblastoma cells.

Evaluation of Olive Fruit Lipoxygenase Extraction Protocols on 9- and 13-Z,E-HPODE Formation.

In plant tissues, enzymes implicated in the lipoxygenase (LOX) pathway are responsible for the hydroperoxydation of polyunsaturated fatty acids, ultimately leading to the production of small chemical species involved in several physiological processes. During industrial olive oil production, these enzymes are activated upon crushing and grinding of olive fruit tissue, subsequently leading to the synthesis of volatile compounds responsible for the positive aroma and flavor of the oil. An investigation of LOX activity during olive fruit ripening and malaxation could assist in the production of oils with favorable aroma and taste. Therefore, a reliable method for olive LOX purification is crucial. Here we report a critical review of six LOX extraction protocols, two of which have shown minimum enzyme activity, possibly leading to misconceptions in the interpretation of experimental data. Future research concerning olive LOX should employ extraction methods that preserve enzyme activity.

The A9 core sequence from NRPS adenylation domain is relevant for thioester formation.

The adenylation (A) domain in nonribosomal peptide synthetases catalyses a two-step reaction in which an amino acid is activated and then transferred to the neighbouring thiolation (T) domain. In this study, we investigated the role of the conserved A9 core sequence of the A-domain of tyrocidine synthetase 1, by analysis of single amino acid mutations in the A9 region. Mutation of an absolutely conserved proline (P490G) significantly reduced the conformational stability of the protein, as evidenced by increased susceptibility to proteolytic cleavage and denaturation. All mutant A-domains were capable of amino acid activation, but the activity in the overall reaction was reduced. Surprisingly, the S491R mutant (mutation at the first residue following the A9 motif) showed elevated overall activity compared to the wild-type protein. Our results suggest that the A9 core sequence plays a role in the second reaction step, in which it could serve as a "clip" for the proper positioning of residues important for the interaction with the T-domain, and/or stabilisation of the thioester-forming conformation.