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Mustard seeds are used as a food, and spice due to their nutritive value, the presence of biologically active compounds, and specific taste. Its morphology was investigated by optical microscopy and scanning electron microscopy before and after isolation by hydrodistillation and microwave assisted distillation. The chemical composition of volatile compounds in different mustard seeds (Brassica juncea L. and Brassica nigra L.) was determined using GC– MS, after applying various techniques (Clevenger hydrodistillation, microwave assisted distillation —MAD, and microwave hydrodiffusion and gravity— MHG). Allyl isothiocyanate, degradation product of allyl glucosinolate, was the main volatile compound in B. juncea essential oils and extract after all applied extraction techniques (91.07– 99.01%). Gastrointestinal stability of allyl isothiocyanate was determined employing two-phase digestion model (gastric and duodenal) by two methods (in vitro digestion method using commercial and ex vivo digestion method using human digestive enzymes). The stability rate of allyl isothiocyanate was higher after the gastric digestion phase by both methods. In B. nigra seeds but- 3-enyl and allyl isothiocyanate were the main compounds after Clevenger hydrodistillation (80.58% and 15.39%, respectively). After MAD, and MHG the main compounds were 4, 5- epithiopentanenitrile (50.70% and 59.93%, respectively), and 3, 4-epithiobutanenitrile (7.61% and 25.97%, respectively), originating from the same glucosinolates, i.e. but-3-enyl and allyl glucosinolate, respectively. Antiproliferative activity of mustard seed EOs and extracts was evaluated against human cancer cell lines (MDA-MB- 231 and TCCSUP). The best antiproliferative activity was shown for B. nigra MHG extract against MDA-MB-231 cell line with IC50 value of 9.1 µg/mL.

The aim of this study was to determine the effects of various processing techniques on the antiproliferative activity of blueberry phenolics against the human breast adenocarcinoma cell line (MDA-MB-321) before and after simulated a two- phase digestion process (stomach and duodenum) based on human digestive juices. Because of the highest content of phenols (TPC), the undigested (122 mg GAE/100 g) and digested freeze-dried blueberry (303.36 mg GAE/100 g) was then tested for its antiproliferative activity against three carcinoma cell lines: cervical cancer cells (HeLa), human colon cancer cells (HCT116), and human osteosarcoma cells (U2OS). The obtained results showed that high-intensity ultrasound treatment and freeze-drying had a better effect (52.67% and 43.41%, respectively) on the cytotoxic activity of blueberry against breast cancer cells than thermal processing (32.31%). Gastric digestion did not decrease the cytotoxic activity in all cancer cell lines, while this activity decreased significantly after the digestion phase in the duodenum.

The influence of freeze-drying, pasteurisation and high-intensity ultrasound on gastrointestinal stabilityand antioxidant activity of cultivated blueberry phenolics was investigated. Quantitative and qualitativeanalyses were done before and after a two-phase in vitro digestion by human digestive enzymes. Antioxi-dant capacity of blueberry phenolics before and after simulated digestion was evaluated using DPPH andthe FRAP assays. After the simulated gastric digestion phase, high stability of phenolics was determined, especially after freeze-drying. During the simulated duodenal digestion, the most affected compounds wereanthocyanins. The average loss of quercetin derivatives was 60%, while in all ultrasound- treated samples, a 3- fold increase in the concentration of phenolic acids was observed. The highest antioxidant activityafter in vitro gastric and duodenal digestion was determined in a freeze- dried sample. The results afterin vitro digestion demonstrated that the use of freeze-drying and high-intensity ultrasound can ensure bet-ter gastrointestinal stability and higher antioxidant activity of blueberry phenolics.

The aim of the present research was to study the chemical profile of volatile and non-volatile compounds in freeze- and spray-dried oregano, thyme, sage, and rosemary as well as to study the gastrointestinal stability and the biological activity of the samples after encapsulation using two proteins (soy and whey). Chemical analysis was performed by HS-SPME/GC-MS and HPLC-PDA techniques. The antioxidant activity (FRAP, DPPH and ORAC assays) and HPLC-PDA analyses were performed before and after simulated two-phase digestion process (gastric and duodenal) using human gastrointestinal enzymes. The effect of protein carriers was depended on the plant matrix and the class of compound. Soy protein is useful for the protection of thymol and carvacrol content in thyme and the increase of the antioxidant activity of sage. Whey protein is useful for flavanols stability in all studied herbs before and after the duodenal digestive phase.

The glucosinolates (GSLs) of Matthiola incana were characterized and quantified as desulfoGSLs in different plant tissues (seeds, siliquae, stems, leaves, and flowers) using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-DAD-MS/MS). The major GSL detected in all plant parts was 4-(methylsulfinyl)but-3-enyl GSL (glucoraphenin, 1), followed by 4-(methylsulfanyl)but-3-enyl GSL (glucoraphasatin, 2), while indol-3-ylmethyl GSL (glucobrassicin, 3) was also found in the flowers. GSL 2 was found to be the dominant in the plants analysed after only 2 days of drying, contrary to all analyses performed after longer storage of plant material, thus suggesting an oxidation of 2 into 1. The stability of GSLs was monitored over a one-month period. The desulfo-counterpart of 1 was unstable in water, showing a dramatic decrease (ca 80%) during the observation time, with spontaneous conversion into cyclic thioimidate N-oxides. Bioaccessibility of GSLs through the simulated ex vivo gastrointestinal digestion was determined using human gastrointestinal juices. Glucoraphenin 1 showed high bioaccessibility in the gastric phase (>90%), while both GSLs 1 and 2 were transformed in the intestinal phase.

Blueberries (Vaccinium corymbosum, L.) are a valuable source of health-promoting bioactive compounds, particularly polyphenols. The beneficial effects of polyphenols on health depend not only on food sources but also on their stability, which can vary depending on the post-harvest processing methods, the matrix in which they are incorporated, and endogenous factors such as microbiota and digestive enzymes. The aim of this study was to investigate the influence of high-intensity ultrasound treatment on in vitro gastrointestinal stability of the total polyphenol content and antioxidant capacity of blueberries. Blueberry puree was treated with high-intensity ultrasound using different amplitudes (30, 45 and 60μm) and treatment times (3, 6 and 9 min), and a part of sample was used as a control (untreated sample). Total phenolic content (TPC) and antioxidant capacity (by DPPH and FRAP methods) were determined prior to and after two-phase in vitro digestion process (gastric and duodenal) using human gastrointestinal enzymes. The results showed a high stability of total blueberry polyphenols and antioxidant activity during simulated gastric digestion step, both in untreated and ultrasound treated samples. The use of high-intensity ultrasound had a significant effect on the stability of polyphenols and their antioxidant activity after simulated digestion in duodenum. Significant increase in polyphenol content after duodenal digestion step was determined in three ultrasound treated samples with longer treatment time (6 and 9 minutes), and only sample with significant reduction of TPC was untreated sample. Modification of the food matrix as a result of cavitation from ultrasound treatment may be the reason for higher stability of polyphenol during in vitro digestion. After in vitro digestion blueberry polyphenols still possessed antioxidant activity that may be directly related to their beneficial effects on health. The highest antioxidant activity measured with DPPH and FRAP method was determined for sample treated with maximum amplitude (60μm) and longest time (9 minutes). Determination of the stability of phenolic compounds is important as it affects their bioavailability for uptake as well as their possible beneficial effects on intestinal epithelial tissue such as reduction of oxidative stress and cancer prevention. Our results showed that ultrasound treatment can preserve the blueberry phenolics and their antioxidant characteristics after in vitro digestion.

The aim of this study was to determine the phenolic profile, antiproliferative, and pro-apoptotic activities of Asparagus acutifolius, Tamus communis, and Ruscus aculeatus aqueous extracts against human bladder (T24) and lung cancer (A549) cell lines. Antiproliferative activity of the extracts at the concentration range from 0.5 to 4 mg/ml was measured by MTT test, after the incubation period of 4, 24, 48, or 72 hr. Pro-apoptotic activity of selected plant extracts against T24 and A549 cells was analyzed by flow cytometry at the concentrations of 0.5, 1, and 2 mg/ml. The highest antiproliferative activity against both cancer cell lines was detected by T. communis aqueous extract. The highest pro-apoptotic effect was achieved by A. acutifolius aqueous extract and its effect was similar against A549 and T24 cells. PRACTICAL APPLICATIONS: Wild edible plants, such as wild asparagus, black bryony, and butcher's broom represent important aspect of the Mediterranean diet. They are rich source of various natural bioactive compounds which express different biological activities (antioxidative, antiproliferative, antibacterial, anti-inflammatory…). The current trend for natural and healthy diet, such as the Mediterranean diet, is in relation to the rapidly growing problem of chronic diseases in a modern society. Wild edible plants have a high direct-use value using them in dishes containing mixed vegetables, in soup preparation, as natural additives in fermented natural products containing cheese or fish, in water infusions (natural drinks).

Spectrophotometric and chromatographic analysis of phenolics in water and ethanolic extracts of wild asparagus, butcher’s broom and black bryony from Croatia was conducted. Their antioxidant capacity (ABTS, DPPH and FRAP assay) and energy content were determined. The gastrointestinal stability of detected phenolics was determined using a two-phase in vitro digestion method with human enzymes. The highest phenolics yield, radical scavenging activity and ferric reducing antioxidant potential were recorded in 40 % ethanolic extract of black bryony, with glycosylated forms of kaempferol as dominant components. Quercetin-3-O-rutinoside and isorhamnetin-3- Orutinoside were dominant phenolics in all wild asparagus extracts, and salicylic acid was predominant in butcher’s broom 40 % ethanolic extract. Phenolic acids of the three species were not stable during gastric and duodenal phases of simulated digestion. Two main black bryony kaempferol glycosides were best preserved after digestion (50 % of each). Black bryony contains more energy than wild asparagus and butcher’s broom. Accordingly, we propose black bryony as a valuable source of antioxidant kaempferol glycosides with relevant gastrointestinal stability and higher energy content than so far more conventional vegetable wild asparagus.

International audience; Market interest in aromatic plants from the Mediterranean is continuously growing mainly due to their medicinal and bioactive compounds (BACs) with other valuable constituents from essential oils (EOs). From ancient times, these plants have been important condiments for traditional Mediterranean cuisine and remedies in folk medicine. Nowadays, they are considered as important factors for food quality and safety, due to prevention of various deteriorative factors like oxidations and microbial spoilage. EOs have different therapeutic benefits (e.g. antioxidant, anti-inflammatory, antimicrobial, and antifungal), while BACs mostly affect nutritive, chemical, microbiological, and sensory quality of foods. Currently, many plant extracts are used for functional (healthy) foods, which additionally fuels consumer and industrial interest in sustainable and non-toxic routes for their production. EO yields from dried plants are below 5%. Their extraction is strongly dependent on the hydrophobic or lipophilic character of target molecules, hence the common use of organic solvents. Similarly, BACs encompass a wide range of substances with varying structures as reflected by their different physical/chemical qualities. Thus, there is a need to identify optimal non-toxic extraction method(s) for isolation/separation of EO/ BCs from plants. Various innovative non-thermal extractions (e.g. ultrasound-, high-pressure-, pulsed electric fields assisted extraction, etc.) have been proposed to overcome the above mentioned limitations. These techniques are “green” in concept, as they are shorter, avoid toxic chemicals, and are able to improve extract yields and quality with reduced consumption of energy and solvents. This research provides an overview of such extractions of both BAC and EOs from Mediterranean herbs, sustained by innovative and non-conventional energy sources.

Four Dalmatian autochthonous olive cultivars (Buhavica, Drobnica, Lastovka and Oblica) were molecularly characterized by analyzing length variability of genomic DNA sequences encompassing 15 microsatellite repeats. Furthermore, several important parameters of olive oils were analyzed in relation to the harvest period. An analysis of major phenolics secoiridoids was done by qNMR, while the fatty acid profile of oils and squalene content was determined by GC–FID. Oxidative stability was evaluated by the Rancimat method and sensory evaluation was carried out by a trained professional panel. The results indicate that the effect of the harvest period on the phenolic profile of oils depends on the olive cultivar and is related to its genetic profile. Drobnica oil from the late harvest contained an extremely high concentration of oleocanthal + oleacein (966 mg/kg). The longest oxidative stability was achieved by Drobnica and Lastovka oils from the early harvest period (20.95 and 18.65 h). Squalene had no effect on the oil oxidative stability. This study shows that the content of phenolic secoiridoids depends mainly on the cultivar. In addition, some cultivars, such as Drobnica did not show significant change of phenolic secoiridoids content in relation to the harvest period.

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.

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

The use of micro- and nanoencapsulation technology represents promising field for the delivery of nutrients, their bioavailability increases and oxidative stability prolongation. The objective of this study was to prepare phenolics extracts from cultivated blueberry (raw, freeze-dried, pasteurized and treated with high intensity ultrasound) and to determine the rate of the putative prolongation of the oxidative stability of the olive oil by phenolics extracts in microemulsions and in liposomes. The droplet size of microemuslions was evaluated using photon correlation spectroscopy (PCS) and it was

Nutritional value of extra virgin olive oil is associated with its complex chemical composition. The aim of this study was to determine phenolic secoiridoids in EVOOs from autochthonous Croatian cultivars (Drobnica, Krvavica, Lastovka, and Oblica) by qNMR, to determine simple phenolics by UPLC, as well as to analyse the fatty acid profile, the antioxidant activity and the oxidative stability of selected oils. This is the first study on chemical and biological characterization of selected autochthonous olives varieties. Drobnica EVOO contained the highest amount of total phenols and major secoiridoid derivatives (oleocanthal, oleacein, oleuropein aglycon, and ligstroside aglycon) compared to other oils. The antioxidant activity of Drobnica phenolics was very high by FRAP and copper-induced LDL oxidation assays, while the oxidative stability of Drobnica oil by Rancimat method was very long (23 h). Practical applications: This study represents the contribution to the research of chemical and biological potential of monovarietal extra virgin olive oil from Croatia. EVOOs from selected Croatian autochthonous cultivars had very high phenolic content that is related to high inhibitory rate of copper-induced oxidation of human LDL as well as the long oxidative stability. Drobnica EVOO showed very long oxidative stability. EFSA approved health claim on olive oil polyphenols (EU, 432/2012) and selected Croatian cultivars, especially Drobnica, are of interest due to its high phenolic content and strong biological potential.

In contrast to the strawberry fruits, strawberry leaves as a source of bioactive compounds with potentially beneficial biological effects have been largely overlooked. In this study we examined direct, dose dependent effects of wild strawberry (Fragaria vesca, L.) leaves aqueous extract, in two experimental models and animal species, the isolated guinea pig hearts and rat aortic rings. Vasodilatory potential of the wild strawberry leaves extract was compared with vasodilatory activity of aqueous extract of hawthorn (Crataegus oxycantha, L) leaves with flowers, which can be regarded as a reference plant extract with a marked vasodilatory activity. The extracts were analysed by their “ phenolic fingerprint” , total phenolic content and antioxidative capacity. Their vasodilatory activity was determined and compared in the intact, isolated aortic rings from 24 rats, that were exposed to the extracts doses of 0.06, 0.6, 6, and 60 mg/100 ml. Both extracts induced similar, dose dependent vasodilation. Maximal relaxation was 72.2 ± ; ; ; ; 4.4 % and 81.3 ± ; ; ; ; 4.5 %, induced by the strawberry and hawthorn extract, respectively. To determine vasodilatory mechanisms of the wild strawberry leaves extract, endothelium– denuded and intact rings exposed to NO synthase inhibitor L-NAME or cyclooxygenase inhibitor indomethacin were used. Removal of the endothelium prevented and exposure to L-NAME or indomethacin strongly diminished the vasodilatatory response to the extract. In the isolated hearts (n=12), the wild strawberry extract was applied at concentrations of 0.06, 0.18, 0.6, and 1.8 mg/100 ml. Each dose was perfused for 3.5 minutes with 15 minutes of washout periods. Heart contractility, electrophysiological activity, coronary flow and oxygen consumption were continuously monitored. The extract did not significantly affect heart rate and contractility, main parameters of the cardiac action that determine oxygen demands, while coronary flow increased up to 45 % over control value with a simultaneous decrease of oxygen extraction by 34 %. The results indicate that the aqueous extract of wild strawberry leaves is an effective direct, endothelium dependent vasodilator, action of which is mediated by NO and cyclooxygenase products and which potency is similar to that of the hawthorn aqueous extract.

In the present study, the chemical composition and biological activity (acetylcholinesterase inhibition and antioxidant activity) of essential oil from endemic species Pinus nigra Arnold ssp. dalmatica (Vis.) Franco was investigated. α-Pinene, β-pinene, germacrene D and s-caryophyllene were identified by GC-FID and GC-MS as dominant components of the oil. P. nigra ssp. dalmatica essential oil showed relatively high inhibitory activity on acetylcholinesterase (AChE), which can be in relation with good AChE inhibitory activity of pure main monoterpene components of the oil (S-a-pinene) and their mixtures (S‑a-pinene:S-s-pinene and R-a-pinene:S-s-pinene, in ratio 3:2). Essential oil from P. nigra ssp. dalmaticadid not show significant antioxidant activity tested by three methods: DPPH radical scavenging method, Ferric Reducing Antioxidant Potential (FRAP) and Thiobarbituric Acid Reactive Species (TBARS) assays. Key words: Acetylcholinesterase inhibition, antioxidant activity, essential oil, Pinus nigrassp. dalmatica, endemic species.

Biološka aktivnost spojeva iz namirnica biljnog podrijetla u organizmu uvjetovana je metaboličkom stabilnošću spojeva iz namirnica i stupnjem njihovih promjena tijekom probavnog procesa. Kapari (Capparis spinosa L.) i petrovac (Crithmum maritimum L.) su aromatične biljke s raznovrsnom primjenom u tzv. mediteranskom modelu prehrane. Zanimljive su zbog zastupljenosti spojeva (flavonoidi i fenolne kiseline) s izraženom biološkom aktivnošću u in vitro uvjetima. Prema rezultatima više različitih modela, vodeni ekstrakti obaju biljaka pokazuju vrlo jako antioksidacijsko djelovanje koje se očituje kroz visok stupanj uklanjanja slobodnog DPPH radikala te kroz sposobnost sprječavanja procesa oksidacije masti u modelima namirnica (metoda izbjeljivanja β -karotena i metoda s tiobarbiturnom kiselinom) i u biološkom modelu (sprječavanje oksidacije lipoproteina male gustoće izoliranog iz krvi čovjeka). Također, vodeni ekstrakt kapara pokazuje vrlo jako antiproliferacijsko djelovanje na stanice tumora debelog crijeva, izazivajući redukciju S faze i povećavajući G2 fazu staničnog ciklusa stanica tumora, tip HT-29. Rezultati simuliranog procesa probave pokazali su da su fenolne kiseline (klorogenska kiselina i njezini derivati), kao dominantni spojevi vodenog ekstrakta petrovca, i flavonoidi (rutin, kemferol-3- O-rutinozid, izoramnetin-3-O-rutinozid), glavne komponente vodenog ekstrakta kapara, izrazito nestabilni u uvjetima visokog pH u tankom crijevu. Ireverzibilne strukturne promjene fenolnih spojeva u vodenim ekstraktima odabranih biljaka rezultirale su potpunim gubitkom antioksidacijske aktivnosti ekstrakata nakon simuliranog procesa probave.